Manuel Lopes Lima PHD Thesis Final

Combining phylogeny,
systematics and
D
ecology to advance
the conservation of
freshwater mussels
(Bivalvia: Unionida)
Manuel Peixoto de Magalhães Lopes Lima
Doutoramento em Biodiversidade, Genética e Evolução
Departamento de Biologia
2020
Orientador
Pedro Beja
CIBIO/InBIO, Laboratório Associado, Universidade do Porto
Coorientador
Ana Filipa Filipe
CIBIO/InBIO, Laboratório Associado, Universidade do Porto
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Combining phylogeny, systematics and ecology to advance the conservation of freshwater mussels (Bivalvia: Unionida)
Acknowledgments
This dissertation would not be possible without the help of my family that was supportive in so
many ways to my “lunacy” in pursuing such an “extraordinary” research field such as biology
and conservation of molluscs. I would like to thank to my supervisors for the help and critical
analyses on the development of the thesis and to all of the members of the “Brave Mussel
Warriors” team that cooperated and worked with me in so many hours of field and laboratory
work, as well as in the writing and elaboration of the manuscripts. Additionally, this work would
not be possible without the participation of all the co-authors of the manuscripts.
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Abstract
The world is currently experiencing a biodiversity crisis, with many species facing the risk of
extinction. This is particularly true for those in freshwater habitats, which are isolated between
land and sea, and generally more exposed to human activities. Therefore, many freshwater
groups of animals are now threatened with extinction and requiring urgent conservation
measures. However, most current conservation efforts remain directed to charismatic
terrestrial vertebrates, like mammals and birds. Although invertebrates dominate on Earth both
in species richness and biomass, knowledge about these taxa is scarce and many groups
need urgent conservation attention. This is the case of freshwater bivalves of the Unionida
order, also known as freshwater mussels (FM). These bivalves are strictly freshwater
inhabitants and originally dominated many freshwater habitats across the planet. They are
important for the aquatic ecosystem functioning, playing key ecological roles and providing
important ecosystem services to humans. They are also very interesting from the biological
point of view, having a series of interesting traits that allow them to live in running water, like
internal fertilization and parental care of their larvae. Especially, they have a unique life cycle
in which FM larvae need to attach to a host (generally a fish) until adulthood, for nutrition but
mainly for upstream dispersion. Another interesting feature is their rare form of mitochondrial
inheritance, also called doubly uniparental inheritance (DUI), where the males inherit
mitochondria from both parents. Male M- and female F-type mitochondrial lineages are highly
divergent and are remarkable for the study of mtDNA evolution.
The overarching goal of this dissertation is to advance the conservation biology of
freshwater mussels by combining research on phylogeny, systematics and ecology, showing
how the integration of multiple research fields has practical implications to preserve highly
endangered taxa. Specifically, the dissertation aims to: (i) understand the geographical
diversity and conservation status patterns of freshwater bivalves, disclosing their main threats,
and needs for conservation and research; (ii) highlight and discuss the importance of basic
biological studies for the conservation of freshwater mussels; and (iii) accurately define species
and integrate evolutionary patterns into conservation planning.
In Chapter 1, I start by introducing the global decline of biodiversity, focusing primarily
on freshwater taxa and especially on the target taxonomic group of the dissertation, the
freshwater mussels (FM). Then, their high ecological and economic importance are
highlighted, as well as their unique biological features. I also discuss the importance of
integrating basic research on species biology, taxonomy and phylogenetic patterns for
conservation. Finally, the chapter presents the general and specific objectives of the thesis.
In Chapter 2, the geographical diversity patterns of freshwater bivalves were revised
and their conservation status, threats, and the main needs for conservation and research are
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analysed and discussed. Chapter 2 reveals that among the several freshwater bivalve groups
analysed, the most threatened by far are FMs, which support the main focus of the following
chapters. We show that freshwater mussel diversity is geographically heterogeneous with two
main hotspots of diversity, the Mississippi basin and associated basins in central North
America, and the Indotropical rivers in Southeast Asia. The main global threats are associated
with habitat degradation, while the most mentioned research needs globally refer to the need
to collect baseline information on distribution, taxonomy, abundance, life-history traits, and
threats. In terms of the needed conservation measures, protection and management of
freshwater habitats are the most cited
The lack of baseline biological information identified in Chapter 2 is then addressed in
Chapter 3, which provides a baseline study on the Iberian dolphin mussel Unio delphinus.
Chapter 3 highlights the importance of basic biological studies for conservation planning and
the potential use of biological traits as environmental indicators. For this, the distribution,
growth, host-fish range and reproductive cycle of this species are described and discussed.
Unio delphinus occupies the western Iberian River basins and, contrary to other well-known
European FM species, is found to grow fast and to be short-lived. Their larvae may attach to
most co-occurring fish species, but only native species were effective hosts. Based on these
results, we reassessed its conservation status and provide recommendations on key
conservation measures.
In Chapters 4-8 the phylogenetic relationships among important groups of FMs are
estimated and analysed, thereby addressing the need to accurately define conservation and
management units, and to include evolutionary patterns in conservation planning. In Chapters
4 and 6, we estimated with a small number of molecular markers, the most comprehensive
phylogenies so far for the most representative families of FMs in the northern hemisphere, the
species-rich Unionidae and the threatened Margaritiferidae. The systematics and taxonomy
within these families are updated with the help of an exhaustive search for diagnostic and
synapomorphic, ecological and morphological characters. The distribution of the main groups
inside those families is mapped and their biogeographic patterns discussed.
Chapter 5 focuses on a contentious group of North American species, that were
originally lumped in a single genus, i.e. Quadrula sensu lato. We estimated its phylogeny and
used molecular species delineation methods complemented by the assessment of ecological,
morphological, and anatomical traits to revise the systematics and taxonomy of these species.
Then we provide conservation guidance based on these results.
In Chapters 7 and 8, we updated the phylogenies within the Unionidae, this time with
a multi-locus approach using whole mitogenomes. Given that many groups of FMs lack
morphological diagnostic characters, it is important to develop new features that help us to
characterize the main evolutionary history of FMs. The mitochondrial genome gene
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arrangement is generally very conserved across taxa and eventual shifts in this order are rare
in many taxa. In chapters 7 and 8 we also explored the use of mitogenome orders to be used
as diagnostic of the higher-order taxonomic groups within FMs.
The present dissertation brings important advances in the basic biology, phylogeny,
biogeography, and conservation of FMs globally. It presents more clear evolutionary
relationships and biogeographical patterns among the main FM groups and highlights the
biodiversity hotspots or areas where their levels of extinction risk, and species richness and
genetic diversity are higher. Finally, this dissertation identifies the main knowledge gaps and
threats for FM species to guide future research and conservation actions.
Keywords
Biogeography, Bivalvia, Conservation, Distribution, Doubly uniparental inheritance,
Freshwater mussels, Growth, Host fish, Macroevolution, Mollusca, Phylogeny, Reproductive
cycle, Species delineation, Taxonomic classification, Unio delphinus.
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Resumo
A biodiversidade mundial está atualmente em crise, com muitas espécies em risco de
extinção. Isto é particularmente verdadeiro para os organismos que habitam ambientes de
água-doce, pois encontram-se isolados entre o mar e a terra e geralmente mais expostos às
atividades humanas. Devido a esta situação, muitos grupos de animais dulçaquícolas estão
agora ameaçados de extinção sendo necessárias medidas urgentes para a sua conservação.
No entanto, a maioria dos esforços de conservação são geralmente direcionados para as
espécies de vertebrados mais carismáticas, tais como os mamíferos e as aves. Embora na
Terra os invertebrados sejam dominantes, tanto em número de espécies quanto em biomassa,
o conhecimento sobre estes grupos é escasso e muitos deles precisam de atenção urgente
no que respeita à sua conservação. Este é o caso dos bivalves de água-doce da ordem
Unionida, também conhecidos como mexilhões de água-doce (MAD). Este grupo de bivalves
ocorre apenas em água-doce e devido às suas elevadas abundâncias originais foram durante
muito tempo um dos grupos taxonómicos dominantes em habitats de água-doce de todo o
planeta. Os MAD são importantes para o funcionamento dos ecossistemas aquáticos pois
desempenham papéis ecológicos cruciais e fornecem importantes serviços ecossistémicos
aos seres humanos. Os MAD são também muito interessantes sob o ponto de vista biológico.
Eles apresentam uma série de características interessantes que lhes permitem viver em água
corrente, tais como a fertilização interna e cuidados parentais das suas larvas mas
especialmente, um ciclo de vida único no qual as suas larvas precisam de se ligar a um
hospedeiro (geralmente um peixe) até à idade adulta, para a sua nutrição, mas principalmente
para dispersão a montante. Outra característica interessante dos MAD é que possuem uma
forma rara de herança mitocondrial, também chamada herança duplamente uniparental, onde
os machos herdam as mitocôndrias de ambos os pais. Essas linhagens mitocondriais
herdadas dos pais (tipo M) e das mães (tipo F) são altamente divergentes e são muito
interessantes para o estudo da evolução do ADN mitocondrial.
O objetivo principal desta dissertação é aumentar o conhecimento atual para a
conservação de mexilhões de água-doce, combinando filogenia, sistemática e ecologia e
mostrar como a integração destes vários campos de investigação tem implicações
importantes na preservação de organismos em risco de extinção. Especificamente, a presente
dissertação visa: (i) representar geograficamente os padrões de diversidade e estatutos de
conservação dos bivalves de água-doce, revelando as suas principais ameaças e
necessidades de conservação e investigação; (ii) destacar e discutir a importância de estudos
biológicos básicos para a conservação de mexilhões de água-doce; e (iii) integrar
metodologias de delimitação de espécies e padrões evolutivos na planificação de ações de
conservação.
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Começo o Capítulo 1, por introduzir o declínio global da biodiversidade, concentrando-

me principalmente nos taxa de água-doce e, principalmente, no grupo taxonómico alvo da
dissertação, os mexilhões de água-doce (MAD). Destaco a seguir, a sua elevada importância
ecológica e económica, bem como as suas características biológicas únicas. Discuto
posteriormente a importância para a conservação de integrar estudos básicos sobre a biologia
de espécies com a taxonomia e padrões filogenéticos. Finalmente, o capítulo apresenta os
objetivos gerais e específicos da tese.
No Capítulo 2, são revistos os padrões de diversidade geográfica dos bivalves de
água-doce, os seus estatutos de conservação, bem como analisadas e discutidas as suas
principais ameaças e as necessidades mais prementes para a sua conservação e
investigação. O Capítulo 2 revela que, dentre os vários grupos de bivalves de água-doce
analisados, de longe os mais ameaçados são os MAD, que são o foco principal dos capítulos
seguintes. Mostramos que a diversidade de mexilhões de água-doce é geograficamente
heterogénea, com dois “hotspots” de diversidade: a bacia do Mississippi e sub-bacias
associadas no centro da América do Norte e os rios Indotropicais no sudeste Asiático. As
principais ameaças globais aos MAD estão associadas principalmente à degradação do
habitat, enquanto os campos de investigação mais mencionados são a necessidade de obter
informações básicas sobre a sua distribuição, taxonomia, abundância, e características da
história de vida, bem como sobre as suas principais ameaças. Em termos das medidas de
conservação necessárias, as mais citadas são a proteção e o a gestão sustentável dos
habitats de água-doce que ocupam.
A falta de informação biológica básica identificada no Capítulo 2 é então abordada no
Capítulo 3, através de estudos sobre o MAD ibérico Unio delphinus. O capítulo 3 destaca a
importância de estudos biológicos básicos para a conservação e o uso potencial de algumas
características biológicas como indicadores ambientais. Para esse efeito, caracterizamos e
analisamos a sua distribuição, crescimento, gama de peixes hospedeiros e ciclo reprodutivo.
O Unio delphinus ocupa as bacias ocidentais da Península Ibérica e, ao contrário de outras
espécies europeias de MAD mais estudadas, cresce rapidamente e tem vida curta. As suas
larvas conseguem afixar-se à maioria das espécies de peixes que ocorrem em simpatria, mas
apenas as espécies de peixes nativos se revelaram hospedeiros eficazes. Com base nestes
resultados, reavaliamos os seu estatuto de conservação e fornecemos recomendações sobre
as principais medidas para a sua conservação.
Nos Capítulos 4-8 foram estimadas e analisadas as relações filogenéticas entre
grupos importantes de MAD, destacando também a necessidade de definir com precisão
eventuais unidades de conservação e gestão e de incluir os padrões evolutivos encontrados,
na sua conservação. Com um pequeno número de marcadores moleculares estimámos, nos
capítulos 4 e 6, as filogenias mais abrangentes até então, para as famílias mais
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representativas de MAD do hemisfério norte, a familia Unionidae que tem a maior riqueza de
espécies, e a Margaritiferidae que contem o maior número de espécies ameaçadas. A
sistemática e a taxonomia dessas famílias foi também atualizada com a ajuda de uma
investigação exaustiva aos seus caracteres diagnósticos e sinapomórficos, ecológicos e
morfológicos. A distribuição dos principais grupos dentro dessas famílias foi também mapeada
e os seus padrões biogeográficos discutidos.
O Capítulo 5 concentra-se num grupo polémico de espécies norte-americanas,
originalmente agrupadas num único género, ou seja, Quadrula sensu lato. Estimamos a sua
filogenia e revemos a sistemática e taxonomia dessas espécies usando métodos moleculares
de delimitação de espécies, complementados pela avaliação de características ecológicas,
morfológicas e anatómicas. Em seguida, fornecemos orientações para a sua conservação
com base nesses resultados.
Nos Capítulos 7 e 8, atualizamos as filogenias dentro dos Unionidae, desta vez com
uma abordagem com vários loci usando mitogenomas inteiros. Dado que muitos grupos de
MAD carecem de caracteres-diagnóstico morfológicos, é importante desenvolver outros
caracteres que nos ajudem a descrever a sua história evolutiva. A ordem em que os genes
estão dispostos no genoma mitocondrial é geralmente muito conservado entre muitos grupos
taxonómicos sendo que eventuais mudanças nessa ordem são geralmente raras. Nos
Capítulos 7 e 8, exploramos então o uso das ordens genéticas dos mitogenomas como
diagnóstico dos principais grupos taxonómicos de MAD.
A presente dissertação providencia importantes avanços para a biologia básica,
filogenia, biogeografia e conservação de MAD a nível global. Clarifica as relações evolutivas
e os padrões biogeográficos entre os principais grupos de MAD, destacando os seus
‘hotspots’ de diversidade. Por fim, esta dissertação identifica as principais lacunas no
conhecimento e as ameaças para as espécies de MAD de forma a orientar ações futuras de
investigação e conservação.
Palavras-chave
Biogeografia, Bivalvia, Conservação, Ciclo reprodutivo, Classificação taxonómica,
Crescimento, Delimitação de espécies, Distribuição, Filogenia, Herança duplamente uni
parental, Macroevolução, Mexilhões de água-doce, Moluscos, Peixe hospedeiro, Unio
delphinus.
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Table of Contents
ACKNOWLEDGMENTS I
ABSTRACT III
KEYWORDS V
RESUMO VI
PALAVRAS-CHAVE VIII
TABLE OF CONTENTS X
INDEX OF FIGURES AND TABLES XIII
LIST OF ABBREVIATIONS XXVII
CHAPTER 1. General introduction 1
1.1 The freshwater biodiversity crisis 1

1.2 Freshwater mussel diversity, importance, and conservation 3
1.3 The need for baseline biological research 6
1.4 Defining species boundaries and integrating phylogenetic diversity patterns in
conservation ranking 7
1.5 Objectives 8
1.6 Thesis structure 9
1.7 References 11
CHAPTER 2. Freshwater bivalves’ conservation, diversity, and research 18
Paper 1. Conservation of freshwater bivalves at the global scale: diversity, threats and
research needs. Lopes-Lima M, Burlakova LE, Karatayev AY, Mehler K, Seddon M,
Sousa R. Hydrobiologia 810, 1-14 (2018).
DOI: 10.1007/s10750-017-3486-7 19
CHAPTER 3. Basic biological traits of Unio delphinus 41
Paper 2. Setting the stage for new ecological indicator species: a holistic case study
on the Iberian dolphin freshwater mussel Unio delphinus Spengler, 1793. Lopes-Lima
M, Hinzmann M, Varandas S, Froufe F, Reis J, Moreira C, Araujo S, Miranda F,
Gonçalves DV, Beja P, Sousa R, Teixeira A. Ecological Indicators 111, 105987 (2020).
DOI: 10.1016/j.ecolind.2019.105987 42
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CHAPTER 4. Phylogeny of the family Unionidae 83
Paper 3. Phylogeny of the most species-rich freshwater bivalve family (Bivalvia:

Unionida: Unionidae): Defining modern subfamilies and tribes. Lopes-Lima M, Froufe
E, Do VT, Ghamizi M, Mock KE, Kebapçi Ü, Klishko O, Kovitvadhi S, Kovitvadhi U,
Paulo OS, Pfeiffer JM, Raley M, Riccardi N, Şereflişan H, Sousa R, Teixeira A,
Varandas S, Wu X, Zanatta DT, Zieritz A, Bogan AE. Molecular Phylogenetics and
Evolution 106, 174-191 (2017).
DOI: 10.1016/j.ympev.2016.08.021 84
CHAPTER 5. Phylogeny, taxonomy and species of the genus Quadrula 148
Paper 4. Revisiting the North American freshwater mussel genus Quadrula sensu lato
(Bivalvia Unionidae): Phylogeny, taxonomy and species delineation. Lopes-Lima M,
Burlakova L, Karatayev A, Gomes-Dos-Santos A, Zieritz A, Froufe E, Bogan AE.
Zoologica Scripta 48, 313-336 (2019).
DOI: 10.1111/zsc.12344 149
CHAPTER 6. Phylogeny of the family Margaritiferidae 277
Paper 5. Expansion and systematics redefinition of the most threatened freshwater

mussel family, the Margaritiferidae. Lopes-Lima M, Bolotov IN, Tu DV, Aldridge DC,
Fonseca MM, Gan HM, Gofarov MY, Kondakov AV, Prié V, Sousa R, Varandas S,
Vikhrev IV, Teixeira A, Wu R-W, Wu X, Zieritz A, Froufe E, Bogan AE. Molecular
Phylogenetics and Evolution 127, 98-118 (2018).
DOI: 10.1016/j.ympev.2018.04.041 278
CHAPTER 7. First male whole mitogenome of a Margaritiferidae species 360
Paper 6. The first Margaritiferidae male (M-type) mitogenome: mitochondrial gene

order as a potential character for determining higher-order phylogeny within Unionida
(Bivalvia). Lopes-Lima M, Fonseca M, Aldridge Dc, Bogan A, Gan Hm, Ghamizi M,
Sousa R, Teixeira A, Varandas S, Zanatta D, Zieritz A, Froufe E. Journal of Molluscan
Studies 83, 249-252 (2017).
DOI: 10.1093/mollus/eyx009 361
CHAPTER 8. Evolution of mitogenome rearrangements in freshwater mussels 371

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Paper 7. Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia:

Unionoidea) macroevolution. Froufe F, Bolotov I, Aldridge DC, Bogan AE, Breton S,
Gan HM, Kovitvadhi U, Kovitvadhi S, Riccardi N, Secci-Petretto G, Sousa R, Teixeira
A, Varandas S, Zanatta D, Zieritz A, Fonseca MM, Lopes-Lima M. Heredity 124, 182-
196 (2020).
DOI: 10.1038/s41437-019-0242-y 372
CHAPTER 9. General Discussion 404
9.1 Main claims and highlights of the dissertation 404

9.2 Freshwater mussel hotspots, main threats, and conservation needs 404
9.3 Linking systematics and phylogeny with conservation 406
9.4 Future research perspectives and conservation implications 409
9.5 References 411
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Index of Figures and Tables
CHAPTER 1. General introduction
Figure 1. Size of the world human population over the last 12,000 years. Adapted from
Roser et al (2019). 1
Figure 2. Changes of Gross domestic product (GDP), domestic material consumption,
and extraction of living biomass (trillion USD at 2010 value) for groups of
countries at distinct development levels. Adapted from IPBES (2019). 2
Figure 3. Evolution of the Living Planet Index (LPI) over the last decades. LPI is a
measure of the state of the world's biological diversity based on population
trends of species from terrestrial, freshwater and marine habitats. Adapted from
WWF (2016). 3
Figure 4. Current extinction risk in different species groups.
Adapted from IPBES (2019). 4
Figure 5. Global distribution map of the freshwater families Unionidae and
Margaritiferidae. Adapted from Lopes-Lima et al (2017a, 2018). 5
Figure 6. Global distribution map of the freshwater families Iridinidae and Mulleriidae.
Adapted from Bogan (2008). 5
Figure 7. Global distribution map of the freshwater families Hyriidae and Etheriidae.
Adapted from Bogan (2008). 6
CHAPTER 2. Freshwater bivalves’ conservation, diversity, and research
Figure 1. Global diversity of freshwater bivalves divided by families. The total number of
species in brackets. 21
Figure 2. Diversity by ecoregions. A All freshwater bivalves; B Unionida; C Sphaeriidae;
D Cyrenidae + remaining freshwater bivalve groups. Ecoregions adapted from
Graf & Cummings (2007) and Haag (2010): NA Nearctic, NT Neotropical, PA
Palaearctic, AF Afrotropical, IN Indotropical, AU Australasian. Glaciated and
desert areas void of mussels in grey. 22
Figure 3. Diversity by ecoregions. A All freshwater bivalves; B Unionida; C Sphaeriidae;
D Cyrenidae + remaining freshwater bivalve groups. Ecoregion subdivisions
adapted from Graf & Cummings (2007) and Haag (2010): NA Nearctic, NT
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Neotropical, PA Palaearctic, AF Afrotropical, IN Indotropical, AU Australasian.

Glaciated and desert areas lacking FBs are in grey. 23
Figure 4. Taxonomic composition and diversity of freshwater bivalves in each ecoregion.
The total number of species in brackets. 24
Figure 5. Map of IUCN Red List conservation status for Unionida freshwater mussels by
ecoregions (bottom of the figure) and global conservation status for freshwater
bivalves and each major freshwater bivalve group (top of the figure). Ecoregion
subdivisions adapted from Graf & Cummings (2007) and Haag (2010): NA
Nearctic, NT Neotropical, PA Palaearctic, AF Afrotropical, IN Indotropical, AU
Australasian. On the scale bar: NE Not evaluated by the IUCN Red List; and the
IUCN Red List categories: DD data deficient, LC least concern, NT near
threatened, VU vulnerable, EN endangered, CR critically endangered, CR (PE)
critically endangered probably extinct, EX extinct. 25
Figure 6. IUCN Red List criteria used for the assessment of freshwater bivalve species
by ecoregions. Ecoregion subdivisions adapted from Graf & Cummings (2007)
and Haag (2010): AF Afrotropical, AU Australasian, IN Indotropical, NA
Nearctic, NT Neotropical, PA Palaearctic. IUCN Red List criteria: A population
size reduction, B geographic range, C small population size and decline, D very
small or restricted populations. 26
Figure 7. Main threats for freshwater bivalves recorded from the IUCN Red List database
by ecoregions. Ecoregion subdivisions were adapted from Graf & Cummings
(2007) and Haag (2010): NA Nearctic, NT Neotropical, PA Palaearctic, AF
Afrotropical, IN Indotropical, AU Australasian. 27
Figure 8. Research needs for freshwater bivalves recorded from the IUCN Red List
database by ecoregions. A All assessed species in the IUCN Red List; B data-
deficient species in the IUCN Red List. Ecoregion subdivisions adapted from
Graf & Cummings (2007) and Haag (2010): NA Nearctic, NT Neotropical, PA
Palaearctic, AF Afrotropical, IN Indotropical, AU Australasian. DD data-deficient
species in the IUCN Red List. 28
Figure 9. Conservation needs for freshwater bivalves extracted from the IUCN Red List
database by ecoregions. Ecoregion subdivisions adapted from Graf &
Cummings (2007) and Haag (2010): NA Nearctic, NT Neotropical, PA
Palaearctic, AF Afrotropical, IN Indotropical, AU Australasian. 29
CHAPTER 3. Basic biological traits of Unio delphinus

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Figure 1. Map showing the known distribution records of Unio delphinus (white circles),
sampling sites for growth and sex-ratio (all red markers), and sampling site for
the evaluation of reproductive cycle (red square). Both maps are represented
using the World Geodetic System 84 (WGS84) projection. 48
Figure 2. A - Size-at-age measurements of shell length; B - size as a function of bivalve
age, modelled by the von Bertalanffy growth function. 52
Figure 3. Histological sections from female gonads of Unio delphinus stained with
Hematoxylin and Eosin (H&E). A - General aspect of female gonads (fa)
organized in acini, in September the acini showing gonads at all development
stages of oogenesis, with several mature oocytes (m) (scale bar 100 μm). B -
Female acinus in September with a predominance of earlier stages of
oogenesis: oogonia (o), previtellogenic oocytes (pvo), pedunculated oocytes
(po), and the germinal epithelium (ge) are visible surrounding the germinative
cells (scale bar 50 μm). C - Female acini in May also presented different
development oogenesis stages, dominantly the earlier previtellogenic oocytes
(pvo) surrounded by the germinative epithelium (scale bar 100 μm). D - Detail
of female acinus in September showing pedunculated oocytes (po) with stalk
(s) visible and mature oocyte in the lumen (l) (scale bar 50 μm). E - Mature
acinus in September, full of mature oocytes (m) in the centre surrounded by
earlier stages and germinative cells (scale bar 100 μm). F - Mature female acini
with mature oocytes (m) released into the lumen (l), and muscle tissue (ms)
(scale bar 100 μm). G - Female acinus in October with only a few mature
oocytes (m) already in the lumen (l), one showing two nucleoli (n) in the nuclei,
presenting still some early stages of oocytes and with several yellow bodies
(yb), indicating early signs of degeneration (scale bar 50 μm). H - Degenerative
female acinus (dfa), surrounded by an undifferentiated epithelium, but still
presenting some stages of oocyte development (scale bar 100 μm). 53
Figure 4. Histological sections from male gonads of Unio delphinus stained with
Hematoxylin and Eosin (H&E). A - General aspect of male gonads (ma)
organized in acini in January, with the acini showing gonads at all development
stages of spermatogenesis, full with mature spermatozoa (s) in the lumen, with
visible muscle tissue (ms) and portions of the ciliated gonoduct (cg) (scale bar
200 μm). B - Partial male acini in January, with male reproductive cells at
different spermatogenesis stages and germinative epithelium (ge) visible, in the
centre the ciliated gonoduct (cg) is full of mature spermatozoa (s) (scale bar 20
μm). C and D - Details of male acinus in October, where is possible to identify
different development spermatogenesis stages, dominantly the earlier
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spermatogonia (sg), spermatocytes (sc), spermatids (st), sperm morulae (sm)

and the last stage spermatozoa (s), not so abundant (scale bar 10 μm). E -
Degenerative male acinus (dma) in August, at the beginning of the post-
spawning period, lumen with already some free spaces, presenting some yellow
bodies (yb) and surrounded by an undifferentiated epithelium, still presenting all
stages of spermatozoa development (scale bar 100 μm). F - Mature
spermatozoa (s) in March, few sperm morulae (sm) and other development
stages (scale bar 10 μm). 56
Figure 5. Histological sections from marsupial female gills of Unio delphinus, without and
with glochidia stained with Hematoxylin and Eosin (H&E) (A and B);
stereoscope images from the gills (C and D) and free glochidia (E). A -
Histological section from marsupial female gill in April, devoid of offspring (scale
bar 500 μm). B - Histological section from marsupial female gills in July, full of
mature glochidia (g) (scale bar 500 μm). C - Marsupial gill at stereoscope, in
March (scale bar 1 mm). D - Detail of gravid gill and feather-like conglutinate full
of eggs (Fs) (scale bar 1 mm). E - Mature glochidia at the microscope, in June
(scale bar 200 μm). 59
Figure 6. A - Mean glochidial infestation (i.e. the number of glochidia per fish and mm of
fish) and B - Effective transformation of glochidia into juveniles (i.e. the number
of juveniles produced per fish and mm of fish), in all fish species. 62
Figure 7. Glochidial transformation rate and attachment periods (shown in bars) per fish
host species. 63
Table 1. Growth parameters for Iberian Unio delphinus populations. L∞ is calculated
from the Wolford equation, L max is the maximum observed length in the field.
The maximum age was estimated from L max. 51
Table 2. Monthly values of all identified Gonadal Development Index (GDI) stages in the
male and female gonads, and presence/absence of eggs and larvae (glochidia)
in the marsupium of Unio delphinus. See text for details on GDI. 57
Table 3. Sex distribution of selected Iberian populations of Unio delphinus. 60
Table 4. Fish species studied and host compatibility test results, including the number
and mean (±SD) length of fish per species, mean initial number of attached
glochidia, mean number of viable juveniles produced and transformation rate
‘Transformation rate’ indicates the proportion of Unio delphinus glochidia that
successfully developed into juvenile mussels. 61
CHAPTER 4. Phylogeny of the family Unionidae

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Figure 1. Phylogenetic tree of the Palaeoheterodonta obtained by Bayesian Inference

(BI) and Maximum likelihood (ML) analyses of the first combined (COI + 28S)
dataset. Support values above the branches are posterior probabilities (BI4) and
bootstrap support (ML4) below. An asterisk (*) indicates nodes with PP95%
posterior probability or bootstrap support. Posterior probability (percentage) or
bootstrap support with P < 50% were omitted for clarity. All subfamily nodes
were collapsed for visual purposes. 97
Figure 2. Phylogenetic tree of the Unionidae obtained by Bayesian Inference (BI) and
Maximum Likelihood (ML) analyses of the second combined (COI + 28S)
dataset. Support values above the branches are posterior probabilities (BI4/BI2)
and bootstrap support (ML4/ML2) below. An asterisk (*) indicates nodes with
PP95% posterior probability or bootstrap support. Posterior probability or
bootstrap support with P < 50% were omitted for clarity. 98
Figure 3. Distribution map of the subfamily Anodontinae. 104
Figure 4. Distribution map of the subfamily Unioninae. 108
Figure 5. Distribution map of the subfamily Rectidentinae. 109
Figure 6. Distribution map of the subfamily Gonideinae. 112
Figure 7. Distribution map of the subfamilies
Ambleminae + Modellnaiinae + Parreysiinae. 115
Table 1. Historical classification systems of the subfamilies and tribes now included in
the Unionidae. (Blue) subfamilies; (red) tribes; ( nn) nomen novum; (*) regional
study; (?) rank uncertain. 87
Table 2. Specimens analysed. (U) Unknown country; (*) not generated from a single
individual. Taxonomy follows Table 3. 92
Table 3. Classification of the Unionidae based on the present analyses. (*) Not included
in the present study. 100
Table C1. List of morphological, anatomical and behavioral characters used in traditional
phylogenetic and systematic analyses of Unionida. (Gln) Glochidial size index;
(Pse) Pseudocardinal teeth; (Lat) Lateral teeth; (L) Left valve; (R) Right valve.
(*) Reported as bilaterally asymmetrical, but see discussion in Pfeiffer and Graf
(2015) (most likely unhooked as other Pseudodon species). Umbo sculpture
classification follows Zieritz et al (2015) 135
CHAPTER 5. Phylogeny, taxonomy, and species of the genus Quadrula

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Figure 1. Map of all sampling sites for the present study; both tissue and shell materials
in red; only shell materials in white. 151
Figure 2. Bayesian consensus tree inferred from the cytochrome c oxidase subunit I (COI)
gene fragment. The values above and below the nodes indicate Bayesian
posterior probability (bpp) percentage and maximum likelihood bootstrap values
(bs), respectively. Values over 95% are represented by an asterisk, and those
<50% are not shown for clarity. 159
Figure 3. Bayesian consensus tree inferred from the NADH dehydrogenase subunit 1
(ND1) gene fragment. The values above and below the nodes indicate Bayesian
posterior probability (bpp) percentage and maximum likelihood bootstrap values
(bs), respectively. Values over 95% are represented by an asterisk, values
below 50% are not shown for clarity. 160
Figure 4. Bayesian consensus tree inferred from the NADH dehydrogenase subunit 1
(ND1) and cytochrome c oxidase I (COI) gene fragments concatenated dataset.
The values above and below the nodes indicate Bayesian posterior probability
(bpp) percentage and maximum likelihood bootstrap values (bs), respectively.
Values over 95% are represented by an asterisk, values below 50% are not
shown for clarity. 161
Figure 5. Haplotype (TCS) networks and uncollapsed Quadrula clade from Figures 2 and
3, showing the relationships of nominal species within the Quadrula quadrula
group for (a) cytochrome c oxidase I (COI) and (b) NADH dehydrogenase
subunit 1 (ND1). 163
Figure 6. Shell outline principal component scores for the first two PC axes obtained from
18 Fourier coefficients of (a) all true species (recognized by molecular species
delineation methods; see results) of Cyclonaias, including a maximum of 50
specimens per species; (b) all nominal species of Cyclonaias pustulosa; (c) only
Cyclonaias kieneriana and Cyclonaias asperata; (d) all nominal species of
Quadrula; (e) all true species (recognized by molecular species delineation
methods; see results) of Theliderma; and (f) only Theliderma metanevra and
Theliderma johnsoni n. sp. Synthetic shell outlines of “extreme” morphotypes
are displayed with the anterior margin facing to the left and the dorsal margin to
the top of the page. 170
Figure 7. Distribution maps of (a) nominal species Cyclonaias asperata and Cyclonaias
kieneriana before the present study and (b) of Cyclonaias kieneriana as
proposed in the present study. 177
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Figure 8. Distribution maps of (a) Cyclonaias petrina before Burlakova et al (2018) and
(b) of C. petrina and Cyclonaias necki after Burlakova et al (2018) and Johnson
et al (2018) findings also supported by the present study. 178
Figure 9. Distribution maps of (a) nominal species within the Cyclonaias pustulosa group
and (b) of C. pustulosa and Cyclonaias succissa as confirmed by Johnson et al
(2018) and the present study. 179
Figure 10. Distribution maps of (a) nominal species within the Quadrula quadrula group
and (b) of Quadrula quadrula as proposed in the present study. 181
Figure 11. Distribution maps of (a) Theliderma metanevra before the present study and (b)
after the present study divided in T. metanevra and
Theliderma johnsoni n. sp 182
Table 1. List of newly sequenced specimens for Cytochrome c oxidase subunit I (COI)
and NADH dehydrogenase subunit 1 (ND1) datasets; nominal taxa, new
identification, site, main basin, and COI and ND1 Haplotype number and
Genbank references. 152
Table 2. List of morphological, anatomical and behavioural characters of Cyclonaias,
Quadrula, Theliderma, and Tritogonia as recognized in the present study.
157
Table 3. Results of repeatability clade analysis (RCA) of main clades corresponding to
the preferred topology. 158
Table 4. Pairwise genetic distance matrixes of nominal quadruline species of the genera
Cyclonaias, Quadrula, Theliderma, and Tritogonia, using the original nominal
taxa. 164
Table 5. Results of molecular species delineation methods. 166
Table 6. Pairwise genetic distance matrixes of quadruline species of the genera
Cyclonaias, Quadrula, Theliderma, and Tritogonia, as recognized in the present
study. 167
Table 7. Historical classification of species formerly assigned to Quadrula.
* extinct. 175
Supplementary Table 1. List of museum lots analysed for the morphometry: taxon, original
identification, new identification, and lot catalogue number. BSGLC (SUNY
Buffalo State College Great Lakes Centre); NCSM (North Carolina Museum of
Natural Sciences). 189
Supplementary Table 2. List of specimens included in the Cytochrome c oxidase subunit
I (COI) dataset; Haplotypes, GenBank references, original identification, new
identification, voucher specimen, and respective study. BSGLC (SUNY Buffalo
State College Great Lakes Centre); FLMNH (Florida Museum of Natural
xx FCUP
History); NCSM (North Carolina Museum of Natural Sciences); UA (University

of Alabama); UAUC (University of Alabama Unionid Collection); UAM (Auburn
University Museum). 193
Supplementary Table 3. List of specimens included in the NADH dehydrogenase subunit
1 (ND1) dataset; Haplotypes, GenBank references, original identification, new
identification, and voucher/specimen and respective study. BSGLC (SUNY
Buffalo State College Great Lakes Centre); NCSM (North Carolina Museum of
Natural Sciences); UA (University of Alabama); UAUC (University of Alabama
Unionid Collection). 216
Supplementary Table 4. List of specimens included in the concatenated Cytochrome c
oxidase subunit I (COI) + NADH dehydrogenase subunit 1 (ND1) dataset;
codes, original identification, new identification,
and Genbank references. 235
Supplementary Table 5. List of morphological, anatomical and behavioural characters
analysed on specimens of Quadrula s.l.. GLN - mean glochidial size index. *
only observed in laboratory conditions.
Superscripts 1 occasionally, 2 shallow. 251
Supplementary Table 6. Species assigned Quadrula sensu lato according the last
comprehensive checklist of the United States (Williams et al 2017),
conservation status by the International Union for Conservation of Nature
(IUCN) Red List and by NaturServe, and legal protection status in the United
States of America. 258
CHAPTER 6. Phylogeny of the family Margaritiferidae
Figure 1. Phylogenetic tree of the Palaeoheterodonta obtained by Bayesian Inference

(BI) and Maximum likelihood (ML) analyses of the combined (COI [3 codons] +
16S + 18S + 28S + H3 [3 codons]) dataset. Support values above the branches
are posterior probabilities and bootstrap support below. Numbers after species
names refer to specimen numbers (see Table 3). 299
Figure 2. Hinge plate and umbo cavity of Margaritiferidae. A - Gibbosula crassa (NCSM
102194.2), B - Cumberlandia monodonta (NCSM 55359.18), C - Margaritifera
margaritifera, (NCSM 5771.1) D - Pseudunio auricularius (NCSM 44514.2). t -
pseudocardinal teeth, u - umbo cavity. 306
Figure 3. Fossil-calibrated ultrametric chronogram of the Margaritiferidae calculated
under a lognormal relaxed clock model and a Yule process speciation
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implemented in BEAST 1.8.4 and obtained for the complete data set of
mitochondrial and nuclear sequences (nine partitions: three codons of COI +
16S rRNA + 18S rDNA + 28S rDNA + three codons of H3). Bars indicate 95%
confidence intervals of the estimated divergence times between lineages (Ma).
Black numbers near nodes are mean ages (Ma). Stratigraphic chart according
to the International Commission on Stratigraphy (2015). 308
Figure 4. Simplified scheme of origin and expansion routes inferred across clades of the
Margaritiferidae. The black numbers show the mean age of putative expansion
events obtained from the multi-locus fossil-calibrated phylogenetic model (see
Fig. 3 for details). Circles indicate the putative places of origin of the family and
several clades. The map was created using ESRI ArcGIS 10 software
(www.esri.com/arcgis); the topographic base of the map was created with ESRI
Data and Maps. 310
Figure 5. Semilogarithmic lineage-through-time (LTT) median plots of chronograms
estimated from 108,004 post-burn-in Bayesian trees for the primary
Margaritiferidae clades, including Gibbosula, Cumberlandia + Pseudunio,
Margaritifera, and the entire family. The grey filling indicates 95% confidence
intervals. 311
Figure 6. Distribution map of the Margaritiferidae. 315
Table 1. Comparison of Margaritiferidae classifications. Fossil genera excluded. (S)
1 2
synonym. Superscripts: under tribe Heudeanini; under subfamily
3 4
Pseudodontinae; under tribe Margaritiferini; under tribe Leguminaiini. 281
1
Table 2. Characters used to define and diagnose Margaritiferidae. papillae present
only; 2 hinge teeth reduced. 283
Table 3. List of specimens analysed, GenBank references, specimen number, locations,
and museum voucher references. *not generated from a single individual. IEBR
- Institute of Ecology and Biological Resources, Hanoi, Vietnam; MNCN - Museo
Nacional de Ciencias Naturales, Madrid, Spain; NCFM - Nanchang Freshwater
Mollusc Collection, Nanchang University, Jiangxi Province, China; IEPN -
Russian Museum of Biodiversity Hotspots; MCZ - Museum of Comparative
Zoology, Harvard University, USA; UAUC - University of Alabama Unionid
Collection, USA; BivAToL - Bivalve Tree of Life Project, USA. 290
Table 4. Biological and ecological characters. (Gln) glochidial size index. Superscripts:
U
unknown; Rrivers; Llakes. 294
Table 5. Results of Repeatability Clade Analysis (RCA) of main clades corresponding to
the preferred topology. High support values (BI posterior probability ≥ 95%, and
ML bootstrap support ≥ 70%) are highlighted in bold. 300
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Table 6. Analysed conchological characters of Margaritiferidae species. Superscripts:

1
W-shaped pustules on umbo and onto disk; 2plications on the posterior slope,
posterior disk; 3plications on the posterior slope, pustules on umbo and disk.
303
Table 7. Anatomical characters. *Not analysed for anatomy. 305
Table 8. The most probable ancestral areas of the primary clades within Margaritiferidae
inferred from three different statistical modelling approaches. High support
values (probability≥70%) are highlighted in bold. *Mediterranean + Eastern
North America. 309
Table 9. Margaritiferidae systematics and taxonomy. 313
Supplementary Figure 1. Gene map of the F-type mitochondrial genome of Gibbosula
crassa. Genes positioned inside the circle are encoded on the heavy strand,
and genes outside the circle are encoded on the light strand. Colour codes for
the: small and large ribosomal RNAs (red), transfer RNAs (purple); F-specific
open reading frame (yellow); protein-coding genes (green). 340
Supplementary Figure 2. Historical biogeography of the Margaritiferidae inferred from
three different statistical modelling approaches, including (A) the combined
results of SDIVA, DEC, and S-DEC; (B) S-DIVA; (C) DEC; and (D) S-DEC,
calculated under a lognormal relaxed clock model and a Yule process
speciation implemented in BEAST 1.8.4 and obtained for the complete data set
of mitochondrial and nuclear sequences (nine partitions: three codons of COI +
16S rRNA + 18S rDNA + 28S rDNA + three codons of H3). Pie chaps near
nodes indicate probabilities of certain ancestral areas. Colour circles on the tip
nodes indicate the range of each species. Black numbers near nodes are BPP
values inferred from BEAST. 341
Supplementary Table 1. Specimens examined for conchological and anatomical features.
ANSP - Academy of Natural Sciences of Drexel University, Philadelphia, PA
USA; MNHN - Muséum National d'Histoire Naturelle, Paris, France; NHMUK -
Natural History Museum, London, UK; NCFM - Nanchang Freshwater Mollusc
Collection, Nanchang University, Nanchang, Jiangxi Province, China; NCSM -
North Carolina Museum of Natural Sciences, Raleigh, NC, USA; RMBH -
Russian Museum of Biodiversity Hotspots, Federal Centre for Integrated Arctic
Research, Russian Academy of Sciences, Arkhangelsk, Russia. 343
Supplementary Table 2. Best-fit models of nucleotide substitution for each partition based
on Bayesian Information Criteria (BIC) using JMODELTEST 2.1.10 (Darriba et
al 2012) for the Bayesian inference analyses. 344
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Supplementary Table 3. List of characteristic examples of fossil records supporting the

primary phylogenetic clades of freshwater bivalves identified in the present
study. 345
Supplementary Table 4. List of fossil calibrations that were used in
BEAST analyses. 349
Supplementary Table 5. Diversification rate statistics for the Margaritiferidae and
Unionidae clades. Superscripts: *variable diversification rate; **Data from
Bolotov et al (2017a); MMekong only. 354
Supplementary Table 6. Margaritiferidae generic names, authorities,
and type species. 355
CHAPTER 7. First male whole mitogenome of a Margaritiferidae species
Figure 1. Diagrams of the five distinct gene orders detected in Unionida. In the female F-
type lineage, three gene orders are depicted: Unionidae F-type 1 (UF1),
Unionidae F-type 2 (UF2) and Margaritiferidae F-type 1 (MF1). In the male M-
type lineage, two gene arrangements are shown: Unionidae M-type 1 (UM1)
and Margaritiferidae M-type 1 (MM1). Continuous lines indicate different
locations of genes between mitogenomes. Grey box highlights the gene
rearrangement region between UF1 and UF2. Yellow boxes indicate the main
differences in gene arrangement between female and male mitogenomes, tRNA
(H) location and rearrangement of ATP8-tRNA(D) region. 365
Figure 2. Phylogenetic (BI-NUC) tree of Unionida estimated from 14 concatenated
individual mtDNA gene sequences (12 protein-coding and 2 rRNA genes).
Values for branch support are represented in the following order: (1) Bayesian
posterior probabilities (PP) for BI-NUC tree, (2) Bayesian PP for BI-AA tree, (3)
ML bootstrap support (BS) values for ML-NUC and (4) ML BS values for ML-AA
tree. Maximum support values (PP = 1, BS = 100) are represented by asterisks.
All five distinct detected gene orders are mapped on the phylogeny branches
(see Fig. 1 for gene order codes). 366
CHAPTER 8. The evolution of mitogenome rearrangements in freshwater

mussels
Figure 1. Gene maps of the F- and M-type mitochondrial genomes of Chamberlainia

hainesiana, Microcondylaea bonellii, Pilsbryoconcha exilis, and Monodontina
xxiv FCUP
vondembuschiana. Genes positioned inside the circle are encoded on the heavy
strand, and genes outside the circle are encoded on the light strand. Colour
codes: Small and large ribosomal RNAs (red), transfer RNAs (purple), FORF F-
specific open reading frame (yellow), MORF M-specific open reading frame
(yellow), PCGs genes (green). 382
Figure 2. Diagrams of the four distinct gene orders known in Unionidae to date. In the F-
type, three gene orders are depicted: UF1, UF2, and UF3. In the male M-type
lineage, the only Unionidae gene arrangement is shown: M-type 1 (UM1). Blue
boxes highlight the gene rearrangement region from UF1 to UF2 (Box A) and
from UF2 to UF3 (Box B). Small and large ribosomal RNAs and transfer RNAs
are depicted by one letter of the amino acid code; Arrow colour codes, follow
Fig. 1. 384
individual mtDNA gene sequences (12 protein-coding and 2 rRNA genes).
Values for branch support are represented in the following order: (1) Bayesian
posterior probabilities (PP) for BI-NUC tree, (2) Bayesian PP for BI-AA tree, (3)
ML bootstrap support (BS) values for ML-NUC and (4) ML BS values for ML-AA
tree. Maximum support values (PP = 1, BS = 100) are represented by asterisks.
Gonideinae subfamily and tribes are highlighted. For details see text. GenBank
codes in Table 1. 385
individual mtDNA gene sequences (24 protein-coding and 4 rRNA genes) of the
first combined Female + Male concatenated data set. Maximum branch support
values (BI-NUC/BI-AA PP = 1; ML-NUC/ML-AA BS = 100) are represented by
asterisks, while # represents the only non-supported branch by ML-AA tree.
Gonideinae subfamily and tribes are highlighted.
GenBank codes in Table 1. 386
Figure 5. Unionidae F-haplotype phylogenetic sub-tree (BI-NUC) used to infer the most
parsimonious putative ancestral gene orders and gene rearrangements
mapped as MF1, UF1, UF2, and UF3 (see text for details). Margaritiferidae and
all subfamily nodes were collapsed for visual purposes. 387
Figure 6. Time-calibrated mitogenomic phylogeny, an example of the three-level
classification scheme (subfamilies, tribes, and subtribes) and evolution of the
mitochondrial gene order in the Unionoidea. Fossil-calibrated ultrametric
chronogram of the Unionoidea calculated under a lognormal relaxed clock
model and a Yule process speciation implemented in BEAST and obtained for
the complete mitogenome data set. The outgroup sample is not shown. Bars
FCUP xxv
indicate 95% confidence intervals of the estimated divergence times between

lineages (Ma). Black numbers near nodes are mean ages (Ma). Colour labels
indicate the mitochondrial gene order (MF1, UF1, UF2, and UF3). Red asterisks
indicate fossil calibrations. Stratigraphic chart according to the International
Commission on Stratigraphy (2015). 388
Figure 7. Historical biogeography of the Unionidae. This combined scenario has been
inferred from three different statistical modelling approaches (S-DIVA, DEC, and
S-DEC) based on the time-calibrated mitogenomic phylogeny (Fig. 6). Pie
charts near nodes indicate probabilities of certain ancestral areas. Colour circles
on the tip nodes indicate the range of each species. Colour labels indicate the
mitochondrial gene order (UF1, UF2, and UF3). 389
Table 1. List of specimens analysed (based on Lopes-Lima et al 2017a,b), GenBank
references, and country. 378
Table 2. Main structural features of the female (above) and male (below) transmitted
mitochondrial genomes of Gonideinae species. 383
Supplementary Table 1. List of fossil calibrations that were used in
BEAST analyses. 403
xxvi FCUP
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List of Abbreviations
16S 16S ribosomal RNA gene
28S 28S ribosomal RNA gene
AF Afrotropical
AU Australasia
BI Bayesian Inference
BIC Bayesian information criterion
COI Cytochrome oxidase subunit 1
DNA Deoxyribonucleic acid
DUI Doubly uniparental inheritance
FM Freshwater mussel
GIS Geographic Information System
H3 Histone H3 gene
IN Indotropical
IUCN International Union for the Conservation of Nature
MCMC Markov-Chain Monte Carlo
ML Maximum Likelihood
mtDNA mitochondrial DNA
NA Nearctic
ND1 NADH dehydrogenase subunit 1
nt nucleotides
NT Neotropical
PA Palaearctic
xxviii FCUP
FCUP 1
CHAPTER 1
General introduction
1.1 The freshwater biodiversity crisis
Never in the world, so many humans used so many natural resources (Figs. 1 & 2). The
exponential human growth over the last 2,000 years is coupled with the increase of
consumptive biological resources per capita, resulting in increasing anthropogenic changes to
natural environments (Sala et al 2000; IPBES 2019), as the major threat to biodiversity and
ecosystem functioning (McKee et al 2003; McShane et al 2011). These impacts have already
caused an extensive contraction of genetic, species and ecological diversity, with gene erosion
and eradication, species extirpations and extinctions, and loss and irreversible transformation
or destruction of many habitats around the globe (Butchart et al 2012; Ceballos et al 2015;
Miraldo et al 2016; IPBES 2019). Therefore, we are living under a biodiversity crisis with
unprecedented severity, since human life on Earth (Barnosky et al 2011). The extinction rates
are far higher than baseline values and closer to those during major extinction events, and we
are potentially experiencing the sixth mass extinction event (Barnosky et al 2011; Pievani
2014).
Figure 1. Size of the world human population over the last 12,000 years. Adapted from Roser
et al (2019).
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Figure 2. Changes of Gross domestic product (GDP), domestic material consumption, and
extraction of living biomass (trillion USD at 2010 value) for groups of countries at distinct
development levels. Adapted from IPBES (2019).
Diversity patterns are not homogeneous across the terrestrial, marine and freshwater realms
(Dawson 2012). Although freshwater habitats only hold 0.01% of the global water volume and
cover around 0.8% of the Earth’s surface, they have a disproportionate species richness when
compared with their terrestrial and marine counterparts (Collen et al 2014). For instance, they
contain 40% of all fish species and a quarter of the global number of vertebrates (Dudgeon et
al 2006). Freshwater habitats are, however, among the most threatened at the global level
(Strayer & Dudgeon 2010; Vörösmarty et al 2010), given that they are rare, isolated by land
and seawater, and generally located downhill of human settlements and therefore exposed to
all kinds of runoff and human wastes (Strayer 2006). Freshwater water bodies have also been
intensively modified for human purposes; for example, many rivers have been intubated,
channelized and re-directed, and lakes dried or redesigned (Strayer & Dudgeon 2010). Water
level, flow, and substrate have been exhaustively impacted by thousands of physical barriers
such as dams, weirs, and floodgates, concrete embankments to prevent the overflow, and
extraction of sands, gravels and other inert materials (Strayer 2006; Reid et al 2019).
Therefore, freshwater habitats are suffering much higher biodiversity declines than those on
marine or terrestrial realms (Dudgeon et al 2006; Reid et al 2019). This pattern can also be
seen using the Living Planet Index developed by the World Wide Fund for Nature (WWF),
where the freshwater species index dropped more sharply between 1970 and 2012 than the
index for the marine or terrestrial populations (Fig. 3; WWF 2016).
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Figure 3. Evolution of the Living Planet Index (LPI) over the last decades. LPI is a measure of
the state of the world's biological diversity based on population trends of species from
terrestrial, freshwater and marine habitats. Adapted from WWF (2016).
1.2 Freshwater mussel diversity, importance, and conservation
Freshwater bivalves of the Unionida order, also known as Freshwater Mussels (FMs),
freshwater clams or naiads, belong to an old (>200 Mya), big monophyletic group of molluscs
that are strictly freshwater inhabitants and, for this reason, have a series of interesting
adaptations to allow them to survive under constant flow (Strayer 2008; Haag 2012). Contrary
to marine bivalves, FMs exhibit internal fertilization, parental care and, more interestingly, their
specialized larvae (glochidia) need to attach to a host (mostly freshwater fish) for dispersion
and nutrition until they metamorphose into juveniles and drop into the substrate (Graf &
Cummings 2006; Barnhart 2008). Freshwater mussels play key ecological roles (e.g. water
filtration, energy, and nutrient cycling, providing bioturbation or sediment mixing), and provide
valuable ecosystem services to humans (e.g. increasing water transparency, source of protein,
pearls and shell materials) (Howard & Cuffey 2006). This group of mussels has a wide
distribution and they often dominate many freshwater habitats regarding both the number of
individuals and biomass (Vaughn 2018). Unfortunately, FMs, like many other freshwater taxa,
have suffered a massive global defaunation over the last centuries (Dirzo et al 2004; Lopes-
Lima et al 2014) and are currently one of the most imperilled groups in the world (Fig. 4;
Lydeard et al 2004; Ferreira-Rodriguez et al 2019).
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Figure 4. Current extinction risk in different species groups. Adapted from IPBES (2019).
This is further exacerbated by the fact that to complete their life cycle, these mussels are
dependent on freshwater fish, which have also shown strong declines globally (Modesto et al
2017). Therefore, decline and extinction estimates of affiliate species such as FMs need to be
recalibrated by taking the host species fluctuations and extinctions into account (Koh et al
2004). Based on the current IUCN Red List and if data deficient species are as threatened as
non-data deficient species, 43% of all assessed FM species are currently threatened, with
13.2% being Critically Endangered and 6.3% already Extinct (Fig. 4; IUCN 2019). This situation
has caused a substantial increase in research and conservation action dedicated to FMs since
the emergence of ecological values during the 1970s (Haag 2012; Lopes-Lima et al 2014).
These research and conservation efforts have been, however, concentrated in a handful of the
more charismatic species (e.g., Margaritifera margaritifera and Unio crassus in Europe, and
Cumberlandia monodonta in North America), and in more economically developed regions
such as North America and Europe (Lopes-Lima et al 2014). Species in other parts of the world
are still poorly known and their conservation status poorly evaluated (Lopes-Lima et al 2014;
Ferreira-Rodriguez et al 2019).
Six families are currently recognized within Unionida based on morphological
characters (Graf & Cummings 2007; Bogan 2008). Two of them are present in the northern
hemisphere (Fig. 5): the Unionidae, which is by far the most speciose family of the order, with
around 600 species, and the Margaritiferidae, which has a much lower species richness but
with most species being at risk of extinction (IUCN 2019).
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Figure 5. Global distribution map of the freshwater families Unionidae and Margaritiferidae.
Adapted from Lopes-Lima et al (2017a, 2018).
Two of the families are present mainly in the southern hemisphere not crossing continental
boundaries (Fig. 6), i.e. the Mulleriidae occurring in South America, and the Iridinidae in Africa.
Figure 6. Global distribution map of the freshwater families Iridinidae and Mulleriidae. Adapted
from Bogan (2008).
From the remaining two families, the Hyriidae is found in both South America and Australia,
while the Etheriidae was originally thought to be composed by less than ten species in South
America, Africa, and Asia, but due to recent revaluation is now considered restricted to Africa
(Fig. 7; Bogan 2008).
6 FCUP
Figure 7. Global distribution map of the freshwater families Hyriidae and Etheriidae. Adapted
from Bogan (2008).
Although several phylogenetic studies have been developed over the last decades (e.g.,
Bogan & Hoeh 2000; Graf & Cummings 2006, 2007; Whelan et al 2011), the phylogeny within
the order is far from stabilized and limited phylogenetic consensus has emerged, especially
regarding to the early evolution of the Unionida (Graf 2013). Phylogenetic patterns within the
families are also poorly understood due to the lack of sequenced taxa and limited robustness
of available phylogenetic analyses (Huff et al 2004; Whelan et al 2011).
1.3 The need for baseline biological research
Accurate conservation status assessment and effective conservation actions require a

profound knowledge about their target taxa and/or habitats (Lopes-Lima et al 2017b).
However, baseline ecological and physiological data on most FM species is still scarce
(Kindsvater et al 2018). Some features are almost unknown across all taxa, such as dispersal
rates of larvae (hitchhiking on fish) or adults, and the mean and maximum distances a male
can fertilize a female (Strayer et al 2004; Lopes-Lima et al 2017b). Other data are only
available for a small number of species or populations and generally on a small-time scale,
such as data on the distribution, population size, structure and trends, and demography (e.g.
recruitment, mortality, and migration) (Ferreira-Rodriguez et al 2019). Also, life-history traits
like lifespan, age at sexual maturity, reproduction stage timing, and fertility are poorly known,
and many times wrongly extrapolated from data on better-known species, such as
Margaritifera margaritifera in Europe or Elliptio complanata in North America (Lopes-Lima et
FCUP 7
al 2014). Additionally, more extrinsic factors like the identification and availability of the fish
host range, the main habitat requirements, and the sensitivity and responses to environmental
stressors like habitat degradation and pollution, are requiring urgent research (Modestro et al
2017; Ferreira Rodriguez et al 2019). This lack of baseline biological data is hindering
conservation efforts. For instance, the lack of knowledge on habitat requirements and
sensitivity to habitat degradation does not allow us to understand how riverine or lacustrine
habitats should be accurately rehabilitated to improve the status of FM populations. Also, the
lack of knowledge on traits involved in the reproductive cycle, such as reproduction timing
(fertilization, spawning, and larvae discharge), age of maturity, fertility, and host fish usage
slows down the implementation of captive propagation programs, necessary for reintroduction
and reinforcement of depleted populations (Patterson et al 2018). Species' baseline data are
not only important for species-focused conservation and research. Meta-analyses and
modelling studies on a wide spatial scale, depend on this type of data for designing bioregions,
prioritizing areas for conservation and evaluating threats and other environmental factors
affecting taxonomic groups, species assemblages and entire ecosystems (Kindsvater et al
2018).
1.4 Defining species boundaries and integrating phylogenetic diversity

patterns in conservation ranking
Given that resources dedicated to conservation are limited, a careful selection of conservation
targets is required (Moilanen & Arponen 2011). Species are globally considered the essential
conservation units by policies and conservation status assessments (Fitzpatrick et al 2015).
Therefore, it is crucial to thoroughly define species boundaries, which is not always an easy
task, especially in FMs (Chong et al 2016; Inoue et al 2018). FMs’ species delineation is many
times difficult due to the lack of clear morphological diagnostic characters, high shell plasticity
and morphological convergence among related species (Froufe et al 2016). For this reason,
molecular analyses have been increasingly used to define species boundaries in these
organisms (e.g. Chong et al 2016; Pfeiffer et al 2016; Inoue et al 2018). However, the molecular
delimitation of cryptic species is fundamentally questioned by the continuous and dynamic
nature of speciation (Chenuil et al 2019). There have been multiple ways and concepts on how
to define species (revised in De Queiroz 2007) but, since the last decades of the 20 th century,
we have mostly used a reproductive isolation approach or the biological species concept (Mayr
1982), which is not always easy to demonstrate. However, many scientists are now
increasingly using the monophyletic isolated lineages approach supported by multiple
arguments or the unified species concept (De Queiroz 2005, 2007) which is easier to
8 FCUP
characterize and easier to validate molecularly. Over the last decade, several analytical
approaches on DNA sequences have been developed to identify species and define species
boundaries (Luo et al 2018). Many of them relied on a standardized segment of the
mitochondrial genome (a region of the cytochrome c oxidase subunit I, COI with around 600
nucleotides) (Hubert & Hanner 2015). The choice of COI over other mitochondrial or nuclear
markers as the main molecular tag or barcode for each species, is due to several reasons: it
has a high discrimination resolution, is generally easy to amplify even from small amounts or
from degraded tissue, and standard protocols are available for amplification in a wide range of
taxa (Hebert et al 2003). More recently, methods applied to coalescent trees are being
increasingly applied not only to COI or single genes, but to multiple nuclear and mitochondrial
markers (Luo et al 2018). The identification of these molecular operational taxonomic units or
MOTUs allows scientists to identify and investigate hidden cryptic diversity and for species
recognition to advance faster than in classical morphological approaches (Kekkonen & Hebert
2014).
Conservation targets and prioritization should also include the evolutionary history
captured by specific sets of species or higher taxa, i.e. their phylogenetic diversity patterns
(Winter et al 2013). For example, phylogenetically unique taxa are generally of higher priority
for conservation; furthermore, metrics have already been developed to include phylogenetic
diversity in species rankings for conservation attention, such as the Evolutionarily Distinct and
Globally Endangered (EDGE) program developed by the London Zoological Society (Redding
& Mooers 2006; Isaac et al 2007).
1.5 Objectives
The overall goal of this dissertation is to advance the conservation biology of freshwater
mussels by combining research on phylogeny, systematics and ecology, showing how the
integration of multiple research fields has practical implications to preserve highly endangered
taxa. The specific objectives of this thesis are:
1. To highlight the biodiversity hotspots of freshwater bivalves, mapping the global

species diversity and conservation status in the main freshwater bivalve ecoregions.
2. To reveal the main gaps of knowledge, conservation and research needs of FMs, using
data from the IUCN Red List database.
FCUP 9
3. To show the importance of basic biological studies for conservation of freshwater

mussels and environmental monitoring, using a case study on several physiological and
ecological traits of a poorly known Iberian endemic species, i.e. Unio delphinus.
4. To demonstrate the importance of systematics and phylogenetic diversity for the

conservation of freshwater mussels, through a series of phylogenetic studies on several
groups within the Unionida order.
5. To test the use of molecular data to define important freshwater mussel taxa for
conservation, estimating the phylogenetic patterns and potential molecular operational
taxonomic units.
6. To test the use of mitochondrial genome arrangements as a diagnostic for freshwater

mussel groups, sequencing and assembling whole mitogenomes of selected species and
mapping the retrieved gene arrangements in whole genome phylogenies.
7. To use information collected during the dissertation to provide recommendations for

freshwater mussel conservation, at national, European and global scales.
1.6 Thesis structure
The dissertation was organised in nine chapters, which together address the general and
specific objectives of the thesis listed in the previous section. The dissertation includes a
general Introduction (Chapter 1) and a General Discussion (Chapter 9), together with seven
chapters that correspond to seven scientific papers already published in international peer-
reviewed journals. Chapter 2 presents a review on the diversity, conservation status and
knowledge gaps. Two of these main gaps are the lack of knowledge about basic biological
traits and systematics clarification that are then addressed in Chapter 3 and Chapters 4-8,
respectively. Below, the main contents of each chapter included in the thesis are summarised.
Chapter 1 is an introduction to the main issues related to the dissertation theme. It
starts by describing the global decline of biodiversity and then focus on the target taxonomic
group of the dissertation – the freshwater mussels (FM) –, discussing its ecological and
economic importance, but also its threatened status and conservation concern. Subsequently,
the importance of basic biological research, taxonomy, and phylogenetic patterns for
conservation are highlighted.
10 FCUP
Chapter 2 addresses Objectives 1 and 2 by presenting a revision of the global diversity

patterns of freshwater bivalves and their conservation status, threats, and research needs.
Based on the results obtained, future paths for freshwater bivalve research are then
suggested.
Chapter 3 addresses Objective 3 highlighting the importance of basic biological studies
for conservation planning and the potential use of biological traits as environmental indicators.
As a case study, the distribution, growth patterns, reproductive cycle, and host fish range of
an Iberian endemic FM, the dolphin freshwater mussel Unio delphinus are presented.
Chapter 4 addresses Objective 4, providing a comprehensive, two marker (COI -
cytochrome oxidase subunit I and the 28S ribosomal RNA) phylogeny of the most diverse
Unionida family, the Unionidae. This phylogeny is complemented with distribution,
morphological, anatomical and behavioural data to revise the systematics of the family.
Chapter 5 addresses Objectives 4 and 5, presenting single and two-marker (mtDNA
COI - cytochrome c oxidase subunit I and ND1 - NADH dehydrogenase subunit 1) phylogenies
of an imperilled group of North American Unionidae species that have historically been placed
under a single genus, i.e. the genus Quadrula. Several molecular species delineation methods
were used to define molecular operational taxonomic units (MOTUs) that were then tested with
an integrative approach including ecological, behavioural and morphometric (Fourier Shape)
analyses, and geographic distribution data.
Chapter 6 addresses Objective 4, revising the systematics of the whole
Margaritiferidae family with five-marker (two mitochondrial: 16S ribosomal RNA and COI
cytochrome c oxidase subunit I, and three nuclear 18S ribosomal RNA, 28S ribosomal RNA,
and the H3 Histone 3 gene) phylogenies, coupled with morphological and ecological
information. The distribution, potential origin and main biogeographic patterns of the family are
also described.
Chapter 7 addresses Objective 4 and 6, providing newly sequenced female (F-) and
male (M-) lineage whole mitochondrial genomes and a whole mitogenome phylogeny. In this
chapter, we present the first published (M-) genome for the Margaritiferidae, revealing that the
gene arrangements of both (F-) and (M-) type mitogenomes are unique within the order
Unionida and can be used as molecular diagnostic characters for the family Margaritiferidae.
Chapter 8 addresses Objective 4 and 6, presenting a comprehensive phylogeny of the
Unionidae using whole mitochondrial genomes. Each distinct mitogenome gene arrangement
is mapped and dated, with most gene rearrangements coinciding with major extinction events.
It is suggested that these evolutionary changes might be related to the pulsed-evolution theory.
These phylogenetic results were then combined with an ancestral area reconstruction to
describe the early diversification patterns and biogeography of the Unionidae. A new
FCUP 11
systematics framework for the classification of the Unionidae was also proposed using the
mitogenome phylogenetic results.
Chapter 9, presents the main conclusions of the dissertation, discussing the main
findings, highlighting the major shortcomings and caveats of the studies, and proposing future
pathways for research and conservation.
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CHAPTER 2
Freshwater bivalves’ conservation, diversity, and
research
Paper I
Conservation of freshwater bivalves at the global scale: diversity, threats
and research needs.
Lopes-Lima M, Burlakova LE, Karatayev AY, Mehler K, Seddon M, Sousa R
Article published in Hydrobiologia 810, 1-14 (2018).
DOI: 10.1007/s10750-017-3486-7
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Conservation of freshwater bivalves at the global scale: diversity, threats

and research needs
Manuel Lopes-Lima1,2,3,*, Lyubov E. Burlakova4, Alexander Y. Karatayev4, Knut Mehler4, Mary

Seddon3, Ronaldo Sousa5
1
CIBIO/InBIO - Research Center in Biodiversity and Genetic Resources, University of Porto, Campus Agrário de
Vairão, Vairão, Portugal
2
CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de
Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal
3
SSC/IUCN-Mollusc Specialist Group, Species Survival Commission, International Union for Conservation of
Nature, c/o The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, United Kingdom
4
Great Lakes Center, Buffalo State College, 1300 Elmwood Ave, Buffalo, NY 14222, USA
5
CBMA - Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus
Gualtar, 4710-057 Braga, Portugal
⁎
Corresponding author: M. Lopes-Lima; e-mail: [email protected]
Abstract
Bivalves are ubiquitous members of freshwater ecosystems and responsible for important
functions and services. The present paper revises freshwater bivalve diversity, conservation
status and threats at the global scale and discusses future research needs and management
actions. The diversity patterns are uneven across the globe with hotspots in the interior basin
in the United States of America (USA), Central America, Indian subcontinent and Southeast
Asia. Freshwater bivalves are affected by multiple threats that vary across the globe; however,
pollution and natural system (habitat) modifications being consistently found as the most
impacting. Freshwater bivalves are among the most threatened groups in the world with 40%
of the species being near threatened, threatened or extinct, and among them the order
Unionida is the most endangered. We suggest that global cooperation between scientists,
managers, politicians and the general public, and application of new technologies (new
generation sequencing and remote sensing, among others) will strengthen the quality of
studies on the natural history and conservation of freshwater bivalves. Finally, we introduce
the articles published in this special issue of Hydrobiologia under the scope of the Second
International Meeting on Biology and Conservation of Freshwater Bivalves held in 2015 in
Buffalo, New York, USA.
Keywords
Bivalvia, Unionida, Venerida, IUCN Red List, Freshwater mussels, Conservation
20 FCUP
Introduction
Freshwater ecosystems are among the most threatened on the planet facing unprecedented
pressures related to the increase of human population and socioeconomic development
(Dudgeon et al 2006; Vörösmarty et al 2010). Increasing anthropogenic pressure worldwide
results in habitat loss, habitat modification and fragmentation, overexploitation of natural
resources (including water), pollution, the introduction of invasive alien species (IAS) and
climate change (Malmqvist & Rundle 2002; Strayer & Dudgeon 2010). The biodiversity crisis
is one of the major consequences of steeply rising human demands, and among the animals
with high extinction rates are freshwater bivalves (FBs) (Lydeard et al 2004; Strayer et al 2004;
Régnier et al 2009; Lopes-Lima et al 2014, 2017a). The future survival of FBs is highly impaired
and considering the large suite of ecosystem services they provide (Vaughn 2017) scientists,
managers, politicians, and the general public need to strengthen their cooperation to conserve
these species.
Whereas over the last years multiple studies have been published concerning the
biology, ecology, and conservation of FBs, most of them were carried out in North America
and Europe (Lopes-Lima et al 2014). Consequently, a great ignorance about basic aspects
(e.g. distribution, diversity, abundance, population structure, and life cycle) concerning species
inhabiting South America, Africa, and Asia persists and much more information is needed for
these continents.
In the present paper, we compile data on FB diversity patterns, conservation status and
threats from the International Union for Conservation of Nature (IUCN) database using a
species list adapted from Graf & Cummings (2017) and mapped them in ecoregions adapted
from Graf & Cummings (2007) and Haag (2010). We also briefly discuss research needs and
urgent management actions that may help conserve these animals and introduce the articles
published in this special issue resulting from the Second International Meeting on Biology and
Conservation of Freshwater Bivalves held in 2015 in Buffalo, United States of America (USA).
Diversity patterns at the global scale

Freshwater bivalves are a polyphyletic group of animals restricted to freshwaters with a little
over 1,200 described species (Bogan 2008; Bogan & Roe 2008; Graf 2013). The main core of
the group (99%) is composed of freshwater mussels of the order Unionida (strictly freshwater)
(72%) and species belonging to 7 families within the order Venerida (27%) (Fig. 1). The
Venerida are composed mainly of families comprising 94% of the species the pea- or fingernail-
clams Sphaeriidae (67%) and the Cyrenidae (27%), which include, for example, the invasive
Asian clam Corbicula fluminea (Müller, 1774). The family Dreissenidae family (3%), well-known
FCUP 21
to contain important invasive alien species (IAS) such as the quagga mussel Dreissena
bugensis Andrusov, 1897 and the zebra mussel Dreissena polymorpha (Pallas, 1771), is also
included in the order Venerida. The remaining handful of FB species are scattered among
other essentially marine orders or families within the order Venerida (Fig. 1).
Figure 1 Global diversity of freshwater bivalves divided by families. The total number of
species in brackets.
Freshwater bivalves are present in all continents except in glaciated (except few sphaeriid
species) and desert areas, but the diversity patterns are not evenly distributed (Fig. 2). The
diversity is higher in the Nearctic (NA), Neotropics (NT) and Indotropics (IN) with ≈25% species
being found in each ecoregion. The Palaearctic (PA) and Afrotropics (AF) have a lower
diversity (≈10%) with Australasia (AU) being the poorest ecoregion (≈5%) (Fig. 2A). There are
also distinct distribution patterns across the main taxonomic groups. The Unionida is similar to
the general pattern for all FBs, with 33% of the species inhabiting the NA and 6% inhabiting
the PA (Fig. 2B). The distribution of pea clams is completely distinct with the hotspots of
diversity being the NT (31%) and the PA (22%), while the remaining diversity is scattered
among the other continents (Fig. 2C). Sphaeriids are also the only FB species that can live at
the higher latitudes of the Arctic, such as the islands of Iceland, Greenland, Baffin, Svalbard
and Novaya Zemlya (Schiøtte & Warén 1992; Bespalaya et al 2017). Finally, for Cyrenidae
and a few other remaining species, the major diversity hotspot is in the IN that contains almost
70% of such species, followed by the PA (18%), while other ecoregions have a much lower
diversity (Fig. 2D).
22 FCUP
Figure 2 Diversity by ecoregions. A All freshwater bivalves; B Unionida; C Sphaeriidae; D

Cyrenidae + remaining freshwater bivalve groups. Ecoregions adapted from Graf & Cummings
(2007) and Haag (2010): NA Nearctic, NT Neotropical, PA Palaearctic, AF Afrotropical, IN
Indotropical, AU Australasian. Glaciated and desert areas void of mussels in grey.
Diversity at an ecoregion scale is also not distributed evenly (Fig. 3). Within NA, the species
diversity is generally higher in the interior basins, while in the NT the diversity is higher in
Central America and the Orinoco, Amazon and Paraguay River basins (Fig. 3A). In the AF, the
Congo River basin is richer in Unionida species (Fig. 3B), and the Nile and Eastern African
River basins have a higher sphaeriid diversity (Fig. 3C). While the Western Palaearctic is quite
diverse in sphaeriids and dreissenids, Laurasia has a higher diversity in the IN, from the Hindu
to the Amur River basin (Figs. 3A, C and D). Within IN, the diversity of sphaeriids is higher in
the Indian subcontinent, while in the Unionida and the remaining groups the diversity is higher
in Indochina and Sundaland (Fig. 3). In AU, a higher number of species is found in the East
(Fig. 3).
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Figure 3 Diversity by ecoregions. A All freshwater bivalves; B Unionida; C Sphaeriidae; D

Cyrenidae + remaining freshwater bivalve groups. Ecoregion subdivisions adapted from Graf
& Cummings (2007) and Haag (2010): NA Nearctic, NT Neotropical, PA Palaearctic, AF
Afrotropical, IN Indotropical, AU Australasian. Glaciated and desert areas lacking FBs are in
grey.
Although specific diversity of FBs is similar in NA, NT and IN, there is a higher taxonomic
diversity in the IN than in NA and NT. In the IN there are representative species of 5 orders
and 10 families compared to the 2 orders and 4 families in the NA and 3 orders and 8 families
in the NT (Fig. 4). Even within the most species-rich FB family, the Unionidae, the IN exhibits
a much higher taxonomic diversity than all the other ecoregions, with representatives of all
subfamilies of Unionidae occurring there, except for the NA Ambleminae.
We would like to stress that diversity patterns described above may be underestimated
and may change substantially as a result of ongoing and future surveys in the less studied
regions of Southeast Asia, Africa, NT, and AU. For example, Bolotov et al (2017) studying the
FBs of a poorly known and remote basin (Sittaung) in Myanmar described two new genera
and seven new species. Also, even in Europe and NA, which are the most well-studied regions,
the knowledge of the diversity of Unionida is still undergoing considerable changes (e.g. Froufe
et al 2016a,b, 2017; Araujo et al 2017; Lopes-Lima et al 2017a; Williams et al 2017; Smith et
al 2018).
24 FCUP
Figure 4 Taxonomic composition and diversity of freshwater bivalves in each ecoregion. The
total number of species in brackets.
Conservation status and major threats

Freshwater bivalves are among the most threatened taxonomic groups in the world, with
almost 40% of the species being near threatened, threatened or extinct (Fig. 5). However, this
high imperilment is mainly due to the contribution of Unionida since not all groups are evenly
threatened or assessed for conservation status (Fig. 5, top). Based on the number of assessed
species, the highest percentage (45%) of near-threatened, threatened and extinct species
(including 25 [2.8%] extinct or probably extinct species) is in Unionida, while only 14.5% of
Sphaeriidae and 8.8% of Cyrenidae (plus all the other remaining species) have a near-
threatened or threatened status (Fig. 5, top part). However, IUCN assessments are not evenly
distributed across taxa and countries and FBs are a good example of this situation (Fig. 5).
FCUP 25
Thus, a higher percentage of large and more conspicuous unionids has been assessed
compared to other FB groups (Fig. 5, top). Some ecoregions (e.g. NA, AF, PA and IN) have a
high percentage of species evaluated, while species from AU and especially NT have a very
low Red List coverage (Fig. 5, bottom part). The percentage of threatened and near-threatened
Unionida species is higher in NA (67%) and PA (52%) than in other ecoregions, with the lowest
percentage (19%) in the IN (Fig. 5, bottom). This does not necessarily mean that fewer species
are threatened in the IN, since this ecoregion has a much higher percentage of data deficient
species, reflecting the lower level of knowledge and data on the threats available for IN
species. On the other hand, almost half of the species have been assessed as of ‘‘least
concern’’ in the AF, which might indicate a more favourable status of freshwater mussels in
this ecoregion.
Figure 5 Map of IUCN Red List conservation status for Unionida freshwater mussels by
ecoregions (bottom of the figure) and global conservation status for freshwater bivalves and
each major freshwater bivalve group (top of the figure). Ecoregion subdivisions adapted from
Graf & Cummings (2007) and Haag (2010): NA Nearctic, NT Neotropical, PA Palaearctic, AF
Afrotropical, IN Indotropical, AU Australasian. On the scale bar: NE Not evaluated by the IUCN
Red List; and the IUCN Red List categories: DD data deficient, LC least concern, NT near
threatened, VU vulnerable, EN endangered, CR critically endangered, CR (PE) critically
endangered probably extinct, EX extinct.
26 FCUP
The IUCN Red List assessments are based on a set of five criteria: (A) population size
reduction, (B) small geographic range, (C) small population size plus decline, (D) very small or
restricted populations and (E) a quantitative analysis of extinction probability (IUCN 2012).
Most of the near-threatened and threatened FB species have been assessed using criteria A
and B and to a much lesser extent using criteria C and D (Fig. 6). Since criterion E needs
comprehensive data on a wide range of features (e.g. demography, life history, habitat
requirements, threats and management options), no FB species was ever evaluated using this
criterion (Fig. 6). Most FB species have been assessed based on their population size
reduction and geographic range contraction compared to a few species with very small
distribution ranges. It is difficult to assign a threatened status using criterion D for most FB
species due to their generally large distribution ranges.
The global pattern is similar in all ecoregions, except the NT and AF (Fig. 6). While the
NT pattern may not be very representative of the ecoregion due to the few assessed species,
in AF it reflects the poor knowledge about the population size and trends. This is due to the
lack of research that is being done in the AF, where survey and monitoring studies are almost
non-existent (Lopes-Lima et al 2014; Sousa et al 2016, 2017).
Figure 6 IUCN Red List criteria used for the assessment of freshwater bivalve species by
ecoregions. Ecoregion subdivisions adapted from Graf & Cummings (2007) and Haag (2010):
AF Afrotropical, AU Australasian, IN Indotropical, NA Nearctic, NT Neotropical, PA Palaearctic.
IUCN Red List criteria: A population size reduction, B geographic range, C small population
size and decline, D very small or restricted populations.
FCUP 27
Freshwater bivalves are affected by multiple threats that range from natural system
modifications to degradation, pollution, introduction of IAS, exploitation and human
disturbance. Within the assessed FB species for the IUCN Red List, pollution is still the most
recorded global threat comprising 42% of all threats (Fig. 7). Natural system (habitat)
modifications such as the construction of dams and channels are the second most cited threat
(20%), followed by urban development, exploitation, agriculture, climate change, mining, and
IAS, together representing less than 10%. Other disturbances such as transport, recreational
activities and geological events only play a minor role.
The relative percentage of recorded threats is generally similar across the main
ecoregions with a few notable exceptions. For instance, in the NA and PA species seem to be
less threatened by climate change than the tropical and southern hemisphere ecoregions.
Conversely, in the more developed areas of the NA and the PA, habitat modifications seem to
negatively affect more species in these ecoregions than in the AF and IN. Exploitation is a
much more detrimental threat in the IN than elsewhere (Fig. 7). Harvesting of mussels for
human consumption in East and Southeast Asia is a major economic activity; for example, in
Vietnam, it may reach up to 50,000 tons per year in each major basin (Köhler et al 2012).
Furthermore, the ratio of agriculture-related threats is higher in AU and PA, mainly due to water
diversion and extraction.
Figure 7 Main threats for freshwater bivalves recorded from the IUCN Red List database by
ecoregions. Ecoregion subdivisions were adapted from Graf & Cummings (2007) and Haag
(2010): NA Nearctic, NT Neotropical, PA Palaearctic, AF Afrotropical, IN Indotropical, AU
Australasian.
28 FCUP
Research and conservation actions needs

Many species of FB are still poorly understood, especially in Central America, Southeast Asia
and Sundaland (Lopes-Lima et al 2014, 2017b). This lack of knowledge hampers their status
assessment.
The IUCN database indicates that research needs are generally lower for the NA and
PA compared to the other ecoregions, especially for the three top research needs, i.e.
population size and distribution, identification of threats and life history (Fig. 8A). This may be
explained by the stronger research effort and higher financial support available for North
American and European studies. However, even in these ecoregions, basic data on
distribution, population size, accurate identification of threats and basic life-history traits are
still lacking for many species. Taxonomical data and knowledge on life-history traits are
particularly needed for AF species. The same general trends in research needs can be seen
for all species assessed by IUCN as well as for data-deficient species (Fig. 8B).
Figure 8 Research needs for freshwater bivalves recorded from the IUCN Red List database
by ecoregions. A All assessed species in the IUCN Red List; B data-deficient species in the
IUCN Red List. Ecoregion subdivisions adapted from Graf & Cummings (2007) and Haag
(2010): NA Nearctic, NT Neotropical, PA Palaearctic, AF Afrotropical, IN Indotropical, AU
Australasian. DD data-deficient species in the IUCN Red List.
Due to the high risk of extinction, many species urgently need worldwide conservation actions.
Land and water protection was found to be the top conservation measure globally and
throughout all ecoregions, but especially for IN species (Fig. 9). Land and water management
is also shown to be one of the top priorities for FB conservation, particularly in the PA and AF
ecoregions (Fig. 9). Other types of conservation actions showed quite distinct patterns among
ecoregions. For example, stronger legislation is likely required for the AF, PA, NA, and AU, but
law enforcement needs to be enhanced only in the AF and PA ecoregions. Moreover,
FCUP 29
increasing awareness of the general public about the importance of conserving FB is quite
essential for the PA and particularly in the IN. Special interest in species ex-situ propagation
and reintroduction programs is exhibited for the NA, emphasising the vast knowledge already
accumulated for many species in the ecoregion (Fig. 9).
Figure 9 Conservation needs for freshwater bivalves extracted from the IUCN Red List
database by ecoregions. Ecoregion subdivisions adapted from Graf & Cummings (2007) and
Haag (2010): NA Nearctic, NT Neotropical, PA Palaearctic, AF Afrotropical, IN Indotropical,
AU Australasian.
Although many research gaps and conservation needs have been identified in the last years,
many recent technological advances can provide us with new insights that are needed for FB
research. For example, new remote sensing techniques like underwater video and side-scan
sonars may help survey FB populations and identify more favourable habitats (Powers et al
2014; Mehler et al 2016). The use of drones in semi-arid regions can aid in tracking and
identifying the remaining pools after droughts where mussels take refuge. These technologies
and the use of environmental DNA analyses may help gathering basic biological and ecological
data on distribution and abundance, which are still missing for many species (Stoeckle et al
2016). More powerful genetics and morphometric tools are also increasingly available, for
instance, new statistical tools for species delimitation using molecular and/or other types of
data such as morphometry and anatomical traits (e.g. Froufe et al 2016b; Pfeiffer et al 2016).
These tools are particularly important because many species present hidden cryptic diversity
30 FCUP
(Froufe et al 2016b; Pfeiffer et al 2016). Additionally, next-generation sequencing is now

allowing for quicker and less expensive robust phylogenies using methods like whole-
transcriptome and whole-mitogenome analyses with a wide range of markers (Guerra et al
2017; Lopes-Lima et al 2017c). Furthermore, using reduced genome representations or snip
analyses, it is now possible to get more information on the phylogeographic patterns of species
and on the definition of conservation units (Catchen et al 2017; Desalle & Amato 2017).
While most of the global protected areas network is aimed at protecting essentially
terrestrial vertebrates, the identification of sites to conserve freshwater vertebrates and
invertebrates such as FB is also of crucial importance (Darwall et al 2011; Maceda-Veiga et al
2017). Using the IUCN Key Biodiversity Areas (KBAs) network (IUCN 2016) or new systematic
conservation planning approaches (e.g. Hermoso et al 2015) may help to promote a better FB
representation within protected area networks.
The proceedings of the Second International Meeting on Biology and

Conservation of Freshwater Bivalves
All the research and conservation needs above summarised, make the facilitation of
cooperation among scientists from different countries and continents particularly important. For
example, recent reviews published by multinational teams of scientists provided vital baseline
information about FB on different continents (e.g. Pereira et al 2014 for South America, Walker
et al 2014 for Australia; Lopes-Lima et al 2017a for Europe; Williams et al 2017 for North
America, and Zieritz et al 2017 for East and Southeast Asia). Additionally, intercontinental
cooperative research is also becoming increasingly common (see for example Zieritz et al
2016; Lopes-Lima et al 2017b). To discuss the current and future research challenges and
needs, the Second International Meeting on Biology and Conservation of Freshwater Bivalves
was hosted by the Great Lakes Centre at SUNY Buffalo State College in Buffalo, New York,
USA, from 4 to 8 October 2015, bringing together over 80 scientists from 19 countries and four
continents (Europe, North America, South America, and Australia) (Burlakova et al 2017).
The present special issue in Hydrobiologia comprises a total of 34 papers (including
this introductory note) summarising some of the information presented in this meeting. These
papers cover a wide variety of topics, from a review of ecosystem services provided by
freshwater mussels (Vaughn 2017) to papers describing the diversity patterns and
conservation of Unionida in East and Southeast Asia (Zieritz et al 2017) as a result of
international collaboration. Seven papers focus on different biological aspects of invasive
bivalve species, including diversity changes by species substitution (Karatayev et al 2017),
physiological aspects (Labecka & Domagala 2016), dispersion (Collas et al 2016), ecological
effects on native bivalve species (Ferreira-Rodríguez et al 2016), low palatability to distinct
FCUP 31
predators (Castro et al 2017), metabolite emission suppression in zebra mussels exposed to

predation stress (Antoł et al 2017) and the use of a new sonar technology and underwater
imagery analysis for the survey of FB in rivers (Mehler et al 2016). Propagation as a
conservation tool was the subject of three studies: one about an improved method of in vitro
culture of glochidia (Ma et al 2016), one introducing short-term breeding of the Endangered
freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) as a new technique for
the augmentation of declining populations (Moorkens 2017) and one revising the challenges
in the conservation progress of Margaritifera auricularia (Spengler, 1793) (Prié et al 2017). Six
papers used molecular tools to describe genetic structure or phylogeographic patterns of
European (Feind et al 2017), North American (Hewitt et al 2016; Mathias et al 2016) and South
American species (da Cruz Santos-Neto et al 2017); to reveal the uncommon doubly
uniparental inheritance of mitochondria in a European species (Soroka and Burzynski 2017)
and the sequencing of transcriptomic resources for an invasive species (Soroka et al 2017).
The interaction between mussels and their host fishes was addressed in three papers that
evaluate the effects of stress (Douda et al 2016), cross-immunity (Chowdhury et al 2017) and
temperature (Schneider et al 2017) on the reproduction of freshwater mussels. Three papers
describe distribution patterns with distinct spatial and temporal scales: the population trends
of Unionidae in Romania (Sîrbu and Benedek 2017), the distribution of freshwater mussels
and their host fishes in Texas (Dascher et al 2017) and a study that reconstructs the historical
range and population size of the threatened species Popenaias popeii (Karatayev et al 2015).
On a smaller scale, a study on the longitudinal variation in freshwater mussel assemblages
within two rivers is presented by Chambers & Woolnough (2016), while Dittman et al (2017)
evaluate the microhabitat and biology of the poorly studied pea clam Sphaerium striatinum.
Two papers assess the growth of M. auricularia (Nakamura et al 2017) and juvenile freshwater
pearl mussels M. margaritifera at the river scale (Cêrná et al 2017). One paper assesses the
shell phenotypic plasticity of Unio crassus (Zajaç et al 2017). The influence of the flood pulses
and near-bed hydrodynamics on freshwater mussels is evaluated by Callil et al (2017) and
Sansom et al (2017), respectively. Finally, toxicology and archaeology are represented by a
study of the effects of polycyclic aromatic hydrocarbons on unionid mussels (Archambault et
al 2017) and the conservation implications of freshwater mussel remains in a Texan river
(Popejoy et al 2016).
Conservation of FB requires urgent collaboration between scientists, managers,
politicians and the general public, to share knowledge and efforts. An example of this
collaboration is the International Meeting on Biology and Conservation of Freshwater Bivalves,
but more efforts are necessary for the transfer of knowledge between scientists and the general
public to raise awareness about the importance of FB conservation. These efforts can include,
but not be limited to the increase in visibility of FB conservation issues in the media, better
32 FCUP
engagement with local communities and stakeholders (e.g. providing training and lifelong
learning opportunities like workshops for public, better information dissemination and
accessibility of collaborative research even integrating participants from civil society into
surveys and research projects), publications and additions to national collections.
Acknowledgements
We thank the Editor-in-Chief Prof. Dr. Koen Martens for his help and support and the
coordinator of Springer Journals Editorial Office, Deepan Selvaraj, for his hard work and
assistance in the editorial process of this Special Issue. This work was supported by FCT-
Foundation for Science and Technology, Project 3599-Promote the Scientific Production and
Technological Development and Thematic 3599-PPCDT by FEDER as part of the project
FRESHCO: multiple implications of invasive species on Freshwater Mussel co-extinction
processes (Contract: PTDC/AGRFOR/1627/2014). FCT also supported MLL under Grant
(SFRH/BD/115728/2016).
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CHAPTER 3
Basic biological traits of Unio delphinus
Paper II
Setting the stage for new ecological indicator species: A holistic case study
on the Iberian dolphin freshwater mussel Unio delphinus Spengler, 1793.
Lopes-Lima M, Hinzmann M, Varandas S, Froufe F, Reis J, Moreira C, Araujo S, Miranda F,
Gonçalves DV, Beja P, Sousa R, Teixeira A
Article published in Ecological Indicators 111, 105987 (2020).
DOI: 10.1016/j.ecolind.2019.105987
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Setting the stage for new ecological indicator species: A holistic case study
on the Iberian dolphin freshwater mussel Unio delphinus Spengler, 1793.
Manuel Lopes-Lima1,2,3,⁎, Mariana Hinzmann2, Simone Varandas4, Elsa Froufe2, Joaquim

Reis5, Claudia Moreira2,6, Sandra Araujo2, Fernando Miranda7, Duarte V.Goncalves1,2,
PedroBeja1,8, RonaldoSousa9, AmilcarTeixeira7
1
CIBIO/InBIO - Research Center in Biodiversity and Genetic Resources, University of Porto, Campus Agrario de
Vairão, 4485-661 Vairão, Portugal
2
CIIMAR/CIMAR - Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto
de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
3
IUCN SSC Mollusc Specialist Group, c/o IUCN, David Attenborough Building, Pembroke St., Cambridge, United
Kingdom
4
CITAB-UTAD - Centre for Research and Technology of Agro-Environment and Biological Sciences, University of
Tras-os-Montes and Alto Douro, Forestry Department, Apartado 1013, 5001-811Vila Real, Portugal
5
MARE - Marine and Environmental Sciences Centre Faculdade de Ciências da Universidade de Lisboa Campo
Grande, 1749-016 Lisboa, Portugal
6
Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto (ICBAS/UP), Rua Jorge Viterbo Ferreira
228, 4050-313 Porto, Portugal
7
CIMO-ESA-IPB - Mountain Research Centre, School of Agriculture, Polytechnic Institute of Braganca, Campus de
Santa Apolonia, Apartado 1172, 5301-854 Bragança, Portugal
8
CIBIO/InBIO - Research Center in Biodiversity and Genetic Resources, Instituto Superior de Agronomia,
Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal
9
⁎
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Abstract
Due to their sensitivity and dramatic declines, freshwater mussels are prime targets for
conservation and environmental monitoring. For this, however, information is needed on life
history and ecological traits, which is lacking in many taxa, including threatened species.
Species recently described or recognized as valid are of particular concern, due to the shortage
of even basic knowledge. A case in point is the recently recognized and Near Threatened
dolphin freshwater mussel Unio delphinus Spengler, 1793, which is endemic to the western
Iberian Peninsula and has suffered marked population declines. To overcome information gaps
for U. delphinus, we carried out a holistic biological study across the species range, aiming to:
i) estimate the area of occupancy (AOO) and extent of occurrence (EOO) based on updated
distribution data taken from the literature and recent surveys; ii) estimate growth patterns from
biometrical (shell dimensions and growth annuli) measurements taken on specimens from
seven populations; iii) estimate sex ratios from gonad tissue biopsies collected on specimens
from eight populations; iv) estimate gametogenesis through histological examination of gonad
and gill tissues collected monthly for a year, from a single population; and v) determine host
species from infestation trials of glochidia with co-occurring fish species. We estimated an
EOO of 706 km2 and an AOO of 61 km2, which together with data on declines assigns the
species to the Endangered category using IUCN criteria. Unio delphinus was found to grow
faster and to be shorter-lived (up to 11 years, maturity at around 2 years old) than other
European freshwater mussels. Growth and life span are similar across the range in lotic
habitats, but different from that in lentic habitats. The larvae of U. delphinus may attach to most
co-occurring fish species, but only native species were effective hosts. Native cyprinids,
especially those from the genus Squalius, seem to be the primary hosts. Overall, the
information provided contributes to a better conservation status assessment, selection of
conservation and rehabilitation areas, guidance for the establishment of propagation programs
and better timing for specimens’ manipulation including monitoring and possible translocations.
The framework presented here highlights the importance of basic biological studies to define
good ecological and physiological status.
Keywords
Conservation, Unionida, Life-history traits, Growth, Host-fish
44 FCUP
Introduction
The definition and requirements of ecological indicators have been subject to some debate
and confusion, but indicator species are undoubtedly important components for ecosystem
quality assessments (Heink and Kowarik 2010). A highly cited review by Carignan and Villard
(2002) identified two ideal general qualities for indicator species: negative association with
human disturbance, and habitat specialisation. Additional characteristics were described from
previous studies (Noss et al 1997): the potential as an early warning system, the discrimination
of the cause of change, the range of responses, and the cost-effectiveness of the survey.
Potential indicator species are often at the same time keystone, area-limited ‘umbrella’,
dispersal-limited, resource-limited, process-limited, or flagship species (Carignan and Villard
2002; Lambeck 1997; Noss et al 1997). However, even when matching all these criteria, there
is still a need for several disparate indicators, since each species reacts to disturbances at
different degrees and scales (Carignan and Villard 2002). Finally, for a species to be useful as
an ecological indicator, it needs first and foremost to be very well studied, so that survey data
allows distinguishing actual disturbance signals from variations that may be unrelated to the
deterioration of ecological integrity (Carignan and Villard 2002).
The bivalves of the Unionida order, also known as freshwater mussels, are key
elements of aquatic ecosystems (Lopes-Lima et al 2014, 2018). They play ecologically
important roles such as bioturbation or sediment mixing, nutrient cycling and energy transfer
from the water column to the bottom, among other processes (Vaughn 2018). However, this
faunal group, like most others in freshwater ecosystems, has been declining dramatically over
the last decades, with several species extinctions and many extirpations being reported
(Lopes-Lima et al 2014, 2018). Freshwater mussels are very sensitive to human activities, but
other intrinsic features increase the probability of extirpation or extinction. For instance, these
organisms generally have a slow metabolism, taking at least a year to reach sexual maturity
(Lopes-Lima et al 2017a). Also, they have a complex life cycle where larvae (glochidia) need
to attach to specific fish hosts (Modesto et al 2018). Given their important ecological role, but
also high sensitivity to habitat, water, and sediment quality, some freshwater mussel species
simultaneously fulfil criteria for indicator, flagship, and umbrella species, making them
important targets for environmental monitoring and conservation (Geist 2010; Lopes-Lima et
al 2017a). Like some freshwater mussel species (e.g. Margaritifera margaritifera), umbrella
species conservation strategies are directed towards wide home range species protecting
other sympatric species (Geist 2010). This is due to the high sensitivity of freshwater mussels
to environmental factors that can arise at different spatial scales, not only local but also regional
such as the land-use and geological influence over the whole catchment area (Strayer et al
2004). Freshwater mussels are also highly valued for their rarity, beauty and interesting
FCUP 45
behaviour (Strayer 2017), which added to their tight interspecific relationships and frequently
high cultural value make them suitable ‘flagship species’ to raise support for freshwater habitat
conservation (Caro 2010). Due to their unique and crucial roles in ecosystem functioning, and
the high biomass in many habitats, they can also be considered ecosystem engineers, given
their large physical effects on the ecosystem (Gutierrez et al 2003).
In Europe, 20 freshwater mussel species are currently recognized (Froufe et al
2016a,b, 2017; Lopes-Lima et al 2017a; Araujo et al 2018). Species richness is higher in
central Europe but southern Europe presents a higher level of endemism and restricted-range
species (Lopes-Lima et al 2017a). This is the case of the dolphin freshwater mussel Unio
delphinus Spengler, 1793, which was considered a subspecies of the widespread and more
common European Unio pictorum until its recent recognition as a valid distinct species (Araujo
et al 2009). Unio delphinus has suffered a 30% range decline over the last decades, mainly
due to habitat degradation, including pollution and changes in the hydrologic regime due to the
presence of dams or other infrastructures, poor river management and water shortage (Araujo
2011). The Iberian Peninsula, as most of the regions within the Mediterranean biodiversity
hotspot (Myers et al 2000), is suffering from water scarcity exacerbated by climate change and
associated instability (Robson et al 2013; Cid et al 2017). Similar negative impacts were
observed on the Iberian populations of other freshwater mussel species (Sousa et al 2012,
2018).
Invasive species are also pointed as one of the main threats to freshwater mussels
(Sousa et al 2014). Introduced predators like mammals, fish, and crayfish are known to
consume freshwater mussels and may cause local declines (Meira et al 2019; Sousa et al
2018, 2019). Zebra mussels Dreissena polymorpha and the Asian clam Corbicula fluminea
can reach high densities in their invasive ranges and may outcompete native mussels,
reducing their fitness and growth and increasing mortality rates (Sousa et al 2011; Bodis et al
2014; Lopes-Lima et al 2017a; Ferreira-Rodriguez et al 2019; Modesto et al 2019). Given that
native mussels seem to depend on specific, and usually native fish host species to complete
their life cycle, changes in the fish fauna can also have deep implications on the mussel
populations (Douda et al 2013; Modesto et al 2018).
Over the last decades, there has been a rising awareness about the need to conserve
freshwater ecosystems and taxa, accompanied by the increase of dedicated conservation
funds, mainly in Europe and North America (Lopes-Lima et al 2017a, 2018). This has also
boosted research on freshwater mussel conservation (Lopes-Lima et al 2014). However, in
Europe, the majority of studies were concentrated on a small number of species present on
the European Union (EU) Habitats Directive, disregarding most of the other freshwater
mussels, especially those that were only recognized after the inception of that EU policy
(Lopes-Lima et al 2018), as is the case for U. delphinus. On the other hand, most recent
46 FCUP
research explores threats, management and conservation methods (Lopes-Lima et al 2014),

but much less effort has been devoted to understand the underlying life-history traits that are
essential for effective conservation planning (Lopes-Lima et al 2014, 2017a). These concerns
have been raised in recent reviews that identify as top conservation research priorities
acquiring information on life-history traits, abundance, distribution, and size structure (Lopes-
Lima et al 2018, Ferreira-Rodriguez et al 2019).
Given the high sensitivity and filtering behaviour of freshwater mussels, they are many
times colloquially mentioned as “aquatic canaries in the coalmine” or “livers of the rivers”
(Cummings et al 2016). Demographic, physiological and behaviour features can be used to
determine the status of a freshwater mussel population and therefore indicate potential
environmental perturbation (Van Hassel & Farris 2007). Basic biological features of freshwater
mussels have already been used to assess environmental disturbance in freshwater habitats,
such as the effects of temperature and heated effluents, sewage, siltation, and impoundment
(reviewed in Van Hassel & Farris 2007 and references therein). Many of the same
characteristics that make freshwater mussels good sentinel organisms (e.g. sedentarism,
large/easy to use, sensitive to disturbance, shells providing historical record, widely distributed,
and bioaccumulation of pollutants) also make them well suited to use as indicators of ecological
integrity in assessments of environmental impact, waterbody status monitoring, and
assessments of environmental history (Van Hassel & Farris 2007). However, studies using
freshwater mussels as biological indicators are still scarce (Lopes-Lima et al 2014) due to the
limited knowledge about their life-history traits.
To face the dearth of life-history trait research and to set up a framework that can serve
both as an example and base for future works, the present work applies a holistic approach to
study U. delphinus and thus improve the efficacy of ongoing and future conservation measures
and their use as environmental indicators. We update the distribution and revise the
conservation status of U. delphinus focusing on eight U. delphinus populations to study the
species growth and lifespan patterns throughout its range, determine and describe its
reproductive cycle and sexuality, and identify its fish hosts.
Materials and methods

Distribution
Distribution data was compiled from the literature and personal data from the authors. The
Extent Of Occurrence (EOO) and Area Of Occupancy (AOO) were estimated by two distinct
methods. For estimating AOO, the number of occupied cells in a uniform 2 × 2 km grid,
covering the entire range of the species taxon, was counted and then obtaining the total area
of all occupied cells. This method is the one proposed in the most recent version of the IUCN
FCUP 47
guidelines (IUCN Standards and Petitions Committee 2019) but, in our opinion, it
overestimates the AOO for linear distributed species such as freshwater mussels. Therefore,
we used another method to estimate AOO which better represents the known area occupied
by the taxon. This method to estimate AOO was first used by Gomes-dos-Santos et al (2019)
and consists in multiplying the mean width of the river by a longitudinal (along the river) 2 km
buffer, for each record point and then tallying up the number of records. The mean river width
was obtained per basin as the average of six equidistant points within the species range. As
for EOO, the first method used was the least convex polygon, as the smallest polygon in which
no internal angle exceeds 180 degrees and which contains all the sites of occurrence, as
suggested by the IUCN guidelines (IUCN Standards and Petitions Committee 2019). However,
since most of the species EOO is on land we feel that the terrestrial range should not be
accounted for, and therefore we used also a second alternative method recently published by
Gomes-dos-Santos et al (2019). This method consisted in multiplying the mean river width by
the sum of the river length between species distribution records in each basin.
Sampling
Live specimens (n ≥ 30 per population) of U. delphinus with varied sizes were collected from
eight populations on Atlantic Iberian river basins (Sabor River: 41.239625, -6.967942; Douro
River: 41.152612, -7.765184; Mira Lagoon: 40.441897, -8.756483; Barrinha Lagoon:
40.450047, -8.797069; Mondego River: 40.204369, -8.361042; Ponsul River: 39.778456, -
7.432322; Guadiana River: 38.831016, -7.085385; and Vascão River: 37.516950, -7.579433)
(Fig. 1), chosen to represent a wide range of latitudes and habitats. All sites contain well-
established and healthy populations of U. delphinus and display a good ecological and
chemical status (Reis 2006; Oliveira et al 2007; SNIRH 2019) and never suffered from known
acute events of pollution.
The shells and growth rings of all specimens were measured (see below Section 2.3
for further details) for seven of these eight populations (excluding the Ponsul River population).
For the seasonal sexual development and determination of the age of maturity, 10 specimens
of U. delphinus were collected from the Sabor River population each month for one year (Fig.
1). The mussels were transported in a refrigerated box and processed within 24 h. The mussels
were anesthetized as described in Hinzmann et al (2013) and euthanized for histological
inspection (see below Section 2.4 for further details). To minimize eventual negative impacts,
the number of animals sacrificed was kept to a minimum.
Gonad tissue biopsies were collected for seven of these eight populations, (excluding
the River Douro Population). All specimens measured and biopsied (see below Section 2.5 for
further details), were then returned to their original locations.
48 FCUP
For the host compatibility experiments, 14 native and 6 non-native fish species,
representing the most common fish taxa with an overlapping distribution with U. delphinus
(Oliveira et al 2007; Lopes-Lima et al 2017a) were collected by electrofishing. For the same
experiment, six gravid mussels were collected from the Douro River population and
transported to the laboratory (see below further details about hosts in Section 2.6).
Figure 1 Map showing the known distribution records of Unio delphinus (white circles),
sampling sites for growth and sex-ratio (all red markers), and sampling site for the evaluation
of reproductive cycle (red square). Both maps are represented using the World Geodetic
System 84 (WGS84) projection.
Shell dimensions (length, width, and height) and the annuli or growth rings were measured
from each individual following Aldridge (1999). Individuals with shell abnormalities, incomplete
annuli, and eroded shell umbos were excluded from the analysis. The length of the annuli
dimensions was used to produce length-age plots for each population. Growth curves were
constructed using the Von Bertalanffy equation (Bauer 1992; Aldridge 1999).
The Equation was used in the form: Lt + m = cLt + d
Where:
Lt is the shell length at time t;
Lt + m is the shell length at time t + m;
m is the measurement collection interval (1 year in the present case, i.e. m = 1);
c = e-km being k the growth constant defining the rate at which the asymptotic length is
reached;
d = L∞ (1 - c), being L∞ the asymptotic length.
FCUP 49
For each population, the longest mussel length was used to calculate the maximum
age at each site, using the following formula t m = -1/k.ln[1 - (Lm/L∞)] (Ziuganov et al 1994).
Analyses of Covariance (ANCOVA) implemented in PAST 3.25 (Hammer et al 2001)
were then used to compare growth models.
Reproductive cycle histological procedure

The gonad and gill tissues were removed from each animal. Sex determination was first made
from smears of fresh gonad tissue (across the whole gonad) observed under the microscope,
and the macroscopic and microscopic aspects of the gonads were recorded. The gonad tissue
samples were then fixed in Bouin’s (Panreac) solution for a week, cut and inserted into
histologic cassettes, and dehydrated in an ethanol gradient followed by xylene and paraffin
impregnation overnight, using a Shandon Citadel 2000 Tissue Processor. To finalize, samples
were included in paraffin blocks using a Shandon HistoCentre 2. Sections of 5-6 μm were
made on a Leica RM2255 microtome and stained using standard H&E coloration (following
Hinzmann et al 2013). Observations were made on an Olympus DX 41 with DP 70 camera. A
division of the main stages of male and female gonadal development or Gonadal Development
Index (GDI) was then established for U. delphinus based on most observed cases in the
respective period, as described in Hinzmann et al (2013). For the determination of the
embryonic development periods, the gills were also inspected.
Sex ratio distribution and age of maturity

All specimens were carefully opened with crossed pliers and four small tissue biopsies were
collected with a biopsy needle across the foot to an Eppendorf tube, fixed and visualized under
the microscope as explained above for the gonad sections. Sex was confirmed by the presence
of male or female cells. The approximate age of maturity was determined using the inverse of
Von Bertalanffy equation on the age of the younger individuals carrying gamete cells. To
assess if sex ratios differed from the 1:1 expectation, a chi-square test was used for each of
the seven populations analysed.
Fish hosts
Experiments assessing the infestation capability of U. delphinus glochidia with native and non-
native fish species were conducted in July 2018. Extraction of the glochidia and infestation
trials followed the methodology described in Douda et al (2013) with a minimal bath volume of
0.5 L per fish individual containing a mean ± SD of 1489 ± 150 viable glochidia, added to each
tank. All fish were then separated by species in 40 L tanks (up to 3 fish per tank) to monitor
the developmental success of U. delphinus glochidia. A 3-mm net was used on the bottom of
each tank to avoid juvenile predation by fish. The tanks were part of a recirculation system
50 FCUP
kept at 20 °C. Fish were fed daily with commercial fish food. Each tank was siphoned daily into
filters (mesh size 100 μm), that were examined for the presence of glochidia and juvenile
mussels. The proportion of successfully transformed juveniles (transformation rate) was
calculated following Douda et al (2013), using the recorded number of juveniles and the initial
number of attached glochidia (determined by the number of dead glochidia + viable juveniles
counted from each tank post infestation). The cumulative number of degree-days was
calculated by the sum of daily temperatures (all at 20 °C by controlled temperature) during
glochidia attachment. This was determined by multiplying the daily temperature by the number
of days during fish infestation. Five days after the last juvenile was recovered, we considered
each trial (N = 56) to be complete. Fish were then checked for residual attached glochidia.
Results
Distribution
Unio delphinus populations were recorded in most Atlantic basins of the Iberian Peninsula,
from the Ulla River in the north to the La Vega River basin in the south, near Gibraltar. It also
occurs in few Mediterranean coastal basins east of Gibraltar until the Guadalhorce River basin
near Malaga (Fig. 1; Appendix 1). The estimated EOO varies between 706 (multiplying the
mean river width by the river length within the extremes of the species distribution in each
basin) and 344,641 (using the minimum convex polygon EOO estimates) km 2. The AOO varies
between 61 and 2,000 km2 using the mean width of the river along the hydrographic network
or 2 × 2 km grid overlay methods, respectively.
Growth and longevity

Individuals of all populations grew approximately up to 20 mm in the first year. Then the annual
growth rate decreased steadily (Fig. 2). The maximum length measured revealed a major
difference between lentic and lotic populations, varying in river populations between about 60-
80 mm, while reaching over 100 mm in both lagoon populations. The von Bertalanffy growth
parameters for all populations are represented in Table 1. The growth constant (k) is similar in
populations from related habitats but are much higher for river than lagoon populations (Fig.
2; Table 1), indicating that the asymptotic length is reached sooner in the former.
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Table 1
Growth parameters for Iberian Unio delphinus populations. L∞ is calculated from the Wolford equation, L max is the maximum observed length in
the field. The maximum age was estimated from Lmax
Growth Asymptotic length Maximum length

Population Wolford plot Maximum age (years)
constant (k) L∞ (mm) Lmax (mm)
River Sabor y = 0.72x + 21.68 (R² = 0.996) 0.33 76.88 69.08 7
River Douro y = 0.68x + 22.18 (R² = 0.993) 0.38 69.85 68.8 11
Mira Lagoon y = 0.81x + 23.98 (R² = 0.996) 0.22 125.04 106.95 9
Barrinha Lagoon y = 0.81x + 23.51 (R² = 0.991) 0.21 125.04 104.94 9
River Mondego y = 0.67x + 24.10 (R² = 0.991) 0.41 71.95 68.66 8
River Guadiana y = 0.71x + 23.39 (R² = 0.984) 0.34 80.34 78.19 11
River Vascão y = 0.65x + 21.69 (R² = 0.998) 0.44 61.63 58.98 7
52 FCUP
As for the maximum age, populations from the larger rivers, i.e. Douro and Guadiana, attained
the longest longevities (≈11 years) followed by the lagoon populations (≈9 years) and finally
by populations colonizing smaller rivers (≈7-8 years) (Table 1). ANCOVA show significant
differences in growth among all populations (F = 9.4, p < 0.01). No significant differences in
growth were detected when considering separately lagoon (ANCOVA, F = 0.07, p > 0.1) and
river (ANCOVA, F = 1.71, p > 0.1) populations.
Figure 2 A - Size-at-age measurements of shell length; B - size as a function of bivalve age,

modelled by the von Bertalanffy growth function.
Reproductive cycle
The general structure of the gonads of Unio delphinus
The gonads of U. delphinus fill most of the foot tissue surrounding the digestive tract. This
species is strictly dioecious, as no case of hermaphroditism was detected. Macroscopically,
gonad tissue presents sex-specific appearance and coloration. The gonad tissues of females
are dark-yellow/orange and are denser and granular, due to the presence of mature oocytes
(Fig. 3). The male gonad tissues have a lighter yellow coloration and were more fluid in terms
of consistency (Fig. 4). The microscopic organization of the gonads consists of highly branched
cell clusters (acini) surrounded by connective and muscular tissue. These acini were found full
of gametes throughout the year, independent of sex.
Oogenesis
Female individuals presented follicles with reproductive cells in all development stages
throughout the year and the reproductive cycle is biannual, continuous and uninterrupted.
However, the prevalence of the different stages of development varies seasonally (see GDI
sub-section below). The oogenesis was mainly divided into five continuous stages, according
to the maturation stage of the gamete cell: oogonia, previtellogenic oocytes, early oocytes,
oocytes and mature oocytes (Fig. 3).
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Figure 3 Histological sections from female gonads of Unio delphinus stained with Hematoxylin
and Eosin (H&E). A - General aspect of female gonads (fa) organized in acini, in September
the acini showing gonads at all development stages of oogenesis, with several mature oocytes
(m) (scale bar 100 μm). B - Female acinus in September with a predominance of earlier stages
of oogenesis: oogonia (o), previtellogenic oocytes (pvo), pedunculated oocytes (po), and the
germinal epithelium (ge) are visible surrounding the germinative cells (scale bar 50 μm). C -
Female acini in May also presented different development oogenesis stages, dominantly the
earlier previtellogenic oocytes (pvo) surrounded by the germinative epithelium (scale bar 100
μm). D - Detail of female acinus in September showing pedunculated oocytes (po) with stalk
(s) visible and mature oocyte in the lumen (l) (scale bar 50 μm). E - Mature acinus in
September, full of mature oocytes (m) in the centre surrounded by earlier stages and
germinative cells (scale bar 100 μm). F - Mature female acini with mature oocytes (m) released
into the lumen (l), and muscle tissue (ms) (scale bar 100 μm). G - Female acinus in October
with only a few mature oocytes (m) already in the lumen (l), one showing two nucleoli (n) in the
nuclei, presenting still some early stages of oocytes and with several yellow bodies (yb),
indicating early signs of degeneration (scale bar 50 μm). H - Degenerative female acinus (dfa),
surrounded by an undifferentiated epithelium, but still presenting some stages of oocyte
development (scale bar 100 μm).
54 FCUP
Oogonia represent the first stage of the gamete development (Fig. 3B), corresponding to the
smallest and rounder cells, located radially along the follicle next to the epithelial cells, with a
diameter between 10 and 13 μm. The nucleus is not visible due to the disperse chromatin and
the intense pink coloration is due to the cytoplasm acidophilic properties. This stage was rare
or inexistent during the peak of maturation and release of mature oocytes (corresponding to
stage 4 of the GDI, described below). In the next stage, previtellogenic oocyte (Fig. 3A and C),
the size of the cells increased, and the nucleus can sometimes be differentiated.
Previtellogenic oocytes still present a very acidophilic cytoplasm, and the size of the cells
varied between 13 and 18 μm. These cells can be found in the periphery or slightly internal
position in the follicle. The vitellogenic oocytes or early oocytes are bigger (20 - 30 μm cell
diameter) than the previous stage, but the main difference is the presence of one or more
(usually two) nucleolus in the nucleus and their localization that is more internal into the lumen
of the follicle. However, early oocytes can still be linked to the germinal epithelium by a
peduncle or stalk, as pedunculated oocytes (Fig. 3B and D). In the following stage, the oocytes,
the shape of the cells become more irregular (Fig. 3A, D, E, and F), at this stage the cell length
can vary between 50 and 60 μm, the nucleus and the nucleoli are visible in a central position,
in some sections is possible to see many vesicles of reserve substances inside the cytoplasm.
Finally, the mature oocytes or eggs can reach up to 100 μm length (cell diameter usually
between 70 and 90 μm: Fig. 3A, D, E, and F). The aspect of these cells is more diffuse,
presenting an acidophilic cytoplasm. The nucleus of mature oocytes is more difficult to observe
in section, but when visible it is smaller and with more basophilic characteristics than the
cytoplasm (Fig. 3G). At this stage, the gametes are concentrated in the lumen and fill the follicle
(Fig. 3D, F, and G), giving little space for the other cell stages. It was also possible to observe
some mature oocytes already in the ciliated gonoduct (not shown). After the major release
events, it is possible to observe a few mature oocytes in the lumen; however, the acini show
degenerative signs, being the integrity of the epithelium compromised (Fig. 3H).
Spermatogenesis
The process of maturation of the gamete cells is here described in four stages, although they
occur simultaneously: spermatogonia, spermatocytes, spermatids, and spermatozoa, which
corresponds to the mobile mature phase of the gametes (Fig. 4). The maturation of the cells
in the male follicle is concentric, with the early stages located more in the periphery and the
mature spermatozoa filling the centre of the lumen (Fig. 4A and E). Each cell stage shows a
tendency to aggregate in clusters, especially the spermatocytes and the spermatids (Fig. 4C).
In the case of spermatids, they were frequently found under the shape of morulae, where
groups of 3-12 cells could be found together (Fig. 4C, D and E). Spermatogonia are the first
stage of maturation of the male gametes (Fig. 4C and D). It corresponds to the larger cells with
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a diameter between 6 and 9 μm and a more irregular shape. They were less frequently found
in the sections. These cells were usually found in the periphery of the follicle, with lighter
basophilic coloration. The interior of the spermatogonia is filled by the nucleus, with condensed
chromatin. The spermatogonia then pass to a second stage, the spermatocyte, through mitotic
division (Fig. 4C and D). With a spherical shape, smaller size, and a diameter between 4 and
6 μm, spermatocytes present dense chromatin that almost fills the whole cell, being the nuclear
membrane hardly visible. These cells develop by meiosis into spermatids which are even
smaller and rounder cells (3-4 μm diameter) (Fig. 4C and D). Spermatids are darkly marked
with the dye, highly basophilic, and present a polyhedral shape and homogenous dark nucleus.
These cells usually have a more internal distribution than the previous ones, and they are
frequently organized in morulae, where clusters of 3 to more than 12 spermatids can be found.
The spermatids develop then into the final stage of maturation, i.e. the spermatozoa (Fig. 4A,
B, E, and F). Spermatozoa were present throughout the year (except in two organisms from
August), but with the prevalence of the different development stages, varying seasonally (see
GDI section below). In fresh samples the flagella were visible, and its activity registered. These
cells present a rod shape in which the body length was approximately 3-5 μm and the flagella
10-20 μm. The spermatozoa with an oval shape had a very basophilic coloration, the cell
diameter varies between 2.0 and 2.5 μm. In many sections, it was possible to observe the
concentration of spermatozoa in the male ciliated gonoduct, ready for spawning (Fig. 4B).
Associated with the reproductive cells it was always possible to observe the presence of
several yellow-brownish bodies or granules in the follicles (Fig. 4C, D and E).
Gonadal Development Index

The Gonadal Development Index (GDI) along the year is summarized in Table 2.
Female
Following the reproductive cycle, it was possible to differentiate five different stages. Often the
same organism presented follicles in different stages, making difficult the representation of cell
maturation through time, which varied among individuals and seasons (Table 2).Stage 1 (early
active) and Stage 2 (late active) correspond in average terms to the initial stages of gametes
development, occurring in very short periods and almost simultaneously, just after the last
stage of development, i.e. stage 5 (resorption) (Table 2). Stage 3 (mature) corresponds to the
phase when gametes reach the maximum maturation. It occurred in two distinct periods, from
January to May and in October (Table 2). This stage is also when follicles reach their maximum
capacity, being full of mature oocytes, awaiting spawning. Stage 4 (spawned) is reached when
many of the mature oocytes were already released (but some can still be found inside the
follicles or the gonoduct).
56 FCUP
Figure 4 Histological sections from male gonads of Unio delphinus stained with Hematoxylin
and Eosin (H&E). A - General aspect of male gonads (ma) organized in acini in January, with
the acini showing gonads at all development stages of spermatogenesis, full with mature
spermatozoa (s) in the lumen, with visible muscle tissue (ms) and portions of the ciliated
gonoduct (cg) (scale bar 200 μm). B - Partial male acini in January, with male reproductive
cells at different spermatogenesis stages and germinative epithelium (ge) visible, in the centre
the ciliated gonoduct (cg) is full of mature spermatozoa (s) (scale bar 20 μm). C and D - Details
of male acinus in October, where is possible to identify different development spermatogenesis
stages, dominantly the earlier spermatogonia (sg), spermatocytes (sc), spermatids (st), sperm
morulae (sm) and the last stage spermatozoa (s), not so abundant (scale bar 10 μm). E -
Degenerative male acinus (dma) in August, at the beginning of the post-spawning period,
lumen with already some free spaces, presenting some yellow bodies (yb) and surrounded by
an undifferentiated epithelium, still presenting all stages of spermatozoa development (scale
bar 100 μm). F - Mature spermatozoa (s) in March, few sperm morulae (sm) and other
development stages (scale bar 10 μm).
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Two peaks of maturation were registered during this stage, one larger from March to July and
a shorter one from November to December (Table 2). Stage 5 (resorption) closely follows the
previous stage, and it is characterized by the presence of empty, destroyed or degenerating
follicles with some yellow bodies present (Table 2). In this last stage, the presence of some
follicles already with gametes in the early stages and some free mature oocytes may also
occur. This stage was dominant in August but occurred from May to September and from
November to December. We only identified some organisms being exclusively in this stage
during September and October (Table 2). Although the gametogenic activity never ceases
completely, there is a decrease in activity during these months (Table 2).
Table 2
Monthly values of all identified Gonadal Development Index (GDI) stages in the male and
female gonads, and presence/absence of eggs and larvae (glochidia) in the marsupium of Unio
delphinus. See text for details on GDI.
Month Female gonad (♀) Male gonad (♂) Gills (♀)
stages stages
January 2, 3 4, 5 empty
February 2, 3 4, 5 empty
March 3, 4 3, 4, 5 empty
April 3, 4 3, 4, 5 eggs (rare)
May 3, 4, 5 3, 4, 5 eggs, glochidia
June 4, 5, 1, 2 2, 3, 4 eggs, glochidia
July 4, 5, 1, 2 2, 3, 4 eggs, glochidia
August 5, 1, 2 4, 5 glochidia
September 5, 1, 2 1, 2 eggs (residual)
October 1, 2, 3 1, 2, 3 eggs (residual)
November 4, 5 3, 4, 5 empty
December 4, 5 3, 4, 5 empty
Male
Due to the extreme variation among contemporaneously sampled individuals and even across
follicles of the same individual, the distinction of the several spermatogenesis stages was more
difficult than for oogenesis. Similarly, as females, all male individuals present mature follicles
with gametes in a continuous cycle. Stage 1 (early active). Given that we never observed a
complete cessation of the reproductive cycle, follicles containing only early stages of male
gametes (spermatogonia and spermatocytes) were never found isolated in this stage. Follicles
from this stage were rarely found in organisms that presented also follicles at stage 5 or in
others that were already in stage 2 or 3 (April to May and September to October). Stage 2 (late
active) corresponds to the period were all stages of maturation of the male gametes are
present, except the mature spermatozoa that can be absent or rare. This stage was only
detected in September and October. Stage 3 (mature) is characterized by the presence of all
58 FCUP
maturation stages in the male follicles, the follicles are full of mature spermatozoa that fill the
lumen. This stage occurred from March to May and from October to November, preceding
spawning. It is characterized by an abundant presence of mature spermatozoa, but also by the
high quantity of all the other stages (more in the periphery) and some yellow bodies. During
this period some free spermatozoa may already be observed in the gonoduct. Stage 4
(spawned) occurred practically throughout the year from January to August (with peaks in
March, May, and June) and from November to December. This stage is characterized by a
decreasing presence of spermatozoa in the follicles and an increase of the other cell
development stages, mainly spermatocytes and spermatids organized in morulae. During this
phase, the gonoduct is full of spermatozoa and the follicles presented empty spaces inside,
but no degenerative follicles were observed. Stage 5 (resorption) is almost inexistent, due to
the continuity of the cycle, occurring only in few organisms in January, March, August,
November, and December. This stage is characterized by the presence of empty follicles or in
degeneration in the male reproductive tissue, by fewer reproductive cells and by the abundant
presence of yellow bodies. There is a decrease in the number of follicles in the tissue, with
some follicles already presenting the early maturation stages of male gametes.
Demibranchs
Unio delphinus females only use the outer pair of the female gills as a brooding chamber, or
marsupium, for glochidia (Fig. 5A-5E). The species only kept glochidia in the marsupium during
short periods (2-3 weeks) and may, therefore, be classified as tachytictic (short term brooders).
When the gills are filled only with eggs, they began to swell, and their coloration is intense
yellow (Fig. 5D). The brooding gills then become lighter and whitish as eggs mature into
glochidia (Fig. 5B and 5E). Eggs were detected from April to July, with two peaks, one in April
and one in June. Glochidia were detected from May to August, with two discharge peaks (May
and August). The organization of the eggs in the marsupial gill is in small conglutinates with a
feather shape (Fig. 5D). These conglutinates are generally composed of a variable content of
eggs and/or glochidia that may change throughout the cycle. When glochidia become
dominant before discharge, conglutinates become more diffuse and less evident.
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Figure 5 Histological sections from marsupial female gills of Unio delphinus, without and with
glochidia stained with Hematoxylin and Eosin (H&E) (A and B); stereoscope images from the
gills (C and D) and free glochidia (E). A - Histological section from marsupial female gill in
April, devoid of offspring (scale bar 500 μm). B - Histological section from marsupial female
gills in July, full of mature glochidia (g) (scale bar 500 μm). C - Marsupial gill at stereoscope,
in March (scale bar 1 mm). D - Detail of gravid gill and feather-like conglutinate full of eggs
(Fs) (scale bar 1 mm). E - Mature glochidia at the microscope, in June (scale bar 200 μm).
Sex ratio distribution and age of maturity

No significant differences were detected from the predicted 1:1 sex ratio in all populations
(Table 3). No gametes were detected in individuals smaller than 41 mm (females) and 31 mm
(males), corresponding to 2.5 years in females and 1.6 years for males, respectively. All
individuals with size and age above these thresholds presented either female or male gametes
in any stage of maturation.
60 FCUP
Table 3
Sex distribution of selected Iberian populations of Unio delphinus
Population ♀% ♂%
Sabor River 47.2 52.8
Barrinha Lagoon 44.4 55.6
Mira Lagoon 50.0 50.0
Mondego River 47.4 52.6
Tejo River 48.6 51.4
Guadiana River 40.6 59.4
Vascão River 48.5 51.5
Hosts
The infestation trials showed that U. delphinus glochidia attach mainly to native cyprinids and
the native brown trout Salmo trutta fario (Table 4; Fig. 6A). Conversely, glochidia were not as
successful in attaching to non-native species, which showed in general much lower infestation
rates (Table 4; Fig. 6A).
Unio delphinus glochidia successfully developed in 11 out of 14 native species tested (79%)
(Table 4; Fig. 6B). In contrast, non-native fish species never produced any viable juvenile
(Table 4; Fig. 6B). The glochidia transformation rates ranged from 0% for the native Southern
Iberian spined-loach Cobitis paludica, the European eel Anguilla Anguilla, the flathead grey
mullet Mugil cephalus and all non-native species, to 48.8% for the Northern Iberian chub
Squalius carolitertii (Table 4; Fig. 7). Fully developed juveniles were collected from the tanks
between 10 and 22 days post infestation, with the sum of daily temperatures during
metamorphosis ranging from 240 to 440 degree-days (Table 4; Fig. 7).
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Table 4
Fish species studied and host compatibility test results, including the number and mean (±SD) length of fish per species, mea n initial number of
attached glochidia, mean number of viable juveniles produced and transformation rate ‘Transformation rate’ indicates the proportion of Unio
delphinus glochidia that successfully developed into juvenile mussels.
Fish Mean ± SD Mean Mean Transformation

Fish Family Fish Species
(N) fish length (mm) (glochidia/fish) (juveniles/fish) rate (%)
NATIVE
ANGUILLIDAE Anguilla anguilla 5 202.0 ± 47.6 5.0 0.0 -
COBITIDAE Cobitis paludica 6 80.8 ± 5.8 16.7 0.0 -
CYPRINIDAE Achondrostoma oligolepis 14 68.6 ± 9.1 24.2 1.5 5.8
Luciobarbus bocagei 8 134.4 ± 23.1 160.8 111.7 41.0
Luciobarbus comizo 16 90.2 ± 16.1 113.8 29.2 20.4
Luciobarbus microcephalus 10 135.0 ± 25.1 138.9 40.1 22.4
Luciobarbus steindachneri 12 133.3 ± 20.1 151.5 37.2 19.7
Pseudochondrostoma duriense 7 132.0 ± 5.7 108.8 17.2 13.7
Pseudochondrostoma polylepis 9 131.5 ± 21.1 89.5 11.9 11.7
Squalius alburnoides 11 75.9 ± 7.0 85.1 50.9 37.4
Squalius carolitertii 4 85.0 ± 9.1 197.2 188.3 48.8
Squalius pyrenaicus 6 130.0 ± 7.9 97.5 80.9 45.3
MUGILIDAE Mugil cephalus 3 160.0 ± 8.7 36.3 0.0 -
SALMONIDAE Salmo trutta fario 5 100.0 ± 15.8 156.7 47.0 23.1
NON-NATIVE
CENTRARCHIDAE Lepomis gibbosus 13 83.8 ± 10.2 103.8 0.0 -
Micropterus salmoides 3 123.3 ± 7.6 37.7 0.0 -
CYPRINIDAE Gobio lozanoi 6 75.8 ± 10.2 31.3 0.0 -
ESOCIDAE Esox lucius 3 171.6 ± 12.6 54.7 0.0 -
ICTALURIDAE Ameiurus melas 3 103.5 ± 6.1 5.2 0.0 -
POECILIIDAE Gambusia holbrooki 13 32.7 ± 3.9 3.1 0.0 -
62 FCUP
Figure 6 A - Mean glochidial infestation (i.e. the number of glochidia per fish and mm of fish)
in all fish species; B - Effective transformation of glochidia into juveniles (i.e. the number of
juveniles produced per fish and mm of fish) in all fish species.
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Figure 7 Glochidial transformation rate and attachment periods (shown in bars) per fish host
species.
Discussion
This study describes for the first time the main life-history traits of the Iberian dolphin freshwater
mussel U. delphinus. This information is vital to increase the basic knowledge about the biology
and ecology of this species, and it may be used for mainstreaming this species as a valuable
environmental indicator and to develop conservation management programs for its
populations.
Distribution
Unio delphinus is widely distributed in the Atlantic coast of the Iberian Peninsula, mainly in its
larger river basins, i.e. Minho, Douro, Tejo, Guadiana, and Guadalquivir (Fig. 1). This
distribution is extended to some smaller river basins north of the Minho and east of the
Guadalquivir. Populations south of the Tejo River basin are highly threatened due to habitat
degradation and fragmentation, and water shortage. As previously described (Gomes-dos-
Santos et al 2019), estimates of EOO and AOO are largely dependent on the method used.
The larger estimates of AOO using the 2 × 2 km grid would allow the species to be listed as
Vulnerable using the B criterion but we follow the suggested method of Gomes-dos-Santos et
al (2019), using the mean length of the river as the best estimation method, that places the
species as Endangered (Appendix 2).
64 FCUP
Growth and longevity

The well-studied freshwater pearl mussel Margaritifera margaritifera and thick-shelled river
mussel Unio crassus exhibit slow growth and may live up to 280 and 90 years, respectively
(Lopes-Lima et al 2017a). The present study shows that the Iberian dolphin freshwater mussel
U. delphinus presents a distinct growth pattern being a short-lived and fast-growing unionid
species. These results corroborate the trends described within the tribe Unionini, where growth
rates are generally faster and life spans shorter than most other unionid groups (Haag & Rypel
2011). A North-South latitudinal gradient has been previously reported for freshwater mussels
growth, with several species showing slower growth but greater longevity for populations living
at higher latitudes (Haag & Rypel 2011), including the European species M. margaritifera (San
Miguel et al 2004) and U. crassus (Helama et al 2017). This pattern might be explained by the
lower temperatures and shorter growth periods of the northern regions (Dunca & Mutvei 2001;
Schone et al 2004). Comparing the obtained values of the growth constant K of the U.
delphinus populations with previously published results from other Unio species, a marked
inter-specific latitudinal gradient is evident: most populations of Unio pictorum from England
and Russia (Aldridge 1999, Rizhinashvili 2008), and U. crassus from Central Europe
(Hochwald 2001; Helama et al 2017) showed lower K values than those obtained here for U.
delphinus and those previously published on the other Iberian Unio species, i.e. Unio
tumidiformis (Reis & Araujo 2016) and the Middle eastern Unio terminalis (Ostrovsky et al
1993). Possibly due to the low number of sites tested and/or the much lower latitudinal
distribution of U. delphinus compared with U. crassus or M. margaritifera, a north-south pattern
was not evident for U. delphinus populations within Iberia, where growth and longevity showed
no significant differences (Table 1). In contrast, the growth constant K was lower and maximum
length higher in the populations from lentic than those from lotic habitats (Fig. 2; Table 1).
Freshwater mussel growth is thought to be influenced by productivity and food availability,
substrate type, water flow and exposure to wind and current (Haag & Rypel 2011 and
references therein). The larger maximum size and lower K values of the Lagoon populations
in Iberia should be related to the very high productivity and hydrological stability of these
habitats (Varandas et al 2014), when comparing with the River populations. Despite the distinct
growth patterns of Lagoon and River populations, the maximum age does not vary
considerably between habitat types, ranging between 7 and 11 years old, average 9; Table 1).
These values are within the lower end for the European Unio species range that has been
reported from 5 to more than 50 and may even reach 90 years old for the U. crassus northern
populations (Lopes-Lima et al 2017a and references therein).
The age of maturity determined in the Sabor population is higher for females (2.5 years)
than for males (1.6 years), with a mean maturity value for the species around 2 years, similar
to previously reported values for other Unio species (Lopes-Lima et al 2017a). Based on the
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maximum longevity and age of maturity, the mean generation length of U. delphinus is about
5-6 years.
Reproductive cycle and sexual strategy

No hermaphroditism was detected, showing that U. delphinus is strictly dioecious, with a
female-male ratio close to 1 but always with a dominance of males (Table 3). Histological
studies on other freshwater mussels show that the sexes are typically separate, though
hermaphroditism has been detected in some species (Van der Schalie 1970; Kat 2009).
Nevertheless, the genus Unio seems to be strictly dioecious since no case of complete
hermaphroditism has been previously detected for any species (e.g. Aldridge 1999; Cek &
Sereflişan 2006; Lopes-Lima et al 2017a).
The reproductive cycle, characterized by the GDI, is continuous and uninterrupted with
both male and female reproductive cells being found throughout the year. This contrasts with
the reproductive cycle of the congeneric U. terminalis and U. tumidiformis that seems to exhibit
a long and single reproductive cycle (Cek & Sereflişan 2006; Reis & Araujo 2016). Brooding
and discharge periods overlap with what is known for most Unio species where brooding is
coincident with the glochidia discharge period in the spring-summer months (i.e. generally
between April and August; Lopes-Lima et al 2017a and references therein). No glochidia have
been found after August which is coherent with results for other Unio species (Lopes-Lima et
al 2017a). This might indicate that we failed to detect a second glochidia discharge period.
Unio delphinus, as all species within the subfamily Unioninae, is ectobranchous (Lopes-
Lima et al 2017b), meaning that the females only use the outer pair of demibranchs as a
brooding chamber for glochidia, also known as marsupium (Fig. 5). Glochidia mature and stay
in the marsupium during short periods (2-3 weeks) and thus U. delphinus can be classified as
tachytictic or short-term brooder. Glochidia are discharged by the exhalant aperture to the
water column entangled in mucous threads, although feather-like constructs full of glochidia
and eggs, also known as conglutinates, are also produced (Fig. 5d). However, as also seen in
the congeneric U. pictorum, these conglutinates contain variable quantities of mature glochidia
and its function is still uncertain, probably being released by females while under hypoxic
stress, to increase ventilation (Aldridge 1999).
Fish hosts
As in all freshwater mussels, the life cycle of the Iberian dolphin freshwater mussel includes a
parasitic stage, in which the larvae (glochidia) need to attach to fish to continue their
development and metamorphose into a young juvenile (Modesto et al 2018). Therefore, it is
crucial to understand the dynamic interactions between freshwater mussels and their fish
hosts. Our study shows that U. delphinus glochidia may attach to all fish species on the trials
66 FCUP
(Fig. 6; Table 4). However, it attached preferentially to native fish species, with the attachment
rates to non-native species being much lower. Furthermore, the effective transformation rate
of glochidia into juveniles only occurred in 10 cyprinid and 1 salmonid native fish species.
Transformation rates were especially high in all fish species from the genus Squalius, followed
by Luciobarbus, Salmo and Pseudochondrostoma species. This indicates a strong co-
evolutionary relationship of U. delphinus with native co-occurring fish and especially with
Squalius species. This link of the freshwater mussel genus Unio with cyprinids and especially
Squalius species has been previously reported (Lopes-Lima et al 2017a). For example, except
for the more divergent Unio tumidus, all European Unio species seem to use at least one
species of Squalius as hosts (Lopes-Lima et al 2017a). Furthermore, Squalius cephalus seems
to be the main host in some U. crassus populations (Taeubert et al 2012) and the Southern
Iberian U. tumidiformis is only able to metamorphose in Squalius species (Reis et al 2014).
The attachment period did not seem to vary much across the effective host species, occurring
between 12 and 22 days post attachment at a constant temperature of 20 °C with the sum of
daily temperatures during metamorphosis ranging from 240 to 440 degree-days (Fig. 7; Table
4). This speed of transformation seems to be lower than the southern Iberian U. tumidiformis
(Reis et al 2014) and more like that reported to the more closely related species the Eastern
Iberian Unio mancus (Araujo et al 2005).
Practical implications for conservation and environmental monitoring

Our results have important implications on future management and conservation actions for
the Iberian dolphin freshwater mussel U. delphinus. The detailed compilation of distribution
data and estimation of the EOO and AOO will allow for a more accurate assessment using
IUCN categories and criteria (Appendix 2). Information about growth, age of maturity and life
span are also especially critical in evaluating the risk of extinction of rare and threatened
species, being important components of the evaluation criteria of IUCN Red-Listing. Unionid
mussels were for long portrayed as long-lived and slow-growing organisms, but this has been
increasingly demystified with species like U. delphinus, presenting distinct growth patterns and
lifespan (Haag & Rypel 2011).
The information about the reproductive cycle here described is very important for future
propagation programs and other conservation actions. For instance, gravid females should be
searched mainly from late Spring to late Summer. Additionally, if the aim is getting a brood
stock of cultivated mussels, only after two years would these mussels be ready for
reproduction.
Although U. delphinus seems to have a continuous reproductive cycle, both oogenesis
and spermatogenesis deaccelerate between August and March. Therefore, translocation and
monitoring programs on this species ideally should be done in September before the rainy
FCUP 67
season to minimize the impact on reproduction, and never during spring/summer where
manipulation and transportation stress may lead to reduced reproductive intensity and ejection
or abortion of larval content. Potential translocations should consider that enough time should
be given to specimens to settle before the heavy torrential floods that generally occur from
October onward, especially in the intermittent rivers in the South. Knowledge about the fish
hosts is also crucial for the future of the species. As already reported in other Mediterranean
species (e.g. Unio foucauldianus; Benaissa et al 2019), especially those with restricted ranges
in southern Europe or North Africa, U. delphinus seems to be unable to transform in any of the
non-native species. This turns the on-going biotic homogenization of the Iberian fish fauna due
to the constant introduction of new non-native fish species in Iberian freshwater habitats
(Clavero et al 2011, 2013; Anastacio et al 2019), one of the major threats to this mussel
species. Furthermore, species like Lepomis gibbosus and Micropterus salmoides are now
major components of many Iberian freshwaters. These fish are piscivorous and very
aggressive, making nests at the banks, where U. delphinus generally aggregates (Authors
pers. obs.). These invasive species maintain other native fish species far from the banks,
potentially decreasing attachment success of U. delphinus larvae. Conversely, most larvae will
attach to the non-native fish that will act as ecological sinks. The laboratory host fish studies
here developed should be complemented with future field experiments to test which fish
species are serving as the effective hosts and to better estimate the impact of invasive species.
Unio delphinus possesses many of the requirements for indicator species (Carignan &
Villard 2002), and here we provide a solid and comprehensive ecological and developmental
data baseline that will allow developing its potential even further. Besides its inherent
conservation importance, information on the growth patterns and life-history traits here
described should now be optimized for use as ecological indicators. This could be achieved,
for instance, by comparing populations exposed to distinct disturbance types and levels. More
comprehensive surveys of U. delphinus populations and varying compositions of co-occurring
fish species could help to develop standardized metrics to assess the status and integrity of
fish communities. Given that this mussel is unable to reproduce in most invasive fish, its decline
or local extirpation can be a direct effect of the decline, extirpation or changes in the fish fauna.
This already occurred in Lake Banyoles (Spain), where despite good abiotic conditions, all
native fish are now extirpated and replaced by many other non-native species, with freshwater
mussel populations declining and disappearing soon after (Garcia-Berthou et al 2000; Araujo
et al 2015).
68 FCUP
Conclusions
Although the study of life-history traits has now become old-fashioned and generally
considered of local/regional interest or purely descriptive, conservation efforts are strongly
hindered by the lack of this basic knowledge. Furthermore, many recent studies, e.g. state of
the art modelling exercises and multispecies biological and biogeographical meta-analyses,
that are attracting a lot of scientific attention, strongly rely on these basic biological data. The
present study makes practical considerations about the conservation of a declining freshwater
species endemic to one of the global biodiversity hotspots and highlights the need to go back
to the basics and to promote the study of life-history traits of poorly studied taxa, especially
those of conservation concern.
Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by Portuguese FCT - Foundation for Science and Technology,
Projects FRESHCO: Multiple implications of invasive species on Freshwater Mussel co-
extinction processes (PTDC/AGR-FOR/1627/2014-04/SAICT/2015), MUSSELFLOW: Host-
dependent evolution, ecology and conservation of freshwater mussels under varying
hydrological conditions: consequences of climate change (PTDC/BIA-EVL/29199/2017), and
ConBiomics: The missing approach for the Conservation of freshwater Bivalves Project N°
NORTE-01-0145- FEDER-030286, co-financed by COMPETE 2020, Portugal 2020 and the
European Union through the ERDF. FCT also supported MLL under the grant
(SFRH/BD/115728/2016), CM under the grant (SFRH/BD/111133/2015) and through Strategic
Funding UID/Multi/04423/2019. PB was supported by EDP Biodiversity Chair.
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Supplementary Material
Appendix 1 Bibliography used to compile the distribution records of Unio

delphinus which were complemented by personal data from all the
authors.
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Appendix 2 Red List assessment of Unio delphinus Spengler, 1793
Scientific Name
Unio delphinus Spengler, 1793
Common Name
Iberian dolphin freshwater mussel
Taxonomic Notes
Unio populations from northwest Iberia were thought to belong to the widespread and more
common European Unio pictorum and the species name Unio delphinus its junior synonym,
until its recent recognition as a valid distinct species (Araujo et al 2009; Lopes-Lima et al 2017).
Unio populations from Morocco, previously thought to be U. delphinus, belong to a separate
species, the Moroccan endemic Unio foucauldianus Pallary, 1936 (Froufe et al 2016).
Red List Assessment

Endangered B2ab(ii,iii,iv)
Justification
The species has been declining significantly up to 30% over the last three generations, being
this reported decline not enough to place the species in one of the threatened categories using
criterion A (Araujo 2009). However, the species is severely fragmented with an estimated Area
of Occupancy (AOO) of 61.45 Km2 and exhibiting an observed continuing decline in terms of
habitat, area of occupancy and number of populations. Therefore, the species is here listed as
Endangered B2ab(ii,iii,iv).
Geographic Range
Unio delphinus populations have been recorded in most Atlantic basins of the Iberian
Peninsula, from the Ulla river in the north to the Guadalquivir in the south and then east to the
La Vega River basin near Gibraltar. It also occurs in few Mediterranean coastal basins east of
Gibraltar until the Guadalhorce River basin near Malaga. Many populations have been recently
extirpated due to dam construction in the Douro and Guadiana basins. Populations on the
south have been disappearing due to decreased water levels.
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Habitat and Ecology

It is generally found in the middle and lower sections of rivers and streams in a great variety of
substrates from silt to sand and gravel, close to the banks (Araujo et al 2009). It may
occasionally occur in standing water habitats like the Ruidera Lakes in Spain and Mira and
Barrinha de Mira lagoons in Portugal. Larvae release occurs continuously from May to August,
with two discharge peaks in May and August. The fish hosts are co-occurring native cyprinids
and the native salmonid species the brown trout Salmo trutta fario.
Threats
Urban pollution, mainly in the coastal areas, has been the main cause of extirpation until the
beginning of the 21st century. The species has been threatened by habitat loss and
fragmentation due to the construction of dams and impoundments that continue to the present.
In the south of Portugal and Spain, water shortage is the main threat due to the increasing
demand for agriculture and urban purposes. This situation is further exacerbated by global
warming scenarios that will likely induce irreversible trends of desertification. Given that U.
delphinus seems to be unable to use non-native fish species as hosts, biotic homogenization
and fish introductions may become a major threat to the species, soon.
Conservation Actions
This species would benefit from the restoration and maintenance of ecological flows in dams,
wastewater treatment, river rehabilitation, and whole catchment management. The species
should benefit from propagation programs for reintroduction in restored habitats.
Research Needs
Long term surveys are needed to monitor the population trends of the most important
populations. Reproduction and ex-situ culture studies are needed for eventual propagation
programs.
References
82 FCUP
e.T195510A8975648.

597-611.

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CHAPTER 4
Phylogeny of the family Unionidae
Paper III
Phylogeny of the most species-rich freshwater bivalve family (Bivalvia:
Unionida: Unionidae): Defining modern subfamilies and tribes
Varandas S, Wu X, Zanatta DT, Zieritz A, Bogan AE
Article published in Molecular Phylogenetics and Evolution 106, 174-191 (2017).
DOI: 10.1016/j.ympev.2016.08.021
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Phylogeny of the most species-rich freshwater bivalve family (Bivalvia:

Unionida: Unionidae): Defining modern subfamilies and tribes
Manuel Lopes-Lima1,2,*,+, Elsa Froufe2,+, Van Tu Do3, Mohamed Ghamizi4, Karen E. Mock5,
Ümit Kebapçı6, Olga Klishko7, Satit Kovitvadhi8, Uthaiwan Kovitvadhi9, Octávio S. Paulo10,
John M. Pfeiffer III11, Morgan Raley12, Nicoletta Riccardi13, Hülya Şereflişan14, Ronaldo
Sousa2,15, Amílcar Teixeira16, Simone Varandas17, Xiaoping Wu18, David T. Zanatta19,
Alexandra Zieritz20, Arthur E. Bogan21,+
1
Vairão, 4485-661 Vairão, Portugal
2
CIIMAR/CIMAR - Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto
de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
3
Department of Aquatic Ecology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and
Technology, 18 Hoang Quoc Viet, Cau Giay, Ha Noi, Viet Nam
4
Muséum d’Histoire Naturelle de Marrakech, Université Cadi Ayyad, Faculté des Sciences, Semlalia, B.P. 2390
Marrakech, Morocco
5
Ecology Center and Department of Wildland Resources, Utah State University, Logan, UT 84322, USA
6
Department of Biology, Faculty of Arts and Sciences, Mehmet Akif Ersoy University, Burdur, Turkey
7
Institute of Natural Resources, Ecology and Cryology, Russian Academy of Sciences Siberian Branch, Chita
672014, Russia
8
Department of Agriculture, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University,
Bangkok 10600, Thailand
9
Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
10
Computational Biology and Population Genomics Group, cE3c - Centre for Ecology Evolution and Environmental
Changes, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal
11
Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA
12
HydroGENomics, Raleigh, NC 27606, USA
13
CNR - Institute for Ecosystems Studies, Verbania Pallanza (VB), Italy
14
Faculty of Marine Sciences and Technology, Ískenderun Technical University, 31200 Iskenderun, Hatay, Turkey
15
16
CIMO/ESA/IPB - Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus
de Santa Apolónia, Apartado 1172, 5301-854 Bragança, Portugal
17
CITAB/UTAD - Centre for Research and Technology of Agro-Environment and Biological Sciences, University of
Trás-os-Montes and Alto Douro, Forestry Department, 5000-801 Vila Real, Portugal
18
School of Life Sciences, Center for Watershed Ecology, Institute of Life Science, Nanchang University, Nanchang
330031, People’s Republic of China
19
Biology Department, Institute for Great Lakes Research, Central Michigan University, Biosciences Bldg. 2408,
Mount Pleasant, MI 48859, USA
20
School of Geography, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Malaysia
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21
Research Laboratory, North Carolina State Museum of Natural Sciences, MSC 1626, Raleigh, NC 27699-1626,
USA
+
These authors contributed equally to the paper.
⁎
Abstract
Freshwater mussels of the order Unionida are key elements of freshwater habitats and are
responsible for important ecological functions and services. Unfortunately, these bivalves are
among the most threatened freshwater taxa in the world. However, conservation planning and
management are hindered by taxonomic problems and a lack of detailed ecological data. This
highlights the urgent need for advances in the areas of systematics and evolutionary
relationships within the Unionida. This study presents the most comprehensive phylogeny to
date of the larger Unionida family, i.e. the Unionidae. The phylogeny is based on a combined
dataset of 1032 bp (COI + 28S) of 70 species in 46 genera, with 7 of these genera being
sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported
subfamilies and 18 tribes, three of which are here named for the first time (i.e. Chamberlainiini
nomen novum, Cristariini nomen novum, and Lanceolariini nomen novum). Molecular analyses
were complemented by investigations of selected morphological, anatomical and behavioural
characters used in traditional phylogenetic studies. No single morphological, anatomical or
behavioural character was diagnostic at the subfamily level and few were useful at the tribe
level. However, within subfamilies, many tribes can be recognized based on a subset of these
characters. The geographical distribution of each of the subfamilies and tribes is also
presented. The present study provides important advances in the systematics of these
extraordinary taxa with implications for future ecological and conservation studies.
Keywords
Mollusca, Systematics, Freshwater mussels, Taxonomy, Classification
86 FCUP
Introduction
Understanding phylogenetic diversity is crucial for conservation prioritization of freshwater
mussels (Bivalvia: Unionida), which are among the most threatened freshwater taxa in the
world (Lydeard et al 2004; IUCN 2015). Due to their ecological and economic importance,
interesting biological traits (e.g. a parasitic life with the reproductive dependence on a host fish
and a particular form of mitochondrial inheritance called double uniparental inheritance; Hoeh
et al 1996, 2002a ; Breton et al 2007; Barnhart et al 2008), scientific research on Unionida has
grown in recent years (Haag 2012; Lopes-Lima et al 2014). However, taxon-based
conservation efforts focused on the Unionidae are hindered by various phylogenetic and
taxonomic uncertainties (e.g. Inoue et al 2014; Pfeiffer et al 2015), and many species,
especially those outside of North America and Western Europe, have been assigned a Data
Deficient status by the IUCN (Bogan & Roe 2008; Kohler et al 2012; IUCN 2015). The
Unionidae is by far the most species-rich family within the order Unionida, with 620 species in
142 genera (Bogan & Roe 2008) widely distributed across the freshwater ecosystems of
Europe, Asia, North America, and Africa. The first classification of the global Unionidae fauna
was attempted by Lea (1836, 1838, 1852, 1870), and later updated by Simpson (1900, 1914).
These works, in which the marsupium (i.e. the gill structure where the eggs and larvae are
brooded), anatomy, larvae type, and umbo sculpture were used as key classification
characters, divided the Unionidae into two subfamilies, Unioninae and Hyriinae (Table 1).
Subsequently, A.E. Ortmann performed a series of studies on North American taxa including
additional anatomical classification characters and divided the Unionidae into three
subfamilies: Unioninae, Anodontinae, and Lampsilinae (Table 1: Ortmann 1910, 1911, 1912,
1919, 1921; Ortmann & Walker 1922). In discussing his classification, Ortmann (1912) noted
the inadequacy of shell characters to define families and subfamilies due to widespread
convergences in shell morphology; a problem that was further discussed by Prashad (1931).
Apart from regional works (e.g. Frierson 1927; Iredale 1934; Haas 1940), little progress was
made on Unionidae classification until the middle of the twentieth century, when Modell and
Haas published their comprehensive classification systems (Table 1: Modell 1942, 1949, 1964;
Haas 1969a,b). Both Haas and Modell classification systems used a set of morphological and
anatomical characters but relied heavily on shell morphology. Haas (1969a,b) classified the
Unionidae into six subfamilies. One of these, i.e. the Hyriinae, combined species from South
America and Australasia and would later be recognized as a distinct family. Modell (1942,
1949, 1964) developed a more complex and inflated classification system, which organized
the Unionidae genera in distinct families and multiple subfamilies. Both authors’ use of highly
variable conchological characters for classification above the genus level led to incoherent
associations.
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Table 1
Historical classification systems of the subfamilies and tribes now included in the Unionidae. (Blue) subfamilies; (red) tribes; (nn) nomen novum;
(*) regional study; (?) rank uncertain.
Simpson Ortmann Modell Haas Heard & Guckert Brandt

(1900/1914) (1912)* (1942) (1969a, 1969b) (1970)* (1974)*
Unionidae Unionidae Unionidae Unionidae Unionidae Unionidae

Unioninae Unioninae Unioninae Alasmidontinae Unioninae Hyriopsinae
Hyriinae Anodontinae Anodontinae Anodontinae Anodontinae Pseudodontinae
Lampsilinae Cafferiinae Hyriinae Lampsilinae Parreysiinae
Coelaturinae Lampsilinae Pleurobeminae Rectidentinae
Contradentinae Quadrulinae Popenaiadinae Modellnaiinae
Hyriinae Unioninae
Hyriopsinae Amblemidae
Lamellidentinae Ambleminae
Lamprotulinae Gonideinae
Nannonaiinae Megalonaiadinae
Parreysiinae
Propehyridellinae
Quadrulinae
Rectidentinae
Elliptionidae
Alasmidontinae
Ambleminae
Elliptioninae
Lampsilinae
Pleurobeminae
Margaritiferidae
Heudeaninae
Pseudodontinae
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Table 1 (cont.)
Graf & Cummings (2007) Bieler et al (2010) Whelan et al (2011) Carter et al (2011) This Study
Unionidae Unionidae Unionidae Unionidae Unionidae

Unioninae Unioninae Unioninae Unioninae Unioninae
Unionini Unionini Unionini Unionini Unionini
Anodontini Anodontini Anodontini Anodontini Ambleminae
Ambleminae Ambleminae Ambleminae Ambleminae Amblemini
Amblemini Amblemini Amblemini Amblemini Lampsilini
Gonideini Lampsilini Lampsilini Lampsilini Pleurobemini
Lampsilini ?Oxynaiini Pleurobemini Pleurobemini Quadrulini
Pleurobemini Pleurobemini Quadrulini Quadrulini Anodontinae
Quadrulini Quadrulini Gonideinae Gonideinae Anodontini
Coelaturinae Modellnaiinae Modellnaiinae Cristariininn
Gonideinae Parreysiinae Parreysiinae Lanceolariininn
Modellnaiinae Coelaturini Rectidentinae Gonideinae
Parreysiinae Oxynaiini Gonideini
Rectidentinae Parreysiini Chamberlainiininn
Lamellidentini Lamprotulini
Rectidentinae Pseudodontini
Parreysiinae
Coelaturini
Oxynaiini
Parreysiini
Lamellidentini
Rectidentinae
Rectidentini
Contradentini
?Modellnaiinae
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Nevertheless, the work by Haas has been widely recognized as the more reliable in terms of
representing generic and sub-generic distinctiveness and is considered fundamental in
establishing the main genera of the Unionida and in particular, the Unionidae (Roe & Hoeh
2003). Concurrent with the work of Haas (1969a,b) and Modell (1942, 1949, 1964), an even
more inflated classification scheme was proposed by Starobogatov (1970) and Zatravkin &
Bogatov (1987), who relied on conchological differences and focused on the curvature of the
frontal section of the valves. This system is merely typological and was disregarded by most
of the western school malacologists (see Graf 2007) and emergent Russian studies (Klishko
et al 2014; Bolotov et al 2015).
A comprehensive molecular phylogenetic study of the Unionidae has not been
attempted to date, primarily due to the difficulties in developing a dataset of enough
geographical and species coverage. The first classification system using a phylogenetic
framework was published by Heard & Guckert (1970; Table 1) for the North American Unionida
fauna. Disregarding shell characters, these authors used a broad anatomical and reproductive
behaviour character set within a phylogenetic context. Their analyses resulted in the division
of the North American Unionidae into two families and several subfamilies. The subsequent
development of powerful molecular and statistical tools, providing a basis for more objective
approaches, has led to the publication of several studies on unionid phylogeny (e.g. Davis et
al 1977, 1981; Davis & Fuller 1981; Davis 1983, 1984; Hoeh et al 1998, 2001, 2002b, 2009;
Roe & Hoeh 2003; Campbell et al 2005; Graf & Cummings 2006; Zanatta & Murphy 2006;
Whelan et al 2011; Campbell & Lydeard 2012a,b; Pfeiffer & Graf 2013, 2015). In many of these
studies, unionid genera or species that had been identified by morphological characters were
not consistent with those revealed through molecular phylogenetic analyses (e.g. Nagel &
Badino 2001; Roe & Hoeh 2003; Campbell & Lydeard 2012a,b). Although the vast majority of
these molecular studies have focused almost exclusively on North American and European
taxa, geographic and taxonomic sampling has recently increased, particularly in Africa
(Whelan et al 2011; Graf 2013; Elderkin et al 2016) and Asia (Huang et al 2002; Zhou et al
2007; Pfeiffer & Graf 2013, 2015; Zieritz et al 2016).
Recent molecular phylogenetic studies have achieved considerable progress in
describing the main divisions within the Unionidae (Graf & Cummings 2006; Whelan et al 2011;
Campbell & Lydeard 2012a, 2012b; Pfeiffer & Graf 2013, 2015). The status of the North
American Ambleminae with four recognized tribes has been recently confirmed (Table 1:
Campbell et al 2005; Campbell & Lydeard 2012a,b). Studies including species from Africa and
the Indotropics examined the subfamily Parreysiinae in detail and recognized several
subfamilies (Table 1: Whelan et al 2011; Pfeiffer & Graf 2015). Despite the considerable recent
progress (Huang et al 2002; Zhou et al 2007; Pfeiffer & Graf 2013, 2015), the vast majority of
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unionid genera from the Eastern Palearctic and the Indotropics have never been analysed in
a modern phylogenetic framework.
Based on bibliographical research, the classification of the Unionidae was recently
reviewed, establishing the currently accepted subdivisions of the Unionidae (Carter et al 2011;
Table 1). This classification divided the family into six subfamilies: the Ambleminae with a North
and Central American distribution; the Parreysiinae with a disjunct distribution primarily in Sub-
Saharan Africa and the Indian subcontinent; the Modellnaiinae with a single species from
Thailand; the Rectidentinae with a Southeast Asian distribution; and two subfamilies, the
Unioninae and Gonideinae, distributed through most of Asia, Europe, North Africa and west
coast of North America.
To increase the success of ongoing and future management efforts and to inform
conservation priorities more effectively, a better understanding of the evolutionary history of
freshwater mussels is necessary. Our objective herein is to improve the understanding of
unionid phylogeny through analysis of a combination of nuclear and mitochondrial molecular
markers from a wide coverage of genera. In detail, this study aims to: (i) resolve the main
phylogenetic relationships within the Unionidae; (ii) discuss the systematics, taxonomy, and
distribution of the recovered unionid subdivisions (subfamilies and tribes); and (iii) compare
the obtained classification with those based on morphological characters.

Taxon sampling
All analysed taxa are listed in Table 2. Taxa were chosen to cover all available genera of
Unionidae subfamilies. Exceptions were made concerning the North American subfamily
Ambleminae (only up to three species per tribe were included) and the African/Asian subfamily
Parreysiinae since both of these subfamilies were studied in detail elsewhere (Campbell &
Lydeard 2012a,b; Campbell et al 2005; Whelan et al 2011). Taxa representatives from all
families of the subclass Palaeoheterodonta were also included (comprising all recognized
Unionida families and from Neotrigonia, the marine sister group of the Unionida) (Giribet &
Wheeler 2002).
DNA extraction, amplification, and sequencing

Whole genomic DNA was extracted from small foot tissue samples preserved in 96% ethanol
using a standard high-salt protocol (Sambrook et al 1989) or the Jetquick tissue DNA Spin Kit
(Genomed) following the manufacturer’s protocol. PCR conditions for both markers, the female
lineages of mitochondrial cytochrome c oxidase subunit 1, COI (LCO22me2 + HCO700dy2;
Walker et al 2006, 2007; and LCO1490 + HCO2198; Folmer et al 1994) and 28S ribosomal
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RNA (28S-RD1.3f and 28S-rD4b; Whiting 2002) were described in Froufe et al (2014).
Annealing temperatures of 48 ºC were used for COI (LCO1490 + HCO2198) and 28S; and 50
ºC for COI (LCO22me2 + HCO700dy2). Amplified DNA templates were purified and
sequenced by the commercial company Macrogen using the same primers.
Phylogenetic analyses
Two concatenated (COI + 28S) data sets were assembled, the Palaeoheterodonta dataset
with representatives from each of the families of the Palaeoheterodonta (Appendix A) and, to
decrease the number of poorly aligned positions of the 28S, the Unionidae dataset with only
representatives of the Unionidae (Appendix B). Both datasets were aligned using the stand-
alone version of GUIDANCE (version 1.5, Penn et al 2010) with the MAFFT multiple sequence
alignment algorithm (version 7, Katoh & Standley 2013). The following GUIDANCE parameters
were used: GUIDANCE score algorithm; 100 bootstrap replicates; a
sequence cut-off score of 0.0 (no sequence removal); a column cut off score of 0.0 (no columns
removal); global pair alignment. Incongruence Length Difference (ILD) tests were performed
to investigate incongruence between them (Farris et al 1994).
The best-fit models of nucleotide substitution under the corrected Akaike Information
Criterion were selected using JModelTest 2.1.8 (Darriba et al 2012) for each partition, of the
subsequent analyses.
For the Palaeoheterodonta dataset (Appendix A), a single scheme with 4 partitions was
applied, model GTR + I + G was optimal for the first and third COI codon positions and the
whole 28S, while model F81 was optimal for the second COI codon positions. For the
Unionidae dataset (Appendix B) more comprehensive analyses were executed including two
distinct partitioning schemes; the first with two partitions corresponding to each gene fragment
(COI and 28S) and the second with four partitions corresponding to the three codon positions
of COI and one for 28S. For the scheme with 2 partitions, model GTR + I + G was optimal for
both. For the scheme with 4 partitions, model GTR + I + G was optimal for the first COI codon
positions and the 28S, while model F81 was optimal for the second COI codon positions.
Finally, model GTR + G was optimal for the third positions of COI.
Two different analyses were then performed on all partitioned schemes of the
concatenated datasets using Bayesian Inference (BI) (BI2: 2 partitions, BI4: four partitions)
and Maximum Likelihood analyses (ML) (ML2: 2 partitions, ML4: 4 partitions). BI analyses were
performed in MrBayes v3.2.5 (Ronquist et al 2012) using the previously selected models.
Analyses were initiated with program-generated trees and four Markov chains with default
incremental heating.
92
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Table 2
Specimens analysed. Uunkonwn country; *not generated from a single individual. Taxonomy follows Table 3.
Taxon COI 28S Country Reference voucher specimen)
ANODONTINAE
ANODONTINI
Alasmidonta marginata Say, 1818 AF156502 AF400688 USA Graf & Foighil 2000; Graf & Cummings 2006
Anodonta anatina (Linnaeus, 1758) KX822632 KX822588 Russia This study
Anodonta cygnea (Linnaeus, 1758) KX822633 KX822589 Italy This study
Anodonta nuttalliana Lea, 1838 KX822634 KX822590 USA This study
Lasmigona compressa (Lea, 1829) AF156503 DQ191414 USA Graf & Foighil 2000; Graf & Cummings 2006
Pseudanodonta complanata (Rossmässler, 1835) KX822661 KX822617 Ukraine This study
Pyganodon grandis (Say, 1829)* AF231734 AF305384 USA Bogan & Hoeh 2000; Graf & Foighil 2000
Simpsonaias ambigua (Say, 1825) KX822666 KX822622 USA This study (NCSM30607)
Strophitus undulatus (Say, 1817)* AF156505 DQ191415 USA Graf & Foighil 2000; Graf & Cummings 2006
CRISTARIINI
Anemina arcaeformis (Heude, 1877) KF667530 KX822587 China An et al 2016; this study
Cristaria plicata (Leach, 1814) KX822637 KX822594 Russia This study
Pletholophus tenuis (Griffith & Pidgeon, 1833) KX822658 KX822614 Vietnam This study (NCSM84924)
Sinanodonta lucida (Heude, 1877) KX822667 KX822624 China This study
Sinanodonta woodiana (Lea, 1834) KX822668 KX822625 Vietnam This study (NCSM84916)
LANCEOLARIINI
Arconaia lanceolata (Lea, 1856) NC_023955 KX822591 China Wang et al 2016; this study
Lanceolaria gladiola (Heude, 1877) KX822648 KX822605 China This study
Lanceolaria grayana (Lea, 1834) KX822649 KX822606 China This study
Lanceolaria grayii (Griffith & Pidgeon, 1833) KX822650 KX822607 Vietnam This study (NCSM84945)
UNIONINAE
UNIONINI
Unio crassus Philipsson, 1788* KC703878 KC703644 France Prié & Puillandre 2014
Unio gibbus Spengler, 1793 KX822671 KX822629 Morocco This study
U
Unio pictorum (Linnaeus, 1758) KC429109 KC429447 (Europe) Sharma et al 2013
Unio tumidus Philipsson, 1788 KX822672 KX822630 Ukraine This study
incertae sedis (UNIONINAE)
Aculamprotula tortuosa (Lea, 1865) KX822631 KX822586 China This study
Cuneopsis heudei (Heude, 1874) KX822638 KX822595 China This study
93
FCUP
Taxon COI 28S Country Reference (voucher specimen)

Cuneopsis pisciculus (Heude, 1874) KX822639 KX822596 China This study
Cuneopsis rufescens (Heude, 1874) KX822640 KX822597 China This study
Nodularia douglasiae (Griffith & Pidgeon, 1833) KX822653 KX822610 China This study
Nodularia nuxpersicae Dunker, 1848 KX822654 KX822611 Vietnam This study (NCSM84990)
Schistodesmus lampreyanus (Baird & Adams, 1867) KX822665 KX822621 China This study
RECTIDENTINAE
CONTRADENTINI
U
Contradens contradens (Lea, 1838) DQ191411 AF400692 (Asia) Graf & Cummings 2006
Contradens semmelinki (Martens, 1891) KX822636 KX822593 Vietnam This study (NCSM84935)
Physunio modelli Brandt, 1974 KX822655 KX822612 Thailand This study
Trapezoideus exolescens (Gould, 1843) KP795036 KP795018 Laos Pfeiffer & Graf 2015
RECTIDENTINI
Ensidens ingallsianus (Lea, 1852) KX822641 KX822598 Laos This study (NCSM84889)
Ensidens sagittarius (Lea, 1856) KP795033 KP795015 Cambodia Pfeiffer & Graf 2015
Ensidens sp. KX822642 KX822599 Laos This study (NCSM84902)
Hyriopsis bialata Simpson, 1900 KX822643 KX822600 Thailand This study
Hyriopsis desowitzi Brandt, 1974 KX822644 KX822601 Thailand This study
Hyriopsis myersiana (Lea, 1856) KX822645 KX822602 Thailand This study
Rectidens sumatrensis (Dunker, 1852) KX822664 KX822620 Malaysia This study
GONIDEINAE
CHAMBERLAINIINI
Chamberlainia hainesiana (Lea, 1856) KX822635 KX822592 Thailand This study
Sinohyriopsis cumingii (Lea, 1852)* HM347668 KX822623 China unpublished; this study
LAMPROTULINI
Lamprotula caveata (Heude, 1877) KX822646 KX822603 China This study
Lamprotula leaii (Griffith & Pidgeon, 1833) KX822647 KX822604 China This study
Potomida littoralis (Cuvier, 1798) JN243905 JN243883 France Whelan et al 2011
Pronodularia japanensis (Lea, 1859) KX822659 KX822615 Japan This study (NCSM27183)
GONIDEINI
Gonidea angulata (Lea, 1838)* DQ272371 AF400691 USA Gustafson & Iwamoto 2005; Graf 2002
Leguminaia wheatleyi (Lea, 1862) KX822651 KX822608 Turkey This study
Microcondylaea bonellii (A. Ferussac 1827) KX822652 KX822609 Italy This study
Solenaia carinata (Heude, 1877) KX822669 KX822626 China This study
94
FCUP
Taxon COI 28S Country Reference (voucher specimen)

Solenaia oleivora (Heude, 1877) KX822670 KX822627 China This study
PSEUDODONTINI
Pilsbryoconcha compressa (Martens, 1860) KX822656 KX822613 Thailand This study
Pilsbryoconcha exilis (Lea, 1838)* KX822657 AF400693 Vietnam Graf 2002; this study
Pseudodon cambodjensis (Petit de la Saussaye, 1865) KX822660 KX822616 Thailand This study
Pseudodon cumingii (Lea, 1850) KX822662 KX822618 Laos This study (NCSM84884)
Pseudodon mouhotii (Lea, 1863) KX822663 KX822619 Laos This study (NCSM84903)
incertae sedis (GONIDEINAE)
Solenaia triangularis (Heude, 1885) KJ434518 KX822628 China This study
AMBLEMINAE
AMBLEMINI
Amblema plicata (Say, 1817) APU56841 AF305385 USA Hoeh et al 1998; Graf 2002
LAMPSILINI
Actinonaias ligamentina (Lamarck, 1819) AF156517 DQ191420 USA Graf & Foighil 2000; Graf & Cummings 2006
Lampsilis cardium Rafinesque, 1820* AF120653 AF305386 USA Giribet & Wheeler 2002; Graf 2002
Villosa iris (Lea, 1829) AF156524 DQ191422 USA Graf & Foighil 2000; Graf & Cummings 2006
PLEUROBEMINI
Elliptio complanata (Lightfoot, 1786)* EU448173 JF899181 USA Unpublished; Distel et al 2011
Elliptio dilatata (Rafinesque, 1820)* AF156507 AF400690 USA Graf & Foighil 2000; Graf 2002
Pleurobema sintoxia (Rafinesque, 1820) AF156509 DQ191418 USA Graf & Foighil 2000; Graf & Cummings 2006
QUADRULINI
Quadrula quadrula (Rafinesque, 1820) AF156511 DQ191417 USA Graf & Foighil 2000; Graf & Cummings 2006
Quadrula verrucosa (Rafinesque, 1820) DQ191413 DQ191416 USA Graf & Cummings 2006
PARREYSIINAE
COELATURINI
Coelatura aegyptiaca (Cailliaud, 1827) JN243892 JN243870 Egypt Whelan et al 2011
LAMELLIDENTINI
Lamellidens corrianus (Lea, 1834) JN243903 JN243881 Burma Whelan et al 2011
OXYNAIINI
Oxynaia pugio (Benson, 1862) JN243899 JN243879 Burma Whelan et al 2011
PARREYSIINI
Parreysia mandelayensis (Theobald, 1873) JN243900 JN243876 Burma Whelan et al 2011
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Two independent runs of 24 × 10 6 generations were sampled at intervals of 1,000 generations

producing a total of 24,000 trees. Burn-in was determined upon the convergence of log-
likelihood and parameter values using Tracer 1.6 (Rambaut et al 2014).
For the ML phylogenetic analyses, sequences were analysed in RaxML 8.0.0
(Stamatakis 2014) where the GTR + G + I model was assumed for each partition with 1000
bootstrap replicates.
Review of morphological, anatomical, and behavioural traits

A table of morphological characters commonly used for Unionidae systematics was
constructed using a compilation of the available literature and direct observations of the
analysed taxa. To characterize and compare glochidial size, the glochidial size index (Gln) was
calculated following Davis & Fuller (1981) where Gln = glochidial shell length (μm) × shell
height (μm) × 10-6. Gln was divided into three size classes: small (≤0.020), medium (>0.020
and ≤0.070) and large (>0.070). These classes were determined using all glochidia
measurements collected for this study (Table C1) and those included in Barnhart et al (2008)
and Hoggarth (1999); the smaller size range of Quadrulini was used to define the class ‘small’,
the larger size range of Anodontini was used to define the class ‘large’, and the ‘medium’ class
size was defined with intermediate Gln values between the two other classes.
Distribution
Distribution maps were constructed using data available from the IUCN database (IUCN 2015),
the Mussel Project website (Graf and Cummings 2016), the North Carolina Museum of Natural
Sciences database (NCMNS 2016), and additional reference works (Zhadin 1938; Haas
1969a,b; Moskvicheva 1973a,b; Brandt 1974; Ðang et al 1980; Clarke 1981; Zatravkin &
Bogatov 1987; Subba Rao 1989; Bogatov & Starobogatov 1992; Howells et al 1996; Klishko
2001, 2003; Prozorova & Bogatov 2006; Cyr et al 2007; Vinarski et al 2007; Kondo 2008;
Nedeau et al 2009; Bogatov 2012; Doucet-Beaupré et al 2012; He & Zhuang 2013). Because
distribution data were gathered and compiled from very distinct sources, ranging from
georeferenced data points, hydrographic basins and geographical regions or countries, the
distributions on the maps are represented with various patterns (e.g. political borders or
hydrographic basins).
Results and discussion

Previous phylogenetic studies of the Unionidae failed to include most of the genera, mainly
those from the Eastern Palearctic and Indotropical ecoregions. We were able to clarify the
96 FCUP
phylogeny within Unionidae by the inclusion of samples from a wide coverage of genera and
geographic distribution.
On both of the following (COI + 28S) datasets, no indels were observed in the COI
alignments and no stop codons were found after translating the sequences to amino acids.
The ILD tests found no significant phylogenetic conflict between the COI and 28S for the
Palaeoheterodonta (p = 0.95) and the Unionidae (p = 0.94) datasets. The Palaeoheterodonta
dataset (COI + 28S) included 81 species in 55 genera, with a total of 1,091 bp (COI: 597 bp,
28S: 494 bp). Since the same topology in the supported nodes was obtained in the resulting
phylogenetic trees, the BI4 (Bayesian Inference with 4 partitions, see methods) topology is
here presented in Fig. 1. These analyses revealed the monophyly of the Unionidae in all
analyses with six supported subfamilies supported by the BI analysis (Anodontinae, Unioninae,
Rectidentinae, Gonideinae, Ambleminae, and Parreysiinae) showing the Parreysiinae as a
sister clade to all the other Unionidae.
The dataset including only Unionidae taxa spanned 70 species in 46 genera, with a
total of 1,032 bp (COI: 597 bp; 28S: 435 bp) aligned nucleotides. All resulting phylogenetic
trees yielded the same topology up to the tribal level, being the BI4 (Bayesian Inference with
4 partitions, see methods) topology presented. Both BI topologies were generally associated
with higher bootstrap support levels than ML topologies. Furthermore, the BI4 topology
resulted in slightly higher bootstrap values than the BI2 topology, presumably due to distinct
COI mutation rates.
The Unionidae is divided into two major clades, which are well supported in all analyses
and partition schemes, i.e. Anodontinae + Unioninae and Rectidentinae + Gonideinae +
Ambleminae (Fig. 2). At the subfamily level most clades are supported by the Bayesian
analyses, with the Rectidentinae also being supported by both ML analyses (Fig. 2). At the
tribal level, the same trend is observed, with all four analyses supporting Contradentini,
Rectidentini, Chamberlainiini, Lamprotulini, with the remaining tribes being supported mostly
by BI analyses only.
The subfamily Anodontinae is divided into three tribes (i.e. Anodontini, Cristariini nomen
novum and Lanceolariini nomen novum), and the subfamily Unioninae is not well resolved,
with Unionini being the only supported tribe. Available tribe names for the currently
unsupported group (sister to the Unionini) include Acuticostinae Starobogatov, 1967 and
Nodulariinae Starobogatov and Zatravkin, 1987. The subfamily Rectidentinae is sister to
Gonideinae + Ambleminae and encompasses two tribes (i.e. Contradentini and Rectidentini).
Both Gonideinae and Ambleminae are divided into four tribes each (see Fig. 2).
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Figure 1 Phylogenetic tree of the Palaeoheterodonta obtained by Bayesian Inference (BI) and
Maximum likelihood (ML) analyses of the first combined (COI + 28S) dataset. Support values
above the branches are posterior probabilities (BI4) and bootstrap support (ML4) below. An
asterisk (*) indicates nodes with P ≥ 95% posterior probability or bootstrap support. Posterior
probability (percentage) or bootstrap support with P < 50% were omitted for clarity. All
subfamily nodes were collapsed for visual purposes.
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Figure 2 Phylogenetic tree of the Unionidae obtained by Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of the second combined
(COI + 28S) dataset. Support values above the branches are posterior probabilities (BI4/BI2) and bootstrap support (ML4/ML2) below. An asterisk
(*) indicates nodes with P ≥ 95% posterior probability or bootstrap support. Posterior probability or bootstrap support with P < 50% were omitted
for clarity.
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In summary, our molecular phylogenetic analyses revealed a division of the Unionidae into 6
subfamilies and 18 tribes, three of which are named here for the first time. Revisions on the
subfamilial and tribal classification within the Unionidae are discussed here along with several
lower-level phylogenetic and taxonomic considerations.
To complement the present molecular analyses, seven morphological, anatomical and
behavioural characters commonly used in traditional classifications of the Unionidae are
summarized for each taxon in Table C1.
Glochidial shape is diagnostic in dividing the Anodontinae + Unioninae (triangular) and
Rectidentinae + Gonideinae + Ambleminae (bilaterally asymmetrical or semi-elliptical) clades
(Table C1). No single morpho-behavioural character analysed herein is diagnostic of
subfamilies within these clades. However, within subfamilies, certain tribes are characterized
by unique diagnostic characters. Within Anodontinae, four characteristics (shell shape, hinge
structure, glochidial size and brooding period) separate the Lanceolariini from the Anodontini
+ Cristariini. Additionally, all taxa within the Lanceolariini analysed are characterized by
nodulous umbo sculpture, although this morphological character is highly variable within all
other subfamilies and tribes (Table C1). Within Rectidentinae, glochidial shape is diagnostic in
separating the Contradentini (bilaterally asymmetrical) and Rectidentini (semi-elliptical).
Among the four tribes in Gonideinae, the Chamberlainiini taxa are unique in exhibiting
ectobranchous marsupia (Table C1).
Classification system
Based on the present results, a new classification of the Unionidae is presented, including the
description of three new tribes: Cristariini Lopes-Lima, Bogan and Froufe 2016; Lanceolariini
Froufe, Lopes-Lima and Bogan 2016; and Chamberlainiini Bogan, Froufe and Lopes-Lima
2016 (Table 3).
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Table 3
Classification of the Unionidae based on the present analyses. (*) Not included in the present
study.
ANODONTINAE Rafinesque, 1820
Anodontini Rafinesque, 1820
+ Alasmidontini Rafinesque, 1820
+ Strophitini Starobogatov, 1970
+ Pseudanodontini Starobogatov, 1970
+ Brachanodonini Bogatov, Sayenko & Starobogatov, 2002
*Arcidens Simpson, 1900 [+ Arkansia Ortmann & Walker 1912]
Alasmidonta Say, 1818
Anodonta Lamarck, 1799
*Anodontoides Simpson in F.C Baker, 1898
Lasmigona Rafinesque, 1831
Pseudanodonta Bourguignat, 1876
Pyganodon Crosse & Fischer, 1894
Simpsonaias, Frierson, 1914
Strophitus Rafinesque, 1820
*Utterbackia F.C. Baker, 1927
Cristariini Lopes-Lima, Bogan & Froufe, Nom. Nov.
Anemina Haas, 1969
Cristaria Schumacher, 1817
Pletholophus Simpson, 1900
Sinanodonta Modell, 1945
Lanceolariini Froufe, Lopes-Lima & Bogan, Nom. Nov.
Arconaia Conrad, 1865
Lanceolaria Conrad, 1853
ANODONTINAE (incertae sedis)
*Pegias Simpson, 1900
*Simpsonella Cockerell, 1903
UNIONINAE Rafinesque, 1820
+ Cafferiinae Modell, 1942
Unionini Rafinesque, 1820
+ Cafferiini Modell, 1942
Unio Philipsson in Retzius, 1788
UNIONINAE (incertae sedis)
Aculamprotula Wu, Liang, Wang & Ouyang, 1998
*Acuticosta Simpson, 1900
Cuneopsis Simpson, 1900
*Inversiunio Habe, 1991
*Lepidodesma Simpson, 1896
Nodularia Conrad, 1853
*Rhombuniopsis Haas, 1920
Schistodesmus Simpson, 1900
RECTIDENTINAE Modell, 1942
+ Hyriopsinae Modell, 1942
Contradentini Modell, 1942
+ Physunioini Starobogatov, 1970
Contradens Haas, 1913
Physunio Simpson, 1900
Trapezoideus Simpson, 1900
Rectidentini Modell, 1942
+ Limnoscaphini Lindholm, 1932
Ensidens Frierson, 1911
FCUP 101
Hyriopsis Conrad, 1853

Rectidens Simpson, 1900
GONIDEINAE Ortmann, 1916
+ Leguminainae Starobogatov, 1970
Chamberlainiini Bogan, Froufe & Lopes-Lima, Nom. Nov.
Chamberlainia Simpson, 1900
Sinohyriopsis Starobogatov, 1970
Lamprotulini Modell, 1942
+ Psilunionini Starobogatov, 1970
Lamprotula Simpson, 1900
Potomida Swainson, 1840
Pronodularia Starobogatov, 1970
Gonideini Ortmann, 1916
+ Leguminaiini Starobogatov, 1970
Gonidea Conrad, 1857
Leguminaia Conrad, 1865
Microcondylaea Vest, 1866
Solenaia Conrad, 1869
Pseudodontini Frierson, 1927
Pseudodon Gould, 1844
Pilsbryoconcha Simpson, 1900
GONIDEINAE (incertae sedis)
*Discomya Simpson, 1900
*Inversidens Haas, 1911
Solenaia triangularis
AMBLEMINAE Rafinesque, 1820
Amblemini Rafinesque, 1820
Amblema Rafinesque, 1820
*Reginaia Campbell & Lydeard, 2012
Lampsilini Ihering 1901
+ Propterini Hannibal, 1912
+ Cyprogeniini Starobogatov, 1970
+ Dromini Starobogatov, 1970
+ Friersoniini Starobogatov, 1970
+ Glebulini, Starobogatov, 1970
+ Medionidinae Starobogatov, 1970
+ Pilaeini Starobogatov, 1970
+ Pileini Bieler et al 2010
+ Popenaiadini Heard & Guckert, 1970
+ Ptychobranchini Starobogatov, 1970
Actinonaias Crosse & Fischer, 1894
*Arotonaias von Martens, 1900
*Cyprogenia Agassiz, 1852
*Cyrtonaias Crosse & Fischer, 1894
*Dromus Simpson, 1900
*Ellipsaria Rafinesque, 1820
*Epioblasma Rafinesque, 1831
*Friersonia Ortmann, 1912
*Glebula Conrad, 1853
*Hamiota Roe & Hartfield, 2005
Lampsilis Rafinesque, 1820
*Lemiox Rafinesque, 1831
*Leptodea Rafinesque, 1820
*Ligumia Swainson, 1840
*Medionidus Simpson, 1900
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*Obliquaria Rafinesque, 1820

*Obovaria Rafinesque, 1819
*Plectomerus Conrad, 1853
*Popenaias Frierson, 1927
*Potamilus Rafinesque, 1818
*Ptychobranchus Simpson, 1900
*Toxolasma Rafinesque, 1831
*Truncilla Rafinesque, 1819
*Venustaconcha Frierson, 1927
Villosa Frierson, 1927
Pleurobemini Hannibal, 1912
+ Elliptionini, Modell, 1942
Elliptio Rafinesque, 1820
*Elliptoideus Frierson, 1927
*Fusconaia Simpson, 1900
*Hemistena Rafinesque, 1820
*Plethobasus Simpson, 1900
Pleurobema Rafinesque, 1819
*Pleuronaia Frierson, 1927
Quadrulini Ihering, 1901
+ Megalonaiadini Heard & Guckert, 1970
*Cyclonaias Pilsbry in Ortmann & Walker, 1922
*Megalonaias Utterback, 1915
Quadrula Rafinesque, 1820
*Tritogonia Agassiz, 1852
*Uniomerus Conrad, 1853
AMBLEMINAE (incertae sedis)
*Barynaias Crosse & Fischer, 1894
*Delpinonaias Crosse & Fischer, 1894
*Disconaias Crosse & Fischer, 1894
*Martinsnaias Frierson, 1927
*Micronaias Simpson, 1900
*Nephritica Frierson, 1927
*Nephronaias Crosse & Fischer, 1894
*Pachynaias Crosse & Fischer, 1894
*Psoronaias Crosse & Fischer, 1894
*Psorula Haas, 1930
*Reticulataus Frierson, 1927
*Sphenonaias Crosse & Fischer, 1894
PARREYSIINAE Henderson 1935
Parreysiini Henderson, 1935
+ Diplasminae Modell, 1942
+ Hemisolasminae Starobogatov, 1970
Parreysia Conrad, 1853
Coelaturini Modell, 1942
+ Brazzaeini Leloup, 1950
+ Dentaspainiini Modell, 1964
+ Mweruellini Pain & F.R. Woodward, 1968
+ Prisodontopsini Pain & F.R. Woodward, 1968
+ Pseudaviculini Modell, 1942 [not available name, Bouchet & Rocroi, 2010]
+ Pseudospathini Leloup, 1950 [not available name, Bouchet & Rocroi, 2010]
+ Pseuodspathinae Starobogatov, 1970
*Brazzaea Bourguignat, 1885
Coelatura Conrad, 1853
*Grandidieria Bourguignat, 1885
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*Mweruella Haas, 1936

*Nitia Pallary, 1924
*Nyassunio Haas, 1936
*Prisodontopsis Tomlin1928
*Pseudospatha Simpson, 1900
Lamellidentini Modell, 1942
Lamellidens. Simpson, 1900
Oxynaiini Starobogatov, 1970
Oxynaia Haas, 1911
*Radiatula Simpson, 1900
*Scabies Haas, 1911
PARREYSIINAE (incertae sedis)
*Germainaia Graf & Cummings, 2009
MODELLNAIINAE Brandt, 1974
*Modellnaia Brandt 1974
UNIONIDAE (incertae sedis)
*Arcidopsis Simpson, 1900 [Arcidopsinae Starobogatov 1970]
*Caudiculatus Simpson, 1900
*Ctenodesma Simpson, 1900
*Diaurora Cockerell, 1903
*Elongaria Haas, 1913
*Gibbosula Simpson, 1900
*Haasodonta McMichael, 1956
*Harmandia Rochebrune, 1882
*Pressidens Haas, 1910
*Prohyriopsis Haas, 1914
*Protunio Haas, 1913
*Pseudodontopsis Kobelt, 1913
*Pseudobaphia Simpson, 1900
*Pseudomulleria Anthony, 1907 [Pseudomulleriinae Starobogatov, 1970]
*Ptychorhynchus Simpson, 1900
*Schepmania Haas, 1912
*Unionetta Haas, 1955
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Anodontinae Rafinesque, 1820 phylogeny and tribal classification

The subfamily status of the Anodontinae was first defined by Rafinesque in 1820 and properly
Latinized by Fleming in 1828. The subfamily status was retained in many of the classical
classifications well into the 20th century (e.g. Ortmann 1910; Modell 1964; Haas 1969a,b;
Heard & Guckert 1970; Davis & Fuller 1981). Subsequent studies demoted Anodontinae to a
tribe within Unioninae due to the shared hooked type and subtriangular external shape of the
glochidia (Graf 2002; Graf & Cummings 2007; Bieler et al 2010; Carter et al 2011). However,
the rank change of Anodontinae into Anodontini has been recently disputed based on
morphology discrepancies in glochidia morphology (Huang et al 2013). Anodontinae and
Unioninae are here recovered as sister clades and due to the ancient divergence of the two
clades are herein considered as subfamilies, following traditional classifications. Within
Anodontinae, we recognize three distinct tribes. In traditional classifications, this subfamily was
characterized by a set of distinctive morphological (e.g. large and ovate thin shells, and large
triangular and hooked glochidia), anatomical (e.g. demibranchs with perforated septa,
secondary water tubes in the outer demibranchs, and marsupium in the external demibranch
pair that distend laterally upon gravidity) and ecological (e.g. most species seem to be
generalists concerning habitat and host fish) characters. Although all the above characters are
found in most of the species within Anodontini and Cristariini, the Lanceolariini present
characters more similar to those of the Unioninae (i.e. shell size and form, glochidial size, and
tachytictia). Members of the Anodontinae have a wide distribution in the Northern Hemisphere,
not occurring in most of the Indotropical, and glaciated or desert regions (Fig. 3).
Figure 3 Distribution map of the subfamily Anodontinae.

FCUP 105
Tribe Anodontini Rafinesque, 1820

Type Genus: Anodonta Lamarck, 1799
Type Species: Mytilus cygneus Linnaeus, 1758
Comments: The Anodontini include one supported clade that contains all analysed
Anodontinae genera from Eastern North America, while the relationships among the Anodonta
and Pseudanodonta species are not well resolved. The Anodontini encompass the genera
Alasmidonta, Anodonta, Lasmigona, Pseudanodonta, Pyganodon, Simpsonaias, Strophitus
(Fig. 2), Anodontoides, Arcidens, and Utterbackia (Table 3; Lydeard et al 1996; Zanatta et al
2007; Breton et al 2011; Inoue et al 2014). Due to the lack of molecular data, two genera
usually assigned to this tribe, i.e. Simpsonella from the Philippines and Pegias from North
America (Haas 1969a,b; Graf and Cummings 2016), are not included in the present molecular
analyses. Placement of both these genera within the tribe thus remains to be tested by
molecular methods. This will be of interest regarding Simpsonella, which has a disjunct
distribution and has been placed within the Contradentini in other studies (Modell 1942, 1964).
We recovered two main clades within the Anodontini: one with Palearctic genera including the
type genus Anodonta that is also present on the West coast of North America and the other
including all East coast North American genera (Figs. 2 and 3). The relationships among and
within genera in each of these clades are not well resolved and should be further investigated.
Diagnosis: Shell is commonly thin and ovate to elongate but with some exceptions, mainly in
Alasmidonta and Lasmigona spp. (Table C1). Hinge is generally toothless or with vestigial
teeth in some genera e.g. Alasmidonta, Lasmigona, and Strophitus. Umbo sculpture is varied
and composed of double-looped and/or pseudo-concentric and/or single-looped ridges, which
are sometimes wrinkled or nodulous. Glochidia are large, triangular, and ventrally hooked with
spines (Table C1).
Distribution: The Anodontini have a disjunct distribution from the Western Palearctic to the
Transbaikalia and on both North American coasts (Fig. 3). Almost all Eastern Asian
Anodontinae species previously ascribed to Anodonta (e.g. A. woodiana and A. arcaeformis)
have later been transferred to other genera that are now placed outside Anodontini (Haas
1969a,b; Kondo 2008). The only Anodonta species still recognized from East Asia, Anodonta
beringiana, should be reassigned to the genus Sinanodonta (Chong et al 2008). The presence
of the tribe Anodontini in Central America and Middle East is pending further evaluation of the
phylogenetic status of Anodonta lurulenta Morelet, 1849, Anodonta pseudodopsis Locard,
1883 and Anodonta vescoiana Bourguignat, 1856.
Tribe Cristariini Lopes-Lima, Bogan and Froufe, nomen novum

Type Genus: Cristaria Schumacher, 1817
106 FCUP
Type Species: Cristaria tuberculata Schumacher, 1817; junior synonym of Dipsas plicata
Leach, 1815.
Comments: The Cristariini includes one supported clade composed by the genera Anemina,
Cristaria, Pletholophus and Sinanodonta (Fig. 2; Table 3). The type genus Cristaria is not
monophyletic in the current analyses and since Cristaria plicata is the type species, Cristaria
tenuis is here reassigned to Pletholophus Simpson, 1900 following Simpson (1900, 1914),
Ðang et al (1980), and He & Zhuang (2013). Many species have been assigned to
Sinanodonta, primarily by the Russian school of nomenclature (Haas 1969a; Graf 2007), but
the validity of these placements should be tested using molecular tools. Sinanodonta lucida
was first described as Anodonta lucida and then assigned to Sinanodonta (Ðang et al 1980)
but both generic attributions are still being used (e.g. Huang et al 2013; Pfeiffer & Graf 2013).
Additionally, recent studies based on morphological data consider S. lucida as a synonym of
S. woodiana (Graf & Cummings 2016; He & Zhuang 2013). Due to the high genetic distance
between these two taxa (12.3%; COI uncorrected p-distance), Sinanodonta woodiana and
Sinanodonta lucida are here recognized as two distinct species. Finally, as mentioned above,
Anodonta beringiana, although not included in the present analysis, should be placed within
the Cristariini though its generic assignment remains to be investigated.
Diagnosis: Shell is usually thin, of elliptical to oval shape, with or without a posterior dorsal
wing. Umbo rather low, sculpture usually consisting of pseudo-concentric folds that are nearly
parallel to growth lines. Periostracum is usually rayed. Hinge is lacking in Anemina and
Sinanodonta but reduced lamellar lateral and pseudocardinal teeth may be present in Cristaria
and Pletholophus.
Distribution: The native range of Cristariini spans from Indochina to China, Korea, Japan, the
Sakhalin Island, Amur Basin, Kamchatka and Chukotka Peninsulas (in Russia) to the Aleutians
and the Pacific Coastal Region of North America, where it may be found as far south as Oregon
(Fig. 3).
Tribe Lanceolariini Froufe, Lopes-Lima and Bogan, nomen novum

Type Genus: Lanceolaria Conrad, 1853
Type Species: Unio grayanus Lea, 1834
Comments: The tribe Lanceolariini is sister to all other Anodontinae. Most of its shell
morphological characteristics appear more similar to the subfamily Unioninae (e.g. well-
developed hinge teeth, medium-sized glochidia, and tachytictia; Table C1). It is therefore not
surprising that all previous classifications placed the genera of this tribe within the Unioninae
rather than in Anodontinae (e.g. Haas 1969a,b; Starobogatov 1970). Lanceolariini
encompasses two genera, i.e. Arconaia Conrad, 1865 and Lanceolaria Conrad, 1853, though
this should be further investigated considering that our results indicate paraphyly of
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Lanceolaria, giving support for the monotypic status of Lanceolariini with Lanceolaria as the
single genus.
Diagnosis: Shell is rather thick, of elongate or lanceolate shape and in some taxa, with
anteroposterior torsion. Umbo is low and positioned near the anterior end. Umbo sculpture is
strictly nodulous and usually restricted to the umbo area but in some cases more widespread.
Pseudocardinal teeth are well developed and long; lateral teeth are straight and thick.
Distribution: Lanceolariini are restricted to Far East Asia, from the Amur River basin (Russia)
to Japan, Korea, the Pacific basins of China and Vietnam (Fig. 3).
Unioninae Rafinesque, 1820 phylogeny and tribal Classification

The Unioninae was one of the first defined subfamilies, and the subfamily level has been
retained in all subsequent classifications of the Unionidae (Table 1). Until the middle of the
20th century, this subfamily encompassed almost all of the unionid genera of Europe, Africa
and Asia except those assigned to Anodontinae (Haas 1969a,b). It later became obvious that
the Unioninae represented a simple collection of very distinct groups that were not related or
similar in most of their characters. In this context, the Unionidae were subdivided by Modell
(1942, 1964) into several subfamilies using umbo sculpture as the main diagnostic character
(Table 1). However, this character alone was unsuitable for this purpose and thus, these
subfamilies were lumped back together until the emergence of modern phylogenetic
approaches (Heard & Guckert 1970; Davis & Fuller 1981). Since then, several Asian and
African genera have been reassigned to other subfamilies based on molecular phylogenetic
analyses and morphology (Liu et al 1979; Huang et al 2002; Zhou et al 2007; Kondo 2008;
Ouyang et al 2011; Pfeiffer & Graf 2013, 2015). Besides, many genera within this subfamily
have never been characterized using a molecular approach.
In the present study, one well-supported clade, i.e. the tribe Unionini, was obtained
within the Unioninae. Phylogenetic relationships among the remaining genera are not well
resolved. The phylogeny recovered Aculamprotula as sister to a clade including Cuneopsis +
Schistodesmus + Nodularia, but with poor support. As a result, Aculamprotula, Cuneopsis,
Schistodesmus, and Nodularia were classified as incertae sedis within Unioninae. If future
phylogenetic analyses that include additional taxa give support to the clade Cuneopsis +
Schistodesmus + Nodularia, the available name would be Nodulariini Starobogatov &
Zatravkin, 1987 since the subfamily name Cuneopsinae Mongin, 1963 is not available (Bieler
et al 2010). Furthermore, if in future studies the genus Acuticosta falls within this clade the
earliest tribe name would change to Acuticostini Starobogatov, 1967. As in the Anodontinae,
the Unioninae present a strict ectobranchous condition but see Araujo et al (2009) and Lopes-
Lima et al (2016) for unusual exceptions in some populations. Marsupial demibranchs lack
specialized characters present in Anodontinae. Hinge teeth are well-defined. Glochidia are
108 FCUP
hooked, triangular and of medium size. The brooding type is tachytictic or short term (Table
C1). The Unioninae is one of the most widely distributed tribes, covering almost all of Europe
and Northwest Africa, as well as Vietnam, China, Far East Russia, Korea, Japan, and the
Sakhalin Island. Besides, two Unio species have disjunct distributions, i.e. Unio abyssinicus in
the Horn of Africa and Unio caffer in South Africa (Fig. 4).
Figure 4 Distribution map of the subfamily Unioninae.
Tribe Unionini Rafinesque, 1820

Type Genus: Unio Philipsson in Retzius, 1788
Type Species: Mya pictorum Linnaeus, 1758
Comments: The Unionini contains only one genus, i.e. Unio. This genus is divided into four
main lineages, i.e. the crassus-, pictorum-, gibbus- and tumidus-lineages (Froufe et al 2016a;
Lopes-Lima et al 2017), all of which are represented in the present phylogeny. Whilst the
crassus- and pictorum-lineages cluster together, relationships among this group and the other
two Unio lineages are not well resolved (Fig. 2).
Diagnosis: The main shared characters of the Unionini are: ectobranchous; marsupial
demibranch without any specialized character; presence of a well-defined hinge structure with
two pseudocardinal and two lateral teeth on the left valve and one or two on the right; umbo
sculpture W-shaped and/or double-looped bars, which in some cases become nodulous or
wrinkled; tachytictia or short term brooding; and the hooked triangular glochidia of intermediate
sizes (Table C1).
FCUP 109
Distribution: The tribe has essentially a western Palearctic distribution, extending from
Western Europe to European Russia and the Caspian basin. Also, three disjunct distributions
are known, i.e. one in the Transbaikal region in Russia and two others in Sub-Saharan Africa
(Fig. 4).
Rectidentinae Modell, 1942 phylogeny and tribal classification

The Rectidentinae originally included Rectidens as the type genus, as well as Physunio and
Ensidens also including some eastern North American and Southeast Asian genera in this
subfamily (e.g. Lastena, Pyganodon, and Pilsbryoconcha) (Modell 1942, 1964), but these were
subsequently reassigned to distinct subfamilies (Haas 1969a). The present phylogeny reveals
two well-supported clades within Rectidentinae, i.e. the tribes Contradentini Modell, 1942 and
Rectidentini Modell, 1942. The Contradentini was first described as a subfamily in the same
study that defined Rectidentinae (Modell 1942). Although the Rectidentinae, Contradentinae,
and Nannonaiinae were all described by Modell (1942), the priority of Rectidentinae was
determined by the First Revisor action (Brandt 1974; Bieler et al 2010). Since the two tribes
within the Rectidentinae show wide variability in morphological and anatomical characters,
none of these characteristics are distinctive on the subfamily level (Table C1). The
Rectidentinae are restricted to South East Asia, i.e. from Eastern India to Myanmar, Thailand,
Laos, Cambodia, and Vietnam, and south to Peninsular Malaysia and the Islands of Sumatra,
Java, Borneo, and Sulawesi (Fig. 5).
Figure 5 Distribution map of the subfamily Rectidentinae.
Tribe Contradentini Modell, 1942

110 FCUP
Type Genus: Contradens Haas, 1911

Type Species: Contradens contradens (Lea, 1838)
Comments: The Contradentini initially included the type genus Contradens, as well as
Caudiculatus, Pressidens and Simpsonella, all from Indochina and the Island of Borneo and
the Philippines (Modell 1942, 1964). Subsequently, all of these genera were reassigned to the
Unioninae, except for Simpsonella, which was placed within the Anodontinae (Haas 1969a,b).
More recently, Caudiculatus and Pressidens were once again placed within the Rectidentinae
(Graf & Cummings 2016). The present analyses recovered three genera in Contradentini, i.e.
Contradens, Physunio, and Trapezoideus. The phylogenetic relationships of the other genera,
i.e. Caudiculatus, Pressidens, and Simpsonella should be further investigated since no
sequence data are available at present. The date of publication of two genera, i.e. Uniandra
Haas, 1912 and Contradens, has been a source of confusion and has been clarified by Bogan
(2015).
Diagnosis: Shell shape is variable, from rounded to elongate. Umbo sculpture ranges from v-
shaped (e.g. in Contradens contradens) to w-shaped/double-looped/nodulous (e.g. in
Physunio superbus) and pseudo-concentric ridges (e.g. in Trapezoideus exolescens). Hinge
plate is well defined, with one lateral and one or two thin pseudocardinal teeth in the left valve,
and one lateral and one pseudocardinal tooth in the right valve. Glochidia are bilaterally
asymmetrical and are quite distinct from any other group of the Unionidae, rendering this trait
diagnostic of the tribe (Pfeiffer & Graf 2015). The brooding type is ectobranchous, but the
brooding period and length are unknown.
Distribution: The Contradentini has the same distribution in South East Asia as described
above for the Rectidentinae (Fig. 5).
Tribe Rectidentini Modell, 1942

Type Genus: Rectidens Simpson, 1900
Type Species: Unio lingulatus Drouet & Chaper, 1892
Comments: The Rectidentini includes the type genus Rectidens as well as Hyriopsis and
Ensidens. Of the four Hyriopsis species included in this study, only Hyriopsis cumingii does
not cluster with the type of the genus Hyriopsis bialata. Thus, Hyriopsis cumingii is here
reassigned to Sinohyriopsis Starobogatov, 1970, with the type species Unio cumingii Lea,
1852 (see Ðặng et al 1980). The remaining Hyriopsis species relationships, i.e. Hyriopsis
bialata, Hyriopsis desowitzi, and Hyriopsis myersiana are still unresolved.
Diagnosis: Shells are usually elongated and, in Hyriopsis, often with evident dorsal wings.
Umbo sculpture is predominantly pseudo-concentric to double-looped or nodulous. Hinge
structure is generally well defined with a variety of teeth numbers and shapes. Glochidia are
of the unhooked elliptical type and intermediate sizes. The brooding type is ectobranchous or
FCUP 111
tetragenous in Hyriopsis and tetragenous in Ensidens and Rectidens (Table C1). The semi-
elliptical unhooked shape of Rectidentini glochidia distinguishes this tribe from the
Contradentini. However, semi elliptical unhooked glochidia are also present in other
subfamilies (i.e. Gonideinae and Ambleminae, Modellnaiinae, and Parreysiinae).
Distribution: Although the distribution of the Rectidentini significantly overlaps with that of the
Contradentini, its range excludes Bangladesh and the Islands of Sulawesi and Sumatra (Fig.
5).
Gonideinae Ortmann, 1916 phylogeny and tribal classification

The Gonideinae was first described including only a single monotypic genus, i.e. Gonidea
angulata (Lea, 1838), which had previously been assigned to Anodontinae (Ortmann 1916).
That species reassignment was based on the distinctive anatomical characters of G. angulata,
which are unique among the North American unionid fauna (Ortmann 1916). Since then, the
phylogenetic position of G. angulata has changed many times. It has been recognized as a
valid subfamily (Ortmann 1916, Heard & Guckert 1970), placed within other subfamilies such
as the Pseudodontinae (Modell 1942) and the Unioninae (Haas 1969a,b), and in a separate
tribe, i.e. Gonideini, within the Ambleminae (Graf 2002; Graf & Cummings 2007), but always
as a monotypic group. Recent molecular phylogenetic analyses have recovered Gonidea in a
clade with several Old-World genera (e.g. Potomida, Pseudodon, and Pronodularia) and
recognized that clade as the Gonideinae (Pfeiffer & Graf 2013, 2015). In the present work, the
Gonideinae is recovered as a monophyletic subfamily that includes the type genus Gonidea
from western North America, three Western Palearctic genera (i.e. Leguminaia,
Microcondylaea, and Potomida) and seven genera from East and Southeast Asia. The
Gonideinae are here divided into two well-supported clades. One includes two sister tribes, i.e.
Chamberlainiini nomen novum and Lamprotulini (Fig. 2).
112 FCUP
Figure 6 Distribution map of the subfamily Gonideinae.
The second clade is composed of two tribes, i.e. the Gonideini and the Pseudodontini, and
one isolated species, i.e. Solenaia triangularis (Fig. 2). No single morphological character is
useful to diagnose the subfamily. All the studied genera have medium-sized semi-elliptical
unhooked glochidia and are tachytictic, though the marsupium location varies among tribes
(i.e. ectobranchous or tetragenous; Table C1). The Gonideinae has a scattered distribution in
the Northern Hemisphere, being present in restricted regions of the Palearctic, Indotropics and
Western Nearctic (Fig. 6).
Tribe Chamberlainiini Bogan, Froufe and Lopes-Lima, nomen novum

Type Genus: Chamberlainia Simpson, 1900
Type Species: Unio hainesianus Lea, 1856
Comments: Chamberlainiini nomen novum is here described for the first time and
encompasses only two genera, i.e. the monotypic Chamberlainia and Sinohyriopsis. The latter
includes Sinohyriopsis cumingii, previously assigned to Hyriopsis (see above), and
Sinohyriopsis schlegelii, previously shown to be related to S. cumingii (Froufe et al 2016b).
Diagnosis: Shell oval, elliptical to rhomboid, often with small anterior wing and posterior dorsal
wing. Posterior ridge is rounded. Umbos are low. Umbo sculpture consisting of well-developed
pseudo-concentric or nodulous ridges. Hinge with single pseudocardinal and lateral tooth in
the right valve, and typically two pseudocardinal and lateral teeth in the left valve. Glochidia,
as in all Gonideinae, are unhooked and semi-elliptical in shape. Brooding type is
ectobranchous and tachytictic. The Chamberlainiini the only ectobranchous tribe within the
Gonideinae (Table C1).
FCUP 113
Distribution: Distribution of the Chamberlainiini is restricted to Indochina, the Huang He River

basin in China, and Japan (Fig. 6).
Tribe Lamprotulini Modell, 1942

Type Genus: Lamprotula Simpson, 1900
Type Species: Chama plumbea Chemnitz, 1795
Comments: In addition to the type genus, the Lamprotulini include the western Palearctic
Potomida and the Far East Asian Pronodularia. The Lamprotulini was first defined as a
subfamily mainly based on characteristics of the umbo sculpture (Modell 1942, 1964). It
originally contained the genus Discomya, for which no genetic information is currently
available, Lamprotula, Potomida, and Pronodularia. Subsequently, all of these genera, except
for Pronodularia, were reassigned to Quadrulinae (Haas 1969a,b). However, Lamprotula,
Pronodularia, and Potomida were recently reassigned back to Gonideinae based on molecular,
morphological and biogeographical studies (Pfeiffer & Graf 2013, 2015; Whelan et al 2011).
The present study confirms the placement of these three genera within Lamprotulini.
Diagnosis: Shells are generally thick, and ovate to triangular. Hinge with well-developed,
strong teeth, generally three pseudocardinals and four laterals. Umbo sculpture consists of W-
shaped to double-looped ridges, which sometimes become nodulous and/or wrinkled.
Glochidia are semi-elliptical and unhooked, and of intermediate sizes (Table C1). The brooding
type is tachytictic and tetragenous except for Pronodularia, which can be ectobranchous or
tetragenous (Kondo 1982) (Table C1). This tribe shares most of the traits with the other
Gonideinae tribes, but all species present strong thick shells and well-developed hinge teeth
(Table C1).
Distribution: The Lamprotulini have a disjunct distribution, with Potomida presenting a patchy
distribution in the Mediterranean region, Lamprotula being distributed from North Vietnam to
North China and Korea, and Pronodularia restricted to Korea and Japan (Fig. 6).
Tribe Gonideini Ortmann, 1916

Type Genus: Gonidea Conrad, 1857
Type Species: Anodon randalli Trask, 1855 (Junior synonym of Anodonta angulata Lea, 1838)
Comments: The Gonideini are divided into two well-supported clades, i.e. one encompassing
the Western North American Gonidea, the Southern European Microcondylaea and the Middle
Eastern Leguminaia, and the other with the Asian Solenaia. Solenaia is not monophyletic, as
Solenaia triangularis was not recovered within the Gonideini (see Pfeiffer & Graf 2015). The
type genus Gonidea, as well as Leguminaia and Microcondylaea were originally placed within
the Pseudodontinae with other Asian genera (Modell 1942), but were all subsequently
reassigned to the Unioninae (Haas 1969a,b). Starobogatov (1970) placed these genera in the
114 FCUP
Pseudodontinae within the Margaritiferidae. Only recently, based on biogeographic and

morphological information, Graf & Cummings (2016) suggested the placement of these genera
within the Gonideinae. In the present study, molecular data confirm the placement of these
three genera within the Gonideini together with some representatives of the genus Solenaia.
Diagnosis: Shell shape is trapezoidal but much more elongated in Solenaia. Hinge teeth are
small, vestigial or absent in Solenaia. Umbo sculpture consists of pseudo-concentric, double-
looped and/or W-shaped ridges, which are sometimes wrinkled. Glochidia are of intermediate
sizes, semi-elliptical and unhooked. The brooding type is tachytictic and tetragenous. Within
the subfamily, the Gonideini are identified by a typical trapezoidal or rectangular shell shape,
and a hinge without teeth or only vestigial teeth (Table C1).
Distribution: The tribe has a curious, disjunct distribution. While Gonidea is restricted to the
west coast of North America, Microcondylaea only occurs from the Italian Peninsula to coastal
Croatia in Europe, and Leguminaia is present in southeast Turkey and the Middle East.
Solenaia occurs from eastern India to Myanmar, Thailand, North Vietnam and China (Fig. 6).
Tribe Pseudodontini Frierson, 1927

Type Genus: Pseudodon Gould, 1844
Type Species: Anodon inoscularis Gould, 1844
Comments: This group was first named as a subfamily, Pseudodontinae, by Frierson (1927)
and included the species Pseudodon cambodjensis and Gonidea angulata. It was then
redefined, mainly using morphological characters, with Pseudodon as the type genus together
with other genera including the North American Gonidea (Modell 1942, 1964). All of these
genera were then subsequently reassigned to the Unioninae subfamily (Subba Rao 1989;
Haas 1969a,b), and only recently were their relationships with the Gonideinae discussed
(Whelan et al 2011; Pfeiffer and Graf 2015). The Pseudodontinae is here demoted to a tribe,
Pseudodontini, within Gonideinae, being composed of only two monophyletic genera, i.e. the
type genus Pseudodon and Pilsbryoconcha (Fig. 2).
Diagnosis: Shell shape is generally ovate in Pseudodon and more elongated in
Pilsbryoconcha. Umbo sculpture is double-looped or W-shaped, with the anterior loops
sometimes fading distally so that only the posterior single-loop or a single row of nodes
remains. The brooding type is tachytictic and tetragenous. Glochidia are unhooked and semi-
elliptical. The representatives of this tribe present a characteristic ‘‘v” shaped fossette present
at the posterior end of the hinge structure with small vestigial teeth, which are completely
absent in Pilsbryoconcha (Table C1).
Distribution: The Pseudodontini are present in Myanmar, Malaysia, Thailand, Cambodia,
Laos, Vietnam, China, and Indonesia including Java, Sumatra, and Borneo (Fig. 6).
FCUP 115
Ambleminae Rafinesque, 1820, Parreysiinae Henderson, 1935 & Modellnaiinae Brandt,

1974
The Ambleminae and Parreysiinae were investigated in detail earlier (Campbell et al 2005;
Whelan et al 2011; Campbell and Lydeard 2012a,b;) and thus not fully explored in the present
study. The Modellnaiinae is a monotypic subfamily defined by Brandt (1974) with Modellnaia
siamensis as the only species. Its status as a subfamily has been retained by posterior
classification systems based on its quite distinct morphological characters (Bieler et al 2010;
Carter et al 2011; Whelan et al 2011; this study). Unfortunately, this species has never been
included in phylogenetic analyses and no sample was available for the present study. Based
on these earlier works and the present classification system, distribution maps are here
presented for Ambleminae, Parreysiinae, and Modellnaiinae (Table 3; Fig. 7). The Ambleminae
are restricted to Canada and the United States east of the Rocky Mountains and extend south
through Mexico to southern Panama. The Parreysiinae has a disjunct distribution in Africa and
Southern Asia. In Africa, Parreysiinae are found in the Nile River basin from the Nile delta
south into East Africa and across sub-Saharan Africa south to Namibia and Mozambique.
Germainaia Graf & Cummings 2009 from northwest Madagascar is treated here as belonging
to the Parreysiinae. In Asia, the Parreysiinae occur in Pakistan, India, Nepal, Myanmar,
Thailand, Indonesia, Cambodia, Laos and Vietnam (Fig. 7). The Modellnaiinae (i.e. Modellnaia
siamensis) is restricted to the middle section of the Mun River in Thailand (Fig. 7).
Figure 7 Distribution map of the subfamilies Ambleminae + Modellnaiinae + Parreysiinae.
Conclusions
116 FCUP
Considering the high levels of the decline of freshwater mussel species worldwide, an
understanding of the phylogenetic diversity is crucial for determining conservation priorities,
especially in poorly explored regions such as Central America and the Indotropics.
Conservation strategies should strive not only to maximize the current levels of biological
diversity but also to include phylogenetic patterns to maximize future levels of biodiversity.
Furthermore, due to the increasing development and biotic homogenization in tropical areas
(e.g. Malaysia and Indonesia) with dramatic negative implications on freshwater habitats,
conservation and management efforts targeting freshwater taxa are urgently needed.
The present study is an important contribution to the definition of freshwater mussel
diversity patterns, especially in the Indotropical and East Asian countries. Here, a phylogeny
of the Unionidae is presented with the greatest generic and geographic coverage to date,
based on a dataset comprising 70 species in 46 genera, 7 of these genera being sequenced
for the first time. Furthermore, it includes 57 species from 35 genera, thereby tripling the
number of analysed taxa from Anodontinae, Unioninae, Rectidentinae, and Gonideinae.
Molecular phylogenetic analyses revealed the presence of 6 subfamilies in the Unionidae,
divided into 18 tribes, 3 of which are described here for the first time. Although we compiled
seven characters traditionally used in Unionidae systematics, no single one was found to be
diagnostic at the subfamily level and few were useful at the tribe level (e.g. larval morphology
for Contradentini). However, within subfamilies, many tribes can be characterized based on a
subset of these characters.
Representing a major international collaborative effort, this study provides important
advances in the systematics of these extraordinary taxa with implications for ecological and
conservation studies (e.g. assessment of conservation status and distribution).
Acknowledgments
Financial support was provided by the Portuguese Foundation for Science and Technology
(FCT) under grants to EF (SFRH/BPD/108445/2015) and MLL (SFRH/BD/115728/2016); the
Vietnam Academy of Science and Technology under grant number IEBR.CBT.TS07/2015 for
the Vietnamese field research; the Central Michigan University Poyang Lake Research
Investment Fund and the Key Laboratory of Poyang Lake Wetland and Watershed Research
Director’s Open Fund of Jiangxi Normal University for procurement of and analysis of some
Chinese specimens. The authors wish to thank: Jamie Smith, Collection manager of Molluscs,
North Carolina Museum of Natural Sciences, Raleigh, North Carolina, for all of her efforts to
ship tissue samples to be used in this project; the Confederated Tribes of the Umatilla Indian
Reservation for providing access to specimens of A. nuttalliana.
FCUP 117
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Appendix A
Concatenated (COI + 28S) FASTA file of the Palaeoheterodonta dataset.
Available online at http://dx.doi.org/10.1016/j.ympev.2016.08.021
Appendix B
Concatenated (COI + 28S) FASTA file of the Unionidae dataset.
Available online at http://dx.doi.org/10.1016/j.ympev.2016.08.021

FCUP 135
Table C1
List of morphological, anatomical and behavioral characters used in traditional phylogenetic and systematic analyses of Unionida. (Gln) Glochidial
size index; (Pse) Pseudocardinal teeth; (Lat) Lateral teeth; (L) Left valve; (R) Right valve. (*) Reported as bilaterally asymmetrical but see
discussion in Pfeiffer & Graf (2015) (mostly likely unhooked like other Pseudodon species). Umbo sculpture classification follows Zieritz et al
(2015).
Brooding Marsupium
Taxon Glochidia shape Gln
Period anatomy
triangular
ANODONTINAE medium/large various ectobranchous
(hooked)
triangular
ANODONTINI large bradytictic ectobranchous
(hooked)
triangular large
Alasmidonta marginata bradytictic ectobranchous
(hooked) (0.115)
triangular large
Anodonta anatina bradytictic ectobranchous
(hooked) (0.122)
triangular large
Anodonta nuttalliana bradytictic ectobranchous
(hooked) (0.092)
triangular large
Anodonta cygnea bradytictic ectobranchous
(hooked) (0.096)
asymmetric large
Lasmigona compressa bradytictic ectobranchous
(hooked) (0.092)
triangular large
Pseudanodonta complanata bradytictic ectobranchous
(hooked) (0.099)
triangular large
Pyganodon grandis bradytictic ectobranchous
(hooked) (0.141)
triangular medium
Simpsonaias ambigua bradytictic ectobranchous
(hooked) (0.0645)
triangular large ectobranchous
Strophitus undulatus bradytictic
(hooked) (0.104) (anterior sections)
triangular
CRISTARIINI large various ectobranchous
(hooked)
136 FCUP
triangular large
Anemina arcaeformis bradytictic ectobranchous
(hooked) (0.126)
triangular large
Cristaria plicata bradytictic ectobranchous
(hooked) (0.080)
triangular large
Pletholophus tenuis unknown unknown
(hooked) (0.072)
triangular large
Sinanodonta lucida unknown ectobranchous
(hooked) (0.190)
triangular large
Sinanodonta woodiana tachytictic ectobranchous
(hooked) (0.156)
triangular
LANCEOLARIINI medium tachytictic ectobranchous
(hooked)
triangular medium
Arconaia lanceolata tachytictic ectobranchous
(hooked) (0.037)
triangular medium
Lanceolaria gladiola tachytictic ectobranchous
(hooked) (0.041)
triangular medium
Lanceolaria grayana tachytictic ectobranchous
(hooked) (0.032)
Lanceolaria grayii unknown unknown unknown ectobranchous

triangular
UNIONINAE medium tachytictic ectobranchous
(hooked)
triangular
UNIONINI medium tachytictic ectobranchous
(hooked)
triangular medium
Unio crassus tachytictic ectobranchous
(hooked) (0.043)
triangular medium
Unio gibbus Unknown ectobranchous
(hooked) (0.044)
triangular medium
Unio pictorum tachytictic ectobranchous
(hooked) (0.042)
FCUP 137
triangular medium
Unio tumidus tachytictic ectobranchous
(hooked) (0.042)
triangular medium
Aculamprotula tortuosa tachytictic ectobranchous
(hooked) (0.059)
triangular medium
Cuneopsis heudei tachytictic ectobranchous
(hooked) (0.054)
triangular medium
Cuneopsis pisciculus tachytictic ectobranchous
(hooked) (0.044)
triangular medium
Cuneopsis rufescens tachytictic ectobranchous
(hooked) (0.054)
triangular medium
Nodularia douglasiae tachytictic ectobranchous
(hooked) (0.043)
Nodularia nuxpersicae unknown unknown unknown ectobranchous

triangular medium
Schistodesmus lampreyanus tachytictic ectobranchous
(hooked) (0.034)
asymmetrical;
ectobranchous;
RECTIDENTINAE semi-elliptical medium unknown
tetragenous
(unhooked)
bilaterally
CONTRADENTINI unknown unknown ectobranchous
asymmetrical
bilaterally
Contradens contradens unknown bradytictic ectobranchous
asymmetrical
Contradens semmelincki unknown unknown unknown unknown

Physunio modelli unknown unknown unknown ectobranchous
bilaterally medium
Trapezoideus exolescens unknown ectobranchous
asymmetrical (0.047)
semi-elliptical ectobranchous;
RECTIDENTINI medium unknown
(unhooked) tetragenous
138 FCUP
semi-elliptical
Ensidens ingallsianus unknown bradytictic tetragenous
(unhooked)
Ensidens sp. unknown unknown unknown tetragenous

Ensidens sagittarius unknown unknown unknown unknown
semi-elliptical
Hyriopsis bialata medium (0.040) tachytictic ectobranchous
(unhooked)
semi-elliptical medium
Hyriopsis desowitzi tachytictic tetragenous
(unhooked) (0.025)
Hyriopsis myersiana bradytictic ectobranchous
(unhooked) (0.040)
semi-elliptical
Rectidens sumatrensis unknown unknown tetragenous
(unhooked)
semi-elliptical ectobranchous;
GONIDEINAE medium tachytictic
(unhooked) tetragenous
semi-elliptical
CHAMBERLAINIINI medium various ectobranchous
(unhooked)
Chamberlainia hainesiana bradytictic ectobranchous
(unhooked) (0.043)
Sinohyriopsis cumingii tachytictic ectobranchous
(unhooked) (0.049)
semi-elliptical
LAMPROTULINI medium tachytictic tetragenous
(unhooked)
Lamprotula caveata tachytictic tetragenous
(unhooked) (0.036)
Lamprotula leai tachytictic tetragenous
(unhooked) (0.036)
FCUP 139
Potomida littoralis tachytictic tetragenous
(unhooked) (0.036)
Pronodularia japanensis tachytictic tetragenous
(unhooked) (0.040)
semi-elliptical
GONIDEINI medium tachytictic tetragenous
(unhooked)
Gonidea angulata tachytictic tetragenous
(unhooked) (0.036)
semi-elliptical
Leguminaia wheatleyi unknown unknown unknown
(unhooked)
Microcondylaea bonellii tachytictic tetragenous
(unhooked) (0.021)
semi-elliptical
Solenaia carinata unknown unknown unknown
(unhooked)
Solenaia oleivora unknown unknown tachytictic tetragenous
semi-elliptical
PSEUDODONTINI unknown unknown tetragenous
(unhooked)
Pilsbryoconcha compressa unknown unknown bradytictic unknown
Pilsbryoconcha exilis unknown tetragenous
(unhooked) (0.024)
Pseudodon cambodjensis* bradytictic unknown
(unhooked) (0.050)
Pseudodon mouhotii unknown unknown unknown unknown
semi-elliptical
Pseudodon cummingii unknown unknown tetragenous
(unhooked)
140 FCUP
Solenaia triangularis unknown unknown unknown unknown

semi-elliptical small; ectobranchous;
AMBLEMINAE various
(unhooked) medium tetragenous
semi-elliptical
AMBLEMINI medium tachytictic tetragenous
(unhooked)
Amblema plicata tachytictic tetragenous
(unhooked) (0.042)
semi-elliptical;
ectobranchous
LAMPSILINI axe-shaped medium bradytictic
(posterior sections)
(unhooked)
semi-elliptical; medium ectobranchous
Actinonaias ligamentina bradytictic
(unhooked) (0.053) (posterior sections)
semi-elliptical medium ectobranchous
Lampsilis cardium bradytictic
semi-elliptical medium ectobranchous
Villosa iris bradytictic
semi-elliptical
PLEUROBEMINI medium tachytictic ectobranchous
(unhooked)
Pleurobema sintoxia tachytictic ectobranchous
(unhooked) (0.023)
Elliptio complanata tachytictic ectobranchous
(unhooked) (0.038)
Elliptio dilatata tachytictic ectobranchous
(unhooked) (0.044)
semi-elliptical
QUADRULINI small tachytictic tetragenous
(unhooked)
semi-elliptical small
Quadrula quadrula tachytictic tetragenous
(unhooked) (0.007)
semi-elliptical small
Quadrula verrucosa tachytictic tetragenous
(unhooked) (0.010)
FCUP 141
Table C1 (cont.)
Taxon Shell shape Umbo sculpture Hinge structure References
ANODONTINAE various various various
ANODONTINI ovate various teeth absent or vestigial

double-looped, Pse: 2L, 1R (small); Barnhart et al 2008;
Alasmidonta marginata triangular
wrinkled Lat: absent William et al 2008
double-looped, Hinzmann et al 2013;
Anodonta anatina ovate pseudo-concentric, teeth absent Pekkarinen & Englund 1995;
wrinkled Wächtler et al 2001
double-looped, Heard 1975;
Anodonta nuttalliana ovate teeth absent
nodulous Nedeau et al 2009
Lima et al 2012;
Anodonta cygnea ovate pseudo-concentric teeth absent Pekkarinen & Englund 1995;
Wächtler et al 2001
Pse: 2L, 1R (smooth, low);
double-looped,
Lasmigona compressa ovate Lat: 2L, 1R (thin); Barnhart et al 2008
wrinkled
Interdental tooth in L valve;
McIvor & Aldridge 2007;
double-looped,
Pseudanodonta complanata ovate teeth absent Pekkarinen & Englund 1995;
nodulous
Wächtler et al 2001
double-looped,
Pyganodon grandis ovate nodulous, teeth absent Barnhart et al 2008
pseudo-concentric
Pse: 1L, 1R (small);
Simpsonaias ambigua elongate double-looped Clarke 1985
Lat: absent or slight ridge
double-looped, Pse: thickening only;
Strophitus undulatus ovate Barnhart et al 2008
pseudo-concentric Lat: absent
CRISTARIINI ovate various teeth absent or vestigial
142 FCUP
He & Zhuang 2013;

Anemina arcaeformis ovate pseudo-concentric teeth absent Jeong et al 1993;
Sayenko 2006
He & Zhuang 2013;
Pse: absent; Heard 1977;
Cristaria plicata ovate pseudo-concentric
Lat: 1L, 1R Sayenko 2006;
Wu et al 1999
He & Zhuang 2013;
double-looped, Pse: 1L, 1R (thin);
Pletholophus tenuis ovate Inaba 1941;
pseudo-concentric Lat: 1L, 1R
Simpson 1914
double-looped, He & Zhuang 2013;
Sinanodonta lucida ovate teeth absent
pseudo-concentric Wu et al 1999
Bogatov & Sayenko 2002;
He & Zhuang 2013;
double-looped,
Sinanodonta woodiana ovate teeth absent Kondo 1987;
pseudo-concentric
Wächtler et al 2001;
Wu et al 1999
Pse: 2L, 2R;
LANCEOLARIINI lanceolate nodulous
Lat: 2L, 1R
lanceolate Pse: 2L, 2R; He & Zhuang 2013;
Arconaia lanceolata nodulous
(twisted) Lat: 2L, 1R Simpson 1914
Pse: 2L, 2R; He & Zhuang 2013;
Lanceolaria gladiola lanceolate nodulous
Lat: 2L, 1R Wu et al 1999
He & Zhuang 2013;
Pse: 2L, 2R;
Lanceolaria grayana lanceolate nodulous Kondo 1987;
Lat: 2L, 1R
Wu et al 1999
Lanceolaria grayii lanceolate nodulous
Lat: 2L, 1R Simpson 1914
UNIONINAE various various various teeth well structured
Pse: 2L, 1/2R;
UNIONINI elongate various
Lat: 2L, 1R
elongate, double-looped, Pse: 2L, 2R (compressed); Pekkarinen & Englund 1995;
Unio crassus
ovate nodulous Lat: 2L, 1R Wächtler et al 2001
FCUP 143
Pse: 2L, 1R (lamellar); Araujo et al 2009;

Unio gibbus ovate nodulous
Lat: 2L, 1R (lamellar) Khalloufi & Boumaïza 2009
Pse: 2L, 2R (compressed); Pekkarinen & Englund 1995;
Unio pictorum elongate nodulous
Lat: 2L, 1R Wächtler et al 2001
w-shaped,
Pse: 2L, 2R (compressed); Pekkarinen & Englund 1995;
Unio tumidus elongate double-looped,
Lat: 2L, 1R Wächtler et al 2001
nodulous, wrinkled
Pse: 2L, 1R;
Aculamprotula tortuosa ovate nodulous He & Zhuang 2013
Lat: 2L, 1R
Pse: 2L, 1R;
Cuneopsis heudei cuneiform pseudo-concentric He & Zhuang 2013
Lat: 2L, 1R
cuneiform Pse: 2L, 2R; He & Zhuang 2013;
Cuneopsis pisciculus nodulous
(twisted) Lat: 2L, 1R Wu et al 1999
cuneiform, Pse: 2L, 1R;
Cuneopsis rufescens nodulous He & Zhuang 2013
elongate Lat: 2L, 1R
w-shaped, He & Zhuang 2013;
elongate, Pse: 2L, 2R;
Nodularia douglasiae double-looped, Kondo 1987;
ovate Lat: 2L, 1R
wrinkled Wu et al 1999
w-shaped,
elongate, Pse: 2L, 2R;
Nodularia nuxpersicae double-looped, He & Zhuang 2013
ovate Lat: 2L, 1R
wrinkled
Schistodesmus lampreyanus triangular nodulous
Lat: 2L, 1R Shu et al 2012
RECTIDENTINAE various various various
CONTRADENTINI various various low number of thin teeth

Brandt 1974;
Pse: 1L, 1R (thin/long); Panha 1990;
Contradens contradens ovate V-shaped
Lat: 1L, 1R (thin) Panha & Eongprakornkeaw
1995
elongate, Pse: 2L, 1R (thin, lamellar); Đặng et al 1980;
Contradens semmelincki V-shaped
ovate Lat: 1R, 1L (thin) Simpson 1914
144 FCUP
Pse: 2L, 1R (thin, lamellar); Brandt 1974;

Physunio modelli ovate W-shaped
Lat: 1R, 2L (thin) Heard 1974
Brandt 1974;
Heard 1974;
Pse: 1L, 1R (thin), Panha & Eongprakornkeaw
Trapezoideus exolescens trapezoidal pseudo-concentric
Lat: 1R, 1L (thin) 1995;
Pfeiffer & Graf 2015;
Simpson 1914;
RECTIDENTINI various various various
Brandt 1974;
Heard 1977;
cuneiform, double-looped, Pse: 1/2L, 2R (thin/long);
Ensidens ingallsianus Panha 1990;
elongate nodulous Lat: 2L, 1R (thin/long)
Panha & Eongprakornkeaw
1995
Brandt 1974; Heard 1977
double-looped, Pse: 1/2L, 2R (thin/long); Panha 1990;
Ensidens sp. elongate
nodulous Lat: 2L, 1R (thin/long) Panha & Eongprakornkeaw
1995
Brandt 1974; Heard 1977
double-looped, Pse: 1/2L, 2R (thin/long); Panha 1990;
Ensidens sagittarius elongate
nodulous Lat: 2L, 1R (thin/long) Panha & Eongprakornkeaw
1995
Brandt 1974;
Chatchavalvanich et al 2006;
Pse: 2L, 2R (crenulate);
elongate, Chumnanpuen et al 2011;
Hyriopsis bialata pseudo-concentric Lat: 2L, 1R;
with wings Panha 1990;
interdentum sculptured
1995
Brandt 1974;
Duangsawang & Kovitvadhi
ovate, Pse: 2L, 2R (parallel ridges);
Hyriopsis desowitzi pseudo-concentric 2009; Duangsawang et al 2008;
with wings Lat: 2L, 1R (short/narrow)
1995
FCUP 145
Brandt 1974;
Ortmann 1916;
ovate, Pse: 1L, 1R;
Hyriopsis myersiana nodulous Panha 1990;
with wings Lat 2L, 1R
1995; Uthaiwan et al 2001
double-looped, Pse: 2L, 2R (thin); Heard 1977;
Rectidens sumatrensis elongate
pseudo-concentric Lat: 1L, 2R Thiele 1934
GONIDEINAE various various various
ovate,
CHAMBERLAINIINI posterior various various
wing
Brandt 1974;
Kovitvadhi & Kovitvadhi 2013;
ovate, Pse: 2L (short, thick), 1R;
Chamberlainia hainesiana nodulous Panha 1993;
with wings Lat: 2L (small), 1R (strong)
1995;
ovate, Pse: 2L, 2R; He & Zhuang 2013;
Sinohyriopsis cumingii pseudo-concentric
with wings Lat: 2L, 1R Wu et al 1999
LAMPROTULINI ovate various various, strong teeth
Lamprotula caveata ovate unknown
Lat: 2L, 1R Wu et al 1999
double-looped, Pse: 2L, 2R;
Lamprotula leai ovate He & Zhuang 2013
nodulous Lat: 2L, 1R
Cek & Sereflişan 2011;
w-shaped, Pse: 2L, 1R (thick);
Potomida littoralis ovate Şereflişan et al 2009;
double-looped Lat: 2L, 1R
Simpson 1914
w-shaped,
Pse: 2L, 1R; Kondo 1982, 2008;
Pronodularia japanensis ovate nodulous,
Lat: 2L, 1R Sayenko 2012
wrinkled
trapezoidal;
GONIDEINI various absent or vestigial teeth
rectangular
Heard 1974;
Gonidea angulata trapezoidal pseudo-concentric Ortmann 1916;
Lat: absent
O’Brien et al 2013
146 FCUP
double-looped, Pse: 1L, 1R (small);

Leguminaia wheatleyi trapezoidal Haas 1969
w-shaped, wrinkled Lat: traces
Pse: 1L, 1R (small); Haas 1969;
Microcondylaea bonellii trapezoidal double-looped
Lat: traces Nagel et al 2007
Pse: absent; He & Zhuang 2013;
Solenaia carinata rectangular unknown
Lat: traces Huang et al 2013
Pse: absent; He & Zhuang 2013;
Solenaia oleivora rectangular unknown
Lat: traces Wang et al 2015
absent or vestigial teeth
PSEUDODONTINI various double-looped
"v" shaped fossette
double-looped,
teeth absent Brandt 1974;
Pilsbryoconcha compressa elongate nodulous,
"v" shaped fossette Panha 1990
single-looped
Brandt 1974;
teeth absent Panha 1990;
Pilsbryoconcha exilis elongate double-looped
"v" shaped fossette Panha & Eongprakornkeaw
1995
Brandt 1974;
rounded, Pse: 1L, 1R (small); Panha 1990;
Pseudodon cambodjensis* unknown
triangular "v" shape fossette Panha & Eongprakornkeaw
1995
Pseudodon mouhotii elongate unknown Brandt 1974
"v" shape fossette
Brandt 1974;
ovate,
double-looped, Pse: 1L, 1R (small); Panha 1990;
Pseudodon cummingii posterior
single-looped "v" shape fossette Panha & Eongprakornkeaw
wing
1995
incertae sedis (GONIDEINAE)
Pse: absent;
Solenaia triangularis triangular unknown He & Zhuang 2013
Lat: traces
Pse: 2L, 1R;
AMBLEMINAE various various
Lat: 2L, 1R
Pse: 2L, 1R;
AMBLEMINI various various
Lat: 2L, 1R
FCUP 147
ovate, double-looped, Pse: 2L, 1R (heavy);

Amblema plicata Barnhart et al 2008
quadrate pseudo-concentric Lat: 2L, 1R
Pse: 2L, 1R;
LAMPSILINI various various
Lat: 2L, 1R
elliptical, Pse: 2L, 1R (large);
Actinonaias ligamentina double-looped Barnhart et al 2008
inflated Lat: 2L, 1R
ovate, Pse: 2L, 1R;
Lampsilis cardium double-looped Barnhart et al 2008
inflated Lat: 2L, 1R
elliptical,
Villosa iris elongate, double-looped Barnhart et al 2008
Lat: 2L, 1R
compressed
Pse: 2L, 1R;
PLEUROBEMINI various various
Lat: 2L, 1R
triagular, double-looped, Pse: 2L, 1R (heavy);
Pleurobema sintoxia William et al 2008
ovate nodulous Lat: 2L (short), 1R
trapezoidal, double-looped, Pse: 2L, 1R (triangular);
Elliptio complanata Barnhart et al 2008
compressed pseudo-concentric Lat: 2L, 1R (long)
double-looped, Pse: 2L, 1R (thick);
Elliptio dilatata elongate Barnhart et al 2008
pseudo-concentric Lat: 2L, 1R (thick)
Pse: 2L, 1R;
QUADRULINI rectangular various
Lat: 2L, 1R
w-shaped,
Pse: 2L (heavy), 1R;
Quadrula quadrula quadrate nodulous, Barnhart et al 2008
Lat: , 2L, 1R (long)
wrinkled
w-shaped,
double-looped, Pse: 2L (large), 1R; Kennedy & Haag 2005;
Quadrula verrucosa rectangular
nodulous, Lat: 2L (large, long), 1R William et al 2008
pseudo-concentric
148 FCUP
CHAPTER 5 Phylogeny, taxonomy and species of

the genus Quadrula
Paper IV
Revisiting the North American freshwater mussel genus Quadrula sensu
lato (Bivalvia Unionidae): Phylogeny, taxonomy and species delineation
Lopes-Lima M
Burlakova L, Karatayev A, Gomes-Dos-Santos A, Zieritz A, Froufe E, Bogan AE
Article published in Zoologica Scripta 48, 313-336 (2019).
DOI: 10.1111/zsc.12344
FCUP 149
Revisiting the North American freshwater mussel genus Quadrula sensu

lato (Bivalvia Unionidae): Phylogeny, taxonomy and species delineation
Manuel Lopes-Lima1,*, Lyubov Burlakova2 , Alexander Karatayev2, Andre Gomes-dos-Santos3,

Alexandra Zieritz4, Elsa Froufe3, Arthur E. Bogan5
1
CIBIO/InBIO - Research Center in Biodiversity and Genetic Resources, University of Porto, Vairão, Portugal
2
Great Lakes Center, Buffalo State College, Buffalo, New York
3
CIIMAR/CIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de
Cruzeiros do Porto de Leixões, Matosinhos, Portugal
4
School of Environmental and Geographical Sciences, University of Nottingham Malaysia Campus, Semenyih,
Malaysia
5
Research Laboratory, North Carolina Museum of Natural Sciences, Raleigh, North Carolina
⁎
Abstract
Freshwater mussels (Bivalvia, Unionidae) have suffered strong declines over the last century.
High morphological plasticity of Unionidae causes disturbances in their systematics and
taxonomy, hampering conservation efforts. Species that have historically been placed under
the North American genus Quadrula have suffered from numerous taxonomic and species
delineation problems since its inception. Four genera are presently recognized within Quadrula
sensu lato, that is, Cyclonaias, Quadrula, Theliderma, and Tritogonia, but their phylogenetic
basis remains incompletely tested. In the present study, we reconstructed several two-marker
(mtDNA cytochrome c oxidase subunit I - COI and NADH dehydrogenase subunit 1 - ND1)
phylogenies with newly collected specimens and all previously available sequences covering
most species within this group. We then delineated the species within the group using an
integrative approach with the application of molecular statistical methods, morphometric
(Fourier Shape) analyses and geographic distribution data. Four clades corresponding to these
genera were consistently recovered in all phylogenies. To validate the generic status of these
clades, molecular analyses were complemented with morphological, anatomical and
ecological data compiled from the literature. Several revisions are here proposed to the current
systematics and taxonomy of these genera, including the synonymization of Cyclonaias
asperata under Cyclonaias kieneriana; the inclusion of Quadrula apiculata and Quadrula
rumphiana under Quadrula quadrula; the placement of Quadrula nobilis under Tritogonia; and
finally the separation of the Mobile River basin populations of Theliderma metanevra as a new
species, that is, Theliderma johnsoni n. sp. The conservation implications of the proposed
changes are then discussed.
150 FCUP
Introduction
Conservation programs and strategies are largely based on species as conservation units,
making species delineation extremely important as a basic conservation tool (Prié et al 2012).
However, taxon-based conservation strategies dedicated to the freshwater mussel family
Unionidae, one of the world's most endangered taxa, are hindered by phylogenetic and
taxonomic uncertainties (Lopes-Lima et al 2017). This is especially true within the most
species-rich Unionidae subfamily, the North American Ambleminae. Across the most recent
systematics studies, the Ambleminae is divided into five tribes (Pfeiffer et al 2019). However,
polyphyly and inappropriate species boundaries have been revealed in some of these tribes,
including the Quadrulini (Lydeard et al 2000; Serb et al 2003; Pfeiffer et al 2016). The
quadruline freshwater mussels are distinctive animals producing thick quadrate shells, some
of which are heavily sculptured. Shell morphology is highly variable within some species from
this group, hindering unambiguous species identification or generic assignment. As shell
morphology has been the original basis for Quadrulini systematics and taxonomy to date, the
systematics and composition of this tribe have suffered a series of changes since its first
description in the early 1900s (see Supporting Information Appendix S1 for an extensive
taxonomic history of the Quadrulini). From the beginning of the 20th century, species that had
been historically placed within the genus Quadrula sensu lato have been divided into four main
species groups, that is, the Quadrula sensu stricto, the pustulosa, the metanevra and the
Tritogonia species groups (Supporting Information Appendix S1). A molecular phylogeny of
these taxa by Serb et al (2003) largely confirmed these groupings and recovered four clades:
Quadrula sensu stricto, the pustulosa species group, the metanevra species group and a fourth
clade including Tritogonia verrucosa and Quadrula nobilis. Although these four clades are
commonly referred to as genera in regional checklists (Parmalee & Bogan 1998; Williams et
al 2008; Howells 2013) the molecular, morphological and ecological evidence supporting these
groups remains limited.
The present study is focused on re-examining the phylogeny, systematics and
taxonomy of Quadrula sensu lato, here defined as including the species from the genera
Quadrula, Theliderma, Cyclonaias and Tritogonia (Williams et al 2017). In detail, this study
aims to: (a) estimate the phylogenetic relationships of specimens collected in Texas with all
published Quadrulini sequences, using a two-marker approach (COI and ND1); (b) perform a
comparative shell morphometry evaluation to complement the molecular results; (c) define
species boundaries with a taxonomic revision of all analysed taxa; (d) test the four classical
generic constructs and their evolutionary significance; and (e) describe the conservation
implications of the obtained results.
FCUP 151
Materials and Methods

Sample collection and materials examined
Specimens of quadruline mussels were collected from 50 sites across the state of Texas from
2003-2011 (Fig. 1). A total of 89 specimens were collected and placed in 99% ethanol for
molecular analyses. Voucher specimens were labelled and deposited in the SUNY Buffalo
State College Great Lakes Center collections, Buffalo, New York (BSGLC). The fieldwork was
carried out with an appropriate Scientific Research Permit SPR-0503-300 issued by the Texas
Parks and Wildlife Department. Additionally, dry shell specimens of the target nominal species
were selected for morphometry from specimens deposited at the North Carolina Museum of
Natural Sciences (NCMS) and BSGLC (See Supporting Information Table S1 for the examined
lot numbers).
Figure 1 Map of all sampling sites for the present study; both tissue and shell materials in red;
only shell materials in white
Sequencing, alignments and phylogenetic analyses

A total of 31 quadruline specimens, including all nominal taxa across the state of Texas, were
selected for molecular analyses (Table 1). For each sample, genomic DNA extraction (Froufe
et al 2014), amplification and bidirectional sequencing were carried out for the F-type mtDNA
cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes.
For COI, the primers LCO_22me and HCO_700dy (Walker et al 2006) were used with
an annealing temperature of 50°C and polymerase chain reaction (PCR) conditions as
described in Froufe et al (2014).
152 FCUP
Table 1.
List of newly sequenced specimens for Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) datasets; nominal taxa,
new identification, site, main basin, and COI and ND1 Haplotype number and Genbank references.
TAXON NEW ID RIVER BASIN GB (COI) GB (ND1)

Quadrula petrina Cyclonaias necki San Marcos S. Antonio/Guadalupe MG969422 MK503297
Quadrula petrina Cyclonaias petrina Concho Colorado MG969416 MK503293
Quadrula petrina Cyclonaias petrina San Saba Colorado MG969419 MK503311
Quadrula aurea Cyclonaias pustulosa San Marcos S. Antonio/Guadalupe MK503268 MK503296
Quadrula aurea Cyclonaias pustulosa San Antonio S. Antonio/Guadalupe MK503269 MK503298
Quadrula aurea Cyclonaias pustulosa San Antonio S. Antonio/Guadalupe - MK503301
Quadrula aurea Cyclonaias pustulosa Guadalupe S. Antonio/Guadalupe MK503272 MK503302
Quadrula aurea Cyclonaias pustulosa Nueces Nueces MK503281 MK503314
Quadrula aurea Cyclonaias pustulosa Nueces Nueces MK503282 MK503315
Quadrula houstonensis Cyclonaias pustulosa Colorado Colorado MK503273 MK503303
FCUP 153

Quadrula mortoni Cyclonaias pustulosa Sandy Creek Neches MK503276 MK503306
Quadrula mortoni Cyclonaias pustulosa Village Creek Neches MK503278 MK503308
Quadrula mortoni Cyclonaias pustulosa Trinity Trinity MK503286 MK503322
Quadrula nobilis Tritogonia nobilis Neches Neches MK503279 MK503309
Quadrula nobilis Tritogonia nobilis Neches Neches MK503280 MK503310
Quadrula nobilis Tritogonia nobilis Trinity Trinity MK503285 MK503321
Quadrula quadrula Quadrula quadrula Ohio Ohio MK503267 MK503291
Theliderma johnsoni Theliderma metanevra Alabama Alabama MK503289 -
Pleurobema riddellii Pleurobema riddellii Village Creek Neches MK503277 MK503307
Pleurobema oviforme Pleurobema oviforme Little Tennessee Mississippi - MK503292
Anodonta nuttalliana Anodonta nuttalliana John Day Columbia MK503266 MK503290
154 FCUP
ND1 was amplified using the PCR conditions and primers (Leu-uurF and LoGlyR) of Serb et
al (2003). Sequences were obtained with the BigDye sequencing protocol (Applied Biosystems
3730xl) by Macrogen Inc., Korea. Forward and reverse sequences were edited and assembled
using ChromasPro 1.7.4 (Technelysium, Tewantin, Australia). All new sequences have been
deposited in GenBank (Table 1 and Supporting Information Tables S2 and S3). Three datasets
were constructed as follows: one for COI, another for ND1 and a third concatenating COI and
ND1. The COI and ND1 datasets included all newly sequenced individuals and all Quadrulini
sequences available in the GenBank database for each gene (Supporting Information Tables
S2-S4). The COI + ND1 dataset included all individuals sequenced for both COI and ND1 plus
GenBank Quadrulini specimens with sequences available for the two genes (Supporting
Information Table S4). For each of the three datasets, sequences of additional specimens were
downloaded from Genbank and/or newly sequenced as outgroup (details in Supporting
Information Tables S2-S4). The three datasets were aligned with the MAFFT multiple
sequence alignment algorithm (Katoh & Standley 2013). Each gene alignment was then
restricted to its unique haplotypes, retrieved using DnaSP v5.1.0.1 (Librado & Rozas 2009).
Phylogenetic analyses were then performed on the three datasets using Bayesian
inference (BI) and maximum likelihood (ML). For the BI analyses, the best-fit models of
nucleotide substitution were selected using JModelTest 2.1.10 (Darriba et al 2012) under the
Bayesian information criterion. For each gene dataset, a three partition scheme was applied,
one per gene codon, with the following selected models: COI (GTR + I + G, HKY, HKY + G),
and ND1 (HKY + G, HKY + G, GTR + I + G). For the COI + ND1 dataset, a six-partition scheme
was applied for the three codons of both COI and ND1 with the same models selected for the
individual gene datasets. BI analyses were performed in MrBayes v3.2.6 (Ronquist et al 2012)
implemented in CIPRES Science Gateway (Miller et al 2010). BI analyses were initiated with
program-generated trees and four Markov chains with default incremental heating. Two
independent runs of 30 × 10 6 generations were sampled at intervals of 1,000 generations
producing a total of 30,000 trees. Burn-in was determined upon the convergence of log-
likelihood and parameter values using Tracer 1.6 (Rambaut et al 2014).
For the ML analyses, the same partitioning scheme was applied for each dataset with
the same model (GTR + G) for all partitions, and sequences were then analysed in RaxML
8.2.10 HPC Black Box (Stamatakis 2014) with 1,000 bootstrap replicates. Haplotype networks
were calculated using TCS 1.21 (Clement et al 2000) with a threshold of 95%.
Molecular based species delineation methods

Five distinct molecular methods were applied to determine the number of molecular
operational taxonomic units (MOTUs). All methods were applied to the COI, ND1 and
concatenated (COI + ND1) datasets, except for the BIN system that relies only on COI. The
FCUP 155
first two are distance-based, that is, the BIN system implemented in BOLD (Ratnasingham &
Hebert 2013) and the Automatic Barcode Gap Discovery (ABGD) (Puillandre et al 2012). For
the BINs system, the COI dataset without the outgroups was analysed with the Cluster
Sequences tool implemented in BOLD 4 (http://v4.boldsystems.org) (Ratnasingham & Hebert
2013). The ABGD species delineation tool was applied to all three datasets without outgroup
using its online version (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html) with the
default settings and the Kimura-2-parameter distance matrix (Puillandre et al 2012).
Two tree-based molecular species delineation methods were applied to all datasets,
that is, the single threshold Generalized Mixed Yule Coalescent (GMYC) model (Fujisawa &
Barraclough 2013) and the Bayesian implementation of the Poisson Tree Processes model
(bPTP) (Zhang et al 2013). For the GMYC method, a Bayesian ultrametric phylogenetic tree
was first generated in BEAST 2.4.6 (Bouckaert et al 2014) with the previously selected models
for each partition and four independent runs of 20 × 10 6 Markov chain Monte Carlo (MCMC)
generations, sampled every 1 × 10 3 generations. Convergence of the parameters was
evaluated using Tracer 1.6 software (Rambaut et al 2014). The consensus tree was annotated
using TreeAnnotator 2.4.6 (Bouckaert et al 2014). The consensus tree was loaded into the R
software package “Species Limits by Threshold Statistics” (Ezard et al 2009) in R 3.2.0 (R
Core Group available at http://www.r-project.org) and analysed using the single threshold
model. For the bPTP, the BI phylogenetic trees previously obtained were used as input trees
in the bPTP webserver (http://species.h-its.org) with 1 × 106 iterations of MCMC and 20% burn-
in. Finally, a 95% statistical parsimony connection limit was used, by using TCS 1.21 (Clement
et al 2000). Sequence divergences (uncorrected p-distance) were assessed using MEGA 7
(Kumar et al 2016).
Morphometry
For a detailed analysis of inter- and intraspecific variation in shell shape within the quadruline
genera Cyclonaias, Quadrula, and Theliderma, we used Fourier Shape Analysis, as developed
and explained by Crampton & Haines (1996). This method decomposes xy-coordinates of a
shell outline into several harmonics, each of which is in turn explained by two Fourier
coefficients. Xy-coordinates of the sagittal shell outline of 1,222 specimens from BSGLC and
NCMS collections (739 specimens of Cyclonaias spp., 254 specimens of Quadrula spp. and
229 specimens of Theliderma spp.; Supporting Information Table S1) were obtained from
digital photographs using the program IMAGEJ (Rasband 2008) and subjected to fast Fourier
transformation using the program HANGLE, applying a smoothing normalization of 3 to
eliminate high-frequency pixel noise. A preliminary analysis indicated that the first 10
harmonics described the outlines with sufficiently high precision. Discarding of the first
harmonic, which does not contain any shape information, resulted in a set of 18 Fourier
156 FCUP
coefficients per individual. Outlines of all specimens within each of the three genera were then
rotated to maximum overlap by program HTREE, resulting in the final set of 18 Fourier
coefficients per individual.
For visual examination of variation in shell shape within and between true and nominal
species, principal component analysis was performed on the 18 Fourier coefficients of (a) all
true species (recognized by the molecular species delineation methods, see results) of
Cyclonaias, including a maximum of 50 specimens per species; (b) all nominal species of
Cyclonaias pustulosa; (c) only Cyclonaias kieneriana and Cyclonaias kleiniana; (d) all nominal
species of Quadrula; (e) all true species (recognized by the molecular species delineation
methods, see results) of Theliderma; and (f) only Theliderma metanevra and Theliderma
johnsoni n. sp. (see Supporting Information Appendix S2 for a detailed description of T.
johnsoni n. sp.). Synthetic outlines of extreme and average shell shapes were drawn using
program HCURVE as explained in Crampton & Haines (1996).
We assessed the rate of accurate identification of true and nominal species based on
shell shape using linear discriminant analysis (LDA) on the 18 Fourier coefficients. To test for
statistically significant differences in sagittal shell shape between species, multivariate
analyses of variance (MANOVA) were run on the 18 Fourier coefficients. Pairwise Hotelling's
post hoc tests were performed to identify significant differences between each pair of
true/nominal species. Statistical analyses were performed in PAST v.3 (Hammer & Harper
2006).
Ecological, morphological and anatomical traits

An extensive bibliographic review of selected ecological, morphological and anatomical traits
was accomplished for all species within Quadrula s.l. (Table 2; Supporting Information Table
S5).
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TABLE 2
List of morphological, anatomical and behavioural characters of Cyclonaias, Quadrula, Theliderma, and Tritogonia as recognized in the present
study
Sexual dimorphism Shell Sulcus Periostracal chevrons Posterior ridge GLN

Cyclonaias NO NO NO low rounded 0.05-0.09
Quadrula NO YES NO well developed 0.005-0.009
low rounded to
Theliderma NO NO2 YES 0.03-0.04
prominent
Tritogonia YES YES NO well developed 0.009
Mantle displays (magazines)
Location Reflexive release Hosts
Morphology Size
(apertures)
Ictaluridae (71%) Centrarchidae (24%)
Cyclonaias stomate-shaped Small Excurrent YES
Acipenseridae (5%)
Quadrula conical (knob-like)1 Large1 Excurrent1 NO* Ictaluridae (67%) Centrarchidae (33%)
Cyprinidae (72%) Centrarchidae (14%)
Theliderma variable shape Small Excurrent YES
Percidae (14%)
Tritogonia slug-shaped* Large* Both* NO* Ictaluridae
a b
Notes. GLN: mean glochidial size index. Only analysed in one species. For most species.
158 FCUP
Results
Alignments and phylogenetic analyses
The COI dataset spanned 582 nucleotides (nt) and included 289 unique haplotypes (232
polymorphic and 192 parsimony informative sites). The ND1 dataset covered 619 bp with 339
unique haplotypes (297 polymorphic and 257 parsimony informative sites). Finally, the
combined COI + ND1 dataset was 1,192 nt long and included 325 individual sequences (501
polymorphic and 427 parsimony informative sites). No insertions or deletions and no stop
codons were observed in any of the datasets after translating all sequences to amino acids.
The results of the BI and ML phylogenetic analyses for the three datasets presented
similar topologies (Table 3), thus only BI phylogenetic trees are shown in Figs. 2-4. In the COI
phylogeny, the Quadrulini clade is monophyletic and well supported in the BI analyses. Within
the Quadrulini clade, the Megalonaias + Uniomerus clade is sister to a clade including three
well-supported subclades corresponding to the genera Quadrula, Tritogonia, and Theliderma,
and a clade including all Cyclonaias sequences (Fig. 2).
TABLE 3
Results of repeatability clade analysis (RCA) of main clades corresponding to the preferred
topology
Clades Analyses COI + ND1 COI ND1
BI 100 100
Quadrulini
ML 74 55
BI 100 100 100
Quadrula sensu lato
ML 98 93 90
BI 100 95 98
Cyclonaias
ML 83 35 68
BI 100 100 100
Quadrula s.s.
ML 100 99 99
BI 100 100 89
Theliderma
ML 100 99 72
BI 100 100 100
Tritogonia
ML 100 98 87
BI 65 97
C. infucata + C. kleiniana + C. kieneriana
ML 55 37
BI 99 99 100
C. petrina + C. nodulata + C. necki
ML 84 51 96
BI 100 100 89
C. pustulosa group
ML 99 64 45
Notes. In bold values higher than 95% (Bayesian Inference) and 70% (Maximum Likelihood).
The ND1 phylogeny recovered similar phylogenetic patterns to that obtained with COI.
However, in these analyses, the Quadrulini is not monophyletic, with the remaining
Ambleminae tribe clades, that is, Amblemini, Pleurobemini and Lampsilini clustering within the
Quadrulini tribe clade (Fig. 3).
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Figure 2 Bayesian consensus tree inferred from the cytochrome c oxidase subunit I (COI) gene fragment. The values above and below the nodes
indicate Bayesian posterior probability (bpp) percentage and maximum likelihood bootstrap values (bs), respectively. Values o ver 95% are
represented by an asterisk, and those <50% are not shown for clarity
160 FCUP
Figure 3 Bayesian consensus tree inferred from the NADH dehydrogenase subunit 1 (ND1) gene fragment. The values above and below the
nodes indicate Bayesian posterior probability (bpp) percentage and maximum likelihood bootstrap values (bs), respectively. Values over 95% are
represented by an asterisk, values below 50% are not shown for clarity
FCUP 161
Figure 4 Bayesian consensus tree inferred from the NADH dehydrogenase subunit 1 (ND1) and cytochrome c oxidase I (COI) gene fragments
concatenated dataset. The values above and below the nodes indicate Bayesian posterior probability (bpp) percentage and maximum likelihood
bootstrap values (bs), respectively. Values over 95% are represented by an asterisk, values below 50% are not shown for clarity
162 FCUP
The Uniomerus clade is sister to a clade containing the four remaining Quadrulini genera (i.e.
Quadrula, Tritogonia, Theliderma, and Cyclonaias; Fig. 3). While Cyclonaias, Quadrula and
Tritogonia are well supported, Theliderma has a low support value (Fig. 3). The COI + ND1
phylogeny shows Quadrulini as monophyletic with Uniomerus being sister to a clade
comprising four well-supported clades (Quadrula, Tritogonia, Theliderma, and Cyclonaias; Fig.
4).
Cyclonaias
Within Cyclonaias, the clade labelled C. pustulosa includes specimens originally identified as
Cyclonaias aurea, Cyclonaias houstonensis, Cyclonaias mortoni, C. pustulosa, and
Cyclonaias refulgens.
Quadrula
All sequences from the nominal species Quadrula quadrula, Quadrula apiculata, and Quadrula
rumphiana cluster within the Q. quadrula clade in all phylogenies (Figs. 2-4). However, both
nominal species Q. apiculata and Q. rumphiana were found to be nested within Q. quadrula
(Figs. 2-4). Both the COI and ND1 95% threshold haplotype networks of the Q. quadrula clade
reveal a low number of mutations among the nominal taxa Q. quadrula, Q. apiculata and Q.
rumphiana (Figs. 5a,b).
Theliderma
Not many COI sequences of Theliderma are represented in the COI dataset, and therefore in
the COI and COI + ND1 phylogenies (Figs. 2 and 4). Nevertheless, in these phylogenies two
distinct clades were obtained in sequences from specimens of T. metanevra: one
corresponding to specimens from the Tennessee basin, and the other with specimens from
the Mobile basin (Figs. 2 and 4). The ND1 phylogeny is better represented with all species
recognized to date except for Theliderma stapes (Fig. 3).
Tritogonia
The sequences of specimens originally identified as Q. nobilis cluster together with those from
T. verrucosa in all phylogenies forming a well-supported clade here assigned to Tritogonia
(Figs. 2-4).
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Figure 5 Haplotype (TCS) networks and uncollapsed Quadrula clade from Figures 2 and 3,
showing the relationships of nominal species within the Quadrula quadrula group for (a)
cytochrome c oxidase I (COI) and (b) NADH dehydrogenase subunit 1 (ND1)
Genetic divergence and species delineation methods
Cyclonaias
Pairwise uncorrected p-distance values among six of the nominal Cyclonaias species, C.
pustulosa, C. aurea, C. houstonensis, C. mortoni, and C. refulgens were low (≤2% for both
COI and ND1: Table 4).
Of the 14 putative Cyclonaias species, only nine were recognized as MOTUs based on
a consensus of all species delineation methods, applied on the COI, ND1, and COI + ND1
datasets (Table 5). The pairwise uncorrected p-distance between these recognized Cyclonaias
MOTUs varied between 2.8% (COI)/3.1% (ND1) and 11.2% (COI)/10.2% (ND1; Table 6). The
uncorrected p-distance within each of the recognized MOTUs was ≤1.2% for COI and ≤1.6%
for ND1 (Table 6).
164 FCUP
Table 4
Pairwise genetic distance matrixes of nominal quadruline species of the genera Cyclonaias, Quadrula, Theliderma, and Tritogonia, using the
original nominal taxa
Within Groups Between Groups
C. houstonensis
C. kieneriana
C. pustulosa
C. kleiniana
C. nodulata
C. asperata
C. infucata
C. mortoni
C. petrina
COI ND1
C. aurea
C. necki
C. asperata 0.012 0.012 0.012 0.082 0.094 0.093 0.102 0.094 0.082 0.082 0.078 0.086
C. kieneriana ---- ---- 0.081 0.094 0.089 0.101 0.093 0.081 0.082 0.077 0.085
C. kleiniana 0.012 0.011 0.080 ---- 0.035 0.099 0.094 0.099 0.083 0.085 0.083 0.090
C. infucata 0.006 0.007 0.082 ---- 0.032 0.097 0.092 0.097 0.087 0.090 0.085 0.092
C. nodulata 0.006 0.009 0.077 ---- 0.088 0.083 0.038 0.040 0.063 0.063 0.064 0.062
C. petrina 0.007 0.006 0.076 ---- 0.095 0.090 0.028 0.047 0.063 0.062 0.064 0.061
C. necki 0.007 0.007 0.077 ---- 0.094 0.084 0.041 0.039 0.064 0.067 0.066 0.066
C. pustulosa 0.010 0.011 0.076 ---- 0.092 0.085 0.052 0.053 0.051 0.017 0.012 0.019
C. aurea 0.011 0.012 0.078 ---- 0.092 0.083 0.050 0.051 0.051 0.014 0.018 0.020
C. houstonensis 0.007 0.008 0.075 ---- 0.088 0.081 0.058 0.059 0.055 0.014 0.017 0.020
C. mortoni 0.013 0.012 0.075 ---- 0.086 0.079 0.052 0.054 0.055 0.020 0.019 0.020
C. refulgens 0.015 0.010 0.074 ---- 0.091 0.084 0.052 0.052 0.052 0.014 0.014 0.017 0.020
C. succissa 0.011 0.011 0.081 ---- 0.094 0.085 0.048 0.044 0.054 0.036 0.033 0.041 0.037
C. tuberculata 0.006 0.006 0.078 ---- 0.088 0.090 0.050 0.056 0.062 0.058 0.056 0.064 0.055
Q. quadrula 0.014 0.012 0.112 ---- 0.110 0.103 0.096 0.097 0.098 0.108 0.104 0.112 0.109
Q. apiculata ---- 0.018 0.105 ---- 0.096 0.096 0.093 0.089 0.095 0.100 0.099 0.103 0.100
Q. rumphiana ---- 0.010 0.105 ---- 0.099 0.095 0.093 0.089 0.095 0.097 0.092 0.100 0.097
T. cylindrica ---- 0.010 ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ----
T. intermedia ---- 0.003 ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ----
T. metanevra 0.017 0.021 0.091 ---- 0.092 0.096 0.094 0.093 0.090 0.084 0.087 0.086 0.088
T. sparsa ---- 0.002 ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ----
T. verrucosa 0.007 0.008 0.096 ---- 0.105 0.093 0.102 0.104 0.098 0.105 0.107 0.104 0.105
T. nobilis 0.009 0.011 0.105 ---- 0.118 0.107 0.108 0.101 0.106 0.102 0.102 0.102 0.106
FCUP 165
Table 4 (cont.)
Between Groups
C. tuberculata
Q. rumphiana
T. intermedia
T. metanevra
T. verrucosa
T. cylindrica
C. refulgens
Q. apiculata
Q. quadrula
C. succissa
T. nobilis
T. sparsa
C. asperata 0.080 0.083 0.094 0.101 0.107 0.114 0.112 0.143 0.111 0.105 0.114 0.115
C. kieneriana 0.079 0.083 0.096 0.101 0.109 0.111 0.116 0.143 0.111 0.106 0.111 0.114
C. kleiniana 0.084 0.092 0.088 0.109 0.116 0.121 0.110 0.143 0.117 0.105 0.112 0.123
C. infucata 0.088 0.095 0.093 0.108 0.110 0.117 0.115 0.139 0.116 0.110 0.107 0.125
C. nodulata 0.059 0.064 0.055 0.123 0.129 0.134 0.129 0.144 0.121 0.113 0.118 0.126
C. petrina 0.058 0.064 0.065 0.127 0.131 0.136 0.125 0.140 0.121 0.110 0.122 0.130
C. necki 0.062 0.070 0.059 0.127 0.131 0.134 0.127 0.147 0.126 0.115 0.116 0.126
C. pustulosa 0.012 0.033 0.054 0.108 0.112 0.116 0.121 0.136 0.115 0.106 0.105 0.119
C. aurea 0.014 0.031 0.051 0.107 0.111 0.115 0.118 0.136 0.119 0.107 0.106 0.118
C. houstonensis 0.013 0.029 0.052 0.103 0.107 0.111 0.116 0.134 0.114 0.105 0.101 0.118
C. mortoni 0.017 0.030 0.050 0.111 0.115 0.119 0.126 0.137 0.118 0.107 0.106 0.118
C. refulgens 0.027 0.049 0.108 0.113 0.116 0.120 0.137 0.116 0.106 0.104 0.116
C. succissa 0.035 0.053 0.109 0.113 0.122 0.124 0.144 0.126 0.113 0.110 0.119
C. tuberculata 0.058 0.053 0.115 0.117 0.120 0.127 0.146 0.126 0.113 0.116 0.121
Q. quadrula 0.108 0.100 0.098 0.017 0.027 0.104 0.139 0.116 0.108 0.109 0.105
Q. apiculata 0.100 0.092 0.085 0.034 0.020 0.109 0.143 0.117 0.112 0.111 0.107
Q. rumphiana 0.097 0.088 0.084 0.034 0.015 0.112 0.145 0.119 0.117 0.110 0.116
T. cylindrica ---- ---- ---- ---- ---- ---- 0.106 0.086 0.079 0.122 0.126
T. intermedia ---- ---- ---- ---- ---- ---- ---- 0.081 0.073 0.135 0.137
T. metanevra 0.088 0.095 0.083 0.101 0.090 0.096 0.040 0.115 0.126
T. sparsa ---- ---- ---- ---- ---- ---- ---- ---- ---- 0.105 0.106
T. verrucosa 0.106 0.100 0.098 0.114 0.116 ---- ---- 0.116 ---- 0.096 0.093
T. nobilis 0.102 0.099 0.114 0.110 0.116 ---- ---- 0.114 ---- 0.114 0.085
166 FCUP
Table 5
Results of molecular species delineation methods
COI ND1 COI + ND1
CONSENSUS
MOTUs
GMYC
GMYC
GMYC
ABGD
ABGD
ABGD
BOLD
(95%)
bPTP
bPTP
bPTP
TCS
TCS
TCS
TAXA
Cyclonaias
C. asperata ✓ ✓ ✓ ✓ ✓     ✓ ✓ ✓ ✓ 
C. kieneriana - - - - - ✓ ✓ ✓ ✓ - - - - ✓
C. infucata ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
C. kleiniana ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
C. nodulata ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
C. petrina ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
C. necki ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
C. pustulosa ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
C. aurea              
C. houstonensis             ✓ 
C. mortoni     ✓    ✓ ✓ ✓  ✓ 
C. refulgens              
C. succissa ✓ ✓ ✓ ✓ ✓  ✓  ✓ ✓ ✓ ✓ ✓ ✓
C. tuberculata ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Quadrula ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Q. quadrula 1 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Q. quadrula 2    ✓       ✓  ✓ 
Q. quadrula 3 ✓ ✓  ✓ ✓         
Q. apiculata ✓ ✓ ✓ ✓ ✓        ✓ 
Q. rumphiana  ✓    ✓ ✓      ✓ 
Theliderma
T. cylindrica - - - - - ✓ ✓ ✓ ✓ - - - - ✓
T. intermedia - - - - - ✓ ✓ ✓ ✓ - - - - ✓
T. metanevra ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
T. johnsoni n. sp. ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
T. sparsa - - - - - ✓ ✓ ✓ ✓ - - - - ✓
Tritogonia ✓ ✓ ✓ ✓
T. verrucosa ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
T. nobilis ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Notes. ✓: recognized as a molecular operational taxonomic unit (MOTU); : not recognized as a MOTU; -: not
analysed.
FCUP 167
Table 6
Pairwise genetic distance matrixes of quadruline species of the genera Cyclonaias, Quadrula,
Theliderma, and Tritogonia, as recognized in the present study
Among groups Between groups
C. kieneriana
C. pustulosa
C. kleiniana
C. nodulata
C. infucata
C. petrina
TAXA COI ND1
C. necki
C. kieneriana 0.012 0.012 0.094 0.082 0.093 0.102 0.094 0.082
C. infucata 0.006 0.007 0.082 0.035 0.097 0.092 0.097 0.089
C. kleiniana 0.012 0.011 0.080 0.032 0.099 0.094 0.099 0.085
C. nodulata 0.006 0.009 0.077 0.083 0.088 0.038 0.04 0.063
C. petrina 0.007 0.006 0.076 0.090 0.095 0.028 0.047 0.062
C. necki 0.007 0.007 0.077 0.084 0.094 0.041 0.039 0.065
C. pustulosa 0.016 0.016 0.076 0.082 0.089 0.052 0.053 0.052
C. succissa 0.011 0.011 0.081 0.085 0.094 0.048 0.044 0.054 0.036
C. tuberculata 0.006 0.006 0.078 0.090 0.088 0.050 0.056 0.062 0.057
Q. quadrula 0.017 0.019 0.112 0.103 0.109 0.096 0.096 0.098 0.107
T. cylindrica ---- 0.010 ---- ---- ---- ---- ---- ---- ----
T. intermedia ---- 0.003 ---- ---- ---- ---- ---- ---- ----
T. metanevra 0.009 0.005 0.090 0.095 0.091 0.093 0.094 0.090 0.086
T. johnsoni ---- 0.002 0.093 0.099 0.096 0.095 0.092 0.088 0.088
T. sparsa ---- 0.002 ---- ---- ---- ---- ---- ---- ----
T. verrucosa 0.007 0.008 0.096 0.093 0.105 0.102 0.104 0.098 0.105
T. nobilis 0.009 0.011 0.105 0.107 0.118 0.108 0.101 0.106 0.103
Between groups
C. tuberculata
T. metanevra
T. intermedia
T. verrucosa
T. cylindrica
Q. quadrula
C. succissa
T. johnsoni
T. sparsa
T. nobilis
TAXA
C. kieneriana 0.083 0.095 0.107 0.112 0.143 0.111 0.111 0.105 0.114 0.115
C. infucata 0.095 0.093 0.112 0.115 0.139 0.117 0.115 0.110 0.107 0.125
C. kleiniana 0.092 0.088 0.115 0.110 0.143 0.118 0.116 0.105 0.112 0.123
C. nodulata 0.064 0.055 0.128 0.129 0.144 0.123 0.117 0.113 0.118 0.126
C. petrina 0.064 0.065 0.131 0.125 0.14 0.125 0.114 0.110 0.122 0.130
C. necki 0.07 0.059 0.13 0.127 0.147 0.129 0.12 0.115 0.116 0.126
C. pustulosa 0.031 0.052 0.112 0.121 0.136 0.119 0.112 0.106 0.105 0.118
C. succissa 0.053 0.114 0.124 0.144 0.129 0.118 0.113 0.11 0.119
C. tuberculata 0.053 0.117 0.127 0.146 0.126 0.126 0.113 0.116 0.121
Q. quadrula 0.100 0.097 0.108 0.141 0.122 0.109 0.112 0.110 0.110
T. cylindrica ---- ---- ---- 0.106 0.088 0.082 0.079 0.122 0.126
T. intermedia ---- ---- ---- ---- 0.084 0.076 0.073 0.135 0.137
T. metanevra 0.096 0.083 0.102 ---- ---- 0.035 0.042 0.117 0.129
T. johnsoni 0.094 0.085 0.094 ---- ---- 0.032 0.036 0.109 0.121
T. sparsa ---- ---- ---- ---- ---- ---- ---- 0.105 0.106
T. verrucosa 0.100 0.098 0.114 ---- ---- 0.096 0.097 ---- 0.093
T. nobilis 0.099 0.114 0.110 ---- ---- 0.115 0.107 ---- 0.085
168 FCUP
Quadrula
The pairwise uncorrected p-distance among all nominal Quadrula species varied from 1.4%
(COI)/1.7% (ND1) to 3.4% (COI)/2.7% (ND1; Table 4). Considering the three datasets, only a
single MOTU was consensually recognized for the Quadrula genus (Table 5) with a within the
p-distance value of 1.7% for COI and 1.9% for ND1 (Table 6).
Theliderma
The pairwise uncorrected p-distance among all the nominal Theliderma species ranged
between 4.0% and 10.6% for ND1(Table 4). The higher within p-distance recorded value was
reached for T. metanevra, 1.7% for COI and 2.1% for ND1 (Table 4).
All originally described Theliderma species are here recognized as MOTUs with T.
metanevra being further divided into two distinct MOTUs, that is, T. metanevra for specimens
from the Tennessee River basin and T. johnsoni n. sp. from the Mobile River basin (Table 5).
The p-distance values among the recognized Theliderma MOTUs varied between 3.5% and
10.1% for ND1 (Table 6). The p-distance within each of the recognized MOTUs was ≤0.9% for
ND1 (Table 6).
Tritogonia
Our analyses revealed a complete consensus of two individual MOTUs within the Tritogonia
genus (Table 5). The two recognized MOTUs T. verrucosa and Tritogonia nobilis exhibited
high interspecific p-distance divergence, 8.5% (COI)/9.3% (ND1), and low intraspecific p-
distance <0.9% for COI and <1.1% ND1 (Table 6).
Morphometry
Cyclonaias
Linear discriminant analysis on the 18 Fourier coefficients extracted through Fourier Shape
Analysis for all Cyclonaias species recognized in this study assigned 75% of individuals to the
correct species (Fig. 6a). Species that are particularly difficult to separate by shell shape are
C. kieneriana and C. pustulosa (16% misidentifications), and Cyclonaias infucata and C.
kleiniana (10%). Also, most true species differed significantly from each other in their shell
shape as approximated by 18 Fourier coefficients, except for C. infucata and C. kleiniana
(MANOVA, pairwise Hotelling's test p = 0.742), and C. infucata and Cyclonaias necki
(MANOVA, pairwise Hotelling's test p = 0.138).
The proportion of C. pustulosa specimens correctly identified to the original nominal
species within the C. pustulosa complex exceeded that of Cyclonaias specimens correctly
identified to species level (see above), with 80% correct identifications (Fig. 6b). All nominal
FCUP 169
species differed significantly from each other in their shell shape as approximated by 18 Fourier
coefficients (MANOVA, pairwise Hotelling's tests p < 0.05).
Using only the nominal species C. kieneriana and Cyclonaias asperata, the Fourier
coefficients differed significantly between C. kieneriana and its synonym C. asperata
(MANOVA: F18,82 = 2.094, p = 0.013), and 95% of specimens were classified correctly based
on shell shape through LDA (Fig. 6c).
Quadrula
Fourier coefficients differed significantly between the nominal species of Quadrula (MANOVA,
pairwise Hotelling's tests p < 0.05; Fig. 6c). Seventy-six percent of specimens were assigned
to the correct nominal species, with 21% and 11% of misidentifications between Q. apiculata
versus Q. quadrula and Q. rumphiana, respectively.
Theliderma
Within the genus Theliderma, 91% of specimens were identified to the correct species (as they
are here recognized) by LDA of Fourier coefficients (Fig. 6e). Theliderma cylindrica,
characterized by its typical elongated-rectangular shape, was 100% correctly identified.
Considerable difficulties in separation by shell shape were present for Theliderma sparsa
versus T. johnsoni n. sp. (21% misidentifications) and T. metanevra (13%), respectively. Most
true Theliderma species pairs differed significantly from each other in their shell shape except
for T. sparsa versus T. johnsoni n. sp. (MANOVA, pairwise Hotelling's test: p = 0.525), T.
sparsa versus T. metanevra (p = 0.227) and T. stapes and T. johnsoni n. sp. (p = 0.427; p-
value could not be computed for the pair T. sparsa vs. T. stapes due to low replicate number).
When including the whole Theliderma dataset in LDA, only 5% of specimens of the pair
T. metanevra/T. johnsoni n. sp. were assigned to the wrong clade (Fig. 6e). When using only
the T. metanevra dataset, 11% of specimens were misidentified (Fig. 6f), though Fourier
coefficients were significantly different between the two species (MANOVA: F18,46 = 3.097, p =
0.001).
Diagnostic characters of the classical genera within Quadrula s.l

Species within Quadrula and Tritogonia share several ecological and morphological traits but
are distinct from those within Cyclonaias and Theliderma (Table 2; Supporting Information
Table S5). Quadrula and Tritogonia species exhibit a marked sulcus that is absent in
Cyclonaias and Theliderma, except for T. sparsa and T. stapes that may display shallow sulci
(Table 2; Supporting Information Table S5).
170 FCUP
FCUP 171
Figure 6 Shell outline principal component scores for the first two PC axes obtained from 18
Fourier coefficients of (a) all true species (recognized by molecular species delineation
methods; see results) of Cyclonaias, including a maximum of 50 specimens per species; (b)
all nominal species of Cyclonaias pustulosa; (c) only Cyclonaias kieneriana and Cyclonaias
asperata; (d) all nominal species of Quadrula; (e) all true species (recognized by molecular
species delineation methods; see results) of Theliderma; and (f) only Theliderma metanevra
and Theliderma johnsoni n. sp. Synthetic shell outlines of “extreme” morphotypes are
displayed with the anterior margin facing to the left and the dorsal margin to the top of the page
Quadrula and Tritogonia glochidial size index are ten times smaller than in Cyclonaias and
Theliderma (Table 2; Supporting Information Table S5). Quadrula and Tritogonia also seem to
share similar morphological and behavioural patterns of the mantle displays, also known as
mantle magazines. While Quadrula and Tritogonia seem to exhibit large mantle displays with
a non-reflexive glochidia release strategy when disturbed, Cyclonaias and Theliderma mantle
displays are small and more inconspicuous and immediately expel their glochidial content
when physically disturbed (Table 2; Supporting Information Table S5). However, some caution
must be taken when interpreting this data since mantle displays were only studied in a small
number of species.
Within Quadrula s.l. some of the analysed characters are exclusive and can be used to
recognize some of the classically recognized genera Cyclonaias, Quadrula, Theliderma, and
Tritogonia (Table 2; Supporting Information Table S5).
The presence of dark chevrons imprinted in the periostracum of shells is a trait that is
exclusive of Theliderma species and can be used to recognize the genus within Quadrulini
(Table 2; Supporting Information Table S5).
The stomate-shaped morphology of the mantle displays seems to be a diagnostic
character for Cyclonaias, but laboratory studies on C. asperata (= C. kieneriana) did not
observe any mantle display for this species (Haag & Staton 2003).
Theliderma hosts are mainly composed of small cyprinids while catfishes are the main
hosts of the other three Quadrula s.l. genera (Table 2; Supporting Information Table S5). The
mantle displays and glochidia of Theliderma are smaller than those of Cyclonaias (Table 2;
Supporting Information Table S5).
Tritogonia verrucosa and T. nobilis are sexually dimorphic in shell shape, a trait that is
unique within the Quadrulini and therefore diagnostic of the genus (Table 2; Supporting
Information Table S5). Also, the mantle display mechanism of T. verrucosa, which involves the
mantle to completely cover both the incurrent and excurrent aperture, is very distinct from
those of all the other Quadrula s.l. species (Supporting Information Table S5). However, this
trait needs to be verified for T. nobilis to be considered diagnostic of the genus.
172 FCUP
Discussion
Phylogenetic relationships within Quadrula and generic support
The three BI and ML phylogenies (COI, ND1, and COI + ND1) obtained in the present study
revealed a well-supported Quadrula sensu lato clade subdivided into four clades (mainly in the
BI analyses), corresponding to the genera Quadrula, Cyclonaias, Theliderma, and Tritogonia
(Figs. 2-4; Table 3). Furthermore, taxa in these clades exhibit coherent combinations of traits
that in our opinion support the validity of their generic status as recently recognized by Williams
et al (2017) (Figs. 2-4; Tables 3 and 5, Supporting Information Table S5).
The current molecular phylogenies cannot strongly support any suprageneric
relationships (probably due to insufficient genetic marker representation) within Quadrula s.l.
However, the morphological and ecological data here presented suggest common evolutionary
origins for the genera Quadrula and Tritogonia, and for Cyclonaias and Theliderma (Table 2;
Supporting Information Table S5). While Quadrula and Tritogonia include large reflexive
mantle displays, miniaturized shell glochidia, and marked shell sulci, Cyclonaias and
Theliderma species have small non-reflexive mantle displays, larger glochidia, and absent or
shallow shell sulci (Table 2; Supporting Information Table S5).
The series of traits shared by Quadrula and Tritogonia are likely associated with
maximizing attachment success to their main hosts, the catfishes (Table 2). These traits
include large conspicuous mantle displays that do not respond to mechanical disturbance (but
probably to another type of stimulus, for example, chemical, that might capitalize on the acute
olfactory sense of their hosts) and miniaturized glochidia. Tritogonia species are the only
Quadrula s.l. species that exhibit marked shell sexual dimorphism. This is probably a result of
the presence of mantle displays that completely cover the incurrent and excurrent apertures
of females, resulting in a distortion of their shells (Table 2, Supporting Information Table S5).
On the other hand, a specialization in attracting small cyprinids and percids seems to have
driven reproductive behaviour and morphology in Theliderma towards females that are
generally completely buried with only the mantle display being visible (Sietman et al 2012).
The displays of Theliderma are also more conspicuously displayed during the day favouring
the visual predatory habits of cyprinids, which contrasts with the other three Quadrula s.l.
genera who are generally displaying at night when feeding activity in catfishes is highest (Hove
et al 2011). Theliderma species are unique within Quadrulini in the production of mucoid
conglutinates (Haag 2012) and by presenting dark chevrons in the shells periostracum (Table
2; Supporting Information Table S5). The glochidia of Theliderma are also much bigger than
those of Tritogonia and Quadrula and more similar in size to most of the other species within
the Ambleminae (Table 2; Barnhart et al 2008). The large size of Theliderma glochidia can be
related to the much lower fecundity of this genus when compared with the other Quadrula s.l.
FCUP 173
genera (Haag 2012). Cyclonaias presents a set of reproductive features that are like those in
Theliderma species. However, glochidial size in Cyclonaias is always larger than in
Theliderma, and Cyclonaias exhibit a prevalence of catfish hosts rather than cyprinids and
percids (Table 2). Adaptation to catfish hosts again is likely associated with the unique
stomate-shaped mantle displays exhibited by Cyclonaias species (Table 2). The miniaturized
glochidia shared by Quadrula and Tritogonia probably represent a derivation from the more
primitive glochidial size of most amblemines (Barnhart et al 2008). On the other hand,
preference for and related adaptations to catfish hosts seem to be ancestral for the Quadrulini,
while preference for small cyprinids and percids in Theliderma is probably the derived state. A
more robust multi-marker molecular approach is needed to get a clearer view of the
evolutionary aspects of these interesting adaptations and to resolve the suprageneric
relationships among Quadrula s.l. genera.
Phylogeny and systematics implications within the four Quadrula sensu lato genera
Cyclonaias
The present results confirm the results of a recent study on this genus (Johnson et al 2018)
recognizing nine of the 14 Cyclonaias species listed by Williams et al (2017) as valid species
(Table 7). However, we here consider C. asperata as a synonym of C. kieneriana due to the
residual genetic divergence between these two taxa (ND1 p-distance <1%) and the fact that
C. kieneriana (Lea, 1852) has priority over C. asperata (Lea, 1861). In contrast, Williams et al
(2017) recognized both species based on their morphological distinctiveness and the fact that
molecular evidence for synonymy was based on only one marker (ND1) from a single
specimen. However, ND1 is a highly representative marker of overall mtDNA evolution in
unionoid mussels (Fonseca et al 2016). Besides, the divergence between C. asperata and C.
kieneriana sequences was very low. As a result, both ND1 (BI and ML) analyses were unable
to resolve both species’ phylogenies, and all ND1 species delineation methods were unable to
separate the two species (Table 5), indicating that C. asperata should be synonymized under
C. kieneriana. The morphometry results supported the distinct morphology of the two nominal
species but very few C. kieneriana shells (n = 4) were available, preventing a comprehensive
analysis (Fig. 6c). Although C. asperata has been reported from a much wider geographic
range than C. kieneriana, both species are sympatric in the whole range of C. kieneriana
suggesting that specimens previously described as C. kieneriana are smooth forms of the
same species (Fig. 7).
Until recently, Cyclonaias archeri has been considered a subspecies of C. asperata
(Turgeon et al 1998). However, since no sequences, tissues or shell specimens of C. archeri
174 FCUP
were available for this study, we rely on Williams et al (2008, 2017) and recognize this species
as separate from C. asperata, based on its distinct morphology.
Cyclonaias necki has recently been separated from Cyclonaias petrina based on
molecular data (COI) and morphology (Burlakova et al 2018; Johnson et al 2018). The specific
rank of C. necki is here confirmed by all species delineation methods used on each of the
datasets (Table 5). The shell shape is also significantly different between C. petrina and C.
necki (Fig. 6a), confirming observations of Burlakova et al (2018) and Johnson et al (2018)
that C. necki shells are thinner, more compressed and more rectangular with a more distinct
and prominent posterior ridge. Distribution ranges of the two species are exclusive, with C.
necki being present only in the San Antonio/Guadalupe River basins, while C. petrina is
restricted to the Colorado basin (Fig. 8; Burlakova et al 2018).
The present paper confirms the inclusion of four nominal species, that is, C. aurea, C.
houstonensis, C. mortoni, C. refulgens, within C. pustulosa (Table 7) and Cyclonaias succissa,
as a related but distinct species, as proposed by Johnson et al (2018). None of the phylogenies
resolved them as monophyletic, and p-distance values among these taxa were very low (Table
4). All nominal species here synonymized with C. pustulosa have distinct and exclusive
geographic distributions (Fig. 9). The molecular results suggest that C. pustulosa is divided
into several morphotypes each in a distinct geographic area. These morphotypes are visible
in the morphometry results and explain why these populations used to be considered distinct
species (Fig. 7b).
The remaining Cyclonaias species recognized in the present study, that is, C. infucata,
C. kleiniana, C. kieneriana, Cyclonaias nodulata, and Cyclonaias tuberculata, were always
retrieved as well supported, divergent clades (Figs. 2-4), and recognized by all species
delineation methods (Table 5). Furthermore, the shell shape is different among all these latter
species, except for the pair C. infucata and C. kleiniana, which might be explained by their
closer genetic relationship (Figs. 2-4; Table 6).
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Table 7
Historical classification of species formerly assigned to Quadrula. * extinct.
Haas (1969a) Graf & Cummings (2007) Bogan (2013)
Quadrula
Quadrula (Quadrula) q. quadrula Quadrula quadrula Quadrula quadrula
Quadrula (Quadrula) q. apiculata Quadrula apiculata Quadrula apiculata
Quadrula (Quadrula) q. rumphiana Quadrula rumphiana Quadrula rumphiana
Quadrula (Pustulosa) couchiana Amphinaias couchiana Quadrula couchiana
Quadrula (s.s.) quadrula fragosa Quadrula fragosa Quadrula fragosa
Cyclonaias
Quadrula (Pustulosa) p. keineriana Quadrula kieneriana
Quadrula (Pustulosa) p. pernodosa Amphinaias asperata Quadrula asperata
Fusconaia succissa succissa Quicucina infucata Quadrula infucata
Quincuncina securiformis kleiniana Quadrula kleiniana
Quadrula (Pustulosa) archeri Amphinaias archeri
Quadrula (Pustulosa) nodulata Amphinaias nodulata Quadrula nodulata
Quadrula (Pustulosa) petrina Amphinaias petrina Quadrula petrina
Quadrula (Pustulosa) p. pustulosa Amphinaias pustulosa Quadrula pustulosa

Quadrula (Pustulosa) aurea Amphinaias aurea Quadrula aurea
Amphinaias houstonensis Quadrula houstonensis
Quadrula (Pustulosa) p. mortoni Quadrula mortoni
Quadrula (Pustulosa) p. refulgens Amphinaias refulgens Quadrula refulgens
Fusconaia succissa succissa Fusconaia succissa Quadrula succissa
Cyclonaias tuberculata tuberculata Cyclonaias tuberculata Cyclonaias tuberculata
Theliderma
Orthonymus cylindricus Theliderma cylindrica Quadrula cylindrica
Orthonymus intermedius Theliderma intermedia Quadrula intermedia
Orthonymus m. metanevrus Theliderma metanevra Quadrula metanevra
Orthonymus m. tuberosus Theliderma tuberosa
Theliderma sparsa Quadrula sparsa

Theliderma stapes
Tritogonia
Tritogonia verrucosa Tritogonia verrucosa Quadrula verrucosa
Quadrula (Quadrula) q. nobilis Quadrula nobilis Quadrula nobilis
176 FCUP
Table 7 (cont.)
Williams et al (2017) This study
Quadrula quadrula 1. Quadrula quadrula

Quadrula apiculata + Quadrula apiculata
Quadrula rumphiana + Quadrula rumphiana
Quadrula couchiana 2. Quadrula couchiana*
Quadrula fragosa 3. Quadrula fragosa
Cyclonaias kieneriana 1. Cyclonaias kieneriana

Cyclonaias asperata + Cyclonaias asperata
Cyclonaias infucata 2. Cyclonaias infucata
Cyclonaias kleiniana 3. Cyclonaias kleiniana
Cyclonaias archeri 4. Cyclonaias archeri
Cyclonaias nodulata 5. Cyclonaias nodulata
Cyclonaias petrina 6. Cyclonaias petrina
7. Cyclonaias necki
Cyclonaias pustulosa 8. Cyclonaias pustulosa
Cyclonaias aurea + Cyclonaias aurea
Cyclonaias + Cyclonaias
houstonensis houstonensis
Cyclonaias mortoni + Cyclonaias mortoni
Cyclonaias refulgens + Cyclonaias refulgens
Cyclonaias succissa 9. Cyclonaias succissa
Cyclonaias
10. Cyclonaias tuberculata
tuberculata
Theliderma cylindrica 1. Theliderma cylindrica

Theliderma intermedia 2. Theliderma intermedia
Theliderma metanevra 3. Theliderma metanevra
4. Theliderma johnsoni n. sp.

Theliderma sparsa 5. Theliderma sparsa
6. Theliderma stapes
Tritogonia verrucosa 1. Tritogonia verrucosa

Quadrula nobilis 2. Tritogonia nobilis
FCUP 177
Figure 7 Distribution maps of (a) nominal species Cyclonaias asperata and Cyclonaias
kieneriana before the present study and (b) of C. kieneriana as proposed in the present study
178 FCUP
Figure 8 Distribution maps of (a) Cyclonaias petrina before Burlakova et al (2018) and (b) of
C. petrina and Cyclonaias necki after Burlakova et al (2018) and Johnson et al (2018) findings
also supported by the present study
FCUP 179
Figure 9 Distribution maps of (a) nominal species within the Cyclonaias pustulosa group and
(b) of C. pustulosa and Cyclonaias succissa as confirmed by Johnson et al (2018) and the
present study
Quadrula
In the absence of genetic data and shell materials for Quadrula couchiana and Quadrula
fragosa, the first being most likely extinct (Williams et al 2017) and the second on the verge of
extinction (Sietman 2003), we make no considerations about their systematics and accept both
as valid within the Quadrula genus following Williams et al (2017).
We here synonymize Q. apiculata and Q. rumphiana under Q. quadrula. Although only
a small number of sequences were available for Q. apiculata and Q. rumphiana, the level of
divergence among these three nominal species is low for both markers (Table 4). Furthermore,
in all phylogenies, Q. quadrula is paraphyletic, with Q. apiculata and Q. rumphiana falling inside
the clade (Figs. 2-4). The level of divergence between these three nominal taxa is lower than
180 FCUP
the divergence between the distinct clades of COI within Q. quadrula sensu stricto identified
by Mathias et al (2018) and also retrieved here in the COI phylogeny and haplotype network
(Figs. 2 and 6a). A specific rank for each of these divergent clades was rejected in that study
due to the existence of gene-flow among them as shown by their microsatellite dataset
(Mathias et al 2018).
The nominal species Q. apiculata, Q. rumphiana and Q. quadrula sensu stricto
presented distinct shell shapes but only 76% of specimens were assigned to the correct
nominal species (Fig. 6d). The slightly distinct shell morphology again suggests that distinct
nominal species were assigned to regional forms despite the relative overlap in the distribution
range of Q. apiculata with both Q. quadrula and Q. rumphiana (Fig. 10) that may also be related
to the considerable overlap among shell shape forms (Fig. 6d).
Theliderma
Only two shells and no genetic material were available for T. stapes, since the species is very
endangered and possibly extinct (NatureServe 2018). Until new evidence emerges, we,
therefore, accept it as valid within the Theliderma genus following Williams et al (2017). Based
on the molecular phylogenies and all species delineation methods, we recognize five additional
species within Theliderma, that is, T. cylindrica, Theliderma intermedia, T. metanevra, T.
johnsoni n. sp., and T. sparsa (Figs. 2-4; Tables 1 and 5). The nominal species T. metanevra
is here divided into two distinct species, the T. metanevra sensu stricto with a Mississippi basin
distribution and T. johnsoni n. sp. distributed within the Mobile basin (Fig. 11). The two species
show high genetic divergence (3.2% for COI and 3.5% for ND1; Table 6). They also differ
morphologically, presenting distinct shell shape with only 5%-11% of specimens being
misidentified by Fourier analysis (Fig. 6e,f) as well as other morphological features (see
Supporting Information Appendix S2).
FCUP 181
Figure 10 Distribution maps of (a) nominal species within the Quadrula quadrula group and
(b) of Quadrula quadrula as proposed in the present study
Tritogonia
The position of T. nobilis could not be resolved in a previous single marker approach (Serb et
al 2003) but in the present study, all phylogenies reveal a well-supported clade comprising T.
nobilis and T. verrucosa. We, therefore, move the nominal species Q. nobilis into Tritogonia
as T. nobilis. Until the end of the 20th century, T. nobilis was not recognized by most authors
as a separate species from Q. Quadrula (Williams et al 2008). However, its placement under
Tritogonia is not new as Simpson (1914) already used this designation. Both T. nobilis and T.
verrucosa exhibit marked sexual dimorphism (Simpson, 1914; Williams et al 2008), which is a
synapomorphy of the genera within the Quadrulini.
182 FCUP
Figure 11 Distribution maps of (a) Theliderma metanevra before the present study and (b)
after the present study divided in T. metanevra and Theliderma johnsoni n. sp
Conservation implications
Cyclonaias
As C. asperata is here synonymized under C. kieneriana, future conservation status
assessment of C. kieneriana should include the distribution of C. asperata sensu stricto (Fig.
7), which would be expected to decrease the extinction risk of the species under the currently
recognized systematics. The separation of C. necki from C. petrina will likely increase the
extinction risk of both species as their distributions are even smaller than previously believed
(Fig. 8) but see Johnson et al (2018) for detailed conservation implications. In contrast, the
secure conservation status of C. pustulosa (Supporting Information Table S6) is here
FCUP 183
strengthened by the inclusion of the nominal taxa C. aurea, C. houstonensis, C. mortoni and
C. refulgens (Fig. 9; Table 7). However, due to their genetic uniqueness, the populations from
Eastern Texas (originally identified as C. mortoni) should be managed independently.
Quadrula
Synonymization of the nominal species Q. rumphiana and Q. apiculata under Q. quadrula does
not affect the conservation status of Q. quadrula due to the wide distribution areas and low
extinction risk of the three forms. That said, subtler potential genetic differences between
populations originally assigned to these species are likely to be revealed in future studies
applying faster-evolving markers.
Theliderma
The conservation status of T. metanevra is currently considered as secure mainly based on
the species’ wide distribution range. However, considering that the Mobile basin populations
represent a separate species (Fig. 11), i.e. T. johnsoni n. sp., the conservation statuses of T.
metanevra and T. johnsoni n. sp. need to be re-assessed separately, and the two species need
to be managed independently.
Acknowledgements
We would like to thank the Editor Dr. Per Ericson and the two reviewers Dr. Ivan Bolotov and
Dr. John Pfeiffer for the help in a funding for Texas surveys was provided by the US Fish and
Wildlife Service State Wildlife Grant Program through the Texas Parks and Wildlife Department
(Contracts: T-15-P/2004-2006; 434301/2006-2007; 187549/2008-2010; 407709/2011-2012),
and by the Texas Water Development Board (Contract: 0604830631/2006-2007) to AYK and
LEB. Molecular analyses were developed under ConBiomics: the missing approach for the
Conservation of Freshwater Bivalves Project No NORTE-01-0145-FEDER-030286, co-
financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and
by FCT through national funds. FCT also supported MLL under Grant
(SFRH/BD/115728/2016).
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FCUP 189
Supplementary Materials
Supplementary Table 1
List of museum lots analysed for the morphometry: taxon, original identification, new identification, and lot catalogue numbe r. BSGLC (SUNY
Buffalo State College Great Lakes Center); NCSM (North Carolina Museum of Natural Sciences).
TAXON ORIGINAL ID NEW ID CATALOGUE NUMBER

Cyclonaias
NCSM: 7117.1, 7117.2, 7117.3, 7117.4, 7117.5, 7118.1, 7118.2, 7118.3, 7118.4, 7118.5,
7118.7, 7118.8, 7166.1, 7166.2, 7166.3, 7166.4, 7324.1, 7324.2, 7324.3, 7324.4,
26973.1, 26973.2, 26973.3, 26973.4, 26973.5, 26973.6, 26973.7, 26973.8, 26973.9,
26981.1, 26981.2, 26981.3, 26983.1, 26983.2, 26983.3, 26983.4, 26983.5, 26983.6,
26983.7, 26983.8, 26987.1, 26987.2, 26994, 27003.1, 27003.2, 27003.3, 27003.4,
C. asperata C. kieneriana 27003.5, 27003.6, 27003.7, 27003.8, 27008.1, 27008.2, 27008.3, 27008.4, 27008.5,
27020, 27155.1, 27155.2, 27155.4, 29403.1, 29403.2, 29403.3, 29403.4, 29630, 30258,
30962.1, 30962.2, 33704.1, 33704.2, 33704.3, 33704.4, 33704.5, 33704.6, 33704.7,
33704.8, 33705.1, 33705.2, 33706.1, 33706.2, 33706.3, 33706.4, 33706.5, 33706.6,
33706.7, 33707.1, 33707.2, 33707.3, 33707.4, 33707.5, 33708, 40672.1, 40672.2,
40672.3, 40672.4, 40672.5, 40830.2, 40830.4
NCSM: 6898.1, 6898.2, 6898.3, 6898.4, 6898.5, 6898.6, 6923.1, 6923.2, 6923.3, 6923.4,
33720.1, 33720.2, 33721
BSGLC: 297, 825, 826, 829, 830, 832, 833, 834, 835, 1047, 1048, 1049, 1050, 1051,
C. aurea C. pustulosa 1360, 1540, 1541, 1542, 1543, 1544, 1545, 1547, 1548, 1549, 1550, 1551, 1552, 1556,
1557, 1565, 1640, 1642, 1643, 1659, 1671, 1681, 1682, 1683, 1684, 1685, 1949, 2215,
2216, 2217, 2227, 2261, 2262, 2263, 2264, 2265, 2266, 2270, 2271, 1553, 1554, 1567,
1568, 1674, 2268, 2269, 3256, 3257
BSGLC: 88, 89, 917, 918, 919, 945, 948, 971, 973, 974, 975, 976, 977, 979, 980, 981,
1007, 1008, 1032, 1711, 1712, 1715, 1716, 1717, 1765, 1766, 2129, 2130, 2131, 2135,
C. houstonensis C. pustulosa
2136, 2137, 2138, 2139, 2178, 2179, 2180, 2181, 2182, 2184, 2185, 2186, 2187, 3246,
3247, 3248
NCSM: 7182.1, 7182.2, 33759.1, 33759.1, 33759.2, 33759.3, 33759.4, 33759.5,
C. infucata C. infucata
33759.6, 33759.7, 33759.8, 33759.9, 46806.1, 46806.4, 48448, 100656
190 FCUP
C. kieneriana C. kieneriana NCSM: 6283.4, 7078.1, 45168.1, 45168.3

NCSM: 5564.1, 5564.2, 5564.3, 5564.4, 5564.5, 5564.6, 7183.1, 7183.1, 7183.11,
C. kleiniana C. kleiniana 7183.12, 7183.13, 7183.14, 7183.15, 7183.2, 7183.3, 7183.5, 7183.6, 7183.7, 7183.8,
7183.9, 7184.1, 7184.2, 7184.3, 7184.4, 48450
NCSM: 5595.1, 5595.1, 5595.2, 5595.3, 5595.4, 5595.5, 5595.6, 5595.7, 5595.8, 5595.9,
5677.1, 5677.1, 5677.11, 5677.13, 5677.14, 5677.2, 5677.3, 5677.4, 5677.5, 5677.6,
5677.7, 5677.8, 5677.9, 7483, 27280, 33732, 33733.1, 33733.2, 33733.3, 33739, 45803,
45804.1, 45804.2, 48451
BSGLC: 36, 37, 38, 39, 44, 45, 46, 63, 64, 90, 92, 94, 152, 345, 363, 364, 434, 436, 449,
C. mortoni C. pustulosa
542, 543, 558, 559, 560, 561, 562, 590, 618, 742, 743, 744, 747, 748, 749, 750, 752,
753, 754, 756, 757, 758, 759, 760, 761, 763, 764, 765, 766, 767, 768, 773, 774, 775,
844, 880, 924, 1231, 1776, 1871, 1872, 1873, 1884, 1885, 1886, 3224, 3225, 3226, 3227,
3228, 3229, 3230, 3231, 3232, 3233, 3234, 3235, 3236, 3237, 3238, 3239, 3240, 3241,
3242, 3243, 3244, 3245, 3249, 3251, 3258, 3259, 3260
BSGLC: 1670, 1672, 1673, 1952, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258,
C. necki C. necki
2259, 2260
NCSM: 7227.1, 7227.3, 7228.1, 7228.2, 7229, 7230.1, 7230.2, 7230.3, 7230.4,
10064.11, 10064.13, 10064.14, 10064.15, 10064.16, 10795.1, 10795.4, 15666, 27412,
C. nodulata C. nodulata 33711.1, 33711.1, 33711.2, 33711.3, 33711.4, 33711.5, 33711.6, 33711.7, 33711.8,
33711.9, 33712.1, 33712.2, 33712.3, 33712.4, 33712.5, 33712.6, 33735, 33738, 40784,
45800.1, 45800.2, 48379.1, 48379.2, 63794.1
BSGLC: 282, 283, 308, 579, 1027, 1028, 1029, 1030, 1031, 1041, 1584, 1585, 1605,
1606, 1615, 1616, 1617, 1619, 1620, 1620, 1622, 1623, 1626, 1627, 1630, 2127, 2128,
C. petrina C. petrina 2133, 2134, 2140, 2142, 2143, 2147, 2148, 2150, 2151, 2153, 2154, 2155, 2156, 2157,
2163, 2164, 2165, 2166, 2167, 2168, 2169, 2172, 2173, 2174, 2175, 2176, 2177, 2233,
2247, 2248, 2249, 2250, 3254, 3255
NCSM: 5975.1, 5975.11, 5975.12, 5975.13, 5975.14, 5975.15, 5975.16, 5975.17,
5975.18, 5975.19, 5975.2, 5975.2, 5975.21, 5975.22, 5975.23, 5975.24, 5975.3, 5975.4,
5975.5, 5975.6, 5975.7, 5975.8, 5975.9, 6085.1, 6085.2, 6085.3, 6085.4, 6085.5, 7036.1,
C. pustulosa C. pustulosa 7036.2, 7036.3, 27322, 27357.1, 27357.3, 27357.4, 30297, 33749, 35289, 43409.1,
43409.4, 43409.5, 43749, 45136.1, 45136.3, 45364.1, 45364.2, 45364.3, 45364.4,
45364.5, 45364.6, 45364.7, 45364.8, 46958, 47039.1, 48298.1, 48298.1, 48298.11,
48298.2, 48298.3, 48298.5, 48298.6, 48298.7, 48298.8, 48298.9, 61971, 63088,
FCUP 191
63088.1, 84539.1, 84539.2, 84539.3, 84541.1, 88100.1, 88100.2, 88100.3, 88100.4,

88100.5
NCSM: 33762, 33760.1, 33760.2, 33760.3, 33760.4, 33760.5, 33760.6, 33760.7,
C. refulgens C. pustulosa
33760.8, 33760.9, 33760.11, 33760.12, 33760.13, 33760.14, 33760.15
NCSM: 7084, 7085.1, 7085.2, 7086.1, 7086.2, 7086.3, 7086.4, 7087.1, 7087.2, 7087.3,
7087.4, 7087.5, 7087.6, 7087.8, 27043.1, 27043.2, 27043.3, 27051.1, 27051.3, 27051.4,
C. succissa C. pustulosa
27051.5, 27051.6, 27051.7, 27051.9, 27118, 27119.1, 27119.2, 27140.2, 48409.1,
48409.2
NCSM: 5563.1, 5563.2, 5690, 5966.1, 5966.1, 5966.11, 5966.2, 5966.3, 5966.4, 5966.5,
5966.6, 5966.7, 5966.8, 5966.9, 6163.2, 6163.3, 6166, 6306, 6345.1, 6345.2, 6345.3,
6528, 6547, 6559.1, 6559.2, 6559.3, 6559.5, 6664.1, 6664.2, 6664.3, 6664.4, 6664.5,
6664.6, 6743.1, 6743.2, 6743.3, 6743.4, 6743.5, 6744, 6745, 6746.1, 6746.2, 6747,
6748, 6750, 6751, 6752, 6754.1, 6754.2, 6754.3, 6754.4, 7418, 7504, 7617.1, 7617.2,
C. tuberculata C. tuberculata 7617.3, 7617.4, 27056, 27067, 27089.1, 27089.2, 27089.3, 27089.4, 27089.5, 27286,
27325.1, 27325.2, 27325.3, 27346.1, 27346.2, 27346.3, 27346.4, 27346.5, 27427.1,
27427.2, 33225.1, 33225.2, 33231, 33232.1, 33232.2, 33234, 43751, 44999.1, 46894.1,
48293.1, 48293.2, 48300.1, 48300.2, 48349, 48351.1, 48351.2, 48384, 63455, 63784.1,
84520.1, 84651, 87507.1, 87507.2, 87507.3, 87508.1, 87508.2, 87508.3, 87508.4,
100603, 102483.1, 102483.2, 102483.3, 102483.4, 102483.5, 102483.6, 102483.7
Quadrula
NCSM: 6090, 6091, 6092, 6095, 6096, 6097, 6098, 6281, 7251, 9876, 26990, 27154,
Q. apiculata Q. quadrula 30365, 33692, 33693, 33694, 33695, 33696, 33697, 33699, 33700, 33701, 33702,
33703, 43664, 45106, 45119, 45131, 48443, 84523, 84542, 84618
NCSM: 6186, 6196, 6200, 7109, 7110, 7112, 7113, 7114, 7115, 7521, 10794, 18085,
20441, 27012, 27077, 27085, 28981, 28998, 33741, 33751, 33752, 33753, 33755,
Q. quadrula Q. quadrula 33756, 33757, 33758, 43411, 44372, 44377, 44389, 44399, 44431, 44458, 46881,
46888, 48368, 48369, 48449, 63086, 63793, 63798, 63799, 63800, 84010, 84390,
84519, 101762
NCSM: 6118, 6128, 6139, 6621, 6886, 6888, 6889, 6891, 6892, 40615, 45142, 45169,
Q. rumphiana Q. quadrula
45199, 47975, 102759
192 FCUP
Theliderma
NCSM: 6402.1, 6402.1, 6402.2, 6402.3, 6402.4, 6402.5, 6402.6, 6402.7, 6402.8, 6500.1,
6500.2, 6500.3, 6500.4, 6500.5, 6906.1, 6906.2, 6954.1, 6954.2, 6954.3, 6957.1, 6957.2,
6959.1, 6959.2, 6959.3, 27227, 48047, 61191.1, 61191.2, 61191.3, 61191.4, 61191.5,
61191.6, 61191.7, 61191.8, 61192.1, 61192.2, 61192.3, 61192.4, 61193.1, 61193.1,
61193.11, 61193.12, 61193.2, 61193.5, 61193.6, 61193.7, 61193.8, 61193.9, 61259.14,
T. cylindrica T. cylindrica 61259.15, 61259.2, 61259.21, 61259.28, 61259.3, 61259.33, 61259.36, 61259.5,
61259.6, 61270.1, 61270.1, 61270.13, 61270.2, 61270.3, 61270.4, 61270.6, 61270.7,
61270.8, 61270.9, 61274.1, 61274.2, 61275.2, 61275.3, 61275.4, 61275.6, 61288.1,
61288.2, 61290.1, 62994.1, 62994.2, 62994.3, 62994.4, 85056.1, 85056.2, 85056.3,
88455.11, 88455.14, 88455.15, 88455.16, 88455.2, 88455.24, 88455.3, 88455.6,
88455.8, 88458.1, 88458.2, 88464.1, 88502
NCSM: 6338.1, 6338.2, 6338.3, 6338.4, 6338.5, 6338.6, 6338.7, 6338.8, 6338.9, 6899.1,
6899.2, 6900.1, 6900.2, 6900.3, 6900.4, 6900.5, 7616.1, 7616.2, 7616.3, 7616.4, 7616.5,
7616.6, 8850.3, 8850.4, 30512, 33715.1, 33715.2, 33716, 40857, 40914.1, 40914.2,
T. intermedia T. intermedia
41039, 43683.2, 47044.2, 48445.1, 48445.2, 48980.1, 63220.1, 63221.1, 63221.2,
63221.3, 63223, 63224.1, 63224.2, 63224.3, 63225.1, 63225.2, 63225.3, 63226.1,
63227.1
NCSM: 7098.1, 7098.2, 7098.3, 7098.4, 7098.5, 7103, 7106.1, 7106.11, 7106.12,
T. metanevra
T. johnsoni 7106.3, 7106.4, 7106.5, 7106.6, 7106.7, 7106.8, 26976, 27153.1, 27153.2, 33714,
(Mobile basin)
33719, 33722, 46889.1, 46889.2
NCSM: 4412.1, 4412.2, 4606, 5972, 6080, 7100.1, 7100.2, 7101, 7102, 7104, 7105.1,
10765.1, 10765.2, 27428, 28995.1, 28995.2, 30298.1, 30298.2, 30298.3, 30522.1,
T. metanevra T. metanevra
30522.2, 30522.3, 30522.4, 30522.5, 33718, 44361, 44776, 45824, 45841.1, 45841.2,
(Tennessee basin)
47051.1, 48447, 63145.2, 63145.4, 88505, 88517.1, 88517.2, 88523.1, 88523.2,
100604.1, 100604.2
NCSM: 6919, 6334.1, 6334.2, 6920, 10188.6, 10188.7, 30916, 40915.1, 40915.2,
T. sparsa T. sparsa
40915.3, 40919, 48362.1, 48362.2, 88695
T. stapes T. stapes NCSM: 101849.1, 101849.2
FCUP 193
List of specimens included in the Cytochrome c oxidase subunit I (COI) dataset; Haplotypes, GenBank references, original identification, new
identification, voucher specimen, and respective study. BSGLC (SUNY Buffalo State College Great Lakes Center); FLMNH (Florida Museum of
Natural History); NCSM (North Carolina Museum of Natural Sciences); UA (University of Alabama); UAUC (University of Alabama Unionid
Collection); UAM (Auburn University Museum).
TAXON HAP REFERENCE ORIGINAL ID NEW ID VOUCHER STUDY
Quadrulini
Cyclonaias
3 AF232805 Quincucina infucata Cyclonaias infucata UAUC919-926 Lydeard et al 2000
4 AF232806 Quincucina infucata Cyclonaias infucata UAUC605 Lydeard et al 2000
5 AF232807 Quincucina infucata Cyclonaias infucata UAUC561 Lydeard et al 2000
257 MH633610 Cyclonaias infucata Cyclonaias infucata QkleOch001 Johnson et al 2018
262 MH633615 Cyclonaias infucata Cyclonaias infucata QinfChi007 Johnson et al 2018
263 MH633616 Cyclonaias infucata Cyclonaias infucata QinfFli009 Johnson et al 2018
265 MH633627 Cyclonaias infucata Cyclonaias infucata QinfOch022 Johnson et al 2018
194 FCUP

255 MH633608 Cyclonaias asperata Cyclonaias kieneriana QaspAla001 Johnson et al 2018
6 AF232808 Quincucina infucata Cyclonaias kleiniana UAUC564 Lydeard et al 2000
7 AF232809 Quincucina infucata Cyclonaias kleiniana UAUC567 Lydeard et al 2000
261 MH633614 Cyclonaias kleiniana Cyclonaias kleiniana QkleSuw004 Johnson et al 2018
15 MH362121 Cyclonaias necki Cyclonaias necki QpetGua004 Johnson et al 2018
15 MH362128 Cyclonaias necki Cyclonaias necki QspeGua004 Johnson et al 2018
15 MG969422 Cyclonaias petrina Cyclonaias necki BSGLC 1672 Burlakova et al 2018
15 MG969423 Cyclonaias petrina Cyclonaias necki NCSM 65378 Burlakova et al 2018
FCUP 195

53 KT285656 Cyclonaias petrina Cyclonaias necki FLMNH 441084 Pfeiffer et al 2016
85 MH362139 Cyclonaias necki Cyclonaias necki QcouGua001 Johnson et al 2018
98 MH362165 Cyclonaias necki Cyclonaias necki FmitGua021 Johnson et al 2018
42 MH362115 Cyclonaias nodulata Cyclonaias nodulata QnodSal022 Johnson et al 2018
42 GU085316 Cyclonaias nodulata Cyclonaias nodulata Qnod1 Boyer et al 2011
42 GU085317 Cyclonaias nodulata Cyclonaias nodulata Qnod2 Boyer et al 2011
196 FCUP

77 MH362105 Cyclonaias mortoni Cyclonaias nodulata QmorRed050 Johnson et al 2018
78 MH362112 Cyclonaias nodulata Cyclonaias nodulata QnodNec017 Johnson et al 2018
78 MH362106 Cyclonaias nodulata Cyclonaias nodulata QaurNec113 Johnson et al 2018
79 MH362107 Cyclonaias nodulata Cyclonaias nodulata QnodOua012 Johnson et al 2018
12 MG969416 Cyclonaias petrina Cyclonaias petrina BSGLC 1617 Burlakova et al 2018
13 MH362135 Cyclonaias petrina Cyclonaias petrina QpetCol011 Johnson et al 2018
FCUP 197

14 MK503268 Cyclonaias aurea Cyclonaias pustulosa BSGLC 1674 This study
16 MH362191 Cyclonaias aurea Cyclonaias pustulosa QaurGua013 Johnson et al 2018
20 MH362255 Cyclonaias hostonensis Cyclonaias pustulosa QhouCol003 Johnson et al 2018
198 FCUP

20 MH362262 Cyclonaias hostonensis Cyclonaias pustulosa QhouBra012 Johnson et al 2018
20 KT285649 Cyclonaias houstonensis Cyclonaias pustulosa FLMNH 441135 Pfeiffer et al 2016
20 MK503273 Cyclonaias houstonensis Cyclonaias pustulosa BSGLC 3246 This study
20 MH362282 Cyclonaias mortoni Cyclonaias pustulosa QmorCol060 Johnson et al 2018
20 MH362267 Cyclonaias pustulosa Cyclonaias pustulosa QpetCol026 Johnson et al 2018
22 MH362289 Cyclonaias mortoni Cyclonaias pustulosa QmorSab006 Johnson et al 2018
22 MH362292 Cyclonaias mortoni Cyclonaias pustulosa QmorTri014 Johnson et al 2018
22 MH362297 Cyclonaias mortoni Cyclonaias pustulosa QmorNec021 Johnson et al 2018
22 MH362345 Cyclonaias mortoni Cyclonaias pustulosa QmorSaJ092 Johnson et al 2018
FCUP 199

22 KT285655 Cyclonaias mortoni Cyclonaias pustulosa FLMNH 441171 Pfeiffer et al 2016
22 MK503276 Cyclonaias mortoni Cyclonaias pustulosa BSGLC 3249 This study
26 MH362211 Cyclonaias aurea Cyclonaias pustulosa QaurNue050 Johnson et al 2018
37 MH362359 Cyclonaias pustulosa Cyclonaias pustulosa QpusStC048 Johnson et al 2018
37 MH362397 Cyclonaias pustulosa Cyclonaias pustulosa QpusNeo062 Johnson et al 2018
37 MH362405 Cyclonaias pustulosa Cyclonaias pustulosa QpusStF072 Johnson et al 2018
37 MH362416 Cyclonaias pustulosa Cyclonaias pustulosa QpusOua088 Johnson et al 2018
37 GU085318 Cyclonaias pustulosa Cyclonaias pustulosa photo PP3 Boyer et al 2011
37 EF033269 Cyclonaias refulgens Cyclonaias pustulosa JC Chapman et al 2008
200 FCUP

FCUP 201

116 MH362328 Cyclonaias mortoni Cyclonaias pustulosa QmorRed055 Johnson et al 2018
116 MH362375 Cyclonaias pustulosa Cyclonaias pustulosa QpusRed025 Johnson et al 2018
128 MH362253 Cyclonaias aurea Cyclonaias pustulosa QaurNec112 Johnson et al 2018
202 FCUP

FCUP 203

204 FCUP

185 MH362357 Cyclonaias pustulosa Cyclonaias pustulosa QpusOhi046 Johnson et al 2018
188 MH362361 Cyclonaias pustulosa Cyclonaias pustulosa QnodRed004 Johnson et al 2018
189 MH362362 Cyclonaias pustulosa Cyclonaias pustulosa QspeTri019 Johnson et al 2018
FCUP 205

215 MH362400 Cyclonaias pustulosa Cyclonaias pustulosa QpusOsa065 Johnson et al 2018
206 FCUP

227 MH362418 Cyclonaias pustulosa Cyclonaias pustulosa QspeOua024 Johnson et al 2018
228 MH362419 Cyclonaias refulgens Cyclonaias pustulosa QrefPas001 Johnson et al 2018
232 MH362424 Cyclonaias refulgens Cyclonaias pustulosa QrefPrl006 Johnson et al 2018
237 MH362429 Cyclonaias succissa Cyclonaias succissa QsucCho001 Johnson et al 2018
241 MH362433 Cyclonaias succissa Cyclonaias succissa QsucEsc013 Johnson et al 2018
242 MH362434 Cyclonaias succissa Cyclonaias succissa QsucYel017 Johnson et al 2018
FCUP 207

38 GU085283 Cyclonaias tuberculata Cyclonaias tuberculata MMTC 150 Boyer et al 2011
39 GU085284 Cyclonaias tuberculata Cyclonaias tuberculata photo Ctub2 Boyer et al 2011
44 HM849070 Cyclonaias tuberculata Cyclonaias tuberculata H2789 Breton et al 2010
44 HM230410 Cyclonaias tuberculata Cyclonaias tuberculata UAM1490 Campbell & Lydeard 2012
44 MH633635 Cyclonaias tuberculata Cyclonaias tuberculata CtubTen003 Johnson et al 2018
Quadrula
1 AF156511 Quadrula quadrula Quadrula quadrula UMMZ 265699 Graf & Foighil 2000
1 MK503266 Quadrula quadrula Quadrula quadrula BIV0364 This study
1 NIMUS820 Quadrula quadrula Quadrula quadrula MUS068.1 Biodiversity I. of Ontario
2 AF231757 Quadrula quadrula Quadrula quadrula Bogan & Hoeh 2000
36 EF033268 Quadrula quadrula Quadrula quadrula H1774 Chapman et al 2008
43 HM230409 Quadrula rumphiana Quadrula quadrula UA20703 Campbell & Lydeard 2012
52 MH633638 Quadrula apiculata Quadrula quadrula QapiRGr081 Johnson et al 2018
52 KT285648 Quadrula apiculata Quadrula quadrula FLMNH 441088 Pfeiffer et al 2016
56 KX853887 Quadrula quadrula Quadrula quadrula BL06QQ07 Mathias et al 2018
208 FCUP

56 KX853900 Quadrula quadrula Quadrula quadrula FLR1QQ04 Mathias et al 2018
56 KX853908 Quadrula quadrula Quadrula quadrula GRMIQQ01 Mathias et al 2018
56 KX853916 Quadrula quadrula Quadrula quadrula IRQRQQ03 Mathias et al 2018
56 KX853925 Quadrula quadrula Quadrula quadrula LMNRQQ08 Mathias et al 2018
56 KX853930 Quadrula quadrula Quadrula quadrula MR1QQ06 Mathias et al 2018
56 KX853947 Quadrula quadrula Quadrula quadrula NFSRQQ03 Mathias et al 2018
56 KX853954 Quadrula quadrula Quadrula quadrula SGMRQQ05 Mathias et al 2018
FCUP 209

56 KX853963 Quadrula quadrula Quadrula quadrula SLR1QQ02 Mathias et al 2018
56 KX853967 Quadrula quadrula Quadrula quadrula SLR117 Mathias et al 2018
59 KX853894 Quadrula quadrula Quadrula quadrula BMC1QQ06 Mathias et al 2018
60 KX853899 Quadrula quadrula Quadrula quadrula EFWRQQ10 Mathias et al 2018
60 KX853938 Quadrula quadrula Quadrula quadrula MSWRQQ11 Mathias et al 2018
60 KX853939 Quadrula quadrula Quadrula quadrula MSWR38 Mathias et al 2018
60 KX853972 Quadrula quadrula Quadrula quadrula SNR1QQ04 Mathias et al 2018
210 FCUP

61 KX853982 Quadrula quadrula Quadrula quadrula WFWRQQ17 Mathias et al 2018
68 KX853937 Quadrula quadrula Quadrula quadrula MSWRQQ09 Mathias et al 2018
FCUP 211

278 MH633643 Quadrula quadrula Quadrula quadrula QquaOhi009 Johnson et al 2018
289 NC_013658 Quadrula quadrula Quadrula quadrula H1773 Breton et al 2009
Theliderma
50 JF326435 Quadrula metanevra Theliderma johnsoni Campbell & Lydeard 2012
50 MK503289 Quadrula metanevra Theliderma johnsoni NCSM 30474.2 This study
8 AF232823 Quadrula quadrula Theliderma metanevra UAUC145 Lydeard et al 2000
40 GU085314 Quadrula metanevra Theliderma metanevra photo Qmet1 Boyer et al 2011
277 MH633642 Quadrula metanevra Theliderma metanevra QmetTen003 Johnson et al 2018
280 MH633646 Quadrula metanevra Theliderma metanevra QmetOhi005 Johnson et al 2018
Tritogonia
24 MK503279 Quadrula nobilis Tritogonia nobilis BSGLC 3253 This study
11 AY655024 Tritogonia verrucosa Tritogonia verrucosa UAUC3195 Campbell et al 2005
35 GU085322 Tritogonia verrucosa Tritogonia verrucosa Boyer et al 2011
35 DQ191413 Tritogonia verrucosa Tritogonia verrucosa UMMZ 62984 Graf & Cummings 2006
35 MH633641 Tritogonia verrucosa Tritogonia verrucosa QverOhi048 Johnson et al 2018
54 KT285657 Tritogonia verrucosa Tritogonia verrucosa FLMNH 441208 Pfeiffer et al 2016
275 MH633639 Tritogonia verrucosa Tritogonia verrucosa QverRed015 Johnson et al 2018
Uniomerus
46 HQ153528 Uniomerus sp. Uniomerus carolinianus wuspCOX01 McCartney et al 2016
212 FCUP

48 HQ153618 Uniomerus carolinianus Uniomerus carolinianus wuspCOX91 McCartney et al 2016
9 AY613846 Uniomerus declivis Uniomerus declivis UAUC3290 Campbell et al 2005
55 KT285659 Uniomerus declivis Uniomerus declivis FLMNH 438312 Pfeiffer et al 2016
51 JF326437 Uniomerus tetralasmus Uniomerus tetralasmus Campbell & Lydeard 2012
273 MH633631 Uniomerus tetralasmus Uniomerus tetralasmus UtetCol005 Johnson et al 2018
286 MH633653 Uniomerus tetralasmus Uniomerus tetralasmus UtetBaP011 Johnson et al 2018
Megalonaias
10 AY655007 Megalonaias nervosa Megalonaias nervosa Campbell et al 2005
266 MH633619 Megalonaias nervosa Megalonaias nervosa MnerGua025 Johnson et al 2018
279 MH633645 Megalonaias nervosa Megalonaias nervosa MnerOhi057 Johnson et al 2018
Amblemini AY654991 Amblema elliottii Amblema elliottii UAUC2511 Campbell et al 2005
DQ648099 Amblema plicata Amblema plicata AP33 Elderkin et al 2007
Lampsilini NC_005335 Lampsilis ornata Lampsilis ornata Serb & Lydeard 2003
NC_028522 Leptodea leptodon Leptodea leptodon Feng et al 2016
Pleurobemini HQ153586 Uniomerus sp. Elliptio crassidens wuspCOX59 McCartney et al 2016
MK503277 Pleurobema riddellii Pleurobema riddellii BIV2457 This study
Anodontini MK503266 Anodonta nuttalliana Anodonta nuttalliana BIV0364 This study
NC_013661 Pyganodon grandis Pyganodon grandis Breton et al 2009
Margaritiferidae NC_034846 Cumberlandia monodonta Cumberlandia monodonta Guerra et al 2017
NC_015476 Margaritifera falcata Margaritifera falcata H2912 Breton et al 2010
FCUP 213
References
Bogan AE. Hoeh WR. 2000. On becoming cemented: evolutionary relationships among the genera in the freshwater bivalve family E theriidae
(Bivalvia: Unionoida). Geological Society, London, Special Publications 177, 159-168.
Boyer SL, Howe AA, Juergens NW, Hove MC. 2011. A DNA-barcoding approach to identifying juvenile freshwater mussels (Bivalvia: Unionidae)
recovered from naturally infested fishes. Journal of the North American Benthological Society 30, 182-194.
Breton S, Beaupre HD, Stewart DT, Piontkivska H, Karmakar M, Bogan AE, Blier PU, Hoeh WR. 2009. Comparative mitochondrial genomics of
freshwater mussels (Bivalvia: Unionoida) with doubly uniparental inheritance of mtDNA: gender-specific open reading frames and putative origins
of replication. Genetics 183, 1575-1589.
Breton S, Stewart DT, Shepardson SP, Trdan RJ, Bogan AE, Chapman EG, Ruminas AJ, Piontkivska H, Hoeh WR. 2011. Novel protein genes
in animal mtDNA: A new sex determination system in freshwater mussels (Bivalvia: Unionoida)? Molecular Biology and Evolution 28, 1645-1659.
Burlakova L, Karatayev A, Froufe E, Bogan AE, Lopes-Lima M. 2018. A new freshwater bivalve species of the genus Cyclonaias from Texas
(Unionidae: Ambleminae: Quadrulini). The Nautilus 132, 45-50.
Campbell DC, Serb JM, Buhay JE, Roe, KJ, Minton RL, Lydeard C. 2005. Phylogeny of North American amblemines (Bivalvia, Uniono ida):
prodigious polyphyly proves pervasive across genera. Invertebrate Biology 124, 131-164.
Campbell DC, Lydeard C. 2012. Molecular systematics of Fusconaia (Bivalvia: Unionidae: Ambleminae). American Malacological Bulletin 30, 1-
17.
214 FCUP
Chapman EG, Piontkivska H, Walker JM, Stewart DT, Curole JP, Hoeh WR. 2008. Extreme primary and secondary protein structure variability in
the chimeric male-transmitted cytochrome c oxidase subunit II protein in freshwater mussels: evidence for an elevated amino acid substitution
rate in the face of domain-specific purifying selection. BMC Evolutionary Biology 8, 165.
Elderkin CL, Christian AD, Vaughn CC, Metcalfe-Smith JL, Berg DJ. 2006. Population genetics of the freshwater mussel, Amblema plicata (Say
1817) (Bivalvia: Unionidae): Evidence of high dispersal and post-glacial colonization. Conservation Genetics 8, 355-372.
Feng L, Zhang X, Zhao G-F. 2016. The complete mitochondrial genome of the scaleshell Leptodea leptodon (Bivalvia: Unionidae). Conservation
Genetics Resources 8, 443-445.
Graf DL, Foighil DÓ. 2000. The Evolution of Brooding Characters among the Freshwater Pearly Mussels (Bivalvia: Unionoidea) of North America.
Journal Molluscan Studies 66, 157-170.
Graf DL, Cummings KS. 2006. Palaeoheterodont diversity (Mollusca: Trigonioida + Unionoida): What we know and what we wish we knew about
freshwater mussel evolution. Zoological Journal of the Linnean Society 148, 343-394.
Guerra D, Plazzi F, Stewart DT, Bogan AE, Hoeh WR, Breton S. 2017. Evolution of sex-dependent mtDNA transmission in freshwater mussels
(Bivalvia: Unionida). Scientific Reports 7, 1551.
Johnson NA, Smith CH, Pfeiffer JM, Randklev CR, Williams JD, Austin JD. 2018. Integrative taxonomy resolves taxonomic uncertainty for
freshwater mussels being considered for protection under the US Endangered Species Act. Scientific Reports 8, 15892.
Lydeard C, Minton RL, Williams JD. 2000. Prodigious polyphyly in imperilled freshwater pearly-mussels (Bivalvia: Unionidae): a phylogenetic test
of species and generic designations. Geological Society, London, Special Publications 177, 145-158.
FCUP 215
Mathias PT, Hoffman JR, Wilson CC, Zanatta DT. 2018. Signature of postglacial colonization on contemporary genetic structure and diversity of
Quadrula quadrula (Bivalvia: Unionidae). Hydrobiologia 810, 207-225.
McCartney MA, Bogan AE, Sommer KM, Wilbur AE. 2016. Phylogenetic Analysis of Lake Waccamaw Endemic Freshwater Mussel Species.
American Malacological Bulletin 34, 109-120.
Pfeiffer III JM, Johnson NA, Randklev CR, Howells RG, Williams JD. 2016. Generic reclassification and species boundaries in the rediscovered
freshwater mussel “Quadrula” mitchelli (Simpson in Dall, 1896). Conservation Genetics 17, 279-292.
Serb JM, Lydeard C. 2003. Complete mtDNA sequence of the North American freshwater mussel, Lampsilis ornata (Unionidae): an examination
of the evolution and phylogenetic utility of mitochondrial genome organization in Bivalvia (Mollusca). Molecular Biology and Evolution 20, 1854-
66.
216 FCUP
List of specimens included in the NADH dehydrogenase subunit 1 (ND1) dataset; Haplotypes, GenBank references, original identification, new
identification, and voucher/specimen and respective study. BSGLC (SUNY Buffalo State College Great Lakes Center); NCSM (North Carolina
Museum of Natural Sciences); UA (University of Alabama); UAUC (University of Alabama Unionid Collection).
Quadrulini
Cyclonaias
51 AY158810 Quincuncina infucata Cyclonaias infucata QiNfu2 Serb et al 2003
53 AY655121 Quincuncina infucata Cyclonaias infucata UAUC3283 Campbell & Lydeard 2012
307 MH633562 Cyclonaias infucata Cyclonaias infucata QkleOch001 Johnson et al 2018
7 AY158757 Cyclonaias asperata Cyclonaias kieneriana aspe792 Serb et al 2003
FCUP 217

19 AY158769 Cyclonaias kieneriana Cyclonaias kieneriana kieN334 Serb et al 2003
41 AY158795 Quincuncina infucata Cyclonaias kleiniana QiNfu57 Serb et al 2003
58 MH361822 Cyclonaias necki Cyclonaias necki FmitGua021 Johnson et al 2018
58 MK503297 Cyclonaias petrina Cyclonaias necki BSGLC 1672 This Study
71 MK503316 Cyclonaias necki Cyclonaias necki NCSM 65378 This Study
72 MK503317 Cyclonaias necki Cyclonaias necki BSGLC 2255 This Study
73 MK503318 Cyclonaias necki Cyclonaias necki BSGLC 2256 This Study
218 FCUP

145 MH361796 Cyclonaias necki Cyclonaias necki QcouGua001 Johnson et al 2018
5 AY158755 Cyclonaias nodulata Cyclonaias nodulata Nodu2595 Serb et al 2003
6 AY158756 Cyclonaias nodulata Cyclonaias nodulata Nodu2592 Serb et al 2003
126 GU085373 Cyclonaias nodulata Cyclonaias nodulata isolate 1 Boyer et al 2011
127 GU085374 Cyclonaias nodulata Cyclonaias nodulata isolate 2 Boyer et al 2011
137 MH361762 Cyclonaias mortoni Cyclonaias nodulata QmorRed050 Johnson et al 2018
138 MH361763 Cyclonaias aurea Cyclonaias nodulata QaurNec113 Johnson et al 2018
FCUP 219

42 AY158798 Cyclonaias petrina Cyclonaias petrina Qpet2546 Serb et al 2003
55 MK503293 Cyclonaias petrina Cyclonaias petrina BSGLC 1617 This Study
220 FCUP

1 AY158745 Cyclonaias aurea Cyclonaias pustulosa aure1083 Serb et al 2003
2 AY158752 Cyclonaias pustulosa Cyclonaias pustulosa pust2587 Serb et al 2003
14 AY158764 Cyclonaias mortoni Cyclonaias pustulosa mort1077 Serb et al 2003
15 AY158765 Cyclonaias aurea Cyclonaias pustulosa aure1085 Serb et al 2003
28 AY158778 Cyclonaias mortoni Cyclonaias pustulosa mort2436 Serb et al 2003
35 AY158788 Cyclonaias refulgens Cyclonaias pustulosa QREF405F Serb et al 2003
57 MK503296 Cyclonaias aurea Cyclonaias pustulosa BSGLC 1674 This Study
63 MK503303 Cyclonaias houstonensis Cyclonaias pustulosa BSGLC 3246 This Study
FCUP 221

64 MH361929 Cyclonaias houstonensis Cyclonaias pustulosa QhouCol029 Johnson et al 2018
65 MH361918 Cyclonaias houstonensis Cyclonaias pustulosa QhouBra011 Johnson et al 2018
66 MK503306 Cyclonaias mortoni Cyclonaias pustulosa BSGLC 3249 This Study
222 FCUP

78 DQ640237 Cyclonaias pustulosa Cyclonaias pustulosa QPVT-ND1-01 Henley et al 2006
78 FJ601230 Cyclonaias pustulosa Cyclonaias pustulosa PP11 Szumowski et al 2012
84 FJ601248 Cyclonaias pustulosa Cyclonaias pustulosa SIP14 Szumowski et al 2012
84 FJ601253 Cyclonaias pustulosa Cyclonaias pustulosa HP1 Szumowski et al 2012
FCUP 223

89 FJ601272 Cyclonaias pustulosa Cyclonaias pustulosa WRP14 Szumowski et al 2012
89 FJ601279 Cyclonaias pustulosa Cyclonaias pustulosa Q1MCOXA20 Szumowski et al 2012
89 HM852934 Cyclonaias pustulosa Cyclonaias pustulosa BM20258 Breton et al 2010
224 FCUP

117 FJ601280 Cyclonaias pustulosa Cyclonaias pustulosa Q22MCOXA19 Szumowski et al 2012
132 HM849266 Cyclonaias houstonensis Cyclonaias pustulosa H2845 Breton et al 2010
133 HM852935 Cyclonaias pustulosa Cyclonaias pustulosa BM20241 Breton et al 2010
FCUP 225

226 FCUP

191 MH361910 Cyclonaias aurea Cyclonaias pustulosa QaurNec112 Johnson et al 2018
FCUP 227

228 FCUP

247 MH362018 Cyclonaias nodulata Cyclonaias pustulosa QnodRed004 Johnson et al 2018
248 MH362019 Cyclonaias pustulosa Cyclonaias pustulosa QspeTri019 Johnson et al 2018
FCUP 229

252 MH362024 Cyclonaias houstonensis Cyclonaias pustulosa QpusOhi054 Johnson et al 2018
230 FCUP

283 MH362075 Cyclonaias pustulosa Cyclonaias pustulosa QspeOua024 Johnson et al 2018
39 AY158792 Fusconaia succissa Cyclonaias succissa Fsucc8 Serb et al 2003
50 AY158809 Fusconaia succissa Cyclonaias succissa Fsuc119 Serb et al 2003
52 AY655088 Cyclonaias tuberculata Cyclonaias tuberculata UAUC3158 Campbell et al 2005
FCUP 231

121 GU085342 Cyclonaias tuberculata Cyclonaias tuberculata Ctub2 Boyer et al 2011
122 GU085343 Cyclonaias tuberculata Cyclonaias tuberculata MMTC 150 Boyer et al 2011
Quadrula
328 MH633590 Quadrula apiculata Quadrula quadrula QapiRGr081 Johnson et al 2018
333 MH633595 Quadrula quadrula Quadrula quadrula QquaOhi009 Johnson et al 2018
20 AY158770 Quadrula rumphiana Quadrula quadrula rump1044 Serb et al 2003
22 AY158772 Quadrula q. clade 2/3 Quadrula quadrula quad902 Serb et al 2003
37 AY158790 Quadrula q. clade 1 Quadrula quadrula QQU1045f Serb et al 2003
47 AY158805 Quadrula apiculata Quadrula quadrula Qapi2620 Serb et al 2003
54 MK503291 Quadrula q. clade 1 Quadrula quadrula This Study
54 HM852936 Quadrula q. clade 1 Quadrula quadrula BM19581 Breton et al 2010
130 HM230421 Quadrula rumphiana Quadrula quadrula UA20703 Campbell & Lydeard 2012
333 NC_013658 Quadrula q. clade 2/3 Quadrula quadrula H1773 Breton et al 2009
Theliderma
33 AY158785 Quadrula cylindrica Theliderma cylindrica QCY2773 Serb et al 2003
43 AY158800 Quadrula cylindrica Theliderma cylindrica Qcstr277 Serb et al 2003
10 AY158760 Quadrula intermedia Theliderma intermedia iNte1512 Serb et al 2003
30 AY158782 Quadrula intermedia Theliderma intermedia QINT2772 Serb et al 2003
31 AY158783 Quadrula intermedia Theliderma intermedia QINT2775 Serb et al 2003
44 AY158802 Quadrula metanevra Theliderma johnsoni Qmet1128 Serb et al 2003
135 JF326448 Quadrula metanevra Theliderma johnsoni Campbell & Lydeard 2012
21 AY158771 Quadrula metanevra Theliderma metanevra Qmet042 Serb et al 2003
45 AY158803 Quadrula metanevra Theliderma metanevra Qmet954 Serb et al 2003
232 FCUP

332 MH633594 Quadrula metanevra Theliderma metanevra QmetTen003 Johnson et al 2018
332 MH633598 Quadrula metanevra Theliderma metanevra QmetOhi005 Johnson et al 2018
11 AY158761 Quadrula sparsa Theliderma sparsa spar1514 Serb et al 2003
32 AY158784 Quadrula sparsa Theliderma sparsa QSP2761F Serb et al 2003
Tritogonia
34 AY158786 Quadrula nobilis Tritogonia nobilis QNOB403 Serb et al 2003
36 AY158789 Quadrula quadrula Tritogonia nobilis QQU145F Serb et al 2003
46 AY158804 Quadrula nobilis Tritogonia nobilis QNobi263 Serb et al 2003
68 MK503309 Quadrula nobilis Tritogonia nobilis BSGLC 3253 This Study
134 JF326447 Quadrula nobilis Tritogonia nobilis Campbell & Lydeard 2012
38 AY158791 Tritogonia verrucosa Tritogonia verrucosa Tver040 Serb et al 2003
49 AY158807 Tritogonia verrucosa Tritogonia verrucosa Tver2753 Serb et al 2003
128 GU085382 Tritogonia verrucosa Tritogonia verrucosa isolate 1 Boyer et al 2011
129 GU085383 Tritogonia verrucosa Tritogonia verrucosa isolate 2 Boyer et al 2011
329 MH633591 Tritogonia verrucosa Tritogonia verrucosa QverRed015 Johnson et al 2018
331 MH633593 Tritogonia verrucosa Tritogonia verrucosa QverOhi048 Johnson et al 2018
Uniomerus
136 JF326451 Uniomerus tetralasmus Uniomerus tetralasmus Campbell & Lydeard 2012
324 MH633583 Uniomerus tetralasmus Uniomerus tetralasmus UtetCol005 Johnson et al 2018
338 MH633605 Uniomerus tetralasmus Uniomerus tetralasmus UtetBaP011 Johnson et al 2018
Megalonaias
40 AY158794 Megalonaias nervosa Megalonaias nervosa MNerv266 Serb et al 2003
123 GU085356 Megalonaias nervosa Megalonaias nervosa isolate 1 Boyer et al 2011
124 GU085357 Megalonaias nervosa Megalonaias nervosa isolate 2 Boyer et al 2011
124 MH633597 Megalonaias nervosa Megalonaias nervosa MnerOhi057 Johnson et al 2018
316 MH633571 Megalonaias nervosa Megalonaias nervosa MnerGua025 Johnson et al 2018
Amblemini
Hap129 AY655086 Amblema elliottii Amblema elliottii UAUC2511 Campbell et al 2005
Hap126 HM852922 Amblema plicata Amblema plicata BM19345 Boyer et al 2011
Lampsilini
Hap123 NC_005335 Lampsilis ornata Lampsilis ornata Serb et al 2003
Hap124 NC_028522 Leptodea leptodon Leptodea leptodon LEPT20150810 Feng et al 2016
FCUP 233
TAXON HAP REFERENCE ORIGINAL ID NEW ID VOUCHER

Pleurobemini
Hap125 MK503292 Pleurobema oviforme Pleurobema oviforme This Study
Hap108 MK503307 Pleurobema riddellii Pleurobema riddellii This Study
Anodontini
Hap127 MK503290 Anodonta nuttalliana Anodonta nuttalliana This Study
Hap095 NC_013661 Pyganodon grandis Pyganodon grandis Breton et al 2009
Margaritiferidae
Hap096 NC_034846 Cumberlandia monodonta Cumberlandia monodonta H3010 Guerra et al 2017
Hap128 NC_015476 Margaritifera falcata Margaritifera falcata Breton et al 2010
References
Boyer SL, Howe AA, Juergens NW, Hove MC. 2011. A DNA-barcoding approach to identifying juvenile freshwater mussels (Bivalvia: Unionidae)
recovered from naturally infested fishes. Journal of the North American Benthological Society 30, 182-194.
Breton S, Beaupre HD, Stewart DT, Piontkivska H, Karmakar M, Bogan AE, Blier PU, Hoeh WR. 2009. Comparative mitochondrial genomics of
freshwater mussels (Bivalvia: Unionoida) with doubly uniparental inheritance of mtDNA: gender-specific open reading frames and putative origins
of replication. Genetics 183, 1575-1589.
Breton S, Stewart DT, Shepardson SP, Trdan RJ, Bogan AE, Chapman EG, Ruminas AJ, Piontkivska H, Hoeh WR. 2011. Novel protein genes
in animal mtDNA: A new sex determination system in freshwater mussels (Bivalvia: Unionoida)? Molecular Biology and Evolution 28, 1645-1659.
Campbell DC, Serb JM, Buhay JE, Roe KJ, Minton RL, Lydeard C. 2005. Phylogeny of North American amblemines (Bivalvia, Unionoida):
prodigious polyphyly proves pervasive across genera. Invertebrate Biology 124, 131-164.
Campbell DC, Lydeard C. 2012. Molecular systematics of Fusconaia (Bivalvia: Unionidae: Ambleminae). American Malacological Bulletin 30, 1-
17.
234 FCUP
Feng L, Zhang X, Zhao G-F. 2016. The complete mitochondrial genome of the scaleshell Leptodea leptodon (Bivalvia: Unionidae). Conservation
Genetics Resources 8, 443-445.
Johnson NA, Smith CH, Pfeiffer JM, Randklev CR, Williams JD, Austin JD. 2018. Integrative taxonomy resolves taxonomic uncerta inty for
freshwater mussels being considered for protection under the US Endangered Species Act. Scientific Reports 8, 15892.
Henley WF, Grobler PJ, Neves RJ. 2006. Non-Invasive Method to Obtain DNA from Freshwater Mussels (Bivalvia: Unionidae). Journal of Shellfish
Research 25, 975-977.
Serb JM, Buhay JE, Lydeard C. 2003. Molecular systematics of the North American freshwater bivalve genus Quadrula (Unionidae: Ambleminae)
based on mitochondrial ND1 sequences. Molecular Phylogenetics and Evolution 28, 1-11.
Szumowski SC, Boyer SL, Hornbach DJ, Hove MC. 2012. Genetic Diversity of Two Common Freshwater Mussel Species, Lampsilis cardium and
Quadrula pustulosa (Bivalvia: Unionidae), in a Large Federally Protected Waterway (St. Croix River, Minnesota/Wisconsin, U.S.A.). American
Malacological Bulletin 30, 59-72.
FCUP 235
List of specimens included in the concatenated Cytochrome c oxidase subunit I (COI) + NADH dehydrogenase subunit 1 (ND1) data set; codes,
original identification, new identification, and Genbank references.
TAXON ORIGINAL ID NEW ID GENBANK (COI) GENBANK (ND1)
Quadrulini
Cyclonaias
Cyclonaias infucata Cyclonaias infucata AF232806 AY655121
Cyclonaias infucata Cyclonaias infucata MH633615 MH633567
236 FCUP

Cyclonaias asperata Cyclonaias kieneriana MH633608 MH633560
Cyclonaias infucata Cyclonaias kleiniana AF232808 AY158795
Quadrula kleiniana Cyclonaias kleiniana MH633614 MH633566
Cyclonaias necki Cyclonaias necki MG969422 MK503297
Cyclonaias necki Cyclonaias necki MH362168 MH361825
FCUP 237

Cyclonaias nodulata Cyclonaias nodulata GU085316 GU085373
Cyclonaias nodulata Cyclonaias nodulata MH362115 MH361772
Cyclonaias nodulata Cyclonaias nodulata GU085317 GU085374
238 FCUP

Cyclonaias petrina Cyclonaias petrina MG969416 MK503293
Cyclonaias petrina Cyclonaias petrina MH362267 MH361924
FCUP 239

Cyclonaias aurea Cyclonaias pustulosa MK503268 MK503296
Cyclonaias houstonensis Cyclonaias pustulosa MK503273 MK503303
Cyclonaias houstonensis Cyclonaias pustulosa MH362262 MH361919
Cyclonaias mortoni Cyclonaias pustulosa MH362282 MH361939
240 FCUP

Cyclonaias mortoni Cyclonaias pustulosa MK503276 MK503306
Cyclonaias aurea Cyclonaias pustulosa MH362211 MH361868
Cyclonaias refulgens Cyclonaias pustulosa EF033269 AY158788
Quadrula pustulosa Cyclonaias pustulosa GU085318 FJ601222
FCUP 241

242 FCUP

FCUP 243

244 FCUP

FCUP 245

Cyclonaias pustulosa Cyclonaias pustulosa MH362361 MH362018
246 FCUP

FCUP 247

Cyclonaias refulgens Cyclonaias pustulosa MH362419 MH362076
248 FCUP

Cyclonaias succissa Cyclonaias succissa MH362429 MH362086
Cyclonaias tuberculata Cyclonaias tuberculata MH633635 MH633587
Cyclonaias tuberculata Cyclonaias tuberculata GU085283 GU085343
Cyclonaias tuberculata Cyclonaias tuberculata GU085284 GU085342
Cyclonaias tuberculata Cyclonaias tuberculata HM849070 HM849213
FCUP 249

Quadrula
Quadrula rumphiana Quadrula quadrula HM230409 HM230421
Quadrula apiculata Quadrula quadrula KT285648 AY158805
Quadrula Quadrula Quadrula quadrula NC_013658 NC_013658
Quadrula Quadrula Quadrula quadrula MK503267 MK503291
Quadrula apiculata Quadrula quadrula MH633638 MH633590
Quadrula Quadrula Quadrula quadrula MH633643 MH633595
Theliderma
Theliderma metanevra Theliderma johnsoni JF326435 JF326448
Theliderma metanevra Theliderma metanevra GU085314 GU085371
Theliderma metanevra Theliderma metanevra GU085315 GU085372
Quadrula metanevra Theliderma metanevra MH633646 MH633598
Quadrula metanevra Theliderma metanevra MH633642 MH633594
Tritogonia
Quadrula nobilis Tritogonia nobilis MK503279 MK503309
Tritogonia verrucosa Tritogonia verrucosa AY655024 AY158791
Tritogonia verrucosa Tritogonia verrucosa GU085322 GU085382
Tritogonia verrucosa Tritogonia verrucosa MH633641 MH633593
Tritogonia verrucosa Tritogonia verrucosa MH633639 MH633591
Uniomerus
Uniomerus tetralasmus Uniomerus tetralasmus JF326437 JF326451
Uniomerus tetralasmus Uniomerus tetralasmus MH633653 MH633605
Uniomerus tetralasmus Uniomerus tetralasmus MH633631 MH633583
Megalonaias
Megalonaias nervosa Megalonaias nervosa AY655007 AY158794
Megalonaias nervosa Megalonaias nervosa MH633619 MH633571
Megalonaias nervosa Megalonaias nervosa MH633645 MH633597
250 FCUP

Amblemini
Amblema elliottii Amblema elliottii AY654991 AY655086
Amblema plicata Amblema plicata DQ648099 HM852922

Lampsilini
Lampsilis ornata Lampsilis ornata NC_005335 NC_005335
Leptodea leptodon Leptodea leptodon NC_028522 NC_028522
Pleurobemini
Pleurobema oviforme Pleurobema oviforme - MK503292
Pleurobema riddellii Pleurobema riddellii MK503277 MK503307
Anodontini
Anodonta nuttalliana Anodonta nuttalliana MK503266 ANBIV0364
Pyganodon grandis Pyganodon grandis NC_013661 NC_013661
Margaritiferidae
Cumberlandia monodonta Cumberlandia monodonta NC_034846 NC_034846
Margaritifera falcata Margaritifera falcata NC_015476 NC_015476
FCUP 251
List of morphological, anatomical and behavioural characters analysed on specimens of Quadrula s.l.. GLN - mean glochidial size index. * only
observed in laboratory conditions. Superscripts 1 occasionally, 2 shallow
Shell sulcus
dimorphism
Periostracal
Mantle displays
chevrons
(magazines)
Sexual
Shell
shape Location
Morphology Size
(apertures)
Quadrula NO NO YES quadrate/rectangular Conical (knob-like) Large Excurrent
1. Q. quadrula NO NO YES quadrate ----- ----- -----
+ Q. apiculata NO NO YES quadrate/rectangular ----- ----- -----
+ Q. rumphiana NO NO YES quadrate ----- ----- -----
2. Q. couchiana NO NO YES ----- ----- -----
3. Q. fragosa NO NO YES quadrate Conical (knob-like) Large Excurrent
Cyclonaias NO NO NO round/oval/quadrate stomate-shaped Small Excurrent
1. C. kieneriana NO NO NO oval/triangular ----- ----- -----
+ C. asperata NO NO NO round/triangular No modification* ----- -----
2. C. infucata NO NO NO rectangular/triangular ----- ----- -----
round/quadrate
3. C. kleiniana NO NO NO ----- ----- -----
triangular
4. C. archeri NO NO NO quadrate ----- ----- -----
5. C. nodulata NO NO NO round/quadrate ----- ----- -----
6. C. petrina NO NO NO quadrate ----- ----- -----
7. C. necki NO NO NO oval/quadrate ----- ----- -----
8. C. pustulosa NO NO NO round/quadrate stomate-shaped Small Excurrent
252 FCUP
+ C. aurea NO NO NO round/quadrate ----- ----- -----

+ C. houstonensis NO NO NO round/quadrate ----- ----- -----
+ C. mortoni NO NO NO quadrate/
+ C. refulgens NO NO NO round/oval ----- ----- -----
9. C. succissa NO NO NO quadrate/rectangular ----- ----- -----
10. C. tuberculata NO NO NO round/quadrate stomate-shaped Small Excurrent
1
Theliderma NO YES OC round/quadrate/rectangular Variate shape Small Excurrent
1. T. cylindrica NO YES NO rectangular white with orange ring Small Excurrent
2. T. intermedia NO YES NO round/quadrate ----- ----- -----
3. T. metanevra NO YES NO quadrate/rectangular polyp-like Small Excurrent
4. T. johnsoni n. sp. NO YES NO quadrate/rectangular ----- ----- -----
5. T. sparsa NO YES OC1 quadrate/rectangular ----- ----- -----
6. T. stapes NO YES YES2 triangular/quadraqte ----- ----- -----
Tritogonia YES NO YES elongate/rectangular slug-shaped Large Both
2
1. T. verrucosa YES NO YES elongate/rectangular slug-shaped Large Both
2. T. nobilis YES NO YES quadrate/rectangular ----- ----- -----
FCUP 253
Supplementary Table 5 (cont.)
Reflexive
Taxa Hosts GLN References
release
Quadrula NO Ictaluridae (67%), Centrarchidae (33%) 0.005-0.009
Howard & Anson 1922
1. Q. quadrula NO Ictaluridae (50%), Centrarchidae (50%) 0.007 Schwebach et al 2002
Barnhart et al 2008
Parmalee & Bogan 1998
+ Q. apiculata ----- ----- 0.005
Barnhart et al 2008
+ Q. rumphiana ----- ----- 0.007 Barnhart et al 2008
2. Q. couchiana ----- ----- -----
Steingraber et al 2004
Barnhart et al .2008
3. Q. fragosa NO Ictaluridae 0.009
Hove et al 2012
Sietman et al 2012
Ictaluridae (71%), Centrarchidae (24%),
Cyclonaias YES 0.05-0.09
Acipenseridae (5%)
1. C. kieneriana ----- ----- -----
Barnhart et al 2008
+ C. asperata ----- Ictaluridae 0.07
Haag & Staton 2003
2. C. infucata ----- ----- 0.07 Williams et al 2008
3. C. kleiniana ----- ----- 0.07 Williams et al 2014
4. C. archeri ----- ----- -----
Coker et al 1921
Surber 1913
5. C. nodulata ----- Ictaluridae (33%), Centrarchidae (67%) 0.05 Howard 1914
Parmalee & Bogan 1998
Barnhart et al 2008
6. C. petrina ----- ----- -----
7. C. necki ----- ----- -----
254 FCUP
Howard 1913, 1914

Ictaluridae (67%), Surber 1913
8. C. pustulosa YES Centrarchidae (17%), 0.07 Coker et al 1921
Acipenseridae (17%) Howard & Anson 1922
Sietman et al 2012
+ C. aurea ----- ----- ----- Howells et al 1996
+ C. houstonensis ----- ----- -----
+ C. mortoni
+ C. refulgens ----- ----- -----
9. C. succissa ----- Ictaluridae 0.06 Williams et al 2014
Hove et al 1994
Barnhart et al 2008
10. C. tuberculata Ictaluridae 0.09
Williams et al 2008
Sietman et al 2012
Cyprinidae (72%), Centrarchidae (14%)
Theliderma YES 0.03-0.04
Percidae (14%)
Yeager & Neves 1986
Cyprinidae (80%),
1. T. cylindrica 0.04 Barnhart et al 2008
Percidae (20%)
Watters et al 2006, 2009
2. T. intermedia Cyprinidae Yeager & Saylor 1995
Cyprinidae (57%), Surber 1913, Howard 1914
3. T. metanevra YES Centrarchidae (29%), 0.03 Crownhart et al 2006
Percidae (14%) Hove et al 2011
4. T. johnsoni n. sp. ----- ----- -----
5. T. sparsa ----- ----- -----
6. T. stapes ----- ----- -----
Tritogonia NO Ictaluridae 0.009
Barnhart et al 2008
1. T. verrucosa NO Ictaluridae 0.009 Hove et al 2011
Sietman et al 2012
Howells 1997
2. T. nobilis ----- Ictaluridae -----
Sietman et al 2012
FCUP 255
References
Barnhart MC, Haag WR, Roston WN. 2008. Adaptations to host infection and larval parasitism in Unionoida. Journal of the North American
Benthological Society 27, 370-394.
Coker RE, Shira AF, Clark HW, Howard AD. 1921. Natural history and propagation of fresh-water mussels. Bulletin of the United States Bureau
of Fisheries 37, 78-181.
Crownhart A, Sietman B, Hove M, Rudh N. 2006. Quadrula metanevra glochidia metamorphose on select minnow species. Ellipsaria 8, 6-7.
Haag WR, Staton JL, Leann Staton J. 2003. Variation in fecundity and other reproductive traits in freshwater mussels. Freshwater Biology 48,
2118-2130.
Hove M, Engleking R, Evers E, Peteler M, Peterson E. 1994. Cyclonaias tuberculata host suitability tests. Triannual Unionid Report 5.
Hove MC, Sietman BE, Bakelaar JE, Bury JA, Heath DJ, Pepi VE, Kurth J, Davis JM, Hornbach DJ, Kapuscinski AR. 2011. Early Life History and
Distribution of Pistolgrip (Tritogonia verrucosa (Rafinesque, 1820)) in Minnesota and Wisconsin. The American Midland Naturalist 165, 338-354.
Hove MC, Steingraeber MT, Newton TJ, Heath DJ, Nelson CL, Bury JA, Kurth JE, Bartsch MR, Thorpe WS, McGill MR, Hornbach DJ. 2012. Early
Life History of the Winged Mapleleaf Mussel (Quadrula fragosa). American Malacological Bulletin 30, 47-57.
Howard AD. 1913. The Catfish as a Host for Fresh-Water Mussels. Transactions of the American Fisheries Society 42, 65-70.
Howard AD. 1914. Experiments in propagation of freshwater mussels of the Quadrula group. Govt. print. off., Washington, USA.
256 FCUP
Howard AD, Anson BJ. 1922. Phases in the Parasitism of the Unionidae. The Journal of Parasitology 9, 68.
Howells RG, Neck RW, Murray HD. 1996. Freshwater Mussels of Texas. Texas Parks and Wildlife Department, Inland Fisheries Division, Austin,
Texas, USA.
Howells RG. 1997. New fish hosts for nine freshwater mussels (Bivalvia: Unionidae) in Texas. Texas Journal of Science 49, 255-258.
Parmalee PW, Bogan AE. 1998. Freshwater mussels of Tennessee. University of Tennessee Press, Knoxville, Tennessee, USA.
Schwebach M, Schriever D, Kanodia V, Dillo, N, Hove M, McGill M, Nelson C, Thomas J, Kapuscinski A. 2002. Channel catfish is a suitable host
species for mapleleaf glochidia. Ellipsaria 4, 12-13.
Sietman BE, Davis JM, Hove MC. 2012. Mantle display and glochidia release behaviors of five quadruline freshwater mussel species (Bivalvia:
Unionidae). American Malacological Bulletin 30, 39-46.
Steingraeber M, Hove M, Bartsch M, Hornbach D, Nelson C, Newton T, Kalas J, Kapuscinski A, Simonsen E. 2004. Two fish species identified
as hosts for Winged Mapleleaf (Quadrula fragosa). Ellipsaria 6, 7-8.
Surber T. 1913. Notes on the natural hosts of fresh-water mussels. Bulletin of the United States Bureau of Fisheries 32, 101-116.
Watters GT, Menker T, Smith B, Harraman K, Kuehnl K. 2006. Host identifications or confirmations. Ellipsaria 8, 8.
Watters GT, Gibson T, Kelly B. 2009. Host identifications or confirmations. Ellipsaria 11, 19.
FCUP 257
Williams JD, Bogan AE, Garner JT. 2008. Freshwater mussels of Alabama and the Mobile basin in Georgia, Mississippi, and Tennessee. The
University of Alabama Press, Tuscaloosa, USA
Williams JD, Butler RS, Warren GL, Johnson NA. 2014. Freshwater mussels of Florida. The University of Alabama Press, Tuscaloosa, USA.
Yeager B, Neves RJ. 1986. Reproductive cycle and fish hosts of the rabbit’s foot mussel, Quadrula cylindrica strigillata (Mollusca: Unionidae) in
the upper Tennessee River drainage. American Midland Naturalist 116, 329-340.
Yeager BL, Saylor CF. 1995. Fish hosts for four species of freshwater mussels (Pelecypoda: Unionidae) in the upper Tennessee River drainage.
American Midland Naturalist 133,1-6.
258 FCUP
Species assigned Quadrula sensu lato according to the last comprehensive checklist of the United States (Williams et al 2017), conservation
status by the International Union for Conservation of Nature (IUCN) Red List and by NaturServe, and legal protection status in the United States
of
America.
TAXON CONSERVATION STATUS PROTECTION
IUCN NATURSERVE USFWS
Cyclonaias Pilsbry in Ortmann and Walker, 1922
Cyclonaias archeri (Frierson, 1905) Tallapoosa Orb T1 - Critically Imperilled petitioned under review
Cyclonaias asperata (Lea, 1861) Alabama Orb NT G4 - Apparently Secure
Cyclonaias aurea (Lea, 1859) Golden Orb NT G1 - Critically Imperilled petitioned candidate
Cyclonaias houstonensis (Lea, 1859) Smooth Pimpleback NT G2 - Imperilled petitioned candidate
Cyclonaias infucata (Conrad, 1834) Sculptured Pigtoe NT G3 - Vulnerable
Cyclonaias kieneriana (Lea, 1852) Coosa Orb G3 - Vulnerable
Cyclonaias kleiniana (Lea, 1852) Florida Mapleleaf G2 - Imperilled not listed
Cyclonaias mortoni (Conrad, 1835) Western Pimpleback T3 - Vulnerable
Cyclonaias nodulata (Rafinesque, 1820) Wartyback LC G4 - Apparently Secure
Cyclonaias petrina (Gould, 1855) Texas Pimpleback G2 - Imperilled petitioned candidate
Cyclonaias pustulosa (Lea, 1831) Pimpleback LC G5 - Secure
Cyclonaias refulgens (Lea, 1868) Purple Pimpleback NT G3 - Vulnerable
Cyclonaias succissa (Lea, 1852) Purple Pigtoe LC G3 - Vulnerable
Cyclonaias tuberculata (Rafinesque, 1820) Purple Wartyback NT G5 - Secure
Quadrula Rafinesque, 1820

Quadrula apiculata (Say, 1829) Southern Mapleleaf G5 - Secure
Quadrula couchiana (Lea, 1860) Rio Grande Monkeyface CR GH - Possibly Extinct
Quadrula fragosa (Conrad, 1835) Winged Mapleleaf CR G1 - Critically Imperilled Endangered
Quadrula nobilis (Conrad, 1854) Gulf Mapleleaf G4 - Apparently Secure
Quadrula quadrula (Rafinesque, 1820) Mapleleaf LC G5 - Secure
Quadrula rumphiana (Lea, 1852) Ridged Mapleleaf LC G4 - Apparently Secure
FCUP 259
Theliderma Swainson, 1840

Theliderma cylindrica (Say, 1817) Rabbitsfoot NT G3 - Vulnerable Threatened
Theliderma intermedia (Conrad, 1836) Cumberland Monkeyface EN G1 - Critically Imperiled Endangered
Theliderma metanevra (Rafinesque, 1820) Monkeyface G4 - Apparently Secure
Theliderma sparsa (Lea, 1841) Appalachian Monkeyface CR G1 - Critically Imperiled Endangered
Theliderma stapes (Lea, 1831) Stirrupshell CR GH - Possibly Extinct Endangered
Tritogonia Agassiz, 1852

Tritogonia verrucosa (Rafinesque, 1820) Pistolgrip G4 - Apparently Secure
260 FCUP
Supplementary Appendix 1. History of Quadrulinae and the included genera
Rafinesque (1820) erected the subgenus Obliquaria (Quadrula) Rafinesque, 1820 and
included eight species but did not designate a type species. Lea (1836, 1838, 1852, 1870)
ignored Quadrula in his four editions of the Synopsis of Unio.
Theliderma Swainson, 1840 was introduced as a subgenus with seven species and no
type species designated. Simpson (1900) subsequently designated Unio lachrymosa Lea,
1828 (=quadrula Rafinesque, 1820) as the type of Theliderma, thus making Theliderma a junior
synonym of Quadrula. However, Simpson (1900) overlooked the prior designation of
metanevra Rafinesque, 1820 as the type species of Theliderma by Gray (1847). Subsequently,
Quadrula, Tritogonia Agassiz, 1852, Orthonymus Agassiz, 1852 and Rotundaria Rafinesque,
1820 were recognized by Agassiz (1852). To add to the confusion, Simpson (1900) used
metanevra as the type species of Quadrula. Later, Simpson (1900, 1914) recognized Quadrula
as a valid genus with Q. metanevra as the type species and placed Quadrula in the Unioninae
Rafinesque, 1820.
The subfamily Quadrulinae was erected by Ihering (1901) in remarks reviewing Simpson’s
(1900) Synopsis. Ihering divided the Unionidae Rafinesque, 1820 into Unioninae, Quadrulinae
Ihering, 1901 and Lampsilinae Ihering, 1901. Ihering in his definition of Quadrulinae included
Quadrula, Pleurobema Rafinesque, 1819, Obovaria Rafinesque, 1819, Cyprogenia Agassiz,
1852, Obliquaria Rafinesque, 1820 and Dromus Simpson, 1900 (Table 1).
Ortmann (1910, 1911, 1912, 1919) and Walker (1918) followed Simpson (1900) and
maintained Quadrula in the Unioninae. Hannibal (1912) recognized Quadrulinae von Ihering
(1901) for Quadrula.
Ortmann & Walker (1922) sorted out some of the confusion surrounding some unionid
names and relied on H.A. Pilsbry as an outside arbiter. They provided a concise history of the
changes in the usage and the type species of Quadrula which had changed from metanevra
to quadrula with the new taxonomic rules regarding absolute tautonymy. Ortmann & Walker
(1922) recognized Quadrula with Quadrula quadrula as the type species and included six
species, remarked that recognition of Tritogonia was a purely taxonomic question. Cyclonaias
Pilsbry in Ortmann & Walker (1922) was erected for Obliquaria tuberculata Rafinesque, 1820.
Since Ortmann & Walker (1922) assumed the type of Theliderma was quadrula, the next
available name for the species group, including metanevra and Unio cylindricus Say, 1817,
was Orthonymus Agassiz, 1852. However, since Gray (1847) designated metanevra as the
type of Theliderma, this is the earliest available name for this species group.
Frierson (1927) placed Quadrula in the Unioninae, recognizing 10 subgenera in
Quadrula including Quadrula, Tritogonia, Bullata Frierson, 1927, Quincuncina Ortmann in
Ortmann & Walker 1922, Orthonymus, and Cyclonaias. The subgenus Bullata Frierson, 1927
FCUP 261
was erected for the Q. pustulosa species group. Frierson (1927) in the printed errata,
recognized Bullata Frierson, 1927 non Jousseaume, 1875 [Gastropoda] was preoccupied and
coined Pustulosa Frierson, 1927 as the replacement name.
Graf & Cummings (2007) followed Haas (1969b) and Starobogatov (1970) in
recognizing Amphinaias Crosse and Fischer in Fischer & Crosse, 1894 with Unio couchiana
Lea, 1860 as the type species. Amphinaias was interpreted to include the taxa in the Quadrula
pustulosa group. This action cannot be accepted as the shell shape of Amphinaias couchiana
is more consistent with the Quadrula quadrula group and Amphinaias is considered a junior
synonym of Quadrula (Williams et al 2017).
Considering Unio pustulosa Lea, 1831 and tuberculata Rafinesque, 1820 are found in
the same clade (see Campbell & Lydeard 2012), the generic name Pustulosa Frierson, 1927
is a junior synonym of Cyclonaias Pilsbry in Ortmann & Walker 1922 (see Williams et al 2017).
Expanding his work on central American taxa, Haas (1929) placed taxa in the Unionidae and
divided the taxa among the subfamilies Lampsilinae, Anodontinae Rafinesque, 1820,
Unioninae, and Quadrulinae. Haas (1929) placed species he assigned to Crenodonta Schülter,
1838 and Rotundaria, but did not describe or erect the subfamily. There was no designation of
a type genus, however, he commented, he was putting the genus Rotundaria in the
Quadrulinae. He provided no discussion of other genera included in the subfamily.
Modell (1942) was the first to clarify which genera might be considered to belong to the
subfamily Quadrulinae Haas, 1929, not the subfamily concept of Ihering (1901) (Table 1).
Haas (1969a) defined the subfamily Quadrulinae Haas, 1929 and included 17 genera
(Table 1). Five genera are Asian, one is European and two are Central American in distribution.
They all have thick shells, often with some pustules and/or plications. This publication was
followed in the same year by the section on Unionoida in the Treatise of Invertebrate
Paleontology (Haas 1969b) where once again, Haas claimed authorship of Quadrulinae.
Modern genera included in the Quadrulinae are the same except that two genera were moved
to subgenera of Amblema Rafinesque, 1820 and one subgenus of Quadrula was changed,
Amphinaias (Table 1).
Starobogatov (1970) treated the Quadrulini as belonging to the Quadrulinae, but placed
it in the Amblemidae Rafinesque, 1820. His concept of the Quadrulinae included three tribes:
Parreysiini Henderson, 1935 [primarily the subcontinent of India], Lamprotulini Modell, 1942
[Asia and southeast Asia] and Quadrulini [North and Central America]. These taxa are well
sculptured, with the Lamprotulini and Quadrulini having sculptured, thick shells often with
pustules and some plications. Only the taxa assigned to the Quadrulini are listed in Table 1.
Arguably, the first phylogenetic analysis of the Unionoidea was presented by Heard & Guckert
(1971). They divided the Unionoidea into the Margaritiferidae Henderson, 1929, Amblemidae
and the Unionidae. Amblemidae was divided among three subfamilies, Ambleminae
262 FCUP
Rafinesque, 1820, Gonideinae Ortmann, 1916 and a new subfamily Megalonaiadinae Heard
& Guckert, 1970. The Amblemidae included the Ambleminae with Quadrulinae Ihering, 1901
as a junior synonym. This subfamily included Amblema Rafinesque, 1820, Elliptoideus
Frierson, 1927, Fusconaia Simpson, 1900, Plectomerus Conrad, 1853, Quadrula, Quincuncina
and Tritogonia Agassiz, 1852.
Graf & Cummings (2007), Bouchet & Rocroi (2010) and Carter et al (2013) correctly used
Quadrulini Ihering, 1901 and included seven genera (Table 1).
Modern molecular work on the unionid fauna has determined that the taxa in the
Quadrulinae are assigned to the Ambleminae, a subfamily restricted to North America
(Campbell & Lydeard 2012). Graf & Cummings (2007) listed eight genera in the Quadrulini.
Asian taxa that historically have been placed in the Quadrulinae (Table 1), have recently been
moved to other subfamilies and/or the Margaritiferidae (Lopes-Lima et al 2017, 2018; Huang
et al 2018; Zieritz et al 2018).
Graf & Cummings (2007) provided a synthesis of published information. (Table 1)
Williams et al (2017) provided the latest revised list of the unionid fauna of the United States
and Canada and listed taxa assigned to the Quadrulini in six genera (Table 1). Graf &
Cummings (2018) have updated the website that now matches Williams et al (2017),
recognizing six genera in the Quadrulini (Table 1).
This study focused on Quadrula sensu lato and recognized four genera in what had been
originally a single genus (Table 1). Currently, two species in this tribe are presumed extinct:
Theliderma stapes (Lea, 1831) and Quadrula couchiana. (Williams et al 2008; Howells 2013).
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Kondakov AV, Prié V, Sousa R, Varandas S, Vikhrev IV, Teixeira A, Wu R-W, Wu X-P, Zieritz
A, Froufe E, Bogan AE. 2018. Expansion and systematics redefinition of the most threatened
98-118.
Modell H. 1942. Das natürliche system der Najaden. Archiv für Molluskenkunde 74, 161-191.
Ortmann AE. 1911. A monograph of the najades of Pennsylvania. Parts I and II. Memoirs of
Ortmann AE. 1912. Notes upon the families and genera of the najades. Annals of the Carnegie
Museum 8, 222-365.
Ortmann AE. 1916. The anatomical structure of Gonidea angulata (Lea). The Nautilus 30, 50-
53.
266 FCUP
Ortmann AE. 1919. A monograph of the naiades of Pennsylvania. Part III: systematic account
of the genera and species. Memoirs of the Carnegie Museum 8, 1-384.
Rafinesque CS. 1819. Prodrome de 70 nouveaux Genres d’Animaux découverts dans

l’intérieur des États-Unis d’Amérique, durant l’année 1818. Journal de Physique, de Chimie,
d’Histoire Naturelle et des Arts 88, 417-429.
Rafinesque CS. 1820. Monographie des coquilles bivalves fluviatiles de la Rivière Ohio,
contenant douze genres et soixante-huit espèces. Annales Générales des Sciences
Physiques a Bruxelles 5, 287-322.
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Encyclopedia or Dictionary of Arts and Sciences, Comprising an Accurate and Popular View
of the Present Improved State of Human Knowledge. Volume 2. First edition. Samuel A.
Mitchel and Horace Ames, Philadelphia, USA.
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nebst Andeutung aller bis jetzt von mir bei Halle gefundenen: Land- und Flussconchylien zur
Erleichterung des Tausches für Freunde der Conchyliologie. Halle Druck der Gebauerschen
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Parts I-III. Bryant Walker, Detroit, Michigan, USA.
Starobogatov YI. 1970. Mollusk fauna and zoogeographical partitioning of continental water
reservoirs of the world. Akademiya Nauk SSSR. Zoologischeskii Instituti Nauka, Leningrad,
Russia.
Swainson W. 1840. A Treatise on Malacology or the Natural Classification of Shells and Shell-
fish. Printed for Longman, Orme, Brown, Green and Longmans, London, UK.
Walker B. 1918. A synopsis of the classification of the fresh-water Mollusca of North America,
north of Mexico, and a catalog of the more recently described species, with notes.
Miscellaneous Publication No. 6, Museum of Zoology, University of Michigan, USA.
FCUP 267
Williams JD, Bogan AE, Garner JT. 2008. Freshwater mussels of Alabama and the Mobile
Basin in Georgia, Mississippi, and Tennessee. University Alabama Press, Tuscaloosa, USA.
Williams JD, Bogan AE, Butler RSS, Cummings K, Garner JT, Harris JL, Johnson NA, Watters
TG. 2017. A revised list of the freshwater mussels (Mollusca: Bivalvia: Unionida) of the United
Zieritz A, Bogan AE, Froufe E, Klishko O, Kondo T, Kovitvadhi U, Kovitvadhi S, Lee JH, Lopes-
Lima M, Pfeiffer JM, Sousa R, Do VT, Vikhrev I, Zanatta DT. 2018. Diversity, biogeography
and conservation of freshwater mussels (Bivalvia: Unionida) in East and Southeast Asia.
268 FCUP
Supplementary Appendix 2. The description of Theliderma johnsoni n. sp.
Class: Bivalvia Linnaeus, 1758

Order: Unionida Gray, 1854
Family: Unionidae Rafinesque, 1820
Subfamily: Ambleminae Rafinesque, 1820
Tribe: Quadrulini von Ihering, 1901
Genus: Theliderma Swainson, 1840
Species: Theliderma johnsoni n. sp. Bogan & Lopes-Lima (Figs. 1-8)
Common name: Southern Monkeyface
Holotype NCSM 30474 (Figs. 1-2); (2 valves, 1 pair; body fixed in formalin and DNA COI
Barcode, GenBank reference: MK503289) Alabama River, ARM 107.5, lower end of Wilcox
Bar, 40 m from left descending bank, point estimated 2.57 air miles SSE centre of Yellow Bluff,
[Coy Quad.]. Wilcox County, Alabama, geographic coordinates (WGS84; 31.93222, -
87.45389); date collected: 19 July 2000; collectors: J.T. Garner, P. Kilpatrick.
Figures 1 (left) and 2 (right). Theliderma johnsoni n. sp. Holotype NCSM 30474, shell length
70.4 mm.
Paratypes: NCSM 7098, (10 valves, 5 pair), Tombigbee River at US 82, [2 air miles] W [centre]
of Columbus, [Columbus South Quad.]. geographic coordinates (WGS84; 33.49342, -
88.46031), 3 September 1978; NCSM 7103, (1 valve), Tombigbee River at US 82[US 45], [2
air] miles W [centre] of Columbus, [Columbus South Quad.], geographic coordinates (WGS84;
33.49342, 88.46031), 27 May 1978; NCSM 7106 (Figs. 3-4), (22 dry valves, 11 pair),
Tombigbee River [Columbus Lake], ca. 0.5 miles above MS 50 crossing, 7.4 air miles NW
[centre] of Columbus, [Columbus North Quad.], geographic coordinates (WGS84; 33.59106,
88.48238), 3 September 1978; NCSM 46889 (Figs. 5-6), (4 dry valves, 2 pair), Tombigbee
River, about 9.5 [air] miles S [centre] of Columbus, 14 [air] miles ENE [centre] of Crawford,
[Trinity Quad.]; geographic coordinates (WGS84; 33.36083, -88.38111), July 1972.; NCSM
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33722, (2 dry valves, 1 pair), Alabama River (ARM 30.4), [ca. 3.7 air miles ESE centre town of
Carlton], [Carlton Quad.]. geographic coordinates (WGS84: 31.33167, - 87.78389), 30 July
1996; NCSM 26976, (2 dry valves, 1 pair), Cahaba River, upstream of AL 14, right descending
bank, [1.24 air miles NW centre Sprott], Sprott Quad. geographic coordinates (WGS84:
32.68700, -87.23883), 16 May 2001. NCSM 27153 (Figs. 7, 8), (4 dry valves, 2 pair), Cahaba
River, 1.5 [air] miles NNE [centre] of Heiberger, just E of CR 47, [Walter C Givhan Bridge],
[Heiberger Quad.], geographic coordinates (WGS84: 32.77631, - 87.27242), 19 September
1990. NCSM 33714, (2 dry valves, 1 pair), Cahaba River, 2.0 [air] miles NNW [centre] Sprott,
[Sprott Quad.], geographic coordinates (WGS84: 32.70699, -87.23769), 13 June 1993; NCSM
33719, (2 dry valves, 1 pair), Cahaba River, at first gravel bar below Walton Creek, [ca. 2.78
air miles SSE centre Harrisburg], [Harrisburg Quad.]; geographic coordinates (WGS84;
32.83972, -87.20361), 15 October 1993.
Figures 3 (left) and 4 (right). Theliderma johnsoni n. sp. Paratype NCSM 7106.10, shell length:
41.5 mm.
46.6 mm.
270 FCUP
80.3
Etymology: Theliderma johnsoni n. sp. is named in honour of Dr. Paul D. Johnson in

recognition of his significant contributions to the conservation, natural history and captive
propagation of freshwater bivalves and freshwater gastropods. Paul is the head of Alabama
Aquatic Biodiversity Center, Alabama Department of Conservation and Natural Resources,
Marion, Alabama. The common name, Southern Monkeyface, is to note the close
conchological similarity with the sister species, Monkeyface, Theliderma metanevra
(Rafinesque, 1820), in the Mississippi River basin.
Diagnosis: Theliderma johnsoni n. sp. is distinguished from other unionid species by a

combination of the following characters: thick shell, inflated, strong hinge teeth, broad
interdentum; pallial line distant from ventral shell margin anteriorly, periostracum varies from
yellowish, greenish-yellow to brown or black, usually without chevrons but may have green
spots. Umbo is elevated above the hinge line, posterior ridge without pustules or knobs,
elevated and narrow near the umbo, becoming broader ventrally, posterior and ventral margins
emarginate anterior and poster to the posterior ridge. Based on high genetic divergence
(uncorrected p-distance ≥3.2% COI and ≥3.5% ND1 from all Theliderma spp.) and Fourier
analysis, (89-95 % of the time separated from T. metanevra) (Figs. 7E and 7F).
Description: Shell's length reaches about 100 mm, thick, quite inflated. Shell outline is
quadrate to rhomboidal, anterior shell margin broadly rounded, dorsal shell margin straight
posterior to the umbo, ventral shell margin broadly-rounded and slightly emarginate anterior to
the posterior ridge, posterior margin straight to emarginate, posterior ridge is narrow and
elevated dorsally, becoming broader posterior ventrally, lacking any large knobs on the ridge
but may have marked growth lines. Posterior slope rather steep, becoming flattened posterior
ventrally with an emargination at the posterior ventral margin of the posterior ridge, posterior
slope usually covered with short plications, but no pustules, extending from posterior ridge to
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the posterior margin. Umbo is narrow and raised dorsally, elevated above the hinge line, umbo
sculpture unknown. Periostracum yellowish, greenish-brown to brown or black, becoming
darker with age, this species may have green marks on the younger shell but not chevrons
and older shells a uniform colour. Shell surface with pustules or elongate pustules covering
disk of shell from the posterior ridge to anterior shell margin in younger specimens, becoming
restricted to the disk near the posterior ridge and in large shells, pustules disappear completely.
Pseudocardinal tooth large and single in right valve, often with a small blade-like tooth anterior
to the main tooth and followed posteriorly by a less prominent denticle posterior to the large
tooth, two large pseudocardinal teeth in left valve. Lateral teeth are short with a single heavy
tooth in the right valve and a pair in the left valve. Interdental area flat and wide. Anterior
adductor muscle scar is deep and smooth, pedal protractor muscle scar posterior and slightly
ventral to anterior adductor muscle scar, separate from the adductor muscle scar, somewhat
deep and smooth, anterior pedal retractor muscle scar located posterior dorsal margin of
anterior adductor muscle scar and etched into the base of the pseudocardinal tooth, posterior
adductor muscle scar shallow and faint, posterior pedal retractor muscle scar faint, may merge
on dorsal edge of posterior adductor muscle scar. Pallial line distant from the ventral margin
on the anterior portion of the shell, well impressed anteriorly becoming faint posteriorly, umbo
cavity deep and open, nacre colour white.
Other Material Examined: Mobile Bay Basin, Tombigbee River. Mississippi, Lowndes
County: NCSM 7103, (length 1 dry left valve), Tombigbee River at US 82[US 45], [2 air] miles
W [centre] of Columbus, [Columbus South Quad.]. 33.49342o N - 88.46031o W, 27 May 1978.
Comparison with Similar Species: Theliderma johnsoni n. sp., resembles the shells of
Quadrula quadrula (Rafinesque, 1820) and Q. apiculata (Say, 1829) [= Q. quadrula] which
have pustules on the posterior ridge lacking in T. johnsoni n. sp., but has a pronounced distinct
poster ridge lacking in these two species of Quadrula while T. johnsoni n. sp. lacks pustules.
Quadrula rumphiana (Lea, 1852) [= Q. quadrula] has a well-defined posterior ridge without
pustules but has a sulcus anterior to the posterior ridge and has pustules in the sulcus and on
the shell disk. Theliderma johnsoni n. sp. lacks the sulcus anterior to the posterior ridge found
in Q. apiculata, Q. quadrula and Q. rumphiana. Theliderma stapes (Lea, 1831) posterior ridge
is smooth but the posterior slope is shorter and steeper than T. johnsoni n. sp.
Distribution: Currently known from the Mobile Bay basin including the Alabama, Cahaba and
Coosa rivers occurring across eastern Mississippi, Alabama and north-western Georgia (See
Williams et al 2008). The distribution of the Mobile Basin populations was reported as restricted
272 FCUP
to the basin typically below the Fall Line in riverine reaches in the Alabama, Tombigbee and
Cahaba rivers, while it extended up the Coosa River basin to Georgia (Williams et al 2008).
Habitat and Biology: Theliderma johnsoni n. sp. is found living from headwaters in the Coosa
River Basin and below the Fall line in the Tombigbee, Cahaba and Alabama rivers. It lives in
flowing waters and inhabits substrates with varying mixtures of gravel and sand.
Conservation Status: Williams et al (1993) listed Quadrula metanevra [now

Theliderma metanevra] as Currently Stable. Since T. johnsoni n. sp. has been split from T.
metanevra, T. johnsoni n. sp. will have to have a new conservation status designated. Williams
et al (2008) reported: “there has been no evidence of robust recruitment in any Alabama
population of Q. metanevra [both Tennessee and Alabama basins] during the past decade.”
This would suggest this species may warrant a listing as threatened.
Comparative material examined:
Quadrula apiculata
Mobile Bay Basin, Alabama River Drainage, Alabama River, Alabama, Monroe County:
NCSM 33694, (8 dry valves, 4 pair), Alabama River, ARM 82.5, [at Haines Island], [5.23 air miles
W centre Franklin], [Franklin Quad.]. 31.72417 N - 87.49973 W, 31 July 1996.
Mobile Bay Basin, Alabama River Drainage, Alabama, Dallas County:

NCSM 45131, (12 dry valves, 6 pair), Bogue Chitto Creek, upstream of SR 22 bridge, [5.4 air
miles ENE centre Safford], [Safford Quad.]. 32.30648 N -87.28007 W, 17 August 2006.
Pearl River Basin, Pearl River Drainage, Mississippi, Pearl River County:
NCSM 33700, (12 dry valves, 6 pair), East Pearl River, Moores Bayou, near Icebox Slough,
[2.32 air miles W centre of Industrial], [Industrial Quad.]. 30.56943 N - 89.80167 W, 21-22
August 1995.
Matagorda Bay Basin, Colorado River Drainage, Texas, Llano County:

NCSM 6090, (2 dry valves,1 pair), Lake Buchanan (upper), [point estimated 4 air miles NE
centre Bluffton], [Lake Buchanan Quad.]. 30.86692 N -98.44973 W, 5 May 1964.
Rio Grande Basin, Rio Grande Drainage, Texas, Webb County:

NCSM 6091, (2 dry valves, 1 pair), Lake Casa Blanca, [ca. 5.27 air miles NE center Laredo],
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[Laredo East Quad.]. 27.54834 N -99.4.3367 W, 5 October 1994.
Quadrula quadrula
Mississippi River Basin, Arkansas River Drainage, Neosho River, Kansas, Woodson
County:
NCSM 28981, (4 dry valves, 2 pair), Neosho River, below low head dam, on exposed bars and
in the channel, in Neosho Falls, [point estimated 0.32 air miles ENE centre town of Neosho
Falls], [Neosho Falls Quad.]. 38.0076 N -95.552 W, 10 October 2003.
Mississippi River Basin, Ohio River Drainage, Ohio River, Kentucky, McCracken
County:
NCSM 7112, (2 dry valves, 1 pair) Ohio River, ca. 4 [air] miles above [NW centre] Paducah,
[Paducah West Quad.]. 37.12126 N - 88.65439 W, 1980.
Mississippi River Basin, Ohio River Drainage, Tennessee River, Alabama, Limestone
County:
NCSM 6186, (2 dry valves, 1 pair), Tennessee River, TRM 306, Decatur Boat Harbour, [point
estimated 1.86 air miles S centre Whiteside], [Decatur Quad.]. 34.60722 N - 86.95805 W, 2
February 2000.
Quadrula rumphiana
Mobile Bay Basin, Alabama River Drainage, Coosa River, Conasauga River, Georgia,
Murry-Whitfield County line:
NCSM 6888, (2 dry valves, 1 pair), Conasauga River at County Route 2, [point estimated at the
town of Beaverdale], [Beaverdale Quad.]. 34.92127 N -84.84189 W, date collected unknown.
Mobile Bay Basin, Alabama River Drainage, Tombigbee River Drainage, Sipsey River,
Alabama, Greene-Pickens County line:
NCSM 6139, (4 dry valves, 2 pair), Sipsey River, 0.5 kilometres downstream of bridge and
railroad, along the right descending side of the river, [6.19 air miles S centre Aliceville],
[Aliceville South Quad.]. 33.04139 N -88.13778 W, 8 June 1999.
Mobile Bay Basin, Alabama River Drainage, Tombigbee River Drainage, Alabama,
Lowndes County:
NCSM 6886, (8 dry valves, 2 pair, 2 right and 2 left valves), Tombigbee River at [US 82/US
274 FCUP
45], [2 air] miles W [centre] of Columbus, [Columbus South Quad.]. 33.49342 N -88.46031 W,
27 May 1978.
Theliderma metanevra
Mississippi River Basin, Tennessee River Drainage, Tennessee River, Tennessee,

Hardin County:
NCSM 33718 (2 dry valves, 1 pair), Tennessee River, River Mile 201.3, [Counce Quad.],
35.1087 N -88.2973 W, 28 July 1988. NCSM 45841 (4 dry valves, 2 pair), [Tennessee River],
TRM 195.5-197.3, Diamond Island area, [point estimated 5.7 air miles to 4.69 air miles NW
centre Nixon], [Pittsburgh Landing Quad.]. start 35.18461 N -88.31265 W end 35.16167 N -
88.3168 W, 14 February 1987.
Mississippi River Basin, Tennessee River Drainage, Tennessee River, Alabama,

Marshall County:
NCSM 7104, (2 dry valves, 1 pair), Wheeler Reservoir [Wheeler Lake], ca. 1 mile below
Guntersville Dam on the left shoreline, ca. 15 ft. below the high pool, Wheeler low plus, [8.2 air
miles NNW centre Guntersville], [Guntersville Dam Quad.]. 34.42916 N -86.40913 W, 5
November 1979.
Mississippi River Basin, Tennessee River Drainage, Tennessee River, Alabama, Colbert
County:
NCSM 6080, (2 dry valves, 1 pair), Tennessee River, TRM 249.0, tail end of 3rd island of the
Buck Island complex, ca. 100 m. from the island, [point estimated 2.4 air miles ENE centre
Pride], [Pride Quad.]. 34.73222 N -87.77861 W, 11 August 1999.
Mississippi River Basin, Black River Drainage, Black River, Missouri, Wayne County:
NCSM 48442, (2 dry valves, 1 pair), Black River, [point estimated at CR 417 crossing, 1.01 air
miles S centre] Williamsville, [Williamsville Quad.]. 36.95672 N -90.54997 W, 4 July 1971.
Theliderma stapes
Mobile Bay Basin: Alabama River Drainage, Tombigbee River, Alabama, Pickens
County:
NCSM 101849, (4 dry valves, 2 pair), Tombigbee River, [point estimated 6.15 air miles S centre
Pickensville], River Mile 324.4, Memphis Landing, [Pickensville Quad.]. 33.13987 N 88.2858
W, 24 October 1976
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Table 1
Measurements of Theliderma johnsoni n. sp. Type series.
Type Museum Catalog

Status acronym number River State Length Height Width
Holotype NCSM 30474 Alabama AL 70.4 59.1 47.0
Paratype NCSM 7098.1 Tombigbee MS 63.9 55.6 42.1
Paratype NCSM 26976 Cahaba AL 95.3 73.8 57.4
Paratype NCSM 27153.1 Cahaba AL 70.6 59.3 37.5
Paratype NCSM 27153.2 Cahaba AL 80.3 68.1 42.5
Paratype NCSM 33722 Alabama AL 66.3 54.1 39.8
References
Gray JE. 1854. A revision of the arrangement of the families of bivalve shells (Conchifera). The
Annals and Magazine of Natural History (Series 2) 13, 408-418.
Ihering H von. 1901. The Unionidae of North America. The Nautilus 15, 37-39, 50-53.
Lea I. 1831. Observations on the naïades, and descriptions of new species of that and other
families. Transactions of the American Philosophical Society 4 (New Series), 63-121.
Lea I. 1852. Descriptions of new species of the family Unionidae. Transactions of the American
Philosophical Society (New Series) (Part 2) 10, 253-294.
276 FCUP
Linnaeus C. 1758. Systema Naturae. Edition X. (Systema naturae per regna tria naturae,
secundum classes, ordines, genera, species cum characteribus, differentiis, synonymis, locis.
Tomus I. Edtio decima, reformata.) Holmiae.
Rafinesque CS. 1820. Monographie des coquilles bivalves fluviatiles de la Rivière Ohio,
contenant douze genres et soixante-huit espèces. Annales Générales des Sciences
Physiques à Bruxelles 5, 287-322.
Say T. 1829. Descriptions of some new terrestrial and fluviatile shells of North America. The
Disseminator of Useful Knowledge; containing hints to the youth of the United States, from the
School of Industry, New Harmony, Indiana, USA.
Swainson W. 1840. A Treatise on Malacology or the Natural Classification of Shells and Shell-
fish. Printed for Longman, Orme, Brown, Green and Longmans, London, UK.
Williams JD, Warren Jr. ML, Cummings KS, Harris JL, Neves RJ. 1993. Conservation status
of the freshwater mussels of the United States and Canada. Fisheries 18, 6-22.
Basin in Georgia, Mississippi, and Tennessee. University of Alabama Press, Tuscaloosa, USA.
FCUP 277
CHAPTER 6
Phylogeny of the family Margaritiferidae
Paper V
Expansion and systematics redefinition of the most threatened freshwater
mussel family, the Margaritiferidae
Froufe E, Bogan AE
Article published in Molecular Phylogenetics and Evolution 127, 98-118 (2018).
DOI: 10.1016/j.ympev.2018.04.041
278 FCUP
Expansion and systematics redefinition of the most threatened freshwater

mussel family, the Margaritiferidae
Manuel Lopes-Lima1,2,3,⁎, Ivan N. Bolotov4,5, Van Tu Do6, David C. Aldridge7, Miguel M.
Fonseca2, Han Ming Gan8, Mikhail Y. Gofarov4,5, Alexander V. Kondakov4,5, Vincent Prié9,
Ronaldo Sousa2,10, Simone Varandas11, Ilya V. Vikhrev4,5, Amílcar Teixeira12, Rui-Wen Wu13,
Xiaoping Wu13, Alexandra Zieritz14, Elsa Froufe2, Arthur E. Bogan15
1
Vairão, Rua Padre Armando Quintas 7, 4485-661 Vairão, Portugal
2
CIIMAR/CIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal
de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos S/N, 4450-208 Matosinhos, Portugal
3
SSC/IUCN - Mollusc Specialist Group, Species Survival Commission, International Union for Conservation of
Nature, c/o The David Attenborough Building, Pembroke Street, CB2 3QZ Cambridge, United Kingdom
4
IBIGER - Institute of Biogeography and Genetic Resources, Federal Center for Integrated Arctic Research,
Russian Academy of Sciences, Severnaya Dvina Emb. 23, 163000 Arkhangelsk, Russian Federation
5
Northern Arctic Federal University, Severnaya Dvina Emb. 17, 163000 Arkhangelsk, Russian Federation
6
Department of Aquatic Ecology and Water Environment, Institute of Ecology and Biological Resources, Vietnam
Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Ha Noi, Viet Nam
7
Department of Zoology, University of Cambridge, The David Attenborough Building, Pembroke Street, Cambridge
CB2 3QY, United Kingdom
8
Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong, 3220
Victoria, Australia
9
Institut de Systématique, Évolution, Biodiversité ISYEB - UMR 7205 - CNRS, MNHN, UPMC, EPHE, Muséum
national d’Histoire Naturelle, Sorbonne Universités, 57 rue Cuvier, CP26, F-75005 Paris, France
10
11
Trás-os-Montes and Alto Douro, Forestry Department, 5000-801 Vila Real, Portugal
12
CIMO-ESA-IPB - Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus
de Santa Apolónia, 5301-854 Bragança, Portugal
13
School of Life Sciences, Center for Watershed Ecology, Institute of Life Science, Nanchang University, Nanchang
330031, People’s Republic of China
14
School of Environmental and Geographical Sciences, University of Nottingham Malaysia Campus, Jalan Broga,
43500 Semenyih, Malaysia
15
Research Laboratory, North Carolina State Museum of Natural Sciences, MSC 1626, Raleigh, NC 27699-1626,
United States.
⁎
Abstract
Two Unionida (freshwater mussel) families are present in the Northern Hemisphere; the
Margaritiferidae, representing the most threatened family, and the Unionidae, which include
FCUP 279
several genera of unresolved taxonomic placement. The recent reassignment of the poorly
studied Lamprotula rochechouartii from the Unionidae to the Margaritiferidae motivated a new
search for other potential species of margaritiferids from members of Gibbosula and
Lamprotula. Based on molecular and morphological analyses conducted on newly collected
specimens from Vietnam, we here assign Gibbosula crassa to the Margaritiferidae.
Additionally, we reanalysed all diagnostic characteristics of the Margaritiferidae and examined
museum specimens of Lamprotula and Gibbosula. As a result, two additional species are also
moved to the Margaritiferidae, i.e. Gibbosula confragosa and Gibbosula polysticta. We
performed a robust five marker phylogeny with all available margaritiferid species and discuss
the taxonomy within the family. The present phylogeny reveals the division of Margaritiferidae
into four ancient clades with distinct morphological, biogeographical and ecological
characteristics that justify the division of the Margaritiferidae into two subfamilies (Gibbosulinae
and Margaritiferinae) and four genera (Gibbosula, Cumberlandia, Margaritifera, and
Pseudunio). The systematics of the Margaritiferidae family is redefined as well as their
distribution, potential origin, and main biogeographic patterns.
Keywords
Unionida, Margaritifera, Lamprotula, Gibbosula, Phylogeny, Bivalvia
280 FCUP
Introduction
Unionida freshwater mussels: diversity and conservation status
The Unionida is the only strictly freshwater order of bivalves (Bogan 2008). It is an old and
widespread order with approximately 800 described species in 180 genera (Bogan 2008). Six
families are currently recognized within Unionida, but only the Unionidae and the
Margaritiferidae are widespread in the Northern Hemisphere (Bogan 2008). While the
Unionidae is extremely diverse (> 600 species), until the present study, only 12 species in one
genus scattered across North America, Europe, North Africa, and Asia had been recognized
within the Margaritiferidae (Bolotov et al 2016; Araujo et al 2017). Additionally, both families
are declining globally and are highly endangered, especially the Margaritiferidae, where all
species assessed with enough data present a near-threatened or threatened conservation
status (IUCN 2018).
Taxonomical history of the Margaritiferidae and its diagnostic characters

Until the end of the twentieth century, the taxonomy and systematics of Unionida had been
based primarily on conchological and anatomical characters (e.g. Haas 1969a; Parmalee &
Bogan 1998; Watters et al 2009). Due to the better availability of Unionida specimens from
North America and Europe, those from tropical and the Southern Hemisphere regions were
relatively poorly studied (Simpson 1900, 1914; Ortmann 1921; McMichael & Hiscock 1958).
Early systematists encompassed all genera of freshwater mussels, including
Margaritana (=Margaritifera) species, within the family Unionidae (Table 1: Lea, 1836, 1838,
1852, 1870; Simpson 1900, 1914; Frierson 1927). However, at the beginning of the twentieth
century, Ortmann (1910) determined that some anatomical characters of some genera were
distinct and of prime systematic value. This author erected a new taxon, first as a sub-family,
Margaritaninae within Unionidae, but immediately after as a separate family, the
Margaritanidae (=Margaritiferidae Henderson 1929, (1910)), both with the genus and species
Margaritana (=Margaritifera) margaritifera (Linnaeus, 1758) as the type. As defined by
Ortmann (1910, 1911a,b, 1912), the Margaritanidae presented distinct anatomical features
from the other Unionidae species, including the lack of discrete apertures separated by mantle
fusions, particular gill and marsupium structure, and glochidial (larval) shape (Table 2).
Although at first other malacologists did not recognize Margaritiferidae as a separate family
(e.g. Simpson 1914), soon it was accepted by most researchers (e.g. Henderson 1929),
including in the comprehensive classification of the Unionida published by Haas (1969a,b).
FCUP 281
Table 1
Comparison of Margaritiferidae classifications. Fossil genera excluded. (S) synonym. Superscripts: 1 Under tribe Heudeanini; 2 under subfamily
Pseudodontinae; 3 under tribe Margaritiferini; 4 under tribe Leguminaiini
Graf & Cummings (2007, 2017)

Ortmann (1911a,b, 1912)
Starobogatov (1970)
Starobogatov (1995)
Bolotov et al (2016)
Araujo et al (2017)
Henderson (1929)
Huff et al (2004)
Ortmann (1910)
Morrison (1975)
Frierson (1927)
Haas (1969a,b)
Modell (1942)
Modell (1949)
Modell (1964)
Thiele (1934)
Smith (2001)
Boss (1982)
This study
Margaritanidae ✓ ✓
Margaritaninae ✓ ✓
Margaritana ✓ ✓ ✓ ✓
(Margaritana) ✓ ✓
Margaritiferidae ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Margaritiferinae ✓ ✓ ✓ ✓ ✓ ✓
Margaritifera ✓ ✓ ✓ ✓ ✓3 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
(Margaritifera) ✓ ✓
Margaritanopsis s ✓ ✓ ✓1 ✓ ✓ s s
(Margaritanopsis) ✓ ✓
Cumberlandia ✓ ✓ ✓ ✓ ✓ ✓3 ✓ ✓ s s s ✓
(Cumberlandia) ✓
Potomida ✓
Pseudunio s ✓3 ✓ ✓ s ✓
(Pseudunio) ✓ ✓
Dahurinaia s ✓1 ✓ s s s
Gibbosula ✓ ✓
Ptychorhynchus ✓ ✓1 ✓1 ✓
Schalienaia ✓3 ✓ s
282 FCUP
Graf & Cummings (2007, 2017)

Ortmann (1911a,b, 1912)
Starobogatov (1970)
Starobogatov (1995)
Bolotov et al (2016)
Araujo et al (2017)
Henderson (1929)
Huff et al (2004)
Ortmann (1910)
Morrison (1975)
Haas (1969a,b)
Frierson (1927)
Modell (1942)
Modell (1949)
Modell (1964)
Thiele (1934)
Smith (2001)
Boss (1982)
This study
Cucumerunioninae ✓ ✓ ✓
Cucumerunio ✓ ✓
Virgus ✓ ✓
Heudeaninae ✓ ✓ ✓ ✓
Heudeana ✓ ✓ ✓ ✓
Schepmania ✓ ✓ ✓ ✓
Ctenodesma ✓ ✓ ✓ ✓ ✓
Pseudodontinae ✓ ✓ ✓ ✓
Pseudodon ✓ ✓ ✓
Monodontina ✓ ✓ ✓ ✓
Nasus ✓ ✓ ✓
Compsopseudodon ✓ ✓
Obovalis ✓ ✓ ✓
Pseudodontopsis ✓ ✓ ✓4
Leguminaia ✓ ✓ ✓ ✓4
Microcondylaea ✓ ✓ ✓4
Leptanodonta ✓ ✓ ✓4
Gonidea ✓ ✓ ✓ ✓
Arcidopsinae ✓
Arcidopsis ✓
Trapezoideus ✓
Solenaia ✓2 ✓
FCUP 283
Table 2
Characters used to define and diagnose Margaritiferidae. 1 papillae present only; 2 hinge teeth reduced.
Ortmann Ortmann Thiele Modell
Character
(1910) (1911a, b) (1934) (1942, 1949, 1964)
1. Diaphragm incomplete formed by gills ✓ ✓
2. Anterior end of inner gills distant from palps ✓ ✓
3. Branchial and anal siphons/apertures ill-defined not closed ✓
4. Supra anal not separate ✓ ✓ ✓ ✓ (1949)
5. Incurrent aperture with bifid or arborescent papillae ✓1 Elongate, unaffected (1949)
6. Gills no water tubes ✓ ✓ ✓ (1949)
7. Gills irregular scattered interlamellar connections ✓ ✓ ✓ ✓ (1949)
8. Gills not fused with mantle posteriorly ✓ ✓ ✓ ✓ (1949)
9. Marsupium in all four gills ✓ ✓ ✓ (1949)
10. Tachytictic
11. Glochidia semilunate, hookless, irregular small teeth ✓ ✓ ✓ (1949)
12. Pedal elevators inconspicuous
13. Anus located dorsal edge posterior adductor muscle
14. Shell elongated ✓ ✓
15. Umbo low ✓
16. Shell mostly compressed
17. Shell with numerous folds/sculpture including pustules some
18. Frequently concave ventral margin
19. Shell with nacre
20. Umbo sculpture angular un-joined chevron-like hooks ✓
21. Umbo sculpture weak concentric ✓ ✓
22. Maximum shell length
23. Umbo cavity shallow
24. Periostracum heavy, blackish ✓ (1911c)
25. Shell aragonite
26. Posterior lateral teeth tend to be reduced ✓2 ✓
27. Mantle attachment scars
28. Conchiolin one layer
29. Complete hinge teeth present ✓
284 FCUP
Table 2 (cont.)
Graf &
Haas Heard & Guckert Boss Smith Araujo
Character Cummings
(1969a,b) (1971) (1982) (2001) et al (2017)
(2006)
On
1. Diaphragm incomplete formed by gills ✓ ✓ ✓ ✓ On mantle
mantle
2. Anterior end of inner gills distant from palps ✓
3. Branchial and anal siphons/apertures ill-defined not closed ✓ ✓ ✓
4. Supra anal not separate ✓ ✓ ✓
5. Incurrent aperture with bifid or arborescent papillae ✓ ✓
6. Gills no water tubes ✓ ✓ ✓
7. Gills irregular scattered interlamellar connections ✓ ✓ ✓ ✓ ✓
8. Gills not fused with mantle posteriorly ✓ ✓ ✓
9. Marsupium in all four gills ✓ ✓ ✓ ✓
10. Tachytictic ✓ ✓
11. Glochidia semi-lunate, hookless, irregular small teeth ✓ ✓
12. Pedal elevators inconspicuous ✓ ✓
13. Anus located dorsal edge posterior adductor muscle ✓ ✓
14. Shell elongated ✓
15. Umbo low
16. Shell mostly compressed ✓ ✓ ✓
17. Shell with numerous folds/sculpture including pustules
18. Frequently concave ventral margin ✓ ✓
19. Shell with nacre ✓
20. Umbo sculpture angular un-joined chevron-like hooks ✓ ✓
21. Umbo sculpture weak concentric
22. Maximum shell length 150mm 200mm
23. Umbo cavity shallow ✓ ✓
24. Periostracum heavy, blackish ✓ ✓
25. Shell aragonite ✓
26. Posterior lateral teeth tend to be reduced ✓ ✓
27. Mantle attachment scars ✓ ✓ ✓
28. Conchiolin one layer ✓ ✓
29. Complete hinge teeth present ✓
FCUP 285
In this fundamental work, the family Margaritiferidae was recognized with nine taxa (five
species and four subspecies) under a single genus, Margaritifera, divided into four subgenera:
Margaritifera, Cumberlandia, Margaritanopsis, and Pseudunio.
During the same period, alternative classifications were published (Modell 1942, 1949,
1964; Starobogatov 1970, 1995; Bogatov et al 2003) based only on few conchological
characters that proposed a much larger number of taxa in the Margaritiferidae (Table 1). These
studies were controversial and subsequently ignored by most malacologists (e.g. Boss 1982;
Smith 2001, Graf & Cummings 2007). Since the beginning of this century, the family
Margaritiferidae has been consistently restricted to around 12 species (Smith 2001; Huff et al
2004; Graf & Cummings 2006). Smith (2001), based on morphological characters only, divided
the Margaritiferidae into three genera: Pseudunio, Margaritifera, and Margaritanopsis. Soon
after, a molecular phylogenetic analysis was published using both nuclear and mitochondrial
markers on seven Margaritiferidae species (Huff et al 2004). Although these phylogenetic
analyses presented three clear clades, these did not agree with the genera previously defined
by Smith (2001), causing Huff et al (2004) to conclude that the generic name Margaritifera
should be considered for all species. In subsequent phylogenetic studies, the Margaritiferidae
has been presented consistently as monophyletic, with a marked genetic structure and divided
into three to four major clades; however, most authors have chosen not to discuss its generic
assignment keeping Margaritifera as the single genus (Huff et al 2004; Graf & Cummings 2007;
Araujo et al 2017). Nevertheless, many North American researchers continued to recognize
Cumberlandia as a valid genus (e.g. Watters et al 2009; Haag 2012).
Recently, two comprehensive five loci molecular phylogenies on the Margaritiferidae
documented several well-supported divergent clades. Bolotov et al (2016) recognized only
three main clades, assigning them as subgenera (Margaritanopsis, Margaritifera, and
Pseudunio) of Margaritifera, resembling the previous classification by Haas (1969a). Shortly
afterward, Araujo et al (2017) described five major divergent clades within the Margaritiferidae
but kept them under the same genus (Margaritifera).
Biogeography and diversification of the Margaritiferidae

The family Margaritiferidae has a broad but disjunct distribution range in the Northern
Hemisphere (Smith 2001). It presents an enigmatic biogeographic pattern with species
aggregations along the western and eastern continental margins and vast distribution gaps in
inland areas (e.g. East Europe, Urals, and Siberia), possibly reflecting vicariance events driven
by plate tectonics (Taylor 1988; Smith 2001; Huff et al 2004). Recently, Bolotov et al (2016)
and Araujo et al (2017) reviewed available biogeographic schemes explaining the origin and
expansion routes of the Margaritiferidae and independently provided new fossil-calibrated
evolutionary models. However, the time and place of origin of the entire family remained
286 FCUP
unclear (Bolotov et al 2016; Araujo et al 2017). The phylogenetic models placed the origin of
the Margaritiferidae in the mid-Cretaceous (Bolotov et al 2016) or even in the Late Triassic
(Araujo et al 2017). The strong temporal discordance between these fossil-calibrated
phylogenies together with significant topological differences and low support values in several
deep nodes suggest that both studies need additional taxon samples. Inclusion of Pseudunio
homsensis from the Orontes River in Turkey, that had been missing from the previous
phylogenetic studies (Bolotov et al 2016; Araujo et al 2017), did not help to obtain a fully
resolved evolutionary reconstruction for the family, as it appears to be a close relative of P.
auricularius (Vikhrev et al 2017). Additionally, previous analyses also lacked Margaritiferidae
taxa from eastern China (i.e. between the Indo-China Peninsula and the Amur River; Smith
2001; Bolotov et al 2015, 2016). As has already been noted (Smith 2001; Bolotov et al 2015),
the inclusion of newly discovered species from this vast range disjunction is crucial for
developing a comprehensive understanding of the biogeography of the Margaritiferidae.
Huang et al (2017) added molecular sequences of Gibbosula rochechouartii to the data set of
Araujo et al (2017) and calculated an updated fossil-calibrated phylogeny placing the origin of
the Margaritiferidae crown group in the Late Cretaceous but were not able to obtain a well-
resolved biogeographic reconstruction.
A large number of fossil specimens assigned to the Margaritiferidae has been
recovered in Europe, Middle Asia, China, Mongolia, Siberia, Japan, North America, and Africa
(e.g. Henderson 1935; Modell 1957; Martinson 1982; Ma 1996; Fang et al 2009; Van Damme
et al 2015; Bolotov et al 2016; Araujo et al 2017). However, recent phylogenetic models were
calculated using a limited set of fossil calibrations because the true phylogenetic affinities of
many fossil taxa remain unclear due to high conchological variability (Bolotov et al 2016; Araujo
et al 2017; Huang et al 2017). The high taxonomic diversity of fossil margaritiferids disagrees
with the limited number of extant taxa and likely reflects a lack of critical revisions in systematic
palaeontology rather than multiple extinction events (Schneider & Prieto 2011; Bolotov et al
2016; Araujo et al 2017). Slow substitution rates in the Margaritiferidae (Bolotov et al 2016)
allow us to expect rather delayed diversification processes within the family, although the
diversification rates in margaritiferids have never been tested to date.
Historical description and classification of some incertae sedis Unionidae taxa

Although recent phylogenetic works have increased our knowledge on the position of many
Unionida genera from the less studied African and Asian countries (e.g. Pfeiffer & Graf 2013
2015; Lopes-Lima et al 2017a; Bolotov et al 2017a,b), the most comprehensive revision of the
Unionidae classification to date placed 42 genera as incertae sedis (Lopes-Lima et al 2017a).
These included Gibbosula (Simpson 1900), whose type species was first described and
illustrated by Wood (1815) as Mya crassa from an unknown locality in China and later classified
FCUP 287
under Gibbosula (i.e. as Gibbosula crassa) within the Unionidae by Simpson (1900). A few
years later, another specimen was found in southern China and described as a new species,
i.e. Unio (Quadrula) mansuyi Dautzenberg & Fischer, 1908. Simpson (1914) placed this
species under Quadrula and did not associate it with G. crassa. A third specimen was
described in 1928 and added to Gibbosula (i.e. Gibbosula confragosa Frierson, 1928) based
on conchological similarities with G. crassa. In his comprehensive classification of the
Unionida, Haas (1969a,b) considered that Gibbosula had been superfluously created by
Simpson and listed it as a synonym of Lamprotula, inside the Unionidae. Additionally, Haas
(1969a) listed Dautzenberg & Fischer's species Unio mansuyi as a synonym of Lamprotula
crassa.
Simpson (1914) was the first to notice that G. crassa presented some typical
margaritiferid conchological features (i.e. mantle attachment scars), but due to other distinct
characters (e.g. heavy shell, well-developed teeth, and deep umbo cavity), it was retained
within the Unionidae. Later, Morrison (1975) also noted that Gibbosula had the same
characters now known to characterize the Margaritiferidae. However, this information was
overlooked by most malacologists who continued to follow Haas (1969a) and kept G. crassa
and G. confragosa under Lamprotula (e.g. Prozorova et al 2005; Graf & Cummings 2007).
Finally, some authors recently described conchological differences between the two Gibbosula
species and Lamprotula and recognized Gibbosula as a separate genus within Unionidae (He
& Zhuang 2013; Graf & Cummings 2018). Furthermore, based on conchological similarities, a
third species of Gibbosula was recently described, i.e. Gibbosula nanningensis (Qian et al
2015).
The genus Lamprotula was recently revealed to be polyphyletic and divided into
Lamprotula s.s. and Aculamprotula (Zhou et al 2007; Pfeiffer & Graf 2013). These authors also
noted that all species of Lamprotula should be comprehensively analysed to clarify their status
and relationships. For instance, based on molecular analyses, Lamprotula rochechouartii has
been moved to Margaritiferidae (Huang et al 2017). Also, morphological and molecular
characteristics of six specimens of G. crassa collected from Bang River, Cao Bang Province,
Vietnam in 2016, suggested that the species did not belong to the Unionidae but to the
Margaritiferidae (Bogan & Do 2016). The reassignment of these two Asian species (i.e. L.
rochechouartii and G. crassa) from the Unionidae to the Margaritiferidae raises the question
of whether there are other overlooked species of Margaritiferidae within this group. To address
this issue, the congeneric G. confragosa and L. rochechouartii shell types were here analysed
as well as other types of Lamprotula sp. for potentially misplaced margaritiferids.
Under these considerations, the present study aimed to: (i) perform a detailed
morphological characterization of collected G. crassa specimens, and available museum
specimens of all Margaritiferidae, Lamprotula, and Gibbosula; (ii) sequence and characterize
288 FCUP
the whole F-type mitogenome of G. crassa; (iii) produce a robust phylogeny of the
Margaritiferidae using five (nuclear and mitochondrial) markers and discuss the systematics
and taxonomy within the family; (iv) compare anatomical, conchological and ecological
characters within and among all retrieved clades; and (v) describe the potential origin and
ancient radiations of the Margaritiferidae and detect the most probable ancestral geographic
areas based on a new multi-locus fossil-calibrated phylogenetic model, using the most
complete sampling of taxa to date and an expanded calibration dataset.

Sampling and museum specimens
Six specimens of G. crassa were collected during a survey in northern Vietnam in the Bang
River, Cao Bang Province, Vietnam, in 2016. Specimens were deposited as vouchers at the
North Carolina Museum of Natural Sciences, United States of America (NCSM 102193,
102194) and the Institute of Ecology and Biological Resources, Hanoi, Vietnam (IEBR-FM 01-
03). Museum specimens of Gibbosula, Lamprotula, and Margaritiferidae, including the type
specimens of Unio mansuyi and G. confragosa, were analysed for morphology and/or genetics
(Table 3 and Supplementary Table 1). Foot tissue samples were collected and preserved in
96% ethanol for DNA extraction.
DNA extractions, sequencing, assembly, and annotation

DNA was extracted from foot samples of two G. crassa individuals and other margaritiferid
specimens (Table 3) following Froufe et al (2016). The complete F-type mitogenome of a single
G. crassa sample was then sequenced and assembled using an established pipeline (Gan et
al 2014). Mitochondrial gene annotations were performed using MITOS (Bernt et al 2013). The
final tRNAs gene limits were rechecked with ARWEN (Laslett & Canbäck 2008). Finally, in-
house scripts were applied to adjust the mtDNA protein-coding limits since MITOS seems to
underestimate gene length. The whole mitogenome sequence has been deposited in GenBank
(MH319826). The mitogenome was then visualized using GenomeVx (Conant and Wolfe 2008)
(Supplementary Fig. 1). The mitochondrial 16S rRNA and Cytochrome c Oxidase I (COI), and
the nuclear 18S rRNA, 28S rRNA, and Histone 3 (H3) gene fragments were amplified from the
extracted gDNAs of both G. crassa and the remaining margaritiferid species, following the
conditions described in Bolotov et al (2016) and Araujo et al (2017).
Individual alignments were performed for each of the five markers: COI - 654 nt, 16S - 475 nt,
18S - 1778 nt, 28S - 307 nt, and H3 - 327 nt. Each alignment was constructed with up to two
FCUP 289
representatives from all available Margaritiferidae species, including GenBank sequences

(Table 3). Representative species from each of the families of the Unionida and Neotrigonia,
Trigoniidae, the marine sister group of the Unionida (Giribet & Wheeler 2002), were included
as outgroups (Table 3). All individual datasets were aligned using the standalone version of
GUIDANCE2 (Sela et al 2015) with the MAFFT multiple sequence global pair alignment
algorithm (Katoh & Standley 2013). The following GUIDANCE parameters were used:
GUIDANCE score algorithm; 100 bootstrap replicates; and a column cut-off score of 0.8.
Substitution saturation tests for all codon positions were accomplished in the protein-coding
loci (COI, and H3) as implemented in DAMBE 6 (Xia 2017). Phylogenetic analyses were then
performed by Bayesian Inference (BI) and Maximum Likelihood (ML) on 13 partitioned
datasets from a single marker to a combination of markers as follows: (1) combined dataset 1:
COI (3 codons) + 16S + 18S + 28S + H3 (3 codons); (2) combined dataset 2: COI + 16S + 18S
+ 28S + H3; (3) mtDNA 1: COI (3 codons) + 16S; (4) mtDNA 2: COI + 16S; (5) COI (3 codons);
(6) COI; (7) 16S; (8) nDNA: 18S + 28S + H3 (3 codons); (9) nDNA: 18S + 28S + H3; (10) 28S;
(11) 18S; (12) H3 (3 codons); and (13) H3. For the BI analyses, the best-fit models of
nucleotide substitution for each partition were previously selected (Supplementary Table 2),
under the Bayesian Information Criterion (BIC) using JModelTest 2.1.10 (Darriba et al 2012).
BI analyses were performed in MrBayes v3.2.6 (Ronquist et al 2012) using the previously
selected models. Analyses were initiated with program-generated trees and four Markov
chains with default incremental heating. Two independent runs of 20 × 106 generations were
sampled at intervals of 1,000 generations producing a total of 20,000 trees. Burn-in was
determined upon the convergence of log-likelihood and parameter values using Tracer 1.6
(Rambaut et al 2014). For the ML phylogenetic analyses, sequences were analysed in RaxML
8.0.0 (Stamatakis 2014) with 1,000 bootstrap replicates, assuming a GTR + G + I model for
each partition.
Morphological and ecological assessments

To evaluate the systematics within Margaritiferidae and detect other potential margaritiferid
species, detailed conchological and anatomical characters were evaluated on newly collected
G. crassa specimens and on museum specimens of Gibbosula, Lamprotula and
Margaritiferidae, including the type specimens of Unio mansuyi and G. confragosa.
Bibliographic data on the major ecological and physiological traits were also compiled for all
margaritiferid species (Table 4). To characterize and compare glochidial size, the glochidial
size index (Gln) was calculated following Lopes-Lima et al (2017a).
290 FCUP
Table 3
List of specimens analysed, GenBank references, specimen number, locations, and museum voucher references. *not generated from a single
individual.
Taxon Specimen COI 16S 18S 28S H3
UNIONIDA
MARGARITIFERIDAE
GIBBOSULINAE
Gibbosula crassa 1 MH293546 MH293536 MH293539 MH293542 MH293549
Gibbosula crassa 2 MH293547 MH293537 MH293540 MH293543 MH293550
Gibbosula laosensis 1 KU763224 KU763193 KU763255 KU763298 KU763342
Gibbosula laosensis 2 KU763225 KU763194 KU763256 KU763299 KU763343
Gibbosula rochechouartii 1 MF072498 MF072505 MF072519 MF072512 MF072526
Gibbosula rochechouartii 2 MF072502 MF072509 MF072523 MF072516 MF072530
MARGARITIFERINAE
Cumberlandia monodonta 1 AY579131 AY579089 AY579105 AY579121 AY579144
Cumberlandia monodonta 2 MH293545 MH293535 MH293538 MH293541 MH293548
Margaritifera dahurica 1 KJ161516 KJ943526 KT343730 KT343738 AY579133
Margaritifera dahurica* 2 KJ161520 KJ943527 KJ943531 MH293544 MH293551
Margaritifera falcata 1 AY579128 AY579085 AY579101 AY579117 AY579141
Margaritifera falcata 2 AY579127 AY579084 AY579100 AY579116 AY579140
Margaritifera hembeli 1 KU763218 KU763189 KU763250 KU763293 KU763336
Margaritifera hembeli 2 KU763219 KU763190 KU763251 KU763294 KU763337
Margaritifera laevis KU763222 KU763192 KU763253 KU763296 KU763340
Margaritifera margaritifera 1 KU763227 KU763196 KU763258 KU763301 KU763345
Margaritifera margaritifera 2 AF303342 AF303301 KU763274 KU763317 KU763360
Margaritifera marrianae KU763243 KU763214 KU763283 KU763326 KU763369
Margaritifera middendorffi 1 AY579124 AY579081 AY579092 AY579108 AY579134
Margaritifera middendorffi 2 KJ161547 KJ943528 KT343726 KT343735 MH293552
Pseudunio auricularius 1 AY579125 AY579083 AY579097 AY579113 AY579137
Pseudunio auricularius 2 AF303309 AF303274 KU763247 KU763290 KU763333
Pseudunio homsensis KX550090 KX550092 KX550088 KX550086 MH293553
Pseudunio marocanus 1 EU429678 EU429689 KU763281 KU763324 KU763367
FCUP 291
Pseudunio marocanus 2 EU429679 EU429691 KU763282 KU763325 KU763368

UNIONIDAE
Lampsilis cardium KX713472 KX713226 KX713305 KX713394 KX713547
Potomida littoralis KP217871 KP217981 KU763287 KU763330 KU763373
Unio pictorum KC429109 KC429266 KC429349 KC429447 KC429186
HYRIIDAE
Hyridella australis KX713467 KX713224 KX713301 KX713389 KX713545
Triplodon corrugatus KX713505 KX713262 KX713352 KX713438 KX713585
Velesunio ambiguus KC429106 KC429263 KC429346 KC429444 KC429183
MULLERIIDAE
Anodontites elongata KX713444 KX713190 KX713268 KX713357 KX713512
Lamproscapha ensiformis KX713471 KX713225 KX713304 KX713393 KX713546
ETHERIIDAE
Etheria elliptica KX713462 KX713219 KX713296 KX713384 KX713540
IRIDINIDAE
Aspatharia pfeifferiana KC429107 KC429264 KC429347 KC429445 KC429184
Chambardia wahlbergi KX713448 KX713202 KX713277 KX713365 KX713520
Mutela hargeri KX713482 KX713237 KX713317 KX713405 KX713559
TRIGONIIDA
TRIGONIIDAE
Neotrigonia lamarckii KC429105 KC429262 KC429345 KC429443 KC429182
Neotrigonia margaritacea U56850 DQ280034 AF411690 AF411689 AY070155
292 FCUP
Table 3 (cont.)
Taxon Specimen Location Voucher

UNIONIDA
MARGARITIFERIDAE
GIBBOSULINAE
Gibbosula crassa 1 Bang River, Cao Bang, Vietnam IEBR-FM GC01
Gibbosula crassa 2 Bang River, Cao Bang, Vietnam IEBR-FM GC03
Gibbosula laosensis 1 Mun River, Thailand
Gibbosula laosensis 2 Luang Prabang, Laos MNCN15.07/12038 (N1687)
Gibbosula rochechouartii 1 Poyang Lake, Yangtze, China
Gibbosula rochechouartii 2 Poyang Lake, Yangtze, China
MARGARITIFERINAE
Cumberlandia monodonta 1 Missouri, USA
Cumberlandia monodonta 2 Meramec River, Missouri, USA
Margaritifera dahurica 1 Ilistaya River, Primorye, Russia IEPN d0088/6
Margaritifera dahurica* 2 Ilistaya River, Primorye, Russia IEPN d0089/2
Margaritifera falcata 1 Idaho, USA MCZ DNA100844
Margaritifera falcata 2 North Umpqua River, Oregon, USA MCZ DNA100699
Margaritifera hembeli 1 Valentine Creek, Louisiana, USA
Margaritifera hembeli 2 Brown Creek, Louisiana, USA
Margaritifera laevis Iwaizumi, Honshu, Japan MNCN-FW1502-2
Margaritifera margaritifera 1 Locust Creek, Pennsylvania, USA
Margaritifera margaritifera 2 Nore River, Ireland MNCN-FW1490-1
Margaritifera marrianae Hunter Creek, Alabama, USA UAUC1651
Margaritifera middendorffi 1 Iturup, Kuril Islands, Russia MCZ DNA100685
Margaritifera middendorffi 2 Nachilova River, Kamchatka, Russia IEPN d0099/6
Pseudunio auricularius 1 Ebro River, Tarragona, Spain MCZ DNA100674
Pseudunio auricularius 2 Canal Imperial, Zaragoza, Spain MNCN-FW1238-12
Pseudunio homsensis Karasu River, Turkey
Pseudunio marocanus 1 Oum Er Rbia River, Morocco MNCN-N1254
Pseudunio marocanus 2 Laabid River, Morocco MNCN-N1264
FCUP 293
UNIONIDAE
Lampsilis cardium Illinois, USA BivAToL-421
Potomida littoralis Cadiz, Spain MNCN-N706
Unio pictorum Thames River, UK BivAToL-204
HYRIIDAE
Hyridella australis New South Wales, Australia BivAToL-378
Triplodon corrugatus Peru BivAToL-380
Velesunio ambiguus New South Wales, Australia BivAToL-379
MULLERIIDAE
Anodontites elongata Peru BivAToL-323
Lamproscapha ensiformis Peru BivAToL-382
ETHERIIDAE
Etheria elliptica Zambia BivAToL-401
IRIDINIDAE
Aspatharia pfeifferiana Chambeshi River, Zambia BivAToL-330
Chambardia wahlbergi Zambia BivAToL-405
Mutela hargeri Zambia BivAToL-401
TRIGONIIDA
TRIGONIIDAE
Neotrigonia lamarckii North Stradbroke Island, Australia BivAToL-97
Neotrigonia margaritacea Tasmania, Australia
294 FCUP
Table 4
Biological and ecological characters. (Gln) glochidial size índex. Superscripts: U unknown; R rivers; L lakes.
Host fish Glochidia size (Gln) Principal Habitats Flow

U U
G. confragosa rivers-floodplainL U
U U
G. crassa mediumR moderate-strong
U U R
G. laosensis headwaters moderate-strong
U U L
G. rochechouartii rivers-floodplain slow-Moderate
U U
G. polysticta rivers-floodplainL slow-Moderate
R
C. monodonta Hiodontidae 0.004 medium-large moderate-strong
R R
M. dahurica Salmonidae 0.006 headwaters -large moderate-strong
M. falcata Salmonidae 0.006 headwatersR-largeR moderate-strong
U R
M. hembeli Esocidae headwaters moderate
R R
M. laevis Salmonidae 0.004 headwaters -large moderate-strong
R R
M. margaritifera Salmonidae 0.005 headwaters -large moderate-strong
M. marrianae Esocidae 0.002 headwatersR slow-moderate
R R
M. middendorffi Salmonidae 0.006 headwaters -large slow-moderate
middle-lower
P. auricularius Acipenseridae, Blenniidae, Gasterosteidae 0.018 moderate-strong
moderate-largeR
U U middle-lower
P. homsensis slow-moderate
moderate-largeR
U U middle-lower
P. marocanus moderate-strong
moderate-largeR
FCUP 295
Table 4 (cont.)
Substrate Water chemistry References

U U
G. confragosa He & Zhuang 2013
G. crassa boulder, cobble hard Bogan & Do 2016
G. laosensis sand, grave, boulder moderate-hard, oligotrophic Bolotov et al 2014
G. rochechouartii hard mud soft-moderate Do 2011a
U
G. polysticta oligotrophic Do 2011b
S. McMurray pers com; Sietman et al 2017
C. monodonta under flat rocks, rock crevices hard
Williams et al 2008
M. dahurica sand, gravel oligotrophic, soft Bolotov et al 2015
M. falcata sand, gravel oligotrophic, soft Nedeau et al 2009
M. hembeli sand, gravel oligotrophic, soft Paul Johnson pers.com.
M. laevis sand, gravel oligotrophic, soft Bolotov et al 2015
M. margaritifera sand, gravel, cobble oligotrophic, soft Lopes-Lima et al 2017c
M. marrianae sand, gravel oligotrophic, soft Paul Johnson pers.com.
M. middendorffi sand, gravel oligotrophic, soft Bolotov et al 2015
P. auricularius sand, gravel hard Prié et al 2010; Prié et al 2018
P. homsensis silt mesotrophic Vikhrev et al 2017
P. marocanus gravel, cobble hard Sousa et al 2016, 2018
296 FCUP
Divergence time estimates

The acceptance of a global molecular clock to our multi-gene data set was estimated using the
maximum likelihood test of MEGA6 (Tamura et al 2013), which revealed that the null
hypothesis of equal evolutionary rate throughout the tree was rejected (p < 0.001). Thus, the
time-calibrated haplotype-level Bayesian phylogeny was reconstructed in BEAST v. 1.8.4
based on multiple fossil calibration points using a lognormal relaxed clock algorithm with the
Yule speciation process as the tree prior (Drummond et al 2006, 2012; Drummond & Rambaut
2007). Calculations were performed at the San Diego Supercomputer Center through the
CIPRES Science Gateway (Miller et al 2010). A fossil-calibrated ultrametric tree was obtained
using BEAST v. 1.8.4. Similar settings were assigned to nine partitions (3 codons of COI + 16S
rRNA + 18S rDNA + 28S rDNA + three codons of H3) as in the MrBayes analyses. The eight
fossil calibrations were used for timing the phylogeny (Supplementary Tables 3 and 4). Priors
for out-group taxa were designated using a “Monophyly” option of BEAUti v. 1.8.4 (Drummond
et al 2012) as follows: (Trigoniidae, (Unionida)). Four replicate BEAST searches were
conducted, each with 30 million generations. The trees were sampled every 1,000th
generation. The log files were checked visually with Tracer v. 1.6 for an assessment of the
convergence of the MCMC chains and the effective sample size of parameters (Rambaut et al
2014). The first 10% of trees were discarded as an appropriate burn-in. Almost all ESS values
were recorded as > 1,000, with a few values as > 250-800 and two values as > 100; the
subsequent distributions were similar to the prior distributions. The resulting tree files from four
independent analyses were compiled with LogCombiner v. 1.8.4 (Drummond et al 2012). The
maximum clade credibility tree was obtained from 108,004 post-burn-in Bayesian trees using
TreeAnnotator v. 1.8.4 (Drummond et al 2012).
Ancestral geographic area reconstructions

Ancestral geographic area patterns were tested using three different approaches, i.e.
Statistical Dispersal-Vicariance Analysis (S-DIVA), Dispersal-Extinction Cladogenesis
(Lagrange configurator, DEC), and Statistical Dispersal-Extinction Cladogenesis (S-DEC)
implemented in RASP v. 3.2 (Yu et al 2015). The set of 108,004 fossil-calibrated binary trees
that were combined from four runs of BEAST v. 1.8.4 (see above), was used for the ancestral
area reconstruction. The user-specified, fossil-calibrated consensus tree, which was obtained
based on this set of trees using TreeAnnotator v. 1.8.4 (see above), was used as a condensed
tree. Outgroup sequences were removed from all datasets, using the appropriate option of
RASP v. 3.2. Only a single sequence for each ingroup species was used for the analyses. Six
possible geographic areas of the in-group taxa were coded as follows: (A) Southeast Asia; (B)
East Asia; (C) western North America; (D) eastern North America; (E) Mediterranean Region
(South Europe, Middle East, and Morocco); and (F) Europe. Seven geographically unreliable
FCUP 297
distribution constraints were excluded from the input matrix as follows: Southeast Asia -
western North America (AC), Southeast Asia - eastern North America (AD), Southeast Asia -
Mediterranean Region (AE), Southeast Asia - Europe (AF), East Asia - eastern North America
(BD), western North America - Mediterranean Region (CE), and western North America -
Europe (CF). Geographic areas were assigned to the species as follows: Southeast Asia -
Gibbosula laosensis, East Asia - G. crassa, G. rochechouartii, Margaritifera dahurica, M.
laevis, and M. middendorffi, western North America - M. falcata, eastern North America -
Cumberlandia monodonta, Margaritifera marrianae, and M. hembeli, and Mediterranean
Region - P. auricularius, P. homsensis, and Pseudunio marocanus. Considering the broad
trans-Atlantic distribution of Margaritifera margaritifera, we assigned the ‘DEF’ range
for this species. The S-DIVA models were calculated with the following parameters: max
areas=2; allow reconstruction with max reconstructions=100; max reconstructions for final
tree=1000; and allowing extinctions. The DEC and S-DEC analyses were run with default
settings and max areas=2. In addition to the evaluations obtained from each analysis
separately, we used generalized results of all three modelling approaches, which were
combined using an algorithm implemented in RASP v. 3.2.
Diversification rate analyses

The diversification rates were assessed based on the combined Bayesian phylogeny across
the primary clades of the Margaritiferidae and the entire family. The set of 108,004 fossil-
calibrated chronograms that were combined from four runs of BEAST v. 1.8.4 (see above) was
used to construct semi-logarithmic lineage-through-time (LTT) plots in R-package ‘ape’ v. 4.0
(Paradis 2012; Popescu et al 2012) with the supplement of ‘paleotree’ v. 2.7 (Bapst 2012). We
did not include a simulation for missing taxa (Pybus & Harvey 2000), because we assumed
that our samples of the margaritiferid clades are nearly complete.
Two tests of a constant diversification rate for the endemic Indo-Chinese clades
outlined above were calculated using ‘ape’ v. 4.0 based on the maximum clade credibility tree
inferred from BEAST (Paradis 2012; Popescu et al 2012). First, the analysis of diversification
with three survival models, i.e. a constant diversification model, a variable diversification rate
through time (Weibull model), and diversification changes at a specified time point (Paradis
1997). The delta parameter from the constant rate model of Paradis (1997) was used as a
mean diversification rate. Additionally, beta values of the Weibull model were tested where β
> 1 suggests declining and β < 1 indicates an increasing rate of diversification. Second, the
gamma statistic of Pybus & Harvey (2000) was applied. The null hypothesis of constant rate is
rejected at the 5% level if a gamma statistic less than -1.645, which suggests a significantly
decreasing rate of diversification through time (Pybus & Harvey 2000).
298 FCUP
Results
Mitogenome characteristics
The length of the newly sequenced female mitogenome haplotype of G. crassa (16,196 nt) is
within the typical range of Unionida. It includes the 13 protein-coding genes, the gender-
specific ORF described for all Unionida mitogenomes with DUI system, 22 transfer RNA (tRNA)
and 2 ribosomal RNA (rRNA) genes (Supplementary Fig. 1).
The datasets included combinations of individual alignments (COI: 654 nt, 16S: 471 nt, 18S:
1778 nt, 28S: 309 nt, H3: 327 nt). No indels were observed and no stop codons were found
after translating the sequences to amino acids in both COI and H3 datasets. All saturation tests
showed significantly lower values of ISS than ISS.C (a critical value determined from
computational simulation) indicating that the evaluated datasets (COI and H3) are not site
saturated and are useful for phylogenetic comparisons. The resulting BI and ML trees of the
concatenated (COI + 16S + 18S+ 28S + H3) datasets generated the same topology within the
ingroup, being presented the topology of the BI with 9 partitions (Fig. 1). Except for the
Iridinidae, paraphyletic in all analyses, all Unionida families are represented by well-supported
monophyletic clades, including the Margaritiferidae (Fig. 1: Table 5). Within the
Margaritiferidae, four well-supported clades can be found, identified here as Gibbosula,
Cumberlandia, Margaritifera, and Pseudunio (Fig. 1; Table 5). In detail, a first division occurs
between a Gibbosula clade (G. rochechouartii + G. crassa + G. laosensis) that is well-
supported in the BI analysis and a clade encompassing all remaining species (Fig. 1; Table 5).
This latter clade is further divided into the Cumberlandia clade (C. monodonta) + the Pseudunio
clade (P. auricularius + P. homsensis + P. marocanus) and the Margaritifera clade (M.
margaritifera, M. dahurica, M. falcata, M. hembeli, M. laevis, M. marrianae, and M.
middendorffi) (Fig. 1; Table 5). The Margaritifera clade is further subdivided in the clade (M.
margaritifera + M. dahurica) sister to the “Pacific” clade (M. falcata + (M. hembeli + M. laevis
+ M. marrianae + M. middendorffi) (Fig. 1; Table 5).
Morphological and ecological analyses

The literature review identified a total of 29 conchological, anatomical and physiological
characters that are common to all analysed Margaritiferid species and can, therefore, be used
to diagnose the family (Table 2).
FCUP 299
Figure 1 Phylogenetic tree of the Palaeoheterodonta obtained by Bayesian Inference (BI) and Maximum likelihood (ML) analyses of the combined
(COI [3 codons] + 16S + 18S + 28S + H3 [3 codons]) dataset. Support values above the branches are posterior probabilities and bootstrap support
below. Numbers after species names refer to specimen numbers (see Table 3).
300 FCUP
Table 5.
Results of Repeatability Clade Analysis (RCA) of main clades corresponding to the preferred topology.
Combined dataset mtDNA
Clades Analyses COI3 + 16S + 18S COI + 16S + 18S +
COI3 + 16S COI + 16S COI3 COI 16S
+ 28S + H33 28S + H3
BI 100 99 100 100 100 99 58
Margaritifera
ML 76 84 90 93 58 78 24
BI 100 100 - - 96 83 -
‘Pacific clade’
ML 86 85 - - 62 61 -
BI 97 99 95 89 - - 78
Gibbosula
ML 74 64 65 - 52 - 61
BI 100 100 100 100 96 75 64
Pseudunio
ML 95 93 78 84 - 79 42
Pseudunio BI 96 99 - - 50 - -
+Cumberlandia ML 38 47 - 50 - 39 -
BI 100 100 100 100 100 100 72
Margaritiferidae
ML 100 100 95 100 94 94 74
BI 100 100 100 100 80 55 99
Unionidae
ML 100 100 97 99 - 69 81
Etheriidae + BI 100 100 100 100 100 100 100
Mulleriidae +
ML 100 100 100 100 100 100 94
Iridinidae
BI 100 100 55 93 76 98 97
Hyriidae
ML 97 98 76 75 70 72 63
FCUP 301
Table 5 (cont.)
Nuclear
Clades
Analyses 18S + 28S + H33 18S + 28S + H3 18S 28S H33 H3
BI - - - - - -
Margaritifera
ML - - - 37 - -
BI 100 100 100 - - -
‘Pacific clade’
ML 37 - 98 - - -
BI 98 99 60 85 - -
Gibbosula
ML - - 40 57 - -
BI 100 100 70 - - -
Pseudunio
ML 68 - - - - -
BI 90 91 93 - - -
Pseudunio + Cumberlandia
ML - - 62 - - -
BI 100 100 100 100 - -
Margaritiferidae
ML 100 100 100 100 - -
BI 100 100 100 100 - -
Unionidae
ML 99 99 99 95 - -
BI 100 100 100 100 100 100
Etheriidae + Mulleriidae + Iridinidae
ML 100 100 100 91 98 97
BI 100 100 84 - 100 100
Hyriidae
ML 93 93 - - 82 91
302 FCUP
Graf & Cummings (2006) listed five morphological synapomorphies for Margaritiferidae,
characters: 7 - gills irregular scattered interlamellar connections; 8 - gills not fused with mantle
posterior; 12 - pedal elevator muscle scars inconspicuous; 13 - anus located dorsal edge of
posterior adductor muscle; and 27 - mantle attachment scars (Table 2). However, only three
historically recognized characters, i.e. characters 7, 13 and 27, are synapomorphies of the
Margaritiferidae since all other characters can be found in other members of the Unionida,
outside the Margaritiferidae. In this study, we identified a new synapomorphy for the
Margaritiferidae, i.e. papillae on the external surface of the excurrent aperture. Also, two
molecular characters are also synapomorphic, i.e. the F- and M- mitogenome gene orders
(Lopes-Lima et al 2017b).
Inspection of the conchological features revealed a few similarities across all species
(Table 6). Mantle attachment scars were found consistently in all analysed specimens and
nacre colour was generally white with the only exceptions being the purple nacre of M. falcata
and M. laevis, and the peach colour in the umbonal region of G. laosensis (Table 6).
Interestingly, most of the inspected characters were distinct and consistent with the four clades
retrieved with the phylogenetic analyses (i.e. Gibbosula, Cumberlandia, Margaritifera, and
Pseudunio; Table 6). While thin shells are typical for Cumberlandia, thin to medium thick shells
can be found in all species of Margaritifera. Except for G. laosensis, the remaining species
belonging to Pseudunio and Gibbosula have ponderous, thick shells. All species within
Cumberlandia, Margaritifera, and Pseudunio have shallow and open umbo cavities (e.g. Fig.
2). Conversely, all species of Gibbosula have deep, compressed umbo cavities (e.g. Fig. 2),
except for G. laosensis (Table 6). Pseudocardinal teeth are also distinct among the clades
(Fig. 2); while Gibbosula and Pseudunio species present large teeth (again except for G.
laosensis), Margaritifera presents peg-like smaller teeth, and those in Cumberlandia are
reduced (Fig. 2). The lateral teeth are consistently well-developed in most species across the
clades, with a few exceptions (Table 6). However, the lateral teeth of species within Pseudunio
and Gibbosula present vertical striations (except for P. auricularius), while this character is
absent or visible only on the posterior end of laterals of Cumberlandia and Margaritifera
species. Shell surface sculpture is also distinct across the genera (Table 6). Species within
Cumberlandia, Pseudunio and Margaritifera are generally smooth, without any sculpture, the
only exceptions being M. hembeli and M. marrianae, which present plications on the posterior
slope and onto the posterior disk. A distinct pattern can be seen in Gibbosula, where all
species, except G. laosensis, are strongly sculptured with pustules, plications or both (Table
6).
FCUP 303
Table 6.
Analysed conchological characters of Margaritiferidae species. Superscripts: 1 W-shaped pustules on umbo and onto disk; 2 plications on posterior
slope, posterior disk; 3 plications on the posterior slope, pustules on umbo and disk.
Shell thickness Mantle attachment scars Umbo pocket Pseudocardinal teeth Lateral teeth
G. confragosa thick present deep open large well-developed
G. crassa thick present deep compressed large well-developed
G. laosensis medium present shallow open peg-like Reduced
G. polysticta thick present deep compressed large well-developed
G. rochechouartii thick present deep compressed large well-developed
C. monodonta thin present shallow open reduced reduced
M. dahurica medium present shallow open peg-like reduced
M. falcata thin-medium present shallow open peg-like well-developed
M. hembeli medium present shallow open peg-like well-developed
M. laevis medium few shallow open peg-like Reduced
M. margaritifera thin-medium present shallow open peg-like well-developed
M. marrianae thin-medium present shallow open peg-like well-developed
M. middendorffi medium present shallow open peg-like well-developed
P. auricularius thick present shallow open large well-developed
P. homsensis thick few shallow open large well-developed
P. marocanus thick present shallow open large well-developed
304 FCUP
Table 6 (cont.)
Surface
Lateral teeth sculpture Umbo sculpture Nacre colour Ventral margin Shell shape
sculpture
G. confragosa reduced unknown white slight convex oval yes1
G. crassa yes unknown white slight convex rectangular yes2
white, peach
G. laosensis yes unknown slight concave elongate no
umbo area
G. polysticta yes unknown white convex oval yes2
G. rochechouartii yes unknown white straight convex rectangular yes3
C. monodonta no Concentric bars white concave elongate no
M. dahurica no unknown white straight elongate no
M. falcata no unknown purple straight slight concave elongate no
M. hembeli posterior end unknown white straight slight concave elongate yes2
M. laevis posterior end unknown white straight slight concave elongate no
M. margaritifera no Concentric bars white straight slight concave elongate no
Concentric almost
M. marrianae posterior end white straight elongate yes2
double looped1
M. middendorffi posterior end unknown white straight elongate no
P. auricularius no concentric bars white concave elongate oval no
P. homsensis yes unknown white straight concave elongate oval no
P. marocanus yes concentric bars white straight concave elongate oval no
FCUP 305
Table 7.
Anatomical characters. *Not analysed for anatomy.
Incurrent Excurrent Gill Gill Labial Foot muscle
Papillae Anal position Diaphragm
aperture aperture attachment structure Palp pigmented
G. confragosa* ----- ----- ----- ----- ----- ----- ----- ----- -----
G. crassa arborescent crenulated yes Posterior dorsal anterior interrupted falcate yes ridge
G. laosensis arborescent crenulated ----- Posterior dorsal anterior Interrupted falcate yes ridge
G. polysticta* ----- ----- ----- ----- ----- ----- ----- ----- -----
G. rochechouartii arborescent crenulated ----- posterior dorsal unknown interrupted unk. yes ridge
C. monodonta arborescent crenulated yes Posterior dorsal anterior interrupted falcate yes ridge
M. dahurica arborescent crenulated yes Posterior dorsal anterior interrupted falcate yes ridge
M. falcata arborescent crenulated yes Posterior dorsal anterior interrupted falcate yes ridge
M. hembeli arborescent crenulated yes Posterior dorsal anterior interrupted falcate yes ridge
M. laevis arborescent crenulated ----- Posterior dorsal anterior Interrupted falcate yes ridge
M. margaritifera arborescent crenulated no Posterior dorsal anterior interrupted falcate yes ridge
M. marrianae arborescent crenulated yes Posterior dorsal anterior interrupted falcate yes ridge
M. middendorffi arborescent crenulated yes Posterior dorsal anterior interrupted falcate yes ridge
P. auricularius arborescent crenulated ----- Posterior dorsal anterior Interrupted falcate yes ridge
P. homsensis arborescent crenulated ----- Posterior dorsal anterior interrupted falcate yes ridge
P. marocanus arborescent crenulated yes Posterior dorsal anterior Interrupted falcate yes ridge
306 FCUP
Figure 2 Hinge plate and umbo cavity of Margaritiferidae. A - Gibbosula crassa (NCSM
102194.2), B - Cumberlandia monodonta (NCSM 55359.18), C - Margaritifera margaritifera,
(NCSM 5771.1) D - Pseudunio auricularius (NCSM 44514.2). t - pseudocardinal teeth, u -
umbo cavities.
Shell shape is also distinct among the four clades: species within Gibbosula present a typically
convex ventral margin and a variable shell shape; Cumberlandia have a concave ventral
margin and elongated shape; Margaritifera shells are elongated and typically straight to slightly
concave ventral margin; and finally Pseudunio shells are elongated-oval with a straight to
concave ventral margin (Table 6). The umbo in most of the examined shells was eroded and
therefore hindered a proper analysis of its sculpture. Nevertheless, concentric bars in the umbo
were present in all species, where this feature was visible (Table 6). All the soft body
anatomical traits were similar in all analysed species (Table 7).
The ecological and other biological characters analysed here also corroborate the
existence of four genera (Table 4). The host fishes of Margaritifera species belong exclusively
to the Salmonidae and the closely related Esocidae, while the hosts for Pseudunio and
Cumberlandia do not belong to these fish families (Table 4). Cumberlandia uses two species
of Hiodontidae, while members of three unrelated families of fish are found to be suitable for
P. auricularius (Table 4). As for the other two species of Pseudunio, no hosts have yet been
identified but no salmonid species occur sympatrically within their current known distribution
(Table 4). The fish hosts for Gibbosula species are all unknown, although at least for the
Southeast Asian taxa (G. laosensis and G. crassa) do certainly not include Salmonidae, since
this family does not occur in this area (Table 4). The glochidia size of P. auricularius is much
larger than those of Margaritifera and Cumberlandia. Since the glochidia of the other two
Pseudunio and all Gibbosula species are undescribed, its utility for systematics still needs to
be confirmed (Table 4). The habitat preferences are also distinct among the genera. While
Margaritifera species prefer oligotrophic soft-water rivers and are more prevalent in
headwaters, Pseudunio generally inhabits the middle to lower sections of moderate to hard-
water mesotrophic rivers. Cumberlandia seems to occur in habitats like those of Pseudunio
(Table 4). However, contrary to all the other genera it is mostly found in a very particular
FCUP 307
microhabitat, i.e. under large flat rocks or in rock crevices (Table 4). Gibbosula seems to be
much more plastic in its habitat preferences (Table 4) although the ecological features of most
species need to be more thoroughly studied.
Origin and ancient radiations of the Margaritiferidae

The combined results of the biogeographic modelling (S-DIVA, DEC, and S-DEC approaches)
based on the fossil-calibrated chronogram obtained from the relaxed molecular clock analyses
returned a robust ancestral area reconstruction for the primary clades of the Margaritiferidae
(Figs. 3 and 4, Supplementary Fig. 2, and Table 8). The model suggests that the
Margaritiferidae Most Recent Common Ancestor (MRCA) was widespread across the eastern
part of Laurasia (probability 55.0%). The S-DIVA, DEC, and S-DEC models support the same
scenario (probability 53.3-58.3%). The origin of the crown group of the family was placed in
the Jurassic (mean age 172 Ma, 95% HPD 168-178 Ma). Based on the combined
biogeographic model, the Gibbosulinae MRCA most likely originated in East Asia (probability
78.6%), with a subsequent vicariance event separating the Southeast Asian species G.
laosensis (probability 79.9%). The origin of the crown group of the subfamily is placed in the
mid-Cretaceous (mean age ~103 Ma, 95% HPD 86-131 Ma).
The Margaritiferinae MRCA most likely evolved in the East Laurasia (East Asia +
Mediterranean Region, probability 62.0%), with the crown group of the subfamily originating in
the Late Jurassic (mean age ~151 Ma, 95% HPD 132-170 Ma). Among Margaritiferinae clades,
the crown group of the Cumberlandia + Pseudunio clade most likely originated in the Early
Cretaceous (mean age ~135 Ma, 95% HPD 129-146 Ma) within the Mediterranean region, with
subsequent dispersal to eastern North America followed by a vicariance event (probability
45.0%). In contrast, S-DIVA model suggests a rather primary broad range of the MRCA across
the Mediterranean Region and eastern North America followed by vicariance (probability
100%). The crown group of Pseudunio originated in the Mediterranean Region (probability
99.9%) in the Eocene (mean age 47 Ma, 95% HPD 35-66 Ma).
The crown group of Margaritifera is of Late Cretaceous origin (mean age 86 Ma, 95%
HPD 51-131 Ma) and most likely evolved in East Asia (probability 52.4%). The sister species
pair of M. dahurica and M. margaritifera diverged in the mid-Eocene (mean age 42 Ma, 95%
HPD 34-57 Ma) via a dispersal event forming a continuous trans-Eurasian range of their MRCA
followed by a vicariance event (probability 70.4%). The origin of the ‘Pacific’ clade, i.e. M.
falcata, M. laevis, M. middendorffi, M. hembeli, and M. marrianae, is placed near the
Palaeocene - Eocene boundary (mean age 57 Ma, 95% HPD 46-73 Ma). The diversification
of this group was largely associated with several dispersal and vicariance events via the
Beringian land bridge (probability 49.2-86.0%).
308 FCUP
Figure 3 Fossil-calibrated ultrametric chronogram of the Margaritiferidae calculated under a lognormal relaxed clock model and a Yule process
speciation implemented in BEAST 1.8.4 and obtained for the complete data set of mitochondrial and nuclear sequences (nine partitions: three
codons of COI + 16S rRNA + 18S rDNA + 28S rDNA + three codons of H3). Bars indicate 95% confidence intervals of the estimated divergence
times between lineages (Ma). Black numbers near nodes are mean ages (Ma). Stratigraphic chart according to the International Commission on
Stratigraphy 2015.
FCUP 309
Table 8.
The most probable ancestral areas of the primary clades within Margaritiferidae inferred from three different statistical modelling approaches.
High support values (probability≥70%) are highlighted in bold. *Mediterranean + Eastern North America.
Probability of ancestral areas (%)
Biogeographic
Clades Ancestral areas Combined
events S-DIVA DEC S-DEC
results
Margaritiferidae E. Asia + Mediterranean Dispersal 58.3 53.3 53.4 55.0
Gibbosulinae (Gibbosula) E. Asia Dispersal 100.0 67.6 68.2 78.6
G. laosensis - G. crassa E. Asia + SE. Asia Vicariance 100.0 71.2 68.6 79.9
Margaritiferinae (Margaritifera +
E. Asia + Mediterranean Vicariance 41.7 73.4 71.0 62.0
Pseudunio + Cumberlandia)
Margaritifera E. Asia Dispersal 65.0 49.1 43.1 52.4
Dispersal +
M. dahurica - M. margaritifera E. Asia + Europe 50.0 81.4 79.9 70.4
Vicariance
M. falcata - M. laevis (Pacific
E. Asia + W. North America Vicariance 100.0 81.4 76.7 86.0
clade)
M. laevis - M. middendorffi E. Asia Dispersal 97.3 63.2 67.3 49.2
Dispersal +
M. middendorffi - M. hembeli E. Asia + W. North America 33.3 66.0 63.8 54.4
Vicariance
W. North America + E. North Dispersal +
M. hembeli - M. marrianae 33.3 40.5 41.9 38.2
America Extinction
Dispersal +
Pseudunio + Cumberlandia Mediterranean 100.0* 64.6 70.5 45.0
Vicariance
Pseudunio Mediterranean Intra-area radiation 100.0 100.0 99.7 99.9
P. auricularius - P. homsensis Mediterranean Intra-area radiation 100.0 100.0 100.0 100.0
310 FCUP
Figure 4 Simplified scheme of origin and expansion routes inferred across clades of the
Margaritiferidae. The black numbers show the mean age of putative expansion events
obtained from the multi-locus fossil-calibrated phylogenetic model (see Fig. 3 for details).
Circles indicate the putative places of origin of the family and several clades. The map was
created using ESRI ArcGIS 10 software (www.esri.com/arcgis); the topographic base of the
map was created with ESRI Data and Maps.
FCUP 311
Figure 5 Semilogarithmic lineage-through-time (LTT) median plots of chronograms estimated

from 108,004 post-burn-in Bayesian trees for the primary Margaritiferidae clades, including
Gibbosula, Cumberlandia + Pseudunio, Margaritifera, and the entire family. The grey filling
indicates 95% confidence intervals.
Diversification rates
The lineage-through-time modelling suggests extremely slow diversification rates in the
Margaritiferidae (Fig. 5). The constant-rate test suggests that all clades diversified under the
pure-birth (constant) model (Supplementary Table 5).
Discussion
Definition of the Margaritiferidae
Since the first definition of the Margaritiferidae by Ortmann, its supposed diagnostic characters
have varied considerably (Table 2). Graf & Cummings (2006), based on a molecular (COI +
312 FCUP
28S) and morphological phylogeny, revised margaritiferid synapomorphies noting that there
was no previous consensus on characters diagnosing the family Margaritiferidae. These
authors retained only five morphological synapomorphies, two conchological (characters 12
and 27, Table 2) and three anatomical (characters 7, 8, and 13, Table 2) characters. All other
analysed characters were considered plesiomorphic (Graf & Cummings 2006). The main
synapomorphies of the family were again re-evaluated by Araujo et al (2017) (Table 2). They
rejected Graf & Cummings (2006) character 12 and considered character 27 as the only
conchological synapomorphy for the Margaritiferidae. These authors retained anatomical
characters 7, 8, and 13, but were not able to fully evaluate the anal position in all taxa (see
Table 2). Other characters previously used to characterize Margaritiferidae were found in other
genera of the Unionidae (Table 2). Finally, a recent mitogenomics study provided the F- and
M- type gene-orders of the Margaritiferidae as two additional synapomorphic diagnostic
characters (Lopes-Lima et al 2017b).
In the present study, 29 analysed characters were common to all margaritiferid species
and therefore can be used to diagnose the family (Table 2). However, only six, i.e. characters
7, 13, and 27 (Table 2), the papillae on the external surface of the excurrent aperture, plus
both mitogenome orders are synapomorphies of the Margaritiferidae. All the other characters
can be found on other members of the Unionida and Neotrigonia, outside the Margaritiferidae.
Expansion of Margaritiferidae
Based on morphological and molecular evidence, the family Margaritiferidae is here expanded
to 16 species and separated into two subfamilies (i.e. Margaritiferinae and Gibbosulinae) and
four genera (i.e. Pseudunio, Cumberlandia, Margaritifera, and Gibbosula) (Fig. 1; Table 9;
Supplementary Table 6).
Until recently, two different species of Gibbosula used to be recognized. Firstly, the
type species G. crassa was described by Wood (1815) from a specimen collected in an
unknown location in China. Since then, only a few specimens of G. crassa or its synonym Unio
mansuyi have been collected, almost a hundred years ago, in the Bang River, Pearl/Zhu River
basin, either in China or Vietnam. During recent surveys, the species was rediscovered but
seems to be quite rare and restricted to the middle stretches of Bang River in Cao Bang
Province, Vietnam. The second previously recognized species within Gibbosula is G.
confragosa, described by Frierson from a single specimen, collected in an uncertain location
in north China. Although Prozorova et al (2005) stated that this species was present in the
Yangtze and other Eastern Chinese basins, there is no current evidence of its occurrence in
the Yangtze basin.
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Table 9
Margaritiferidae systematics and taxonomy.
Margaritiferidae Henderson, 1929
Gibbosulinae Bogan, Bolotov, Froufe, Lopes-Lima, nom. nov.
Gibbosula Simpson, 1900,

Gibbosula confragosa Frierson, 1928
Gibbosula crassa (Wood, 1815)
Gibbosula laosensis (Lea, 1863), comb. nov.
Gibbosula polysticta (Heude, 1877), comb. nov.
Gibbosula rochechouartii (Heude, 1875), comb. nov.
,
Margaritiferinae Henderson, 1929
Cumberlandia Ortmann, 1912

Cumberlandia monodonta (Say, 1829)
Margaritifera Schumacher, 1816

Margaritifera dahurica (Middendorff, 1850)
Margaritifera falcata (Gould, 1850)
Margaritifera hembeli (Conrad, 1838)
Margaritifera laevis (Haas,1910)
Margaritifera margaritifera (Linnaeus, 1758)
Margaritifera marrianae Johnson, 1983
Margaritifera middendorffi (Rosen, 1926)
Pseudunio Haas, 1910

Pseudunio auricularius (Spengler, 1793)
Pseudunio homsensis (Lea, 1864)
Pseudunio marocanus (Pallary, 1928)
Since G. confragosa original description, only one specimen has been collected and described,
i.e. a specimen from Lake Baiyangdian, Hai River basin, Hebei province, northern China,
previously incorrectly labelled as U. microstictus (He & Zhuang 2013). Besides the shell
surface sculpture differences, the disjunct distribution of G. confragosa suggests a distinct
specific rank. The newly found specimens and shells of G. crassa from Vietnam, here analysed
in detail, feature the characteristics diagnostic and synapomorphies of the Margaritiferidae
(Tables 6 and 7). Additionally, the F-type whole mitogenome sequence of one of the specimens
collected revealed the typical gene order of the Margaritiferidae (Supplementary Fig. 1), which
is unique to this family (Lopes-Lima et al 2017b). The phylogenetic analyses also confirm the
inclusion of G. crassa in the Margaritiferidae family, forming a well-supported clade (BI only)
with G. laosensis and G. rochechouartii. The shells of G. confragosa and G. polysticta present
mantle attachment scars exclusive to the Margaritiferidae and were therefore included in the
Margaritiferidae (Fig. 1; Table 6) and assigned to Gibbosula due to similarities in shell
characteristics with the type species, G. crassa (Table 6). An additional Gibbosula species was
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recently described, Gibbosula nanningensis (Qian et al 2015). No specimens of this species

were available for evaluation, but based on the description, i.e. the absence of mantle
attachment scars and its distinct morphology, we reject its assignment to Gibbosula and
therefore to the Margaritiferidae. A detailed systematics description of the species within
Gibbosula is presented in Supplementary Appendix 1. Most of the earlier works on the
systematics of margaritiferid genera have failed to retrieve monophyletic clades based on
morphological characters alone (Huff et al 2004). More recently, authors showed that previous
generic assignments were inconsistent with the molecular phylogenetic patterns (Huff et al
2004; Bolotov et al 2016; Araujo et al 2017). Whilst all these studies recognized Margaritifera
as the single genus within the Margaritiferidae, the rationale for this generic assignment is not
always clear. Bolotov et al (2016) suggested that the clades found should be assigned to
distinct subgenera but maintained Margaritifera as a monotypic genus due to the
morphological similarity and moderate level of genetic divergence among the clades.
In the present study, four well-supported clades (mainly in the BI analyses) were
consistently retrieved using the most comprehensive Margaritiferidae data set analysed to date
(Fig. 1, Table 5). The divergence of these clades, corresponding to the subgenera identified
by Bolotov et al (2016), is older (from late Jurassic to early Cretaceous) than previously
believed due to the inclusion of new species and improvements in the fossil calibration (see
details below). The present study further revealed a set of consistent morphological, biological
and ecological features characteristic to each of the clades. Based on these results, each clade
was assigned to a separate generic rank (Fig. 1). The genus Gibbosula includes the species
G. crassa, G. confragosa, G. laosensis, G. polysticta, and G. rochechouartii (Fig. 1;
Supplementary Table 6). The morphological and ecological features of Gibbosula are
consistently more distinct from the other three genera (Tables 6 and 7). This agrees with the
molecular phylogeny developed here, which presents two main clades, one with all Gibbosula
species and another including (Margaritifera + (Cumberlandia + Pseudunio) (Fig. 1; Table 5).
Due to their old divergence (late Jurassic, see below) and clear morphological differences, a
subfamily rank was assigned to each of these two clades, i.e. Margaritiferinae and
Gibbosulinae Bogan, Bolotov, Froufe, Lopes-Lima, new subfamily. Distribution of the two
Margaritiferidae subfamilies is mutually exclusive, with the Gibbosulinae being restricted to
East and Southeast Asia, while the Margaritiferinae are widespread throughout the rest of the
Holarctic (Fig. 6).
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Figure 6 Distribution map of the Margaritiferidae.
Systematics
Margaritiferidae Henderson, 1929 (Ortmann, 1910)
Type genus: Margaritifera Schumacher, 1816
Type species: Mya margaritifera Linnaeus, 1758
Type Locality: “Habitat in totius orbis arctici cataractis” [Arctic habitat in the entire world
cataracts] (Linnaeus, 1758).
Comments: This family was split from the Unionidae and four more species were moved from
the Unionidae, refining the definition of the family and the variation in shell shape, anatomy
and geographic distribution.
Diagnosis: Shell shape varies from elongated to rectangular or oval, shell thickness varies
from thin to very thick. The posterior ridge of the shell varies from low and rounded to well-
developed and posterior slope with or without plications, maximum shell length about 200 mm.
Umbo sculpture presents angular un-joined chevron-like hooks but Zieritz et al (2015) have
referred to this sculpture as double looped. Periostracum colour varies from a dark green to
typically black. Lateral teeth vary from vestigial to well-defined with vertical sculptures on all or
the posterior portion of the teeth. Pseudocardinal teeth vary from peg-like in both valves to
thick and massive. Umbo pocket varies from shallow and open to deep and compressed (Fig.
2). Lateral mantle attachment scars are present in varying numbers inside of the pallial line.
Nacre varies from white to purple. Mantle free around edges of the animal. Apertures open
without any mantle fusions to separate the incurrent, excurrent or supra-anal apertures.
Branchial and supra-branchial areas not separated posteriorly by gills, but by a diaphragm
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comprised of a ridge of mantle tissue. Incurrent aperture with arborescent papillae and in at
least one species has simple papillae on the external side of incurrent aperture mantle surface
typically along the length of the aperture. Excurrent aperture smooth or crenulated, lacking
papillae, external side of excurrent aperture mantle surface typically has small papillae along
the length of the aperture. Gills attached to the visceral mass only anteriorly. Labial palps
falcate in outline. Interlamellar gill connections are “irregularly scattered or forming irregular
oblique row, or incomplete septa which run obliquely to the direction of the gill filaments” (Heard
& Guckert 1970). Gills lack water tubes. Marsupium occupies all four gills. Muscular section of
the food pigmented either dark red or black. Anus is located on the posterior dorsal margin of
the posterior adductor muscle. This family is a short term brooder or tachytictic. Most species
are dioecious with only a few listed as hermaphroditic or having hermaphroditic populations.
Fish hosts, when known, are Salmonidae, Esocidae, Acipenseridae, Blenniidae,
Gasterosteidae, and Hiodontidae, with each margaritiferid genus being restricted to a single or
few host fish families. Female and male mitochondrial genome orders are unique for
Margaritiferidae and different from Unionidae.
Distribution: The family is found in North America north of Mexico, Western and Northern
Europe, western North Africa in Morocco, western Middle-East in Syria, Turkey and Lebanon,
Southeast Asia and north to eastern Russia and Japan (Fig. 6).
Subfamily Margaritiferinae Henderson, 1929

Type genus: Margaritifera Schumacher, 1816
Type Locality: “Habitat in totius orbis arctici cataractis” [Arctic habitat in the entire world
cataracts] (Linnaeus, 1758).
Comments: This subfamily contains three genera: Margaritifera, Cumberlandia and
Pseudunio. Species of Cumberlandia and Margaritifera have thin to medium-thick, elongated
shells, while Pseudunio has thick shells and well-developed teeth. All have a shallow open
umbo cavity. The three genera use different fish families as hosts.
Diagnosis: Shell shape elongate, with a concave or straight ventral margin. Shell thin to
moderately thick or thick, posterior ridge rounded. Shell surface smooth or with plications on
the posterior slope and the posterior edge of the shell disk. Umbo sculpture is listed as
concentric bars but usually eroded. Umbo pocket shallow and open (Fig. 2). Nacre colour
usually white but may also be purple. Lateral teeth usually well-developed but may be reduced;
some species have vertical sculpture. Pseudocardinal teeth are peg-like to large (Fig. 2). Fish
hosts when known are Salmonidae, Esocidae, Acipenseridae, Blenniidae, Gasterosteidae,
and Hiodontidae, with host fish families being mutually exclusive to each margaritiferine genus.
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Distribution: This subfamily is Holarctic in distribution including North America, Europe,

Morocco, Turkey, Syria and Lebanon, China, Japan, and eastern Russia (Fig. 6).
Cumberlandia Ortmann, 1912

Type species: Unio monodonta Say, 1829
Type locality: “at the falls of the Ohio, on the rocky flats which are exposed in a low state of
the water” (Say, 1829).
Type specimen: The type specimen of Unio monodonta appears to be lost (Watters et al
2009).
Comments: This large, arcuate shell is distinctive in shape, being very thin shelled and living
in fast water usually under large flat rocks. It has been recognized as different from the typical
Margaritifera and based on the gill structure, Heard & Guckert (1971) erected a subfamily for
this genus.
Diagnosis: Shell shape elongate usually with a convex ventral margin, shell is thin, shell
surface is smooth except for growth arrest line, posterior ridge rounded. Lateral teeth reduced
to a slight rounded ridge. Pseudocardinal teeth are reduced (Fig. 2). Umbo cavity open and
shallow (Fig. 2). Interlamellar gill connections were described as “scattered and in interrupted
rows but developed as continuous septa which run obliquely forward” (Heard & Guckert 1970).
Fish hosts are Hiodontidae.
Distribution: “Cumberlandia monodonta occurs in the Mississippi Basin from southern
Minnesota and Wisconsin south to the Ouachita River drainage in south-central Arkansas, and
in the Ohio River drainage from Ohio and West Virginia downstream to the mouth of the Ohio
River, including some tributaries” such as the Tennessee and Cumberland River drainages
(Williams et al 2008) (Fig. 6).
Margaritifera Schumacher, 1816

Type locality: “Habitat in totius orbis arctici cataractis” [Arctic habitat in the entire world
cataracts]. (Linnaeus, 1758).
Type specimens: There exists a specimen in the Linnean Society of London, Box No. LSL
22, Dance label image Ref. G-M 00101251. Dance was uncertain this was a Linnean
specimen, so the listing by Graf & Cummings (2018) may be invalid. There are two additional
lots in the Linnean Collection, Uppsala University, Museum of Evolution, Zoology Section
(Uppsala University 1999) which are potentially part of the syntype series (UUZM 2018).
Comments: Margaritifera is the most widespread genus within the family with a Pacific,
Atlantic and central Eurasian distribution. Since Bolotov et al (2016), the Japanese endemic
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M. togakushiensis (Kondo & Kobayashi 2005) has been considered a synonym of M.

middendorffi based on morphology and phylogenetic data.
Diagnosis: Shell shape elongate, usually with concave ventral margin. Shell is thin to
moderately thick. Posterior ridge rounded. Shell surface smooth except for growth arrest lines.
Lateral teeth are distinct and peg-like. Pseudocardinal teeth vary from well-developed to
reduced (Fig. 2). Umbo cavity shallow and open (Fig. 2). Nacre colour typically white but purple
in M. falcata and in some M. laevis individuals. Host fish are species of the Salmonidae or
Esocidae for two species restricted to the Gulf Coast of the United States. (Table 4).
Distribution: The genus Margaritifera is widespread across North America, Western Europe,
China, Japan and eastern Russia (Fig. 6).
Pseudunio Haas, 1910

Type species: Unio sinuata Lamarck, 1819 = Unio auricularius Spengler, 1793
Type locality: “Habite dans le Rhin, la Loire, et les autres grandes rivières du continent
européen tempéré et austral” [Lives in the Rhine, the Loire and other great rivers of continental
Europe] (Lamarck, 1819).
Type specimen: The Mollusc Collection, Muséum d’Histoire Naturelle, Genève contains one
valid syntype of Unio sinuata Lamarck, 1819 MHNG-MOLL-50572 and 3 possible syntypes
MHNG-MOLL-50573. Lamarck had only three specimens in total so at least one of these
specimens is not a valid type. Dr. Tardy noted the specimens in lot 50573 measured 104 to
117mm while Lamarck listed a range of sizes from 140 to 145mm (Tardy, Pers. Comm.). The
type of Unio auricularius was first listed and figured by Lister (1685) and is pre-Linnean.
Spengler (1793) validated this species. There is a lectotype in lot ZMUC Biv-315 (Knudsen et
al 2003). [Zoological Museum, University of Copenhagen, Copenhagen, Denmark].
Comments: Placement of the three species here assigned to Pseudunio have often been
assigned to Margaritifera. However, in the phylogeny presented herein, they form a separate
clade from Margaritifera, using a different suite of host fish families.
Diagnosis: Shell shape elongate oval. Shells thick. Posterior ridge rounded. Umbo sculpture
is concentric bars. Posterior slope smooth. Shell surface is smooth. Lateral teeth are well
developed, and most have vertical striations. Pseudocardinal teeth are large and well
developed (Fig. 2). Umbo cavity open and shallow (Fig. 2). Fish hosts include species of the
Acipenseridae, Blenniidae, and Gasterosteidae (Table 4).
Distribution: Species assigned to Pseudunio presently occur in rivers in northern Morocco,
the Iberian Peninsula, France, southern Turkey, Syria, Lebanon, and formerly part of England,
Italy, Germany and the Netherlands (Fig. 6).
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Subfamily Gibbosulinae Bogan, Bolotov, Froufe and Lopes-Lima, new subfamily

Type genus: Gibbosula Simpson, 1900
Type species: Mya crassa Wood, 1815
Type locality: unknown (Wood, 1815); but listed as China, freshwater (Wood, 1825)
Comments: All the taxa included in this subfamily clade except for G. laosensis were
historically included in the Unionidae. The only previous reference recognizing that Gibbosula
belonged in the Margaritiferidae was by Morrison (1975). Transferring these four taxa from the
Unionidae to the Margaritiferidae has changed our understanding of the range in morphological
characteristics (including shell shape and anatomy) within this family.
Diagnosis: Shell shape ranges from elongate to rectangular or oval. Shell moderately thick to
thick. Posterior ridge rounded to rather sharp. Shell surface is smooth with growth arrest rings
or with the posterior slope marked with heavy plications and the disk of the shell covered with
pustules or w-shaped nodules. Umbo sculpture is unknown. Lateral teeth well developed with
vertical sculpture. Pseudocardinal teeth well developed and large (Fig. 2). Umbo pocket deep
and compressed (Fig. 2) and one species with the pocket shallow and open. Nacre colour is
white to some with peach colour. Fish hosts for this subfamily are unknown (Table 6).
Distribution: Species assigned to Gibbosula occur or used to occur in the upper Mekong River
basin in Thailand, Laos, Vietnam, the Bang River in the Pearl River basin of Vietnam, the
middle Sittaung River basin in Myanmar, the Yangtze River basin of southern China and one
species from North China (Fig. 6).
Gibbosula Simpson, 1900

Type species: Mya crassa Wood, 1815
Type locality: unknown (Wood, 1815); but listed as China, freshwater (Wood, 1825:12)
Type specimens: Mya crassa types are unknown; Unio (Quadrula) mansuyi Dautzenberg &
Fischer 1908, a junior synonym, lectotype MNHN-MP-0136 here designated.
Comments: Gibbosula now contains five species, is restricted to Southeast Asia and northeast
China. Margaritanopsis laosensis is included in Gibbosula, but conchologically resembles
Margaritifera and Cumberlandia with a thin, elongate smooth shell rather than the thick
rectangular or oval sculptured shells of the other species assigned to this genus. As Gibbosula
nanningensis Qian, Fang & He 2015, does not conform to the diagnosis of Gibbosula and has
simple papillae and not arborescent papillae in the incurrent aperture, it is here transferred to
the genus Lamprotula, Unionidae.
Diagnosis: Shell shape varies from rectangular, oval to elongate in G. laosensis. Ventral
margin varies from concave in G. laosensis to rounded or convex. Shell thickness ranges from
medium-thick in G. laosensis to thick. Posterior ridge varies from rounded especially in G.
laosensis to rather sharp. Umbo sculpture is unknown. Posterior slope has plications but is
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smooth in G. laosensis. Shell surface is smooth, with plications or covered with pustules of
various shapes. Lateral teeth are typically well developed except for the reduced teeth in G.
laosensis and have vertical striations. Pseudocardinal teeth are usually large and well
developed (Fig. 2), except in G. laosensis where they are peg-like. Umbo cavity deep and
compressed (Fig. 2) or open and shallow as in G. laosensis. Nacre colour is typically white.
Fish hosts are unknown (Table 4).
Distribution: Species assigned to Gibbosula occur in rivers of northern Thailand, Laos, central
Myanmar, western Vietnam, northern Vietnam in the headwaters of Pearl River system,
tributaries of the Yangtze River basin in southern China, and north China (Fig. 6).
Origin and diversification of the Margaritiferidae

In this study, we provide an updated fossil-calibrated phylogeny of the Margaritiferidae, which
includes almost all known members of the family, except for G. confragosa and G. polysticta.
These new results suggest that East Asia was the most likely place of origin of the
Margaritiferidae. Although the statistical biogeographic models assume that the crown group
of the family was widely distributed across the East Laurasia (East Asia + Mediterranean), the
fossil evidence shows an East Asian origin for both the stem and the crown group (e.g. Chen
1984; Jingshan et al 1993; Ma 1994, 1996; Jiang et al 2005; Pan and Sha 2009; Fang et al
2009; Yao et al 2011), i.e. the region of the Yangtze Plate and the adjoining complex of small
terraces that formed the present Tibetan Plateau (Van Damme et al 2015). Additionally,
†Shifangella margaritiferiformis Liu & Luo 1981 from the Late Triassic deposits of China (Fang
et al 2009) is here proposed as a fossil member of the crown group of Margaritiferidae +
Unionidae, most likely representing a separate ancestral family (Supplementary Tables 3 and
4). This agrees with Graf et al (2015) and Skawina & Dzik (2011), who suggested that pre-
Jurassic freshwater bivalves may represent the stem-groups of modern unionoid clades.
Bolotov et al (2017a) showed that the Unionidae most likely originated in East and Southeast
Asia, which is consistent with the hypothesis of an Asian origin for both families.
Concerning the combined results of our fossil-calibrated and biogeographic modelling,
we suggest that the Margaritiferidae family originated in East Asia (Figs. 3 and 4) in the mid-
Jurassic, most likely simultaneously with the Unionidae (Bolotov et al 2017a). We advance that
†Palaeomargaritifera guangyuanensis Ma, 1984 comb. res. from the Middle Jurassic deposits
of Sichuan is the earliest known fossil member of the family (Supplementary Tables 3 and 4).
This dating is not consistent with the three earlier fossil-calibrated models (Bolotov et al 2016;
Araujo et al 2017; Huang et al 2017). Bolotov et al (2016) placed the origin of Margaritiferidae
in the mid-Cretaceous but did not use any fossil calibrations for the deep nodes, which led to
a possible underestimation of the family age. In contrast, Araujo et al (2017) suggested that
the family originated in the Late Triassic based on the age of †Shifangella, which is the most
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probable MRCA of Margaritiferidae and Unionidae (Supplementary Tables 3 and 4). Huang et
al (2017) assigned †Shifangella as a stem calibration for the Margaritiferidae and placed the
origin of the family crown group in the Late Cretaceous that is close to the dating of Bolotov et
al (2016).
The divergence between Gibbosulinae and Margaritiferinae in the Late Jurassic
represented the earliest split within the Margaritiferidae. The Gibbosulinae, a local clade of
East Asian origin, diversified during the Late Cretaceous possibly via connections between the
paleo-river systems of East and Southeast Asia. We suggest that †Gibbosula tibetica (Gu,
1976) comb. nov. from the Late Cretaceous deposits of the Tibetan Plateau could be
considered the earliest known fossil member of the Gibbosulinae (Supplementary Tables 3
and 4). Whilst Bolotov et al (2016) hypothesized that G. laosensis clustered with C. monodonta,
this was not confirmed in our phylogeny. This discrepancy can be explained by the absence
of other members of the Gibbosulinae in the reconstruction by Bolotov et al (2016). The
external resemblance between G. laosensis and C. monodonta that was a subject of long-term
discussion (Walker 1910; Smith 2001; Bolotov et al 2016) is surely a result of morphological
convergence. Interestingly, both clades (Gibbosulinae and Pseudunio + Cumberlandia)
include species with narrow, elongated shells (G. laosensis and C. monodonta) as well as
broad, rounded shells (G. crassa, G. rochechouartii, P. homsensis).
The Margaritiferinae MRCA had a continuous range from East Asia to the
Mediterranean Region in the Late Jurassic, which was most likely facilitated by host fish
dispersal within a continuous paleo-river system or along the Tethys coastal line (Hou and Li
2017). The earliest history of this clade is well documented via fossil records from Jurassic
deposits of North Africa and Europe (Delvene et al 2013, 2016; Van Damme et al 2015). †
“Margaritifera” crosthwaitei (Newton, 1909) from the Late Jurassic deposits of Egypt and
†Asturianaia soudanensis (Van Damme & Bogan, 2015) comb. nov. from the Middle to Late
Jurassic deposits of Niger are the earliest fossil members from North Africa that could be
assigned to this clade (Van Damme et al 2015). Fossils identified as “Margaritifera” cf.
valdensis (Mantell, 1844) are known from the Late Jurassic deposits of Spain (Delvene et al
2013, 2016). Three additional Late Jurassic margaritiferid species were recently described
from Spain: †Asturianaia colunghensis Delvene, Munt, Piñuela & García-Ramos, 2016, †A.
lastrensis Delvene, Munt, Piñuela & García-Ramos, 2016 and †“Margaritifera” lagriega
Delvene, Munt, Piñuela & García-Ramos, 2016 (Delvene et al 2016).
The MRCA of Pseudunio + Cumberlandia clade most likely originated in the
Mediterranean Region and dispersed to eastern North America with a subsequent vicariant
event in the Early Cretaceous. †Paraheudeana idubedae (Palacios & Sánchez, 1885) from the
Early Cretaceous deposits of Spain appears to be the earliest known member of the crown
group of this clade (Supplementary Tables 3 and 4). The evolutionary history of Pseudunio
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was associated with the intra-Mediterranean radiation from the mid-Eocene to mid-Miocene.
Our results support the assumption of Bolotov et al (2016) that the split between P. auricularius
and P. marocanus was well before the Messinian Salinity Crisis (MSC). Additionally, the new
model indicates that the split between P. auricularius and P. homsensis most likely preceded
this paleogeographic event. In contrast, the divergence between Unio species in Morocco and
Iberia was coincident with the MSC (Froufe et al 2016). The earliest fossils resembling the
extant Cumberlandia are known from the Early Cretaceous deposits in North Africa: †C.
rhazensis (Mongin, 1968) comb. nov. and †C. saharica (Mongin, 1968) comb. nov. (Van
Damme et al 2015).
Margaritifera is the most widespread and diverse group of recent margaritiferids. This
clade most likely originated in East Asia in the Late Cretaceous. The earliest fossils that may
belong to this clade are known from the mid-Cretaceous deposits of Mongolia: †Margaritifera
elongata (Martinson, 1982) comb. nov., †M. sainshandensis (Martinson, 1982) comb. nov. and
†M. glabra (Kolesnikov, 1956) comb. nov. (Supplementary Table 3). However, the first two
species together with nine additional fossil taxa from Mongolia were considered synonyms of
†Unio longus (Zhu, 1976) from China (Sha et al 2006). A detailed discussion of the fossil taxa
taxonomy is beyond the scope of the present investigation, but it should be mentioned that
Sha et al (2006) provided their revision without studies of the type series of the synonymized
species. Our reconstruction of the diversification patterns within this clade is largely congruent
with the multiple trans-Beringian exchange model developed by Bolotov et al (2015, 2016) and
is supported by numerous fossil records (Supplementary Table 3). An expanded sampling of
species from the ‘Pacific’ clade (M. falcata, M. laevis, M. middendorffi, M. hembeli, and M.
marrianae) indicates the possibility of an extinction event that closes the gap between East
Asian M. middendorffi and its relatives from southeastern North America, i.e. M. hembeli and
M. marrianae. Previously, Bolotov et al (2016) suggested that an additional Margaritifera
species could be within this gap following the hypothesis of Taylor (1988) regarding vicariate
forms of Margaritiferidae on both sides of the Pacific. However, Taylor’s unnamed taxon is a
morphological form of M. falcata, which differs by nacre colour (white with salmon spots) but
is not genetically different from the typical violet-nacre form (our unpubl. data). The new fossil-
calibrated model also supports the hypothesis that the Mekong and Yangtze unionoid faunas
must have developed as independent radiations during the entire Cenozoic (Schneider et al
2013; Bolotov et al 2017a,b) because G. laosensis (Mekong River basin) and G. crassa (Pearl
River basin) split ~65 Ma ago, and the G. laosensis + G. crassa subclade diverged from G.
rochechouartii (Yangtze) ~103 Ma ago. The two largest paleo-Mekong radiations in the
Unionidae most likely originated in the Early Cenozoic (mean age=51-55 Ma) or even pre-
Cenozoic (mean age=65-71 Ma) (Bolotov et al 2017a,b). These results are following the
concept of long-lived (ancient) rivers, suggesting that several large rivers on Earth may have
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existed for long-term periods comparable with geological epochs (Bolotov et al 2017a). The
present results highlight that the placement of several Jurassic and Early Cretaceous
margaritiferid species within the genus Margaritifera (e.g. Delvene et al 2013, 2016; Van
Damme et al 2015) needs to be revised because these taxa most likely represent ancestral
fossil lineages that are not directly associated with the crown group of the latter genus despite
their morphological similarity. The description of two fossil species from the same deposit
based on small conchological differences, a common procedure in systematic palaeontology
(e.g. Delvene et al 2016), most likely leads to the overestimation of the actual diversity of fossil
taxa, e.g. Margaritiferidae, because the sympatric occurrence of several closely related
species is an unusual phenomenon. The co-occurrence of M. laevis and M. middendorffi in
several rivers of Japan, South Kuriles and Sakhalin Island (Bolotov et al 2015, 2016; Araujo et
al 2017) is the only example of such a secondary sympatry known to date, whereas distribution
ranges of the other species reflect a drainage-dependent allopatric speciation model without
clear secondary contact zones. This evolutionary pattern suggests a limited number of
ancestral fossil lineages not only by the single confirmed extinction event but also by the slow
substitution and diversification rates within the family. Modelling results suggest delayed
diversification rates in the Margaritiferidae (Fig. 5 and Supplementary Table 5) that are
consistent with findings for the Indo-Chinese Unionidae, which also reveal slow diversification
rates (Bolotov et al 2017a). Indeed, the rates in margaritiferids are ∼2.5 times slower compared
with the Unionidae (Bolotov et al 2016). These results may be associated with slower rates of
molecular evolution in the Margaritiferidae, which support the hypothesis of a possible link
between delayed diversification and slow molecular evolution in freshwater mussels (Bolotov
et al 2017a), although this enigmatic pattern requires further investigation.
Conclusions
The current study supports the increase of extant margaritiferid species to 16 and suggests
their division into two subfamilies and four genera. Since a better understanding of
phylogenetic diversity is central for determining conservation priorities (Lopes-Lima et al
2017c, 2018), the results reported here may be important in the definition of future
management strategies devoted to the conservation of margaritiferid species. The inclusion of
G. crassa, G. polysticta, G. rochechouartii, and G. confragosa in the Margaritiferidae, confirms
the family as the most threatened among unionoids (IUCN 2018). The first three mentioned
species have a threatened status (IUCN 2018), while G. confragosa has never been evaluated
(IUCN 2018). All four “new” margaritiferids seem to have small distribution ranges and are
affected by multiple impacts (IUCN 2018). Further studies on the Margaritiferidae should
include basic ecological and physiological research, collecting data on distribution, abundance,
324 FCUP
habitat preferences, host fish identification, and reproductive cycles, as well as a phylogenomic
approach to complement the current phylogenetic evaluation. Finally, a complete revision of
numerous fossil margaritiferid taxa is necessary for the future development of reliable
phylogenetic, phylogenomic and biogeographic reconstructions.
Acknowledgments
The authors would like to thank the editor Dr. Perez-Losada and two anonymous reviewers for
their valuable suggestions and comments. M. Caballer MNHN e-RECOLNAT (ANR-11-INBS-
0004) and Ms. Virginie Heros, molluscs, Museum National d’Histoire Naturelle, Paris are
acknowledged for the photographs of the types of Unio mansuyi, Unio rochechouartii, and Unio
affinis used in this paper and their assistance with specimens in their collection. Ms. Krasimira
(Kasey) Seizova, Drexel University Co-op student working in the Malacology Department,
Academy of Natural Sciences of Drexel University, Philadelphia took the photographs of the
type specimen of Gibbosula confragosa. These pictures were taken for the ANSP type project
and used with the permission of Dr. Gary Rosenberg. Pictures of the NCSM specimen of
Gibbosula crassa were taken by Ms. Raquel Fagundo and Ms. Joanna Cox; those of the
Margaritiferid hinge plates were taken by Macauley Whiting, all in the Non-Molluscan
Invertebrate Unit, North Carolina Museum of Natural Sciences, Raleigh, NC with permission
of Dr. Bronwyn Williams. Jeffrey T. Garner, Florence, Alabama assisted with the USNM
ledgers. He Jing, Shell Discoveries and ConchBooks are acknowledged for the permission to
reproduce the figures from Qian et al 2015 for the description of Gibbosula nanningensis. This
work was supported by FCT - Foundation for Science and Technology, Project 3599 - Promote
the Scientific Production and Technological Development and Thematic 3599-PPCDT by
FEDER as part of the project FRESHCO: Multiple implications of invasive species on
Freshwater Mussel co-extinction processes (contract: PTDC/AGRFOR/1627/2014). FCT also
supported MLL (SFRH/BD/115728/2016) and EF (SFRH/BPD/108445/2015). The Russian
Ministry of Education and Science (project no. 6.2343.2017/4.6), the Federal Agency for
Scientific Organizations (project no. 0409-2015-0143), the Presidium of the Russian Academy
of Sciences (scientific program no. 52), and the Russian Foundation for Basic Research, RFBR
(project no. 17-45-290066) supported INB, MYG, AVK, and IVV. The Nagao Natural
Environment Foundation (NEF) supported for VTD research project “The study of Unionoida
biodiversity and conservation status in Bang and Ky Cung River basins in Northeast Vietnam”.
FCUP 325
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338 FCUP
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Supplementary material
Supplementary Figure 1 Gene map of the F-type mitochondrial genome of Gibbosula crassa.
Genes positioned inside the circle are encoded on the heavy strand, and genes outside the
circle are encoded on the light strand. Colour codes: small and large ribosomal RNAs (red),
transfer RNAs (purple); F-specific open reading frame (yellow); protein-coding genes (green).
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342 FCUP
Supplementary Figure 2 Historical biogeography of the Margaritiferidae inferred from three different statistical modelling approaches, including
(A) the combined results of SDIVA, DEC, and S-DEC; (B) S-DIVA; (C) DEC; and (D) S-DEC, calculated under a lognormal relaxed clock model
and a Yule process speciation implemented in BEAST 1.8.4 and obtained for the complete data set of mitochondrial and nuclear sequences (nine
partitions: three codons of COI + 16S rRNA + 18S rDNA + 28S rDNA + three codons of H3). Pie chaps near nodes indicate probabilities of certain
ancestral areas. Colour circles on the tip nodes indicate the range of each species. Black numbers near nodes are BPP values inferred from
BEAST.
FCUP 343
Supplementary Table 1.
Specimens examined for conchological and anatomical features. ANSP - Academy of Natural Sciences of Drexel University, Philadelphia, PA,
USA; MNHN - Muséum national d'Histoire Naturelle, Paris, France; NHMUK - Natural History Museum, London, UK; NCFM - Nanchang
Freshwater Mollusk Collection, Nanchang University, Nanchang, Jiangxi Province, China; NCSM - North Carolina Museum of Natural Sciences,
Raleigh, NC, USA; RMBH - Russian Museum of Biodiversity Hotspots, Federal Center for Integrated Arctic Research, Russian Academy of
Sciences, Arkhangelsk, Russia.
Taxa Conchological Anatomical
Gibbosula confragosa ANSP 145237 -----
Gibbosula crassa MNHN-IM-2000-1743, 33100, 33101; NCSM 102193, 102194 NCSM 102193, 102194
Gibbosula laosensis NCSM 83059 RMBH biv0136/3
Gibbosula polysticta MNHN MP 3387 MNHN MP 3387
Gibbosula rochechouartii NCSM 6468, NCFM 092142 NCFM 092142
Cumberlandia monodonta NCSM 28549, 33041, 55345, 55350, 55359 NCSM 30377, 34969, 87784, 100543
Margaritifera dahurica NCSM 27972 RMBH biv0165/2; NCSM 27972
Margaritifera falcata NCSM 48386, 48393 NCSM 27974, 29455, 41056, 41059
Margaritifera hembeli NCSM 28946, 45395 NCSM 100550
Margaritifera laevis NCSM 102192 RMBH d0078/9
Margaritifera margaritifera NCSM 5771,7329 NCSM 28288, 28944, 83212
Margaritifera marrianae NCSM 33045, 62914, 62915 NCSM 30343, 30371, 46348
Margaritifera middendorffi
NCSM 48955 RMBH biv0167/1; NCSM 48955
+ M. togakushiensis
Pseudunio auricularius NCSM 44514 MNHN-IM-2013-62643
Pseudunio homsensis RMBH biv0176 RMBH biv0176; NHMUK 1936.10.3
Pseudunio marocanus NCSM 85240, 102909, 102910 NCSM 1012909, 102910
344 FCUP
Best-fit models of nucleotide substitution for each partition based on Bayesian Information
Criteria (BIC) using JMODELTEST 2.1.10 (Darriba et al 2012) for the Bayesian inference
analyses.
Partition Model
COI HKY + G + I
COI codon 1 GTR + G + I
COI codon 2 F81 + G
COI codon 3 HKY + G
16S GTR + G + I
18S GTR + G + I
28S GTR + G + I
H3 K80 + G
H3 codon 1 JC
H3 codon 2 JC
H3 codon 3 K80 + G
FCUP 345
List of characteristic examples of fossil records supporting the primary phylogenetic clades of freshwater bivalves identified in the present study.
Group
Ancestral genera
Clades [dating following our fossil-calibrated
[stratigraphic dating]
model]
Unionida Crown [near Permian - Triassic boundary] †Silesunio Skawina & Dzik, 2011 [early Late
Triassic]
Margaritiferidae + Unionidae Crown [Late Triassic] †Shifangella Liu & Luo, 1981 [Late Triassic]
Margaritiferidae Crown [Middle Jurassic] †Palaeomargaritifera Ma, 1984 stat. res.
[Middle Jurassic]
Gibbosulinae (Gibbosula) Crown [mid-Cretaceous] Gibbosula Simpson, 1900 [mid-Cretaceous]
G. laosensis - G. crassa n/a Gibbosula Simpson, 1900
Margaritiferinae Stem/Crown [Middle to Late Jurassic] †”Margaritifera” [Late Jurassic]
(Margaritifera + Pseudunio + Cumberlandia)
Pseudunio + Cumberlandia Stem [Late Jurassic to Early Cretaceous] †Asturianaia Delvene, Munt, Piñuela &
García-Ramos, 2016 [Late Jurassic]
Crown [Early Cretaceous] †Paraheudeana Starobogatov, 1970
?†Protelliptio Russell, 1934 [Early
Cretaceous]
Cumberlandia Stem [from Early Cretaceous] ?Cumberlandia Ortmann, 1912 [Early

Cretaceous]
Pseudunio Crown [Eocene] Pseudunio Haas, 1910 [Lower Oligocene]
P. auricularius - P. homsensis Crown [Miocene] Pseudunio Haas, 1910
Margaritifera Crown [Late Cretaceous] Margaritifera Schumacher, 1816 [Late
Cretaceous]
M. dahurica - M. margaritifera Crown [Eocene] Margaritifera Schumacher, 1816
Pacific clade Crown [near Paleocene - Eocene boundary] Margaritifera Schumacher, 1816
(M. laevis - M. falcata)
M. laevis - M. middendorffi Crown [near Eocene - Oligocene boundary] Margaritifera Schumacher, 1816
M. middendorffi - M. hembeli Crown [Oligocene] Margaritifera Schumacher, 1816
M. hembeli - M. marrianae Crown [Miocene] Margaritifera Schumacher, 1816
Supplementary Table 3 (cont.)
346 FCUP
Clades Examples of ancestral species* Reference

Unionida †S. parvus Skawina & Dzik, 2011** Skawina & Dzik (2011)
Margaritiferidae + Unionidae †S. margaritiferiformis Liu & Luo, 1981** Fang et al (2009)
Margaritiferidae †P. guangyuanensis Ma, 1984 comb. res.**, P. angulata (Ma, Ma (1996),
1996) comb. nov. Fang et al (2009)
Gibbosulinae (Gibbosula) †G. tibetica (Gu, 1976) comb. nov.** Ma (1996)
G. laosensis - G. crassa n/a n/a
Margaritiferinae (Margaritifera + †”M.” crosthwaitei (Newton, 1909) Van Damme et al (2015)
Pseudunio + Cumberlandia)
Pseudunio + Cumberlandia †A. colunghensis Delvene, Munt, Piñuela & García-Ramos, 2016, Van Damme et al (2015),
†A. lastrensis Delvene, Munt, Piñuela & García-Ramos, 2016, †A. Delvene et al (2016)
soudanensis (Van Damme & Bogan, 2015) comb. nov.
†P. valdensis (Mantell, 1844), †P. idubedae (Palacios & Sánchez, Van Damme et al (2015)
1885)**
P. biornatus (Russell, 1932), P. douglassi (Stanton, 1903), P. hamili
(McLearn, 1929) Skawina & Dzik (2011)
Cumberlandia †C. rhazensis (Mongin, 1968) comb. nov., †C. saharica (Mongin, Van Damme et al (2015)
1968) comb. nov.
Pseudunio †Pseudunio sp.** Schneider & Prieto 2011),
Araujo et al (2017)
P. auricularius - P. homsensis †P. flabellatus (Goldfuss, 1837) comb. nov.**, †P. flabellatiformis Bolotov et al (2016)
(Grigorowitch-Beresowski, 1915) comb. nov.
Margaritifera †M. sainshandensis (Martinson, 1982) comb. nov., †M. elongata Martinson (1982)
(Martinson, 1982) comb. nov. [primary homonym of Unio elongata
Lamarck, 1819], †M. glabra (Kolesnikov, 1956) comb. nov.
M. dahurica - M. margaritifera †M. occulta Maderny, 1972, †M. martinsoni Modell, 1964** Bolotov et al (2016)
Pacific clade †M. perdahurica (Yokoyama, 1932)**, †M. otatumei (Suzuki, 1942), Henderson (1935);
(M. laevis - M. falcata) †M. owadaensis Noda, 1970, †M. sinopae (Cockerell, 1915), †M. Modell (1957);
herrei (Hannibal, 1912) Bolotov et al (2016)
M. hembeli - M. marrianae M. condoni (White, 1885) Modell (1957)
*The revision of numerous fossil species of the Margaritiferidae appears to be a complicated task and is well beyond the scope of the present
study. Here we list characteristic examples of possible ancestral lineages supporting each primary clade of the family and provide several minor
FCUP 347
taxonomic changes. Considering our new data on the extremely low diversification rates in the Margaritiferidae, high diversity of fossil species
may be an artifact caused by shell shape variability. **Fossil calibrations (see Supplementary Table 4 for details). n/a - not available.
References
Araujo R, Schneider S, Roe KJ, Erpenbeck D, Machordom A. 2017. The origin and phylogeny of Margaritiferidae (Bivalvia, Unionoida): a synthesis
of molecular and fossil data. Zoologica Scripta 46, 289-307.
Bolotov IN, Vikhrev IV, Bespalaya YV, Gofarov MY, Kondakov AV, Konopleva ES, Bolotov NN, Lyubas AA. 2016. Multi-locus fossil-calibrated
phylogeny, biogeography and a subgeneric revision of the Margaritiferidae (Mollusca: Bivalvia: Unionoida). Molecular Phylogenetics and Evolution
103, 104-121.
Delvene G, Munt MC, Piñuela L, García-Ramos JC. 2016. New Unionida (Bivalvia) from the Kimmeridgian (Late Jurassic) of Asturias, Spain, and
their palaeobiogeographical implications. Papers in Palaeontology 2, 265-285.
Fang ZJ, Chen JH, Chen CZ, Sha JG, Lan X, Wen SX. 2009. Supraspecific taxa of the Bivalvia first named, described, and published in China
(1927-2007). University of Kansas Paleontological Contributions (New Series) 17, 1-157.
Henderson J. 1935. Fossil non-marine mollusca of North America. Geological Society of America Special Papers 3, 1-290.
Ma Q. 1996. Revision of Mesozoic Margaritiferidae in China and their development. Acta Palaeontologica Sinica 35, 408-429.
Martinson GG. 1982. Late Cretaceous Molluscs of Mongolia. Nauka Publ., Moscow, 82 pp.
Modell VH. 1957. Die fossilen Najaden Nordamerikas. Archiv für Molluskenkunde 86, 183-200.
348 FCUP
Schneider S, Prieto J. 2011. First record of an autochthonous community of fluviatile freshwater molluscs from the Middle/Late Miocene Upper
Freshwater Molasse (southern Germany). Archiv für Molluskenkunde 140, 1-18.
Skawina A, Dzik J. 2011. Umbonal musculature and relationships of the Late Triassic filibranch unionoid bivalves. Zoological Journal of the
Linnean Society 163, 863-883.
Van Damme D, Bogan AE, Dierick M. 2015. A revision of the Mesozoic naiads (Unionoida) of Africa and the biogeographic implications. Earth-
Science Reviews 147, 141-200.
FCUP 349
Supplementary Table 4. List of fossil calibrations that were used in BEAST analyses
Calibration
MRCA Description Reference
no.
Hard minimum age: 230 Ma, †Silesunio parvus Skawina & Dzik (2011) (Unionida:
Silesunionidae).
Diagnosis and phylogenetic placement: Elongated shell of small size does not exceed
50 mm and generalized morphology, with juvenile stage bearing concentric ribs parallel
with the mantle margin (Skawina & Dzik 2011). Umbonal muscles tend to disperse over
the anterior slope of the beaks. The Silesunionidae is a prospective earliest member of
the order Unionida (Skawina & Dzik 2011).
Present study:
Stratigraphic horizon and locality: Lacustrine grey claystone and red finely grained
Calibration 1 Unionida New crown
mudstone bed within red-coloured fluviatile series of Late Carnian calcareous
calibration
mudstones, Krasiejów, Opole Silesia, southern Poland (Skawina & Dzik 2011).
Absolute age estimate: Late Carnian, 230 Ma, based on stratigraphy; 95% soft upper
bound 273 Ma based on the age of †Lyroschizodus orbicularis Newell & Boyd 1975
(Trigoniida: Trigoniidae), the earliest known member of Trigoniidae (Newell & Boyd
1975).
Prior settings: exponential distribution, mean (lambda) = 11.6, MRCA: Unio pictorum -
Velesunio ambiguus.
Hard minimum age: 201 Ma, †Shifangella margaritiferiformis Liu & Luo, 1981 in Liu
(1981) (Unionida: unnamed ancestral family).
Diagnosis and phylogenetic placement: Shell thin, large to very large; transversely
elongate, ventral margin concave; elongate-oval to Margaritifera-shaped; equivalve,
inequilateral; moderately to rather inflated. Posterior ridge strong, edge-form (Fang et
al 2009). Umbonal region compressively flattened; beaks broad, not projecting or
slightly rising above hinge margin, prosogyrous, situated rather anteriorly. Surface
Present study:
Margaritiferidae ornamented with regularly spaced concentric rings and growth lines, but with regular
Calibration 2 New crown
+ Unionidae concentric rings only in preadult specimens; lunule absent; escutcheon developed,
calibration
relatively wide; ligament opisthodetic. Hinge plate narrow, with unionid dentition, but
teeth fine and smooth, parallel to hinge margin. Anterior adductor scar shallow,
rounded, accompanied by a pedal scar at the upper posterior side. The genus was
considered a member of Margaritiferidae (Fang et al 2009), but it seems to be rather a
prospective ancestral lineage of both families.
Stratigraphic horizon and locality: Second Member, Wuzhongshan Formation, Upper
Triassic, Jinhe, Shifang, Sichuan, southwestern China (Fang et al 2009).
350 FCUP
Calibration
no.
Absolute age estimate: Triassic/Jurassic boundary, 201 Ma, based on stratigraphy;
95% soft upper bound 230 Ma based on calibration 2.
Prior settings: exponential distribution, mean (lambda) = 7.9, MRCA: Margaritifera
marrianae - Potomida littoralis.
Hard minimum age: 168 Ma, †Palaeomargaritifera guangyuanensis (Ma, 1984) comb.
res.
Diagnosis and phylogenetic placement: Shell large; greatly elongate, with largest shell
about 120 mm long and 44 mm high; length about 2.7 times as long as height; shell
width about one-fifth as long as height; anterior margin rounded; posterodorsal margin
nearly straight, obliquely passing into rounded posterior margin; posteroventral end
prominently protracting backward; ventral margin long, with wide, shallow sinus; umbo
broad and low, positioned at about two-ninths shell length from anterior; posterior
ridge obtuse (Fang et al 2009). Shell flat, surface with irregular commarginal lines.
Present study:
Anterior pseudocardinal teeth strong, two in left valve and one in right valve; posterior
Calibration 3 Margaritiferidae New stem
lateral lamellar teeth, one in left valve and seemingly two in right valve; anterior
calibration
adductor scar deep, elongate-oval, with arborescent-like striations; upper pedal scar
deeper, lower one isolated and shallower; posterior adductor scar also shallow (Fang
et al 2009). It seems to be a prospective stem lineage of extant Margaritiferidae.
Stratigraphic horizon and locality: Third Member, Guangyuan Group, Middle Jurassic,
Nanshan, Guangyuan, Sichuan, southwestern China (Fang et al 2009).
Absolute age estimate: Bajocian/Bathonian boundary, 168 Ma, based on stratigraphy;
Prior settings: exponential distribution, mean (lambda) = 9, MRCA: Gibbosula crassa -
Margaritifera hembeli.
Hard minimum age: 129 Ma, †Paraheudeana idubedae (Palacios & Sánchez, 1885).
Diagnosis and phylogenetic placement: The assignation of this species to the genus
is based on the shell shape, notable mantle attachment scars on the inner side of the
valve and the arborescent rugosity of the muscle adductor scars (Delvene & Araujo Present study:
Pseudunio +
Calibration 4 2009; Van Damme et al 2015). It seems to be a prospective crown lineage of the New crown
Cumberlandia
extant Pseudunio + Cumberlandia clade. calibration
Stratigraphic horizon and locality: Hauterivian-Barremian Urbión Group, Valdehierro
and Valdemadera sites, La Rioja Province, Cameros Basin, Spain (Delvene & Araujo
2009).
FCUP 351
Calibration
no.
Absolute age estimate: Hauterivian/Barremian boundary, 129 Ma, based on
stratigraphy; 95% soft upper bound 168 Ma based on calibration 4.
Prior settings: exponential distribution, mean (lambda) = 7.8, MRCA: Pseudunio
homsensis - Cumberlandia monodonta.
Absolute age estimate: 46 Ma; 95% soft upper bound 92 Ma (twice the age of the
M. falcata - M. Bolotov et al (2016):
Calibration 5 fossil). Prior settings: exponential distribution, mean (lambda) = 12.5, MRCA:
laevis Crown calibration
Margaritifera falcata - M. laevis.
M. Absolute age estimate: 34 Ma; 95% soft upper bound 68 Ma (twice the age of the
Bolotov et al (2016):
Calibration 6 margaritifera - fossil). Prior settings: exponential distribution, mean (lambda) = 9.3, MRCA:
Crown calibration
M. dahurica Margaritifera margaritifera - M. dahurica.
Absolute age estimate: 35 Ma; 95% soft upper bound 70 Ma (twice the age of the
P. auricularius - Araujo et al (2017):
Calibration 7 fossil). Prior settings: exponential distribution, mean (lambda) = 9.5, MRCA:
P. marocanus Crown calibration
Pseudunio auricularius - P. marocanus.
Hard minimum age: 86 Ma, † Gibbosula tibetica (Gu, 1976) comb. nov.
Diagnosis and phylogenetic placement: The species differs by thick shell, massive
hinge plate, well-developed pseudocardinal teeth, and the arborescent rugosity of the
muscle adductor scars (Ma 1996; Data Base of Paleontological Fossils of Nanjing
Institute of Geology and Palaeontology, Chinese Academy of Sciences: lot nos.
30963, 30964, 30965, 30967, 30968, and 30969). It seems to be a crown lineage of Present study:
Calibration 8 Gibbosula the extant Gibbosula. New crown
Stratigraphic horizon and locality: Late Cretaceous Shigatse Group, Zhaxilin village, calibration
Shigatse Region, Tibet, China.
Absolute age estimate: Coniacian/Santonian boundary, 86 Ma, based on stratigraphy;
Prior settings: exponential distribution, mean (lambda) = 22.3, MRCA: Gibbosula
rochechouartii - G. laosensis.
References
Araujo R, Schneider S, Roe KJ, Erpenbeck D, Machordom A. 2017. The origin and phylogeny of Margaritiferidae (Bivalvia, Unionoida): a synthesis
of molecular and fossil data. Zoologica Scripta 46, 289-307.
352 FCUP
Bolotov IN, Vikhrev IV, Bespalaya YV, Gofarov MY, Kondakov AV, Konopleva ES, Bolotov NN, Lyubas AA. 2016. Multi-locus fossil-calibrated
phylogeny, biogeography and a subgeneric revision of the Margaritiferidae (Mollusca: Bivalvia: Unionoida). Molecular Phylogenetics and Evolution
103, 104-121.
Delvene G, Munt MC, Piñuela L., García-Ramos JC. 2016. New Unionida (Bivalvia) from the Kimmeridgian (Late Jurassic) of Asturias, Spain,
and their palaeobiogeographical implications. Papers in Palaeontology 2, 265-285.
Fang ZJ, Chen JH, Chen CZ, Sha JG, Lan X, Wen SX. 2009. Supraspecific taxa of the Bivalvia first named, described, and published in China
(1927-2007). University of Kansas Paleontological Contributions (New Series) 17, 1-157.
Gu ZW, Huang BY, Chen CZ, Wen S, Ma Q, Lan X, Xu J, Liu L, Wang D, Qui R, Huang Z, Zhang Z, Chen J, Wu P. 1976. Fossil Lamellibranchiata
of China. Science Press, Beijing, China.
Liu X-Z. 1981. On some newly discovered non-marine pelecypods from the Late Triassic Wuzhongshan Formation in Sichuan Basin. Bulletin of
the Chengdu Institute of Geology and Mineral Resources, Chinese Academy of Geological Sciences 2, 121-136.
Ma Q-H. 1984. Jurassic-Lower Cretaceous Lamellibranchiata from Sichuan Basin. In Editorial group on Continental Mesozoic Stratigraphy and
Palaeontology of Sichuan Basin (eds), Continental Mesozoic Stratigraphy and Palaeontology in Sichuan Basin of China. People’s Publishing
House of Sichuan, Chengdu, China.
Ma Q. 1996. Revision of Mesozoic Margaritiferidae in China and their development. Acta Palaeontologica Sinica 35, 408-429.
Newell ND, Boyd DW. 1975. Parallel evolution in early trigonacean bivalves. Bulletin of the American Museum of Natural History 154, 55-162.
FCUP 353
Palacios P, Sánchez R, 1885. La formación Wealdense en las provincias de Soria y Logroño. Boletín de la Comisión del Mapa Geológico de
España 12, 1-32.
Skawina A, Dzik J. 2011. Umbonal musculature and relationships of the Late Triassic filibranch unionoid bivalves. Zoological Journal of the
Linnean Society 163, 863-883.
Van Damme D, Bogan AE, Dierick M. 2015. A revision of the Mesozoic naiads (Unionoida) of Africa and the biogeographic implications. Earth-
Science Reviews 147, 141-200.
354 FCUP
Diversification rate statistics for the Margaritiferidae and Unionidae clades.
Paradis’s test of diversification with three survival models The constant-rates test
LRT p-value Beta parameter
Clade Div. rate Selected model (by Gamma p-value
(constant rate model of the Weibull
(delta ± s.e.) AIC) statistic (two-sided)
vs. Weibull model) model (± s.e.)
Margaritiferidae 0.014±0.004 0.096 1.488±0.322 Weibull (variable rate -0.628 0.530
through time)
Gibbosula 0.012±0.008 0.037* 5.250±2.097 Weibull (variable rate -0.767 0.443
through time)
Pseudunio + 0.015±0.009 0.703 1.203±0.566 Constant rate 0.945 0.345
Cumberlandia
Margaritifera 0.023±0.009 0.042* 2.135±0.618 Weibull (variable rate -0.838 0.402
through time)
Rectidentinae** 0.037±0.008 0.013* 1.603±0.256 Weibull (variable rate -1.612 0.107
through time)
Rectidentini**,M 0.036±0.011 0.018* 2.744±0.573 Weibull (variable rate -2.306 0.021*
through time)
ContradentiniM 0.047±0.014 0.102 1.520±0.345 Constant rate -0.747 0.455
Pseudodontinae**,M 0.038±0.010 0.022* 1.683±0.324

Weibull (variable rate -1.448 0.148
through time)
M
Superscripts: *variable diversification rate; **Data from Bolotov et al (2017a); Mekong only.
References
Bolotov IN, Kondakov AV, Vikhrev IV, Aksenova OV, Bespalaya YV Gofarov MY, Kolosova YS, Konopleva ES, Spitsyn VM, Tanmuangpak K &
Tumpeesuwan S. 2017. Ancient river inference explains exceptional Oriental freshwater mussel radiations. Scientific Reports 7, 2135.
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Margaritiferidae generic names, authorities, and type species.
Genus name and authority Type species
Gibbosula Simpson, 1900 Mya crassa Wood, 1815
+Margaritanopsis Haas, 1910b1 Unio laosensis Lea, 1863
+(Odhnerella) Modell, 1964 Unio rochechouartii Heude, 1875
Cumberlandia Ortmann, 1912 Unio monodonta Say, 1829
Margaritifera Schumacher 18162 Mya margaritifera Linn., 17582
+Margaritana Schumacher, 18172 Mya margaritifera Linn., 17582
+Damaris “Leach” Swainson, 1823 Mya margaritifera Linn., 1758
+Damalis Leech, 1847 non Fabricius, 1805 Mya margaritifera Linn., 1758
+Baphia Mörch, 1853 non Gray, 1847 Mya margaritifera Linn., 1758
+Margaritiferana Fagot, 1893 Unio elongata Lamarck, 1819 = Mya margaritifera Linn., 1758
+Kurilinaia Bogatov & Zatravkin, 1988 Dahurinaia kurilensis Zatravkin & Starobogatov, 1984 = M. laevis
+Dahurinaia Starobogatov, 1970 Unio dahuricus Middendorff, 1850
+Schalienaia Starobogatov, 1970 Unio hembeli Conrad, 1838
+Baryana Locard, 18893 [nomen nudum] Unio baryus “Bourg.” Locard, 1889 [nomen nudum]
Pseudunio Haas, 1910a Unio sinuata Lamarck, 1819 = U. auricularius Spengler, 1793
+Potamida Agassiz, 1846, non Brongniart, 1810 Unio sinuata Lamarck, 1819 = U. auricularius Spengler, 1793
+Potodoma Haas, 1969b, non Meigen, 1800 Unio sinuata Lamarck, 1819 = U. auricularius Spengler, 1793
1
The date of publication for Margaritanopsis Haas has been used as 1912 (e.g. Haas 1969a; Smith 2001) and Haas 1969b and Graf & Cummings
(2017) used 1910. The correct date of publication is based on the first publication of the generic name in association with a species name that
occurs on Plate 12, figures 1-2 of Haas (1910) published in Lieferung 546, dated 1910 and signature 5, dated 13 August 1910 (see Bogan 2015).
The text description of Margaritanopsis appears on pages 121-122 which was published in Lieferung 559, dated 1912 and signature 16 dated 13
February 1912 (see Bogan 2015).
2
Spelling of Margaritifera and its type species, suppression of the generic name Margaritana and the use of Margaritiferidae have been stabilized
by the ICZN Opinion 495 [1957].
3
Baryana Locard, 1889 (Locard 1889:18) erected the group of Baryana “Bourguignat” in the genus Unio and declared the type of the group as”
Le type de ce groupe est l’Unio baryus Bourg. de l’Euphrate.” Unio baryus is the type species by original designation. The work is credited by
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Locard to a Bourguignat manuscript. Unio baryus Bourg. in Locard is a manuscript name and is a nomen nudum. Graf (2010) noted Unio sinuata
Lamarck, 1819 was the only other included species and observed this genus is invalid and a nomen dubium. Baryana has been used with Unio
sinuata Lamarck as the type species in error, which would make it a senior synonym of Pseudunio Haas 1910 (Graf & Cummings 2017). Vinarski
& Kantor (2016) include Baryana as a synonym of Margaritifera without comment on its nomenclatural status. All generic names were checked
against the database of Neave (2018).
References
Agassiz L. 1846. Nomenclatoris zoologici. Index universalis, continens nomina systematic classium, ordinum, familiarum et generum animalium
omnium. Jent et Gassmann, Soloduri, Switzerland.
Bogan AE. 2015. Determining the date of publication for Contradens Haas and Uniandra Haas (Bivalvia: Unionidae). The Nautilus 129, 175-178.
Bogatov VV, Zatravkin MN. 1988. New species of the order Unioniformes (Mollusca: Bivalvia) from the south of the Soviet Far E ast. Trudy
Zoologicheskogo instituta 187, 155-168.
Conrad TA. 1853. A synopsis of the family of Naïades of North America, with notes, and a table of some of the genera and sub-genera of the
family, according to their geographical distribution, and descriptions of genera and sub-genera. Proceedings of the Academy of Natural Sciences
of Philadelphia 6, 243-269.
Fagot P. 1893. Histoire malacologique des Pyrénées Françaises et Espagnoles. Bulletin de la Société Ramond 28, 247-262.
Graf DL. 2010. Funeral for the Nouvelle École -iana generic names introduced for freshwater mussels (Mollusca: Bivalvia: Unionoida).
Proceedings of the Academy of Natural Sciences of Philadelphia 159, 1-23.
Graf DL. Cummings KS. 2018. The MUSSEL Project Database. Available at http://musselproject.uwsp.edu/db (accessed 4 Jan 18).
FCUP 357
Gray JE. 1847. A list of the genera of Recent Mollusca, their synonyma and types. Proceedings of the Zoological Society of London 15, 129-206.
Haas F. 1910a. Pseudunio, neues Genus für Unio sinuatus Lam. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft 42, 181-183.
Haas F. 1910b-1920. Die Unioniden. Neubearbeitung und Fortsetzung der Küsterschen und Clessinschen Monographien von Unio und Anodonta.
In Küster HC (ed), Systematisches Conchylien-Cabinet von Martini und Chemnitz 9, 1-344.
Haas F. 1969. Superfamilia Unionacea. Das Tierrich, 88. Walter de Gruyter, Berlin, Germany.
Haas F. 1969b. Superfamily Unionacea. In Moore RC (ed), Treatise on Invertebrate Paleontology Part N, Volume 1, Mollusca 6. Bivalvia, pp.
N411-N470. Geological Society of America, The University of Kansas, Kansas, USA.
ICZN. 1957. Opinion 495. Designation under the plenary powers of a type species in harmony with accustomed usage for the nominal genus
“Unio” Philipsson, 1788 (Class Pelecypoda) and validation under the same powers of the family-group name “Margaritiferidae” Haas, 1940.
Opinions and Declarations Rendered by the International Commission on Zoological Nomenclature 17, 287-322.
Lamarck JBPA. 1819. Les nayades. In Histoire naturelle des Animaux sans Vertébres Vol. 6, pp. 67-100. Paris.
Lea I. 1863. Description of a new species of Unio and a Monocondylaea. Proceedings of the Academy of Natural Sciences of Philadelphia 15,
190.
Leach WE. 1847. The classification of the British Mollusca. The Annals and Magazine of Natural History (Series 1) 20, 267-273.
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Linnaeus C. 1758. Systema Naturae. Edition X. (Systema naturae per regna tria naturae, secundum classes, ordines, genera, species cum
characteribus, differentiis, synonymis, locis. Tomus I. Edtio decima, reformata.) Holmiae.
Locard A. 1889. Catalogue des espèces Française appartenant aux genres Margaritana et Unio connues jusqu’a ce jour. Contribution a la Frauna
Malacologique Française, vol. 13, Pitrat aine, Imprimeus, Lyon, France.
Middendorff AT. 1850. Beschreibung einiger Mollusken-arten, nebsteinem Blicke auf der geographischen Character de Land- und Süsswasser-
Mollusken Nord-Asiens. Bulletin de la Classe Physico-Mathématique de l'Académie Impériale des Sciences de Saint-Pétersbourg 9, 108-112.
Mörch OAL. 1852-1853. Catalogus conchyliorum quae reliquit D. Alphonso D’Aguirra & Gadea, Comes de Yoldi, regis daniae cubiculariorum
princeps, ordinis dannebrogici in prima classe & ordinis caroli tertii eques. Hafniae, Typis Ludovici Kleini, Copenhagen, Danmark.
Neave 2018. Neave Nomenclator zoologicus. Available at http://ubio.org/NZ (accessed 4 Jan 2018).
Ortmann AE. 1912. Cumberlandia, a new genus of naiades. The Nautilus 26, 13-14.
Say T. 1829. Descriptions of some new terrestrial and fluviatile shells of North America. The disseminator of useful knowledge; containing hints
to the youth of the United States, from the School of Industry, New Harmony, Indiana, USA
Schumacher CF. 1815 (1816). Afhandling over conchyliologiske Systemer, og om nogle toskallende Conchylier. Oversigt over det Kongelige
Danske Videnskabernes Selskabs Forhandlinger og dets Medlemmers Arbeider i de sidst to Aar.
Schumacher CF. 1817. Essai d’un nouveau système des habitations des vers testacés avec XXII planches. De l’Imprimerie de Mr. le directeur
Schultz, Copenhagen, Denmark.
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Simpson CT. 1900. Synopsis of the Naiades: or pearly fresh-water mussels. Proceedings of the United States National Museum 22, 501-1044.
Smith DG. 2001. Systematics and Distribution of the Recent Margaritiferidae. In Bauer G, Wächtler K (eds), Ecology and Evolution of the
Freshwater Mussels Unionoida, Ecological Studies, pp. 33-49. Springer Berlin Heidelberg, Berlin, Heidelberg, Germany.
Spengler L. 1793. Beskrivelse over et nyt Slægt af de toskallede Konkylier, forhen af mig kaldet Chaena, saa og over det Linnéiske Slægt Mya,
hvilket nøiere bestemmes, og inddeles i tvende Slægter. Skrivter af Natur-historie-Selskabet 3, 16-69.
Starobogatov YI. 1970. Mollusk fauna and zoogeographical partitioning of continental water reservoirs of the world. Zoologischeskii Instituti Nauka,
Akademiya Nauk, Leningrad, Russia.
Swainson W. 1823. Zoological illustrations, or original figures and descriptions of new, rare, or interesting animals, selected chiefly from the
classes of Ornithology, Entomology, and Conchology, and arranged on the principles of Cuvier and other modern zoologists. James Moyes,
London, UK.
Vinarski MN, Kantor Y. 2016. Analytical catalogue of fresh and brackish water molluscs of Russia and adjacent countries. A.N. Severtsov Institute
of Ecology and Evolution of Russian Academy of Sciences, Moscow, Russia.
Wood W. 1815. General conchology: or a description of shells arranged according to the Linnean system and illustrated with plates drawn and
coloured from nature. Vol. 1. John Booth, London, UK.
Zatravkin MN. Starobogatov YI. 1984. New species of the superfamily Unionoidea (Bivalvia, Unioniformes) from the Soviet Far East.
Zoologicheskii Zhurnal 63, 1785-1791.
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CHAPTER 7
The unique mitogenomes of Margaritiferidae
Paper VI
The first Margaritiferidae male (M-type) mitogenome: mitochondrial gene
order as a potential character for determining higher-order phylogeny
within Unionida (Bivalvia)
Lopes-Lima M, Fonseca M, Aldridge Dc, Bogan A, Gan Hm, Ghamizi M, Sousa R, Teixeira
A, Varandas S, Zanatta D, Zieritz A, Froufe E
Article published in Journal of Molluscan Studies 83, 249-252 (2017).
DOI: 10.1093/mollus/eyx009
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The first Margaritiferidae male (M-type) mitogenome: mitochondrial gene

order as a potential character for determining higher-order phylogeny
within Unionida (Bivalvia)
Manuel Lopes-Lima1,2, Miguel M. Fonseca1,2,3, David C. Aldridge4, Arthur E. Bogan5, Han Ming
Gan6,7, Mohamed Ghamizi8, Ronaldo Sousa9, Amílcar Teixeira10, Simone Varandas11, David
Zanatta12, Alexandra Zieritz13, and Elsa Froufe1
1
CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Matosinhos,
Portugal.
2
CIBIO/InBIO-Research Center in Biodiversity and Genetic Resources, Universidade do Porto, 4485-661 Vairão,
Portugal.
3
Department of Biochemistry, Genetics, and Immunology, University of Vigo, Vigo 36310, Spain.
4
Aquatic Ecology Group, Department of Zoology, University of Cambridge, Cambridge CB2 3QZ, United Kingdom.
5
North Carolina Museum of Natural Sciences, MSC 1626, Raleigh, NC 27699-1626, USA.
6
Genomics Facility, Tropical and Medicine Biology Multidisciplinary Platform, Monash University Malaysia, 47500
Petaling Jaya, Selangor, Malaysia.
7
School of Science, Monash University Malaysia, 47500 Petaling Jaya, Selangor, Malaysia.
8
Muséum d’Histoire Naturelle de Marrakech, Faculté des Sciences, Université Cadi Ayyad, Semlalia, B.P. 2390
Marrakech, Morocco.
9
CBMA-Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, 4710-057
Braga, Portugal.
10
CIMO-ESA-IPB-Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus de
Santa Apolónia, 5301-854 Bragança, Portugal.
11
CITAB-UTAD-Centre for Research and Technology of Agro-Environment and Biological Sciences, University of
Trás-os-Montes and Alto Douro,5001-811 Vila Real, Portugal.
12
Biology Department, Institute for Great Lakes Research, Central Michigan University, Mount Pleasant, MI 48859,
USA.
13
School of Environmental and Geographical Sciences, Faculty of Science, University of Nottingham Malaysia
Campus, Semenyih, Selangor, Malaysia
⁎
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Introduction
The unionid family Margaritiferidae, comprising 12 extant species, is widely distributed across
the northern hemisphere in North America, Europe and Asia (Bolotov et al 2016). Most species
in this family have dramatically declined over the last century, with nine of the 12 species
assessed as threatened in the most recent IUCN Red List (IUCN 2016). Among these is the
Moroccan pearl mussel Margaritifera marocana (Pallary, 1918), considered one of the 100
most threatened species on the planet (Baillie & Butcher 2012). This species is now restricted
to two small streams in the Oum Er Rbia and Sebou basins and conservation measures are
urgently needed (Sousa et al 2016). Beyond the conservation concern, Unionida are also
biologically interesting. They present an unusual mechanism of mitochondrial inheritance
called doubly uniparental inheritance (DUI), in which all individuals have the typical maternally
transmitted mtDNA (F-type), but the males possess in their germ cells a paternally inherited
mtDNA instead (M-type) (Zouros et al 1994; Breton et al 2009). So far, DUI has been observed
in over 100 species from four bivalve orders (Gusman, Azuelos & Breton 2017), including three
families within Unionida, i.e. Unionidae, Hyriidae and Margaritiferidae (Walker et al 2006).
However, to date, no whole M-type mitogenome has been published for any species belonging
to the last two of these families.
The gene arrangement within mitogenomes is highly conserved in many taxonomic
groups. For example, most vertebrates share the same gene order (Pereira 2000). In other
faunal groups, like Bivalvia, the mitochondrial genome arrangement is more variable, although
not many distinct gene orders have been described so far (Serb & Lydeard 2003). Still, in
unionoids, mitogenome rearrangements seem to be rare events that are unlikely to be
homoplastic. In this context, the mitogenome gene order might be used as an additional
character for phylogenetic inference. However, its utility for the Unionida phylogeny has never
been tested.
The order Unionida has 6 recognized families with around 800 species (Lopes-Lima et
al 2014), but the phylogenetic relationships among these families are still not fully resolved
(Graf 2013). This lack of coherence among studies has been consistently attributed to the low
number of molecular markers used and insufficient taxon sampling (Bogan & Roe 2008; Graf
2013; Fonseca et al 2016).
Under the above-mentioned assumptions, the aims of the present study are to (1)
sequence and analyse the whole M- and F-type mitogenomes of M. marocana, (2) infer the
phylogenetic relationships among Unionoidea species using all both the F- and M-type mtDNA
sequences publicly available and (3) determine the gene order of all analysed mitogenomes
and evaluate its phylogenetic utility.
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Materials and Methods

One male specimen deposited in the Muséum d’Histoire Naturelle de Marrakech (voucher
MHNM16ZMB23) from the Laabid River (GPS WGS84: 32.142334, -7.027595) was dissected
for the sampling of gonadal and mantle tissue. DNA extractions followed Froufe et al (2016).
The complete M- and F-type mitogenomes were then sequenced, assembled and annotated
using an established pipeline (Gan, Schultz & Austin 2014). The F and M mitogenomes have
been deposited in the GenBank database under the accession numbers KY131953 and
KY131954, respectively.
The two newly obtained M. marocana mitogenome sequences were aligned with all
(43) M- and F-type Unionida mitogenome sequences available on GenBank as of March 2016,
as well as with the F- and M-type mitogenomes of Mytilus galloprovincialis as outgroup (list of
genomes and respective accession numbers used supplied on request). DNA (NUC) and
amino acid (AA) sequences of all mtDNA protein-coding genes (PCGs) except ATP8, and the
gender-specific open reading frames (M-ORF, H-ORF, and F-ORF) were used in the
phylogenetic analyses. The sequences of each gene were aligned using MAFFT v. 7.304
(Katoh & Standley 2013) and trimmed with GUIDANCE v. 1.5 (Penn et al 2010; see Froufe et
al 2016, for parameters used). The gene alignments were then concatenated, resulting in two
alignments with the following length: 14,350 aligned nucleotide positions or 6,246 aligned
amino acid + nucleotide positions (4,085 aligned amino acids positions and 2,161 aligned
nucleotide positions from the rRNAs genes). The optimal partitioning scheme (i.e. the best set
of nonoverlapping partitions that cover the whole alignment) for each alignment was selected
using PartitionFinder v. 1.1.1 (Lanfear et al 2012) under the greedy algorithm with proportional
branch lengths across partitions. The best substitution models of DNA and protein evolution
for each partition were selected under the BIC ranking method (Schwarz 1978). The codon
positions of the PCG and each rRNA were defined as the initial data blocks for the partitioning
schemes search. Maximum likelihood (ML) phylogenetic inference was performed using
RAxML v. 8.0.0 (Stamatakis 2014) with 100 rapid bootstrap replicates and 20 ML searches.
Bayesian Inference (BI) was applied using MrBayes v. 3.2.1 (Ronquist et al 2012) with two
independent runs (1 × 10 7 generations with a sampling frequency of 1 tree for every 100
generations), each with four chains (three hot and one cold). All runs reached convergence
(average standard deviation of split frequencies < 0.01). The posterior distribution of trees was
summarized in a 50% majority-rule consensus tree (burn-in of 25%).
Results and Discussion

The length of the two newly sequenced mitogenomes of M. marocana, 16,001 nt for the female
haplotype and 17,562 nt for the male haplotype, is within the typical range for each sex-specific
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haplotype within Unionida. The sequenced haplotypes include the 13 PCGs typically found in
metazoan mitochondrial genomes, the sex-specific ORF described for all Unionida
mitogenomes with DUI system (Breton et al 2009, 2011a) and 22 transfer RNA (tRNA) and
two ribosomal RNA (rRNA) genes. The M-type genome is the largest sequenced to date within
the Unionida. M-type genomes are generally larger than F-type genomes due to the larger size
of the PCG COX2 and M-ORF in M-type genomes compared with COX2 and F-ORF in F-type
genomes (Breton et al 2009). Four intergenic regions were identified in the M. marocana M-
type genome between the following gene pairs: NAD3-tRNA (A) 106 nt, tRNA(H)-tRNA(Q) 411
nt, ND4L-tRNA(D) 255 nt and tRNA(D)-ATP8 498 nt. These regions were analysed to search
for the M-ORF. The results of the blast search (Altschul et al 1997) retrieved a significant hit
with another Margaritiferidae M-ORF (Margaritifera monodonta, E-value = 4e-34) and a Fickett
test score of 1.201 (Fickett 1982; a score > 0.95 means the sequence is probably coding),
suggesting that the M-ORF is located in the region between the genes ND4L and tRNA(D).
The M-type mitogenome of M. marocana presents a novel gene order within Unionida (Fig. 1).
The F-type mitogenome gene order is the same as already observed for the two previously
available Margaritiferidae F-type mitogenomes (Breton et al 2011b; Yang et al 2015). All the
phylogenies inferred in this study support the reciprocal monophyly of both (Unionidae +
Margaritiferidae) F- and M-type lineages (Fig. 2 shows the topology of the BI-NUC tree; all
other phylogenetic trees figures supplied on request). Additionally, the monophyly of Unionidae
(both F- and M-type), Margaritiferidae (F-type) and all represented Unionidae subfamilies are
well supported in all inferred mtDNA trees, except for the Unioninae, for which monophyly was
only well supported in the BINUC tree. The remaining phylogenetic trees (BI-AA, ML-NUC, and
ML-AA) showed conflicting results regarding the position of the clade comprising Arconaia
lanceolata and Lanceolaria grayana (Fig. 2). These conflicting results have also been found in
previous studies where different mitogenome phylogenetic methodologies revealed distinct
tree topologies (Huang et al 2013; Fonseca et al 2016). Five distinct mtDNA gene orders have
been detected in the present dataset, three in the F-type lineage and two in the M-type lineage.
In the F-type lineage, gene order UF1 is shared by the Unionidae subfamilies Anodontinae,
Ambleminae, and Unioninae, whereas gene orders UF2 and MF1 are found in the represented
species of the subfamily Gonideinae and the family Margaritiferidae, respectively (Figs 1, 2).
In the female lineage, there is only a difference between UF1 and MF1 in the location of
tRNA(E) (Fig. 1). The gene order of UF2 is more distinct and might have resulted from a
tandem duplication of the gene region between COX2 and tRNA(W) followed by random
deletion of segments of the duplicated gene region (Doucet-Beaupré et al 2010). Between the
M and F mitogenomes, the differences are in the location of tRNA(H) and the inversion of the
ATP8-tRNA(D) region. An additional distinct location of tRNA(S1) is also found in margaritiferid
M mitogenomes (Fig. 1).
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Figure 1 Diagrams of the five distinct gene orders detected in Unionida. In the female F-type lineage, three gene orders are depicted: Unionidae
F-type 1 (UF1), Unionidae F-type 2 (UF2) and Margaritiferidae F-type 1 (MF1). In the male M-type lineage, two gene arrangements are shown:
Unionidae M-type 1 (UM1) and Margaritiferidae M-type 1 (MM1). Continuous lines indicate different locations of genes between mitogenomes.
Grey box highlights the gene rearrangement region between UF1 and UF2. Yellow boxes indicate the main differences in gene arrangement
between female and male mitogenomes, tRNA (H) location and rearrangement of ATP8-tRNA(D) region.
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Figure 2 Phylogenetic (BI-NUC) tree of Unionida estimated from 14 concatenated individual mtDNA gene sequences (12 protein-coding and 2
rRNA genes). Values for branch support are represented in the following order: (1) Bayesian posterior probabilities (PP) for BI-NUC tree, (2)
Bayesian PP for BI-AA tree, (3) ML bootstrap support (BS) values for ML-NUC and (4) ML BS values for ML-AA tree. Maximum support values
(PP = 1, BS = 100) are represented by asterisks. All five distinct detected gene orders are mapped on the phylogeny branches (see Fig. 1 for
gene order codes).
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Mapping gene orders over the inferred mtDNA phylogeny suggests that UF1 might be
ancestral within Unionidae and UF2 derived in the ancestral lineage of the Gonideinae.
However, these hypotheses have limited support, because no mitogenome sequences, and
therefore no gene order information, are available for three of the seven presently recognized
unionid subfamilies. Future inclusion of mtDNA gene orders of these currently unrepresented
subfamilies could change the inference of the ancestral and derived mtDNA gene orders within
Unionidae. In the M-type, only one gene arrangement per family is obtained: UM1 for the
Unionidae and MM1 for the Margaritiferidae. Since the Unionida are a very old order (Graf &
Cummings 2007), and as a consequence of the several distinct mitogenome gene
arrangements already found, it is likely that as novel mitogenomes from additional unionoid
families and subfamilies become available, the corresponding gene orders might be useful to
resolve their phylogenetic relationships within the order.
Acknowledgements
This work was partially supported by the Mohamed bin Zayed Species Conservation Fund
under project ‘Biodiversity and conservation of the critically endangered freshwater mussels in
Morocco: ecogeographic, genetic and physiological information’ (Reference 15256799), by the
IUCN SOS Save Our Species fund under project ‘Breeding the most endangered bivalve on
Earth: Margaritifera marocana’ (Ref. 2015B-015) and by the Portuguese Foundation for
Science and Technology (FCT) under grants SFRH/BPD/70654/2010 (MMF),
SFRH/BPD/108445/2015 (EF), and SFRH/BD/115728/2016 (MLL).
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of mitochondrial genome organization in Bivalvia (Mollusca). Molecular Biology and Evolution
20, 1854-1866.

(Margaritifera marocana) in Morocco: Conservation status of the rarest bivalve in African fresh
Yang S, Mi Z, Tao G, Liu X, Wei M, Wang H. 2015. The complete mitochondrial genome
sequence of Margaritiana dahurica Middendorff. Mitochondrial DNA 26, 716-717.
Zouros E, Ball AO, Saavedra C, Freeman KR. 1994. An unusual type of mitochondrial DNA
inheritance in the blue mussel Mytilus. Proceedings of the National Academy of Sciences 91,
7463-7467.
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CHAPTER 8
Evolution of mitogenome rearrangements in
freshwater mussels
Paper VII
Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia:
Unionoidea) macroevolution
Froufe F, Bolotov I, Aldridge DC, Bogan AE, Breton S, Gan HM, Kovitvadhi U, Kovitvadhi S,
Riccardi N, Secci-Petretto G, Sousa R, Teixeira A, Varandas S, Zanatta D, Zieritz A, Fonseca
MM, Lopes-Lima M
Article published in Heredity 124, 182-196 (2020).
DOI: 10.1038/s41437-019-0242-y
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Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia:

Unionoidea) macroevolution
Elsa Froufe1, Ivan Bolotov2,3, David C. Aldridge4, Arthur E. Bogan5, Sophie Breton6, Han Ming
Gan7, Uthaiwan Kovitvadhi8, Satit Kovitvadhi9, Nicoletta Riccardi10, Giulia Secci-Petretto1,
Ronaldo Sousa11, Amilcar Teixeira12, Simone Varandas13, David Zanatta14, Alexandra Zieritz15,
Miguel M. Fonseca1, Manuel Lopes-Lima1,16,17
1
CIIMAR/CIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de
Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, Matosinhos 4450-208, Portugal
2
IBIGER - Institute of Biogeography and Genetic Resources, Federal Center for Integrated Arctic Research,
Russian Academy of Sciences, Severnaya Dvina Emb. 23, Arkhangelsk 163000, Russian Federation
3
Northern Arctic Federal University, Severnaya Dvina Emb. 17, Arkhangelsk 163000, Russian Federation
4
Department of Zoology, University of Cambridge, The David Attenborough Building, Pembroke Street, Cambridge
CB2 3QZ, United Kingdom
5
North Carolina State Museum of Natural Sciences, 11 West Jones St., Raleigh, NC 27601, USA
6
Département de Sciences Biologiques, Université de Montréal, Montréal, QC H2V 2S9, Canada
7
Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong 3220 VIC,
Australia
8
Department of Zoology, Faculty of Science, Kasetsart University Bangkok 10900, Thailand
9
Department of Agriculture, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University,
Bangkok 10600, Thailand
10
CNR - Institute for Ecosystems Studies, Verbania Pallanza (VB), Italy
11
Gualtar, Braga 4710-057, Portugal
12
CIMO/ESA/IPB - Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus
de Santa Apolónia, Apartado 1172, Bragança 5301-854, Portugal
13
Trás-os-Montes and Alto Douro, Forestry Department, Vila Real 5000-801, Portugal
14
Biology Department, Institute for Great Lakes Research, Central Michigan University, Biosciences Bldg. 2408,
Mount Pleasant, MI 48859, USA
15
School of Environmental and Geographical Sciences, University of Nottingham Malaysia Campus, Jalan Broga,
Semenyih 43500, Malaysia
16
Vairão, Rua Padre Armando Quintas 7, Vairão, Porto 4485-661, Portugal
17
SSC/IUCN - Mollusc Specialist Group, Species Survival Commission, International Union for Conservation of
Nature, c/o The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, United Kingdom
Abstract
Using a new fossil-calibrated mitogenome-based approach, we identified macroevolutionary
shifts in mitochondrial gene order among the freshwater mussels (Unionoidea). We show that
the early Mesozoic divergence of the two Unionoidea clades, Margaritiferidae and Unionidae,
was accompanied by a synchronous split in the gene arrangement in the female mitogenome
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(i.e. gene orders MF1 and UF1). Our results suggest that this macroevolutionary jump was
completed within a relatively short time interval (95% HPD 201-226 Ma) that coincided with the
Triassic-Jurassic mass extinction. Both gene orders have persisted within these clades for
~200 Ma. The monophyly of the so-called “problematic” Gonideinae taxa was supported by all
the inferred phylogenies in this study using, for the first time, the M- and F-type mitogenomes
either singly or combined. Within Gonideinae, two additional splits in the gene order (UF1 to
UF2, UF2 to UF3) occurred in the Mesozoic and have persisted for ~150 and ~100 Ma,
respectively. Finally, the mitogenomic results suggest ancient connections between freshwater
basins of East Asia and Europe near the Cretaceous-Paleogene boundary, probably via a
continuous paleo-river system or along the Tethys coastal line, which are well supported by at
least three independent but almost synchronous divergence events.
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Introduction
The tempo, timing, and mode of evolution have attracted considerable debate among
evolutionary biologists. Here we use a new approach using mitogenome rearrangements to
investigate changes at the geological time scale in the speciose and imperilled freshwater
mussels.
In many taxonomic groups, the gene arrangement within mitogenomes is highly
conserved, e.g. many vertebrate groups share the same gene order (Pereira 2000). Other
faunal groups, such as the Bivalvia, exhibit several different mitochondrial gene arrangements
(e.g. Yuan et al 2012), which are the result of different mechanisms such as tandem duplication
followed by gene loss (Boore 2000). Although local homoplastic arrangements have been
identified in some invertebrate groups (e.g. Flook & Rowel 1995; Dowton & Austin 1999),
complete gene orders generally remain unique and represent signatures with diagnostic value
(Basso et al 2017), providing a powerful signal for inferring ancient evolutionary relationships
(Boore 2000).
Among freshwater mussels of the order Unionida, which spans about 900 species and
represents the major bivalve radiation in the freshwater environment (Lopes-Lima et al 2017a,
2018a), five mitogenome rearrangements have been described so far (Lopes-Lima et al
2017b). Within the superfamily Unionoidea (Margaritiferidae + Unionidae), the mitochondria
are furthermore unusual in that two highly divergent mtDNA molecules exist in males (Female
or F- and Male or M-type) as a result of Doubly Uniparental Inheritance (DUI) (Zouros et al
1994; Breton et al 2009). This contrasts with most animal taxa, which inherit their mtDNA
exclusively through the maternal lineage and thus exhibit only F-type mtDNA. In Unionoidea
males, M-type mtDNA is restricted to the gonadal tissue inherited from the paternal lineage,
and F-type mtDNA is present in all somatic tissues transmitted from the maternal lineage and
also in female gonadal tissue (Breton et al 2009; Froufe et al 2016; Fonseca et al 2016; Lopes-
Lima et al 2017b).
In recent decades, complete mitochondrial genome sequences have been published
for a wide range of taxa, enabling reconstruction of shallow and deep phylogenies in both
vertebrates and invertebrates (e.g. Jacobsen et al 2014; Liu et al 2016). However, the number
of available mitogenomes for Unionida is low, particularly for M-type genomes (Froufe et al
2016; Fonseca et al 2016; Lopes-Lima et al 2017b; Huang et al 2019). A further shortcoming
is that published mitogenomes are restricted to only a few higher Unionida taxa, with no
mitogenomes being available for several families and subfamilies. In fact, of the six recognized
Unionida families (Lopes-Lima et al 2014), published mitogenomes are essentially restricted
to the Unionoidea (Unionidae + Margaritiferidae) with a distribution predominantly within the
Northern Hemisphere. While some studies have questioned the monophyly of the Unionoidea
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(e.g. Combosch et al 2017; Whelan et al 2011) the most comprehensive recent studies, using
either full mitogenomes (Huang et al 2019; Wu et al 2019) or hundreds of nuclear loci (Pfeiffer
et al 2019) support its monophyletic status. Moreover, mitogenome-based Unionida
phylogenies reconstructed to date have been based on either F- or M-type mitogenomes
(Froufe et al 2016; Fonseca et al 2016; Lopes-Lima et al 2017b). Although in these studies the
highly divergent F- and M-type mitogenomes recovered identical phylogenies, concatenated
phylogenetic analyses of M- and F-type datasets would be expected to recover a more robust
phylogeny.
The Unionidae is the most species-rich Unionida family, comprising 620 species in
several subfamilies and distributed widely (Lopes-Lima et al 2017a). However, phylogenetic
relationships within and between Unionidae subfamilies are still contentious and different
phylogenies have been resolved with different analysed markers (e.g. Lopes-Lima et al 2017a;
Bolotov et al 2017a).
One of the least studied Unionidae subfamilies, the Gonideinae, has a scattered
distribution in the Northern Hemisphere (Lopes-Lima et al 2017a). Species in this subfamily
have suffered major declines, and half of the assessed Gonideinae species are currently listed
as Near Threatened or Threatened (IUCN 2019). Moreover, 70% of recognized Gonideinae
species have either never been assessed or are listed as Data Deficient by the IUCN Red List
(IUCN 2019), indicating an urgent need for research into this family’s diversity, distribution, and
ecology.
Another outcome of the general lack of data on Gonideinae is their unresolved
phylogeny. Monophyly of this sub-family is disputed. The first molecular study to include the
so-called “problematic” Gonideinae taxa (Graf 2002) only examined the type species, i.e.
Gonidea angulata (Lea 1838). Subsequent studies included several additional Gonideinae
taxa but the clade Gonideinae was never recovered as monophyletic (Graf & Cummings 2006;
Whelan et al 2011; Pfeiffer & Graf 2013). More recently, multi-marker and mitogenomic
approaches have consistently recovered Gonideinae as monophyletic (Huang et al 2013;
Pfeiffer & Graf 2015; Fonseca et al 2016; Froufe et al 2016; Lopes-Lima et al 2017a,b). Bolotov
et al (2017a,b) subsequently elevated one of the four Gonideinae tribes established by Lopes-
Lima et al (2017a), i.e. Pseudodontini, to the subfamily level (i.e. Pseudodontinae).
A good understanding of the evolutionary biogeography of the Gonideinae can be
fundamental for reconstructing patterns of connections of freshwater systems through space
and time on a global scale. Our knowledge in this respect is still far from complete. The first
biogeographic scenarios developed using Unionida data (e.g. Starobogatov 1970; Banarescu
1991) proved highly inaccurate, as they were mostly descriptive and based solely on the (dis-
)similarity between unionid faunas. Furthermore, these scenarios were generated at a time
when unionid taxonomy was poorly resolved and included numerous paraphyletic higher-order
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taxa as well as nominal taxa, determined by shell shape rather than reliable indicators of true
biological species (e.g. Bolotov et al 2017a; Konopleva et al 2017). Modern paleontology-
based models seem to be much more reliable. Based on the fossil record from Vietnam,
Schneider et al (2013) developed the hypothesis of the independent development of Unionida
faunas in the Yangtze and Mekong basins, at least during the entire Cenozoic. Also, Van
Damme et al (2015) showed that the African Early Cretaceous Unionida are representatives
of Asian/Eurasian taxa with the lack of Gondwanan elements, while the African Jurassic
assemblages are distinctly related to those in Eurasia.
Recently, a first statistical biogeographic model for the Unionidae at the global level
indicated that the Unionidae most likely originated in Southeast and East Asia in the Jurassic,
with the earliest expansions into North America and Africa (since the Albian), following the
colonization of Europe and India (Bolotov et al 2017a). However, the Jurassic fossil record of
western North America (for a review see Watters 2001) and Africa (Van Damme et al 2015)
indicate that these continents were colonized before the Cretaceous. Additionally, two species-
rich monophyletic mussel radiations with an early Cenozoic or even pre-Cenozoic origin were
discovered within the paleo-Mekong catchment (Bolotov et al 2017a,b). These findings
revealed that the largest river systems (e.g. the Mekong, Yangtze, and Mississippi) may
represent ancient evolutionary hotspots of freshwater mussels (Scholz & Glaubrecht 2004;
Wesselingh 2007).
Based on the most comprehensive data set of mitogenomes sampled to date, including
eight newly sequenced mitogenomes, this paper aims to improve our understanding of the
higher-order phylogeny and classification of Unionidae by the following: (1) testing the
monophyly of the poorly known Gonideinae subfamily using both full F- and M- mitogenomes
and, for the first time, mitogenomes concatenated; (2) estimating macroevolutionary patterns
in freshwater mussels of the Unionidae using, for the first time, a fossil-calibrated mitogenomic
approach; (3) estimating the timing of major divergence events and comparing them to those
of mitogenome rearrangements; and (4) developing an updated integrative approach to the
systematics of Unionidae, based on the mitogenomic results. This will allow the reconstruction
of the potential origin and ancient radiations of the Unionidae and detect the most probable
ancestral areas.
Methods
Sampling, DNA extractions, sequencing, assembly, and annotation
One male specimen of Chamberlainia hainesiana, Microcondylaea bonellii, Pilsbryoconcha
exilis and Monodontina vondembuschiana were dissected for the sampling of gonadal (to
recover M-type mtDNA) and mantle (to recover F-type mtDNA) tissues. DNA extractions
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followed Froufe et al (2016). The complete M- and F-type mitogenome sequencing and
assemblage followed Gan et al (2014), while annotations were performed using MITOS (Bernt
et al 2013). The final limits of tRNA genes were rechecked with ARWEN (Laslett & Canbäck
2008). All F- and M-mitogenomes have been deposited in the GenBank database under the
accession numbers MK994770-MK994777 and were visualized using GenomeVx (Conant &
Wolfe 2008).
DNA (NUC) and amino acid (AA) sequences of all mtDNA protein-coding genes (PCG),
except ATP8 and the gender-specific open reading frames (M-ORF, H-ORF, and F-ORF;
Breton et al 2011), were used in the phylogenetic analyses. The sequences of each gene were
aligned using MAFFT software (version 7.304, Katoh & Standley 2013) and trimmed with
GUIDANCE2 (Sela et al 2015; see Froufe et al (2016) for the parameters used).
The gene alignments were then concatenated, resulting in two alignments with the
following length: 13,449 aligned nucleotide positions and 3,870 aligned amino acid positions +
1,889 aligned nucleotide positions from the rRNA genes. The optimal partitioning scheme for
each alignment was selected using PartitionFinder v. 2.1.1 software (Lanfear et al 2016) under
the greedy algorithm with proportional branch lengths across partitions. The best substitution
models of DNA and protein evolution for each partition were selected under the BIC ranking
method (Schwarz 1978). The codon positions of the protein-coding genes and each rRNA were
defined as the initial data blocks for the partitioning schemes search.
An additional data set was also created, concatenating both F- and M-type gene
alignments, with the following length: 26,780 aligned nucleotide positions and 7,661 aligned
amino acid positions + 3,797 aligned nucleotide positions from the rRNA genes. This alignment
included 45 Unionida species plus Mytilus galloprovincialis as an outgroup (Table 1) using the
same partitioning method and model selection as described above.
All phylogenetic analyses were performed using both Maximum Likelihood (ML) and Bayesian
Inference (BI) methods. ML analyses were performed using RAxML (v. 8.0.0, Stamatakis
2014) with 100 rapid bootstrap replicates and 20 ML searches. The BI was applied using
MrBayes v. 3.2.7a (Ronquist et al 2012) with two independent runs (10 7 generations with a
sampling frequency of 1 tree for every 100 generations), each with four chains (3 hot and 1
cold). All runs reached convergence (average standard deviation of split frequencies below
0.01). The posterior distribution of trees was summarized in a 50% majority-rule consensus
tree (burn-in of 25%).
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Table 1
List of specimens analysed (based on Lopes-Lima et al 2017a,b), GenBank references, and country
TAXON CODE F-TYPE GenBank M-TYPE GenBank COUNTRY
UNIONIDA
UNIONIDAE
AMBLEMINAE
Lampsilis ornata LamOrn NC_005335 - USA
Leptodea leptodon LepLeo NC_028522 - China (Introduced)
Potamilus alatus PotAla KU559011 KU559010 China (Introduced)
Quadrula quadrula QuaQua NC_013658 FJ809751 USA
Toxolasma parvum TaxPar NC_015483 - USA
Venustaconcha ellipsiformis VenEll FJ809753 NC_013659 USA
GONIDEINAE
CHAMBERLAINIINI
Chamberlainia hainesiana ChaHai MK994770 MK994771 Thailand
Sinohyriopsis cumingii SinCum NC_011763 KC150028 China
Sinohyriopsis schlegelii SinSch NC_015110 - China (Introduced)
GONIDEINI
Microcondylaea bonellii MicBon MK994772 MK994773 Italy
Ptychorhynchus pfisteri PtyPfi KY067440 - China
Solenaia carinata SolCar NC_023250 KC848655 China
Solenaia oleivora SolOle NC_022701 - China
LAMPROTULINI
Lamprotula leai LamLea NC_023346 - China
Lamprotula scripta LamScr NC_030258 - China
Potomida littoralis PotLit NC_030073 KT247375 Portugal
Pronodularia japanensis ProJap AB055625 AB055624 Japan
PILSBRYOCONCHINI
Pilsbryoconcha exilis PilExi MK994776 MK994777 Malaysia
Monodontina vondembuschiana PseVon MK994774 MK994775 Malaysia
UNIONINAE
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Aculamprotula tientsinensis AcuTie NC_029210 - China

Aculamprotula coreana AcuCor NC_026035 - South Korea
Aculamprotula tortuosa AcuTor NC_021404 - China
Anemina arcaeformis AneArc NC_026674 - China
Anemina euscaphys AneEus NC_026792 - China
Anodonta anatina AnoAna NC_022803 KF030962 Poland
Cristaria plicata CriPli NC_012716 - China
Cuneopsis pisciculus CunPis NC_026306 - China
‘Lamprotula gottschei’* LamGot NC_023806 - China
Lanceolaria grayana LanGra NC_026686 - China
Lanceolaria lanceolata ArcLan NC_023955 - China
Lasmigona compressa LasCom NC_015481 - USA
Lepidodesma languilati LepLan NC_029491 - China
Nodularia douglasiae NodDou NC_026111 - China
Pyganodon grandis PygGra NC_013661 FJ809755 USA
Sinanodonta lucida SinLuc NC_026673 - China
Sinanodonta woodiana SinWoo HQ283348 KM434235 China
Unio crassus UniCra KY290447 KY290450 Poland
Unio delphinus UniDel KT326917 KT326918 Portugal
Unio pictorum UniPic NC_015310 - Poland
Unio tumidus UniTum KY021076 KY021073 Poland
Utterbackia imbecillis UttImb NC_015479 - USA
Utterbackia peninsularis UttPen HM856636 NC_015477 USA
MARGARITIFERIDAE
Margaritifera dahurica MarDah NC_023942 - China
Margaritifera falcata MarFal NC_015476 - USA
Pseudunio marocanus PseMrc KY131953 KY131954 Morocco
MYTILIDA
Mytilus galloprovincialis MytGal AY497292 AY363687 Greece
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Divergence time estimates

The time-calibrated mitogenomic phylogeny was reconstructed in BEAST v. 1.8.4 based on
two reliable fossil calibrations (Supplementary Table 1) using a lognormal relaxed clock
algorithm with the Yule speciation process as the tree prior (Drummond et al 2006, 2012;
Drummond & Rambaut 2007). Calculations were performed at the San Diego Supercomputer
Center through the CIPRES Science Gateway (Miller et al 2010). The sample of M-type
mitogenomes was used as an outgroup. Similar settings to each gene partition as in the
MrBayes analyses were specified but using a simplified evolutionary model (HKY; see Bolotov
et al 2017a for details). Five replicate BEAST searches were conducted, each with 5 × 10 7
generations and a tree sampling every 5,000th generation. The log files were checked visually
with Tracer v. 1.7 for an assessment of the convergence of the MCMC chains and the effective
sample size of parameters (Rambaut et al 2018). The chains in one run did not reach the
convergence and were excluded, the other runs were compiled with LogCombiner v. 1.8.4
(Drummond et al 2012) using an appropriate burn-in depending on the start of convergence of
MCMC chains in each run. Most of ESS values were recorded as > 300, with a few ESS values
> 100. The maximum clade credibility tree was obtained from the post-burn-in trees using
TreeAnnotator v. 1.8.4 (Drummond et al 2012).
Ancestral gene order and ancestral area reconstructions

TreeREx (Bernt et al 2008) was used for inferring the most parsimonious putative ancestral
gene orders and gene rearrangements along the obtained Unionidae F-haplotype phylogenetic
sub-tree with the default settings (http://pacosy.informatik.uni-leipzig.de/185-0-TreeREx.html).
Ancestral area reconstruction models were calculated for the Unionidae using three different
approaches, i.e. Statistical Dispersal-Vicariance Analysis (S-DIVA), Dispersal-Extinction
Cladogenesis (Lagrange configurator, DEC), and Statistical Dispersal-Extinction Cladogenesis
(S-DEC) implemented in RASP v. 3.2 (Yu et al 2015) following Bolotov et al (2017a).
Margaritiferidae were not used in this analysis due to the limited number of available
mitogenomes. Four possible distribution areas of the in-group taxa were coded as follows: (A)
Southeast Asia, (B) East Asia, (C) North America, and (D) Europe. From the input matrix, two
geographically unreliable constrains (AC and AD) were excluded.
Results
Mitogenome characteristics and gene arrangements
All eight sequenced haplotypes include the 13 protein-coding genes (PCGs) typically found in
metazoan mitochondrial genomes, the sex-specific ORF described for all Unionida
mitogenomes with DUI system (Breton et al 2009, 2011) and 22 transfer RNA (tRNA) and two
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ribosomal RNA (rRNA) genes (Fig. 1). As expected, the length of the four newly sequenced
M-type mitogenomes is larger than the corresponding F-type (Breton et al 2009), ranging from
16,267 bp in P. exilis to 17,465 bp in C. hainesiana, while the F-type ranged from 16,020 bp in
M. bonellii to 16,746 bp in C. hainesiana (Table 2). The A + T content, and GC and AT skews
are similar in all sequenced species in both F- and M- mtDNA types, averaging around 60%,
37 (+) and -0.23 (+), respectively (Table 2).
The gene arrangements of Microcondylaea bonellii, P. exilis, and Monodontina
vondembuschiana are identical to all Gonideinae mitogenomes available on GenBank (2018),
named UF2 (Lopes-Lima et al 2017b). However, C. hainesiana has a new and distinct gene
arrangement, here named UF3 (Fig. 2).
All the phylogenies inferred in this study that are based on M and F mitogenomes alone (i.e.
not combined) support the monophyly of Gonideinae (Fig. 3). Moreover, the four tribes
Chamberlainiini, Gonideini, Lamprotulini, and Pilsbryoconchini, are also monophyletic in both
M- and F-type trees (Fig. 3). The same results were obtained when using for the first time the
M and F mitogenomes combined, despite the lower number of species (Fig. 4). The only
unsupported result on the topology is seen in the relationship among the tribes Gonideini,
Pilsbryoconchini and Lamprotulini in the ML AA data set (Fig. 4).
Ancestral gene order and ancestral area reconstructions

The TreeREx analysis indicated that the evolution of gene orders in the Unionidae F-type
mtDNA is characterized by two independent events of tandem duplication and random loss
(TDRL), with every ancestral gene order showing the highest consistency scores. The analysis
suggests that the ancestral gene order for Unionidae F mitogenome is UF1, which is also found
in the contemporary species of the subfamilies Ambleminae and Unioninae (Fig. 5). The fossil-
calibrated mitogenomic analysis placed the split between the UF1 and MF1 gene orders in the
Late Triassic (mean age = 208 Ma, 95% high posterior density (HPD) 201-226 Ma) (Fig. 6).
The ancestral gene order of the Gonideinae species represented in our study is UF2, which
results from a TDRL event of an mtDNA segment involving nad3, trnH, trnA, trnS2, trnS1, trnE,
nad2, and trnM (Fig. 2 Box A). In UF2, the genes trnH, trnS1, nad2, and trnM pertain to the
original segment, while the remaining genes-nad3, trnA, trnS2, and trnE-are present in the
duplicated segment (Fig. 2 Box A). The fossil-calibrated model developed suggests that the
UF1 and UF2 gene orders split near the Jurassic-Cretaceous boundary (mean age=149 Ma,
95% HPD 138-162Ma) (Fig. 6).
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Figure 1 Gene maps of the F- and M-type mitochondrial genomes of Chamberlainia

hainesiana, Microcondylaea bonellii, Pilsbryoconcha exilis, and Monodontina
vondembuschiana. Genes positioned inside the circle are encoded on the heavy strand, and
genes outside the circle are encoded on the light strand. Colour codes: Small and large
ribosomal RNAs (red), transfer RNAs (purple), FORF F-specific open reading frame (yellow),
MORF M-specific open reading frame (yellow), PCGs genes (green)
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Table 2
Main structural features of the female (above) and male (below) transmitted mitochondrial genomes of Gonideinae species
Chamberlainiini Gonideini
♀ C. hainesiana S. cumingii S. schlegelii M. bonellii P. pfisteri S. carinata S. oleivora

Tot. size (pb) 16,746 15,954 15,939 16,020 16,040 16,716 16,392
A+T% 58.10 60.24 60.30 62.00 60.77 60.89 59.93
GC (+) skew 0.37 0.36 0.35 0.35 0.36 0.39 0.37
AT (+) skew -0.29 -0.23 -0.23 -0.20 -0.22 -0.22 -0.22
♂ C. hainesiana S. cumingii M. bonellii S. carinata
Tot. size (pb) 17,465 17,100 16,737 17,102
A+T% 62.35 59.71 59.79 61.01
GC (+) skew 0.43 0.41 0.35 0.38
AT (+) skew -0.24 -0.27 -0.26 -0.27
Lamprotulini Pilsbryoconchini
♀ L. leai L. scripta P. littoralis P. japanensis P. exilis M. vondembuschiana
Tot. size (pb) 16,530 16,250 15,789 16,826 16,168 16,028
A+T% 60.28 58.95 58.23 57.20 60.72 58.97
GC (+) skew 0.37 0.36 0.36 0.36 0.37 0.38
AT (+) skew -0.21 -0.23 -0.23 -0.21 -0.22 -0.24
♂ P. littoralis P. japanensis P. exilis M. vondembuschiana
Tot. size (pb) 16,451 16,967 16,267 16,364
A+T% 58.93 57.12 61.90 59.55
GC (+) skew 0.34 0.36 0.35 0.37
AT (+) skew -0.24 -0.25 -0.25 -0.26
Newly sequenced species are presented in bold
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Figure 2 Diagrams of the four distinct gene orders known in Unionidae to date. In the F-type, three gene orders are depicted: UF1, UF2, and
UF3. In the male M-type lineage, the only Unionidae gene arrangement is shown: M-type 1 (UM1). Blue boxes highlight the gene rearrangement
region from UF1 to UF2 (Box A) and from UF2 to UF3 (Box B). Small and large ribosomal RNAs and transfer RNAs are depicted by one letter of
the amino acid code; Arrow colour codes, follow Fig. 1
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Figure 3 Phylogenetic (BI-NUC) tree of Unionida estimated from 14 concatenated individual mtDNA gene sequences (12 protein-coding and 2
rRNA genes). Values for branch support are represented in the following order: (1) Bayesian posterior probabilities (PP) for BI-NUC tree, (2)
Bayesian PP for BI-AA tree, (3) ML bootstrap support (BS) values for ML-NUC and (4) ML BS values for ML-AA tree. Maximum support values
(PP = 1, BS = 100) are represented by asterisks. Gonideinae subfamily and tribes are highlighted. For details see text. GenBank codes in Table
1
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Finally, the UF3 gene order also arises after a TDRL event within Gonideinae (Fig. 2 Box B).
It involved an mtDNA segment containing twelve genes: trnQ, trnC, trnI, trnV, trnL2, nad1,
trnG, nad6, nad4, nad4l, atp8 and trnD. In UF3, the genes trnC, trnI, trnV, trnG, nad6, atp8,
and trnD are retained in the original segment, whilst genes trnQ, trnL2, nad1, nad4, and nad4l
were not lost in the duplicated one (Fig. 2 Box B). The fossil-calibrated model placed the split
between the UF2 and UF3 gene orders in the Cretaceous near the Albian-Cenomanian
boundary (mean age = 102 Ma, 95% HPD 77-124 Ma) (Fig. 6).
Figure 4 Phylogenetic (BI-NUC) tree of Unionida estimated from 28 concatenated individual

mtDNA gene sequences (24 protein-coding and 4 rRNA genes) of the first combined Female
+ Male concatenated data set. Maximum branch support values (BI-NUC/BI-AA PP = 1; ML-
NUC/ML-AA BS = 100) are represented by asterisks, while # represents the only non-
supported branch by ML-AA tree. Gonideinae subfamily and tribes are highlighted. GenBank
codes in Table 1
The combined ancestral area reconstruction model suggests that the Most Recent Common
Ancestor (MRCA) of the crown group of the Ambleminae + (Gonideinae + Unioninae) clade
used to be widely distributed across the supercontinent of Laurasia (probability 100%) (Fig. 7).
The earliest split was between the Laurentian (Ambleminae) and Eurasian (Gonideinae +
Unioninae) taxa. This vicariance event is placed in the Late Jurassic (mean age = 159 Ma,
95% HPD 155-170 Ma). Early diversification of the Gonideinae + Unioninae clade is placed
within East Asia (probability 100%; Fig. 7). The origin of the MRCA of this large clade (mean
age = 149 Ma, 95% HPD 138-162 Ma) and subsequent splitting into two subclades (mean
ages of crown groups = 137 and 106 Ma and 95% HPD 123-152 and 90-124 Ma for Gonideinae
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and Unioninae, respectively) most likely resulted from an intra-area radiation (probability 100%
in each case) during the early Cretaceous. The Yangtze and Mekong unionid faunas have
likely been separated since the Albian (mean ages = 95-102 Ma, 95% HPD 77-124 Ma) (Fig.
7).
Figure 5 Unionidae F-haplotype phylogenetic sub-tree (BI-NUC) used to infer the most
parsimonious putative ancestral gene orders and gene rearrangements mapped as MF1, UF1,
UF2, and UF3 (see text for details). Margaritiferidae and all subfamily nodes were collapsed
for visual purposes
Discussion
Phylogenetic patterns
The new mitogenomic results presented here place the Pilsbryoconchina subtribe (previously
under the Pseudodontinae as described by Bolotov et al 2017a) as a subclade within the
monophyletic Gonideinae in both the M- and F-type phylogenies. Our results are thus in
agreement with the phylogeny recovered by Lopes-Lima et al (2017a), which is also supported
by morphological data. However, the recovered results contradict that of Bolotov et al
(2017a,b), which suggested elevation of the Pseudodontini to the subfamily level. Our results
further indicate that the number of recognized subfamilies within the Unionidae is most likely
lower than has been suggested by recent phylogenetic studies (Lopes-Lima et al 2017a,b;
Bolotov et al 2017a,b).
388 FCUP
Figure 6 Time-calibrated mitogenomic phylogeny, an example of the three-level classification scheme (subfamilies, tribes, and subtribes) and
evolution of the mitochondrial gene order in the Unionoidea. Fossil-calibrated ultrametric chronogram of the Unionoidea calculated under a
lognormal relaxed clock model and a Yule process speciation implemented in BEAST and obtained for the complete mitogenome dat a set. The
outgroup sample is not shown. Bars indicate 95% confidence intervals of the estimated divergence times between lineages (Ma). Black numbers
near nodes are mean ages (Ma). Colour labels indicate the mitochondrial gene order (MF1, UF1, UF2, and UF3). Red asterisks indicate fossil
calibrations (Supplementary Table 1). Stratigraphic chart according to the International Commission on Stratigraphy 2015
FCUP 389
Figure 7 Historical biogeography of the Unionidae. This combined scenario has been inferred
from three different statistical modelling approaches (S-DIVA, DEC, and S-DEC) based on the
time-calibrated mitogenomic phylogeny (Fig. 6). Pie charts near nodes indicate probabilities of
certain ancestral areas. Colour circles on the tip nodes indicate the range of each species.
Colour labels indicate the mitochondrial gene order (UF1, UF2, and UF3)
The mitogenomic results fully support three large subfamily-level clades: Ambleminae,
Gonideinae, and Unioninae. It is important to note that our analyses did not include members
of the Parreysiinae and Rectidentinae. Nor did it include sequences of Modellnaia siamensis,
the only species of the monotypic Modellnaiinae, which is characterized by several
morphological and anatomical autapomorphies suggesting its separation within the Unionidae
as a “phylogenetic relic” (Brandt 1974; Heard & Hanning 1978). Future studies including full
mitogenomes of several taxa from Parreysiinae, Rectidentinae, and Modellnaiinae are needed
to fully resolve the higher-level phylogeny of the global Unionidae. Our results highlight that
resolving the systematics of a large, species-rich clade such as the Unionidae is a complex
task. Previous taxonomic schemes for the Unionidae included only two levels of family-group
names, i.e. subfamilies and tribes (reviews: Lopes-Lima et al 2017a; Bolotov et al 2017a,b).
However, our whole mitogenome analyses reveal that despite the limited number of taxa
390 FCUP
included, the Unionidae classification scheme could be better explained by including three
levels of family-group names (i.e. subfamilies, tribes, and subtribes) to accurately reflect the
presence of several levels of highly divergent clades within this family (Fig. 6). Subfamilies
represent the largest clades that are fully supported by the mitogenomic approach (Fig. 7);
some of which may be characterized by unique morphological synapomorphies, although
several subfamilies have been supported by molecular data only (e.g. Lopes-Lima et al 2017a).
The most recent nuclear-based Unionoida phylogeny (using hundreds of nuclear
protein-coding loci; Pfeiffer et al 2019) shows strong similarity to our findings concerning the
relationships of both families and subfamilies. Moreover, mitogenome data currently available
suggest that the Unionidae comprise seven (Lopes-Lima et al 2017a) or eight (Bolotov et al
2017a) subfamily clades. Of these, the Gonideinae (encompassing Pseudodontinae),
Unioninae (encompassing the Anodontinae) and Ambleminae were well supported in the
mitogenomic results obtained herein, whilst the validity and placement of the Parreysiinae,
Rectidentinae and Modellnaiinae clades are yet to be confirmed by mitogenomic analyses.
The largest monophyletic clades, within each subfamily, exhibiting significant
morphological synapomorphies and fully supported by the present mitogenomic results, are
herein considered as tribes. Therefore, using these criteria, the Gonideinae comprise two
tribes, i.e. Gonideini (trapezoidal to rectangular shells with none or only vestigial hinge teeth
and tetragenous brooding type) and Chamberlainiini (round to oval shells, with a well-
developed hinge structure and ectobranchous brooding type).
The subtribes represent smaller but distant clades within the tribes, comprising several
genera or even a single highly divergent genus that usually does not reveal any unique
synapomorphies but can be distinguished based on molecular characters. Based on data
available to date, including the present results, the Gonideini comprise at least five subtribes,
i.e. Chamberlainiina, Gonideina, Lamprotulina, Pilsbryoconchina and Pseudodontina (Lopes-
Lima et al 2017a; Bolotov et al 2017a,b).
Macroevolutionary patterns of the Unionidae

The new mitogenomic analysis presented herein supports the hypothesis of an ancient
Mesozoic origin and diversification of the Unionoidea (Taylor 1988; Ma 1996; Van Damme et
al 2015; Bolotov et al 2016; Araujo et al 2017; Bolotov et al 2017a,b). The new results indicate
that the Late Triassic split between the Margaritiferidae and Unionidae coincided
approximately with the Triassic-Jurassic extinction that was one of the largest mass extinction
events in the Phanerozoic (Watters 2001; Hesselbo et al 2002; Bogan & Weaver 2012; Percival
et al 2017; Smithwick & Stubbs 2018). The divergence event between the two families was
associated with the TDRL event leading to the formation of the two stable mitochondrial gene
orders, i.e. MF1 and UF1, which have persisted without changes for ~200 Ma. However, there
FCUP 391
were at least two additional Mesozoic splits in the mitochondrial gene order (i.e. UF1 vs. UF2
and UF2 vs. UF3) within the Unionidae, with UF2 and UF3 being restricted to a single
subfamily, the Gonideinae. The first split coincided with the origin of this subfamily but the UF3
is a third, new and distinct gene arrangement derived from UF2 present in a single species,
Chamberlainia hainesiana. These two mitochondrial gene orders have also persisted for long-
term periods of ~150 and ~100 Ma for UF2 and UF3, respectively.
At least two splits in the mitochondrial gene order were associated with the origin of the
MRCAs of large and diverse clades at the family (Unionidae vs. Margaritiferidae) or subfamily
(Unioninae vs. Gonideinae) levels. Concerning this evidence, these TDRL events could be
considered progressive evolutionary innovations because they lead to the formation of stable
gene orders that have persisted within widely distributed and diverse clades for ~150-200 Ma.
As for the mitogenome gene order, our ancestral state analyses suggest UF1 (in the
Unionidae) as the ancestral gene order, which is maintained in the subfamilies Ambleminae
and Unioninae sensu lato (Fig. 6). Additionally, it indicates that the evolution of F-type mtDNA
gene orders is characterized by two independent events of TDRL (Moritz et al 1987; Boore
2000). One resulted in the evolution of UF2, present in the Gonideinae, and the other in UF3,
within Gonideinae but restricted to Chamberlainia hainesiana. In contrast, all sequenced M-
type Unionidae mitogenomes to date present the same gene order, i.e. UM1 (Lopes-Lima et
al 2017b) (Fig. 2). Possibly this could be explained by the higher natural selection pressure
and/or due to the tight control of the DUI system on the paternal mitochondrial inheritance. In
summary, our results reveal that each TDRL event was followed by the stable long-term
persistence of a mitochondrial gene order through evolving lineages (or even a single lineage,
although the Chamberlainia clade may be under-sampled) and corresponds to the first reliable
mitogenomic evidence supporting the evolutionary stasis in molecular traits of freshwater
bivalves. However, this should be further explored using an expanded data set of mitochondrial
genomes that may facilitate the understanding of how evolutionary rates have shifted across
multiple genetic loci and how that corresponds to ecologically relevant traits.
Diversification and Biogeography

Combining our new fossil-calibrated mitogenomic analyses with robust ancestral area
reconstruction provides new insights into early diversification patterns and biogeography of the
Unionidae. According to our results, the Ambleminae + (Gonideinae + Unioninae) clade
originated in the late Jurassic, with their MRCA distributed across Laurentia and Eurasia of the
supercontinent of Laurasia. The split between the Ambleminae and Gonideinae + Unioninae
clades was likely associated with a vicariance event driven by plate tectonics, i.e. the formation
of the early Jurassic Transcontinental Laurasian Seaway (Bjerrum et al 2001). The
Ambleminae is an entirely Laurentian subfamily, which diversified primarily through radiation
392 FCUP
within the Mississippi drainage basin from the Early Cretaceous (Bolotov et al 2017a). In this
context, a peculiar Unionidae fauna from the Late Jurassic of western North America (Watters
2001) appears to be ancestral lineages and stem groups of the Ambleminae + (Gonideinae +
Unioninae) clade. The Gonideinae and the Unioninae (Unionini, Anodontini, Lanceolariini, and
Lepidodesmini) (Fig. 6) originated in East Asia, most likely via intra-area radiation within the
paleo-Yangtze River system during the Cretaceous (Fang et al 2009; Wang et al 2018). The
Southeast Asian Gonideinae taxa (Mekong basin) were separated via several vicariance
events in the Albian - Cenomanian, which may indicate the drainage rearrangement of paleo
river systems of the Indochina Peninsula and surrounding terrains during this period (Wang et
al 2018). The mitogenomic results suggest ancient connections between freshwater basins of
East Asia and Europe near the Cretaceous-Paleogene boundary, probably via a continuous
paleo-river system or along the Tethys coastal line (Hou & Li 2018), and this is also depicted
in the Margaritiferinae subfamily within Margaritiferidae (Lopes-Lima et al 2018b). This pattern
is well supported by at least three independent but almost synchronous divergence events:
Potomida vs. Lamprotula and Pronodularia, Microcondylaea vs. Solenaia, and Unio vs.
Nodularia and its relatives. During the same period, faunal exchange via the Beringian Land
Bridge with subsequent vicariance events may also have started. The question of the origin of
the family-clade, i.e. Unionidae, remains unanswered due to the lack of available mitogenomes
of Parreysiinae and Rectidentinae, although combined COI, 28S and 16S data indicated that
this family most likely originated within East or Southeast Asia (Bolotov et al 2017a).
The new results presented herein support the hypothesis that several of the largest
river basins on Earth may represent so-called ancient (long-lived) rivers, the Unionida faunas
of which have existed throughout long-term periods comparable with geological epochs
(Bolotov et al 2017a; Lopes-Lima et al 2018b). The mitogenomic results suggest that the
MRCA of the entire Gonideinae + Unioninae clade may have originated within the paleo-
Yangtze drainage basin. This indicates that the modern Yangtze may be a system of at least
Late Jurassic origin and a stable refugium for very ancient, relic lineages that have existed for
approximately 150 Ma. The unionid fauna of the paleo- Mississippi system seems to be of
Early Cretaceous origin (mean age of the crown group in our model) that has diversified for at
least 120 Ma. The paleo-Mekong fauna appears to be younger as it likely separated from the
paleo-Yangtze fauna in the Albian - Cenomanian, and its two largest monophyletic unionid
radiations may have had a Late Cretaceous or Palaeocene origin (Bolotov et al 2017a,b).
These results agree with the dating of divergence between two primary clades of the Southeast
Asian cave spitting spiders that were separated at ~55 Ma by the paleo-Mekong River, which
served as a biogeographic barrier (Luo & Li 2017).
FCUP 393
Systematics
Based on the morphological evidence, we propose the putative MRCA of the Unionidae and
Margaritiferidae as a new fossil family-level taxon in the Unionoidea.
Superfamily Unionoidea Rafinesque, 1820
Family †Shifangellidae Bolotov, Bogan, Lopes-Lima & Froufe fam. nov.
Type genus: †Shifangella Liu & Luo in Liu (1981)
Diagnosis: The Margaritiferidae and Unionidae are the most conchologically similar families
to the †Shifangellidae. However, †Shifangellidae can be distinguished from the
Margaritiferidae by having a weakly developed, narrow hinge plate (vs. typically well-
developed and rather thick) and a shallow, smooth anterior adductor scar (vs. deep with
arborescent-like striations), and from the Unionidae by an elongated Margaritifera-like shell
with strongly concave ventral margin (vs. typically straight, rounded or slightly concave).
Distribution: Late Triassic, southwestern China (Sichuan).
Biology: This ancestral family likely had parasitic glochidial larvae like its descendants
(ancestral state reconstruction, probability 100%).
Comments: Synonymy of the genus †Palaeomargaritifera Ma 1996 (Middle Jurassic, China)
with †Shifangella suggested by Fang et al (2009) most likely erroneous because
†Palaeomargaritifera has a well-developed, thick hinge plate, strong pseudocardinal teeth, and
deep anterior adductor scar with arborescent-like striations supporting its original placement
within the Margaritiferidae. The genus †Dianoconcha Guo, 1988 (Middle Jurassic, China),
another synonym of †Shifangella proposed by Fang et al (2009), differs by a subtrapezoid,
elongate-elliptical or rhomboid shell. This feature together with a narrow hinge plate and an
observable but shallow anterior adductor scar suggest that it most likely belongs to the
Unionidae. Concerning their age and diagnostic features mentioned above,
†Palaeomargaritifera and †Dianoconcha appear to be the MRCAs of the crown groups of the
Margaritiferidae and Unionidae, respectively. The family-level placement of several unionoid
genera described from the Early Jurassic of China (e.g. †Pseudomargaritifera Ma 1996 and
†Solenoides Ma 1996) is unclear and requires further revision; some of them might be
members of the †Shifangellidae.
Conclusions
All the phylogenies inferred in this study using, for the first time, both the M- and F-
mitogenomes individually and combined support the monophyly of the so-called “problematic”
Gonideinae taxa. Moreover, the new mitogenomic results place the Pseudodontinae, as
previously described by Bolotov et al (2017a), as a subclade within the monophyletic
Gonideinae in both M- and F-type phylogenies. Additionally, the present work supports the
394 FCUP
hypothesis of an ancient Mesozoic origin and diversification of the Unionoidea and reveals that
each TDRL event was followed by the stable, long-term persistence of a mitochondrial gene
order through evolving lineages and corresponds to the first reliable mitogenomic evidence
supporting the evolutionary stasis in molecular traits of freshwater mussels. Finally, we
propose a new systematics framework with three infrafamilial levels (i.e. subfamilies, tribes,
and subtribes) that better explains the evolutionary patterns within the Unionidae. Future
application of the phylogenetic mitogenome-based approach outlined here to Parreysiinae,
Rectidentinae, and Modellnaiinae will be an important step to further resolve current taxonomic
classification uncertainties within the Unionidae. Moreover, this study demonstrates the
considerable potential for using comparative genomic techniques for unravelling patterns in
the tempo, timing, and mode of evolutionary processes.
Data archiving
Sequence data have been submitted to GenBank accession numbers: MK994770-MK994777.
Acknowledgements
The authors wish to thank the Editor and the three anonymous reviewers for helpful remarks
and suggestions that improved the quality of the manuscript. This research was developed
under ConBiomics: the missing approach for the Conservation of Freshwater Bivalves Project
N° NORTE-01-0145-FEDER-030286, co-financed by COMPETE 2020, Portugal 2020 and the
European Union through the ERDF, and by FCT through national funds
(UID/Multi/04423/2019). FCT also supported MLL (SFRH/BD/115728/2016). The Russian
Ministry of Education and Science (project no. 6.2343.2017/4.6), the Federal Agency for
Scientific Organizations (project no. 0409-2015-0143), the Presidium of the Russian Academy
of Sciences (scientific program no. 52), and the Russian Foundation for Basic Research, RFBR
(project no. 17-45-290066) supported INB. Permits for fieldwork and sampling in Malaysia
were issued by the Malaysian Ministry of Higher Education (FRGS/1/2015/WAB13/UNIM//1).
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List of fossil calibrations that were used in BEAST analyses
Calibration
no.
Calibration 1 Margaritiferidae + Hard minimum age: 201 Ma, †Shifangella margaritiferiformis Liu & Luo, 1981 in Present study:
Unionidae Liu (1981) (Unionoidea: †Shifangellidae Bolotov, Bogan, Lopes-Lima & Froufe New crown
fam. nov.). calibration
Diagnosis and phylogenetic placement: A putative ancestor of the
Margaritiferidae and Unionidae. The Margaritiferidae and Unionidae are the most
conchologically similar families to the †Shifangellidae. However, †Shifangellidae
can be distinguished from the Margaritiferidae by having a weakly developed,
narrow hinge plate (vs. well-developed and rather hick) and a shallow smooth
anterior adductor scar (vs. deep with arborescent-like striations), and from the
Unionidae by an elongated Margaritifera-like shell with strongly concave ventral
margin (vs. straight, rounded or slightly concave).
Stratigraphic horizon and locality: Second Member, Wuzhongshan Formation,
Upper Triassic, Jinhe, Shifang, Sichuan, southwestern China (Fang et al 2009).
Absolute age estimate: Triassic/Jurassic boundary, 201 Ma, based on stratigraphy;
95% soft upper bound 230 Ma based on the age of †Silesunionidae, a prospective
earliest member in the order Unionida (Skawina & Dzik 2011).
Prior settings: exponential distribution, mean (lambda) = 7.9, MRCA:
Margaritifera marocana – Potomida littoralis.
Calibration 2 Ambleminae – Hard minimum age: 155 Ma, †Hadrodon jurassicus Yen, 1952 (Unionoidea: Graf et al
(Gonideinae + Unionidae). 2015: Crown
Unioninae) Diagnosis and phylogenetic placement: A putative ancestral lineage of the calibration
Ambleminae – (Gonideinae + Unioninae) clade. This Laurentian unionid fossil is
characterized by a broad hinge plate and strong lateral teeth in combination with
its conspicuous undulating external surface (Yen 1952).
Absolute age estimate: Late Jurassic, Morrison formation, Montrose County,
Colorado, 155 Ma, based on stratigraphy (Yen 1952); 95% soft upper bound 201
Ma based on calibration 1.
Prior settings: exponential distribution, mean (lambda) = 12.5, MRCA: Quadrula
quadrula – Unio crassus.
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CHAPTER 9
General Discussion
9.1 Main claims and highlights of the dissertation
This dissertation provides and integrates new knowledge about the biology, ecology, and
distribution of freshwater mussels (FM), also providing considerable scientific advances on
their phylogeny and systematics, thereby contributing to facilitate and guide future
conservation planning and actions on FM. The FM biodiversity hotspots are delineated in the
present dissertation and their main global threats, research, and conservation needs identified.
One of these identified research needs, basic biological studies, is further demonstrated to be
essential not only for conservation but also for the potential use of FM for environmental
indication. We then show the importance of integrated morphological, ecological and molecular
approaches to establish the evolutionary relationships among FM taxa at different taxonomic
levels. Finally, we use the information gathered to provide guidance on future conservation
actions.
9.2 Freshwater mussel hotspots, main threats, and conservation needs
The study of the diversity patterns of all freshwater bivalves highlighted the importance of
several areas as diversity hotspots for the most diverse and threatened group, the Unionida or
FMs (Chapter 2), as the Indotropical river basins that host a high taxonomic diversity of FMs.
Over the last decades, this area has suffered from accelerated deforestation and freshwater
habitat degradation, and thus deserves urgent conservation attention (Gallardo et al 2018).
Furthermore, recent surveys indicate that FM species diversity is much higher than previously
thought, making FM research in this area urgent (Bolotov et al 2017, 2019). The main
knowledge gaps were also unveiled. Baseline data such as distribution, life-history, and
taxonomy were the most cited research needs for FM species across the whole distribution,
but especially in poorly studied areas (Chapter 2). In North America and Europe, FMs are
being increasingly studied and protected (Lopes-Lima et al 2014). Nevertheless, long-term
monitoring data to evaluate FM population trends and demographic features are still lacking,
even in more developed regions (Chapter 2).
Most of the major identified threats to FMs were directly or indirectly connected to
habitat degradation or modification, with pollution being generally pointed out as the main
cause (Chapter 2). Although water quality and ecological status have been improving in many
FCUP 405
developed countries, FM populations are still not reversing their decline status, with whole river
catchment management producing better results (Lopes-Lima et al 2017). Nevertheless, in
less developed areas with none or poor wastewater treatment, pollution is still a major cause
of FM decline. Invasive species and climate change were still not considered as a major threat
(Chapter 2). However, this should change shortly, given that recent reports show that these
factors can have a major impact on freshwater mussels (Gallardo et al 2016). For instance,
many restricted and threatened FM species are specialized in their host-fish usage (Modesto
et al 2018). This was also shown for the Iberian dolphin mussel Unio delphinus which is not
able to complete its life-cycle in any of the tested non-native fish (Chapter 3). Therefore, the
increased biotic homogenization and freshwater fish introductions around the globe may
become a serious threat to these mussels (Modesto et al 2018). Additional invasive species
like other freshwater bivalves, mammals and crayfish have also been shown to have a
deleterious effect on bivalves, either by competition or predation (Burlakova et al 2000;
Skyrienė & Paulauskas 2012; Meira et al 2019). Species at the edge of their distribution or
those living in semi-arid conditions have been shown to be seriously threatened. For instance,
the Moroccan pearl mussel Pseudunio marocanus is now almost extinct due to water shortage
(Sousa et al 2016). This species and others in northwest Africa are likely to be increasingly
threatened due to the intensification of water consumption, combined with the on-going trends
of increasing temperature, decreasing rainfall and instable climate (Sousa et al 2016).
Freshwater mussel populations occupying their southern distribution limits, such as
Margaritifera margaritifera in the Iberian Peninsula, are also particularly exposed to climate
changes (Santos et al 2015) and associated increases in seasonal climatic instability (Sousa
et al 2012, 2018; Nogueira et al 2019).
Ranking on the top of conservation needs, research on basic biological and ecological
features is essential to effectively conserve and manage a species (Chapter 2). We
demonstrated this in a case study on an Iberian FM species (Chapter 3). It was shown that
the study of basic life-history traits and other basic biological and ecological features allows for
a better estimation of FM species conservation status (Chapter 3) and that these traits can be
used for ecological indication of freshwater habitats and communities. Furthermore, based on
the study of these features, clear suggestions were given to improve the conservation success
of potential actions on the target species, such as how to properly conduct population
translocations or establish propagation programs and reintroductions.
The results of Chapters 2 and 3 were not without potential limitations and
shortcomings. The species richness patterns reviewed in Chapter 2 were mapped on
biogeographic regions adapted from Graf & Cummings (2007) for the world and revised for
North America using Haag (2010). Although for North America a thorough compilation of
species richness across the country coupled with a hierarchical cluster analysis produced
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robust FMs’ bioregions (Haag 2010), for the rest of the world the division made by Graf &
Cummings (2007) is not clearly explained but seems to be based on the classical
zoogeographic divisions subdivided by the main freshwater mussel assemblages. A more
robust statistical analysis of the global diversity patterns was not attempted by these authors,
probably due to the many inconsistencies and gaps of knowledge that still existed on the global
FM phylogeny and taxonomy. Therefore, some of the bioregions chosen to map the diversity,
threats, research, and conservation needs were possibly not accurate and should be revised
with a proper statistical analysis.
Another potential limitation is the use of IUCN Red List data. Although around half of
the freshwater bivalve species were already red listed at least once, their assessments are not
homogeneous across regions. This means, for instance, that results obtained for South
America or Australia were only based on very few species. Another limitation might be the lack
of standardization of Red List assessments over the last decades. The Red List categories
and criteria have changed since its inception, and the way species were analysed and the
information included in each assessment changed considerably over the years, with recent
assessments generally containing much more data, e.g. detailed distributions, threats,
ecosystem services provided, among others.
For Chapter 3 the conservation status of U. delphinus was updated based only on the
species range and plausible threats using the B criterion of IUCN. Given that only very scarce
information regarding population trends was available, it was not possible to use criteria A or
C (Chapter 2). Although most of the fish species that co-occur with U. delphinus were used
for the determination of its hosts, we were not able to collect a few of those fish species, being
possible that other native or non-native species could also be good hosts for the reproduction
of U. delphinus. Furthermore, all of the host fish studies were made under laboratory conditions
and although the best hosts were species within the genus Squalius, field studies should be
made in the future to indicate if larvae prefer these fishes in situ or if other species are more
used. The growth experiments should have also been controlled for factors known to affect
this parameter, like nutrients and calcium concentration, temperature, pH, etc. Since these
parameters were not assessed, the differences in growth pattern evidenced by the lotic and
lentic populations could only be inferred and speculated. This chapter also discusses its
potential use for ecological indication, but this still needs to be tested with populations under
different stresses or environmental conditions.
9.3 Linking systematics and phylogeny with conservation

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The present dissertation also brings important advances in our knowledge about the
evolutionary relationships among the major groups within the most speciose and the most
threatened families of freshwater mussels, the Unionidae and the Margaritiferidae respectively.
Due to their sessile condition and very low dispersal ability, freshwater mussels are interesting
model taxa to use on biogeographic studies and reconstruction of historical hydrologic and
geologic changes, such as the formation and development of the current river basins or the
movement and connections of the Earth’s landmasses (Graf & Cummings 2006). Therefore, it
is very useful to use several layers of taxonomic complexity that help to explain and date these
events and the biogeography of these organisms. Until the present dissertation, phylogenetic
studies on FMs used a taxonomy with five layers within each family, i.e. species, genus, tribe,
sub-family, and family (Graf & Cummings 2016). However, due to the lack of sampled species
and access to whole specimens (for many species only shells are generally available in
museum collections), many of them were left as incertae sedis (Graf & Cummings 2006;
Whelan et al 2011). This classification framework was here applied to the results from the most
comprehensive phylogeny performed until now in terms of sampled taxa, to revise the higher
classification within both families. This classification divided the Unionidae into 6 subfamilies
and 18 tribes, and the Margaritiferidae into two subfamilies (Chapters 4 and 6). The
distribution of each subfamily and tribe was then mapped to highlight biogeographic patterns.
We could see, with a few exceptions, that the Unionidae are divided into Eastern and Western
Palaearctic subgroups by the Ural Mountains, with most tribes not sharing taxa from both sides
(Chapter 4). A strong biogeographic divide was also seen in the Mekong basin that is not
crossed by some major groups (Chapter 4). The Margaritiferidae also showed a biogeographic
differentiation among its two subfamilies, with one occurring in southeast Asia and the other
with a wide distribution area, probably due to the vagile nature of their migratory host fishes
(Chapter 6). Considering these biogeographic patterns and risk of extinction, Margaritiferidae
species are of extreme concern, mainly those within the genera Gibbosula in South-East Asia,
Pseudunio around the Mediterranean, and Cumberlandia with a restricted range in North
America, all highly threatened and of high phylogenetic distinctiveness (Chapter 6). Almost
nothing is known regarding the population status, ecology and life-history traits of Gibbosula
species which are very rare and not easily found (Do 2011a, 2011b; Thi Dieu Phuong 2011).
At a lower taxonomic scale, the phylogeny and species diversity of a North American
group, that historically belonged to the Quadrula genus, were revised (Chapter 5). This group
of species was traditionally lumped together under the genus Quadrula due to shell
morphological similarities. However, until now there were many uncertainties regarding the
identity, number of species and their generic assignment within this group. Our molecular
results, complemented with a compilation of traits from the literature, supported the existence
of 21 species in 4 genera. A new species to science, i.e. Theliderma johnsoni, was described
408 FCUP
and important conservation implications of the taxonomical revision on the group were
highlighted.
Some freshwater mussels have a very interesting and unique form of mitochondrial
inheritance (only shared with few other bivalve groups), also called doubly uniparental
inheritance (DUI), where males inherit mitochondria from both parents (Zouros et al 1994;
Hoeh et al 2002). Each male has (F-) type mitochondria inherited from the mother in all cells,
except in those from the germinative tissue that have (M-) type mitochondria and are inherited
from the father (Breton et al 2007). In FMs, both F- and M- mitochondrial genome sequences
are very divergent within a single individual, and even the gene arrangement of each
mitogenome sequence is quite distinct (Doucet-Beaupré et al 2010). In this dissertation, all
morphological and anatomical synapomorphies of the Margaritiferidae were compiled
(Chapter 6), including the gene arrangement of both F- and M-type mitogenomes, which are
unique to the family and may be used as a molecular diagnostic tool (Chapter 7). It was
discovered that mitogenome rearrangements in FMs were rare events that occurred mainly
during short periods and coincided with extinction events, stabilising thereafter during long
periods (Chapter 8). The phylogenetic patterns obtained within the family Unionidae were
consistent with other genomic phylogenies recently published by other authors in the meantime
using hundreds of nuclear loci (Pfeiffer et al 2019). This suggests that we are getting closer to
have a stabilized phylogeny of the main groups within Unionidae. Due to this reason, the
classification system for this family was again revised introducing subtribes as an additional
taxonomical layer (Chapter 8).
Several shortcomings and caveats can be identified from the results of Chapters 4 to
8, though they are unlikely to have affected significantly its results and key conclusions. These
chapters provide a good phylogenetic basis on the major groups of the Unionidae and
Margaritiferidae, which are complemented with other traits. However, some of the phylogenetic
relationships among the higher taxa were not well supported, especially the relationships
among the subfamilies. This should be related to the low number of molecular markers used,
suggesting that additional markers are needed to reveal these ancient evolutionary
relationships. Other recently produced phylogenies using the same low number of markers
showed differences in these relationships that seem to be dependent on the number and
composition of taxa (Pfeiffer et al 2019). Besides the low number of markers, taxa from several
regions were never included in the Unionidae phylogeny and our knowledge of the whole group
is still incomplete, with many species being still considered as incertae sedis. Furthermore, the
biogeographic inferences accomplished in Chapters 6 and 8 only considered the few taxa
used for the phylogenetic analyses, and these patterns might change if more taxa are added
to the models.
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The present whole mitogenome analyses indicated that gene arrangements might be
diagnostic of specific taxonomic groups, such as the families Unionidae and Margaritiferidae
or specific subfamilies of the Unionidae (Chapters 7 and 8). However, whole mitogenome
sequences are only available from a small number of species, and so these trends need to be
confirmed with additional taxa from other regions and taxonomic groups. The phylogeny
presented using whole mitogenomes (Chapter 8) was well supported but revealed a distinct
topology regarding the main relationships among the Unionidae subfamilies captured in
Chapter 4. Nevertheless, the mitogenome phylogenetic analyses are concordant with another
recently published study with hundreds of nuclear markers across the whole genome (Pfeiffer
et al 2019), indicating that the phylogeny should be stabilising, and we are closer to the true
species tree.
Since more recent evolutionary diversification events needed to be retrieved, in the
phylogenetic study of the species historically placed within Quadrula (Chapter 5), only
mitochondrial markers were used. Given that mtDNA may suffer from introgression and
hybridization events, with mtDNA evolution sometimes being distinct from the species
evolution, all phylogenetic assumptions and molecular species delineation methods should
now be confirmed with nuclear markers displaying distinct evolutionary rates.
9.4 Future research perspectives and conservation implications
The results of this thesis bring important conservation implications that vary across the globe.
Wide and extensive surveys are needed in many regions, not only in less studied regions such
as Africa, South America and Sundaland, but even in Europe and North America, where
species inventories and population information is scarce and uneven across regions (Lopes-
Lima et al 2014, 2017). To better estimate conservation status and to distinguish between
natural fluctuations and anthropogenic declines, population trends should also be researched
globally, emphasizing the need to establish long term monitoring programs for important FM
populations, such as those included in the Long Term Ecological Research (LTER) Network
(Knapp et al 2012).
FM species on the southern hemisphere in Africa, South America, and Australia have
been neglected and need to be studied urgently to prevent future extinctions and extirpations.
Basic biological traits and population information are almost non-existent for species in these
regions and genetic diversity studies almost absent. The application of the framework here
developed for the study of basic biological traits (chapter 3) in species from these regions
should be pursued to better understand the ecological requirements and features of this poorly
known fauna.
410 FCUP
The use of FMs to monitor the environmental health of both freshwater habitats and
their terrestrial surroundings has a great potential and should be developed significantly in the
future. The efficacy of these traits and other potential biomarkers, e.g. biochemical or genetic,
should then be tested in distinct impacted environments or to evaluate the success of eventual
rehabilitation actions.
Another field of research that requires immediate attention is the selection of protected
areas that include freshwater diversity (Suski & Cooke 2007). It has been shown that protected
areas developed for terrestrial taxa fail to protect freshwater organisms (Darwall et al 2011).
Also, there is a wide mismatch between the areas important for freshwater conservation and
those within protected areas networks (Abraham & Kelkar 2012; Hermoso et al 2015).
Therefore, FMs and other freshwater taxa should be included in future systematic conservation
planning exercises to design more effective conservation or protected areas for freshwater
diversity.
To help in the selection of the most diverse and important global areas for freshwater
mussels, we should try to maximise species richness, but also to capture the higher
phylogenetic diversity possible (Faith et al 2004). For that, we first need a robust phylogeny of
the order using multiple markers and the higher number of taxa possible. The whole
mitogenome phylogenies here presented and the multi-nuclear marker resources recently
published by Pfeiffer et al (2019), seem to be close to the true species phylogeny. Therefore,
a combined multi-marker approach using these two techniques should be accomplished in the
future, with selected taxa from most genera within the order. At a higher resolution taxonomical
scale, reduced genome representations like restriction site associated DNA markers (RAD-Seq)
or genotyping by sequence (GBS) should be applied to reveal hidden cryptic diversity and test
intraspecific mitochondrial lineages to have a clearer molecular definition of species. Given
that in developing countries freshwater habitats are becoming increasingly impacted, it is
essential to rapidly invest in the identification of species, evolutionarily significant units, and
management units in these countries, especially on the identified hotspots such as the
Indotropical region.
Knowing the higher taxa phylogeny and having most species identified will allow for the
elaboration of robust biogeographic models and infer historical geological and hydrological
patterns. Furthermore, the evolutionary relationships, distributions and conservation status
patterns here obtained may be coupled with equivalent data from other freshwater taxa to
select important areas for conservation and protection at several geographic levels.
By presenting an integrative approach combining ecological, distribution,
morphological and genetic data, the present dissertation provides important advances on
several research fields in freshwater mussels, from evolution and ecology to environmental
FCUP 411
indication with important contributions for the global conservation planning of these highly
threatened molluscs.
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Clausnitzer V, Cumberlidge N, Cuttelod A, Dijkstra K‐DB, Diop MD, García N, Seddon MB,
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Combining phylogeny,

Começo o Capítulo 1, por introduzir o declínio global da biodiversidade, concentrando-

CHAPTER 1. General introduction 1

1.1 The freshwater biodiversity crisis 1

CHAPTER 2. Freshwater bivalves’ conservation, diversity, and research 18

CHAPTER 3. Basic biological traits of Unio delphinus 41

CHAPTER 4. Phylogeny of the family Unionidae 83

Paper 3. Phylogeny of the most species-rich freshwater bivalve family (Bivalvia:

CHAPTER 5. Phylogeny, taxonomy and species of the genus Quadrula 148

CHAPTER 6. Phylogeny of the family Margaritiferidae 277

Paper 5. Expansion and systematics redefinition of the most threatened freshwater

CHAPTER 7. First male whole mitogenome of a Margaritiferidae species 360

Paper 6. The first Margaritiferidae male (M-type) mitogenome: mitochondrial gene

CHAPTER 8. Evolution of mitogenome rearrangements in freshwater mussels 371

Paper 7. Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia:

CHAPTER 9. General Discussion 404

9.1 Main claims and highlights of the dissertation 404

Index of Figures and Tables

CHAPTER 1. General introduction

CHAPTER 2. Freshwater bivalves’ conservation, diversity, and research

Neotropical, PA Palaearctic, AF Afrotropical, IN Indotropical, AU Australasian.

CHAPTER 3. Basic biological traits of Unio delphinus

spermatogonia (sg), spermatocytes (sc), spermatids (st), sperm morulae (sm)

CHAPTER 4. Phylogeny of the family Unionidae

Figure 1. Phylogenetic tree of the Palaeoheterodonta obtained by Bayesian Inference

CHAPTER 5. Phylogeny, taxonomy, and species of the genus Quadrula

History); NCSM (North Carolina Museum of Natural Sciences); UA (University

CHAPTER 6. Phylogeny of the family Margaritiferidae

Figure 1. Phylogenetic tree of the Palaeoheterodonta obtained by Bayesian Inference

Table 6. Analysed conchological characters of Margaritiferidae species. Superscripts:

Supplementary Table 3. List of characteristic examples of fossil records supporting the

CHAPTER 7. First male whole mitogenome of a Margaritiferidae species

CHAPTER 8. The evolution of mitogenome rearrangements in freshwater

Figure 1. Gene maps of the F- and M-type mitochondrial genomes of Chamberlainia

indicate 95% confidence intervals of the estimated divergence times between

1.1 The freshwater biodiversity crisis

1.2 Freshwater mussel diversity, importance, and conservation

1.3 The need for baseline biological research

Accurate conservation status assessment and effective conservation actions require a

1.4 Defining species boundaries and integrating phylogenetic diversity

1. To highlight the biodiversity hotspots of freshwater bivalves, mapping the global

3. To show the importance of basic biological studies for conservation of freshwater

4. To demonstrate the importance of systematics and phylogenetic diversity for the

6. To test the use of mitochondrial genome arrangements as a diagnostic for freshwater

7. To use information collected during the dissertation to provide recommendations for

1.6 Thesis structure

Chapter 2 addresses Objectives 1 and 2 by presenting a revision of the global diversity

Froufe E, Gonçalves DV, Teixeira A, Sousa R, Varandas S, Ghamizi M, Zieritz A, Lopes-Lima

Lopes-Lima M, Teixeira A, Froufe E, Lopes A, Varandas S, Sousa R. 2014. Biology and

Lopes-Lima M, Froufe E, Do VT, Ghamizi M, Mock KE, Kebapçi Ü, Klishko O, Kovitvadhi S,

Lopes-Lima M, Sousa R, Geist J, Aldridge DC, Araujo R, Bergengren J, Bespalaja Y, Bódis E,

Miraldo A, Li S, Borregaard MK, Flórez-Rodríguez A, Gopalakrishnan S, Rizvanovic M, Wang

Moilanen A, Arponen A. 2011. Setting conservation targets under budgetary constraints.

Roser M, Ritchie H, Ortiz-Ospina E. 2019. World Population Growth. Available at

Winter M, Devictor V, Schweiger O. 2013. Phylogenetic diversity and nature conservation:

Conservation of freshwater bivalves at the global scale: diversity, threats

Manuel Lopes-Lima1,2,3,*, Lyubov E. Burlakova4, Alexander Y. Karatayev4, Knut Mehler4, Mary

Diversity patterns at the global scale

Figure 2 Diversity by ecoregions. A All freshwater bivalves; B Unionida; C Sphaeriidae; D