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The cane or marine toad, Rhinella marina (Anura, Bufonidae): Two genetically
and morphologically distinct species

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DOI: 10.11646/zootaxa.4103.6.7

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 Zootaxa 4103 (6): 574–586 ISSN 1175-5326 (print edition)
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Copyright © 2016 Magnolia Press
Article ZOOTAXA
ISSN 1175-5334 (online edition)
http://doi.org/10.11646/zootaxa.4103.6.7
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The cane or marine toad, Rhinella marina (Anura, Bufonidae): two genetically
and morphologically distinct species

ALDEMAR A. ACEVEDO1,2, MARGARITA LAMPO1,* & ROBERTO CIPRIANI3,4

1
Centro de Ecología, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas, 1020-A, Venezuela.
E-mail: [email protected]
2
Grupo de Investigación en Ecología y Biogeografía (GIEB), Facultad de Ciencias
Básicas, Universidad de Pamplona, Pamplona, Colombia. E-mail: [email protected]
3
Department of Biological Science, California State University, Fullerton, CA 92831-3599. E-mail: [email protected]
4
MyLife.com, 1100 Glendon Ave., Los Angeles, CA 90024
*corresponding author email: [email protected]

Abstract

Rhinella marina is a Neotropical toad that has been introduced widely worldwide. Its toxic effects to frog-eating predators
threaten the native and domestic fauna of some regions where it has been introduced. Despite previous studies suggesting
two genetically distinct cryptic species within R. marina, one east and one west of the Andes, its taxonomic status re-
mained unresolved due to the absence of morphological complementary evidence. For the first time, data from two mito-
chondrial genes (ND3 and CR) and 23 morphometric landmarks are combined to evaluate the taxonomic status of this
species. Our results support the hypothesis of two separate evolutionary lineages within R. marina and demonstrate that
these lineages have significantly diverged in skull shape. We identified two distinct morphotypes, one eastern and one An-
dean western, with no overlapping morphospaces. The geographic pattern of genetic variation was consistent with a stable
structured population with no evidence of recent demographic or geographic expansions. The concordance between the
observed geographic patterns in morphometric and genic traits calls for the recognition of two species under R. marina
name.

Key words: biological control, mitochondrial DNA, morphometric geometry, phylogeny, taxonomy, Venezuela

Resumen

Rhinella marina es un sapo neotropical que se ha esparcido globalmente. Los efectos tóxicos que produce la ingesta de
sus adultos en sus depredadores han convertido a esta especie en una amenaza para la fauna silvestre y para los animales
domésticos en algunas regiones en donde esta especie ha sido introducida. A pesar de que estudios genéticos sugieren que
R. marina incluye al menos dos especies crípticas, una al este y la otra al oeste de los Andes, su estatus taxonómico sigue
sin resolverse debido a la ausencia de evidencia morfológica que complemente a la genética. Con base en el análisis de
dos genes mitocondriales (ND3 y CR) y de 23 hitos morfométricos evaluamos el estatus taxonómico de poblaciones de
R. marina. Nuestros resultados apoyan la hipótesis de la existencia de dos linajes evolutivamente independientes en R.
marina y demuestran que ambos linajes han divergido significativamente en la forma del cráneo. Identificamos dos mor-
fotipos, uno al este y otro al oeste de la cordillera Andina, cuyos morfoespacios no se solapan. Encontramos una estructura
geográfica de la variación genética concordante con la de dos poblaciones estables sin evidencias de expansiones de-
mográficas recientes. La coincidencia en la variación geográfica entre las características morfométricas y genéticas de es-
tas poblaciones abogan por el reconocimiento de dos especies de R. marina.

Introduction

The marine or cane toad, Rhinella marina (formerly Bufo marinus) (Linnaeus), is one of the most cosmopolitan
amphibian species of the world. Although its natural range extends from South Texas to Central Amazonia, cane

574 Accepted by M. Vences: 17 Mar. 2016; published: 18 Apr. 2016

toads have also established populations in central United States, Australia, Japan, Philippines, Taiwan, Papua New
Guinea and several islands across the Caribbean and the Pacific, as a result of translocations (Easteal 1981, 1985;
Lever 2001). Due to the toxicity of adult frogs to predators and their potential for large-scale impact on many wild
and domestic animals, cane toads are recognized by the International Union for the Conservation of Nature (IUCN)
and the Invasive Species Group (ISG) as one of the 100 worst invasive species of the world (Global Invasive
Species Database 2005).
Despite being among the most studied amphibian species (e.g. regarding its physiology and ecology) (Shine
2010), the taxonomic identity of populations under R. marina name remains unresolved. Its widespread natural
distribution has suggested a complex of cryptic species, but the delimitation of these species has been difficult. The
thirty-one synonyms proposed along its taxonomic history (Frost 2015) has evidenced the taxonomic complexity of
this taxon. Morphological traits can be uninformative in recognizing species boundaries (i.e. cryptic species) if i)
species have evolved under a strong stabilizing selection that reduces morphological variations or ii) mate
recognition relies on non-morphological characteristics (e.g. vocalizations) as most often occurs in anurans
(Bickford et al. 2007; Borkin et al. 2004; Stuart et al. 2006). Among anurans, the genus Rhinella has been
considered as morphologically conserved (Graybeal 1997; Pramuk 2006). Although unique unreversed
synapomorphies can be used to diagnose some South American species groups of toads (Rhinella margaritifera,
Rhinella marina, Rhinella granulosa, Rhaebo guttatus and Incilius valliceps), the paucity of variation in
osteological characters have challenged taxonomist attempting to separate some species within these groups
(Pramuk 2006).
Few genetic studies have shown evidence of cryptic species within R. marina. Based on sequence analyses of
the ND3 gene and the flanking tRNA genes, Slade & Moritz (1998) detected a phylogenetic break within R. marina
separating populations east and west of the Venezuelan Andes. Additional evidence based on the 12S rRNA, 16S
rRNA and cytochrome b (Cytb) mitochondrial genes summed to the increasing evidence of two paraphyletic
groups of R. marina, one from Central America and Ecuador (west of the Andes) and those from the Amazon Basin
(east of the Andes) (Vallinoto et al. 2010). However, the lack of complementary evidence on nuclear genes or
morphological diagnostic characters have postponed the reevaluation of the taxon because mitochondrial
phylogenies may not always reflect the species phylogeny (Avise et al. 1983). For example, conflicting signals
between the mitochondrial and nuclear-based phylogenies of R. marina populations across the Amazon river have
been associated with recent hybridization with closely related species (Sequeira et al. 2011).
The development of geometric morphometry in the last few decades has substantially improved the statistical
power for distinguishing shapes, offering new avenues for the study of systematics of organisms using morphology
(Adams et al. 2004; Adams et al. 2013; Rohlf & Marcus 1993). Based on the use of landmarks, these methods
overcome some of the limitations of the classic morphometry based on linear distances, e.g. the high correlation
existing between linear measurements and size, by removing the non-shape variation of scale, position and
orientation of the landmark configuration (Kendall 1977). Despite its widespread use in the analysis of complex
cranial morphologies, a feature commonly used for studying the systematics of bufonids (Pramuk 2006), landmark-
based geometric morphometry has not been widely used in taxonomy of anurans, although it has proved useful in
complementing molecular evidence for phylogenetic inferences (Kaliontzopoulou 2011)
Using a fine-scale sampling and combining evidence from two mitochondrial genetic markers and from the
shape of cranial structures, we reevaluate the taxonomic status of populations of R. marina east and west of the
Andean cordillera in Venezuela.

Materials and methods

Ethics statement. All animal procedures were conducted in accordance with international guidelines. Animal
transport followed guidelines for transport of live animals by the Convention on International Trade in Endangered
Species of Wild Fauna and Flora (CITES) and euthanasia was conducted according to the recommendations of the
American Association for Veterinary Medicine (AMVA). The study was approved by the Comisión Interministerial
para el Acceso a Recursos Genéticos del Ministerio del Poder Popular para el Ambiente (Venezuela), a
governmental office that grants access to genetic resources and evaluates bioethic issues related with the use of
wildlife. The study did not involve protected or endangered species. Rhinella marina is currently listed by the
International Union for the Conservation of Nature (IUCN) in the Least Concern category.

RHINELLA MARINA: TWO DISTINCT SPECIES Zootaxa 4103 (6) © 2016 Magnolia Press · 575
TABLE 1. Locality, geographic coordinates, region, source, GenBank accession numbers of mitochondrial haplotypes,
and sample size used in this study.
Locality Geographic Region Source GenBank Accession Numbers
coordinate (Number of samples)
ND3 CR
VE1 8.500828 N West This study KP704669–KP704677 KP995007–KP995014
71.392431 W (9) (8)
VE2 8.612631 N West This study KP704678–KP704684 KP995015–
71.662886 W (7) KP995020
(6)
VE3 8.62375 N West This study KP704685–KP704691 KP995021–KP995026
71.650028 W (7) (6)
VE4 8.566028 N West This study KP704692–KP704693 KP995027–KP995028
71.367583 W (2) (2)
VE5 11.866508 N West This study KP704694–KP704696 KP995029–KP995030
70.031294 W (3) (2)
VE6 7.8655 N Southern This study KP704697–KP704698 KP995031–KP995032
72.4321 W depression (2) (2)
VE7 10.575506 N East This study KP704699–KP704670 KP995033–KP995034
64.228825 W (2) (2)
VE8 10.3775 N East This study KP979778 KP995035
68.7725 W (1) (1)
VE9 7.5287 N East This study KP979779– KP995036–KP995042
68.236783 W KP979781;KP704701– (7)
KP704713
(16)
VE10 7.835372 N East This study KP704714–KP704723 KP995043–KP995053
68.917461 W (10) (11)
VE11 8.930939 N, East This study KP704724 KP9955054
68.044225 W (1) (1)
VE12 10.015892 N Northern This study KP704725–KP704734 KP995055–KP995062
69.677606 W depression (10) (8)
VE13 Lake Maracaibo West (Slade & Moritz (2)
1998)
VE14 Higuerote East (Slade & Moritz (2)
1998)
VE15 Güiria East (Slade & Moritz (1)
1998)
Texas, West (Slade & Moritz (1)
USA 1998) –
Mexico West (Slade & Moritz (5)
1998) –
Costa Rica West (Slade & Moritz (3)
1998)
Panama West (Slade & Moritz (1)
1998) –
French East (Slade & Moritz (2)
Guyana 1998) –

Peru East (Slade & Moritz (1)

1998) –

576 · Zootaxa 4103 (6) © 2016 Magnolia Press ACEVEDO ET AL.

 Sample collection. We selected 14 localities around the Andean cordillera; six on the east side, six on the west
side, one on the northern depression and one on the southern depressions (Figure 1). Geographic coordinates for all
localities are given in Table 1. Seven to ten adults were collected at each locality during 2010, totalizing 80
specimens. All specimens were collected under permit No. 394 issued on January 2010 to Margarita Lampo by the
Venezuelan Ministry for the Environment. Toads were transported alive to the laboratory in plastic containers with
water and ventilation holes. In the laboratory, they were euthanized using intradermal benzocaine injection
(182mg/kg) (Birchard 2002). From each specimen, 50g of muscle tissue were cut from the inguinal area and stored
in 95% ethanol. The specimens were fixed in formalin and stored in 70% ethanol for subsequent morphometric
analyses. All voucher specimens were deposited at Museo de Historia Natural La Salle (MHNLS) in Caracas.

FIGURE 1. Geographic distribution of Rhinella marina populations sampled for two mitochondrial markers and
morphometric landmarks across the Andean cordillera in northern Venezuela. The circles indicate locations of specimens
sequenced in this study and the squares indicate locations associated to previously published sequences used in this study.
Black circles and squares correspond to eastern populations and white circles and squares to western populations. The
geographic coordinates of localities are given in Table 1. The genetic distances between western and eastern populations (grey)
are greater than between western populations (black) or between eastern populations (white) (see text for details).

DNA extraction and PCR amplification. DNA was extracted from tissue samples using phenol-chloroform
with ethanol precipitation (Sambrook & Russel 2001) and stored at -20oC. We selected DNA fragments for the
subunit III of NADH dehydrogenase (ND3) and the Control Region (CR) using conventional PCR assays. We used
primers ND3.1, ND3.2, MVZ25-L (Moritz et al. 1992, Slade & Moritz 1998), CytbA-L and ControlP-H (Goebel et
al. 1999).
Polymerase chain reaction for ND3 and CR amplification were carried out in a final volume of 25 µL volumes,
each containing 2µL of DNA, 0.2µL of Taq polymerase, 1.25 µL MgCl2 50mM, a total of 0.5µL of
dATP+dGTP+dTTP+dCTP, 0.25 µL of each primer, 2.5µL 10x buffer and distilled water to 18.05µL.
Amplifications were performed in a MJ Research thermocycler programmed as follows. Initial denaturation cycle
at 95o C (30 sec), annealing at 50o C (1 min) and extension at 72o C (2 min) for 41 cycles. Amplification products

RHINELLA MARINA: TWO DISTINCT SPECIES Zootaxa 4103 (6) © 2016 Magnolia Press · 577
were separated by gel electrophoresis on 2% agarose gels in TAE buffers and PCR products were visualized after
staining with ethidium bromide (Sambrook & Russel 2001). Prior to sequencing, PCR products were purified using
the commercial kit Concert GIBCO®. Sequences were resolved using an automated DNA sequencer (ABI3130XL)
by the Unit for Genetic and Forensic Studies at Instituto Venezolano de Investigaciones Científicas (IVIC).
Genetic divergence. Alignment of sequences was performed with the ClustalW algorithm using no gap
penalty implemented in Bioedit v.7.0.5.1, and adjusted manually. The number of segregating sites (S), haplotipic
diversity (Hd), and nucleotide diversity (p) were estimated using the program DnaSP v.5.10.01 (Librado & Rozas
2009) for the concatenated ND3+CR fragment. Tajima´s D (Tajima 1989) and Fu´s F tests (Fu 1997) were used to
examine the selective neutrality of mitochondrial fragments and detect departures from the mutation-drift
equilibrium due to of recent demographic expansions. Significance of these indices was assessed using confidence
limits generated under a coalescent model assuming constant population size and the number of segregating sites
(Rozas et al. 2003). A hierarchical analysis of molecular variance (AMOVA) of the concatenated (ND3+CR)
fragment was also performed using Arlequin 3.5 to partition the genetic differentiation attributable to differences
between regions (eastern vs. western) (FCT), to differences among localities within regions (FSC), and to variance
among localities in both regions (FST). We also examined the percent genetic distances among populations for ND3,
including sequences previously published (Slade & Moritz 1998), using the k2p model implemented in program
MEGA v.5.0.
Phylogenetic and haplotype network reconstructions. Saturation indices were estimated using DAMBE
2.2.13 in order to assess whether particular positions or classes of substitutions needed to be weighted or excluded
prior to the phylogenetic analyses. Phylogenetic analyses were conducted for the separated ND3 and CR markers
using maximum parsimony (MP) and maximum likelihood (ML) algorithms in PAUP* v.4.0b10 and the Bayesian
analysis in MrBayes v.3.1. Trees were rooted on Rhinella granulosa for ND3 and on Rhinella rubescens and
Rhinella crucifer for CR. The MP analyses were performed after excluding non-informative characters using
heuristic searches with TBR branch swapping and 1000 random addition sequence replicates (Swofford & Olsen
1990). Support for each branch was assessed for the MP, ML and BI analyses by bootstrap proportions based on
1000 pseudoreplicates. We used the Akaike Information Criterion (AIC) and the Bayesian inference criterion (BIC)
implemented in software Jmodeltest v.0.1.1(Posada 2008) to choose the substitution model that best fit our data.
Each Bayesian analyses consisted of 5 x 106 generations with a random starting tree and four Markov chains
sampled every 1,000 generations. Median-joining networks were reconstructed for the combined data sets (ND3 +
CR) by program Network v.4.6 (Bandelt et al. 1999).
Geometric morphometry. Landmark configuration. Morphometric analyses were based on digital
mammogram images (Mamoray Agfa HT300) of preserved carcasses of 65 males with snout-vent lengths between
15 and 18 cm. All specimens were exposed to 30 kilovolts/7 milliamps for 0.8 seconds at a focal distance of
105mm. A 5x5 cm plastic underlying mesh was used as a reference scale. We selected 23 landmarks on anatomical
homolog structures of the cranial area of all specimens (Figure 2). These were informative sites frequently used in
amphibian taxonomy (Duellman & Trueb 1994; Zelditch et al. 2004) (Table 2).
Shape variation analysis. We performed a generalized Procrustes analysis (GPA) to separate size and shape
components of form variation (Bookstein 1991). Shape coordinates were computed by standardizing each configuration
to a unit centroid size and by minimizing differences in translation and rotation of all specimens using a least-square
algorithm implemented in tpsDig software (v.2.12). To plot the data on a Euclidean space the resulting coordinates were
projected orthogonally onto a plane tangential to the shape space using the software tpsRelwarp v.1.46 (Rohlf 2008). A
consensus shape configuration for each group (east and west) was calculated by averaging the coordinates for each
group, and these were used to derive a series of Principal Warps (PW). We initially used unweighed PWs (α=0), and then
assigned a greater weight to distant PWs (α=1) (Rohlf & Marcus 1993) to explore shape changes at larger scales as
suggested by (Bookstein 1989). A matrix of Partial Warps scores was generated by projecting each specimen
configuration onto the Principal Warps.
A principal component analysis and a canonical variate analysis implemented in MorphoJ (v. 1.02) were
performed on the matrix of Partial Warp scores, to obtain the RelativeWarps (RW) and their associated scores, and
to test the hypothesis of shape differences between groups (east vs. west) (Klingenberg 2011). Percent
misclassifications were estimated based on the identification function. Each group was plotted onto the RW axes,
and thin-plate spline (TPS) deformation grids were obtained using tpsRelwarp v1.46 (Bookstein 1991; Zelditch et
al. 2004).

578 · Zootaxa 4103 (6) © 2016 Magnolia Press ACEVEDO ET AL.

TABLE 2. Anatomical landmarks used for the geometric morphometry analysis of skulls of adult males of Rhinella
marina from northern Venezuela.
Landmark Anatomical structure
1 Left Maximum point in squamosal bone
2 Left Nasal-maxilla posterior joint
3 Left Maxilla-quadratojugal joint
4 Left Pre maxilla
5 Left Nasal-maxilla anterior joint
6 Right Nasal-maxilla anterior joint
7 Right Pre maxilla
8 Right Maxilla-quadratojugal joint
9 Right Nasal-maxilla posterior joint
10 Right Maximum point in squamosal bone
11 Left Nasal bone-anterior rim of eye joint
12 Right Nasal bone-anterior rim of eye joint
13 Left Superior edge of postorbital crest
14 Left Occiptal condyle
15 Right Occipital condyle
16 Right Superior edge of postorbital crest
17 Left Squamosal-occipital suture
18 Left Frontoparietal-squamosal suture
19 Left Nasal-frontoparietal junction at the orbit
20 Central Nasal-frontoparietal junction along midline
21 Right Nasal-frontoparietal junction at the orbit
22 Right Frontoparietal-squamosal suture
23 Right Squamosal-occipital suture

Results

Genetic divergence and phylogenetic relationships. We obtained 70 sequences of 429 bp for ND3 and 56
sequences for CR, although amplification fragments for CR differed significantly in their lengths between eastern
(707 bp) and western (737 bp) samples due to the presence of gaps. Geographic coordinates of collection sites,
GenBank accession numbers for sequences obtained in this study and the source for previously published
sequences are given in Table 1.
Saturation indices for the two markers (0.1862 for ND3 and 0.3267 for CR) were significantly lower than the
critical values (p<0.001). For the concatenated fragment, haplotype diversity was high while nucleotide diversity
was low (Table 3). Non-significant Tajima's D and Fu's F indicated no evidence of an excess of low-frequency
haplotypes in any of the lineages that could suggest a recent demographic expansion. The AMOVA attributed the
largest variance component to differences between regions (eastern vs. western) (97.54%). Variations among
populations within regions and within populations only accounted for a small fraction of the variance component
(1.05 and 1.42%, respectively) (Table 4). The k2p genetic distances estimates indicated large percent divergences
of 7.2—10.3 between regions compared to 0–6 within regions (Figure 1). Uncorrected p-distances were slightly
lower (6.3–8.7 between regions and 0–5.6 within regions) but showed similar variation pattern. The haplotype
network for the combined markers (ND3+CR) depicted a clear separation between two haplo-groups, eastern and
western, with no common haplotypes between these groups (Figure 3). Haplotypes from both depressions were
also unique, and grouped with different haplo-groups. The northern depression haplotypes (Hap21 and Hap22)
grouped with the eastern haplo-group (<1% divergence), while those from the southern depression (Hap7) grouped

RHINELLA MARINA: TWO DISTINCT SPECIES Zootaxa 4103 (6) © 2016 Magnolia Press · 579
with the western haplo-group (<1% divergence). In the one hand, we could not identify a contact zone where both
haplotypes were present. On the other hand, we found no evidence of star-like structures with frequent haplotypes
connected to low frequency haplotypes that could suggest recent demographic expansions.

TABLE 3. Sample size (N), number of segregating sites (S), haplotype diversity (Hd), nucleotide diversity (p), Tajima’s
(D), Fu’s (F) and raggedness index (r) for eastern and western lineages of Rhinella marina from Venezuela based on the
concatenated ND3 +CR datasets.
Lineage N S Hd π D F
Eastern 30 36 0.926 0.00805 -0.0989 -0.518
Western 26 12 0.927 0.00270 -0.0491 -0.539
*
p<0.05

TABLE 4. Analysis of Molecular Variance (AMOVA) based on 821 informative sites for the concatenated ND3+CR
mitochondrial markers of Rhinella marina collected east and west of the Venezuelan Andes.
Source of variation df Variance Percent variation FST
components
Between regions 1 143.366 97.54 0.975**
Among populations within regions 9 1.536 1.05 0.425**
Within populations 45 2.082 1.42 0.985**
Total 55 100.0

**p<0.05

The ML and BI phylogenetic reconstructions produced similar topologies: a phylogenetic break within R.
marina separating populations into two major clades, an eastern and a western clade. The ND3 and CR optimal
trees were congruent in terms of the split of the eastern and western clades (Figure 4). The ML analysis for the CR
fragment yield six trees with a score of 77 steps, consistency and rescaled consistency indices of 0.8617 and 0.8584
respectively, and high support bootstrap values for both clades (Figure 4a). For the ND3, the analysis produced
nine trees with a score of 94 steps, consistency and rescaled consistency indices of 0.8731 and 0.8420 respectively,
although bootstrap values were lower than for CR. The inclusion of previously published ND3 sequences from
Central America and other South American countries resulted in the grouping of samples from Panama, Costa
Rica, Mexico and United States with the Venezuelan western clade and samples from Peru and the French Guyana
with the eastern clade (Figure 4b).
Morphological divergence. The first two PCA axes summarized 74 per cent of the total amount of variation in
the data set. Thus, between-group variation described from here refers to the morphospaces delimited by these two
axes. The resulting polygons defining the morphospaces of the groups showed no overlap (Figure 5b). The first two
CVA axes produced an unequivocal separation of the eastern and western groups (λ= 0.1372, χ2=79.4456, df =42,
p=0.00042) with no misclassifications. The thin-plate splines grids showed deformations for each group in
opposite directions mainly around the pre-maxillar and nasal bones (landmarks 4, 5, 6, 7, 11, 12, 19, 20, 21) and the
occipital regions (landmarks 13, 14, 15, 16, 17, 18, 22, 23) (Figure 5a). These landmarks allow for the unequivocal
separation of morphotypes (i.e. no misclassifications) and can be used as diagnostic characters. Landmarks 5 and 6
on the anterior joint of the nasal bone with the upper maxilla, 2 and 9 on the posterior joints between the nasal bone
and maxilla, and 14 and 15 on the occipital condyles showed the largest contributions to the observed shape
changes. In western populations, a forward displacement of the occipital and the pre-maxilla regions results in a
shortening of the occipital region. The longitudinal distance between the maxilla-quadratojugal joints (landmark 3
and 8) and the occipital condyles (landmarks 14 and 15) is significantly shorter in western populations compared
with eastern populations (Student-t=5.187, df=51.6, p=0.0000036).

580 · Zootaxa 4103 (6) © 2016 Magnolia Press ACEVEDO ET AL.

FIGURE 2. Dorsal x-ray images of Rhinella marina skulls from one western and one eastern population. Circles show the
anatomical landmarks described in Table 2. The red circles correspond to landmarks showing the greatest contributions to the
shape differences between eastern and western populations. The red arrows indicate the direction of displacement of
landmarks. The white vertical bars indicate differences in measurements in externally visible landmarks between eastern and
western populations.

FIGURE 3. Statistical parsimony network for the Rhinella marina species complex in northern Venezuela based on
concatenated ND3+CR mitochondrial haplotypes. The left branches correspond to western populations and the right branches
to eastern populations. Connections between haplotypes correspond to one mutational step and small dots indicate additional
mutational steps.

RHINELLA MARINA: TWO DISTINCT SPECIES Zootaxa 4103 (6) © 2016 Magnolia Press · 581
FIGURE 4. Trees showing phylogenetic relationships for the Rhinella marina species complex in northern Venezuela.
Phylogenetic relationships are inferred from 429bp fragment of the ND3 (a) and 737bp for CR (b). The names refer to the
location where the haplotype was collected and east and west to their location in relation the Andean Cordillera, as in Figure 1.
The numbers at the nodes indicate maximum likelihood, maximum parsimony and Bayesian inference bootstrap values (ML/
MP/BI).

582 · Zootaxa 4103 (6) © 2016 Magnolia Press ACEVEDO ET AL.

FIGURE 5. Morphological differences in skull shape between east and west populations of Rhinella marina. Changes with
respect to the average on the deformation grids (a) and morphospaces in the canonical variates (b) resulting from comparisons
between eastern and western populations.

Discussion

The combined morphometric and molecular evidence allowed for the identification of two cryptic species within
Rhinella marina. The mitochondrial DNA datasets from ND3 and CR confirmed the phylogenetic break between
populations located east and west of the Andean cordillera identified earlier (Sequeira et al. 2011; Slade & Moritz
1998; Vallinoto et al. 2010), with more than 97% of the observed genetic variation attributed to differences
between regions. In agreement with the genetic evidence, the morphometric geometry analysis revealed, for the
first time, a divergent pattern of skull evolution in R. marina. The identification of an eastern and a western
morphotype with no overlapping morphospaces suggests independent evolutionary histories of morphological
traits across the cis and trans-Andean cordillera. Our fine-scale survey revealed no common haplotypes or
morphotypes between regions, despite the proximity between some eastern and western locations, suggesting that
the two lineages may be allopatric. This concordance between the observed geographic patterns in morphometric
and genic traits calls for the taxonomic revision and the recognition of two species R. marina, one east and one
west to the Andean cordillera.
Previous molecular studies based on mtDNA suggested that the divergence of R. marina across the Andes
predated the speciation of some other species of the R. marina group in South America (Slade & Moritz 1998;
Vallinoto et al. 2010). According to molecular clocks using mitochondrial DNA, eastern and western populations

RHINELLA MARINA: TWO DISTINCT SPECIES Zootaxa 4103 (6) © 2016 Magnolia Press · 583
could have split during the uplift of the Andean cordillera (early Pleistocene) (Slade & Moritz 1998) or earlier
during marine incursions and the formation of the Pebas system (middle-late Miocene) (Vallinoto et al. 2010). The
fine-scale geographic pattern of genetic variation of mitochondrial markers observed in this study showed no
evidence of recent geographic or demographic expansions in R. marina populations around the Andean cordillera
(east or west), in contrast to populations at the southern banks of the Amazon river (Sequeira et al. 2011). These
authors hypothesize a recent southward expansion of populations located south of the Amazon as a result of a
southward re-establishment of the Cerrado during the Holocene.
Cranial morphology in bufonids is highly variable and, for the most part, reflects the evolutionary history of
major taxonomic lineages within this group (Pramuk 2006). At lower taxonomic levels, however, large
phylogenetic breaks are often associated with subtle or indistinguishable changes in morphology, a disparity that
results in the frequent occurrence of cryptic species in bufonids. For example, molecular markers have revealed
cryptic speciation in Anaxyrus punctatus (Jaeger et al. 2005), Rhinella margaritifera (Fouquet et al. 2007) and in
the Dendrophryniscus, Amazophrynella (Fouquet et al. 2012), Frostius (Junca et al. 2012) and Peltophryne genera
(Alonso et al. 2012). Most of these species, however, remain taxonomically cryptic because the assignment of valid
names requires establishing links with voucher type specimens that are frequently unsuitable for molecular studies.
Thus, the identification of diagnostic morphological characters is necessary for the taxonomic revaluation of
species.
Among South American toads, the species belonging to the R. marina group show distinctively large parotoid
glands and, internally, the medial ramus of the pterygoid and the parasphenoid alae join forming a jagged suture
(Pramuk 2006). Within this group, R. marina lacks of tibial glands and has particularly large parotoid glands that
protrude from the outline of the lateral surface of the head (Kwet et al. 2006). However, a depression of the pre-
maxillar region and a posterior elongation of the frontoparietal and occipital regions (e.g. condyles and
sphenethmoids) of R. marina adults from eastern populations result in a slightly rounder head compared to western
populations. In western populations, the quadratojugal bones tend to be closer to the transverse line formed by the
occipital condyles than in eastern populations (Figure 2). These shape differences can unequivocally separate the
eastern from the western lineage and can be captured by the position of diagnostic landmarks that, despite
involving non-visible internal structures, can be used for the evaluation of voucher specimens with non-destructive
x-ray or ecography techniques.
The type locality for the first description of Rhinella marina [Rana marina (Linneus 1758)] was probably
located in Surinam (Muller & Hellmich 1936), east to the Andean cordillera. Later, at least three synonyms were
proposed for R. marina specimens collected west of the Andes: Bufo horribilis (Wiegmann 1833), B. angustipes
(Taylor and Smith 1945), B. pithecodactylus (Rivero 1961) and B. marinus horribilis (Lynch and Fulger 1965). The
earliest description from a western location Bufo horribilis corresponds to a specimen from Mexico (Veracruz).
These suggest that eastern populations should maintain the Rhinella marina nomenclature and the name Rhinella
horribilis should be revalidated for western populations, as suggested earlier (Frost 2015), until further studies
integrate a specimens from its complete geographic distribution. Given the possibility of using non-distructive x-
ray imaging techniques in voucher specimens, landmark-based geometric morphometry is likely to open new
avenues for solving the taxonomy of R. marina.

Acknowledgements

We thank Ivonne Rivas for providing the mammogram images, and Marcos Méndez and Celsa Señaris for assisting
with the molecular and the morphological analysis, respectively. We are also grateful to Dinora Sánchez, Francisco
Nava, Javier Valera. Jorge Novoa y Antonio Pérez for assistance in the field.

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