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Strain survey on three continents confirms the polyphyly of the genus

Pediastrum (Hydrodictyaceae, Chlorophyceae)

Article  in  Journal of the Czech Phycological Society · March 2014

DOI: 10.5507/fot.2014.005

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Fottea, Olomouc, 14(1): 63–76, 2014 63

Strain survey on three continents confirms the polyphyly of the genus

Pediastrum (Hydrodictyaceae, Chlorophyceae)

Mrutyunjay Jena1,2,3, Christina Bock1,4, Chhandashree Behera 1,2, Siba P. Adhikary5

& Lothar Krienitz1*

1
Leibniz–Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhütte2, D–16775 Stechlin–
Neuglobsow, Germany; *e–mail: krie@igb–berlin.de, tel.: 033082 69926, fax: 033082 69917
2
Department of Botany, Utkal University, Bhubaneswar–756014, Odisha, India
3
Department of Botany, Berhampur University, Berhampur–760007, Odisha, India
4
University of Essen, Faculty of Biology, D–45141 Essen, Germany
5
Department of Biotechnology, Visva–Bharati Santiniketan–731235, West Bengal, India

Abstract: Approximately 100 new strains of the Pediastrum–phenotype were isolated from inland waters
of India, eastern and southwestern Africa, and Germany and were studied by light and scanning electron
microscopy. The 18S rRNA and ITS genes of 28 strains were sequenced and compared with 25 sequences of
Hydrodictyaceae from the GenBank database. Phylogenetic analyses confirmed the polyphyly of Pediastrum.
The new species Sorastrum pediastriforme was described. This species resembled morphologically Pediastrum,
however, the phylogenetic analyses revealed its affiliation within the genus Sorastrum. Furthermore, the
following new combinations: Monactinus sturmii, Pseudopediastrum alternans, Pp. brevicorne, Pp. integrum,
and Pp. pearsonii were established.

Key words: CBC, ITS, Lacunastrum, Monactinus, morphology, Pediastrum, phylogeny, Pseudopediastrum,
Sorastrum, SSU, Stauridium

Introduction strains of Hydrodictyon, 29 strains of Pediastrum

and three strains of Sorastrum revealed a pattern of
The family Hydrodictyaceae (Chlorophyceae) is colony–form evolution within the family from two–
from a morphological point of view, one of the most dimensionality to three–dimensionality (McManus &
diverse natural lineages of coccoid green algae and Lewis 2005). Another molecular phylogenetic study
comprises microscopic colonies of Pediastrum Meyen, of 28 hydrodictyacean strains resulted in taxonomic
Euastropsis Lagerh. and Sorastrum Kütz. as well as revisions (Buchheim et al. 2005). It was shown that the
macroscopic colonies of Hydrodictyon Roth. Inspired genus Pediastrum is of polyphyletic origin. Therefore,
by members of the genus Pediastrum, systematists beside Pediastrum the genera Monactinus (Turpin)
have established more than 350 taxa (Parra 1979). E. Hegewald, Stauridium (Printz) E. Hegewald,
A flood of observations and revisions were published Pseudopediastrum E. Hegewald and Parapediastrum
about Pediastrum and summarized by Komárek & Fott E. Hegewald were delineated. Nevertheless, the
(1983). The most recent review reduced the number number of strains analysed was very low, for example
of Pediastrum–species to 24 (Komárek & Jankovská the genera Monactinus (formerly Pediastrum simplex)
2001). The other genera included smaller numbers of and Parapediastrum (P. biradiatum) were described
species: one of Euastropsis, seven of Sorastrum, and from a single strain (Buchheim et al. 2005). Members
five of Hydrodictyon (according to Komárek & Fott of the unicellular genus Tetraedron Kütz. were linked
1983). phylogenetically to the Hydrodictyaceae (Buchheim
The first molecular phylogenetic studies et al. 2005; McManus & Lewis 2005) however, the
including only two members of Hydrodictyaceae relative position was not clear from these analyses.
showed the close relationship of Hydrodictyon The morphospecies P. duplex was shown within
reticulatum (L.) Lagerh. and Pediastrum duplex different phylogenetic lineages of Hydrodictyaceae
Meyen (Wilcox et al. 1992; Lewis 1997) and their (McManus & Lewis 2005). Therefore, one of these
placement within Sphaeropleales (Buchheim et al. lineages was established as new genus Lacunastrum
2001). An expanded phylogenetic analysis of 26S H. McManus (McManus et al. 2011). More detailed
rRNA, 5.8S + ITS2 genes using sequences of seven phylogenetic analyses based on 26S rRNA and rbcL
64 Jena et al.: The polyphyly of the genus Pediastrum

genes on P. duplex and its comparison to other members were compared with 24 sequences of Hydrodictyaceae and
of Hydrodictyaceae were provided by Mc Manus & one (outgroup) Scenedesmus–sequence from the GenBank
Lewis (2011). database (National Center for Biotechnology Information
We have isolated ca. 100 strains of [NCBI] http://www.ncbi.nlm.nih.gov/). The origin and
accession numbers of all these 53 strains are listed in Table 1.
Hydrodictyaceae from three different continents
– Africa, Asia and Europe – and studied them Phylogenetic analyses. Based on a concatenated data set (18S
morphologically. From this collection, we subjected rRNA and ITS–2 rRNA) of 47 sequences with 1872 aligned
28 strains to molecular phylogenetic analyses by base positions (excluding intron) phylogenetic analyses were
sequencing the nuclear 18S rRNA gene and ITS performed. The Sequence alignment was done manually
genes and the analyses confirmed the polyphyly of the according to their secondary structure (Coleman 2003) using
genus Pediastrum. For species delineation, we applied the SequentiX Alignment Editor (Hepperle 2004). The ITS–1
the CBC concept (Coleman 2003). In the frame of rRNA was not unambiguously alignable and was, therefore,
this study we described the new species Sorastrum excluded from the data set. In total, 28 strains that include
representatives of the genera Pediastrum, Pseudopediastrum,
pediastriforme Jena, C. Bock, Behera et Krienitz,
Parapediastrum, Monactinus and Sorastrum were
and established five new combinations in the genera choosen from the GenBank database (National Center for
Pseudopediastrum and Monactinus. Biotechnology Information [NCBI] http://www.ncbi.nlm.
nih.gov/). The selection of additional sequences were done
based on the publications of Buchheim et al. (2005) and
McManus & Lewis (2005, 2011). Scenedesmus obliquus
Material and Methods UTEX 393 was selected as outgroup. The phylogenetic tree
in Figure 1 was infered from a concatenated dataset using
Algal cultures. About 100 unialgal strains of Pediastrum– Treefinder (Jobb 2008). The applied models were chosen
phenotype were isolated from field samples of inland as proposed by Treefinder: For 18S rRNA (1640 bases) the
waters of India, Israel, Kenya, Namibia, and Germany by Tamura Nei model with Gamma distribution (TN:G:5), and
microcapillaries and grown in a modified Waris solution for ITS–2 the transitional model with Gamma distribution
(McFadden & Melkonian 1986). All strains were maintained (J2:G:5) were chosen.
at the algal strain collection of the Leibniz–Institute of To confirm the tree topology, bootstrap analyses
Freshwater Ecology and Inland Fisheries (IGB) at Stechlin, were calculated by distance (neighbor–joining; NJ; 1000
Germany, and grown in suspensions at 15 °C under a 14:10 replicates), parsimony (MP; 1000 replicates) using PAUP*,
h light–dark regime. The strain of the newly described version 4.0b10 (Swofford 2002) and maximum likelihood
Sorastrum pediastriforme and the strains representing the five (ML; 1000 replicates; settings as described above) criteria.
new combinations were deposited at the Culture Collection Bayesian inference (Bayesian posterior probabilities [BPP];
of Algae at the University of Göttingen (SAG, Germany). using the covarion model; 1 million generations) were
calculated using MrBayes version 3.1 (Huelsenbeck &
Microscopy. The algae were examined using a Nikon Eclipse Ronquist 2001). The Bayesian analyses were carried out
E600 light microscope (LM) with differential interference until the average standard deviations of split frequencies
contrast. Photomicrographs (Figs 3–6) were taken with a between two runs was lower than 0.01. The first 25% of the
Nikon Digital Camera DS–Fi1, and Nikon software NIS– calculated trees was discarded as burn–in. A 50% majority–
Elements D (Nikon Corporation, Tokyo, Japan). For scanning rule consensus tree was calculated for posterior probabilities
electron microscopy (SEM) the colonies of cultured material (PP).
were fixed in 2.5% glutaraldehyde in culture medium for 30
min at 5 °C, washed three times in 3 mM Hepes buffer (pH Secondary structures analysis. Secondary structures of
7.0), and postfixed in 1% osmium tetroxide in a 1:1 mixture ITS–2 were constructed with the help of mfold (Zucker
of 2% OsO4 and 3 mM Hepes for 1 h. The fixed cells were 2003).The program 4SALE (Seibel et al. 2006, 2008; Schultz
then washed three times in Hepes buffer and dehydrated in & Wolf 2009) was used for comparision of ITS–2 among
a graded series of ethanol dilutions. Following dehydration, the strains to locate compensatory base changes (CBCs)
the material was dried at the critical point using aBAL– including Hemi–CBCs in the aligned ITS–2 sequences.
TEC CPD 030 critical point dryer (Balzers, Schalkmühle, The overview graphic was performed with PseudoViewer
Germany). Dried material was mounted on aluminium stubs, (Byun & Han 2006). In order to find molecular criteria to
coated with gold/palladium by a Polaron CA 508 sputter– discriminate the species, these secondary structures were
coater (Fisons Instruments, VG Microtech, Uckfield, UK) checked for Compensatory Base Changes (CBCs) including
and examined under using the field–emission SEM Hitachi Hemi–CBCs using 4SALE (Seibel et al. 2006, 2008).
S–4500 (Hitachi Corporation, Tokyo, Japan) at 5 kV, 10 µA.

DNA Isolation, PCR and Sequencing. A total of 28 strains

were selected for molecular phylogenetic analyses. Algal
cells were mechanically disrupted in the presence of glass
beads (~ 0.5 mm in diameter, Carl Roth GmbH + Co. KG,
Results
Karlsruhe, Germany) using the Retsch MM 2000 (Retsch,
Hahn, Germany) bead disruptor. Total Genomic DNA was The concatenated set of 18S rRNA and ITS–2
extracted according to Krienitz et al. (2012). The 18S rRNA sequences of 47 strains was included in the analyses
and ITS–2 rRNA genes were amplified and sequenced as to assess the phylogeny of Hydrodictyaceae. The
described by Bock et al. (2011). The newly sequenced strains 28 newly sequenced strains were placed in seven
Fottea, Olomouc, 14(1): 63–76, 2014 65

subclades of the phylogenetic tree (Fig. 1). Generally, the genus Pseudopediastrum. For the essential new
the family of Hydrodictyaceae formed a monophyletic combinations see taxonomic conclusions.
group well supported in the statistical analyses, only Members of the former morphospecies
the ML analyses resulted in a lower bootstrap value Pediastrum duplex clustered in two different clades
of 67. Members of the genus Pediastrum sensu lato, of the tree. Six of these strains remained in P. duplex,
which were in the focus of this study, were grouped whereas two other strains were determined as
into different lineages. According to suggestions Lacunastrum gracillimum (Figs 6a–d). These strains
of Buchheim et al. (2005) these lineages were had relatively long processes and the ultrastructure of
regarded as genera and designated as Pediastrum, the cell wall was rippled, smooth, but not net–shaped
Pseudopediastrum, Monactinus, Stauridium and and granular. These results confirmed the findings of
Parapediastrum. Two strains belonged to the recently McManus et al. (2011). Five new strains of Stauridium
erected genus Lacunastrum (McManus et al. 2011). tetras (Ehrenb.) E. Hegewald from Kenya and India
Hydrodictyon was closely related to Pediastrum, and S. privum (Printz) E. Hegewald from Kenya
and Sorastrum is placed on the base of the tree as and Germany clustered in the clade of Stauridium.
sister to all other hydrodictyacean subclades. The One strain of Parapediastrum biradiatum (Meyen)
bootstrap support of different genera was high, only E. Hegewald from Germany clustered together with
the Pseudopediastrum clade earned low support. The a strain from Sweden in the Parapediastrum clade.
position of Tetraedron remained ambiguous. These results confirmed the findings of Buchheim et al.
The strain MJ 2008/46 (SAG 2392) from a dam (2005).
near Nyahururu in Kenya clustered in the Sorastrum To test the results of the topology of the
clade. Members of the genus Sorastrum normally, phylogenetic tree, the secondary structure of the ITS–2
were characterized by three–dimensional colonies. was studied, with the main goal to find CBCs and hemi–
However, the MJ 2008/46 (SAG 2392) strain showed CBCs associated with individual taxa. The secondary
flat coenobia (Figs 2a–d) resembling morphospecies structure of the taxa studied exhibited the common
of the genus Pediastrum, in particular those of P. pattern with four helices, in which the helix I was
duplex. Only few cells displayed a three–dimensional branched, shown here using Sorastrum pediastriforme
(tetrahedric) shape (Fig. 2d). This material was (Fig. 7) as exemplar. To establish an overview, we made
described as new species Sorastrum pediastriforme a pairwise comparison of the CBCs of the conserved
(see taxonomic conclusions). regions of all helices between the species which were
Members of the genus Monactinus were found discovered to be the closest relatives to each other
in India, Kenya and Germany. Whereas the strain from according to the tree. Sorastrum pediastriforme differs
Germany exhibited the typical morphology of M. from S. spinulosum (UTEX 37) by 2 CBCs in Helix
simplex var. simplex with large irregular holes within I (not shown). Monactinus simplex and M. sturmii
the coenobium (Figs 3a–b), the four tropical strains differed by 2 CBCs and 5 or 6 hemi–CBCs in helix III
resembled M. simplex var. sturmii with stocky cells and (Table 2). The species of Pseudopediastrum differed
smaller holes (Figs 3c–d). in the following numbers of CBC (Table 3): Pp.
Six new strains clustered in the boryanum differed from Pp. kawraiskyi by 2 CBCs in
Pseudopediastrum clade. These strains showed helix III. Pp. alternans differed from Pp. brevicorne
characteristic differences (Figs 4a–d, 5a–d). Whereas only by hemi–CBCs, 3 in helix I and 1 in helix III. Pp.
the strains MJ 2008/33 and MJ 2008/35 from Lake integrum differed from Pp. kawraiskyi by 2 CBCs in
Dollgow in Germany fully met the characteristics of helix III. Pp. pearsonii differed from Pp. integrum by
Pseudopediastrum boryanum (Turp.) Menegh. (not 1 CBC in helix I and 1 CBC in helix III (not shown).
shown), the others exhibited features of other taxa.
Strain MJ 2008/83 from the river Cuando in Namibia
possesses long conical lobes and deep incisions, Discussion
resembling with Pseudopediastrum alternans Nyg.
(Figs 4a–b). Strain MJ 2008/28 from Lake Dollgow in Our study confirmed the polyphyletic origin of members
Germany had short lobes with shallow incisions and of the genus Pediastrum which was already shown by
resembled Pp. boryanum var. brevicorne A. Braun Buchheim et al. (2005) and McManus & Lewis (2005).
(Figs 4c, d). Strain MJ 2008/86 from a pond in Namibia Furthermore, we confirmed the generic conception
with short rounded apices and very shallow incisions of Hydrodictyaceae and studied new strains from all
resembled Pseudopediastrum integrum Nägeli (Figs genera of the relationship of Pediastrum sensu lato
5a, b). Strain KR 2006/01 from Lake Naivasha, Kenya, (Parapediastrum, Pseudopediastrum, Lacunastrum,
similar in morphology but different in molecular Monactinus, and Stauridium) as well as Sorastrum.
criteria, resembled Pp. integrum var. pearsonii (G.S. Generally, the differentiation between the genera of
West) Fritsch (Figs 5c, d) known from tropical Hydrodictyaceae is validated by both, morphological
regions. Because of the clustering of these strains in and molecular criteria. However, an interesting
the same clade, they all were considered as species of example was uncovered contrasting with this general
66 Jena et al.: The polyphyly of the genus Pediastrum

Fig. 1. Phylogeny according to the concatenated SSU and ITS–2 rRNA gene data set of the studied Hydrodictyaceae. Values at the nodes
indicate statistical support estimated by four methods – Bayesian support (MB BPP), maximum likelihood (ML), maximum parsimony (MP)
and neighbor joining (NJ). Hyphens indicate support below 50% for ML, MP, NJ and below 0.95 for MB BPP.
Fottea, Olomouc, 14(1): 63–76, 2014 67

Fig. 2. Micrographs of Sorastrum pediastriforme (MJ 2008/46) in culture: (a) LM; (b–d) SEM. The arrow in (d) indicates a tetrahedric cell.
Scale bar 2 µm (a); 14 µm (b); 6 µm (c), 10 µm (d).

Fig. 3. Micrographs of Monactinus in culture: (a, b) M. simplex (MJ 2008/34); (c, d) M. sturmii (MJ 2009/172). Scale bar 10 µm (a); 20 µm
(b); 15 µm (c, d).
68 Jena et al.: The polyphyly of the genus Pediastrum

Fig. 4. Micrographs of different Pseudopediastrum strains: (a, b) Pp. alternans (MJ 2008/83); (c, d) Pp. brevicorne (MJ 2008/28). Scale bar
15 µm (a); 12 µm (b); 10 µm (c, d).

Fig. 5. Micrographs of different Pseudopediastrum strains: (a, b) Pp. integrum (MJ 2008/86); (c, d) P. pearsonii (KR 2006/01). Scale bar 12
µm (a); 20 µm (b); 10 µm (c); 12 µm (d).
Fottea, Olomouc, 14(1): 63–76, 2014 69

Fig. 6. Micrographs of Lacunastrum gracillimum strains: (a, b) MJ 2009/123; (c, d) MJ 2009/162. Scale bar 25 µm (a); 36 µm (b); 20 µm (c);
22 µm (d).

view. The new species Sorastrum pediastriforme

which morphologically resembles Pediastrum but
molecular phylogenetic analyses reveals its alliance
with Sorastrum.
Studying two strains of Lacunastrum
gracillimum from India, we confirmed the polyphyly
of the Pediastrum duplex phenotype revealed by
McManus et al. (2005) and the delineation of the
genus Lacunastrum by McManus et al. (2011). It was
shown that phylogenetic findings sharpen the eyes
for subsequent careful morphometric evaluation of
diagnostic features to discriminate the two very similar
phenotypes of P. duplex and L. gracillimum (McManus
et al. 2011).
In the present state we are still working
to clarify the interplay between morphology and
phylogeny in Hydrodictyaceae with reference to the
other two main clades of planktonic Chlorophyceae,
the Scenedesmaceae and Selenastraceae. The
Fig. 7. Secondary structure of the ITS–2 of Sorastrum pediastriforme interpretation of the state widely depends on
strain SAG 2392. Sorastrum spinulosum strain UTEX LB2452 as exemplarily treated groups. In Scenedesmaceae an
core struture, intraspecific base changes between strain SAG 2392
extraordinary level of morphological variability must
and UTEX LB2452 are marked in circles.
be reconciled to a scarcity of molecular peculiarities
such as compensatory base changes (Vanormelingen
et al. 2007). On the other side, the relatively uniforme
spindle–shaped phenotype of Acutodesmus represents
70 Jena et al.: The polyphyly of the genus Pediastrum

Fig. 8. Drawings of Sorastrum pediastriforme (MJ 2008/46). Scale bar 10µm (a, b, e, f); 6 µm (c, d, g, h). (a) iconotype.

different phylogenetic clades (Hegewald & Wolf found only in Kenya. Probably, this is the exception
2003; Lewis & Flechtner 2004; Hegewald et al that proves the rule of cosmopolitan distribution.
2013). In Selenastraceae similar phenotypes such as
the needle–shaped or crescent–shaped morphology
hide a multitude of phylotypes (Fawley et al. 2007; Taxonomic conclusions
Krienitz et al. 2011). In the Hydrodictyaceae members
of the genus Pseudopediastrum are characterized both Sorastrum pediastriforme Jena et C. Bock sp. nov.
by high morphological diversity and phylogenetic (Figs 2a–d, 9a–h)
divergence resulting in four new combinations in this Desriptio: Coenobia plus minusve planae multicellularis,
study. Regardless of the molecular background that 4–8–16 cellulae continens. Cellulae externae per duos lobis
formae, rare trilobates. Lobis complanes, elongates cum
has revealed overwhelming phenotypic diversity of the
apicibus coronatis. Cellulae interiorae quadricornutae per
Hydrodictyaceae, it seems unlikely that more genus– foramen inter cellulae. Chloroplastus unicus, parietale,
level diversity will be uncovered. It is suspected that massivus, cum pyrenoido. Propagatio zoosporis.
the complicated Pediastrum–like phenotype of most A speciebus ceteris generis ordine nucleotidorum in 18S and
members of this family has evolved infrequently, ITS rRNA differt.
at best, and independently than easier constructed
phenotypes such as spheres which undergo convergent Coenobia more or less flat, multicellular, containing
evolution (Potter et al. 1997; Krienitz & Bock 2012). 4–8–16 cells. Outer cells two–lobated, seldom
Our survey of Pediastrum and related genera trilobated. Lobae elongated with crown–like apices.
from three continents did not provide arguments for Inner cells four–lobated with holes between the cells.
endemism of this group, because most of the taxa were One parietal, massive chloroplast per cell with a
found in different geographic regions. Even species pyrenoid. Propagation by zoospores. The species differs
formerly known only from tropical or subtropical from other species by the order of the nucleotides in
regions like Monactinus (Pediastrum) simplex SSU and ITS rRNA gene sequences.
extended their distribution area into the temperate zone Holotype: an air–dried sample of the authentic strain
(Coesel & Krienitz 2008). The species P. pearsonii SAG 2392 (MJ 2008/46) deposited at the Botanical
known from tropics (Komárek & Jankovská 2001) we Museum at Berlin–Dahlem, Germany under the
Fottea, Olomouc, 14(1): 63–76, 2014 71

designation B40 0040819. University of Göttingen (SAG, Germany).

Iconotype: our Fig. 9a Pseudopediastrum pearsonii (G.S. West) Jena et C.
Authentic strain: SAG 2392 deposited at the Culture Bock comb. nov.
Collection of Algae at the University of Göttingen Basionym: Pediastrum pearsonii G.S. West 1912. Ann. S. Afr. Mus.
9, p. 79, figs. 30–32.
(SAG, Germany). Synonym: Pediastrum integrum var. pearsonii (G.S. West) Fritsch in
Type locality: Kenya, dam near Nyahururu Fritsch et Stephens 1921.
Etymology: The species epithet was selected because
of the morphological similarity of the colonies to Epitype (designated here): An air–dried sample of the
species of the genus Pediastrum. strain KR 2006/01 deposited at the Botanical Museum
at Berlin–Dahlem under the designation B40 0040818.
Authentic strain: SAG NA2013.011 (KR 2006/01)
Monactinus sturmii (Reinsch) Jena et C. Bock deposited at the Culture Collection of Algae at the
comb. nov. University of Göttingen (SAG, Germany).
Basionym: Pediastrum sturmii Reinsch 1867. Die Algenflora des
mittleren Theiles von Franken, p. 90, pl. VII, fig. Ia–d.
Synonym: Pediastrum simplex var. sturmii (Reinsch) Wolle 1887.
Acknowledgements
This work was supported in part by a fellowship grant of the German
Epitype (designated here): An air–dried as well as a Academic Exchange Service for Mrutyunjay Jena. We thank
formaldehyde–fixed sample of the strain MJ 2009/172, Reingard Rossberg for joint work at the SEM and Monika Papke for
deposited at the Botanical Museum at Berlin–Dahlem technical assistance.
under the designation B40 0040814.
Authentic strain: SAG NA2013.007 (MJ 2009/172)
deposited at the Culture Collection of Algae at the
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strain MJ 2008/86 deposited at the Botanical Museum substitution. – Mol. Biol. Evol. 19: 698–707.
Huelsenbeck, J. P. & Ronquist, F. (2001): MRBAYES: Bayesian
at Berlin–Dahlem under the designation B40 0040817. inference of phylogenetic trees. – Bioinformatics 17: 754–
Authentic strain: SAG NA2013.010 (MJ 2008/86) 755.
deposited at the Culture Collection of Algae at the Komárek, J. & Fott, B. (1983): Chlorophyceae (Grünalgen)
Table 1. List of strains used in this study.
72

b)
Strain a) Species Origin Accession number Reference

UTEX LB782 Hydrodictyon africanum South Africa, Cape Flats AY663031 Buchheim et al. 2005
AY779861
CBS Hydrodictyon reticulatum USA, Indiana AY779858 Buchheim et al. 2005
AY779862
MJ 2009/123 Lacunastrum gracillimum India, Orissa, pond at Athagarh HM021304 this study
MJ 2009/162 Lacunastrum gracillimum India, Orissa, pond at Chadneswar HM021305 this study
UTEX 1601 Monactinus simplex unknown AY780664 Buchheim et al. 2005
AY780655
KR 2008/06 Monactinus sturmii India, Kerala, pond at Kochi HM021323 this study
MJ 2008/34 Monactinus sturmii Germany, Lake Dollgow HM021323 this study
MJ 2009/169 Monactinus sturmii India, Orissa, pond at Chadneswar HM021322 this study
MJ 2009/172 Monactinus sturmii India, Orissa, River Mohanadi HM021321 this study
SAG NA2013.007
MJ 2009/284 Monactinus sturmii Kenya, Lake Baringo HM021325 this study
UTEX 37 Parapediastrum biradiatum Sweden AY663034 Buchheim et al. 2005
AY779865
MJ 2008/60 Parapediastrum biradiatum Germany, Lake Schwanter See HM021320 this study
UTEX LB1366 Pediastrum angulosum Czech Republic, Podbansko pool, High Tatra Mountains AY663032 Buchheim et al. 2005
AY779863
UTEX LB1370 Pediastrum angulosum Czechoslovakia, Doksy, Machovo jezero pool AY663033 Buchheim et al. 2005
AY779864
LN0201NC Pediastrum duplex USA, North Carolina, Lake Norman AY577744 McManus & Lewis 2005
SAG 28.83 Pediastrum duplex Germany, Pfälzer Wald, forest pond AY780662 Buchheim et al. 2005
AY780653
SAG 84.80 Pediastrum duplex Germany, Schöhsee, Plön AY577748 McManus & Lewis 2005
SF0202NY Pediastrum duplex USA, New York, Stockpot Flats AY577757 McManus & Lewis 2005
KR 2005/327 Pediastrum duplex Israel, Lake Kinneret HM021298 this study
Jena et al.: The polyphyly of the genus Pediastrum
Table 1 Cont.
MJ 2008/01 Pediastrum duplex Germany, Lake Dagow HM021300 this study
MJ 2008/06 Pediastrum duplex Kenya, dam near Nyahururu HM021301 this study
MJ 2008/24 Pediastrum duplex Germany, Lake Geron HM021303 this study
MJ 2008/54 Pediastrum duplex Kenya, dam near Nyahururu HM021299 this study
MJ 2009/107 Pediastrum duplex India, Orissa, pond at Bhubaneswar HM021302 this study
RL0201FR Pediastrum sp. France, Reilhac Lake AY577749 McManus & Lewis 2005
MJ 2008/83 Pseudopediastrum alternans Namibia, River Cuando near Kongola HM021311 this study
Fottea, Olomouc, 14(1): 63–76, 2014

SAG NA2013.008
OL0301MN Pseudopediastrum boryanum USA, Minnesota, Otter Lake AY577759 McManus & Lewis 2005
EL0203CT Pseudopediastrum boryanum USA, Storrs, Eagleville Lake Connectcut AY577742 McManus & Lewis 2005
UTEX LB1372 Pseudopediastrum boryanum v. longicorne Czech Republic, pond Brehyne, Doksy AY663036 Buchheim et al. 2005
AY779867
MJ 2008/28 Pseudopediastrum brevicorne Germany, Lake Dollgow HM021310 this study
SAG
NA2013.009

Heg 1976-16 Pseudopediastrum boryanum unknown AY780661 Buchheim et al. 2005

AY780652
UTEX LB470 Pseudopediastrum boryanum UK, Malham Tarn, Yorkshire AY663035 Buchheim et al. 2005
AY779866
MJ 2008/33 Pseudopediastrum boryanum Germany, Lake Dollgow HM021306 this study
MJ 2008/35 Pseudopediastrum boryanum Germany, Lake Dollgow HM021307 this study
MJ 2008/86 Pseudopediastrum integrum Namibia, pond at Mariental HM021309 this study
SAG NA2013.010
SAG 35.81 Pseudopediastrum kawraiskyi Germany, Steinhuder Meer AY780663 Buchheim et al. 2005
AY780654
KR 2006/1 Pseudopediastrum pearsonii Kenya, Lake Naivasha HM021308 this study
SAG NA2013.011
SAG 13.94 Sorastrum americanum USA, Minnesota, Lake Itasca State Park AY780657 Buchheim et al. 2005
MJ 2008/46 Sorastrum pediastriforme Kenya, dam near Nyahururu HM021312 this study
SAG 2392
73
 74

Table 1 Cont.
UTEX LB2452 Sorastrum spinulosum Finland, pond AY663041 Buchheim et al. 2005
AY779872
UTEX LB785 Sorastrum sp. USA, Bloomington, Indiana AY663040 Buchheim et al. 2005
AY779871
SAG 36.81 Stauridium privum Finland, Kuusjärvi, Saukkolahti near Jyväskylä AY780666 Buchheim et al. 2005
AY780656
MJ 2008/47 Stauridium privum Kenya, dam near Nyahururu HM021319 this study
MJ 2008/74 Stauridium privum Germany, Lake Stechlin HM021318 this study
SAG 37.81 Stauridium tetras unknown AY779891 Buchheim et al. 2005
AY780656
UTEX 38 Stauridium tetras Czechoslovakia AY779892 Buchheim et al. 2005
AY779869
KR 2006/04 Stauridium tetras Kenya, Lake Naivasha HM021317 this study
KR 2008/04 Stauridium tetras India, Kerala, pond at Kochi HM021316 this study
MJ 2008/07 Stauridium tetras Kenya, dam near Nyahururu HM021315 this study
MJ 2008/51 Stauridium tetras Kenya, dam near Nyahururu HM021314 this study
MJ 2009/269 Stauridium tetras Kenya, Thika, ricefield HM021313 this study
UTEX 1367 Tetraedron minimum Slovakia. Rezabinec pond, Razice AY663042 Buchheim et al. 2005
AY779875
KR 1979/12 Tetraedron minimum Germany, pond at Schortewitz AY780670 Buchheim et al. 2005
AY780659
a)
Boldface + underlined: holo- or epitypes; b)Accession numbers in bold: sequenced in the frame of this study
Jena et al.: The polyphyly of the genus Pediastrum
Fottea, Olomouc, 14(1): 63–76, 2014 75

Table 2. Number of CBCs and Hemi-CBCs (in brackets) in conserved regions of ITS-2 of Monactinus.

Taxa compared Helix 1 Helix II Helix IIIa Helix IV

UTEX 1601 vs MJ 2008/34 0 (2) 0 (0) 2 (5) 0 (0)

UTEX 1601 vs KR 2008/06 0 (2) 0 (0) 2 (6) 0 (0)
UTEX 1601 vs MJ 2009/284 0 (2) 0 (0) 2 (6) 0 (0)
UTEX 1601 vs MJ 2009/169 0 (3) 0 (0) 2 (6) 0 (0)
UTEX 1601 vs MJ 2009/172 0 (2) 0 (0) 2 (6) 0 (0)

Table 3. Number of CBCs and Hemi-CBCs (in brackets) in conserved regions of ITS-2 of Pseudopediastrum.

Taxa compared Helix 1 Helix II Helix III Helix IV

P. boryanum MJ 2008/33 vs P. boryanum Hg 1976-16 0 (0) 0 (0) 0 (0) 0 (0)

MJ 2008/33 vs P. boryanum var. cornutum (UTEX 470) 0 (2) 0 (0) 0 (0) 0 (0)

MJ 2008/33 vs P. kawraiskyi 35.81 0 (1) 0 (1) 2 (0) 0 (0)

MJ 2008/33 vs MJ 2008/35 0 (0) 0 (0) 0 (0) 0 (1)

MJ 2008/33 vs MJ 2008/86 0 (1) 0 (0) 0 (3) 0 (0)

MJ 2008/33 vs KR 2006/01 1 (1) 0 (1) 1 (2) 0 (0)

MJ 2008/33 vs MJ 2008/83 1 (2) 0 (1) 0 (3) 0 (1)

MJ 2008/33 vs MJ 2008/28 1 (0) 0 (1) 0 (4) 0 (1)

P. alternans MJ 2008/83 vs P. boryanum Hg 1976-16 1 (2) 0 (1) 0 (3) 0 (1)

MJ 2008/83 vs P. boryanum var. cornutum (UTEX 470) 0 (4) 0 (1) 0 (3) 0 (1)

MJ 2008/83 vs P. kawraiskyi 35.81 0 (1) 0 (2) 1 (2) 0 (0)

MJ 2008/83 vs MJ 2008/28 0 (3) 0 (0) 0 (1) 0 (0)

P. brevicorne MJ 2008/28 vs P. boryanum Hg 1976-16 1 (0) 0 (1) 0 (4) 0 (1)

MJ 2008/28 vs P. boryanum var. cornutum UTEX 470 1 (2) 0 (1) 0 (4) 0 (1)

MJ 2008/28 vs P. kawraiskyi 35.81 0 (4) 0 (2) 2 (1) 0 (0)

P. integrum MJ 2008/86 vs P. boryanum Hg 1976-16 1 (1) 0 (0) 0 (3) 0 (0)

MJ 2008/86 vs P. boryanum var. cornutum UTEX 470 0 (3) 0 (0) 0 (3) 0 (0)

MJ 2008/86 vs P. kawraiskyi 35.81 0 (0) 0 (1) 2 (1) 0 (0)

MJ 2008/86 vs KR 2006/01 1 (0) 0 (1) 1 (1) 0 (0)

MJ 2008/86 vs MJ 2008/83 1 (1) 0 (1) 0 (2) 0 (0)

MJ 2008/86 vs MJ 2008/28 0 (4) 0 (1) 0 (4) 0 (0)

 76 Jena et al.: The polyphyly of the genus Pediastrum

Table 3 Cont.

P. pearsonii KR 2006/01 vs P. boryanum Hg 1976-16 1(1) 0 (1) 1 (2) 0 (0)

KR 2006/01 vs P. boryanum var. cornutum UTEX 470 1 (3) 0 (1) 1 (2) 0 (0)
KR 2006/01 vs P. kawraiskyi 35.81 1 (0) 0 (2) 2 (0) 0 (0)
KR 2006/01 vs MJ 2008/83 1 (1) 0 (1) 1 (1) 0 (0)
KR 2006/01 vs MJ 2008/28 1 (2) 0 (0) 1 (1) 0 (0)

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