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(American Journal of Botany. 2008;95:1079-1095.) doi: 10.3732/ajb.0800046 © 2008 Botanical Society of America, Inc. |
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Phycology |
Botanisches Institut, Lehrstuhl I, Universität zu Köln, Gyrhofstr. 15, D-50931 Köln, Germany
Received for publication 7 February 2008. Accepted for publication 11 June 2008.
ABSTRACT
Nuclear-encoded small subunit (SSU) rDNA, 1506 group I introns, and chloroplast rbcL genes were sequenced from 97 strains representing the largest desmid genus Cosmarium (45 spp.), its putative relatives Actinotaenium (5 spp.), Xanthidium (4 spp.), Euastrum (9 spp.), Staurodesmus (13 spp.), and other Desmidiaceae (Zygnematophyceae, Streptophyta) and used to assess phylogenetic relationships in the family. Analyses of single genes and of a concatenated data set (3260 nt) established 10 well-supported clades in the family with Cosmarium species distributed in six clades and one nonsupported assemblage. Most of the clades contained representatives of at least two genera highlighting the polyphyletic nature of the genera Cosmarium, Euastrum, Staurodesmus, and Actinotaenium. To enhance resolution between clades, we extended the data set by sequencing the slowly evolving chloroplast-encoded large subunit (LSU) rRNA gene from 40 taxa. Phylogenetic analyses of a concatenated data set (5509 nt) suggested a sister relationship between two clades that consisted mainly of Cosmarium species and included C. undulatum, the type species of the genus. We describe molecular signatures in the SSU rRNA for two clades and conclude that more studies involving new isolates, additional molecular markers, and reanalyses of morphological traits are necessary before the taxonomic revision of the genus Cosmarium can be attempted.
Key Words: Actinotaenium • clades • Cosmarium • Desmidiaceae • Euastrum • molecular phylogeny • molecular signatures • polyphyly • taxonomy
The conjugating green algae (Zygnematophyceae, Viridiplantae) represent the most species-rich lineage in the Streptophyta except for the embryophytic land plants (Gerrath, 1993
). They share with most embryophytes not only a common ancestry but also the absence of flagellate reproductive stages. A peculiar mode of sexual reproduction (i.e., conjugation) sets the class apart from other streptophytes and may have contributed to their successful diversification (Brook, 1981). On the basis of ultrastructural analyses of mitosis and cytokinesis, zygnematophycean algae have been recognized as members of the streptophyte algae known also as Charophyceae sensu Stewart and Mattox (Pickett-Heaps, 1975). Molecular phylogenetic analyses of the Zygnematophyceae corroborated these results (Chapman and Buchheim, 1992; Surek et al., 1994; McCourt et al., 2000; Gontcharov et al., 2003), although the phylogenetic position of the class among the streptophyte algae still remains unresolved (Chapman et al., 1998; Karol et al., 2001; Lewis and McCourt, 2004; Turmel et al., 2007).
Phylogenetic analyses of the Zygnematophyceae using a broad taxon sampling and multigene data sets have more recently led to the conclusion that many traditional genera of the class are polyphyletic, suggesting that the characters used to delineate these taxa are either plesiomorphic, homoplasious or unreliable (Gontcharov et al., 2003, 2004; Gontcharov and Melkonian, 2005; Hall et al., 2008). This situation is well known in microalgal systematics in which descriptions of genera have often been based on single or very few morphological characters visible in the light microscope without a careful investigation of phylogenetic significance (one related example is that of the coccoid green algal genus Chlorella and its relatives; Krienitz et al., 2004; Luo et al., 2006). We have therefore started to evaluate the genus concept in the most species-rich family of the Zygnematophyceae, the Desmidiaceae (desmids), using taxon-rich sampling and multigene phylogenetic analyses. Although the traditional genus Staurastrum Meyen ex Ralfs was shown to be polyphyletic, a monophyletic core of the genus could be identified using such an approach (Gontcharov and Melkonian, 2005). It is anticipated that once a phylogenetic framework of a desmid genus has been established, a reinvestigation of its morphological traits should lead to the recognition of hitherto overlooked, but more sound and reliable generic morphological characters.
Here, we address the phylogenetic status of the genus Cosmarium Corda ex Ralfs (Desmidiaceae, Zygnematophyceae), the most species-rich desmid genus with more than 1000 species described (Gerrath, 1993). Together with Staurastrum (
700 spp.) it constitutes about half of the total number of species in the otherwise species-poor streptophyte green algae. It should be mentioned that Cosmarium has always been regarded as an artificial genus and thus taxonomically problematic (West and West, 1905, 1908; Fritsch, 1953, Hirano, 1959a; Krieger and Gerloff, 1962, 1965, 1969; Prescott et al., 1981, Croasdale and Flint, 1988; Brook and Johnson, 2002; Gerrath, 2003). It was poorly circumscribed by a vague diagnosis (Ralfs, 1848) and linked morphologically to other genera such as Euastrum Ehr. ex Ralfs and Xanthidium Ehr. ex Ralfs. Thus, although over the 160 years since its description, numerous taxa have been added to Cosmarium, the definition of the genus ("the fronds are minute, simple, constricted in the middle; the segments are generally broader than long and inflato-compressed, but in some species orbicular or cylindrical; they are neither emarginate at the end nor lobed at the sides, and have no spines or processes"; Ralfs, 1848, p. 91) has not changed and its discriminatory power diminished. Although widely acknowledged to be polyphyletic, the genus is still adopted to date in its original sense (Lenzenweger, 1999; Brook and Johnson, 2002; Gerrath, 2003; Coesel and Meesters, 2007). Unfortunately, attempts during the 19th century to resolve the taxonomic problems in Cosmarium and establish more "natural" (morphologically uniform) taxonomic entities were unsuccessful (e.g., Nägeli, 1849; de Bary, 1858; Lundell, 1871; Kirchner, 1878; Gay, 1884; Hansgirg, 1888; De Toni, 1889; Raciborski, 1889; Turner, 1892). The novel taxa were based on simple morphological features such as cell and semicell shape, ornamentation of the cell surface, degree of cell constriction, and chloroplast shape that occur in any combination in the genus. In 1954, Teiling established a new genus, Actinotaenium Teil., for taxa with smooth-walled, elongated cells, circular in apical view, and displaying a shallow sinus. Although Actinotaenium is often regarded as a "natural group" (Prescott et al., 1981, p. 1), its members are distinct only in the combination of characters that individually occur in many Cosmarium taxa. Another consequence of the unsatisfactory taxonomic status of Cosmarium is the fact that only one unfinished attempt of a monography of the genus dealing with fewer than half of the described species exists (Krieger and Gerloff, 1962,1965, 1969). The lack of type material and the inaccessibility or vagueness of many original descriptions further complicates a critical assessment of species that are distinguished largely on the basis of the shapes of cells, semicells, and chloroplasts; features of cell wall ornamentation; and zygospore shape. The extent of the variability of these characters is poorly known, and their taxonomic significance has never been assessed within a cladistic framework.
The first tests of the genus concept of Cosmarium with molecular tools confirmed the expected polyphyly of the genus, but this result was based on a very limited taxon sampling, and the phylogeny was affected by long-branched taxa and the limited phylogenetic resolution of the marker used (nuclear-encoded small subunit [SSU] rDNA). Six Cosmarium sequences representing distinct morphotypes within the genus had no relationship to each other; instead, some species formed clades with members of other genera (Gontcharov et al., 2003). Conversely, phylogenetic analyses of two taxa of Cosmarium with the same morphotype, revealed only a distant relationship between them, questioning the validity of morphological characters traditionally used in the taxonomy of the genus but confirming a previously reported relationship between some smooth-celled species of Cosmarium and spine-bearing Staurodesmus Teil. taxa (Gontcharov et al., 2003; Gontcharov and Melkonian, 2005).
The primary goal of this study was to define major monophyletic lineages within the traditional genus Cosmarium, related genera, and the family Desmidiaceae and generate a hypothesis of their phylogenetic relationship. To this end, we sampled more than 120 species, representing major morphotypes of Cosmarium, and its putative relatives, Staurodesmus, Euastrum, Xanthidium, and Actinotaenium. Nuclear-encoded SSU rDNA, noncoding 1506 group I introns, chloroplast large subunit (LSU) rDNA and protein-coding rbcL gene sequences were obtained from these taxa and analyzed individually and in concatenation using different phylogenetic methods. We identified 10 clades in the Desmidiaceae, with the genus Cosmarium distributed over six of these. Furthermore, Cosmarium, Euastrum, Staurodesmus, and Actinotaenium were shown to be polyphyletic. Mapping traditional morphological traits that discriminate these genera on the phylogenetic tree revealed extensive homoplasy, calling into question the current genus concept in the Desmidiaceae.
MATERIALS AND METHODS
Cultures
One hundred twenty-seven strains of Desmidiaceae and Peniaceae used for this study were obtained from different sources (Appendix 1) and grown in modified WARIS-H culture medium (McFadden and Melkonian, 1986) at 15°C with a photon fluence rate of 40 µmol•m–2•s–1 in a 14/10 h light/dark cycle. The taxonomic designation of all strains was verified by light microscopy prior to DNA extraction (Krieger and Gerloff, 1962, 1965, 1969; Prescott et al., 1981; Croasdale and Flint, 1988; Brook and Johnson, 2002; Coesel and Meesters, 2007).
DNA extraction, amplification, and sequencing
After mild ultrasonication to remove mucilage, total genomic DNA was extracted using the Qiagen (Hilden, Germany) DNeasy Plant Mini Kit. Nuclear-encoded (nu) SSU rDNA (including the 1506 group I intron) and chloroplast-encoded (cp) rbcL and LSU rDNA were amplified by polymerase chain reactions (PCR) using published protocols and 5'-biotinylated PCR primers (Marin et al., 1998, 2005; Gontcharov, et al., 2004). PCR products were purified with the Dynabeads M-280 system (Dynal Biotech, Oslo, Norway) and used for bidirectional sequencing reactions (for protocols, see Hoef-Emden et al., 2002). Gels were run on a Li-Cor IR2 DNA sequencer (Li-Cor, Lincoln, Nebraska, USA).
Sequence alignments and tree reconstructions
Sequences were manually aligned using the SeaView program (Galtier et al., 1996). For coding regions of the nu SSU rDNA, cp LSU rDNA, and noncoding 1506 group I introns, the alignment was guided by primary and secondary structure conservation (Bhattacharya et al., 1994, 1996; Wuyts et al., 2000, 2001; Gillespie et al., 2006). All three codon positions of the rbcL gene were used for analyses. The alignments are available at http://srs.ebi.ac.uk/, accessions ALIGN_001252, ALIGN_001253 and ALIGN_001254.
The amount of phylogenetic signal vs. noise in our nu SSU rDNA, rbcL, and cp LSU rDNA data were assessed by plotting the uncorrected against corrected distances determined with the respective model of sequence evolution estimated by the program MODELTEST version 3.06 (Posada and Crandall, 1998). The selected models and model parameters are summarized in Table 1. Also, the measure of skewness (g1-value calculated for 10c000 randomly selected trees in the program PAUP* version 4.0b10; Swofford, 2002) was compared with the empirical threshold values (Hillis and Huelsenbeck, 1992) to verify the nonrandom structuring of the data. To quantify the extent of substitution saturation in data sets, we calculated the Iss statistic for the individual and combined data sets with the program DAMBE (Xia and Xie, 2001).
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). Evolutionary models (for ML and NJ analyses) were selected by the Akaike information criterion in MODELTEST. ML and MP analyses used heuristic searches with a branch-swapping algorithm (tree bisection-reconnection); distances for NJ analyses were calculated by ML. In BI, two parallel MCMC runs were carried out for two million generations sampling every 100 generations for a total of 20000 samples. The first 500–1500 samples were discarded as burn-in, and the remaining samples were analyzed using the sumt command in MrBayes. The robustness of the trees was estimated by bootstrap percentages (BP; Felsenstein, 1985) using 1000 (NJ and MP) or 100 (ML) replications and by posterior probabilities (PP) for BI. BP < 50% and PP < 0.95 were not taken into account. In MP, the stepwise addition option (10 heuristic searches with random taxon input order) was used for each bootstrap replicate. The ML bootstrap used a single heuristic search (starting tree via stepwise addition) per replicate.
Previous molecular phylogenetic studies resolved the families Peniaceae (the genus Penium) and Desmidiaceae as sisters and have shown that they evolved with comparable evolutionary rates unlike the more distantly related, fast-evolving desmid families Gonatozygaceae and Closteriaceae (Besendahl and Bhattacharya, 1999; McCourt et al., 2000; Denboh et al., 2001; Gontcharov et al., 2003). Later Penium was found to not be monophyletic, and only one sublineage was sister to the Desmidiaceae (Gontcharov et al, 2004; Hall et al., 2008). Therefore, only P. margaritaceum and P. spirostriolatum, comprising this distinct clade, were used as an outgroup for the Desmidiaceae in our analyses, and three more species of Penium were regarded as ingroup taxa (see Results).
Combined analyses
For concatenated analyses, partitions were fused and analyzed using a single "concatenated model" with averaged parameters. Before that, models for individual partitions (Table 1), ML topologies, and ML/NJ(ML)/MP bootstrap support (Table 2) were obtained and compared to reveal significant discrepancies. We also assessed incongruence between the data sets by the incongruence length difference (ILD) test (Farris et al., 1994) in PAUP* (partition homogeneity test with 1000 replicates).
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RESULTS
Quality of the molecular data
Apart from the differences in the alignment length, base and substitution frequencies, number of parsimony-informative positions, pattern of substitution distribution (G parameter), and proportion of invariable sites between our data sets, the most complex GTR+I+G model of sequence evolution was identified by the MODELTEST as the best model fitting the data (Table 1). A test of the data sets for substitution saturation (distribution of the uncorrected vs. corrected distances; Fig. 1) revealed a nearly linear correlation in the SSU rDNA data indicating low saturation. The saturation plot of rbcL was somewhat leveled off, suggesting the presence of some saturation that would be expected from the third codon position of this protein-coding gene (Gontcharov et al., 2004). However, according to the Iss statistics, neither of the data sets was saturated (P < 0,001; Table 2).
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) showed that the length distributions were considerably left-skewed, indicating that the alignments were significantly more structured than random data and likely contained a strong phylogenetic signal (Table 1). Noise assessment in the data sets without the outgroup (two Penium species) yielded results identical to those obtained with the complete data sets, suggesting that the outgroup did not interfere with the phylogenetic signal (not shown).
rbcL data set (127 taxa)
ML analyses of 127 rbcL sequences (1339 nt, GTR+I+G model; Table 1) yielded the phylogenetic tree in Fig. 2. Although resolution of internal branches was mostly low, 11 terminal clades that included most of the taxa studied were resolved and designated STD1, STD2, CO1, CO2, CO3, CO4, ARTHR, Euastrum, Micrasterias, omniradiate, and CAP (Fig. 2). Most clades were well supported; only STD2, Euastrum, omniradiate, and CAP attained weak to moderate support (Fig. 2; Table 2). In addition, two lineages containing most of the multicellular (filamentous or colonial) taxa analyzed and some Cosmarium species were recovered, the larger multicellular 2 assemblage, and a long-branched Spondylosium planum/S. secedens pair, multicellular 1. Species of the genus Xanthidium (including Staurastrum tumidum; Gontcharov et al., 2003; Gontcharov and Melkonian, 2005) were also split between several individual branches and one clade. Finally, Haplotaenium minutum formed an unresolved assemblage with two Staurastrum species; however, bootstrap analyses favored monophyly of Staurastrum with weak support (Fig. 2; Table 2).
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Cosmarium was most prominent (26 of 68 species analyzed) in clade CO2. Strain SVCK482, identified as C. undulatum, the type species of the genus (Silva, 1952; Gerrath, 1993), was also a member of this clade. Beside Cosmarium, this species-rich clade accommodated three Actinotaenium and four Euastrum species (Fig. 2). CO2 was further divided into several subclades and some single branches, but their relationships were largely unresolved likely due to low sequence divergence in the clade. Cosmarium species also formed the bulk of the taxa in clade omniradiate, that also included Spondylosium panduriforme and two additional Actinotaenium species (Fig. 2). In the omniradiate clade, the Cosmarium species formed four well-supported subclades whose relationships, however, were unresolved in the rbcL phylogeny (Fig. 2).
Our analyses also revealed well-supported relationships between some Cosmarium and Staurodesmus species. Members of Staurodesmus were recovered in three distinct clades (STD1, STD2, and ARTHR), and two of these clades also contained species of Cosmarium. In both STD2 and ARTHR, Cosmarium species diverged in paraphyletic succession before the Staurodesmus taxa. Only in STD2 clade the derived position of the Staurodesmus species was supported by high bootstrap values (Fig. 2).
Two Cosmarium strains, M 2717 and SVCK 570, both identified as C. punctulatum, were found in two distantly related clades, CO2 and CO3, respectively. Light microscopic examination revealed minor differences in cell dimensions and patterns of the cell wall ornamentation between the strains, which, nevertheless, fitted the rather broad species diagnosis (Prescott et al., 1982). A similar discrepancy between taxonomic designation and phylogenetic position in the tree was observed for Staurodesmus extensus and S. extensus var. joshuae that showed little affinity to each other in clade STD1 (Fig. 2). It is likely that the small difference in cell morphology between these conspecific strains masked the conspicuous genetic distances between the two taxa that should warrant the status of separate species
Concatenated data set (SSU rDNA + rbcL)
To increase phylogenetic resolution and probe the clades established in the rbcL analyses with another marker, we combined SSU rDNA (including 1506 group I introns) and rbcL sequences obtained from the same strain. The taxon sampling was reduced to 97 species in this data set because in some taxa the SSU rRNA gene could not be amplified using various primer combinations (e.g., most members of the CO3 and CO4 clades) or their sequences formed extremely long branches in the SSU rDNA phylogeny (e.g., Cosmarium ovale and Euastrum moebii). Concatenation of SSU rDNA and rbcL sequences resulted in a data set consisting of 3260 nt (Table 1).
Comparison of the SSU rDNA- and rbcL-based topologies for the 97 taxa data set and their bootstrap supports (Table 2) showed general agreement between the markers (results not shown). Only two clades attained somewhat weaker or no support in the SSU rDNA phylogeny compared to the rbcL data set (CO3 and STD2, respectively). Both partitions recovered two nonsupported assemblages, one containing eight of nine Euastrum species, the other the multicellular species and associated Cosmarium taxa (Table 2). Most members of these assemblages had accelerated evolutionary rates in the SSU rRNA gene or in both genes that likely affected bootstrap support for their grouping. Different placement of these unresolved taxa/branches in the SSU rDNA and rbcL trees was responsible for failure of the combined data set to pass the ILD test (P = 0.003). Only when long branches such as C. depressum, Actinotaenium cruciferum, and members of the Euastrum and multicellular assemblages were removed from the data set did it pass the test (P > 0,005). This result suggests that the nuclear and chloroplast data sets are congruent for the data set as a whole. Problems with the ILD test are well known (e.g., Dolphin et al., 2000; Yoder et al., 2001; Darlu and Lecointre, 2002; Dowton and Austin, 2002; Quicke et al., 2007), so failures due to localized cases of incongruence are not surprising (Thornton and DeSalle, 2000). Phylogenetic analyses of the data set without the long-branched taxa mentioned revealed that they have no effect on the support levels of other terminal clades and the general tree topology.
In the combined analyses the clades ARTHR, STD1, CO1, CO2, and CO3 attained 100%BP/1.00 PP in all analyses (Fig. 3; Table 2). The support increased also from weak or moderate to high (>90% BP) for the omniradiate and STD2 clades. The earlier unresolved Xanthidium lineage attained moderate bootstrap values, whereas the two multicellular assemblages remained without support. The more divergent SSU rDNA sequences contributed also to the resolution of the terminal branches within some clades, in particular CO2 (Fig. 3).
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The PCR products obtained by amplification of cp LSU rDNA with primers that bound in the B19 and G20 domains of the gene (nomenclature after De Rijk et al., 2000), varied significantly in length between species. This inconsistency was due to the presence of a single intron in 16 taxa that occurred at four different insertion sites (Fig. 4). BLAST searches indicated that the introns in the cp LSU rDNA of desmids are similar to group IA1, IA3, and IB4 introns from other green algal chloroplasts and likely contain group I homing endonucleases (Turmel et al., 1993, 1995).
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Mapping morphological characters that define genera on the phylogenetic tree
Most of the 10 clades identified in the Desmidiaceae by our molecular phylogenetic analyses, comprised representatives of several desmid genera demonstrating the artificial nature of these taxa and the inadequacy of the morphological features used to define them. As a first step to characterize the new clades, we mapped morphological features of the cell and chloroplast of each species, on the rbcL topology (NJ[ML] bootstrap consensus; Fig. 2) in all clades/assemblages that contain Cosmarium species (Fig. 5). The following morphological characters were computed and mapped: (1) apical view of the cell (circular [omniradiate], elliptical [biradiate] or triangular), (2) ornamentation of the cell wall (smooth-walled, ornamented [granular/spiny] or with [a few] stout spines), (3) width of the isthmus (narrow [<1/2 of the cell width] or broad [>1/2 of the cell width]), and (4) type of the chloroplast (axial or parietal). In addition, distinct features of specific genera, e.g., a lobed cell outline (L), incision of the apical lobe (I), and presence of filaments (F) or colonies (C) were also recorded (Fig. 5).
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, Höftberger and Meindl (1993)) or cell/semicell shape are also not confined to single clades (Fig. 5). Characters are mostly nonlinked and occur together in various combinations. However, the 12 taxa characterized by circular in vertical view cells (and distributed over five clades/assemblages and five genera: Cosmarium, Actinotaenium, Bambusina, Groenbladia, and Spondylosium) all had cells with a wide isthmus, whereas the opposite does not apply (Fig. 5).
Synapomorphies in the SSU rRNA
Mapping morphological characters traditionally used to distinguish desmid genera on the phylogenetic tree clearly demonstrated that these characters cannot be used to circumscribe the clades identified in this study (Fig. 5). To initiate a molecular circumscription of the clades, we searched for the presence of molecular signatures, i.e., nonhomoplasious synapomorphies (NHS; according to Marin et al., 2003) in the SSU rRNA. We restricted the analysis to clades that contained a large number of Cosmarium species. Screening of the secondary structure-based alignment revealed several substitutions in the SSU rRNA that characterize the omniradiate and CO2 clades (Fig. 6).
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; nucleotides 30 (G
A) and 36 (AU); Fig. 5A), whereas all other members of the Desmidiaceae (and also the Desmidiales) reveal the plesiomorphic state (G, A) at these positions. The two substitutions in the clade omniradiate thus represent nonhomoplasious synapomorphies of this clade.
A compensatory base change (A-UG-C) in base pair 38 of Helix 49 (bp 47 of H1399; according to Gillespie et al. (2006)) differentiated all members of clade CO2 from the rest of the family (Fig. 6B). The A-U pair is conserved across the family and the order Desmidiales making the CBC in clade CO2 an NHS for this clade.
DISCUSSION
In this study, two ribosomal genes (nu SSU rDNA and cp LSU rDNA), the protein-coding rbcL, and the noncoding 1506 group I intron of the SSU rDNA were used to assess phylogenetic relationships among species of the Desmidiaceae (Zygnematophyceae, Streptophyta). The phylogenetic structure of the most species-rich genus in the family, Cosmarium, and its putative relatives, Actinotaenium, Euastrum, Staurodesmus, and Xanthidium was the major focus of our analyses. A large data set and extensive taxon sampling revealed 10 distinct clades in the Desmidiaceae.
Most of the clades established here were well supported (Figs. 2, 3). Comparison of individual topologies and their support values showed that the different markers used contained a similar phylogenetic signal and yielded largely congruent phylogenetic relationships between desmid taxa (Table 2). In general, the rbcL gene provided better support for most of the clades but was less informative in resolving relationships among species within clades (e.g., CO2), whereas divergence of the nu SSU rDNA and the 1506 group I intron was sometimes too high to recover a clade (e.g., CO3, STD2; Table 2). Differences in evolutionary rates between markers were particularly pronounced in clade CO2, in which divergence of rbcL at the species level was particularly low compared to SSU rDNA (compare Figs. 2 and 3). The concatenated analyses of SSU rDNA and rbcL resulted in increased support for clades compared to phylogenetic analyses of individual genes (Table 2). Relationships among clades, however, remained largely unresolved even when fast and slow evolving sequences were combined (Fig. 4). In the latter case, though, taxon sampling was smaller, and phylogenetic resolution may be much better, when taxon sampling is increased to the same level as in the concatenated SSU rDNA and rbcL analyses.
Phylogeny of Cosmarium
Not surprisingly, the results presented here confirmed the long-anticipated artificial nature of the genus (e.g., West and West, 1905, 1908; Fritsch, 1953, Krieger and Gerloff, 1962; Prescott et al., 1981). Early molecular phylogenetic studies were indicative in this respect, but the limited taxon sampling, presence of long-branched taxa, and the relatively low phylogenetic signal of the markers made the conclusions preliminary (Lee, 2001; Nam and Lee, 2001; Gontcharov et al., 2003; Moon and Lee, 2003; Gontcharov and Melkonian, 2005; Hall et al., 2008). In the current study, 45 (68 in the rbcL analyses) species/strains of Cosmarium were distributed over six clades, one nonsupported assemblage (multicellular), and a long-branched basal lineage (Fig. 3). Four of the six clades also included species of other desmid genera. In the rbcL analyses with 127 taxa, additional species of Cosmarium associated with other clades (Euastrum, Micrasterias) or formed a novel clade (CO4).
One reason for the rampant polyphyly of the genus Cosmarium lies in its vague diagnosis that lacks any distinct synapomorphic or even typological characters. When describing Cosmarium, Ralfs (1848) stressed the lack of some characters typical for other genera (e.g., incision at the apex, lateral lobes, spines or processes), but he did not present a single character or a combination of features that is unique to the genus. We have shown that the morphology of the genus Cosmarium as defined by Ralfs is one of the most common in the family and has likely arisen independently in several lineages (Fig. 5). Its evolutionary status (ancestral vs. derived) could be also different. Noteworthy in this respect is the close relationship between smooth-walled Cosmarium and spine-bearing Staurodesmus taxa in their common clades. It is very likely that in the STD2 clade, a triradiate cell with a stout spine at each angle, typical for the genus Staurodesmus, was derived from a smooth-walled biradiate "Cosmarium" taxon (Figs. 2–4
), whereas in the ARTHR clade spines may have been lost in some members, in which case the "Cosmarium"-type morphology may be the derived state (Fig. 2).
The presence of several morphotypes in most Cosmarium-containing clades demonstrates homoplasy of morphological features thought to be important for desmid taxonomy. Traditionally, semicell shape, degree of radiation (biradiate vs. omniradiate), cell wall features (smooth vs. ornamented), and chloroplast morphology have been used to distinguish intrageneric taxa in Cosmarium (de Bary, 1858; Lundell, 1871; Hansgirg, 1888; De Toni, 1889; Raciborski, 1889; Turner, 1892; West and West, 1905; Hirano, 1959a, 1959b; Bourrelly, 1966). However, our study revealed that each of these morphological features has a mosaic distribution in the tree and does not explicitly characterize a specific clade (Fig. 5). Our phylogenies revealed a patchy distribution of the omniradiate morphotype over our trees in several unrelated clades (Figs. 1–4). Moreover, we found that some species that differ in their degree of radiation have nearly identical sequences (e.g., Cosmarium portianum [biradiate]/C. bisphaericum [omniradiate] in the omniradiate clade; C. lundelli and C. pachydermum [biradiate]/Actinotaenium turgidum [omniradiate] in CO2 (Fig. 2), and C. connatum [biradiate]/C. pseudoconnatum [pseudo-omniradiate] in CO3, Fig. 2). The latter case is a good example for the likely derived nature of the omniradiate morphotype at least in some Cosmarium strains. In conclusion, the degree of radiation is a homoplasious feature in the family Desmidiaceae and its taxonomic significance has been largely overestimated.
Morphological heterogeneity of all clades containing Cosmarium taxa (Fig. 5) and lack of information on other phenotypical features currently does not allow conclusions to be drawn on the morphological characters that may unite their members or, more importantly, that differentiate the clades. The characters traditionally used to define taxa above the species level are not suitable for this purpose, in particular features of cell wall ornamentation and chloroplast morphology (Fig. 5). Many Cosmarium species have distinct patterns of granules, spines, or warts on the cell surface, and it is not clear whether these types and patterns are homologous and what features would represent character states here. Also very little is known about such a distinct characteristic of the desmid cell wall as the pores, their types, functions, significance of different distribution patterns. Preliminary studies already revealed quite a diversity of pore patterns (Couté and Tell, 1981; Neuhaus and Kiermayer, 1981; Coesel, 1984; Gontcharov et al., 2002), however, their possible suitability for desmid taxonomy above the species level is virtually unknown.
Our knowledge about the diversity of chloroplast structures in desmids is also very limited and has not been extended significantly beyond the careful early studies of Lütkemüller (1893, 1895) and Carter (1919a, 1919b, 1920a, 1920b). Teiling (1952) typified chloroplast shapes of desmids and presented a scenario of their hypothetical evolution correlating it with cell morphology, mostly radiation, but chloroplast morphology is still poorly known in many Cosmarium species.
Type species of Cosmarium
Affiliation of a type species is crucial for the identity of a genus. In Cosmarium this issue is complicated by the uncertainty with the designation of the type species. The Index Nominum Genericorum (ING; http://botany.si.edu/ing/) recognizes C. margaritiferum as the type although in the earlier version of ING C. undulatum had been suggested (Silva, 1952). The designation of C. margaritiferum as the type is credited to Nägeli (1849, p. 114). However, Nägeli considered Cosmarium as the subgenus of Euastrum and referred to E. margaritiferum Ehr. Ehrenberg (1835) regarded his alga identical with Ursinella margaritifera Turpin (1820), so the correct citation should be Euastrum margaritiferum (Turpin) Ehr. Because the publications by Turpin and Ehrenberg were published before the starting point of desmid taxonomy (Ralfs, 1848), Nägeli’s designation of the type species is invalid (ICBN, Art. 7.7). Obviously, Nägeli was not familiar with Ralfs’s publication at that time, and his typification had no relation to the genus Cosmarium Corda ex Ralfs.
Moreover, the alga described by Turpin is not identifiable and obviously not identical to two or likely three species illustrated by Ralfs under the name C. margaritiferum (1848, tables XVI, XXXIII; figs. 2a–d, 3a, b). In contrast, the choice of C. undulatum as the type of Cosmarium was prompted by the fact that it is the most clearly known of the species included in the genus by Corda (Silva, 1952). We agree with Silva that Nägeli’s typification should be rejected because it is based on an invalidly published name, and following Silva (1952), we regard C. undulatum as the type species of the genus Cosmarium.
Our analyses placed C. undulatum in the CO2 clade (Figs. 2–4) linking the genus name to this clade. The CO2 clade is a member of a large, weakly supported assemblage that unites two more clades, CO3 and the genus Xanthidium (Fig. 4). The species richness of the CO2 and CO3 clades and the diversity of morphotypes in the clades suggest that these lineages will accommodate the majority of the existing Cosmarium species.
Cosmarium species from other clades that have no affinity to CO2/CO3 should in the future either be classified together with representatives of the genera in their clades or recognized as new genera.
The omniradiate clade is phylogenetically most distant from the rest of the genus Cosmarium and is likely one of the basal branches in the Desmidiaceae (Gontcharov et al., 2003, 2004; this study). Like other clades containing Cosmarium spp., this clade includes a number of morphotypes distinct in cell/semicell shape, chloroplast morphology, and cell wall ornamentation (Fig. 5). Membership of two omniradiate Actinotaenium (formerly Penium) species with elongated, weakly constricted cells and Spondylosium (formerly Cosmarium) panduriforme forming short filaments further complicates the circumscription of the clade. However, we discovered several nonhomoplasious molecular synapomorphies (NHS; for definition, see Marin et al., 2003) in the SSU rDNA that discriminate members of the omniradiate and CO2 clades from all other taxa in the family Desmidiaceae and that might be useful in future taxonomic revisions (Fig. 6).
The taxonomic affinity of Cosmarium species belonging to the clades ARTHR and STD2, and the nonsupported assemblage multicellular, is not yet clear. The distinctness of the ARTHR and STD2 clades from STD1 that contains the type species of the genus Staurodesmus, Std. triangularis (Gontcharov and Melkonian, 2005), received further support with the extended taxon sampling and the larger data sets in this study. The current lack of synapomorphic phenotypic characters for these clades calls for further phenotypic study before taxonomic conclusions should be made.
Within the assemblage of multicellular desmids, the Cosmarium species either showed affinity to the colonial Heimansia (C. sinostegos), the filamentous Spondylosium pulchellum (C. regnellii) or formed an independent lineage (C. difficile, C. dilatatum; Figs. 2, 3). Most taxa comprising this cluster are characterized by fast evolutionary rates in all three genes and were long-branched in the individual as well as the concatenated analyses (Figs. 2–4). One may hypothesize that the morphological diversification of Desmidiaceae from a unicellular to a multicellular life habit, a consequence of a peculiar cell division mode (Gerrath, 1970, 1973; Krupp and Lang, 1985a, , 1985b), may have been accompanied by accelerated rates of evolution in several genes. These long branches likely affected topologies and may have been responsible for lack of support for this assemblage in almost all data sets (Table 2).
Identification of monophyletic entities consisting of species of the traditional genus Cosmarium should stimulate the search for synapomorphic phenotypic characters distinguishing these lineages.
Euastrum
The current study substantiated the polyphyletic nature of the traditional genus Euastrum (Hall et al., 2008) assigning its species to several clades (see Results). Most Euastrum taxa studied formed an assemblage that received low or no support with the different data sets (Figs. 2–4; Table 2). The relatively high divergence of Euastrum sequences may have been responsible for the uncertain status of this assemblage, so that it requires further study with a more comprehensive taxon sampling. It appears that the Euastrum assemblage is split into two morphologically distinct lineages. One lineage comprises large-celled taxa (>50–60 µm long) with smooth cell walls and a more or less regular porous cell surface often provided with several large facial protrusions and excavations, e.g., E. oblongum and E. affine. In these species, the polar lobe has a narrow vertical incision with parallel margins. A similar morphology is typical for a number of other Euastrum taxa, and their affiliation with this lineage is anticipated (Gontcharov et al., 2003).
The second lineage contains relatively small-celled (typically <50 µm long) and variously ornamented taxa. Their apical incision is less pronounced and often appears as a V-shaped invagination.
Euastrum taxa placed outside the Euastrum assemblage are large-celled, with granules or spines arranged in specific patterns. Their semicells are divided into one or two basal lobes and a polar lobe, as is typical for the genus, but the polar lobe is concave to nearly straight and lacks an apical incision. Species with these characteristics were distributed between two putatively related clades consisting mostly of Cosmarium taxa, i.e., CO2 and CO4. In most cases, their phylogenetic position could be accessed with rbcL only; therefore, we consider their position as tentative. However, high support values for the clades that include Euastrum substellatum, E. verrucosum, E. germanicum, E. spinulosum, E. moebii, and E. prowsei (Fig. 2) suggest that these species are distinct from other Euastrum taxa.
Actinotaenium
This study confirmed the polyphyletic status of the genus Actinotaenium as suggested previously (Gontcharov et al., 2003). In our analyses, six Actinotaenium species were distributed among three only distantly related lineages including the genus Penium (e.g., A. cruciferum; Figs. 2–4). Their position in the tree highlights the homoplasious nature of the characters used to define the genus (elongated, omniradiate cells with two stellate [A. cucurbita, A. cf. wollei] or numerous parietal [A. turgidum] chloroplasts and smooth cell walls (Teiling, 1954).
The status of phenotypic characters in A. phymatosporum and A. silvae-nigrae, members of the omniradiate clade, and particularly A. cruciferum, having a weak affinity to one of the Penium clades (Figs. 2–4), is unclear. The morphology of the former species (elongate-elliptical, omniradiate cells with a very weak median constriction and axial chloroplasts) is similar to that of the genus Penium in which they were placed until relatively recently (Kouwets and Coesel, 1984), and these characteristics may be pesiomorphic.
The affiliation of the type species of the genus, A. curtum, remains currently unknown (Gontcharov et al., 2003), and taxonomic revision of Actinotaenium must await clarification of its phylogenetic position.
Conclusions
Phylogenetic analyses based on several genes from two genomes and extensive taxon sampling confirmed the anticipated artificial nature of the desmid genera Cosmarium, Euastrum, Staurodesmus, and Actinotaenium. Our results highlight the inadequacy of morphological features such as cell and semicell shape, degree of radiation, chloroplast morphology, and cell wall ornamentation, traditionally used to discriminate these genera and revealed numerous cases of homoplasy in distantly related lineages. Many clades established during this study deserve to be recognized as new genera but cannot be formally described in the framework of the current morphology-based taxonomic concept of desmids because either their phenotypical features are insufficiently known or they do not differ from those of other clades and genera. Molecular signatures may be used to circumscribe the new taxa in the future, but their utility is not straightforward in a group that accounts for more than 2000 species. Although many desmid strains are available in culture collections, the strains still represent only a minute fraction of the taxa described, the unknown diversity of desmids that may exist in the environment notwithstanding. Many more isolates of desmids are needed as well as refined analyses of morphological traits and additional molecular markers. At the current state of knowledge, desmid systematics has arrived at the beginning.
Appendix 1. Strain information and EMBL/GenBank accession numbers for taxa used in this study. A dash (—) indicates the region was not sampled. New sequences are boldfaced. ACOI = Coimbra Collection of Algae, University of Coimbra, Portugal (http://www1.ci.uc.pt/botanica/ACOI.htm); ASW = Sammlung von Algen-Kulturen, University of Vienna, Austria (Kusel-Fetzmann and Schagerl, 1992); CCAC = Culture Collection of Algae at the University of Cologne, Germany (http://www.ccac.uni-koeln.de); M = Culture Collection Melkonian, Botanical Institute, University of Cologne, Germany (strains available upon request); NIES = Microbial Culture Collection at National Institute for Environmental Studies, Tsukuba, Japan (http://www.nies.go.jp/biology/mcc/home.htm); SAG = Sammlung von Algenkulturen, University of Göttingen, Germany (http://www.epsag.uni-goettingen.de/html/sag.html); SVCK = Sammlung von Conjugaten-Kulturen, University of Hamburg, Germany (http://www.biologie.uni-hamburg.de/b-online/d44_1/44_1.htm). Taxon names in parentheses correspond to those used in the culture collection catalogue.
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FOOTNOTES
1 The authors thank P. C. Silva and F. A. C. Kouwets for discussion on the type species of Cosmarium. This work was supported by DFG grant ME-658/26-1. 
2 Author for correspondence (e-mail: gontcharov{at}biosoil.ru); permanent address: Institute of Biology and Soil Science, 690022, Vladivostok-22, Russia.
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50%) and posterior probabilities (PP) (
