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rotist, Vol. 162, 253–267, April 2011ttp://www.elsevier.de/protisublished online date 23 October 2010

RIGINAL PAPER

Study of Conflict between Molecular Phylogenynd Taxonomy in the Desmidiaceae (Streptophyta,iridiplantae): Analyses of 291 rbcL Sequences

ndrey A. Gontcharova,b,1, and Michael Melkonianb

Institute of Biology and Soil Science, 690022, Vladivostok-22, RussiaBiowissenschaftliches Zentrum, Universität zu Köln, Zülpicher Str. 47b, D-50674 Köln, Germany

ubmitted February 15, 2010; Accepted July 9, 2010onitoring Editor: David Moreira

olecular phylogenetic analyses of 93 new and 198 non-redundant GenBank rbcL sequences of the fam-ly Desmidiaceae (Zygnematophyceae, Streptophyta) established 22 mostly highly supported clades,n addition to four non-supported lineages and eight single-taxon branches within the family. Nineovel clades and single-taxon branches were identified, suggesting that current taxon sampling hasot reached saturation in the family. The highly polyphyletic nature of most desmid genera corrobo-ated in this study using a large taxon set, calls for re-evaluation of the genus concept in the familyesmidiaceae that traditionally relied on features of cell morphology. Molecular phylogenetic data havehown that these morphological characters are highly homoplastic or plesiomorphic and thus cannote used to delineate genera. The dramatic discrepancy between the currently practised systematic

reatment of the family and the composition of the clades based on sequence comparisons requiresmendation of almost all existing genera and description of a larger number of novel genera. The clades

dentified during this study provide a framework for the future emendation/description of genera in theesmidiaceae.2010 Elsevier GmbH. All rights reserved.

ey words: clades; Desmidiaceae; phylogeny; polyphyly; rbcL; taxonomy.

ntroduction

he family Desmidiaceae (Zygnematophyceae) ishe most species-rich and taxonomically com-lex group of streptophyte green algae, the latterepresenting those green algae from which thembryophyte land plants evolved about 470–450a ago (Becker and Marin 2009). The family is dis-

inct in cell wall architecture (each cell consists ofwo almost symmetrical overlapping halves (semi-ells) with a system of complex pores penetrating

Corresponding author; fax +7 4232 310 193-mail gontcharov@biosoil.ru (A.A. Gontcharov).

the secondary cell wall, the primary cell wall hav-ing been shed after cell division) and is regardedas the most derived in the order Desmidiales andthe whole class Zygnematophyceae (Brook 1981;Lütkemüller 1902; Mix 1972; West and West 1904).Molecular phylogenetic studies have confirmed thisassertion and resolved the Desmidiaceae as the‘crown’ lineage of the class preceded by the para-phyletic Peniaceae (Gontcharov et al. 2003, 2004;Hall et al. 2008a; McCourt et al. 2000).

In the family Desmidiaceae about 2,500 specieshave been validly described (Gerrath 1993). Manyof these species, however, contain additionalinfraspecific taxa (subspecies, varieties and forms)

2010 Elsevier GmbH. All rights reserved.doi:10.1016/j.protis.2010.08.003

254 A.A. Gontcharov, M. Melkonian

that are either doubtful or may represent distinct(morpho)species (Kouwets 2008); the real taxo-nomic diversity of the family is thus not knownwith any certainty. The Desmidiaceae comprisesc. 30–33 genera, most of which were describedby Ralfs (1848) and his contemporaries (Bailey1851; Brébisson 1856; Kützing 1849; Nägeli 1849;Nordstedt 1877; Wallich 1860) during the mid-19th century. The genera established by theseearly scholars covered the overall morphologicaldiversity in the family and additional genera wereonly added through critical reassessment of knownmorphological traits (Bando 1988; Bourrelly 1964;Coesel 1993; Teiling 1948, 1952, 1954) or floris-tic surveys of tropical regions (Comperè 1976;Gauthier-Lièvre 1958; Gontcharov and Watanabe1999; Grönblad 1954; Iyengar and Ramanathan1942; Scott and Prescott 1956; West and West1897). Features of cell morphology such as celloutline in side and apical views, cell organiza-tional level (unicellular, filamentous or colonial),presence/absence of cell processes, lobes andincisions, cell wall ornamentation (granules, verru-cae) and rarely chloroplast shape were the majorfeatures used to distinguish genera in the Desmidi-aceae.

Species are unevenly distributed among desmidgenera. Particularly species-rich are two genera,Cosmarium (more than 1000 spp) and Stauras-trum (c. 800 spp), comprising two-thirds of the totalnumber of species in the family. They are followedby Euastrum (>260 spp), Xanthidium (>110 spp),Staurodesmus (>100 spp), and Micrasterias (c. 60spp), leaving less than 200 species in the remainingc. 25 genera. Early in the 20th century it was recog-nized that the ever increasing number of species inthe family Desmidiaceae had blurred the morpho-logical boundaries between many of the traditionalgenera and that the genus concept required revi-sion (West and West 1905, 1912). Several attemptswere made to rectify this situation by dividingthe most species-rich genera into smaller entities(Gay 1884; Kirchner 1878; Nägeli 1849; Palamar-Mordvintseva 1976, 1982; Turner 1892), However,only the proposals of the Swedish desmidiolo-gist Teiling (Teiling 1948, 1954), who separatedsome taxa of Cosmarium and Staurastrum intothe new genera Actinotaenium and Staurodesmusrespectively, gained recognition by most of the tax-onomic experts in the field (Brook and Johnson2002; Coesel and Meesters 2007; Croasdale andFlint 1988; Croasdale et al. 1994; Gerrath 2003;Lenzenweger 1997; Prescott et al. 1981, 1982).All other desmid genera essentially remained unal-tered since their description (Ralfs 1848).

Recent assessments of the genus concept withmolecular tools confirmed the polyphyletic natureof almost all traditional genera in the Desmidiaceaeand highlighted the problem of their taxonomic cir-cumscription (Gontcharov et al. 2003; Gontcharovand Melkonian 2005, 2008; Hall et al. 2008a,b).Species of most genera were scattered over sev-eral clades and such clades usually contained alsospecies assigned to other genera. This rampantpolyphyly occurred both in species-rich as wellas species-poor genera, and in genera with mor-phologically more or less uniform species (e.g.Spondylosium). The most complex pattern of phylo-genetic relationships was revealed in the traditionalgenus Cosmarium with its species being distributedamong 11 mostly well-supported clades. Sevenof these clades also included representativesof Actinotaenium, Staurodesmus, Euastrum andother genera (Gontcharov and Melkonian 2008).Overall, comparison of 127 rbcL sequences repre-senting 14 traditional genera recovered 17 mostlypolygeneric clades, non-supported lineages andsingle-taxon branches in the family Desmidiaceae.Thus, the results of the molecular phylogeneticstudies suggested that the number of clades in theDesmidiaceae may exceed the number of generacurrently recognized in the family. However, pre-vious studies were mostly focused on resolvingphylogenetic relationships above the family levelor were confined to a particular genus or a groupof putatively related genera while a global analysisof the molecular phylogeny of the family Desmidi-aceae at the genus level is still lacking. Moreover,it is not clear what criteria should guide taxonsampling and what minimal number of sequenceswould be required for such an analysis becauseneither traditional taxonomy nor the known phe-notypic characters obviously reflect phylogeneticrelationships among species in the Desmidiaceae(Gontcharov and Melkonian 2008).

To test whether additional taxon (sequence) sam-pling would lead to the discovery of novel cladesor whether the previous sampling effort had beensufficient to identify clade diversity in the fam-ily, we more than doubled the number of taxa(sequences) analyzed. A data set of 291 rbcLsequences was generated and their molecular phy-logeny compared with that previously obtainedusing a smaller taxon set (127 rbcL sequences;Gontcharov and Melkonian 2008). Selection of themolecular marker was guided by the availability ofrbcL sequences in the data bases, easy amplifi-cation, sequencing and alignment procedures andsufficient phylogenetic signal above the specieslevel (Källersjö et al. 1998), although phyloge-

rbcL Phylogeny of Desmidiaceae (Streptophyta) 255

netic resolution at deeper nodes is clearly limitedusing a single, organellar gene. We resolved 34clades, non-supported lineages and single-taxonbranches in the family Desmidiaceae (9 of theseare novel), a clade representing a monophyleticgroup of sequences/strains/taxa above the level ofa morphospecies (i.e. a genus, family, etc.).

Results

Sequence Sampling

Two hundred ninety one (198 from Genbank)non-redundant (>3 positions difference) sequencesrepresenting 23 traditional desmid genera wereincluded in our data set. In this alignment the genusCosmarium accounted for 130 sequences, Stau-rodesmus – 30, Staurastrum and Euastrum – 22each, Actinotaenium – 19, and Xanthidium – 8sequences. In addition, ten sequences represent-ing two paraphyletic lineages of the genus Penium(Peniaceae), the closest relative of the Desmidi-aceae (Gontcharov et al. 2003, 2004; Gontcharovand Melkonian 2008; Hall et al. 2008a) wereincluded in the data set. Of these only the mostdistant Penium margaritaceum (4 sequences) andP. spirostriolatum (2 sequences) were selected asoutgroup.

Comparison of sequences obtained from mul-tiple isolates of several distinct morphospecies(e. g. Cosmarium amoenum, C. elegantissimum,C. pseudopyramidatum, Actinotaenium cucurbita,Pleurotaenium trabecula, etc.) has shown thatthese strains differ from each other in more than4 substitutions. Therefore we regard our limit inrbcL sequence divergence (>3 positions difference)as sufficient to discriminate closely related desmidstrains.

Quality of the Molecular Data

The most complex GTR+I+� model of sequenceevolution was identified by Modeltest as the bestmodel fitting the data. A test of the data set forsubstitution saturation (distribution of the uncor-rected vs. corrected distances; results not shown)revealed a somewhat leveled off correlation indicat-ing the presence of some saturation which wouldbe expected from the third codon position of thisprotein-coding gene (Gontcharov et al. 2004).

Comparison of the skewness of the tree lengthdistribution (g1 value; results not shown) of randomtrees of our data set with the empirical threshold val-ues (Hillis and Huelsenbeck 1992) demonstratedthat the length distributions were considerably

left-skewed, indicating that the alignment was sig-nificantly more structured than random data andlikely contained a strong phylogenetic signal. Noiseassessment in the data sets without the outgroup(two Penium species) yielded results identical tothose obtained with the complete data sets, sug-gesting that the outgroup did not interfere with thephylogenetic signal (not shown).

Phylogeny

ML analyses of our data set (291 sequences,1339 nt) produced the phylogenetic tree shown inFigures 1–3 .

Twenty two terminal clades that included mostof the sequences analyzed were resolved. Thesewere the clades “omniradiate”, Haplotaenium,STD1-4, CO1-3, CO5, Cosmarium pseudopyrami-datum, ARTHR, Euastrum1, Euastrum2, Euastrumpectinatum, Tetmemorus, Pleurotaenium, Actino-taenium cucurbita, Actinotaenium curtum, multicel-lular1, multicellular2 and CAP (Fig. 1). Most cladesof the tree were well-supported (>95%BP, 0.97PP;Table 1), only the clades “omniradiate”, multicellu-lar2, STD2, and CAP attained weak to moderatebootstrap support (50–85% BP).

Four Cosmarium and two Euastrum sequencesformed a paraphyletic lineage CO4 (Fig. 3) which,however, was resolved as a clade with mod-erate support in NJ and MP (64% and 88%BP, respectively). Similarly, the paraphyletic lin-eage Xanthidium (8 sequences) attained moderatesupport only in NJ and MP (64–81% BP). Thenon-supported lineage Micrasterias comprised rep-resentatives of this genus as well as Cosmariumralfsii, Staurodesmus dickei and Triploceras gracile.Finally, an assemblage containing 20 out of 22species of the genus Staurastrum was resolvedwithout support. Only S. turgidum (Xanthidiumlineage) and S. orbiculare (clade STD2) wereexcluded from the assemblage Staurastrum withstrong support (Figs 1, 2).

Eight species, Docidium undulatum, Stau-rodesmus bulnheimii, Cosmarium mesikommerii,C. bioculatum M3032, C. decedens, C. ovale, Xan-thidium armatum and Phymatodocis nordstedtianahad affinity to none of the clades or non-supportedlineages and branched individually (single-taxonbranches).

The branching pattern among the clades andnon-supported lineages remained largely unre-solved. Only the basal position of the clade CAPand of Phymatodocis attained weak support bybootstrap percentages (56–68%BP; Fig. 1).

256 A.A. Gontcharov, M. Melkonian

Micrasterias

omniradiate

multicellular2

Xanthidium

Staurastrum

Euastrum2

Euastrum1

ARTHR

STD1

STD2

CO1

CO2

CO3

CAP

PleurotaeniumSTD3

STD4Haplotaenium

CO4

multicellular1

54/-/1.00

-/-/1.00

100/99/1.00

50/-/1.00

100/99/1.00

98/97/1.00

97/91/1.00

-/-/0.98

85/64/0.97

54/-/0.97

99/99/1.00

67/59/1.00

96/87/1.00

78/76/-

67/60/-

68/56/-

56/51/-

Phymatodocis nordstedtiana

Euastrum pectinatum

TetmemorusCosmarium pseudopyramidatumActinotaenium cucurbita

57/-/-

97/73/1.00

92/84/1.00

outgroup (Pen. margaritaceum/spirostriolatum)

Euastrumassemblage

CO5

Actinotaenium curtum

STD2assemblage

0.005substitutions/site

rbcL Phylogeny of Desmidiaceae (Streptophyta) 257

Sequences were unevenly distributed amongthe clades. Most clades included 10 or moresequences, generally corresponding to distinctmorphological species of the desmids. The cladesCO2 (44 sequences from 3 traditional genera),multicellular2 (31 sequences, 8 genera), “omnira-diate” (26 sequences, 3 genera), and CO3 (25sequences, 2 genera) were the most species-rich. These large clades were further split intoseveral well-supported subclades (Figs 2, 3).Some clades, namely Haplotaenium, Tetmemorus,STD3, STD4, Cosmarium pseudopyramidatum,Euastrum pectinatum, Actinotaenium cucrbita, andActinotaenium curtum comprised only two tofour very similar sequences, mostly represent-ing the same or closely related morphospecies(Figs 1–3).

Members of the best represented genus Cos-marium (130 sequences, >10% of the totalspecies number) were distributed among 12 clades(“omniradiate”, ARTHR, Euastrum1, Cosmariumpseudopyramidatum, multicellular1, multicellular2,CO1-CO3, CO5, STD2, and CAP) and two non-supported lineages (Micrasterias and CO4). Exceptfor CO5 and C. pseudopyramidatum, none ofthese clades consisted exclusively of Cosmar-ium species. Most frequently Cosmarium specieswere found together with species of Actinotae-nium (clades “omniradiate”, CO2, CO3, and CAP),Euastrum (Euastrum1, CO4 and CO2), or Stau-rodesmus (ARTHR and STD2). In the clades“omniradiate”, STD2, CO2, and CO3 as well asin the lineage CO4, Cosmarium taxa representedthe majority of their members, whereas in theclade ARTHR, species of Staurodesmus and Cos-marium were nearly equally represented. Theclade Euastrum1 accommodated three distinctivemorphospecies of Cosmarium and the lineageMicrasterias and clade CAP included a single Cos-marium species each, C. ralfsii and C. tinctum,respectively (Figs 2, 3).

Species of the genus Staurodesmus were dis-tributed over six well-supported clades of the tree,STD1-STD4, ARTHR and CO1 (Figs 1–3). Threeof these clades (ARTHR, STD2, and CO1) alsoincluded Cosmarium spp, and clade STD1 accom-modated Xanthidium octocorne (Fig. 2). Whereas

in clade ARTHR some Staurodesmus and Cosmar-ium spp. formed a subclade in which the speciesof both genera mixed and could not be sepa-rated, the six sequences of Staurodesmus fromclade STD2 constituted a well-defined subcladeexcluding Cosmarium spp. (Fig. 2). A single Stau-rodesmus species, S. clepsidra var. sibiricum, wasresolved as a basal divergence in the clade CO1(>95% BP, 1.00 PP; Fig. 3).

Desmids of multicellular organization (9 tradi-tional genera) formed two relatively large clades,multicellular1 and multicellular2 that containedmost of the long-branch sequences in the data set.Along with filamentous and colonial forms theseclades also included Cosmarium species, mostlyas basal divergences (Figs 2, 3). The species-rich clade multicellular2 (31 sequences; 51–56%BP) contained several well-supported subclades(Fig. 2). In two of these subclades, filamentous andunicellular forms represented well-supported sis-ter groups (Heimansia pusilla with C. sinostegosand Spondylosium pulchellum with C. regnelli/C.norimbergense, respectively). In the clade multicel-lular1 (11 sequences; >97% BP, 1.00PP) a singleCosmarium representative, C. regnellii (SVCK465),was resolved as a sister to a subclade (>95%BP, 1.00 PP) comprising the colonial Cosmo-cladium perissum, the filamentous Spondylosiumtetragonum, and several strains of Teilingia granu-lata (Fig. 3).

Thirty nine morphospecies were representedin our data set by more than one sequence(Appendix). Noteworthy, multiple sequences of lessthan a half of these species (17) formed mono-phyletic lineages. Most of these were desmidswith distinctive morphological features that madespecies identification unequivocal (e. g. Cosmar-ium amoenum, C. elegantissimum, Cosmocladiumperissum, Euastrum pectinatum, Penium spirostri-olatum). More often, sequences originating fromdifferent strains and assigned to the same mor-phospecies were either distantly placed within aclade (e.g. C. contractum, Staurastrum punctula-tum, Staurodesmus convergens, C. blyttii, Teilingiagranulata) or scattered over different clades of thetree (e.g. C. laeve, C. subprotumidum, C. subtu-midum).

➛

Figure 1. Overview of phylogeny of desmids (Desmidiaceae, Zygnematophyceae) based on comparisons of291 rbcL sequences (1339 nt). The tree topology was inferred by maximum likelihood (ML) using the GTR+I+�model. Penium margaritaceum and P. spirostriolatum were used as outgroup. Support [bootstrap percentages(BP) ≥ 50% and posterior probabilities (PP) ≥ 0.95: NJ(ML)/MP/BI] is shown only for major clades and basalbranches. Branches with 100% BP in all methods and 1.00 PP are shown bold-face. For clade names seeResults.

258 A.A. Gontcharov, M. Melkonian

M1172 AM911322SVCK14 FN432054 Actinotaenium cucurbitinum ACOI901 EF371279

C. elegantissimumM1887 AM911271ACOI345 FN432062

C. debaryi SVCK454 AY964173

C. quadratum SVCK10 FN432078C. subcucumis ACOI103 AM911312

C. isthmium SVCK229 AY964169C. bisphaericum SVCK436 AY964172

C. portianum M2560 AM911273 Actinotaenium phymatosporum M1368 AM911233A. silvae-nigrae SVCK295 AM911234

A. inconspicuum M3022 FN432048Actinotaenium sp.2 FN432053

Staurodesmus brevispinus JH0180 EF371349St. gladiosum JH0132 EF371347

St. polytrichum JH0015 EF371344

St. punctulatum SAG679.1 FN432117St. subgemmulatum AF428112

St. polymorphum JH0053 EF371345St. punctulatum AF425771

St. margaritaceum AF425774

St. sebaldi M1129 AM911332St. tetracerum UTCC348 EF371282

St. pseudosuecicum JH0010 EF371342St. galeatum AF428113

St. pingue UTEX1606 AF203506St. crenulatum AF428111

St. natator AF428110St. quadricornutum AF428114

St. quadricornutum AF426168St. leptocladum M998 FN432116

St. arctiscon JH0014 EF371343St. lunatum SVCK15 AJ553971

Micrasterias furcata JH0064 EF371313Cosmarium ralfsii SVCK300 AM911324

Std. dickiei SVCK38 FN432123M. rotata UTEX1941 AF203500

M. foliacea NIES297 EF371311Triploceras gracile SAG24.82 EF371354

Micrasterias thomasiana M2253 FN432110Docidium undulatum NIES285 FN432102

Haplotaenium sp. M3063 FN432109H. minutum SVCK302 AJ553947

Std. isthmosus M3096 FN432124Std. mamillatus JH0090 EF371348

Std. convergens UTEX2508 EF371281Std. subulatus SVCK106 FN432127Cosmarium cf. contractum NIES452 AM911283

C. moniliferum M3047 FN432067C. contractum SVCK396 AJ553937M2558 FN432120

M1886 AM911344C contractum M3039 FN432060

Std. validus SVCK457 AM911343Cosmarium prominulum M3030 FN432071

Euastrum subalpinum ACOI855 AM911246E. gayanum M3050 FN432105

Cosmarium dilatatum SVCK463 FN432061E. bidentatum ACOI282 FN432103E. divaricatum SVCK156 AM911242E. dubium M3064 FN432104

C. decedens ACOI794 AM911330

Cosmarium quadratulum FN432077E. biverrucosum SVCK464 AM911244

E. binale ACOI488 AM911245UTEX1748 AF203497

SVCK203 FN432106 Euastrum pectinatum

Tetmemorus laevis SVCK 227 EF371353SVCK409 EF371352SVCK214 FN432133 T. brebissonii

M3060 FN432074M3033 FN432075

M3021 FN432045Actinotaenium cucurbitaSVCK259 AY964170

M3023 FN432044Euastrum intermedium JH0159 EF371302E. vigrense SVCK380 FN432108E. crassum JH0018 EF371300

E. affine SVCK185 AM911240E. subhexalobum ACOI1671 AM911253

E. oblongum ASW07018 AM911239

E. trigibberum ACOI1174 AM911241

P. ehrenbergii JH0331 EF371329P. baculoides JH0008 EF371327

P. constrictum JH0135 EF371328M1369 FN432113

FN432114UTEX489 AF203503 P. trabecula

Staurodesmus dejectus M3062 FN432121Std. dejectus AF425772

Std. megacanthus M3065 FN432126Std. dejectus CCAP681/1 FN432122

Cosmarium bioculatum M3032 FN432056S. extensus ACOI1000 AM911339

S. extensus JH0386 EF371317Staurodesmus sp. M2404 FN432131Xanthidium octocorne M3057 FN432135

Std. isthmosus SVCK466 FN432125Std. extensus ACOI956 AM911337Std. triangularis SVCK280 AM911336

Staurodesmus sp. FN432130Staurodesmus sp. M3069 FN432128

Staurodesmus sp. M3068 FN432129Std. omearii M0751 AM911333

Std. spencerianus ACOI735 AM911335Std. bulnheimii SVCK84 FN432118

Cosmarium messikommeri M3041 FN432066Cosmarium sp. M2856 AM911327

C. difficile ACOI403 AM911326Cosmarium sp. M3066 FN432097

C. norimbergense ACKU1115 AY964160C. regnellii M2947 AM911276

JH0368 EF463096 Spondylosium pulchellumCCAP680/1 AF203505

SVCK365 AM911261C. laeve M3099 FN432065

Onychonema filiforme JH0420 EF463094Onychonema sp. UTEX832 AF203501

Spondylosium pulchrumJH0269 EF371341 SVCK331 AJ553970

Desmidium baileyi JH0228 EF371299

Desmidium sp. UTEX612 AF203495D. aptogonum JH0184 EF371298

D. aptogonum SVCK108 EF463091D. baileyi ACOI1062 FN432101

Hyalotheca dissiliens UTEX476 AF203499H. mucosa JH0055 EF371305

D. grevillei SVCK113 EF463090JH0199 EF371283

Bambusina borreriCCAC0045 AJ553935Groenbladia taylori JH0339 EF463093

G. neglecta SVCK478 AJ553943Cosmarium cf. tenue M3036 FN432094Heimansia pusilla SVCK428 EF371291

Cosmarium sinostegos ACOI406 AM911275M3053 FN432057

M3048 FN432058AM911274

C. bioculatum

C. granatumM2127 AM911282M3102 FN432063C. subtumidum ACKU1066 AY964166

C. subgranatum M2629 AM911281C. obtusatum ACKU1173 AY964181

C. laeve SVCK35 AM911280C. angulosum ACOI378 AM911328

C. subtumidum SAG612-8a FN432090Cosmarium sp. M3058 FN432095

C. tenue ACKU1104 AY964176C. subprotumidum M3049 FN432086

C. regnellii M3100 FN432080Std. mucronatus M1394 AM911342

Std. dickiei AF425773Std. bienianus M1130 AM911341

Std. brevispinus ACOI881 AM911340Staurastrum orbiculareAF425770

M2217 AM911331C. laeve CCAP612/21 AF425770

C. boergesenii ACKU1047 AY964162C. laeve ACKU1032 AY964180C. angulosum ACKU1041 AY964154C. impressulum SVCK58 AM911279C. meneghinii SVCK59 AM911284C. polygonum CCAP612/20 FN432069

SVCK382 AY964171SVCK163 FN432046

Spondylosium panduriforme SAG52.88 AJ553969

C. amoenum

C. biretum SAG44.86 AM911267C. pseudoholmii SAG15.87 FN432072

C. broomei M2075 AM911269Spondylosium luetkemuelleri FN432115

C. quadrum M3045 FN432079C. reniforme SVCK34 AY964179C. quadrum var. sublatum f. dilatatum ACOI368 AM911272

Actinotaenium diplosporum M3028 FN432047

C. cucumis M2715 AM911270C. depressum ACOI1030 AM911325

omniradiate

Staurastrum

Micrasterias

multicellular2

Euastrum2

Euastrum1

ARTHR

STD1

STD2

Pleurotaenium

STD3

STD4Haplotaenium

CO5

56/-/1.00

-/-/1.00

68/56/-

100/99/1.00

50/-/1.00

56/51/-

100/99/1.00

85/64/0.9754/-/0.97

80/79/0.99

99/97/1.00

93/96/1.00

-/57/1.00

75/85/1.0089/90/1.00

-/55/0.96

-/56/0.97

90/95/1.00

77/70/0.95

87/52/-

87/92/1.00

85/87/1.0083/90/0.99

92/98/1.00

77/-/-

99/97/1.00

64/67/-

98/98/1.00

97/93/1.0097/98/1.00

59/85/0.99

92/84/1.0098/96/1.00

88/96/1.00

93/62/-73/67/1.00

67/-/-

97/73/1.00

57/-/-

Tetmemorus

100/98/1.00

83/92/1.00

97/100/1.00

64/66/1.00 68/85/1.0078/85/1.00

54/75/0.99

99/98/1.0097/99/1.00

88/72/0.99

92/84/0.99

98/95/1.00

79/56/1.00 98/92/1.00

69/95/1.00

66/74/-

Cosmarium pseudopyramidatum

Actinotaenium curtum

Std. convergens

-/72/-

100/100/1.00[NJ(ML)/MP/BI]

0,005substitutions/site

STD2assemblage

Euastrumassemblage

rbcL Phylogeny of Desmidiaceae (Streptophyta) 259

RbcL Sequence Divergence

Taking into account the large taxon sampling, it wasnot surprising to find considerable variation in totalbranch lengths over all taxa in the tree, likely reflect-ing different evolutionary rates of gene sequences.Particularly short branches (Fig. 4) were observedin the clades ARTHR and Euastrum2, that com-prised taxa with rather uniform morphotypes(despite the Staurodesmus/Cosmarium intermixingin clade ARTHR). In contrast, several mostly poly-generic clades (e. g. multicellular1, multicellular2,CO1, “omniradiate” and CAP) were characterizedby long branches. These and other long-branchclades/lineages, in general, had little effect ontree topology and clade support, although whenthe long-branch Haplotaenium and Docidium wereexcluded from the analyses, the non-supportedMicrasterias lineage gained moderate support in NJand MP (77–80% BP, results not shown).

The gene rbcL provided relatively few autapo-morphic characters for the terminal branches ofthe tree and within most clades/subclades theaverage divergence (p-distance) between any twosequences did not exceed 3–4% (Fig. 4). In con-trast, a number of synapomorphies were revealedat the level of clades/subclades yielding relativelylong common branches and strong support formost clades/subclades (Figs 2, 3; Table 1). Towardsthe root of the tree, though, relationships amongclades remained largely unresolved because therbcL gene contained too few informative sites at thisdeep level of divergence.

Discussion

Ninety three novel and 198 additional Genbanksequences of the chloroplast-encoded rbcL genewere used to investigate the molecular phylogenyof the species-rich and taxonomically complex fam-ily Desmidiaceae (Zygnematophyceae). Based onthe number of sequences and genera sampled,the present analysis is the most comprehensiveto date. Sequences displaying less than 3 posi-tions difference were regarded as redundant andare represented in the data set by only one arbi-trarily selected sequence. Analyses of sequencedivergence among multiple isolates of the same

distinctive morphospecies showed that such athreshold still allows discrimination of different iso-lates of a single morphospecies that may representdifferent ecotypes. Using this threshold a taxonsampling that consisted of 285 sequences repre-senting 215 morphospecies of 23 traditional generaof the family Desmidiaceae and as an outgroup sixPenium sequences was created.

While it had previously been established, thatmost of the traditional desmid genera are poly-phyletic (Gontcharov et al. 2003; Gontcharov andMelkonian 2005, 2008; Hall et al. 2008a,b), an esti-mation of the total number of clades comprising thefamily has not yet been attempted due to limitedtaxon sampling. A primary goal of the present studywas therefore, to test whether increasing the taxon(sequence) sampling in the species-rich generawould uncover novel clades that could lead to a bet-ter understanding of the genetic diversity presentin this ecologically important group of streptophytegreen algae. The results presented here, indeedindicate that clade sampling is far from saturated inthe Desmidiaceae. Analyses of 291 non-redundantrbcL sequences representing 23 traditional desmidgenera recovered 34 clades/lineages/branchesin the family (in a previous study using 127rbcL sequences representing 14 genera, 17clades/lineages/branches were found in the family;Gontcharov and Melkonian 2008).

Analyses of this large taxon (sequence) setresolved 22 mostly well-supported clades in thefamily (Figs 1–3; Results). In addition, four non-supported lineages, previously established asclades in multi-gene phylogenies (Gontcharovand Melkonian 2008), were recovered only topo-logically (Micrasterias and Staurastrum), or asparaphyletic groups (CO4 and Xanthidium) andeight single-branch sequences remained unas-signed (Figs 1–3). Comparison of phylogenetictrees of the family Desmidiaceae (including Peniumsequences as outgroup) based on 291 and 127rbcL sequences (Gontcharov and Melkonian 2008)showed that most new sequences were placed intoalready known clades but also revealed a num-ber of novel clades (Table 1). Five novel clades,mostly accommodating few sequences belongingto the same or closely related morphospecies, i.e.Cosmarium pseudopyramidatum, Actinotaeniumcucurbita, STD3, STD4 and Actinotaenium curtum

➛

Figure 2. Enlarged portion of ML tree from Figure 1 showing structure and composition of the clades of theDesmidiaceae. See the legend of Figure 1 for details. Strain numbers and accession numbers of strains areprovided as given in the list of strains (Supplementary Table). Sequences obtained for this study are shownbold-face.

260 A.A. Gontcharov, M. Melkonian

Cosmarium regnellii SVCK465 FN432081Cosmocladium perissumUTEX2447 AF203494

ACOI342 FN432100Spondylosium tetragonum JH0281 EF371336

JH0140 EF371351M3103 FN432132

SVCK24 EF463097SAG41.81 AM911260

UTCC284 AF203504SAG25.80 EF371350

SVCK31 AM911259 Staurodesmus clepsydra var. sibiricum SVCK87 FN432119

Cosmarium melanosporum JH0011 EF371289C. bioculatum CCAP612/17 AM911265

C. sphagnicolum SAG3.94 FN432085C. tinctum M2301 AM911278

C. sphagnicolum M3035 FN432084

C. tesselatum SVCK381 AM911320C. ovale SVCK342 AM911309

C. maculatum SVCK422 AM911315C. askenasyi SVCK403 FN432055

Euastrum moebii SVCK358 AM911248

C. margaritiferum SVCK88 AM911311C. praemorsum M3043 FN432070

C. subtumidum M3067 FN432088C. subtumidum M3031 FN432089C. dentatum SVCK149 AY964175C. depressum ACKU1212 AY964164C. perforatum SVCK109 AM911318C. auriculatum ACKU1142 AY964156

C. pseudoconnatum JH0264 EF371280Actinotaenium wollei ACKU4010 AY964159

C. pseudonitidulum M3055 FN432073C. pseudoconnatum SVCK150 AY964168

C. connatum FN432059C. pseudoconnatum M1272 AM911316C. subprotumidum ACKU1083 AY964157C. obsoletum ACKU1183 AY964177

C. obsoletum M2303 AM911277C. ornatum SVCK569 AM911288

C. vogesiacum M3052 FN432092Cosmarium sp.1 FN432098

C. subprotumidum FN432087C. punctulatum SVCK570 AM911289

C. caelatum ACOI826 AM911319C. punctulatum SAG139.80 FN432076

Cosmarium sp.2 FN432099S. tumidum SVCK85 AJ553972

Xanthidium cristatum SVCK426 AM911263SVCK147 AM911264

JH0261 EF371355SVCK281 FN432134X. subhastiferum CCAP690/1 AF203507

X. brebissonii SVCK379 DQ026261X. armatum ASW07059 DQ026262

Cosmarium sp. ACKU1232 AY964161C. ocellatum M3059 FN432068

C. phaceolus M2302 AM911299C. binum ACOI896 AM911329

E. germanicum SVCK461 AM911251E. . spinulosum ACOI1092 AM911252

E. substellatum SVCK364 AM911247E. verrucosum SVCK798 AM911250

C. protractum SVCK460 AM911298C. tetraophthalmum SVCK220 AM911314

A. turgidum M1192 AM911238C. lundellii SVCK357 AM911310

C. undulatum SVCK482 AM911297C. subochthodes ACOI377 AM911296C. botrytis SVCK274 AM911295

C. pseudonitidulum ACOI1160 AM911294C. ochthodes M1205 AM911293

Cosmarium sp. M3107 FN432096C. trachypleurum ACOI935 AM911307C. holmii M1211 AM911303

C. sexangulare ACKU1300 AY964174C. cyclicum M1208 AM911304

C. retusiforme FN432082C. hammeri ACOI349 AM911302

C. trilobulatum ACOI866 AM911301Cosmarium sp. M3061 FN432093

C. costatum ACKU1082 AY964155C. tumens M2736 FN432091

C. blyttii ACKU1079 AY964167C. humile M3051 FN432064

C. subprotumidum ACKU1011 AY964158C. subprotumidum SVCK373 AM911292

C. sphaeroideum SAG141.80 FN432083C. blyttii CCAP612/19 AM911290

C. crenatum M2164 AM911268C. notabile ACOI936 AM911287

C. punctulatum M2717 AM911300C. levinotabile ACKU1108 AY964163

C. subcrenatum M1200 AM911286

A. cucurbita M1199 AM911236Actinotaenium cf. subglobosum M3026 FN432049

Actinotaenium sp.1 FN432052Actinotaenium sp. M2390 FN432051

Actinotaenium sp. M3025 FN432050Phymatodocis nordstedtiana SVCK327 AJ553962

Cosmarium tinctum AM911285Actinotaenium cruciferum M2025 AM911235

Penium polymorphum M2335 AM911255P. cf. didymocarpum JH0212 EF371316

P. exiguum M2159 AJ553960P. cylindrus ACOI780 AJ553959

P. spirostriolatumSVCK189 AJ553961SVCK332 FN432111

P. margaritaceumUTEX600 AF203502

ACOI330 EF371322M1016 FN432112

SAG22.82 AM911254

Xanthidium

CO1

CO2

CO3

CAP

CO4

multicellular167/60/-

78/76/-87/54/1.00

98/97/1.0099/95/1.00

98/100/1.00

89/91/0.99

85/92/1.00

97/91/1.0096/98/1.00

93/91/1.00 97/98/0.99

96/87/0.99

98/98/1.00

99/97/1.00

64/56/-96/95/1.00

85/92/1.00

86/78/1.00

79/83/1.00

99/99/1.00

93/94/1.00

97/93/1.00

95/92/1.00

99/99/1.00

99/99/1.00

98/99/1.00

83/88/1.00

98/98/1.00

outgroup100/100/1.00 [NJ(ML)/MP/BI]

0,005 substitutions/site

-/-/0.98 Teilingia granulata

E. prowsei SVCK353 FN43210767/59/1.00

X. antilopaeum

Figure 3. Enlarged portion of ML tree from Figure 1 showing structure and composition of the clades of theDesmidiaceae. See the legend of Figures 1 and 2 for details.

as well as four novel single-taxon branches, Docid-ium undulatum, Cosmarium bioculatum M3032,C. messikommeri, and Staurodesmus bulnheimii(Figs 1–3) were discovered during this study.

Although two more clades, Pleurotaenium and Tet-memorus, that were not sampled by Gontcharovand Melkonian (2008) have been identified dur-ing this study, they are not novel, since they were

rbcL Phylogeny of Desmidiaceae (Streptophyta) 261

Table 1. Support (NJ(ML)/MP/BI) for the clades in different analyses. Number of taxa (sequences) analyzedare provided in brackets before the support values.

Clade/assemblage 127 sequences (Gontcharov andMelkonian 2008)

291 sequences (Figs 1–3)

“omniradiate” (16) 54/-/0.99 (26) 56/-/1.00Staurastrum (2) -/-/- (20) -/-/-Micrasterias (4) 86/78/1.00 (7) -/-/-Haplotaenium (1) (2) 100STD4 N/A (2) 100ARTHR (4) 100 (9) 100Euastrum assemblage (10) 60/55/0.99 (28) -/-/-

Euastrum1 (6) 96/96/1.00 (11) 92/84/1.00Euastrum pectinatum N/A (2) 100Tetmemorus N/A (3) 97/73/1.00Cosmarium pseudopyramidatum N/A (2) 100Actinotaenium cucurbita N/A (3) 100Euastrum2 (3) 100 (6) 100

Pleurotaenium N/A (6) 100/99/1.00STD3 N/A (4) 100STD1 (7) 100 (12) 100multicellular 2 (10) 54/55/- (31) 56/51/-STD2 assemblage (10) 77/84/- (27) 54/-/0.97

CO5 (4) 100 (7) 100/99/1.00STD2 (6) 97/91/1.00 (18) 85/64/0.97Actinotaenium curtum N/A (2) 100

multicellular 1 (2) 100 (6) 98/97/1.00CO1 (2) 100 (6) 97/91/1.00CO4 (5) 92/85/0.99 (6) -/-/-CO3 (8) 100 (25) 99/99/1.00Xanthidium (4) -/-/- (8) -/-/-CO2 (33) 96/78/0.99 (44) 96/87/0.99CAP (5) 74/59/0.96 (6) 78/76/-basal position of the clade

“omniradiate” in the family51/-/0.95 -/-/-

basal position of Phymatodocisand the clade CAP

66/-/- 67/60/-

1N/A: not accessed.100 = 100/100/1.00.

previously recovered as clades in a multi-gene phy-logeny by Hall et al. (2008a).

The significant expansion of the known cladesin general had little effect on their support val-ues. This refers to both, strongly supported(e.g. CO2, CO3, ARTHR, STD1, multicellular1)and weakly supported clades (e.g. multicel-lular2 and “omniradiate”), in which the newsequences were mostly assigned to alreadyexisting subclades/branches (Table 1). In afew cases the new sequences formed novelclades disrupting existing clades which receivedno support anymore (e.g. the assemblagesSTD2, and Euastrum; Fig. 2, broken lines;Table 1).

Our study demonstrated that the traditionalgeneric boundaries of the Desmidiaceae that arelargely based on overall cell morphology areclearly inadequate and do not reflect phyloge-netic relationships among desmids. Less thanhalf of the 23 traditional desmid genera analyzedwere resolved as independent clades (Pleurotae-nium and Haplotaenium), distinct subclades ofpolygeneric clades (Cosmocladium, Onychonema,Hyalotheca and Groenbladia) or as single-taxonbranches (Docidium and Phymatodocis). It islikely that their generic circumscription, which isbased on unique sets of phenotypic traits, maynot require revision (although additional taxonsampling may invalidate this conclusion in the

262 A.A. Gontcharov, M. Melkonian

“omniradiate”

Staurastrum

Micrasterias

Haplotaenium

STD4ARTHR

Euastrum1

E. pectinatum

Tetmemorus

C. pseudopyramidatum

A. cucurbita

Euastrum2

Pleurotaenium

STD3STD1

multicellular2

STD2CO5

A. curtum

multicellular1

CO1CO4

CO3Xanthidium

CO2CAP

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08

p-di

stan

ces

Figure 4. Mean pairwise genetic divergences (%) between any two sequences within the clades and non-supported lineages recovered by the molecular phylogenetic analyses (Fig. 1). The divergence was calculatedas an uncorrected p-distance using Mega4 (Tamura et al. 2007) with gaps and missing data treated with pairwisedeletion.

future).Two additional, apparently monophyleticgenera, Bambusina and Teilingia, will likely needto incorporate their sister taxa, Desmidium gre-villei and Spondylosium tetragonum, respectively(Figs 2, 3). The distinct features of cell divisionshared by members of each sister pair (Hall et al.2008b) favors emendation of the diagnoses of Bam-busina and Teilingia against description of two newmonotypic genera for D. grevillei and S. tetragonum.Finally, monophyly of the genus Tetmemorus wasalso supported by placing its three sequences (twospecies) into a clade (73–97% BP, 1.00 PP) embed-ded in the non-supported Euastrum lineage (Fig. 2;see also Hall et al. 2008a). Due to the currentlyunclear status of the Euastrum lineage, definiteconclusions regarding the validity of the genericstatus of Tetmemorus cannot be made because thisclade could be part of a larger Euastrum clade. Tet-memorus spp, in fact, share with some elongatedsmooth-celled species of Euastrum (putative mem-bers of the clade Euastrum2) the apical incision ofthe semicells typical for the genus Tetmemorus.

The remaining 12 desmid genera were resolvedas polyphyletic (see Results) either because theirspecies were distributed among several unre-lated clades in the tree and/or the respectiveclades/lineages accommodated representatives ofother genera (e. g. Micrasterias and Xanthidium),often with strong support. The genus Cosmariumwas most problematical in this respect becauseits species were distributed among 17 divergencesof the tree (clades, non-supported lineages andsingle-taxon branches). Members of the tradi-tional genus Staurodesmus and Actinotaeniumoccurred in six clades/branches, and Euastrumin five. In most cases these clades included rep-resentatives of more than one genus and onlythe clades STD3, STD4, Actinotaenium cucurbita,Actinotaenium curtum, Euastrum2, Cosmariumpseudopyramidatum, and CO5 were limited tospecies of Staurodesmus, Actinotaenium, Euas-trum and Cosmarium, respectively (see Results).

The discordance between taxonomy and phy-logeny of the Desmidiaceae revealed in molecular

rbcL Phylogeny of Desmidiaceae (Streptophyta) 263

phylogenetic analyses can only be resolved byrecognizing clades, that are well-supported bybootstrap values and synapomorphic morpholog-ical/molecular traits, as genera. Although mostclades currently appear to be well-supported byphylogenetic analyses (also involving additionalgenes such as rDNA; Gontcharov and Melkonian2008), evaluation of their phenotypic traits needs afresh approach in search of characters not inval-idated by the rampant homoplasies that aboundamong traditional characters such as cell shape,cell processes, radiation, cell wall ornamenta-tion and organizational level (Gontcharov andMelkonian 2008). Until such novel phenotypic traitsunfold, the current confusion in desmid systemat-ics and taxonomy is unlikely to disappear. Based oncurrent knowledge about the molecular phylogenyof the Desmidiaceae up to 18 novel genera mayeventually have to be recognized. Some candidatesof clades that could represent such novel generaare discussed in the following.

Clade “omniradiate”

The clade originally comprised only four specieswith omniradiate (circular) cells in apical view(Gontcharov et al. 2003, 2004). Later, the clade wasextended to 16 sequences representing three tradi-tional desmid genera, Cosmarium, Actinotaeniumand Spondylosium (Gontcharov and Melkonian2008). While the clade was only poorly sup-ported in the rbcL phylogeny (Gontcharov andMelkonian 2008; Fig. 2), this study with a totalof 26 sequences), multigene analyses stronglyincreased support for this clade (Gontcharov andMelkonian 2008). The clade was positioned asthe most basal divergence in the family. Mor-phologically, the clade is quite heterogeneousand accommodates taxa with omni-(Actinotaeniumspp, Spondylosium luetkemuelleri and some Cos-marium spp), bi- (Cosmarium spp) or triradiate(Spondylosium panduriforme) cells in apical view;with deeply (most Cosmarium spp) or weakly(Actinotaenium spp, some Cosmarium spp) con-stricted semicells; or with a smooth or ornamentedcell walls. The two Spondylosium species assignedto this clade (Fig. 2) share a filamentous organiza-tional level but are otherwise not closely related.The taxonomic status of the clade “omniradiate”remains unclear. Apparently the clade cannot belinked to any of the three traditional genera compris-ing it and either represents a single novel genus ormay have to be split into several new genera. A non-homoplasious synapomorphy -two substitutions inthe spacer of Helix 25 of the SSU rRNA - shared

by the 16 clade members for which this gene wassequenced, distinguished the clade from all otherDesmidiaceae (Gontcharov and Melkonian 2008)suggesting that a search for novel synapomorphicphenotypic traits in the clade may also yield results.

Clade CO1

This clade (91–97% BP, 1.00 PP) included fivesmall-sized Cosmarium species that formed tworobust long-branch subclades and Staurodesmusclepsidra var. sibiricum occupying a basal posi-tion in the clade (96–98% BP, 1.00 PP; Fig. 3).This lineage could either represent a single genus(for which no synapomorphies are yet known) or,the pronounced morphological differences amongthe three groups of species (spines in the Stau-rodesmus taxon, papillae at the lateral sides of C.sphagnicolum and a smooth cell wall in both C.bioculatum CCAP 612/17 and C. melanosporum),may indicate that each group constitutes a novelgenus.

Clades CO2 and CO3

The most species rich (44 sequences in ouranalyses) clade CO2 (96–96% BP, 1.00 PP) cor-responded to the genus Cosmarium because itincluded the type species of the genus, C. undu-latum. Beside Cosmarium spp it also containedseveral Euastrum and Actinotaenium species.Apart from diversity of morphotypes in cladeCO2 (Gontcharov and Melkonian 2008), the inter-nal sequence diversity (p-distance < 2%; Fig. 4)was surprisingly low. Most species found in thisclade display cell wall ornamentations althoughsmooth-walled taxa also occur (e.g. Actinotae-nium spp, C. subprotumidum). Clade CO2 waspreviously characterized by a nonhomoplasiousmolecular synapomorphy (a compensatory basechange A–U=>G–C in the 3’ terminus of Helix 49 ofSSU rRNA; Gontcharov and Melkonian 2008) andthis feature could be used as a defining characterfor the genus Cosmarium s. str.

Morphological diversity within the secondCosmarium-rich clade, CO3 (25 sequences;99%BP, 1.00 PP; Fig. 3), was similar to that in CO2(Gontcharov and Melkonian 2008) and these twolineages comprised basically similar morphotypes.Results of multigene analyses have suggestedthat CO2 and CO3 may be sister groups, butsupport for this was low and in the rbcL phylogenythis relationship was not recovered (Gontcharovand Melkonian 2008). Based on the distributionpattern of 133 arbitrarily sampled Cosmariumsequences among clades, it is anticipated that

264 A.A. Gontcharov, M. Melkonian

CO2 (=Cosmarium s. str.) and CO3 (likely a newgenus) will accommodate most members of thetraditional genus Cosmarium.

Clades STD1-STD4 and ARTHR

The traditional genus Staurodesmus was amongthe best sampled desmid genera (30 sequencesrepresenting 20 morphospecies or c. 20% of thetotal number of species) in the present study. RbcLsequence comparisons resolved Staurodesmus ashighly polyphyletic with its members distributedamong six mostly robust clades, STD1-STD4,ARTHR and CO1, that showed no affinity to eachother (Figs 1–3). One additional species of thegenus, Std. bulnheimii, remained unassigned andformed a single-taxon branch. The artificial natureof the traditional genus Staurodesmus was furtheremphasized by the fact that four of its six cladesalso included representatives of other desmid gen-era, Cosmarium in ARTHR, CO1 and STD2 andXanthidium in STD1 (Figs 2, 3). The latter cladeincluded Std. triangularis, the type species of thegenus, thus clade STD1 likely represents Stau-rodesmus s. str. (X. octocorne should be transferredto Staurodesmus) while other clades containingspecies of Staurodesmus but showing no affin-ity to STD1 likely represent novel genera yetto be defined phenotypically and described. Theoccurrence of taxa with a single spine at thelateral sides of each semicell (a distinctive fea-ture of the traditional genus Staurodesmus) inseveral distinct clades and their close relation-ship with smooth-walled species of Cosmarium ortaxa with two spines at the lateral sides of eachsemicell (X. octocorne) strongly suggests that thediagnosis of Staurodesmus s. str. (clade STD1)needs to be revised. It is obvious that the pres-ence/absence of spines at the lateral sides of eachsemicell is highly homoplastic and in some taxamay in fact be environmentally regulated (Hoff-mann, Gontcharov and Melkonian, unpublishedobservations).

Clades multicellular1 and multicellular2

The phylogenetic relationship between twoclades/lineages containing desmids with a fila-mentous or colonial organizational level (cladesmulticellular1 and 2) remains unclear. They wereresolved as members of one clade in somemulti-gene analyses (Hall et al. 2008a,b), orformed a poorly supported clade (Gontcharov andMelkonian 2008), and in single gene phylogeniesthey represented independent lineages in theDesmidiaceae (Gontcharov et al. 2003; and this

study). Since most taxa in multicellular 1 and 2display long-branches, caution is required whenrelationships of the sequences to each other areto be addressed. The basal position of Cosmariumspecies in both multicellular1 and multicellular2(Fig. 2), however, suggests that this morphotypeand the type of cell division typical for Cosmariummay have given rise to a range of filamentous andcolonial forms characterized by distinct patterns ofcell division (Hall et al. 2008b).

Clade CAP

This moderately supported basal clade, along withPhymatodocis nordstedtiana, fills the evolutionarygap between the Peniaceae, characterized by aplesiomorphic state of the cell wall ultrastructure,and the Desmidiaceae, which are distinct in theapomorphic state of the same character. NeitherA. cruciferum and C. tinctum, nor the small-celledPenium spp. allied to them, have been studied ultra-structurally, thus characteristics of the cell wall inmembers of the clade CAP remains unknown. Itis very likely that this clade consists of three novelgenera.

Conclusions

Comparison of 285 rbcL sequences of theDesmidiaceae, the most species-rich taxon inthe streptophyte green algae, gave clear evi-dence that current taxon sampling has not yetreached saturation: the number of novel cladesidentified significantly increased upon addition ofnew sequences. At the same time, the poly-phyletic nature of the majority of the traditionalgenera in the family, already documented in pre-vious studies, was corroborated using the largertaxon set. The conflict between molecular phy-logeny and traditional taxonomy thus disclosed canonly be resolved by a complete overhaul of thegenus concept in this well-known group of greenalgae that dates back to the 19th century. In ourestimation, this requires a re-evaluation of pheno-typic traits using a fresh, unbiased approach insearch for traits not invalidated by homoplasies,plesiomorphies or phenotypic plasticity. A likelyresult of such a “polyphasic approach” (e.g. Hoef-Emden and Melkonian 2003; Marin et al. 2003;Pröschold et al. 2001) to the systematics of theDesmidiaceae will be the description of mono-phyletic genera, based on both morphological andmolecular synapomorphies, a goal that should beworth the considerable effort involved in such anendeavor.

rbcL Phylogeny of Desmidiaceae (Streptophyta) 265

Methods

Cultures: Ninety three strains of Desmidiaceae and Peni-aceae used for this study were obtained from differentsources (Appendix) and grown in modified WARIS-H cul-ture medium (McFadden and Melkonian 1986) at 15 ◦Cwith a photon fluence rate of 40 �mol m−2 s−1 in a 14/10hr light/dark cycle. The taxonomic identity of the strainssequenced during this study was verified by light microscopy(voucher photographs of strains isolated during this studyare available from the respective culture collection) prior toDNA extraction using standard literature (Brook and Johnson2002; Coesel and Meesters 2007; Croasdale and Flint 1988;Krieger and Gerloff 1962, 1965, 1969; Prescott et al. 1981).All database sequences referred to previously publishedpapers, in which procedures for identification of strains weredescribed.

DNA extraction, amplification and sequencing: After mildultrasonication to remove mucilage, total genomic DNA wasextracted using the QIAGEN DNeasy Plant Mini Kit (QIAGEN,Hilden, Germany). The chloroplast-encoded rbcL gene wasamplified by polymerase chain reactions (PCR) using publishedprotocols and 5’-biotinylated PCR primers (Gontcharov et al.2004). PCR products were purified with the Dynabeads M-280system (DYNAL BIOTECH, Oslo, Norway) and used for bidi-rectional sequencing reactions (for protocols, see Hoef-Emdenet al. 2002). Gels were run on a Li-Cor IR2 DNA sequencer(LI-COR Inc, Lincoln, NE, USA).

Sequence alignments and tree reconstructions:Sequences were aligned using the SeaView program (Galtieret al. 1996) and all three codon positions of the rbcLgene were used for analyses. The amount of phylogeneticsignal versus noise in our data was assessed by plottingthe uncorrected against corrected distances determinedwith the respective model of sequence evolution estimatedby Modeltest 3.06 (Posada and Crandall 1998). Also, themeasure of skewness (g1-value calculated for 10,000 ran-domly selected trees in PAUP 4.0b10; Swofford 2002) wascompared with the empirical threshold values (Hillis andHuelsenbeck 1992) to verify the non-random structuring ofthe data. To quantify the extent of substitution saturationin data sets, the Iss statistic was calculated with DAMBE(Xia and Xie 2001) for the individual and combined datasets.

Phylogenetic trees were inferred with ML, distance (NJ) andMP optimality criteria using PAUP 4.0b10 and Bayesian infer-ence (BI) using MrBayes 3.1.2 (Huelsenbeck and Ronquist2001). Evolutionary models (for ML and NJ analyses) wereselected by the Akaike Information Criterion in Modeltest. MLand MP analyses used heuristic searches with a branch-swapping algorithm (tree bisection-reconnection); distances forNJ analyses were calculated by ML. In BI, two parallel MCMCruns were carried out for two million generations samplingevery 100 generations for a total of 20,000 samples. Con-vergence of the two chains was checked, stationarity wasdetermined (the first 1100 samples were discarded as “burn-in”) according to the ‘sump’ plot and the posterior probabiliteswere calculated from the trees sampled during the station-ary phase. The robustness of the trees was estimated bybootstrap percentages (BP; Felsenstein 1985) in NJ and MPusing 1000 replications and by posterior probabilities (PP) inBI. BP < 50% and PP < 0.95 were not taken into account.In MP, the stepwise addition option (10 heuristic searcheswith random taxon input order) was used for each bootstrapreplicate.

Acknowledgements

This work was supported by DFG grant ME-658/26-1 and grants from RFBR 09-04-00270а and-00621а.

Appendix A. Supplementary data

Supplementary data associated with this arti-cle can be found, in the online version, atdoi:10.1016/j.protis.2010.08.003.

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