American Journal of Botany 95(9): 1079–1095. 2008. I N SEARCH OF MONOPHYLETIC TAXA IN THE FAMILY DESMIDIACEAE (ZYGNEMATOPHYCEAE, VIRIDIPLANTAE): THE GENUS COSMARIUM 1 Andrey A. Gontcharov 2 and Michael Melkonian Botanisches Institut, Lehrstuhl I, Universit ä t zu K ö ln, Gyrhofstr. 15, D-50931 K ö ln, Germany 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 phylo- genetic 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 con- tained representatives of at least two genera highlighting the polyphyletic nature of the genera Cosmarium , Euastrum , Staurodes- mus , 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 in- volving 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; poly- phyly; taxonomy. The conjugating green algae (Zygnematophyceae, Viri- known in microalgal systematics in which descriptions of gen- diplantae) represent the most species-rich lineage in the Strepto- era have often been based on single or very few morphological phyta except for the embryophytic land plants ( Gerrath, 1993 ). characters visible in the light microscope without a careful in- They share with most embryophytes not only a common ances- vestigation of phylogenetic signifi cance (one related example is try but also the absence of fl agellate reproductive stages. A pe- that of the coccoid green algal genus Chlorella and its relatives; culiar mode of sexual reproduction (i.e., conjugation) sets the Krienitz et al., 2004 ; Luo et al., 2006 ). We have therefore started class apart from other streptophytes and may have contributed to evaluate the genus concept in the most species-rich family to their successful diversifi cation (Brook, 1981). On the basis of of the Zygnematophyceae, the Desmidiaceae (desmids), using ultrastructural analyses of mitosis and cytokinesis, zygnemato- taxon-rich sampling and multigene phylogenetic analyses. Al- phycean algae have been recognized as members of the strepto- though the traditional genus Staurastrum Meyen ex Ralfs was phyte algae known also as Charophyceae sensu Stewart and shown to be polyphyletic, a monophyletic core of the genus Mattox ( Pickett-Heaps, 1975 ). Molecular phylogenetic analy- could be identifi ed using such an approach ( Gontcharov and ses of the Zygnematophyceae corroborated these results Melkonian, 2005 ). It is anticipated that once a phylogenetic ( Chapman and Buchheim, 1992 ; Surek et al., 1994 ; McCourt framework of a desmid genus has been established, a reinvesti- et al., 2000; Gontcharov et al., 2003 ), although the phylogenetic gation of its morphological traits should lead to the recognition position of the class among the streptophyte algae still remains of hitherto overlooked, but more sound and reliable generic unresolved ( Chapman et al., 1998 ; Karol et al., 2001 ; Lewis and morphological characters. McCourt, 2004 ; Turmel et al., 2007 ). Here, we address the phylogenetic status of the genus Cos- Phylogenetic analyses of the Zygnematophyceae using a marium Corda ex Ralfs (Desmidiaceae, Zygnematophyceae), broad taxon sampling and multigene data sets have more re- the most species-rich desmid genus with more than 1000 spe- cently led to the conclusion that many traditional genera of the cies described ( Gerrath, 1993 ). Together with Staurastrum class are polyphyletic, suggesting that the characters used to (~700 spp.) it constitutes about half of the total number of spe- delineate these taxa are either plesiomorphic, homoplasious or cies in the otherwise species-poor streptophyte green algae. It unreliable ( Gontcharov et al., 2003 , 2004 ; Gontcharov and should be mentioned that Cosmarium has always been regarded Melkonian, 2005 ; Hall et al., 2008 ). This situation is well as an artifi cial 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 1 Manuscript received 7 February 2008; revision accepted 11 June 2008. was poorly circumscribed by a vague diagnosis ( Ralfs, 1848 ) The authors thank P. C. Silva and F. A. C. Kouwets for discussion on the and linked morphologically to other genera such as Euastrum type species of Cosmarium . This work was supported by DFG grant Ehr. ex Ralfs and Xanthidium Ehr. ex Ralfs. Thus, although ME-658/26-1. 2 Author for correspondence (e-mail: [email protected]); over the 160 years since its description, numerous taxa have permanent address: Institute of Biology and Soil Science, 690022, been added to Cosmarium , the defi nition of the genus ( “ the Vladivostok-22, Russia. fronds are minute, simple, constricted in the middle; the seg- ments are generally broader than long and infl ato-compressed, doi:10.3732/ajb.0800046 but in some species orbicular or cylindrical; they are neither 1079 1080 American Journal of Botany [Vol. 95 emarginate at the end nor lobed at the sides, and have no spines criminate these genera on the phylogenetic tree revealed exten- or processes ” ; Ralfs, 1848 , p. 91) has not changed and its dis- sive homoplasy, calling into question the current genus concept criminatory power diminished. Although widely acknowledged in the Desmidiaceae. to be polyphyletic, the genus is still adopted to date in its origi- nal sense ( Lenzenweger, 1999 ; Brook and Johnson, 2002 ; Gerrath, 2003 ; Coesel and Meesters, 2007 ). Unfortunately, attempts MATERIALS AND METHODS during the 19th century to resolve the taxonomic problems in Cosmarium and establish more “ natural ” (morphologically uni- Cultures— One hundred twenty-seven strains of Desmidiaceae and Pe- form) taxonomic entities were unsuccessful (e.g., N ä geli, 1849 ; de niaceae used for this study were obtained from different sources (Appendix 1) Bary, 1858 ; Lundell, 1871 ; Kirchner, 1878 ; Gay, 1884 ; Hansgirg, and grown in modifi ed WARIS-H culture medium ( McFadden and Melkonian, − 2 − 1 1888 ; de Toni, 1889 ; Raciborski, 1889 ; Turner, 1892 ). The 1986 ) at 15° C with a photon fl uence rate of 40 µ mol⋅ m ⋅ s in a 14/10 h light/ dark cycle. The taxonomic designation of all strains was verifi ed by light mi- novel taxa were based on simple morphological features such croscopy prior to DNA extraction ( Krieger and Gerloff, 1962 , 1965 , 1969 ; as cell and semicell shape, ornamentation of the cell surface, Prescott et al., 1981 ; Croasdale and Flint, 1988 ; Brook and Johnson, 2002 ; degree of cell constriction, and chloroplast shape that occur in Coesel and Meesters, 2007 ). any combination in the genus. In 1954, Teiling established a new genus, Actinotaenium Teil ., for taxa with smooth-walled, DNA extraction, amplifi cation, and sequencing— After mild ultrasonica- elongated cells, circular in apical view, and displaying a shal- tion to remove mucilage, total genomic DNA was extracted using the Qiagen low sinus. Although Actinotaenium is often regarded as a “ nat- (Hilden, Germany) DNeasy Plant Mini Kit. Nuclear-encoded (nu) SSU rDNA ural group ” ( Prescott et al., 1981 , p. 1), its members are distinct (including the 1506 group I intron) and chloroplast-encoded (cp) rbcL and LSU only in the combination of characters that individually occur in rDNA were amplifi ed by polymerase chain reactions (PCR) using published protocols and 5 ′ -biotinylated PCR primers ( Marin et al., 1998, 2005 ; many Cosmarium taxa. Another consequence of the unsatisfac- Gontcharov, et al., 2004 ). PCR products were purifi ed with the Dynabeads tory taxonomic status of Cosmarium is the fact that only one M-280 system (Dynal Biotech, Oslo, Norway) and used for bidirectional se- unfi nished attempt of a monography of the genus dealing with quencing reactions (for protocols, see Hoef-Emden et al., 2002 ). Gels were run fewer than half of the described species exists ( Krieger and on a Li-Cor IR 2 DNA sequencer (Li-Cor, Lincoln, Nebraska, USA). Gerloff, 1962, 1965, 1969). The lack of type material and the inaccessibility or vagueness of many original descriptions further Sequence alignments and tree reconstructions— Sequences were manually complicates a critical assessment of species that are distin- aligned using the SeaView program ( Galtier et al., 1996 ). For coding regions of guished largely on the basis of the shapes of cells, semicells, the nu SSU rDNA, cp LSU rDNA, and noncoding 1506 group I introns, the and chloroplasts; features of cell wall ornamentation; and