J. Phycol. 36, 747–758 (2000) PHYLOGENY OF THE CONJUGATING GREEN ALGAE (ZYGNEMOPHYCEAE) BASED ON rbc L SEQUENCES1 Richard M. McCourt 2 Department of Botany, Academy of Natural Sciences, 1900 Benjamin Franklin Parkway, Philadelphia, Pennsylvania 19103 Kenneth G. Karol Cell Biology and Molecular Genetics/Plant Biology, H. J. Patterson Hall, Building 073, University of Maryland, College Park, Maryland 20742 Jeremy Bell, Kathleen M. Helm-Bychowski Department of Chemistry, DePaul University, 1036 W. Belden, Chicago, Illinois 60614 Anna Grajewska Department of Biological Sciences, DePaul University, 1036 W. Belden, Chicago, Illinois 60614 Martin F. Wojciechowski Section of Evolution and Ecology, University of California, One Shields Avenue, Davis, California 95616, and Museum of Paleontology and University/Jepson Herbaria, University of California, Berkeley, California 94720 and Robert W. Hoshaw Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721 Sequences of the gene encoding the large subunit analyses using first plus second positions versus third of RUBISCO (rbcL) for 30 genera in the six currently position differed only in topology of branches with recognized families of conjugating green algae (Des- poor bootstrap support. The tree derived from third midiaceae, Gonatozygaceae, Mesotaeniaceae, Peni- positions only was more resolved than the tree derived aceae, and Zygnemataceae) were analyzed using maxi- from first and second positions. The rbcL-based phy- mum parsimony and maximum likelihood; bootstrap logeny is largely congruent with published analyses of replications were performed as a measure of support small subunit rDNA sequences for the Zygnematales. for clades. Other Charophyceae sensu Mattox and The molecular data do not support hypotheses of Stewart and representative land plants were used as monophyly for groups of extant unicellular and fila- outgroups. All analyses supported the monophyly of mentous or colonial desmid genera exhibiting a com- the conjugating green algae. The Desmidiales, or pla- mon cell shape. A trend is evident from simple om- coderm desmids, constitute a monophyletic group, niradiate cell shapes to taxa with lobed cell and plastid with moderate to strong support for the four compo- shapes, which supports the hypothesis that chloroplast nent families of this assemblage (Closteriaceae, Des- shape evolved generally from simple to complex. The midiaceae, Gonatozygaceae, and Peniaceae). The anal- data imply that multicellular placoderm desmids are yses showed that the two families of Zygnematales monophyletic. Several anomalous placements of gen- (Mesotaeniaceae, Zygnemataceae), which have plesio- era were found, including the saccoderm desmid Roya morphic, unornamented and unsegmented cell walls, in the Gonatozygaceae and the zygnematacean Entran- are not monophyletic. However, combined taxa of sia in the Coleochaetales. The former is strongly sup- these two traditional families may constitute a mono- ported, although the latter is not, and Entransia’s phy- phyletic group. Partitioning the data by codon position logenetic position warrants further study. revealed no significant differences across all positions Key index words: Charophyceae; Desmidiales; green or between partitions of positions one and two versus algal phylogeny; molecular systematics, rbcL, Zygne- position three. The trees resulting from parsimony matales, Zygnemophyceae Abbreviations: CI, consistency index; GTR, general time-reversible; ML, maximum likelihood; MP, max- imum parsimony; rbcL, large subunit of RUBISCO; 1 Received 9 June 1999. Accepted 16 April 2000. SSU, small subunit 2Author for correspondence: e-mail [email protected]. 747 748 RICHARD M. McCOURT ET AL. I can find only one paper dealing with the question of family, the Mesotaeniaceae or saccoderm desmids (six the general evolution of Desmids . Altogether it is a to seven genera; Prescott et al. 1972, Gerrath 1993), production testifying to the scant knowledge of the comprises taxa of unicells or loosely joined cells. A author with regard to the Desmidieae in general. third group, known as the placoderm desmids, exhib- West (1899) its the greatest range of morphological diversity. Cell The classification of desmids is not altogether easy. walls in this group are segmented and have more elab- West and Fritsch (1927) orate wall ornamentation than the Zygnemataceae or After 300 years of microscopy and, of late, intensive in- Mesotaeniaceae. Some (Fritsch 1935, West and Fritsch vestigations, desmids continue to bewilder and in- 1927) placed placoderms (approximately 36 genera) trigue. along with the saccoderms in one family, the Desmidi- Prescott et al. (1972) aceae. But the generally accepted classification now recognizes saccoderms as a separate family, the Meso- Organisms that have elicited such comments, rang- taeniaceae, and moves some genera from the Desmidi- ing from derision by colleagues to sympathetic under- aceae into three additional, smaller families: Closteri- standing to wonder, not surprisingly present interest- aceae (two genera), Gonatozygaceae (two genera), ing classification problems. The conjugating green and Peniaceae (one genus) (Gerrath 1993). Kouwets algae (Orders Desmidiales and Zygnematales) are so and Coesel (1984) merged the two genera of the Go- distinctive that they have occasionally been put in a natozygaceae into the Peniaceae. class separate from other green algae (Fott 1971, Some authors have preferred to group all conjugating Round 1971, Bold and Wynne 1985). The uniqueness green algae within one order, the Zygnematales (Bold of the group has been reinforced in a modern cladis- and Wynne 1985). However, placoderm desmids are of- tic framework (Kenrick and Crane 1997). However, ten treated as members of the order Desmidiales the relationships of the families and genera within the (Gerrath 1993), with the Zygnemataceae and Mesotaeni- conjugating green algae have experienced a bewilder- aceae classified separately in the Zygnematales. Whether ing series of rearrangements (West 1904, West and one or two orders are used, the conjugating green algae Fritsch 1927, Transeau 1951, Randhawa 1959, Yama- are considered either members of a monophyletic group gishi 1963, Prescott et al. 1972, Mix 1973a, Hoshaw and within the class Charophyceae (sensu Mattox and Stewart McCourt 1988, Gerrath 1993). Moreover, the place- 1984) or, because the latter group is paraphyletic, as ment of the conjugating green algae among green al- members of a separate class, the Zygnemophyceae (Ken- gae and plants is still unclear, although they are consid- rick and Crane 1997). ered one of several green algal groups monophyletic Sequence data for rbcL have been used in a wide vari- with land plants, with the entire group referred to as ety of phylogenetic analyses of green plants and algae the Streptophyta (Mishler and Churchill 1985, Bremer (Chase et al. 1993, Olmstead and Palmer 1994, Manhart 1985). Molecular and morphological analyses have sup- 1994, Daugbjerg et al. 1994, Nozaki et al. 1995, 1997a,b,c, ported the monophyly of the conjugating green algae 1999, Chapman et al. 1998). For this study, our analyses and their relationship to other streptophytes (Hoshaw included rbcL sequences for 30 genera of conjugating and McCourt 1988, Mishler et al. 1994, Surek et al. green algae, including representatives of all six families 1994, McCourt 1995, Melkonian and Surek 1995, along with 12 outgroup genera, to test hypotheses of re- Kranz and Huss 1996, Qiu and Palmer 1999) but have lationships among genera, families, and orders. Prelimi- not explored phylogeny within the group in detail nary results based on these rbcL data were cited as part (Bhattacharya et al. 1994, McCourt et al. 1995, Park et of a review of molecular systematics of green algae al. 1996). The present paper uses DNA sequence data (Chapman et al. 1998); the present study includes an for rbcL, the chloroplast-encoded gene for the large additional ingroup genus (Entransia) and presents an subunit of the RUBISCO protein, in a broad sample expanded analysis of the sequence data. of genera in all six families and the two orders of the conjugating green algae. Classification schemes of the group have been gen- methods erally based on morphology and, more recently, ultra- Specimens and culture methods. Genera used in this study (Ta- structure (Transeau 1951, Prescott et al. 1972, Mix ble 1) included (percentage of total genera in family is in pa- rentheses): one Closteriaceae (50%), 14 Desmidiaceae (38%), 1973a, Hoshaw and McCourt 1988, Gerrath 1993). two Gonatozygaceae (100%), five Mesotaeniaceae (71%), one Schemes based on cell wall and thallus structure (e.g. Peniaceae (100%), and seven Zygnemataceae (58%). The data Prescott et al. 1972, 1975, 1981, Mix 1973a) have gen- set comprises 19 new sequences of conjugating green algae plus erally recognized three groups in the conjugating 11 previously published sequences (McCourt et al. 1995, Park et al. 1996). Published rbcL sequences of 16 other streptophyte green algae. Two of these groups have been recog- green algae and land plants were used as outgroups, specifically nized as families, whose members exhibit smooth cell six genera (seven species) of Characeae, two genera (four spe- walls consisting of a single piece and lacking pores or cies) of Coleochaetales, and five embryophytes (Pickett-Heaps other ornamentation (Transeau 1951, Prescott et al. 1975, Mishler and Churchill 1985, Graham 1993, Mishler et al. 1972).