/ Phycol. 28, 375-380 (1992) CONCORDANCE OF MOLECULAR AND ULTRASTRUCTURAL DATA IN THE STUDY OF ZOOSPORIC CHLOROCOCCALEAN GREEN ALGAE' Louise A. Lewis,^ Lee W. Wilcox, Paul A. Fuerst,* and Gary L. Floyd^ Departments of Plant Biology and *Molecular Genetics, The Ohio State University, Columbus, Ohio 43210 ABSTRACT Ultrastructural studies have led to reconsidera- Alternative evolutionary hypotheses generated from fea- tion of several morphologically based groups and tures of vegetative cell morphology and motile cell ultra- resulted in the separation of the green algae into structure were investigated using a molecular data set. five classes (Pickett-Heaps 1975, Moestrup 1978, Complete nuclear-encoded small subunit (18S) ribosomai Stewart and Mattox 1978, Melkonian 1982, Floyd RNA (rRNA) gene sequences were determined for six spe- and O'Kelly 1984, Mattox and Stewart 1984, O'Kel- cies (three each) of the chlorococcalean green algae "Neo- ly and Floyd 1984, Sluiman 1989). Through these chloris" and Characium. Based on motile cell ultra- studies it became apparent that many of the tradi- structure, it was previously shown that both genera could tional groupings based on vegetative cell morphol- be separated into three distinct groups possibly represent- ogy contained genera that cut across the newly pro- ing three separate orders and two classes of green algae. posed class boundaries and that certain characters 18S rRNA gene sequences were also obtained for three probably reflected parallel changes. additional taxa, Dunaliella parva Lerche, Pediastrum Two recent examples of the apparent discordance duplex Meyen, and Friedmannia israelensis Chanta- between vegetative morphological and ultrastruc- nachat and Bold. These organisms were selected because tural classifications involve chlorococcalean genera each, in turn, is a representative of one of the three ul- that are vegetatively non-motile but produce motile trastructural groups into which the Neochloris and zoospores. Watanabe and Floyd (1989) found that Characium species are separable. Phylogenetic analyses Neochloris species could be separated into three utilizing the molecular data fully support the ultrastruc- groups based on ultrastructural features of motile tural findings, suggesting that the similar vegetative cell cells. The primary feature delimiting the three morphologies observed in these organisms have resulted groups is the orientation of the basal bodies in the from convergence. flagellar apparatus (FA), i.e. whether they are clock- wise (CW), directly opposed (DO), or counterclock- Key index words: Characium; Chlorococcales; Chloro- wise (CCW), when the cells are viewed "top-down." phyceae; Chlorophyta; flagellar apparatus; Neochloris; Watanabe and Floyd suggested that Neochloris spe- ribosomai RNA cies falling into the first two FA groups have affin- ities to the Chlorophyceae and that the species in Systematists have traditionally relied on morpho- the CCW group better belonged in another class, logical characters in their attempts to reconstruct the Pleurastrophyceae (sensu Mattox and Stewart the evolutionary history of organisms. For green 1984), As in Neochloris, the genus Characium also algae, the characters used have heen relatively few appears to be paraphyletic, with the species falling in numher and primarily involve vegetative cell mor- into the same three ultrastructural groups as the phology (e.g. cell shape, numher of nuclei per cell, Neochloris species (Floyd and Watanabe 1990). plastid shape, growth requirements; see Bold and Wynne 1985). Characters such as cell/colony shape Information that is independent of vegetative have heen shown to be misleading in some instances morphology and FA ultrastructure should help to due to morphological plasticity (Trainor et al. 1971). determine which of the two character sets most ac- Realizing the limitations of such characters, a num- curately reflects the true phylogenetic relationships. ber of workers over the past 20 years have focused Here we present a molecular data set obtained from their efforts on characters believed to be evolution- complete DNA sequences of the 18S ribosomai RNA arily conserved, including ultrastructural features of (rRNA) genes of representative species from each the mitotic spindle, cytokinetic apparatus, and fla- of the three ultrastructural groups into which Neo- gellar apparatus. These are assumed to be less en- chloris and Characium species can be separated. Be- vironmentally influenced than are vegetative cell cause of the unavailability of appropriate sequences characteristics because they are involved in the vital with which to compare those of the Neochloris and functions of cell division, reproduction, and motil- Characium species, we also obtained sequence data ity. for three additional taxa, one from each of the three ultrastructural groups. With the acquisition of molecular data on these ' Received 23 December 1991, Accepted 24 February 1992, ' Present address: Department of Botany, Duke University, taxa, we were able to compare the evolutionary re- Durham, North Carolina 27706, lationships suggested by three different character ' Address for reprint requests. sets obtained from vegetative cell morphology, fla- 375 376 LOUISE A, LEWIS ET AL, TABLE I, Basal body orientation and source of chlorococcalean taxa examined. B.B,O," Reference Source** "Neochloris"-like taxa' Ettlia minuta CW Watanabe and Floyd 1989 776 Neochloris aquatica DO Watanabe and Floyd 1989 138 Parietochloris pseudoalveolaris CCW Watanabe and Floyd 1989 975 "Characium"-MVe taxa C, vacuolatum CW Floyd and Watanabe 1990 2111 C. hindakii DO Eloyd and Watanabe 1990 2098 C, perforatum CCW Eloyd and Watanabe 1990 2104 Additional taxa in flagellar apparatus groups Dunaliella parva CW Cbappell and Eloyd (unpubl, obs,) 1983 Pediastrum duplex DO Wilcox and Eloyd 1988 — Friedmannia israelensis CCW Melkonian and Berns 1983 1181 ' Basal body orientation, CW = clockwise, DO = directly opposite, CCW = counter-clockwise, I" Unialgai or axenic cultures were obtained from UTEX, the University of Texas Culture Collection (Starr and Zeikus 1987) except for Pediastrum duplex, which was collected from Lake Mendota, Wisconsin, by L,W,W, ' Nomenclature follows Deason et al, 1991, gellar apparatus ultrastructure, and 18S rRNA se- excluded from the analyses. The total number of aligned nucle- otide sites (including insertions/deletions) compared for all spe- quence data. cies in the study was 1713, Three group I introns (Michel and Westhof 1990) were found MATERIALS AND METHODS in the 18S rRNA genes of two of the taxa included in this analysis. We sequenced the 18S rRNA genes from the "Neochloris" and Their locations with respect to the Glycine max sequence are be- Characium species and from three other green algae with known tween positions 567 and 568 (Neochloris aquatica Starr), 1172 and flagellar apparatus ultrastructure: Dunaliella parva Lerche, Ped- 1173 {Dunaliella parva), and 1781 and 1782 {D. parva). Further iastrum duplex Meyen, and Friedmannia israelensis Chantanachat characterization of the introns will be published elsewhere, and Bold (see Table 1), Pairwise nucleotide distance (for all sites) was estimated using We employed the 18S rDNA sequence from Glycine max (Eck- the program DNADIST and PHYLIP, with the Kimura correc- enrode et al, 1985) as an outgroup taxon. An initial analysis (data tion (Felsenstein 1990), and relationships between taxa were then not shown) incorporating the 18S sequence from the protist Acan- determined from the nucleotide distances using the FITCH tree- thamoeba (Gunderson and Sogin 1986) was used to root the tree building program of PHYLIP (Fitch and Margoliash 1967, Fel- and to verify G, max as an outlier with respect to the algal se- senstein 1990), quences. Also, inclusion of unpublished sequences from the char- One hundred thirty-six nucleotide sites representing all phy- ophycean genera, Coleochaete, Klebsormidium, and Chlorokybus, logenetically informative positions in the data set were analyzed yielded the same tree topology as when Glycine alone was em- by a parsimony approach using PAUP (Swofford 1990), Because ployed as an outgroup taxon, rRNA has a specific secondary structure that may cause non- Growih of organisms and isolation of DNA. Eor most strains, the independence of sites, compensatory changes in stems were down- cells were grown in liquid medium (9:1; Starr and Zeikus 1987) weighted by one half (see Swofford and Olsen 1990), The branch or on 1,5% 9:1 agar plates, Dunaliella was maintained in E/2 and bound procedure of PAUP was used to find the shortest tree. (Guillard and Ryther 1962), DNA was extracted from either The reliability of the resulting clades was then tested by a boot- vegetative (non-motile) or motile cells. The vegetative cells were strap analysis (heuristic search using MULPARS, TBR branch ground with liquid nitrogen and/or sterile sand in UNSET buffer swapping, random stepwise addition, 50% majority rule, 100 rep- (Garriga et al, 1984), followed by phenol: chloroform extraction lications) and by a "decay" study, where trees of progressively and ethanol precipitation (Maniatis et al, 1982), Wall-less motile longer lengths are examined in order to assess how easily branch- cells were Iysed directly in UNSET, and DNA was extracted from es collapse (Mishler et al, 1991), The consistency index (CI; Kluge the lysates, and Farris 1969) was also calculated. DNA amplification, cloning, and sequencing. To obtain suflicient Sequence availability. The