J. Phycol. 34, 306±311 (1998) POLYPHYLY OF TETRASPORALEAN GREEN ALGAE INFERRED FROM NUCLEAR SMALL-SUBUNIT RIBOSOMAL DNA1 Gregory C. Booton Department of Molecular Genetics Gary L. Floyd Department of Plant Biology and Paul A. Fuerst 2 Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210 ABSTRACT DO), led O'Kelly and Floyd (1984) to propose that Chaetopeltis is an intermediate between algae with Ultrastructural studies of tetrasporalean green algae counterclockwise (CCW) absolute orientation (e.g. have suggested the order is polyphyletic. These features, in- Hafniomonas and ulvophycean zoospores) and algae cluding the absolute orientation of the ¯agellar apparatus with clockwise (CW) absolute orientation (e.g. and the bi- versus quadri¯agellated motile cell morphology, Chlamydomonas and zoospores of Tetraspora). Then, suggest that Chaetopeltis as well as a number of others, because Tetraspora produces naked gametes in gam- may be ancestral to a group that includes Tetraspora. In etangia similar to Chaetopeltis, they suggested that this study, we examine the phylogenetic relationships of se- Tetraspora might have been derived from a Chaeto- lected tetrasporalean taxa based on analysis of 18S ribo- peltis-like alga and that the early Chlamydomonas-like somal RNA gene sequences. Results show that the tetra- organisms would have originated as a zoospore re- sporalean taxa are polyphyletic. Bi¯agellated genera group leased from a Tetraspora-like alga. O'Kelly (1992) has with bi¯agellated volvocalean taxa, whereas the quadri¯a- reviewed these arguments with the same conclu- gellated species compose a distinct monophyletic clade not sions. closely related to the bi¯agellated taxa. In addition, tetra- Recently, O'Kelly et al. (1994) examined the ul- sporalean taxa group with other chlorophycean algal spe- trastructure of motile cells of four other quadri¯a- cies with similar ¯agellar apparatus absolute orientation, gellate chlorophycean algae. Zoospores of Hormotil- but the quadri¯agellated Tetrasporales do not appear to be opsis gelatinosa, H. tetravacuolaris, Planophila terrestris, ancestral to the entire Chlorophyceae. These results are con- and Phyllogloea ®mbriata all had cruciate apparatuses, cordant with previous conclusions drawn from ultrastruc- pyrenoids traversed by cytoplasmic channels, and tural data and further con®rm the utility of (small-sub- scales present on the surfaces of the cells. Because unit) ribosomal RNA gene sequences to discern green algal these features are essentially identical to those of the evolutionary relationships. Chaetopeltis zoospores, the authors created the new order Chaetopeltidales. Key index words: Chlorophyta evolution; SSU rRNA; In this study, we examine the relationship of se- Tetrasporales lected tetrasporalean taxa using small-subunit ribo- somal DNA (SSU rDNA). This ubiquitous molecule Utilizing primarily ultrastructural features of the has been widely used to examine the evolution of ¯agellar apparatus of motile cells, O'Kelly and Floyd algae in a number of studies (e.g. Gunderson and (1984) proposed that Tetraspora may be more prim- Sogin 1986, Huss and Sogin 1990, Rausch et al. itive than Chlamydomonas. They also proposed that 1989, Bhattacharya et al. 1996). More recent studies Chaetopeltis is one of the ancestral chlorophycean assessing relationships of various chlorophycean al- green algae because body scales on the surface of, gae with 18S rDNA data have clearly supported pro- and proximal sheaths in, the zoospores of Chaetopel- posed relationships based primarily on ultrastruc- tis are quite similar to those known for members of tural features of ¯agellar apparatuses (Lewis et al. the Ulvophyceae. These features, plus the nearly 1992, Wilcox et al. 1992, Friedl and Zeltner 1994, cruciate ¯agellar apparatus (i.e. directly opposed 5 Friedl 1995). The present molecular work was con- ducted to test hypotheses suggested by the previous 1 Received 3 January 1997. Accepted 10 November 1997. ultrastructural studies. The four primary hypotheses 2 Author for reprint requests; e-mail [email protected]. tested were: (1) the Tetrasporales is polyphyletic; 306 POLYPHYLY OF TETRASPORALEAN ALGAE 307 quadri¯agellate and bi¯agellate zoospore producing TABLE 1. Taxonomic authorities and GenBank references for species taxa will cluster as separate, coherent groups; (2) whose ribosomal sequences were utilized in this study. some bi¯agellate, zoospore-producing tetraspora- GenBank SSU reference accession lean taxa will cluster with taxa from the Volvocales; a (3) taxa that produce quadri¯agellate zoospores, in- Species (or UTEX culture no.) no. cluding Chaetopeltis, which appears to possess the Ankistrodesmus stipitatus (5 A. Huss and Sogin X56100 falcatus var. stipitatus) 1990 largest number of putative primitive ¯agellar appa- (Chod.) Lemm ratus features of any known chlorophycean organ- Asteromonas gracilis Artari (5 Wilcox et al. 1993 M95614 ism, comprise the most ancient group within the Stephanoptera gracilis) Artari Chlorophyceae; and (4) phylogenetic reconstruc- Chaetopeltis orbicularis Berthold (422) this study U83125 tion would support an evolutionary history of abso- Characium hindakii Lee & Bold Lewis et al. 1992 M63000 Chlamydomonas reinhardtii Dang Gunderson et al. M32703 lute ¯agellar orientation as follows: 1987 Chlamydopodium vacuolatum (Kor- Lewis et al. 1992 M63001 CCW → DO → CW. schikoff) Ettl et KomaÂrek Chlorella fusca Shihara & Krauss Huss and Sogin X56104 MATERIALS AND METHODS 1990 Tetrasporalean taxa included in this study were Chaetopeltis or- Chlorella kessleri Fott & NovaÂkova Huss and Sogin X56105 bicularis, Hormotilopsis gelatinosa, Hormotilopsis tetravacuolaris, Plan- 1990 ophila terrestris, Gloeococcus maximus, Hormotila blennista, Paulschulzia Chlorella minutissima Fott & No- Huss and Sogin X56102 pseudovolvox, and Tetraspora sp. Algal cultures (Table 1) were ob- vaÂkova 1990 tained from UTEXÐThe Culture Collection of Algae at The Uni- Ettlia minuta (Ance et Bold) Lewis et al. 1992 M62996 versity of Texas at Austin (Starr and Zeikus 1987) and were main- KomaÂrek tained in culture prior to DNA extraction in liquid media. Se- Dunaliella salina (Dunal) Teod. Lewis et al. 1992 M84320 quences of other taxa used in this study were determined previ- Fusochloris perforata (Lee & Lewis et al. 1992 M62999 ously in our laboratory or were obtained from GenBank. Bold) Floyd, Watanabe, and DNA extractions, PCR ampli®cations, and sequencing. Algal cultur- Deason ing, DNA extractions, and PCR ampli®cation conditions were per- Gloeotilopsis planctonica Iyengar Friedl and Zeltner Z28970 formed as previously reported in Booton et al. (1997). 18S rRNA & Phillipose 1994 genes were ampli®ed in two overlapping halves. The 59 portion Gloeococcus maximus (Mainx) (166) this study U83122 was ampli®ed using primers CRN5 and 1262. These are forward Fott & Novakova and reverse primers respectively. The 39 section of the gene was Hormotila blennista Trainor & (1239) this study U83123 ampli®ed using primers 892C and SSU2. All primers used in PCR Hilton ampli®cation and subsequent 18S rDNA sequencing reactions are Hormotilopsis gelatinosa Trainor (104) this study U83126 shown in Table 2. The PCR reactions were carried out as follows: & Bold denaturation at 948±978 C for 1 min, annealing at 508±538 C for Hormotilopsis tetravacuolaris Trai- (946) this study U83124 1.5 min, and extension at 728 C for 3 min; 35 cycles. Reactions nor & Bold were performed in a Perkin-Elmer (Norwalk, Connecticut) Cetus Hydrodictyon reticulatum (L.) Wilcox et al. 1992a M74497 TC1 thermocycler. Ten microliters of PCR products were electro- Lagerh. phoresed on a 1% agarose gel to determine size and approximate Myrmecia israelensis (Chantana- Lewis et al. 1992 M62995 quantity before sequencing. Separate PCR reactions from the chat et Bold) Friedl same individual that produced a band of the correct size were Neochloris aquatica Starr Lewis et al. 1992 M62861 pooled and used directly for subsequent sequencing reactions. Nephroselmis olivacea Stein SteinkoÈtter et al. X74754 Sequencing primers were g32P-labeled (ICN Inc., Irvine, Califor- 1994 nia) and 3 mL of PCR product was sequenced directly by double- Nitella sp. KuÈtz Wilcox et al. 1993 M95615 stranded cycle sequencing using a commercial kit following man- Paulschulzia pseudovolvox Skuja (167) this study U83120 ufacturer's instructions (Gibco BRL dsDNA Cycle Sequencing Sys- Pediastrum duplex Meyen Lewis et al. 1992 M62997 tem, Gibco BRL, Inc., Gaithersburg, Maryland). Planophila terrestris Groove & (1709) this study U83127 Phylogenetic reconstruction and data analysis. The primary se- Hofstetter quences of Chaetopeltis orbicularis, Hormotilopsis gelatinosa, H. tetra- Protoderma sarcinoidea (Grover Bhattacharya et al. Z47998 vacuolaris, Planophila terrestris, Gloeococcus maximus, Hormotila blen- & Bold) Tupa 1996 nista, Paulschulzia pseudovolvox, and Tetraspora sp. were aligned Pseudoscour®eldia marina SteinkoÈtter et al. X75565 with other 18S rRNA gene sequences examined in this study us- (Throndsen) KuÈtz. 1994 ing the sequence alignment program Eyeball Sequence Editor for Scenedesmus obliquus (Turp.) Huss and Sogin X56103 the PC (ESEE, Cabot and Beckenbach 1989). The proposed sec- KuÈtz 1990 ondary structure of the SSU ribosomal RNA (rRNA) was used to Tetraspora sp. Link (234) this study U83121 determine the homology of sites within the alignment. The ®nal Volvox carteri Iyengar Rausch et al. 1989 X53904 alignment contained a charophycean