Origin and Evolution of the Colonial Volvocales (Chlorophyceae) as Inferred from Multiple, Chloroplast Gene Sequences

Molecular Phylogenetics and Evolution Vol. 17, No. 2, November, pp. 256 –268, 2000 doi:10.1006/mpev.2000.0831, available online at http://www.idealibrary.com on

Origin and Evolution of the Colonial Volvocales (Chlorophyceae) as Inferred from Multiple, Chloroplast Gene Sequences Hisayoshi Nozaki,* ,1 Kazuharu Misawa,* Tadashi Kajita,† Masahiro Kato,* Seiichi Nohara,‡ and Makoto M. Watanabe‡ *Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; †Botanical Garden, Graduate School of Science, University of Tokyo, Hakusan, Bunkyo-ku, Tokyo 112-0001, Japan; and ‡National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba-shi, Ibaraki 305-0053, Japan Received December 16, 1999; revised June 22, 2000

A combined data set of DNA sequences (6021 bp) from five protein-coding genes of the chloroplast genome (rbcL, atpB, psaA, psaB, and psbC genes) were analyzed for 42 strains representing 30 species of the colonial Volvocales (Volvox and its relatives) and 5 related species of green algae to deduce robust phylogenetic relationships within the colonial green flagellates. The 4-celled family Tetrabaenaceae was robustly resolved as the most basal group within the colonial Volvocales. The sequence data also suggested that all five volvocacean genera with 32 or more cells in a vegetative colony (all four of the anisogamous/oogamous genera, Eudorina, Platydorina, Pleodorina, and Volvox, plus the isogamous genus Yamagishiella) constituted a large monophyletic group, in which 2 Pleodorina species were positioned distally to 3 species of Volvox. Therefore, most of the evolution of the colonial Volvocales appears to constitute a gradual progression in colonial complexity and in types of sexual reproduction, as in the traditional volvocine lineage hypothesis, although reverse evolution must be considered for the origin of certain species of Pleodorina. Data presented here also provide robust support for a monophyletic family Goniaceae consisting of two genera: Gonium and Astrephomene. © 2000 Academic Press

INTRODUCTION Ever since van Leeuwenhoek (1700) first described it, Volvox has been an object of fascination for many biologists because of its distinctive organization in the asexual phase (a rotating sphere containing more than 500 cells that are differentiated into somatic and reproductive cells) and its oogamous sexual reproduction. Traditionally, it has been assumed that Volvox evolved from a simpler colonial green flagellate in the family Volvocaceae when the latter underwent a proTo whom correspondence should be addressed. Fax: ⫹81-3-58028747. E-mail: [email protected]. 1

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gressive increase in the number of Chlamydomonaslike cells per colony, increasing in tendency to differentiate somatic from reproductive cells and increasing in tendency to produce visibly different types of gametes (see Smith, 1950; Pickett-Heaps, 1975; Kirk, 1998). One clear assumption of this sort of volvocine lineage hypothesis was that Volvox evolved from an ancestor resembling modern Pleodorina and represented the endpoint of this evolutionary pathway (e.g., Pickett-Heaps, 1975; Larson et al., 1992; Kirk, 1998). Larson et al. (1992) used a molecular phylogenetic analysis of partial rRNA sequences of 11 taxa in 8 colonial genera to provide support for a monophyletic family Volvocaceae (including Gonium), while simultaneously casting doubt on the monophyly of the genus Volvox and on the traditional assumption that evolution within the Volvocaceae had occurred by a simple, linear progression in size an developmental complexity. The apparent complexity of the group increased when cladistic analyses of morphological data for 26 colonial volvocalean species in 11 genera led to the proposal that certain colonial flagellates that others had included in the family Volvocaceae should be transferred to two new/reappraised, closely related families: the Tetrabaenaceae and the Goniaceae (Nozaki and Ito, 1994; Nozaki et al., 1996). However, although subsequent molecular phylogenetic studies based on coding sequences of chloroplast genes (rbcL, which encodes the large subunit of Rubisco, and/or atpB, which encodes the beta subunit of ATP synthase) of more than 20 colonial species supported the concept of a monophyletic family Tetrabaenaceae, they left the position of the Goniaceae—as well as relationships among Volvox and certain other members of the family Volvocaceae—ambiguous (Nozaki et al., 1997a, 1999), indicating that additional data would be required to resolve such ambiguities and construct a robust phylogeny of the colonial Volvocales. Very recently, Coleman (1999) analyzed internal transcribed spacer (ITS) sequences of multiple isolates representing es-

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sentially all genera and species of the colonial Volvocales and cast doubt on the validity of the Goniaceae and the Tetrabaenaceae. Chloroplast-encoded, protein-coding genes, such as rbcL, are useful for phylogenetic studies of photoautotrophs (see Soltis et al., 1990; Clegg, 1993), because they are single-copy genes with slow evolutionary rates that generally lack introns (but see Nozaki et al., 1998) and therefore have sequences that are readily aligned. Recently, complete DNA sequences of the chloroplast genome have been determined for various groups of photosynthetic eukaryotes (e.g., Ohyama et al., 1986; Kowallik et al., 1995; Reith and Munholland, 1995; Wakasugi et al., 1997), which has facilitated the design of polymerase chain reaction (PCR) primers useful for amplifying various chloroplast protein-coding genes of other photoautotrophs. In the present study, degenerate PCR primers were newly established for amplifying psaA (P700 chlorophyll a-apoprotein A1), psaB (P700 chlorophyll a-apoprotein A2), and psbC (photosystem II CP43 apoprotein) genes of the colonial Volvocales. Also, in an attempt to deduce robust phylogenetic relationships among these colonial green flagellates and to elucidate probable stages in the origin of the genus Volvox, 6021 bp in the coding regions of the combined data set from five chloroplast genes (rbcL, atpB, psaA, psaB, and psbC) were analyzed for 42 strains representing 30 species of colonial volvocaleans and 5 species of other green algae (Table 1). MATERIALS AND METHODS The 42 colonial volvocaleans and 5 related green algae analyzed in this study are listed in Table 1. The methods for culture, preparation of total DNA, and direct sequencing of the PCR products of the five chloroplast genes were essentially the same as those previously described (Nozaki et al., 1995, 1997a,c, 1999), except for the following two aspects. (1) Degenerate PCR primers for amplification and direct sequencing of the psaA, psaB, and psbC genes (Tables 2– 4) were designed on the basis of aligned amino acid sequences encoded by the corresponding genes of various eukaryotic photoautotrophs, after the deduced amino acid sequences of these genes had been aligned using CLUSTAL W (Thompson et al., 1994). (2) After DNA samples from strains that had not previously been studied were extracted with phenol and chloroform (see Nozaki et al., 1995, 1997c), they were further purified using the GFX Genomic Blood Purification Kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The rbcL, atpB, psaA, psaB, and/or psbC genes of some of the operational taxonomic units (OTUs) analyzed in this study contained group I or II introns (Table 5), but only presumptive coding regions were used in this study. The coding regions of the chloroplast genes and the splice sites of the presumptive

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group I or II introns were determined following Cech (1988) and Lambowitz and Belfort (1993), using the GENETYX computer program (Software Development Co., Tokyo, Japan). For phylogenetic analysis, 6021 bp in the combined coding regions of the five chloroplast genes (1128, 1128, 1491, 1494, and 780 bp of rbcL, atpB, psaA, psaB, and psbC genes, respectively; Table 5) of all 47 OTUs (Table 1) could be unambiguously aligned by deleting only a one-codon insertion in the Pandorina morum UTEX 854 psaA gene between nucleotides corresponding to positions 1875 and 1876 of the Chlorella vulgaris psaA gene (Wakasugi et al., 1997). The aligned sequences from 44 OTUs (all but the three strains of Astrephomene; see below) were subjected to unweighted maximum-parsimony (MP) analysis, including a bootstrap analysis (Felsenstein, 1985) based on 1000 replications of the general heuristic search (full heuristic type), using PAUP* 4.0 (Swofford, 1998). Based on the same alignment data, a maximum-likelihood (ML) analysis (based on the HKY85 model with empirical base frequencies and transition/transversion set to 2.0) was carried out using PAUP* 4.0 to estimate quartet puzzling support (QPS) values (which have the same practical meaning as the bootstrap values) for internal branches of the phylogenetic tree with 1000 puzzling steps (comparable to the number of bootstrap replicates) (see Strimmer and von Haeseler, 1996). Since Tajima and Takezaki (1994) suggest that phylogenetic analyses based on the Tajima–Takezaki distances give a more nearly correct tree topology than analyses using Jukes–Cantor (Jukes and Cantor, 1969) or Kimura (Kimura, 1980) distances, we used the Tajima– Takezaki distances to construct neighbor-joining (NJ) trees. In addition, because the first and second nucleotides of codons (1st–2nd) generally exhibit much lower substitution rates than the third nucleotide positions (3rd), a weighted-distance method (K. Misawa and F. Tajima, unpublished) was carried out to calculate Tajima–Takezaki distances based on the same alignment as in the MP analysis. The calculated weights for the 1st–2nd rbcL, 3rd rbcL, 1st–2nd atpB, 3rd atpB, 1st– 2nd psaA, 3rd psaA, 1st–2nd psaB, 3rd psaB, 1st–2nd psbC, and 3rd psbC were 0.744, 0.338, 0.743, 0.327, 0.980, 0.426, 0.981, 0.426, 0.513, and 0.228, respectively. With the NJ algorithm of Saitou and Nei (1987) a phylogenetic tree was constructed, and the robustness of the resulting lineages was tested by a bootstrap analysis with 1000 replications. In these three types of phylogenetic methods, Paulschulzia pseudovolvox and Lobomonas monstruosa served as an outgroup, since these species are members of lineages lying just outside the colonial Volvocales according to phylogenetic trees based on the rbcL and atpB genes (Nozaki et al., 1999, unpublished data). In addition, phylogenetic trees based only on the first and second nucleotides of codons in the combined sequence data set from the five

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TABLE 1 List of the Colonial Volvocalean Taxa/Strains and Related Species Included in the Phylogenetic Analysis and DDBJ/EMBL/GenBank Accession Nos. of the Five Chloroplast Genes Accession No. Taxa Tetrabaenaceae a Tetrabaena socialis (⫽ Gonium sociale) Basichlamys sacculifera (⫽ Gonium sacculiferum) Goniaceae a Gonium pectorale Gonium octonarium Gonium quadratum

Strain designation

atpB

rbcL

psaA

psaB

psbC

NIES b-571

AB014014 c

D63443 d

AB044415

AB044466

AB044525

NIES-566

AB014015 c

D63430 d

AB044416

AB044467 & AB044468

AB044526

NIES-569

D63437 d

AB044242

AB044463

AB044521

GO-LC-1⫹ NIES-653

AB014016 c & AB014017 c AB014018 c AB014019 c

D63436 d D63438 d

AB044241 AB044243

AB044462 AB044464

Gonium multicoccum

UTEX e 2580

AB014020 c

D63435 d

AB044461

Gonium viridistellatum Astrephomene gubernaculifera

UTEX 2519 NIES-418

D86831 f D63428 d

UTEX 1394

AB014021 c AB014022 c & AB014023 c AB044181

AB044239 & AB044240 AB044244 AB044233 & AB044234 AB044235

AB044520 AB044522 & AB044523 AB044481

Astrephomene perforata

NIES-564

AB014024 c

D63429 d

AB044236 & AB044237 & AB044238

AB044460

Volvocaceae a Pandorina morum

NIES-574

D63442 d

AB044226

AB044452

AB044505

UTEX 854

AB014025 c & AB014026 c AB044180

AB044167

AB044231

AB044456

UTEX 880

AB044179

AB044166

AB044455

UTEX 1727 UTEX 2326

AB044178 AB044177

AB044165 AB044164

AB044229 & AB44230 AB04428 AB044227

AB044510 & AB044511 AB044509

Pandorina colemaniae Volvulina pringsheimii Volvulina compacta

NIES-572 UTEX 1020 NIES-582

AB014027 c AB014028 c AB014029 c

D63441 d D63444 d D86832 f

AB044457 AB044447 AB044446

Volvulina steinii

UTEX 1525 UTEX 1531

AB044174 AB044175

AB044160 AB044161

AB044232 AB044220 AB044217 & AB044218 & AB044219 AB044223 AB044224

NIES-545

AB044173

AB044159

AB044448

Volvulina boldii

UTEX 2185

AB044176

AB044451

AB044504

Yamagishiella unicocca

UTEX 2428 UTEX 2430

AB014030 c AB014031 c

AB044162 & AB044163 D86823 f D86825 f

AB044221 & AB044222 AB044225

AB044501 AB044502 & AB044503 AB044500

AB044443 AB044444

AB044495 AB044496

Platydorina caudata

Nozaki E-5 UTEX 1658

AB044172 AB014032 c

AB044168 D86828 f

AB044445 AB044442

AB044497 AB044494

Eudorina cylindrica Eudorina unicocca

UTEX 1197 UTEX 1215

AB014033 c AB014007 c

D86833 f D63434 d

AB044441 AB044440

AB044493 AB044491 & AB044492

UTEX 737

AB014008 c

D86829 g

AB044439

AB044489 & AB044490

AB044169 & AB044170

AB044213 AB044214 & AB044215 AB044216 AB044211 & AB044212 AB044210 AB044207 & AB044208 & AB044209 AB044204 & AB044205 & AB044206

AB044465 AB044458 AB044459

AB044454 AB044453

AB044449 AB044450

AB044524 AB044513 & AB044514 AB044515 & AB044516 & AB044517 AB044518 & AB044519

AB044508 AB044506 & AB044507 AB044512 AB044499 AB044498

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TABLE 1—Continued Accession No. Taxa Eudorina elegans

Strain designation

atpB

rbcL

NIES-456 UTEX 1205

AB014009 c AB014010 c

D63432 d D88805 g

UTEX 1212

AB014012 c

D88806 g

c

d

psaA

psaB

AB044199 AB044200 & AB044201 AB044202 & AB044203 AB0440198 AB044190 & AB044191 & AB044192 AB044193 & AB044194 AB044195 & AB044196 & AB044197 AB044182

AB044435 AB044436 & AB044437 AB044438

psbC AB044485 AB044486

AB044434 AB044430

AB044487 & AB044488 AB044484 AB044480

AB044431

AB044482

AB044432 & AB044433

AB044483

AB044424

AB044474

AB044184 & AB044185

AB044425

AB044475

D63447 d

AB044183

AB044426

AB044476

AB014003 c AB014001 c AB014002 c

D63448 d D86835 f D86836 f

AB044188 AB044186 AB044187

AB044429 AB044427 AB044428

AB044479 AB044477 AB044478

137C

M13704 h

J01399 i

AB044419

AB044470

AB044528

UTEX 1344

AB014034 c

D86838 f

AB044469

AB044527

Nozaki S-4 NIES-474

AB014036 c AB044533

AB014041 c AB044171

AB044417 & AB044418 AB044420 AB044421

AB044471 AB044472

AB044529 AB044530

UTEX 167

AB014040 c

D86837 f

AB044473

AB044531 & AB044532

Eudorina illinoisensis Pleodorina californica

NIES-460 UTEX 809

AB014013 AB014004 c

D63433 D63439 d

Pleodorina japonica

UTEX 2523

AB014005 c

D63440 d

Pleodorina indica

UTEX 1990

AB014006 c

D86834 f

Volvox (sect. Janetosphaera) aureus Volvox (sect. Merrillosphaera) carteri Volvox (sect. Copelandosphaera) dissipatrix Volvox (sect. Volvox) rousseletii barberi globator Chlamydomonadaceae Chlamydomonas reinhardtii Chlamydomonas debaryana Vitreochlamys ordinata Lobomonas monstruosa Tetrasporaceae Paulschulzia pseudovolvox

NIES-541

AB013998 c

D63445 d

NIES-905

AB013999 c

D63446 d

UTEX 2184

AB014000 c

UTEX 1862 UTEX 804 UTEX 955

AB044422 & AB044423

a

Colonial volvocalean families proposed by Nozaki et al. (1996). Microbial Culture Collection at the National Institute for Environmental Studies (Watanabe and Hiroki, 1997). c Data from Nozaki et al. (1999). d Data from Nozaki et al. (1995). e Culture Collection of Algae at the University of Texas at Austin (Starr and Zeikus, 1993). f Data from Nozaki et al. (1997a). g Data from Nozaki et al. (1997b). h Data from Woessner et al. (1986). i Data from Dron et al. (1982). b

chloroplast genes were also constructed using all 47 OTUs (Table 1) and the methods described above. In the weighted-distance method (K. Misawa and F. Tajima, unpublished), the calculated weights for 1st–2nd rbcL, 1st–2nd atpB, 1st–2nd psaA, 1st–2nd psaB, and 1st–2nd psbC were 0.744, 0.742, 0.980, 0.981, and 0.513, respectively. Pairwise distances (p-distances) based on the combined data set from five chloroplast genes between the ingroup OTUs were calculated by PAUP* 4.0.

RESULTS Substitutions of Nucleotides P-distances between 45 ingroup OTUs based on the first and second nucleotides of the codons in the 6021 bp of combined coding sequences for the five chloroplast genes were less than 0.05, whereas those for the third nucleotides of the codons generally ranged from 0.1 to 0.3. Furthermore, the latter type of p-distances between Astrephomene and other genera were espe-

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TABLE 2 Primers Used for Amplifications and Sequencing of the PsaA Genes Designations

Positions a

Sequence (5⬘–3⬘)

psaA-F2 psaA-F5 psaA-R6 psaA-F8 psaA-R9 psaA-F7 psaA-R3 psaA-F11 psaA-R12 psaA-R4

532–551 1012–1031 1097–1076 b 1192–1214 1259–1238 b 1282–1304 1385–1366 b 1552–1574 1793–1772 b 2096–2077 b

TGGTT(TC)CA(TC)TA(TC)CA(TC)AA(AG)GC CA(CT)GT(TA)GGTTTATA(TC)GA(AG)AT AT(AG)AT(TA)GATAA(TC)GA(AC)CC(AG)AATA GGTGGTTT(CT)TGTATTGTIGGTGC GT(AT)GGATC(AG)TA(GA)TCACG(AT)ACCA GA(CT)CGTGT(AT)AT(ATC)CG(TC)CACCGTGA ATIGT(AG)TC(AG)TT(AG)TGIAT(AG)TA GG(TC)GG(TC)AAAGT(AT)GCIATGATGCC GG(AG)CC(AG)TCACA(AT)GGGAA(AG)CGGA TCIATIA(AG)(TC)TC(TC)TGCCA(AG)TA

a b

Coordinate number from the Chlorella vulgaris psaA gene (Wakasugi et al. 1997). Reverse primer.

cially high: they ranged from 0.241 to 0.315 (88% were more than 0.26), whereas such p-distances among the other 42 ingroup OTUs were all less than 0.270, and 98% were less than 0.26. To examine whether the third nucleotides of codons in these five genes exhibit saturation of substitutions, p-distances among ingroup OTUs based on the third nucleotides were compared with those of the first and second nucleotides of the same codons. The resulting data indicate that these third nucleotide positions have been saturated with substitutions in the genus Astrephomene, but not in the other genera (Fig. 1). Consequently, only the first and second nucleotides were considered for phylogenetic analyses in which the genus Astrephomene was included, whereas all three positions were considered in phylogenetic analyses in which only the other 44 OTUs were included. Phylogenetic Analyses Bootstrap 50% majority-rule consensus NJ trees based on two kinds of sequence data are presented in Figs. 2 and 3, and branches that are resolved with 50% or more bootstrap or QPS values by the MP and/or ML analyses are indicated. Based on all 6021 bp in the five chloroplast genes from 44 OTUs (excluding Astrephomene), high boot-

strap/QPS values (96, 89, or 82% in the NJ, MP, or ML analysis, respectively) suggested that the three colonial volvocalean families (the Volvocaceae, Goniaceae, and Tetrabaenaceae) constituted a large monophyletic group, in which the Tetrabaenaceae (Tetrabaena and Basichlamys) was positioned most basally with high bootstrap/QPS values (97, 94, or 71% in the NJ, MP, or ML analysis, respectively) (Fig. 2). Furthermore, the family Volvocaceae was resolved as a monophyletic group with relatively high bootstrap values (69 – 88%) in the NJ and MP analyses, and this family was shown to be sister to the genus Gonium (Fig. 2). The family Volvocaceae was further subdivided into two monophyletic groups. One was composed of the two isogamous genera Pandorina and Volvulina, and its monophyly was supported by high bootstrap/QPS values (77–98%) in the NJ, MP, and ML methods (Fig. 2). The other clade (Volvox–Yamagishiella group) was resolved with relatively low bootstrap/QPS values (56 – 69%) in NJ and ML calculations and consisted of all of the members of five volvocacean genera, Yamagishiella, Eudorina, Platydorina, Pleodorina, and Volvox. In the Volvox–Yamagishiella group, Platydorina and Volvox section Volvox were positioned most basally (although their robust phylogenetic positions were not

TABLE 3 Primers Used for Amplifications and Sequencing of the PsaB Genes Designations

Positions a

Sequence (5⬘–3⬘)

psaB-F1 psaB-R4 psaB-F3 psaB-F5 psaB-R2 psaB-R6

205–224 635–616 b 869–890 989–1010 1114–1095 b 1760–1741 b

GCITGGCA(AG)GGIAA(TC)TT(TC)GA AA(AG)TTATCCCAACC(AT)AC(AG)TG TGTA(CT)CG(TC)AC(TG)AA(CT)TTTGGTAT TACA(CT)TTCCAATTAGG(TC)TTAGC AT(AG)TA(TC)TG(AG)TG(AG)TGIGT(AG)TA ATIGT(AG)TTIA(AG)CATCCA(AG)AA

a b

Coordinate number from the Chlorella vulgaris psaB gene (Wakasugi et al., 1997). Reverse primer.

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TABLE 4 Primers Used for Amplifications and Sequencing of the PsbC Genes Designations

Positions a

Sequence (5⬘–3⬘)

psbC-F1 psbC-F3 psbC-R2 psbC-R4 psbC-F5 psbC-R6 psbC-R8 psbC-F7

154–173 233–254 1079–1060 b 1199–1180 b 593–614 893–872 b 704–685 b 742–761

GCICA(TC)GTICA(TC)GCIGG A(AG)AA(AG)CCIATGTA(TC)GA(AG)CA(AG)GG TCCCA(AG)AAIC(GT)CATIGT(TC)TC GGIGC(AG)TGIGTCAT(AG)TA(TC)TC T(TC)AT(TC)AC(AT)AA(TC)CCAAC(AT)ACAAA GG(GA)TA(AT)GC(AT)GT(GA)TT(AG)TT(AG)AACC CC(AG)ATAATATCTTCCA(TG)(AG)TT GG(TG)ATTTGGCACATTTATAC

a b

Coordinate number from the Chlorella vulgaris psbC gene (Wakasugi et al., 1997). Reverse primer.

resolved), and the genus Yamagishiella was resolved as a sister group to the Eudorina group (which was composed of Eudorina, Pleodorina, and Volvox, excluding the section Volvox; see Nozaki et al., 1999) with relatively high bootstrap/QPS values (76 – 85%) in the NJ and ML analyses (Fig. 2). Furthermore, the Eudorina group was subdivided into two subgroups: one subgroup was robustly monophyletic and composed of three species of Volvox (excluding the section Volvox) and two species of Pleodorina (P. californica and P. japonica), whereas the other subgroup was basal or paraphyletic, comprising Pleodorina indica and all of the members of the genus Eudorina that were studied. In the former subgroup, Volvox dissipatrix was positioned most basally and Volvox aureus was sister to the clade composed of the two Pleodorina species (Fig. 2). When only the first and second nucleotides of each codon from all 47 OTUs were analyzed, most of the phylogenetic relationships resolved were essentially consistent with the results obtained using all three nucleotides of each codon. However, the phylogenetic relationships derived using only the first and second nucleotides of each codon were generally less robust (Fig. 3) than those derived using all three nucleotides (Fig. 2), possibly because of limited phylogenetic information as a consequence of the low substitution rates in the first and second nucleotides (see Fig. 1). The NJ and ML methods suggested with 61– 82% bootstrap/ QPS values that all three colonial families constituted a monophyletic group in which the Tetrabaenaceae was positioned most basally (Fig. 3). Monophyly of the Volvocaceae was resolved with a bootstrap value of only 66% in the NJ calculation, whereas somewhat higher bootstrap/QPS values (73–74% in the NJ and ML analyses) supported the concept of a monophyletic family Goniaceae, in which Gonium and Astrephomene are sister taxa (Fig. 3). In addition, 66 –72% bootstrap/ QPS values in the NJ and ML calculations suggested that the genus Yamagishiella is a sister group to the Eudorina group, in which three species of Volvox (excluding the section Volvox) and two species of Pleodo-

rina (P. californica and P. japonica) are resolved as a monophyletic group by all three phylogenetic methods, although with relatively low bootstrap/QPS values (57–76%) (Fig. 3). Distribution of Presumptive Group I and II Introns Inserted in the Five Genes In total, 58 intervening sequences or introns were recognized in the five chloroplast genes of the 47 OTUs analyzed in this study. They were classified into 52 group I and 6 group II introns (Table 5) on the basis of presumptive splice sites of the genes (Cech, 1988; Lambowitz and Belfort, 1993). The highest frequency of such introns was detected in the psaA genes (22 group I and 2 group II introns) among the five chloroplast genes, whereas only 3 group I introns were recognized in the psaB genes, and the atpB genes contained only 3 group II introns (Table 5). Splice sites of group I or II introns in certain OTUs were identical in each of the five chloroplast genes (Table 5). However, such introns seem to be almost discrete in distribution of the present phylogenetic trees (Figs. 2 and 3), suggesting recent horizontal transmission of the introns within the colonial Volvocales (see Nozaki et al., 1998). DISCUSSION Early Evolutionary Events of the Colonial Volvocales Nozaki and Ito (1994), on the basis of the cladistic analysis of morphological data, proposed a new family, the Tetrabaenaceae, to encompass the four-celled species Tetrabaena socialis (⫽ Gonium sociale). Later, Nozaki et al. (1996) assigned another four-celled alga Basichlamys sacculifera (⫽ Gonium sacculiferum) to this family, based on their further cladistic analysis of morphological data. However, the phylogenetic position of the Tetrabaenaceae within the colonial Volvocales has been ambiguous in previous molecular phylogenetic studies of the colonial Volvocales based on the rbcL and/or atpB gene sequences (Nozaki et al., 1995,

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TABLE 5 Positions of Nucleotide Sequences Analyzed in This Study for Five Chloroplast Genes and Distribution of Presumptive Groups I and II Introns Inserted in the Genes Chloroplast genes [position analyzed] a RbcL Gene [31–1158]

AtpB Gene [229–1356]

PsaA Gene [568–2058]

PsaB Gene [238–1731]

PsbC Gene [265–1044]

OTUs and presumptive introns inserted in the genes (5⬘ position of insertion in the coding regions) a Volvulina compacta: Group I (462, 699) b Volvulina boldii: Group I (699) Pleodorina californica: Group I (462) c Pleodorina japonica: Group I (462) c Pleodorina indica: Group I (462, 699) b Gonium multicoccum: Group I (462, 699) c Gonium viridistellatum: Group I (462, 699) b Astrephomene gubernaculifera NIES 418: Group I (462) c UTEX 1394: Group I (462) Pandorina morum NIES-574: Group II (958) d Astrephomene gubernaculifera NIES 418: Group II (958) d Gonium pectorale: Group II (958) d Volvox carteri: Group I (1605) Pleodorina californica: Group I (1104, 1410) Pleodorina japonica: Group I (1605) Pleodorina indica: Group I (1104, 1605) Eudorina elegans UTEX 1205: Group I (1605) Eudorina elegans UTEX 1212: Group I (1104) Eurodina unicocca UTEX 737: Group I (1104, 1605) UTEX 1215: Group I (1104, 1605) Platydorina caudata: Group II (1797) Yamagishiella unicocca UTEX 2430: Group I (1104) Volvulina steinii NIES-545: Group I (1104) Volvulina compacta: Group I (1104, 1605) Pandorina morum UTEX 880: Group I (1104) Gonium multicoccum: Group I (1605) Astrephomene gubernaculifera NIES-418: Group I (844) Astrephomene perforata: Group I (844) & Group II (1797) Chlamydomonas debaryana: Group I (1605) Paulschulzia pseudovolox: Group I (991) Pleodorina indica: Group I (294) Eudorina elegans UTEX 1205: Group I (294) Basichlamys sacculifera: Group I (1050) Eudorina elegans UTEX 1212: Group I (708) Eudorina unicocca UTEX 737: Group I (579) UTEX 1215: Group I (579) Pandorina morum UTEX 2326: Group I (296) Pandorina morum UTEX 854: Group I (918) Volvulina steinii UTEX 1531: Group I (579) Astrephomene gubernaculifera NIES-418: Group I (579) UTEX 1394: Group I (708, 918) Astrephomene perforata: Group II (625) Gonium multicoccum: Group I (579, 708, 918) Gonium quadratum: Group I (708) Paulschulzia pseudovolox: Group I (579)

a

Coordinate number from the Chlorella vulgaris rbcL, atpB, psaA, psaB, and psbC genes (Wakasugi et al., 1997). Data from Nozaki et al. (1997a). c Data from Nozaki et al. (1995). d Data from Nozaki et al. (1999). b

1997a, 1999). In the present study, the 6021 bp of the combined data set from five chloroplast genes suggested that the three families of the colonial Volvocales (the Volvocaceae, Goniaceae, and Tetrabaenaceae), taken together, are monophyletic, and the Tetrabaen-

aceae was resolved as a sister group to the monophyletic group composed of the Volvocaceae and the Goniaceae (Figs. 2 and 3). This is consistent with the cladistic analyses of morphological data (Nozaki and Ito, 1994; Nozaki et al., 1996) and the recent study of

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FIG. 1. Comparison of p-distances between ingroup OTUs related to the three genera of the colonial Volvocales (Eudorina [E. cylindrica and E. elegans NIES-456], Gonium [G. pectorale and G. quadratum], and Astrephomene [A. gubernaculifera NIES-418 and A. perforata] based on the 1st–2nd nucleotides of codons and on the 3rd nucleotides of codons, in the 6021/3 codons of the combined data set from five chloroplast genes used for the present phylogenetic analyses (Table 5). P-distances between Astrephomene and Eudorina/Gonium are presented by “Astrephomene”. Arrow indicates p-distance between NIES-418 and UTEX 1394 of Astrephomene gubernaculifera.

the distribution of the xanthophyll loroxanthin within the colonial Volvocales (Schagerl and Angeler, 1998) which supports the separation of the Tetrabaenaceae from the Goniaceae. All of the isogamous members of the Volvocaceae and Goniaceae have eight or more cells in a vegetative colony and exhibit tubular mating structures (bilateral mating papillae) in each of the two conjugating gametes, except for Gonium multicoccum, which has no tubular mating structures at all (Nozaki and Kuroiwa, 1991; Nozaki and Ito, 1994). In contrast, only one of the conjugating isogametes bears tubular mating structures (unilateral mating papilla) in Tetrabaena socialis (Nozaki, 1986) and the unicellular green alga Chlamydomonas reinhardtii (see Harris, 1989), which assume basal positions in the present phylogenetic trees (Figs. 2 and 3). Therefore, after four-celled colonial forms similar to the Tetrabaenaceae had appeared, an increase in colony cell number (to eight or more) and a transition from unilateral to bilateral mating papillae appears to have occurred in the common ancestor of the Volvocaceae and Goniaceae. This advanced type of mating structure might somehow have accelerated the further increase in colony cell number and colonial complexity during subsequent evolution. On the basis of ITS sequences, Coleman (1999) demonstrated phylogenetic relationships of the colonial Volvocales which are inconsistent with those of the present molecular phylogenetic analyses in certain aspects (Figs. 2 and 3). She resolved with 50 –75% bootstrap values that the Tetrabaenaceae and the genus

Gonium are sister taxa and that Astrephomene is positioned most basally within the colonial Volvocales. This discrepancy in the basal phylogenetic relationships of the colonial Volvocales may result from the difference in the unicellular OTUs analyzed but does not relate to the lack of Astrephomene in the present molecular phylogenetic analyses using all three nucleotides in each codon. Coleman (1999) analyzed only a single unicellular OTU (Chlamydomonas reinhardtii), whereas the present phylogenetic study used C. reinhardtii and three other unicellular species, of which C. debaryana and Vitreochlamys ordinata were robustly resolved as a sister group to the whole colonial Volvocales (Fig. 2). When Astrephomene was included in the present molecular phylogenetic analyses using all three nucleotides of the codons, the most basal position of the Tetrabaenaceae within the colonial Volvocales was also robustly resolved, but Astrephomene was positioned basally to the Pandorina–Volvulina lineage (not shown). Origin and Evolution of the Anisogamous/Oogamous Volvocacean Genera The volvocacean algae have traditionally been considered to exhibit evolution of sexual reproduction from isogamy to anisogamy to oogamy (e.g., Smith, 1955; Pickett-Heaps, 1975). They can be classified into three isogamous genera (Pandorina, Volvulina, and Yamagishiella), three anisogamous genera (Eudorina, Platydorina, and Pleodorina), and one oogamous genus (Vol-

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FIG. 2. Bootstrap 50% majority-rule consensus NJ tree (based on the Tajima–Takezaki distance calculated by the weighted-distance method) using all 6021 nucleotides in the combined data set from the coding regions of rbcL, atpB, psaA, psaB, and psbC genes of 44 OTUs (excluding only the three strains of Astrephomene) (Table 1). Numbers above the branches are the bootstrap values (50% or more) based on 1000 replications of the NJ analysis. Branches also resolved with 50% or more bootstrap or QPS values (following 1000 replications) by the MP and/or ML analyses are also shown by numbers under the branches without brackets or with brackets, respectively.

vox) (e.g., Nozaki, 1996). Nozaki and Ito (1994), employing a cladistic analysis of morphological data, concluded that all of the anisogamous/oogamous members of the Volvocaceae constitute a monophyletic group and suggested that anisogamy with sperm packets (bundles of male gametes/sperm) evolved only once in this family. However, molecular phylogenetic studies of a large number of the colonial volvocalean species based on the rbcL and/or atpB gene sequences (Nozaki et al., 1997a, 1999) and ITS sequences (Coleman, 1999) did not robustly resolve phylogenetic relationships be-

tween the isogamous and the anisogamous/oogamous members of the Volvocaceae, although three anisogamous/oogamous phylogenetic groups (Volvox section Volvox, the Eudorina group, and Platydorina) were suggested in the rbcL–atpB gene phylogeny (Nozaki et al., 1997a, 1999). The present phylogenetic analyses based on multiple chloroplast genes resolved a monophyletic group (the Volvox–Yamagishiella group) composed of Volvox section Volvox, Platydorina, the Eudorina group, and the isogamous genus Yamagishiella (Fig. 2). All five genera belonging to the Volvox–Ya-

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FIG. 3. Bootstrap 50% majority-rule consensus NJ tree (based on the Tajima–Takezaki distance calculated by the weighted-distance method) using only the first and second nucleotides of codons in the combined data set from the coding regions of rbcL, atpB, psaA, psaB, and psbC genes of all 47 OTUs (Table 1). Numbers above the branches are the bootstrap values (50% or more) based on 1000 replications of the NJ analysis. Branches also resolved with 50% or more bootstrap or QPS values (following 1000 replications) by the MP and/or ML analyses are also shown by numbers under the branches without brackets or with brackets, respectively.

magishiella group have vegetative colonies with 32 or more cells enclosed by the cellular envelopes, whereas other volvocacean genera, Pandorina and Volvulina, have up to 16 cells in a colony without cellular envelopes (Nozaki and Kuroiwa, 1992; Nozaki and Ito, 1994). Therefore, after a 32-celled colony with cellular envelopes had evolved, multiple (at least two) phylogenetic lines giving rise to the oogamous genus Volvox might have appeared within the Volvocaceae. The present sequence data suggest that the isogamous genus Yamagishiella is sister to the Eudorina group within the Volvox–Yamagishiella group (Fig. 2), although the phylogenetic relationships between Vol-

vox section Volvox, Platydorina, and the clade composed of Yamagishiella and the Eudorina group remain ambiguous (Fig. 2). In addition, Eudorina exhibits paraphyletic or basal phylogenetic status within the Eudorina group (Fig. 2). Both Yamagishiella and Eudorina have 32-celled, spheroidal vegetative colonies without differentiation of obligately somatic cells (Nozaki and Kuroiwa, 1992). Therefore, Yamagishiella can be considered to be an ancestral form of the Eudorina group, and parallel evolution of anisogamy/oogamy with sperm packets might have occurred in the common ancestor of the Eudorina group, as well as in the ancestors of Volvox section Volvox and

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Platydorina. Alternatively, evolution of anisogamy with sperm packets might have occurred only once in the common ancestor of the Volvox–Yamagishiella group, and reverse evolution from anisogamy with sperm packets to isogamy might have occurred in the origin of the genus Yamagishiella. Phylogenetic Status of the Volvocaceae and the Goniaceae The colonial volvocalean algae analyzed in this study were traditionally classified into the Volvocaceae and the Astrephomenaceae (e.g., Starr, 1980). However, Matsuda et al. (1987) suggested a close phylogenetic relationship between Gonium (including the fourcelled species now assigned to the Tetrabaenaceae) and Astrephomene on the basis of their common sensitivity to a cell wall lytic enzyme of Chlamydomonas reinhardtii, to which other colonial Volvocales were insensitive. Based on the vegetative ultrastructure of the extracellular matrix. Nozaki and Kuroiwa (1992) removed the genus Gonium from the Volvocaceae and classified Astrephomene and Gonium in a single family, the Goniaceae. Subsequently, Nozaki and Ito (1994), on the basis of the cladistic analysis of morphological data, resolved two monophyletic groups essentially consistent with the Volvocaceae and the Goniaceae, from which the four-celled species was removed to the Tetrabaenaceae (Nozaki and Ito, 1994). However, the phylogenetic relationships of these three colonial families were ambiguous in previous molecular phylogenetic studies of the colonial Volvocales (Larson et al., 1992; Buchheim et al., 1994; Nozaki et al., 1995, 1997a, 1999), although Coleman (1999) very recently resolved the monophyly of the Volvocaceae but the nonmonophyly of the Goniaceae (with the most basal position of Astrephomene within the colonial Volvocales; see above) with 50 –75% bootstrap values. In the present study, robust phylogenetic status for the Volvocaceae and the Goniaceae was resolved with sequence data from five chloroplast genes. Since saturation of substitutions in the third nucleotides in the sequenced codons apparently has occurred in the genus Astrephomene (Fig. 1), phylogenetic analyses based on only the first and second nucleotides of the codons were carried out, with the results that monophyly of the Goniaceae was resolved with relatively high bootstrap/ QPS values in the NJ and ML analyses (Fig. 3). Although the family Volvocaceae was resolved robustly as a monophyletic group only in the NJ method that was based on the first and second nucleotides of the codons (Fig. 3), the NJ and MP analyses (excluding Astrephomene) using all nucleotides in these codons resolved the monophyly of this family with relatively high bootstrap values (Fig. 2). Therefore, the separation of these two colonial families, as well as the Tetrabaenaceae that was proposed on the basis of previous morphological studies (Nozaki and Ito, 1994; Nozaki et

al., 1996), is supported by the present molecular phylogenetic analyses. Evolution of the Genus Pleodorina Since the anisogamous genus Pleodorina has 64 or 128 cells in a vegetative colony and exhibits differentiation of obligately small somatic cells (see Nozaki and Kuroiwa, 1992), it represents the closest ancestral form of the oogamous genus Volvox in the traditional volvocine hypothesis (e.g., Larson et al., 1992; Kirk, 1998). However, the present sequence data resolved that three species of Volvox (excluding section Volvox), Pleodorina californica, and P. japonica constitute a robust clade in which the two Pleodorina species are positioned distally and Volvox dissipatrix exhibits the most basal position (Fig. 2). Since no morphological features supporting such phylogenetic relationships seem to be available, the distal phylogenetic position of the Pleodorina species seems unusual. However, phylogenetic analysis based on only the first and second nucleotides of all codons also resolved such a relationship between the two Pleodorina species and the three species of Volvox (Fig. 3). Similarly, 18S and 28S rRNA sequence data suggested a phylogenetic position of P. californica distal to several taxa of Volvox (Larson et al., 1992; Kirk, 1998). Therefore, it appears that P. californica and P. japonica might have originated from a Volvox-like alga via a decrease in colony cell number and size, as well as by reverse evolution from oogamy to anisogamy. On the other hand, the present molecular phylogenetic analyses suggested that Pleodorina indica was positioned distally to the genus Eudorina and separated from two other species of Pleodorina (Fig. 2). Therefore, P. indica might have evolved from a Eudorina-like, 32-celled alga by an increase in colony cell number and differentiation of obligately somatic cells. Thus, on the basis of the present molecular phylogenetic analysis, neither of these two separate phylogenetic lines of the genus Pleodorina appears to represent the direct ancestral form of any member of the genus Volvox. As revealed by our molecular phylogenetic analyses, most of the evolutionary tendencies in the colonial Volvocales appear to be consistent with the traditional volvocine lineage hypothesis, in that the overall evolution of the group shows a gradual progression in colonial complexity and in types of sexual reproduction, especially in the origin of the Volvocaceae–Goniaceae and the Volvox–Yamagishiella group (Fig. 2). However, reverse evolution from a Volvox-like alga should be considered with respect to the origin of P. californica and P. japonica within the Volvocaceae (Figs. 2 and 3). Therefore, these Pleodorina species may represent one of the limits of the colonial development in the volvocine series of the colonial Volvocales.

MOLECULAR PHYLOGENY OF THE COLONIAL VOLVOCALES

ACKNOWLEDGMENTS We are grateful to anonymous reviewers who kindly and dramatically improved the text. This study was in part supported by a Grant-in-Aid for Scientific Research (No. 11440241 to H.N.) from the Ministry of Education, Science and Culture, Japan, and by a Grantin-Aid for a Research Project on Conservation Methods for Subtropical Island Ecosystems (to S.N.) from the Environmental Agency, Japan.

REFERENCES Buchheim, M. A., McAuley, M. A., Zimmer, E. A., Theriot, E. C., and Chapman, R. L. (1994). Multiple origins of colonial green flagellates from unicells: Evidence from molecular and organismal characters. Mol. Phylogenet. Evol. 3: 322–343. Cech, T. R. (1988). Conserved sequences and structures of group I introns: Building an active site for RNA catalysis—Review. Gene 73: 259 –271. Clegg, M. T. (1993). Chloroplast gene sequences and the study of plant evolution. Proc. Nat. Acad. Sci. USA 90: 363–367. Coleman, A. W. (1999). Phylogenetic analysis of “Volvocaceae” for comparative genetic studies. Proc. Nat. Acad. Sci. USA 96: 13892– 13897. Dron, M., Rahire, M., and Rochaix, J.-D. (1982). Sequence of the chloroplast DNA region of Chlamydomonas reinhardii containing the gene of the large subunit of ribulose bisphosphate carboxylase and parts of its flanking genes. J. Mol. Biol. 162: 775–793. Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using bootstrap. Evolution 38: 16 –24. Harris, E. H. (1989). “The Chlamydomonas Sourcebook,” Academic Press, San Diego. Jukes, T. H., and Cantor, C. R. (1969). Evolution of protein molecules. In “Mammalian Protein Metabolism” (H. N. Munro, Ed.), pp. 21–132. Academic Press, New York. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111–120. Kirk, D. L. 1998. “Volvox: Molecular Genetic Origins of Multicellularity and Cellular Differentiation,” Cambridge Univ. Press, Cambridge, UK. Kowallik, K. V., Stoebe, B., Schaffran, I., Kroth-Pancic, P., and Freier, U. (1995). The chloroplast genome of a chlorophyll a ⫹ c-containing alga, Odontella sinensis. Plant Mol. Biol. Rep. 13: 336 –342. Lambowitz, A. M., and Belfort, M. (1993). Introns as mobile genetic elements. Annu. Rev. Biochem. 62: 587– 622. Larson, A., Kirk, M. M., and Kirk, D. L. (1992). Molecular phylogeny of the volvocine flagellates. Mol. Biol. Evol. 9: 85–105. Matsuda, Y., Musgrave, A., van den Ende, H., and Roberts, K. (1987). Cell wall of algae in the Volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide of Chlamydomonas reinhardtii. Bot. Mag. Tokyo 100: 373–384. Nozaki, H. (1986). Sexual reproduction in Gonium sociale (Chlorophyta, Volvocales). Phycologia 25: 29 –35. Nozaki, H. (1996). Morphology and evolution of sexual reproduction in the Volvocaceae (Chlorophyta). J. Plant Res. 109: 353–361. Nozaki, H., and Ito, M. (1994). Phylogenetic relationships within the colonial Volvocales (Chlorophyta) inferred from cladistic analysis based on morphological data. J. Phycol. 30: 353–365. Nozaki, H., Ito, M., Sano, R., Uchida, H., Watanabe, M. M., and Kuroiwa, T. (1995). Phylogenetic relationships within the colonial Volvocales (Chlorophyta) inferred from rbcL gene sequence data. J. Phycol. 31: 970 –979.

267

Nozaki, H., Ito, M., Sano, R., Uchida, H., Watanabe, M. M., Takahashi, H., and Kuroiwa, T. (1997a). Phylogenetic analysis of Yamagishiella and Platydorina (Volvocaceae Chlorophyta) based on rbcL gene sequences. J. Phycol. 33: 272–278. Nozaki, H., Ito, M., Uchida, H., Watanabe, M. M., and Kuroiwa, T. (1997b). Phylogenetic analysis of Eudorina species (Volvocaceae, Chlorophyta) based on rbcL gene sequences. J. Phycol. 33: 859 – 863. Nozaki, H., Ito, M., Watanabe, M. M., and Kuroiwa, T. (1996). Ultrastructure of the vegetative colonies and phylogenetic position of Basichlamys (Volvocales, Chlorophyta). Eur. J. Phycol. 31: 67–72. Nozaki, H., Ito, M., Watanabe, M. M., Takano, H., and Kuroiwa, T. (1997c). Phylogenetic analysis of morphological species of Carteria (Volvocales, Chlorophyta) based on rbcL gene sequences. J. Phycol. 33: 864 – 867. Nozaki, H., and Kuroiwa, T. (1991). Morphology and sexual reproduction of Gonium multicoccum (Volvocales, Chlorophyta) from Nepal. Phycologia 30: 381–393. Nozaki, H., and Kuroiwa, T. (1992). Ultrastructure of the extracellular matrix and taxonomy of Eudorina, Pleodorina and Yamagishiella gen. nov. (Volvocaceae, Chlorophyta). Phycologia 31: 529 –541. Nozaki, H., Ohta, N., Takano, H., and Watanabe, M. M. (1999). Reexamination of phylogenetic relationships within the colonial Volvocales (Chlorophyta): An analysis of atpB and rbcL gene sequences. J. Phycol. 35: 104 –112. Nozaki, H., Ohta, N., Yamada, T., and Takano, H. (1998). Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae). Plant Mol. Biol. 37: 77– 85. Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T., Sano, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S., Inokuchi, H., and Ozeki, H. (1986). Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574. Pickett-Heaps, J. D. (1975). “Green Algae: Structure, Reproduction and Evolution in Selected Genera,” Sinauer, Sunderland, MA. Reith, M. E., and Munholland, J. (1995). Complete nucleotide sequence of the Porphyra purpurea chloroplast genome. Plant Mol. Biol. Rep. 13: 333–335. Saitou, N., and Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406 – 425. Schagerl, M., and Angeler, D. G. (1998). The distribution of the xanthophyll loroxanthin and its systematic significance in the colonial Volvocales. Phycologia 37: 79 – 83. Smith, G. M. (1950). “The Fresh-water Algae of the United States,” 2nd ed., McGraw–Hill, New York. Smith, G. M. (1955). “Cryptogamic Botany,” Vol. 1, McGraw–Hill, New York. Soltis, D. E., Soltis, P. S., Clegg, M. T., and Durbin, M. (1990). RbcL sequence divergence and phylogenetic relationships in Saxifragaceae sensu lato. Proc. Nat. Acad. Sci. USA 87: 4640 – 4644. Starr, R. C. (1980). Colonial chlorophytes. In “Phytoflagellates: Developments in Marine Biology” (R. E. Cox, Ed.), Vol. 2, pp. 147– 163. Elsevier North Holland, New York. Starr, R. C., and Zeikus, J. A. (1993). UTEX-The Culture Collection of Algae at the University of Texas at Austin. J. Phycol. 29(Suppl.): 1–106. Strimmer, K., and von Haeseler, A. (1996). Quartet puzzling: A quartet maximum-likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13: 964 –969. Swofford, D. L. (1998). “PAUP* 4.0: Phylogenetic Analysis Using Parsimony, version 4.0b2,” Computer program distributed by Sinauer, Fitchburg, MA.

268

NOZAKI ET AL.

Tajima, F., and Takezaki, N. (1994). Estimation of evolutionary distance for reconstructing molecular phylogenetic trees. Mol. Biol. Evol. 11: 278 –286. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res. 22: 4673– 4680. van Leeuwenhoek, A. (1700). Concerning the worms in sheeps, livers, gnats and animalicula in the excrement frogs. Phil. Trans. R. Soc. Lond. 22: 509 –518. Wakasugi, T., Nagai, T., Kapoor, M., Sugita, M., Ito, M., Ito, S.,

Tsudzuki, J., Nakashima, K., Tsudzuki, T., Suzuki, Y., Hamada, A., Ohta, T., Inamura, A., Yoshinaga, K., and Sugiura, M. (1997). Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: The existence of genes possibly involved in chloroplast division. Proc. Natl. Acad. Sci. USA 94: 5967–5972. Watanabe, M. M., and Hiroki, M. (1997). “NIES-Collection. List of strains. Algae and Protozoa,” 5th ed., National Institute for Environmental Studies, Tsukuba. Woessner, J. P., Gillham, N. W., and Boynton, J. E. (1986). The sequence of the chloroplast atpB gene and its flanking regions in Chlamydomonas reinhardtii. Gene 44: 17–28.