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J. Jpn. Bot. 91 Suppl.: 345–354 (2016)

18S Ribosomal RNA Gene Phylogeny of a Colonial Volvocalean Lineage (Tetrabaenaceae--, Volvocales, ) and Its Close Relatives

a,b, a,b a,b Takashi Nakada *, Takuro Ito and Masaru Tomita

aInstitute for Advanced Biosciences, Keio University, Kakuganji, Tsuruoka, Yamagata, 997-0052 JAPAN; bSystems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, 252-0882 JAPAN; *Corresponding author: [email protected]

(Accepted on January 19, 2016)

The lineage of colonial green consisting of Tetrabaenaceae, Goniaceae, and Volvocaceae (TGV-clade) belongs to the clade Reinhardtinia within Volvocales (Chlorophyceae). Reinhardtinia is closely related to some in the unicellular genera and . Although 18S rRNA gene sequences are preferred phylogenetic markers for many volvocalean species, phylogenetic relationships among the TGV-clade and its relatives have been examined mainly based on chloroplast genes and ITS2 sequences. To determine the candidate unicellular sister, 18S rRNA gene sequences of 41 species of the TGV-clade and its relatives were newly determined, and single and 6-gene phylogenetic analyses performed. No unicellular sister was determined by 18S rRNA gene analyses, but 6 unicellular clades and 11 ribospecies were recognized as candidates. Five of the candidate lineages and 27 taxa of the TGV-clade were examined by 6-gene phylogeny, revealing one clade including Chlamydomonas reinhardtii, Chlamydomonas debaryana, and Vitreochlamys ordinata to be more closely related than that containing Vitreochlamys aulata and Vitreochlamys pinguis.

Key words: 18S rRNA, colonial, , molecular phylogeny, unicellular, Volvocales.

Tetrabaenaceae, Goniaceae, and cells) 8- to 50,000-celled genera (, Volvocaceae constitute a colonial green , , Colemanosphaera, algal clade (TGV-clade) within Volvocales , , , and (Chlorophyceae), and include simple to ). Only colonies of Volvocaceae have a complex colonial forms (e.g., Nozaki 2003a, shared colonial envelope. Nozaki et al. 2014, Nakada and Nozaki 2015). Within Volvocales, the TGV-clade belongs Tetrabaenaceae includes undifferentiated to the clade Reinhardtinia (Nakada et al. 2008, 4-celled genera (, ), 2010a). To understand the evolutionary origin Goniaceae undifferentiated 8- to 128-celled of coloniality of the TGV-clade, determination genera (, ), and of its unicellular sister is first required. Previous Volvocaceae both undifferentiated and phylogenetic analyses showed that the TGV- differentiated (with somatic and reproductive clade is more closely related to some species

—345— 346 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue of Chlamydomonas and Vitreochlamys To determine the candidate(s) unicellular (Vitreochlamys aulata (Pascher) Batko, sister of the TGV-clade, 18S rRNA gene Vitreochlamys pinguis Nakazawa, Vitreochlamys sequences of 36 strains of core-Reinhardtinia ordinata (Skuja) Nakazawa, and Vitreochlamys (15 unicellular and 21 colonial) and five species gloeocystiformis (O. Dill) Nakazawa) than to a closely related to Paulschulzia pseudovolvox clade consisting of Paulschulzia pseudovolvox were newly determined, and single gene 18S (P. Schulz) Skuja, rostrata Hazen, rRNA and 6-gene combined 18S rRNA-atpB- and two Vitreochlamys species (Vitreochlamys rbcL-psaA-psaB-psbC phylogenetic analyses fluviatilis (F. Stein) Batko and Vitreochlamys performed. nekrassovii (Korshikov) Nakazawa) (e.g., Nozaki et al. 1999, Nakazawa et al. 2001, Materials and Methods Pröschold et al. 2001, Nozaki et al. 2003, The methods of DNA extraction, PCR, Pröschold et al. 2005, Herron et al. 2009, Nakada and sequencing were as described previously et al. 2008, 2010a, 2010b; the most inclusive (Nakada et al. 2007, 2010c). The primers for clade containing F. Stein, but not PCR and sequencing were 18S-FA, RB, FC, Paulschulzia pseudovolvox, is termed here as RD, FE, RF, FA2, and RB2 (Nakazawa and “core-Reinhardtinia”). However, phylogenetic Nozaki 2004, Nakada et al. 2007, 2010b). relationships among unicellular and colonial The 18S rRNA gene sequences obtained species/strains of core-Reinhardtinia have yet in this study (LC086332–LC086372) and to be resolved, partly because of inconsistencies several recently published sequences were in the phylogenetic marker genes used for unambiguously aligned with sequences of the unicellular and colonial species. clade Reinhardtinia (Nakada et al. 2008, 2010a, The phylogenetic relationships within the 2010b). For 18S rRNA single gene analyses, TGV-clade have been resolved mainly based the root of the clade Reinhardtinia was placed on chloroplast genes such as atpB, rbcL, psaA, on the branch leading to Heterochlamydomonas psaB, and psbC (e.g., Nozaki et al. 1995, 1997, according to Nakada et al. (2008). 1999, 2000, Nozaki 2003b, Nozaki et al. 2003, For 18S rRNA-atpB-rbcL-psaA-psaB-psbC 2014), while the phylogeny of Chlamydomonas 6-gene analyses, operational taxonomic units species tend to have been analyzed using 18S for which these five chloroplast gene sequences rRNA genes (e.g., Buchheim et al. 1990, 1996, were unavailable were excluded. The chloroplast 1997a, 1997b, Pröschold et al. 2001, Hoham 5-gene alignment was unambiguously et al. 2002, Nakada et al. 2008, Yumoto et al. constructed according to Nozaki et al. (2000) 2013). Additionally, Nakazawa et al. (2001) and combined with the 18S rRNA alignment analyzed phylogenetic relationships among (Table 1). For Bayesian phylogenetic analyses, Vitreochlamys species using rbcL genes, the alignment was divided into four partitions: showing the polyphyletic nature of the genus. 18S rRNA gene (18S), and first, second, and Pröschold et al. (2005) also showed the poorly third codon positions of the protein coding resolved ITS2 phylogeny of core-Reinhardtinia; chloroplast genes (cp1–cp3). however, only four colonial strains were The homogeneity of base frequencies was included in the analyses. Herron et al. (2009) tested for 18S rRNA and each codon position of provided the combined 6-gene phylogeny of the protein coding genes using the chi-squared core-Reinhardtinia using 18S rRNA and five test with PAUP* 4.0b10 (Swofford 2002). chloroplast genes, but 18S rRNA gene data Because homogeneity was rejected only for were missing or only partially included in the the cp3 partition (p < 0.05) when third codon analyses. positions of the atpB, psaA, or psaB genes December 2016 Nakada et al.: Unicellular relatives of colonial volvocaleans 347

Table 1. Species, genes and strains used in the 6-gene phylogenetic analyses. Sequences obtained in this study are shown in boldface Species Accession numberb (Strain designationa) 18S rRNA atpB rbcL psaA psaB psbC Core-Reinhardtinia: Unicellular Chlamydomonas debaryana AB542922 AB014034 D86838 AB044417–8 AB044469 AB044527 (SAG 15.72c = UTEX 1344d) Chlamydomonas reinhardtii M32703 M13704 J01399 AB044419 AB044470 AB044528 (UTEX 1061c, 137Cd) Vitreochlamys aulata LC086342 AB076122 AB050492 AB076144 AB076159 AB076180 (SAG 69.72c, d) Vitreochlamys ordinata LC086344 AB014036 AB014041 AB044420 AB044471 AB044529 (NIES-882c = Nozaki S-4d) Vitreochlamys pinguis LC086345 AB076120 AB050491 AB076142 AB076157 AB076174 (NIES-883c = Spha-12/1998-7-16d) Core-Reinhardtinia: Colonial Astrephomene gubernaculifera LC086347 AB014022–3 D63428 AB044234 AB044458 AB044513–4 (NIES-418c, d) Astrephomene perforata LC086348 AB014024 D63429 AB044238 AB044460 AB044518–9 (NIES-564c, d) Basichlamys sacculifera LC086349 AB014015 D63430 AB044416 AB044467–8 AB044526 (NIES-566c, d) Colemanosphaera charkoviensis LC086350 AB905589 AB905591 AB905593 AB905595 AB905597 (NIES-3388c, Isa 7-1d = NIES-3383) Eudorina elegans var. elegans LC086352 AB014009 D63432 AB044199 AB044435 AB044485 (NIES-456c, d) Eudorina elegans var. carteri LC086351 AB014012 D88806 AB044203 AB044438 AB044487–8 (NIES-721c = UTEX 1212d) Eudorina illinoisensis LC086353 AB014013 D63433 AB044198 AB044434 AB044484 (NIES-460c, d) Eudorina peripheralis LC086354 AB014007 D63434 AB044209 AB044440 AB044491–2 (NIES-725c = UTEX 1215d) Eudorina unicocca AB511841 AB014008 D86829 AB044206 AB044439 AB044489–90 (TKI-C-2c = NIES-1858, UTEX 737d) Gonium multicoccum LC086355 AB014020 D63435 AB044240 AB044461 AB044481 (NIES-737c = UTEX 2580d) Gonium pectorale LC066324 AB014016–7 D63437 AB044242 AB044463 AB044521 (NIES-569c, d) Gonium viridistellatum LC086356 AB014021 D86831 AB044244 AB044465 AB044524 (NIES-654c = UTEX 2519d) Pandorina morum LC066325 AB044179 AB044166 AB044230 AB044455 AB044509 (NIES-887c = UTEX 880d) Platydorina caudata LC086357 AB014032 D86828 AB044212 AB044442 AB044494 (NIES-728c = UTEX 1658d) Pleodorina californica LC086358 AB014004 D63439 AB044192 AB044430 AB044480 (NIES-735c = UTEX 809d) Pleodorina japonica AB688627 AB014005 D63440 AB044194 AB044431 AB044482 (NIES-577c = UTEX 2523d) aAbbreviations of the culture collections are as following: NIES. Microbial Culture Collection at the National Institute for Environmental Studies (http://mcc.nies.go.jp/top.jsp). SAG. Sammlung von Algenkulturen at the University of Göttingen (http://www.epsag.uni-goettingen.de/). UTEX. Culture Collection of Algae at the University of Texas at Austin (https:// utex.org). bAccession numbers of the genes from the International Nucleotide Sequence Database Collaboration (http://www.insdc. org). c18S rRNA gene sequences. dChloroplast gene sequences (atpB, rbcL, psaA, psaB, and psbC). 348 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Table. 1. Continued Species Accession numberb (Strain designationa) 18S rRNA atpB rbcL psaA psaB psbC Core-Reinhardtinia: Colonial Pleodorina starrii LC086359 AB214424 AB214427 AB214430 AB214432 AB214434 (NIES-1362c = 2000-602-P14d) Pleodorina thompsonii LC086360 AB214407 AB214408 AB214410–1 AB214412 AB214413 (“RT” c, d = UTEX 2804) Tetrabaena socialis LC086361 AB014014 D63443 AB044415 AB044466 AB044525 (NIES-571c, d) Volvox aureus var. aureus LC086362 AB013998 D63445 AB044182 AB044424 AB044474 (NIES-541c, d) Volvox carteri f. nagariensis X53904 AB076109 AB076099 AB076128 AB076148 AB076163 (UTEX 1885c, d) Volvox gigas LC086363 AB076112 AB076084 AB076132 AB076150 AB076165 (NIES-867c = UTEX 1895d) AB542923 AB014002 D86836 AB044187 AB044428 AB044478 (SAG 199.80c = UTEX 955d) Volvox tertius LC086364 AB076107 AB076098 AB076126 AB076147 AB076162 (NIES-544c = UTEX 132d) Volvulina compacta LC086365 AB593420 AB593419 AB593421 AB593422 AB593423 (TN-0205-2-Pn-1c, d = NIES-2567) Volvulina steinii LC086366 AB044173 AB044159 AB044222 AB044448 AB044500 (NIES-545c, d) Yamagishiella unicocca LC086367 AB014030 D86823 AB044213 AB044443 AB044495 (NIES-666c = UTEX 2428d) Outgroup Lobomonas mostruosa LC086368 AB044533 AB044171 AB044421 AB044472 AB044530 (NIES-474c, d) Paulschulzia pseudovolvox AF408246 AB014040 D86837 AB044422–3 AB044473 AB044531–2 (UTEX 167c, d) aAbbreviations of the culture collections are as following: NIES. Microbial Culture Collection at the National Institute for Environmental Studies (http://mcc.nies.go.jp/top.jsp). SAG. Sammlung von Algenkulturen at the University of Göttingen (http://www.epsag.uni-goettingen.de/). UTEX. Culture Collection of Algae at the University of Texas at Austin (https:// utex.org). bAccession numbers of the genes from the International Nucleotide Sequence Database Collaboration (http://www.insdc. org). c18S rRNA gene sequences. dChloroplast gene sequences (atpB, rbcL, psaA, psaB, and psbC). were included, these third codon positions for every 1,000,000 generation, discarding the were excluded from further analyses. For the first 25% of generations as burn-in, and the 6-gene analyses, Paulschulzia pseudovolvox iterations were automatically stopped when the and L. monstruosa Korshikov were treated as average standard deviations were below 0.01, the outgroup according to the results of the 18S indicating convergence. Consequently, 8,000,000 rRNA analyses. and 2,000,000 generations of iterations were Bayesian phylogenetic analyses were performed for the 18S rRNA single gene and performed using MrBayes 3.2.1 (Ronquist et al. combined 6-gene analyses, respectively. The 2012). Convergences of Markov chain Monte evolutionary models for the Bayesian inference Carlo iterations were evaluated based on the (GTR + I + G for 18S [both for single- and average standard deviation of split frequencies 6-gene analyses]; GTR + I + G for cp1; F81 + I December 2016 Nakada et al.: Unicellular relatives of colonial volvocaleans 349

Fig. 1. Bayesian phylogenetic tree of the clade Reinhardtinia (Volvocales, Chlorophyceae) based on 18S rRNA gene sequences. The corresponding posterior probabilities (≥0.90; top left) and the bootstrap proportions (≥50%) from maximum likelihood (top right) and neighbor-joining (bottom) analyses are shown next to the branches. Branch lengths and scale bars represent the expected number of nucleotide substitutions per site. Accession numbers of the genes from the International Nucleotide Sequence Database Collaboration (http://www.insdc.org) are indicated in parentheses, and sequences obtained in this study are shown in boldface). Unicellular clades (UC-)A–F are indicated on the right. The other unicellular species/strains are indicated by U“ ”. Authentic strains (strains used in the original publications) of some species are indicated by “A”. 350 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Fig. 2. Bayesian phylogenetic tree of the core Reinhardtinia based on combined 18S rRNA-atpB-rbcL-psaA-psaB- psbC gene sequences, excluding the third codon positions of atpB, psaA, and psaB. The corresponding posterior probabilities (≥0.90; top left) and the bootstrap proportions (≥50%) from maximum likelihood (top right), maximum parsimony (bottom left), and neighbor-joining (bottom right) analyses are shown next to the branches. Branch lengths and scale bars represent the expected number of nucleotide substitutions per site.

+ G for cp2; GTR + I + G for cp3) were selected Jukes and Cantor distances) analyses were using MrModeltest 2.2 (Nylander 2004), and performed (maximum parsimony analysis was those for four partitions were unlinked in the not performed because heuristic searches were Bayesian inference for 6-gene analyses. not completed within a reasonable time in the Bootstrap analyses (Felsenstein 1985) were preliminary analyses). For 6-gene analyses, performed using PAUP* 4.0b10 (Swofford maximum likelihood (100 replications, nearest 2002). For 18S rRNA analyses, maximum neighbor interchange heuristic searches started likelihood (100 replications, nearest neighbor with a neighbor joining tree), maximum interchange heuristic searches started with a parsimony (1,000 replications, tree-bisection- neighbor joining tree) and neighbor-joining reconnection heuristic searches started with (Saitou and Nei 1987; 1,000 replications using stepwise addition algorithms), and neighbor- December 2016 Nakada et al.: Unicellular relatives of colonial volvocaleans 351 joining (Saitou and Nei 1987; 1,000 replications Chlamydomonas marvanii H. Ettl SAG 73.81; using Jukes and Cantor distances) analyses were and L. rostrata SAG 45-1 and L. sphaerica E. G. performed. The evolutionary models for the Pringsh. SAG 45-2. maximum likelihood analyses (TrN + I + G for Phylogenetic relationships within core- both single- and 6-gene analyses) were selected Reinhardtinia were barely resolved with using Modeltest 3.7 (Posada and Crandall 1998). 18S rRNA single gene phylogeny (Fig. 1). The monophyly of Goniaceae, Volvocaceae, Results and TGV-clade was not recovered, but The 18S rRNA gene sequence of that of Tetrabaenaceae (Tetrabaena and “ oogama” SAG 9.79 (the strain is Basichlamys) was robustly supported (100% not actually Chloromonas, because the cell has a posterior probability [PP] and 100% bootstrap pyrenoid; unpublished observation) (LC086341) proportions [BPs]). In the phylogenetic tree, was found to be different from one previously six unicellular clades (UC-A to UC-F) and 12 published sequence (U70793), with several sequences (ribospecies) from unicellular strains unidentified bases, but identical to another were recognized. The posterior probability (JN904005). The sequence of Chlamydomonas supporting UC-D was low (0.70), but this cribrum H. Ettl SAG 13.72 (LC086333) clade was supported by maximum likelihood differed from the previously published sequence and neighbor-joining bootstrap proportions (AF517099) by 7 bp including indels. The (72% and 92%, respectively). Otherwise, sequences of Pleodorina californica W. Shaw each unicellular clade was robustly supported NIES-735 (LC086358) and Volvox tertius (≥0.99 PPs and ≥90% BPs). “Chlamydomonas Art. Mey. NIES-544 (LC086364) were also reinhardtii” JinCheon1 (KF864473) was not previously published, but that of Pleodorina closely related to Chlamydomonas reinhardtii californica UTEX 809 (= NIES-735; FJ610145) P. A. Dang. UTEX 1061 (conspecific with the lacked a central region of ca. 400 bp while epitype strain of Chlamydomonas reinhardtii; that of Volvox tertius UTEX 132 (= NIES- Nakada et al. 2010b), but was sister to the 544; FJ610144) lacked a central 16-bp region, colonial species Eudorina illinoisensis (Kof.) and both contained additional inconsistencies. Pascher NIES-460, with moderate statistical Previously published sequences inconsistent support (0.85 PP and 97–100% BPs). UC-A, -B, with the present sequences were not used for -C, and Vitreochlamys aulata were also related further analyses. to colonial species, but the statistical support was The 18S rRNA gene sequence of low (0.78–0.97 PPs and <60% BPs). Chlamydomonas sp. SAG 11-45 (formerly In the 6-gene Bayesian phylogeny (Fig. 2), “petasus”; Nakada et al. 2010b) was identical the monophyletic nature of Tetrabaenaceae, to that of “Chlamydomonas reinhardtii” CCAP Goniaceae, and Volvocaceae were respectively 11/45 (FR865586), while those of Tetrabaena recovered. The monophyly of Tetrabaenaceae socialis (Dujard.) Nozaki & Mot. Ito NIES-571 was strongly supported (1.00 PP and 100% and SAG 12-3 (as contaminant of BPs), but statistical support for the monophyly sp.; Nakada and Nozaki 2007) were also of Goniaceae and Volvocaceae was low (1.00 identical. Additionally, some 18S rRNA PP and <50% BPs for Goniaceae; <0.90 PP and sequences from different species were identical; <50% BPs for Volvocaceae). Statistical support Chlamydomonas parallelistriata Korshikov for the TGV-clade was also low (0.93 PP and (erroneously labelled “parallestriata”) SAG 2.73 <80% BPs). The sister group of the TGV-clade and Chlamydomonas nasuta Korshikov SAG was moderately resolved as the clade consisting 15.84; Chlamydomonas cribrum SAG 13.72 and of Chlamydomonas debaryana Gorozh. (UC- 352 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

A in Fig. 1), Vitreochlamys ordinata (UC-C), A, UC-C, and UC-D as more closely related and Chlamydomonas reinhardtii (UC-D) (0.95 to the TGV-clade (0.95 PP and 70–89 BPs) PP and 70–89% BPs). The clade consisting of than Vitreochlamys aulata and Vitreochlamys Vitreochlamys aulata and Vitreochlamys pinguis pinguis. However, phylogenetic positions of was more distantly related to the TGV-clade. the remaining three clades and nine ribospecies remain uncertain, and multigene analyses Discussion including these clades/ribospecies are required. Phylogenetic relationships among major Several close relationships between strains lineages of core-Reinhardtinia were not resolved with different species assignments were in the 18S rRNA gene phylogeny, suggesting found, and some differently labeled strains early divergence of core-Reinhardtinia within were found to have identical 18S rRNA gene a relatively short period. The monophyly of sequences. This synonymy therefore requires the known colonial clade, the TGV-clade, was evaluation based on morphological comparisons not recovered, and five unicellular lineages of culture strains such as: Chlamydomonas (“Chlamydomonas reinhardtii” JinCheon1, angulosa O. Dill with Chlamydomonas Vitreochlamys aulata, UC-A, UC-B, and UC- debaryana; Chlamydomonas typica Deason & C) were found to be specifically related to H. C. Bold with Chlamydomonas sphaeroides some colonial species. While statistical support Gerloff; Chlamydomonas fasciata H. Ettl connecting UC-A to Gonium, and Vitreochlamys with Chlamydomonas parallelistriata and aulata, UC-B, and UC-C to Tetrabaenaceae, Chlamydomonas nasuta; Chlamydomonas was weak (0.78–0.97 PPs and <60% BPs), cribrum with Chlamydomonas marvanii; and “Chlamydomonas reinhardtii” JinCheon1 L. rostrata and L. sphaerica. Note that some was found to be moderately related to E. similarly close pairs of species have been illinoisensis NIES-460 (0.85 PP and 97–100% examined using culture strains and species- BPs). Moreover, the strain JinCheon1 was level differences confirmed; for example, E. distantly related to authentic Chlamydomonas unicocca G. M. Sm. from E. peripheralis (M. reinhardtii, and the original identification as E. Goldst.) T. K. Yamada (Yamada et al. 2008); Chlamydomonas reinhardtii (KF864473) T. socialis from B. sacculifera (Scherff.) Skuja unsupported; however, if this strain is indeed (Stein 1959); Chlamydomonas reinhardtii from unicellular, it could be the first example of Chlamydomonas globosa J. Snow (Nakada et secondary unicellular algae in the TGV-clade. al. 2010b); and Vitreochlamys ordinata from However, it should also be noted that old Vitreochlamys gloeocystiformis (Nakazawa et al. cultures of Tetrabaenaceae and Goniaceae often 2001). contain unicellular individuals (e.g., Mogi et al. In the present study, 18S rRNA gene 2012). sequences from several authentic or well- Six unicellular clades and 11 unicellular characterized strains were determined (Fig. 1). ribospecies (excluding JinCheon1) were Publication of these sequences will help promote found to be good candidate unicellular sisters DNA barcoding of unicellular and colonial for the TGV-clade (they are also candidates volvocaleans using 18S rRNA gene sequences of secondary unicellular algae). Only alone. 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a,b a,b a,b 仲田崇志 ，伊藤卓朗 ，冨田 勝 ：群体性オオヒ ゲマワリ系統（緑藻綱，オオヒゲマワリ目，テトラバエ ナ科・ヒラタヒゲマワリ科・オオヒゲマワリ科）および 近縁種の 18S リボソーム RNA 遺伝子系統 テ ト ラ バ エ ナ 科， ヒ ラ タ ヒ ゲ マ ワ リ 科， オ オ ヒ 統群の姉妹群は特定されず，その候補として 6 系統群と ゲ マ ワ リ 科 か ら な る 群 体 性 系 統（TGV 系 統 群 ） は 11 種が見出された．この内 5 系統群／種と主な TGV 系 Reinhardtinia 系統群（緑藻綱，オオヒゲマワリ目）に属 統群について 6 遺伝子系統解析を行ったところ，コナミ し，単細胞性のコナミドリムシ属 (Chlamydomonas) や ドリムシ (Chlamydomonas reinhardtii)，Chlamydomonas Vitreochlamys のいくつかの種と近縁である．TGV 系統 debaryana，Vitreochlamys ordinata を含む単系統群が， 群と近縁種では葉緑体遺伝子や ITS2 配列が系統解析に Vitreochlamys aulataと Vitreochlamys pinguis よりも TGV 使われていたが，TGV 系統群の姉妹群は特定されてい 系統群に近いことが示唆された． なかった．そこで本研究では TGV 系統群と近縁種 41 （a慶應義塾大学先端生命科学研究所， 種について 18S rRNA 遺伝子配列を新たに決定し，系 b慶應義塾大学大学院政策・メディア研究科） 統解析に用いた．18S rRNA 遺伝子の解析では TGV 系