植物研究雑誌 J. J. Jpn. Bo t. 79:255-261 79:255-261 (2004)

Phylogenetic Phylogenetic Analysis of the Tetrasporalean Genus Asterococcus () sased on 18S 18S Ribosomal RNA Gene Sequences

Atsushi Atsushi NAKAZA WA and Hisayoshi NOZAKI

Department Department of Biological Sciences ，Graduate School of Science ，University of Tokyo ， Hongo Hongo 7-3-1 ，Bunkyo-ku ，Tokyo ，113 ・0033 JAPAN (Received (Received on October 30 ，2003)

Nucleotide Nucleotide sequences (1642 bp) from 18S ribosomal RNA genes were analyzed for 100 100 strains of the clockwise (CW) group of Chlorophyceae to deduce the phylogenetic position position of the immotile colonial genus Asterococcus Scherffel ， which is classified in the Palmellopsidaceae of . We found that the genus Asterococcus and two uni- cellular ， volvocalean genera ， Lobochlamys Proschold & al. and Oogamochlamys Proschold Proschold & al.， formed a robust monophyletic group ， which was separated from two te 位asporalean clades ， one composed of Link and Sk 吋a and the other other containing the other palme l1 0psidacean genus Chlamydocaps αFot t. Therefore ， the Tetrasporales Tetrasporales in the CW group is clearly polyphyletic and taxonomic revision of the order order and the Palmellopsidaceae is needed.

Key words: 18S rRNA gene ，Asterococcus ，Palmellopsidaceae ，phylogeny ，Tetraspor- ales. ales.

Asterococcus Asterococcus Scherffel (1908) is a colo- Recently ， Ettl and Gartner (1 988) included nial nial green algal genus that is characterized Asterococcus in the family Palmello- by an asteroid chloroplast in the cell and psidaceae ， because cells of this genus have swollen swollen gelatinous layers surrounding the contractile vacuoles and lack pseudoflagella immotile immotile colony (e. g. ， Ettl and Gartner in the nor トattached vegetative colony. 1988). 1988). This genus is generally assigned to Patricia et al. (2000) revealed that the the Tetrasporales (e. g. ，Lemmermann 1915 ， Asterococcus belongs to the clockwise (CW) Tiffany Tiffany 1934 ，Smith 1950 ， Ettl 1964 ， Fott group of Chlorophyceae ， based on the CW 1971 ， Ettl and Gartner 1988). However ， the orientation of the flagellar apparatus in A. systematic systematic position of Asterococcus at the superbus (Cienkowski) Scherffe l. Nakazawa family family level varies according to the author. et al. (2004) carried out a taxonomic study of Lemmermann (1915) classified Asterococcus Asterococcus based on the comparative mor- in in the . Korshikov (1 953) con- phology of many strains and molecular sidered sidered this genus a member of the dendroid phylogenetic analyses of the Rubisco 1紅 ge family family Chlorangiellaceae because his alga subunit (rbcL) gene sequences. However ， the produced dendroid ，gelatinous colonies. Fott phylogenetic position of this genus within (1 971) distinguished Asterococcus from the CW group was ambiguous ， possibly be- other other members of the Tetrasporales and es- cause of the limited numbers of operational tablished tablished a new far 凶ly ， the Asterococcaceae. taxonomic units (OTUs) analyzed (Naka-

-255- 256 植物研究雑誌第79 巻第4号 平成16 年8月 zawa et al. 2004). Therefore ， sequence data quencing of 18S rRNA genes (Table 2). of of Asterococcus 紅 e needed for genes that Ninety five OTU s in the CW group exam- have have been determined in many taxa in the ined in this study were the same as those of CW group. Although Pr りschold et al. (2001) Proschold et al. (2001) ， and five additional studied studied the phylogeny of 95 strains of the CW OTU s are listed in Table 1. Seven OTU s CW group based on 18S ribosomal (r) RNA of the directly opposed (DO) group (Fig. 1) genes ，Asterococcus sequences were not ana- were selected from Proschold et al. (2001). lyzed. lyzed. Therefore we evaluated the phylo- The alignment of the 18S rRNA genes from genetic genetic position of the genus Asterococcus the 95 CW and seven DO OTUs was ex- based based on 18S rRNA sequences. Our results tracted from that of Proschold et al. (2001) suggest suggest that Asterococcus is separated from and the five additional OTU s (Table 1) were other other members of the Tetrasporales within realigned using Clustal X (Thompson et al. the the CW group. 1997). The alignment (1 642 bp; see Proschold Material Material and Methods et al. 2001) was subjected to unweighted Two. strains of Asterococcus were selected maximum p紅 simony (MP) analysis ，includ- as as representatives of the genus since they ing bootstrap analysis (Felsenstein 1985) have have different colonial forms and belong to with 100 replications of a full heuristic

the the two subgroups constituting the mono 田 search based on the nearest-neighbor inter 司 phyletic phyletic Asterococcus group in the rbcL change (NNI) branch-swapping algorithm ， gene gene phylogeny (Nakazawa et al. 2004). using PAUP 4.0bl0 (Swofford 2002). They were A. superbus IAM C ・299 with a Following the guidelines for topology con- spherical spherical colony and A. korschikoffii Ettl struction with distance methods (N ei and ACOI 326 with a dendroid colony (Naka- Kumar 2000) ，we selected Jukes-Cantor dis- zawa et al. 2004) (Table 1). tances (Jukes and Cantor 1969) to construct

The methods for DNA extraction and di 聞 (NJ) neighbor-joining trees. From the align- rect rect sequencing of polymerase chain reaction ment used for the MP analysis ，a Jukes- (PCR) products were the same as those of a Cantor distance matrix was calculated (J ukes previous previous study (Nakazawa et al. 2004) ，ex- and Cantor 1969) using PAUP 4.0bl0. With cept cept for the primers used for PCR and se- the NJ algorithm (Saitou and Nei 1987) ，a

Table Table 1. Additional OTUs of CW group (Proschold et al. 2001) used for phylogenetic analysis using the18S rRNA genes genes

Origin Origin of 18S DDBJAMB L/ GenBank Species Strain designation sequence sequence data accession number

Asterococcus Asterococcus superbus IAM 1 C-299 The present study AB175836 2 Asterococcus Asterococcus korschikoffii ACOI 326 The present study AB175837 3 Characiochloris Characiochloris acuminata UTEX 2095 Buchheim et al. (2002) AF395435 4 Hafniomonas montana NIES -257 Nozaki et al. (2003) AB101517 Hφ uomonas mon ωna NIES-656 Nozaki et al. (2003) AB101518 lIAM Culture Collection at the University of Tokyo (Sugiyama et al. 1998). 2The 2The Culture Collection of Algae at the Department of Botany ，University of Coimbra (Santos and Mesquita 1986 ， Santos Santos 1988 ， Santos et al. 1993). 3Culture 3Culture Collection of Algae at the University of Texas at Austin (Starr and Zeikus 1993). 4Microbial 4Microbial Culture Collection at the National Institute for Environmental Studies (Watanabe et al. 2000). August August 2004 Journal of Japanese' Botany Vo l. 79 No. 4 257

Table Table 2. Primers used for amplifications and sequencing of the18S rRNA genes

Designation Designation Positions 1 Sequence (5'-3') 18S-FA 18S-FA 1-21 AACCTGGTTGATCCTGCCAGT 18S-RD 57 0- 550 2 GCTGGCACCAGACTTGCCCTC 18S-FG 18S-FG 557-577 AGTCTGGTGCCAGCAGCCGCG 18S-FE 18S-FE 1112-1132 GGGAGTATGGTCGCAAGGCTG 18S-RF 18S-RF 1202-1182 2 CCCGTGTTGAGTCAAATTAAG 2 18S-RB 18S-RB 1799 ー1774 TGATCCTTCTGCAGGTTCACCTAC lCoordinate lCoordinate number from the Chlorella vulgaris Beijerink 18S rRNA gene (Huss et al. 1990). 2Reverse 2Reverse primer.

phylogenetic 位ee was constructed ，also using lineages of other tetrasporalean algae ， one PAUP 4.0bl0. The robustness of the result- containing a palmellopsidacean species ing ing lineages was tested by bootstrap analysis [Chlamydoc αrps α maxima (Mainx) Ettl &

with with 1000 replications using PAUP 4.0bl0. Gartner] and the other composed of two co 四 U sing the same alignment data ，a maximum lonial genera in the likelihood likelihood (ML) analysis was carried out (Tetraspora Link and Paulschulzia Sk 吋a). using using PUZZLE in PAUP 4.0bl0 ， with the By contrast ， the genus Asterococc α us consti- HKY model (Hasegawa et al. 1985) to esti- tuted a robust monophyletic group with two mate mate qu 紅 tet puzzling support values ， which unicellular volvoca 叫lean genera Oog αル. have have the same practical meaning as bootstrap mηlochla α mηlyS Pr め.。凸 S印chold & al. (ο2001) and values values (Strimmer and von Haeseler 1996) for Loboch μα l， m. ηη ly y戸 internal internal branches of the phylogenetic tree high boot 臼st 住rap/QPS values (79-99 %) with with with 1000 puzzling steps (comp 訂 able to the the NJ ，MP ， and ML methods. Within this number of bootstrap replicates). In these group ， the MP and ML analyses suggested phylogenetic phylogenetic analyses ， the seven species in that Asterococcus and Oog αmochlamys the the DO group (Fig. 1) were designated the formed a clade with 71-75 % bootstrap/QPS outgroup outgroup since the DO group is sister to the values (not shown) ， whereas the NJ analysis CW group containing volvocalean and did not support this clade. biflagellate biflagellate te 住asporalean algae (Booton et et al. 1998). Discussion Tetrasporales Tetrasporales is traditionally characterized Results Results as having a gelatinous structure (gelatinous The NJ tree of the 18S rRNA sequences is matrix an d/ or pseudoflagella) in the vegeta- shown in Fig. 1， and branches with 50 % or tive phase in which cells can be converted greater greater bootstrap/QPS values in the NJ ，MP into reproductive cells without preceding cell

an d/ or ML 紅 e shown. A robust mono 闘 divisions (e. g. ， Ettl and Gartner 1988). phyletic phyletic group consisting of the genus However ， the validity of this order has been Asterococcus Asterococcus (A. superbus and A. kor- doubted because no common characteristics schikoffii) schikoffii) was resolved with high boot- clearly distinguish the Tetrasporales from the strap/QPS strap/QPS values (92-100 %) using the NJ ， volvocalean an d/ or chlorococcalean green MP ， and ML methods. This genus was dis- algae (e. g. ， van den Hoek et al. 1995). Based tant tant from the ‘Reinhardtii' -Clade (Proschold on the 18S rRNA gene phylogeny ， Booton et et al. 2001) ， which included two separate et al. (1998) demonstrated that the Tetra- 258 植物研究雑誌第79 巻第4号 平成16 年8月

Ettlia minuta Botryococcus braunii

'Reinhardti i'- Clade Clade

Polytomella Polytomella parva

一一 0.005 substitutions/site August August 2004 Joumal of Japanese Botany Vo l. 79 No. 4 259 sporales sporales is polyphyletic ，composed of a in the CW group (e. g. ，Nakazawa et al. biflagellate/CW biflagellate/CW group and a quadriflage ト 200 1). Patricia et al. (2000) and Nakazawa late lD O group within the Chlorophyceae. et al. (2004) reported details of the cell struc- We clearly resolved three sep 紅 ate lineages ture of Asterococcus using transmission elec- of of the tetrasporalean algae within the CW 住on microscopy. However ，there 紅 'e no group group (Fig. 1) ， and concluded that morphological studies of Oogamochlamys Te 甘asporales is polyphyletic even within the and Lo bochlamys based on ultrastructural CW group. Therefore ， an immotile vegeta- observations. Further electron microscopic tive tive phase with gelatinous structures and the studies of these two genera may resolve direct direct formation of the reproductive cells ultrastructural features characterizing characterizing characterizing the Tetrasporales likely Asterococcus and Oogamochlamys/ Lo bo- evolved evolved in multiple phylogenetic positions in chlamys and contribute to reconstruction of a the the CW group ，and the taxonomic sep 訂 ation natural taxonomic system of the CW green of of Tetraspor a1 es from Volvocales/Chlorococ- algae traditionally classified as members of cales cales does not represent the natural phylo- the Tetrasporales. genetic genetic relationships within the . Based on this study ， Asterococcus and two We 紅 e grateful to Dr. T. Proschold for biflagellate biflagellate unicellular genera Ooga- providing the alignment of the 18S rRNA mochlamys and Lobochlamys (Volvocales) genes. This study was supported in pぽ tby a form a robust monophyletic group within the grant from the Institute for Fermentation (to CW group; However ，no morphological fea- HN). tures tures seem to characterize Oogamochlamys/ Lobochlamys and Asterococcus at the light References microscopic microscopic level (Proschold et al. 2001 ， Booton G. C. ，Fl oyd G. L. and Fuerst P. A. 1998. Nakazawa et al. 2004). Although the muci- Polyphyly of te 住asporalean green algae inferred lage lage layer around the cell of Lobochlamys from nuclear small-subunit ribosomal DN A. J. Phyco l. 34: 30 6- 31 1. (Proschold (Proschold et al. 2001) seems to be homolo- Buchheim M. A. ，Buchheim J. A. ， Carlson T. and gous gous to the colonial gelatinous layers of Kugrens P. 2002. Phylogeny of Lo bocharacium Asterococcus ， such a layer is also recognized (Chlorophyceae) and allies: a study of 18S and 26S in in another unicellular genus ， Vitreochlamys rDNA data. J. Phyco l. 38: 37 6- 383.

Fig. Fig. 1. Distance tree based on aligned nucleotide sequences for 1642 bp in the 18S rRNA genes for two strains Asterococcus of Asterococcus and 98 other strains of CW group and seven DO algae. The tree was derived with the neigh- bor-joining bor-joining method (Saitou and Nei 1987) based on the Jukes-Cantor distance (Jukes and Cantor 1969) in- dicated dicated by the scale bar below the tree. Numbers above branches 訂 e the bootstrap values (50 % or more)

based based on 1000 replications. Numbers in brackets below the branches 町 e bootstrap values resolved in the ma- jority-rule jority-rule (50 %) consensus t回 e of a bootstrap analysis based on 100 replications of most p町 simonious

analysis analysis by PAUP 4.0b lO. Numbers in p紅 entheses below the branches are quartet puzzling support (QPS) values values (50 % or more) of a maximum likelihood analysis with 1000 puzzling steps. Bootstrap/QPS values

at at distal branches within Oogamochlamys ，Lo bochlamys and Chloromonas 紅 e not shown. Designations of species species names and the alignment of 18S rRNA genes are almost based on those of Proschold et al. (200 1). ACOI: The Culture Collection of Algae at the Dep 訂 tment of Botany ，University of Coimbra (Santos and Mesquita Mesquita 1986 ， Santos 1988 ， Santos et al. 1993). V 山1: IAM Culture Collection at the of University Tokyo (Sugiyama (Sugiyama et al. 1998). NIES: Microbial Culture Collection at the National Institute for Environmental Studies Studies (Watanabe et al. 2000). SAG: Sammlung von Algenkulturen at the of University Gottingen

(Schlδsser (Schlδsser 1994). UTEX: Culture Co Il ection of Algae at the University of Texas at Austin (Sta 町 and Zeikus 1993). 1993). 260 260 植物研究雑誌第79 巻第4号 平成16 年8月

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中沢敦，野崎久義: 18S rRNA 遺伝子配列を用 いたヨツメモ目 Asterococcus 属(緑藻綱)の分子 系統解析 Asterococcus Asterococcus Scherffel は放射状の葉緑体と細胞 伝子1642 塩基対を用いた系統解析を行った.その 群体を包む膨潤した寒天状基質層により特徴付け 結果，Asterococcus は遊泳性の単細胞性ボルボッ られる不動性微細藻であり，緑藻綱，ヨツメモ目， クス目の Lo bochlamys 及び Oogamochlamys と高い パルメロプシス科に分類されている.筆者らは本 ブートストラップ確率で単系統をなし ， Tetraspora 属の緑藻綱の CW グループ(鞭毛基部の配置が時 を含むヨツメモ目のクレード及び，パルメロプシス 計回り型のボルボックス目，クロロコックム目の 科に属する maxima (M 泊nx) Ettl & 一部， 2 鞭毛性ヨツメモ目を含む一群)内におけ Gartner と分離することが示された. る系統的位置を明らかにするため， 18S rRNA 遺 (東京大学大学院理学系研究科生物科学専攻)