J. Jpn. Bot. 85: 364–369 (2010)

Volvulina compacta (, ), New to Japan, and Its Phylogenetic Position

a, b, a c Takashi Nakada *, Masaru Tomita and Hisayoshi Nozaki

aInstitute for Advanced Biosciences, Keio University, 246-2, Mizukami, Kakuganji, Tsuruoka, Yamagata, 997-0052 JAPAN; bGraduate School of Media and Governance, Keio University, Fujisawa, 252-8520 JAPAN; cDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033 JAPAN *Corresponding author: [email protected]

(Received on June 30, 2010)

Volvulina Playfair is a colonial volvocalean genus with usually 8 or 16 cells forming hollow ellipsoidal colonies. In this study, a strain of Volvulina was isolated from Lake Tsukui, Kanagawa Prefecture, Japan. This strain was morphologically identified as Volvulina compacta Nozaki, which was previously known only from strains isolated from Nepal. multigene analyses using rbcL, atpB, psaA, psaB and psbC genes showed that the Japanese strain of V. compacta is closely related to the Nepalese strain of V. compacta and to an American strain of Volvulina pringsheimii Starr, but only distantly to Volvulina steinii Playfair previously reported from Japan. Although the Japanese strain was more closely related to V. pringsheimii than to Nepalese V. compacta, it was clearly distinguished from V. pringsheimii by its square shape in the top view. These results indicate the existence of cryptic species within V. compacta.

Key words: Molecular phylogeny, , Volvocaceae, Volvulina compacta.

Volvulina Playfair is a colonial volvocalean 1990). genus with usually 8 or 16 cells forming hollow In 2002, a single strain of Volvulina was ellipsoidal colonies (Playfair 1915, Ettl 1983). isolated from Lake Tsukui in Kanagawa Prefecture, Three species have been recognized in this genus Japan. This strain was morphologically identified (V. steinii Playfair [=V. playfairiana Skvortzov], as V. compacta and its molecular phylogeny was V. pringsheimii Starr and V. compacta Nozaki), examined based on five chloroplast genes. and all have been studied based on culture strains. Strains of “V. boldii” nom. nud. are also Materials and Methods known (online catalogue of UTEX: http://web. A strain of Volvulina, TN-0205-2-Pn-1, was biosci.utexas.edu/utex/). Only one species has isolated from a water sample collected from been reported from Japan, namely, V. steinii Lake Tsukui (35°36′5″N, 139°13′50″E), Mii, (Nozaki 1982, Tsukii 2010), with the remaining Midori-ku, Sagamihara, Kanagawa Prefecture, two known only from two single countries (V. Japan, on 30 May 2002. The strain was isolated pringsheimii from the USA and V. compacta using the pipette-washing method (Pringsheim from Nepal; Starr 1962, Nozaki and Kuroiwa 1946). Cultures were grown in screw-cap tubes

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(18 × 150 mm; Fujimoto Rika, Tokyo, Japan) p-values (BPs; Felsenstein 1985) of the containing 9–10 mL of MG medium (Kasai et maximum likelihood (100 replications; nearest al. 2009) at about 20°C with an alternating 14 neighbor interchange [NNI] heuristic searches h light / 10 h dark cycle at a light intensity of started with a neighbor-joining tree; GTR + I + about 200–300 µmol·m-2·s-1 provided by cool G model, selected by Modeltest 3.7 [Posada and white fluorescent lamps. Light microscopy was Crandall 1998]), maximum parsimony (1,000 carried out using a Leica DM 2500 microscope replications), and neighbor-joining (Saitou and equipped with Nomarski interference optics Nei 1987; 1,000 replications using Jukes and (Leica, Wetzlar, Germany) and an Olympus DP Cantor [JC] distances) analyses were calculated 71 digital camera (Olympus, Tokyo, Japan). using PAUP * 4.0b10 (Swofford 2002). Five chloroplast genes of the strain TN-0205- The strain used in this study (TN-0205- 2-Pn-1, rbcL, atpB, psaA, psaB and psbC, were 2-Pn-1) is deposited in the Microbial Culture determined (AB593419, AB593420, AB593421, Collection at the National Institute for AB593422 and AB593423, respectively) for Environmental Studies (NIES-MCC; Kasai et al. the phylogenetic analyses. DNA extraction, 2009) as NIES-2567. polymerase chain reaction (PCR), and sequencing methods were as described Results previously (Nakada et al. 2007, 2010, Nozaki The vegetative colonies were usually et al. 2000). The PCR and sequencing reactions composed of 8 or 16 cells on the periphery were performed using previously published of spherical to broad ovoid, broad ellipsoidal primers (Nozaki et al. 1995, 1997, 1999, 2000). colonies (Figs. 1–3). Cells were biflagellate and The phylogenetic analyses were performed rounded inverted-triangular in the longitudinal with 22 operational taxonomic units (OTUs) of section, and a rounded square in the top view colonial Volvocales (Tetrabaenaceae, (Figs. 1–4). Each cell was enclosed by a and Volvocaceae), including 13 and keystone-shaped gelatinous structure directly Volvulina OTUs. Tetrabaenaceae was treated extending from the parental colonial envelope as the outgroup (Nozaki et al. 2000). The to form “individual sheaths.” There were no alignment was constructed according to Nozaki prominent intercellular spaces on the surface et al. (2000) and was unambiguous. No introns (Figs. 1, 3, 4). The chloroplast was large and were found in the amplified regions of rbcL, cup-shaped, with radiating ridges on the surface, atpB, psaA, psaB or psbC genes of the strain and with a single in the basal part TN-0205-2-Pn-1. The homogeneity of base (Figs. 2, 4). The pyrenoid was surrounded by frequencies was tested for each codon position fragmented starch grains. Each anterior cell using PAUP* 4.0b10 (Swofford 2002) and was had a larger stigma than posterior cells, and not significantly rejected (p > 0.05). Bayesian the posterior cells sometimes lacked stigmata inference (BI) was performed using MrBayes 3.1.2 (Figs. 1, 3). The stigma was positioned slightly (Ronquist and Huelsenbeck 2003) as described posterior to the center of the cell (Figs. 1, 3, 4). previously (Nakada and Nozaki 2009; 1,000,000 Two contractile vacuoles were located at the base generations of Markov chain Monte Carlo of the flagella (Fig. 4). The colonies were 11–42 iterations; the first 25% were discarded as burn- µm in width and 14–52 µm in length. Cells in). The evolutionary models of the 1st (GTR + I were 7–21 µm in diameter from the top view. + G), 2nd (F81 + I + G) and 3rd codon positions Asexual reproduction occurred by autocolony (GTR + I + G) of the protein coding genes formation (Figs. 5, 6). Each cell formed a plakea were selected using MrModeltest 2.2 (Nylander (Fig. 6) that developed into a daughter colony 2004), and were unlinked in the BI. Bootstrap after inversion. Daughter colonies were not 366 植物研究雑誌 第 85 巻 第 6 号 2010 年 12 月

Figs. 1–6. Volvulina compacta Nozaki. Nomarski interference microscopy after 3-day culture in MG medium. Arrowheads indicate stigmata. Figs. 1–4. Vegetative colonies. Anterior parts of the colonies are shown towards the top. Adjustments of contrast and brightness were carried out with Adobe Photoshop Element 4.0. Figs. 1–2. 16-celled colonies. Fig. 1. Surface view of the lateral side. Fig. 2. Optical section showing the hollow interior. Fig. 3. Surface view of the lateral side of an 8-celled colony. Fig. 4. Top view of a cell in a vegetative colony. Figs. 5–6. Asexual reproduction. Fig. 5. Parental colony stained with methylene blue. Fig. 6. Plakea in a parental colony. cv. Contractile vacuole. p. Pyrenoid. Scale bars = 10 µm. surrounded by a tightly surrounding parental pringsheimii (USA) with high statistical support cellular envelope (see Nozaki and Kuroiwa (1.00 PP and 100% BP), but only distantly to 1992), but rather were partitioned from each the Japanese strain of V. steinii NIES-545. The other by parental “individual sheaths” (Fig. 5). Japanese strain of V. compacta was more closely In the phylogenetic trees based on combined related to V. pringsheimii than to the Nepalese chloroplast rbcL, atpB, psaA, psaB and strain of V. compacta (1.00 PP and 84–95% BP). psbC genes (Fig. 7), the Japanese strain of V. compacta belonged to a clade comprising Discussion species of Pandorina and Volvulina with strong Among the colonial volvocaleans within the statistical support (1.00 posterior probability clade Reinhardtinia (sensu Nakada et al. 2008), [PP] and 83–99% BP). Within the clade, there are four genera that form 8- or 16-celled Japanese V. compacta was closely related to the ellipsoidal colonies, namely, Pandorina, Nepalese strain of V. compacta and a strain of V. Volvulina, and (Ettl December 2010 Journal of Japanese Botany Vol. 85 No.6 367

1.00/95 0.05 84/86 Volvulina pringsheimii UTEX 1020 (USA) 1.00/100 Volvulina compacta TN-0205-2-Pn-1 (Japan) 100/100 Volvulina compacta NIES-582 (Nepal) 1.00/90 -/68 73/88 Pandorina morum UTEX 2326 53/- 1.00/100 Pandorina morum NIES-574 100/100 Pandorina morum UTEX 854 “Volvulina boldii” UTEX 2185

1.00/85 1.00/100 Volvocaceae 72/85 1.00/92 99/100 Pandorina morum UTEX 880 83/99 1.00/100 Pandorina morum UTEX 1727 98/100 1.00/100 Pandorina colemaniae NIES-572 100/100 1.00/100 Volvulina steinii UTEX 1531 1.00/100 100/100 Volvulina steinii UTEX 1525 100/100 1.00/77 Volvulina steinii NIES-545 78/97 caudata UTEX 1658

1.00/95 1.00/100 carteri NIES-732 71/93 100/100 Eudorina unicocca UTEX 737 Yamagishiella unicocca UTEX 2430 UTEX 955 multicoccum UTEX 2580 Goniaceae 1.00/100 Gonium pectorale NIES-569 98/99 Basichlamys sacculifera NIES-566 1.00/100 Tetrabaenaceae 100/100 Tetrabaena socialis NIES-571

Fig. 7. Bayesian phylogenetic tree of colonial Volvocales (Tetrabaenaceae, Goniaceae and Volvocaceae) based on combined rbcL, atpB, psaA, psaB, and psbC gene sequences (6,021 bp). Tetrabaenaceae was treated as an outgroup. The corresponding posterior probabilities (≥ 0.90) are shown near nodes (top left). The bootstrap p-values (≥ 50%) from maximum likelihood (top right), maximum parsimony (bottom left), and neighbor- joining (bottom right) analyses are also shown. Branch lengths and the scale bar represent the expected number of nucleotide substitutions per site.

1983, Nozaki and Kuroiwa 1992, Nozaki vacuoles (Playfair 1915, Korshikov 1938, 2003). The present Japanese strain (TN-0205- Pocock 1953, Stein 1958, Nozaki 1982, Nozaki 2-Pn-1) has hollow vegetative colonies and et al. 1987) and are clearly distinguished from the was distinguishable from Pandorina, whose Japanese and Nepalese strains of V. compacta, colonies are generally contiguous in the center whose cells have two apical contractile vacuoles (Ettl 1983, Nozaki 2003). The present strain (Nozaki and Kuroiwa 1990). The vegetative was also distinguishable from Eudorina and cells of V. pringsheimii are similar to those of Yamagishiella through the lack of a tightly V. compacta in that they have two contractile surrounding cellular envelope (Nozaki and vacuoles (Starr 1966, Nozaki et al. 1987). Kuroiwa 1992). However, they are nearly circular in the top view Currently three validly published species of and distant from each other in the colony, while Volvulina are recognized (Playfair 1915, Starr the cells of V. compacta are square and in close 1962, Nozaki et al. 1987, Nozaki and Kuroiwa contact in the top view (this study; Nozaki and 1990). Cells of Volvulina steinii are circular in Kuroiwa 1990). Furthermore, the surface of V. the top view with more than two contractile pringsheimii zygotes are reportedly spiny (Starr 368 植物研究雑誌 第 85 巻 第 6 号 2010 年 12 月

1962), while those of Nepalese V. compacta Prefectural Government and Tsuruoka City, and are smooth (Nozaki and Kuroiwa 1990). Both by a Grant-in-Aid for Young Scientists (B; No. smooth and spiny-walled zygotes of V. steinii 22770086 to T.N.). have been reported (Carefoot 1966, Nozaki 1982). Because only a single strain of Japanese References V. compacta is available, the morphology of Carefoot J. R. 1966. and intercrossing its zygotes is currently unknown. Some strains in Volvulina steinii. J. Phycol. 2: 150–156. Coleman A. W. 1977. Sexual and genetic isolation in the of Volvulina are referred to as “V. boldii” nom. cosmopolitan algal species Pandorina morum. Am. J. nud. and are distinguished from other species Bot. 64: 361–368. by multiple in the chloroplast (online Coleman A. W. 2001. Biogeography and speciation in the catalogue of UTEX, http://web.biosci.utexas. Pandorina/Volvulina () superclade. J. Phycol. 37: 836–851. edu/utex/; see also Coleman 2001). Ettl H. 1983. Chlorophyta I. Phytomonadina. In: Ettl The Japanese strain of V. compacta was H., Gerloff J., Heynig H. and Mollenhauer D. (ed.), distantly related to the strains of V. steinii and “V. Süßwasserflora von Mitteleuropa, vol. 9. pp. 1–807. boldii.” Although in the phylogenetic analyses Gustav Fischer, Stuttgart. it was more closely related to V. pringsheimii Felsenstein J. 1985. Confidence limits on phylogenies: an approach using bootstrap. Evolution 39: 783–791. than the Nepalese strain, no morphological Kasai F., Kawachi M., Erata M., Mori F., Yumoto K., Sato differences were found between the Japanese M. and Ishimoto M. (eds.) 2009. NIES-Collection list and Nepalese strains of V. compacta. Japanese V. of strains, 8th edition. Jpn. J. Phycol. (Sôrui) 57 Suppl.: compacta and V. pringsheimiii were also similar 1–350. Korshikov A. A. 1938. On the occurrence of Volvulina in the absence of group I introns in rbcL and steinii in Ukraina. Bull. Soc. Nat. Moscou 47: 56–63 psaA, unlike Nepalese V. compacta, which has (in Russian with English abstract). two group I introns in both the rbcL and psaA Nakada T., Misawa K. and Nozaki H. 2008. Molecular gene (Nozaki et al. 1997, 2000). It is therefore systematics of Volvocales (Chlorophyceae, likely that the Nepalese and Japanese strains of Chlorophyta) based on exhaustive 18S rRNA phylogenetic analyses. Mol. Phylogenet. Evol. 48: V. compacta represent two independent cryptic 281–291. species, since several sexually isolated groups Nakada T. and Nozaki H. 2009. Taxonomic study of have been reported within single volvocacean two new genera of fusiform green , Tabris species such as V. steinii (Carefoot 1966) and gen. nov. and Hamakko gen. nov. (Volvocales, Chlorophyceae). J. Phycol. 45: 482–492. Pandorina morum (O. F. Müll.) Bory (Coleman Nakada T., Soga T. and Tomita M. 2010. Phylogenetic 1977). However, several “P. morum” strains position of a rare loricated green alga, Cephalomonas closely related to V. compacta have also been granulata N. L. Higinb. (Volvocales, Chlorophyceae). isolated from Nepal, Thailand, South Africa Phycol. Res. 58: 62–68. and USA (Coleman 2001), and thus, detailed Nakada T., Suda S. and Nozaki H. 2007. A taxonomic study of Hafniomonas (Chlorophyceae) based on morphological comparisons and examination of a comparative examination of cultured material. J. sexual compatibilities using worldwide strains Phycol. 43: 397–411. of “P. morum,” V. compacta and V. pringsheimii Nozaki H. 1982. Morphology and reproduction of Japanese are needed before formal taxonomic division of Volvulina steinii (Chlorophyta, Volvocales). J. Jpn. Bot. 57: 105–113. V. compacta. Nozaki H. 2003. Flagellated Green . In: Wehr J. D. and Sheath R. G. (ed.), Freshwater Algae of North We are grateful to Ms. Suzue Arii (The America: Ecology and Classification. pp. 225–252. Kanagawa Prefectural Water Works, Tanigahara, Academic Press, San Diego. Japan) for her kind help in collecting the water Nozaki H., Hara Y. and Kasaki H. 1987. Light and electron microscopy of pyrenoids and species delimitation in samples from Lake Tsukui. This study was Volvulina (Chlorophyta, Volvocaceae). J. Phycol. 23: supported in part by grants from Yamagata 359–364. December 2010 Journal of Japanese Botany Vol. 85 No.6 369

Nozaki H., Itoh M., Sano R., Uchida H., Watanabe M. Playfair G. H. 1915. Freshwater algae of the Lismore M. and Kuroiwa T. 1995. Phylogenetic relationships district: with an appendix on the algal fungi and within the colonial Volvocales (Chlorophyta) inferred Schizomycetes. Proc. Linn. Soc. New South Wales 40: from rbcL gene sequence data. J. Phycol. 31: 970–979. 310–362. Nozaki H., Ito M., Sano R., Uchida H., Watanabe M. M., Pocock M. A. 1953. Two multicellular motile , Takahashi H. and Kuroiwa T. 1997. Phylogenetic Volvulina Playfair and , a new genus. analysis of Yamagishiella and Platydorina Trans. R. Soc. South Africa 34: 103–127. (Volvocaceae, Chlorophyta) based on rbcL gene Posada D. and Crandall K. A. 1998. Modeltest: testing the sequences. J. Phycol. 33: 272–278. model DNA substitution. Bioinformatics 14: 817–818. Nozaki H. and Kuroiwa T. 1990. Volvulina compacta Pringsheim E. G. 1946. Pure Cultures of Algae. Cambridge sp. nov. (Volvocaceae, Chlorophyta) from Nepal. University Press, Cambridge. Phycologia 29: 410–417. Ronquist F. and Huelsenbeck J. P. 2003. MrBayes 3: Nozaki H. and Kuroiwa T. 1992. Ultrastructure of the Bayesian phylogenetic inference under mixed models. extracellular matrix and taxonomy of Eudorina, Bioinformatics 19: 1572–1574. and Yamagishiella gen. nov. (Volvocaceae, Saitou N. and Nei M. 1987. The neighbor-joining method: Chlorophyta). Phycologia 31: 529–541. a new method for reconstructing phylogenetic trees. Nozaki H., Misawa K., Kajita T., Kato M., Nohara S. and Mol. Biol. Evol. 4: 406–425. Watanabe M. M. 2000. Origin and evolution of the Starr R. C. 1962. A new species of Volvulina Playfair. Arch. colonial Volvocales (Chlorophyceae) as inferred from Mikrobiol. 42: 130–137. multiple, chloroplast gene sequences. Mol. Phylogenet. Stein J. R. 1958. A morphological study of Astrephomene Evol. 17: 256–268. gubernaculifera and Voluvulina steinii. Am. J. Bot. 45: Nozaki H., Ohta N., Takano H. and Watanabe M. M. 1999. 388–397. Reexamination of phylogenetic relationships within the Swofford D. L. 2002. PAUP*: Phylogenetic Analysis Using colonial Volvocales (Chlorophyta): An analysis of atpB Parsimony (* and Other Methods). Version 4.0b10. and rbcL gene sequences. J. Phycol. 35: 104–112. Sinauer, Sunderland. Nylander J. A. A. 2004. MrModeltest 2.2. Program Tsukii Y. 2010. Picture Book of Freshwater distributed by the author. Evolutionary Biology Centre, Microorganisms: Visual Guide Book of Protists. Uppsala University. Seibundo Shinkosha, Tokyo (in Japanese).

a, b, a c 仲田崇志 *, 冨田 勝 , 野崎久義 ：日本新産ヨリソイマ ユダマモ Volvulina compacta（緑藻綱オオヒゲマワリ科） とその系統的位置 Volvulina（新称；マユダマモ属）は主に 8 または 16 縁で，本邦から知られていた V. steinii（新称：スタイン 細胞性の群体性オオヒゲマワリ類で，中空で楕円体状の マユダマモ）とは系統的に離れていた．日本産ヨリソイマ 群体を形成する．本研究において，マユダマモ属の株 ユダマモはスタインマユダマモよりも V. pringsheimii に が本邦神奈川県津久井湖より分離され，形態に基づき より近縁だったが，V. pringsheimii とは細胞が頂面観で Volvulina compacta（新称：ヨリソイマユダマモ）と同 四角形であることから明確に区別された．これらの結果か 定された．ヨリソイマユダマモはこれまでネパールからし ら，ヨリソイマユダマモに隠蔽種が含まれることが示唆さ か知られておらず，本邦では初の報告となる．葉緑体の れた． rbcL, atpB, psaA, psaB および psbC 遺伝子を用いた複 （a慶應義塾大学先端生命科学研究所， 数遺伝子解析によると，ヨリソイマユダマモの日本産株は b慶應義塾大学政策・メディア研究科， ネパール産株および V. pringsheimii のアメリカ産株と近 c東京大学大学院理学系研究科生物科学専攻）