Volvocaceae, Chlorophyceae), New to Japan, and Its Phylogenetic Position
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J. Jpn. Bot. 85: 364–369 (2010) Volvulina compacta (Volvocaceae, Chlorophyceae), 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. Chloroplast 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 cell shape in the top view. These results indicate the existence of cryptic species within V. compacta. Key words: Molecular phylogeny, taxonomy, 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 —364— December 2010 Journal of Japanese Botany Vol. 85 No.6 365 (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, Goniaceae (Figs. 1–4). Each cell was enclosed by a and Volvocaceae), including 13 Pandorina 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 pyrenoid 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, Eudorina and Yamagishiella (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 Platydorina caudata UTEX 1658 1.00/95 1.00/100 Volvox carteri NIES-732 71/93 100/100 Eudorina unicocca UTEX 737 Yamagishiella unicocca UTEX 2430 Volvox globator UTEX 955 Gonium 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.