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Page 1 植物研究雜誌 J. Jpn. Bot 81: 139–147 (2006) Morphology And 植物研究雑誌 J. J. Jpn. Bo t. 81: 81: 139-147(2006) Morphology and Molecular Phylogeny of Gonium multicoccum (Volvocales ， Chlorophyceae) Newly Found in Japan a b a b Toshihiro Toshihiro K. YAMADA *， Takashi NAKADA ，Kazuyuki 乱1IYAJI and Hisayoshi NOZAKI aDepartment aDepartment of Biology ， Faculty of Science ，Toho University ， 2-2-1 ，Miyama ，Funabashi ，Chiba ， 274-8510 JAPAN; 'Present 'Present address: Department of Biological Sciences ， Graduate School of Science ， University University of Tokyo ，7-3-1 ，Hongo ，Bunkyo-ku ，Tokyo ， 113-0033 JAPAN E-mail: E-mail: toshikan@bio l. s.u-tokyo.ac.jp bDepartment bDepartment of Biological Sciences ， Graduate School of Science ，University of Tokyo ， 7-3-1 ，Hongo ，Bunkyo-ku ，Tokyo ， 113-0033 JAPAN (Received 00 December 5，2005) Strains Strains of Gonium were isolated from a soil sample collected in Fukuoka Prefecture ， Japan. Morphological observations identified the strains as Gonium multicoccum multicoccum Pocock ， which has not been reported previously from Japan. Molecular phylogenetic phylogenetic analyses based on the plastid rbcL gene sequences suggested a close rela- tionship tionship between Japanese and Nepalese strains of the species. Key words: Chlorophyceae ，Gonium multicoccum ，ITS2 ，rbcL ，Volvocales. The genus Gonium O. F. 島1ull. (Gonia- Prefecture ， J apan. Here ，we report the mor- ceae ，Volvocales ， Chlorophyceae) is charac- phology ， reproduction ， and molecular terized terized by a flattened colony with 8，16 ， or phylogeny of these strains. 32 biflagellate cells a汀 anged in a single layer layer (Nozaki and It o 1994 ， Nozaki 2003). 島iaterials and 島iethods There There are seven species in the genus The soil samples used in this study were (Watanabe (Watanabe 1977 ， Ettl 1983 ， Nozaki 1989). collected in a paddy field at Chikuzen- There There are detailed morphological reports on machi ， Asakura-gun ，Fukuoka Prefecture ， the the strains of four species grown in culture Japan in March 2004. Clonal cultures [G. [G. pectorale O. F. Mull. (Stein 1958 ， (Asa.Goni.84 ，Asa.Goni.6 ，and AsCl-l) were Kusumoto et al. 1978) ， G. multicoccum established using the pipette-washing Pocock (Nozaki and Kuroiwa 1991) ， G. method (Pringsheim 1946) from Petri dishes quadratum E. G. Pringsh. ex N ozaki (N ozaki (90 x 20 mm) in which a small amount of 1993) ， and G. viridistellatum M. Watanabe dried soil sample had been rewetted with dis- (Watanabe (Watanabe 1977 ，Nozaki 1989)]. The follow- tilled water. The cultures were grown in ing ing species have been recorded in Japan: G. screw-cap tubes (18 x 150 mm) containing pectorale pectorale (Kusumoto et al. 1978 ， Nozaki about 11 mL of AF-6 medium (Kato 1982) 1984) ， G. viridistellatum (Watanabe 1977 ， modified by the elimination of CaC03 and Nozaki Nozaki 1989) ，and G. formosum Pascher the addition of 400 mg.L- 1 MES (Kasai et al. (Akiyama et al. 1977). 2004). The cultivation temperature was Recently ，strains of G. multicoccum were about 25 0 C ， with altemating periods of 10 h isolated isolated from soils collected in Fukuoka darkness and 14 h light at an intensity of -139- 140 140 植物研究雑誌第81 巻第3号 平成18 年6月 150-200μmol.m- 2 .s- 1 provided by cool-white 2.0 mL of AFM medium (Nakazawa et al. fluorescent fluorescent lamps. 2001) were added to the condensed culture To observe the vegetative morphology and in a watch glass (60 mm in diameter) sup- asexual asexual reproduction ， about 0.5 mL of an ac- ported on a glass depression in Petri dishes. tively tively growing culture was inoculated into To minimize evaporation from the watch fresh fresh medium every seven to eight days. To glasses ， about 5.0 mL of distilled water were induce induce sexual reproduction ，22 mL of four- added to the bottoms of the Petri dishes. The day-old day-old cultured material were condensed to Petri dishes were exposed to the growing 0.5- 1. 0 mL by centrifugation. Subsequently ， conditions described above. Light micros- Table Table 1. Li st of rbcL gene and ITS2 sequences used in this study Origin Origin and DDBJAMB L/ GenBank Taxa Strain designation accession nurnber rbcL rbcL ITS2 Goniaceae Goniaceae Gonium multicoccum UTEX* 2580 Nozaki et al. (1 995) Mai and Colernan (1997) D63435 U66966 UTEX 783 Nozaki et al. (2002) Mai and Coleman (1997) AB076102 & AB076103 U66967 NIES** ー1038 This study This study AB246187 AB246191 Asa.Goni.84 Asa.Goni.84 This study This study (NIES-1708) (NIES-1708) AB246188 AB246192 Gonium octonarium NIES-851 Nozaki et al. (1 995) D63436 Gonium pectorale NIES-569 Nozaki et al. (1 995) D63437 Kita.Goni.3*** Kita.Goni.3*** This study (NIES-1713) (NIES-1713) AB246189 Kaneko4** キヰ This study (NIES-1711) (NIES-1711) AB246190 Gonium quadratum NIES-653 Nozaki et al. (1 995) D63438 Gonium viridistellatum NIES-289 Nozaki et al. (2002) AB076091 NIES-654 Nozaki et al. (1 995) D86831 NIES-857 Nozaki et al. (2002) AB076092 & AB076093 Volvocaceae Volvocaceae Pandorina Pandorina morum UTEX 880 Nozaki et al. (2000) AB044166 Pandorina Pandorina colemaniae NIES-572 Nozaki et al. (1 995) D63441 *Culture *Culture Collection of Algae at the University of Texas at Austin (Starr and Zeikus 1993). **Microbial **Microbial Culture Collection at the National Institute for Enviorornental Studies (Kasai et al. 2004). **キ Isolated from soil collected in a paddy field ，Ki tahiroshirna ，Hokkaido ，Japan ， in March 2004. ****Isolated ****Isolated frorn soil collected in a paddy field ，Ki n-cho ，Okinawa ，Japan ， in March 2000. June June 2006 Joumal of Japanese Botany Vo 1. 81 No. 3 141 copy copy was carried out using an Olympus multicoccum (Asa.Goni.84 and NIES-I038) BX60 microscope equipped with Nomarski were sequenced ， as described by Coleman interference interference optics. et al. (1 994). The methods for extracting total DNA and sequencing sequencing the large subunit of the Rubisco Results (rbcL) (rbcL) gene (including inte 汀 upted group 1 The vegetative colonies were composed of introns) introns) of two strains of G. multicoccum 8，16 ， or 32 cells. In four- to eight-day-old (Asa.Gon i. 84 and NIES-I038) and two new cultures ，16-celled colonies were most abun- isolates isolates of G. pectorale (Kita.Goni.3 and dan t. In culture more than 10 days old ，32- Kaneko4) Kaneko4) (Table 1) were essentially as de- celled colonies were not formed ， but 8-celled scribed scribed previously (Nozaki et al. 1995 ，1998 ， colonies became frequen t. The 16-celled 2000 ， 2002). The region sequenced co 町 b colonies contained 12 peripheral and four sponded sponded to positions 31-1158 of the central cells a町 anged in a curved ， rhomboid Chlorella Chlorella vulgaris Beij. rbcL gene to square plate ， measuring up to 99μm wide (Y oshinaga et al. 1988 ，Wakasugi et al. (Fig. 1). The 32-celled colonies were octago- 1997). 1997). The new Japanese strain of G. nal in outline ，radially symme 佐ical ，and con- multicoccum multicoccum described here had two inter- tained 4 central ， 10 median ， and 18 rupted rupted group 1 introns in the same exon posi- peripheral cells in a plate measuring up to tions tions as the Nepalese strain (UTEX 2580) of 135 阿n wide (Figs. 2， 1 1). The cell 紅 range- G. G. multicoccum (Nozaki et al. 2002). The ment of the 8-celled colonies may be classi- coding coding regions (1 128 bp) of the sequences fied into two types. Approximately 95 % of from from these four strains were aligned with the 8-celled colonies had cells a町 anged in those those of eight other Gonium strains and two four zigzag rows of two cells each (Fig. 3). strains strains of the related genus P α ndorina (Table In the other 8-celled colonies ， the cells were 1). 1). From this alignment ，a distance matrix arranged in two rows of three cells each ， was calculated by applying the two- with am 訂 ginal (Fig. 4) or central (Fig. 5) parameter parameter method (Kimura 1980) in Clustal row of two cells. X (Thompson et al. 1997). A phylogenetic The cellS were nearly spherical ， elongate tree tree was constructed using the neighbor- bell-shaped ， or angular in shape ， and were up joining joining (NJ) algorithm (Saito and Nei 1987) ， to 21μm long. Each cell was biflagellate and also also using Clustal X ， and the robustness of had a stigma in the anterior portion ，two con 闇 the the resulting lineages was tested using a tractile vacuoles near the base of the flagella ， bootstrap bootstrap analysis (Felsenstein 1985) with and a massive cup-shaped chloroplas t. In the 1，000 replications. In addition ，a maximum young colonies ， each cell contained one to parsimony parsimony (MP) analysis [including a boot 聞 three pyrenoids ， whereas in the mature colo- strap strap analysis based on 1，000 replications of nies ，two to eight pyrenoids were seen in the the the general heuristic search using the tree- chloroplast (Figs. 1-5). bisection-reconnection bisection-reconnection (TBR) branch-swapp 同 Asexual reproduction was accomplished ing ing algorithm] was performed using PAUP* by autocolony formation. Each cell of the 8- ， 4.0bl0 4.0bl0 (Swofford 2003). Since the genus 16 ・， or 32-celled colonies divided three ，four ， Gonium has been resolved as a monophyletic or five times successively to form an 8- ，16- ， group group (Nozaki et al. 2000 ，2002) ， the two or 32-celled daughter colony ，respectively ， Pandorina Pandorina strains (Table 1) were designated within the parental individual gelatinous as as the outgroup. In addition ， the intemal sheath. transcribed transcribed spacer (ITS) 2 regions of ribo- The strains were homothallic and exhib- somal somal DNA from two strains of G. ited isogamous sexual reproduction. The 142 142 植物研究雑誌第81 巻第3号 平成 18 年6月 Figs. Figs. 1-10. Nomarski interference microscopy of Japanese strain of Gonium multicoccum Pocock (Asa.Gon i.
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