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. 84). Fig. 1. 16-celled vegetative colony. Note multiple pyrenoids (arrows). Fig. 2.32 ・・ celled vege- tative tative colony. Figs. 3-5. Three types of 8-celled vegetative colonies. Note multiple pyrenoids (arrows). Figs. 6- 10. Sexual reproduction. Fig. 6. Late stage of plasmogamy. Fig. 7. Quadriflagellate zygote. Fig. 8. One- day-old day-old aplanozygote. Fig. 9. Six-day-old aplanozygotes surrounded by watery gelatinous matrix. lndian ink

prep 訂 ation. Fig. 10. Eight-day-old mature aplanozygotes. June June 2006 Joumal of Japanese Botany Vo l. 81 No. 3 143

-20μm Fig. Fig. 11. Line drawing of vegetative colony of Japanese strain of Gonium multicoccum multicoccum Pococ k. mating mating reaction occurred within 2 days after In the phylogenetic analyses of the rbcL transferring transferring the colonies to AFM medium. gene sequences ， the Japanese strain and three The colonies dissociated into individual other strains of G. multicoccum formed a cells ， from which biflagellate protoplasts es- monophyletic group (with 93 % and 86 % caped caped to become gametes. After gametic bootstrap values in NJ and MP analyses ，re- union union (Fig. 6) ，a quadriflagellate planozygote spectively) that is separated from four other was formed (Fig. 7). The zygotes then settled species of Gonium. G. multicoccum was sub- and and secreted a cell wall (Fig. 8). A broad ge- divided into two clades (Fig. 13). One com- latinous latinous matrix could be seen around them prised two North American strains (NIES- (Fig. (Fig. 9). After about one week ， the hypno- 1038 from Texas and UTEX 783 from zygotes zygotes became reddish-brown in colo r. Califomia) ， while the other contained the They measured 14-26 ドm in diameter (Fig. Japanese and Nepalese strains (UTEX 2580). 10). 10). Both clades were supported with 100 % Our Japanese strain (Asa.Goni.84) and a bootstrap values in the NJ and MP methods strain strain of G. multicoccum from Nepal (UTEX (Fig. 13).

2580; 2580; Nozaki et al. 2002) had identical se 聞 quences quences of the rbcL exon (1 128 bp) and Discussion group group 1 introns (844 bp). However ， the ITS2 Pocock (1955) characterized Gonium sequences sequences (22 4- 226 bp) differed (Fig. 12). multicoccum as having 8- ，16- ， or 32-celled 144 144 植物研究雑誌第81 巻第3号 平成18 年6月

20 40 Asa.Goni.84 AATACTCGCTCCCTATATACATG 一一一一一一一一 GGGAACGGAACTG 議CTGTC UTEX2580 AATACTCGCTCCCTATATACATG 一一一一一一一一 GGGAACGGAACTG 窓CTGTC UTEX783 AATACTCGCTCCCTCCAACTCCGGTTGGTTAGGGAACGGAACTG 議CTGTC NIES-1038 AATACTCGCTCCCTCCAACTCCGGTTGGTTAGGGAACGGAACTG&CTGTC

60 80 100 Asa.Goni.84 TCGGCGGTTAATTTAA- 一一 TTAACTGCCGGGTCAGTTGAAGTATGCAGAG UTEX2580 TCGGCGGTTA 丙TTTAA---TTAACTGCCGGGTCAGTTGAAGTATGCAGAG UTEX783 TCGGCAGTTA 益-CTAACCGTTAACTGCCGGGTCAGCTGAAGTGTGCAGAG NIES-1038 TCGGCAGTTAA-CTAACCGTTAACTGCCGGGTCAGCTGAAGTGTGCAGAG

120 140 Asa.Goni.84 GTTATTGCATGGACCCGTTTATGGGCCTCTACTGGGTAGGCAACTTGC-T UTEX2580 GTTATTGCATGGACCCGTTTATGGGCCTCTACTGGGTAGGCAACTTGC-T UTEX783 GTTGATGCATGGACCCGCTCATGGGCCTCTACTGGGTAGGCAATTAACTT NIES-1038 GTTGATGCATGGACCCGCTCATGGGCCTCTACTGGGTAGGCAATTAACTT

160 180 200 Asa.Goni.84 TGCTAATGCTTTAGTAGATGGCCTGGGACCGTGTGTGTTTTAACCCAAAC UTEX2580 TGCTAATGCTTTAGTAGATGGCCTGGGACCGTGTGTGTTTTAACCCAAAC UTEX783 TGCTAATGCTTTAGTAGATGGCTTGGGACTGTGCCTGTC--GGCCCAAAC N 工ES-1038 TGCTAATGCTTTAGTAGATGGCTTGGGACTGTGCCTGTC 一一 GGCCCAAAC

220 240 Asa.Goni.84 CAGGAACTCAAT-ATTT 思ATTGAGCAACTGT 一一 TTATTTTC UTEX2580 CAGGAACTCAAT 一一 TTTCDATTGAGCAACTGT 一一 TTATTTTC UTEX783 CAGGAACTCAGTT[TACG t3 ACTGAGCAACTGTACCTATTT 一C NIES-1038 CAGGAACTCAGTT:TACG 鰐ACTGAGCAACTGTACCTATTTTC

Fig. Fig. 12. Alignment of ITS2 sequences from four strains of Gonium multicoccum Pocock (Table 1). Nucleotides which are different between Asa.Goni.84 and UTEX 2580 are indicated by shading. vegetative vegetative colonies with multipyrenoid cells. Pocock (1955) reported the frequent pro- Although Although G. discoideum Prescott (1 942) has duction of 8-celled colonies ， in which the 32-ce l1 ed colonies ， the number of pyrenoids cells were a町 anged in two rows of three ， and in in the chloroplast and the shape of vegetative a row of two. By con 仕ast ， our J apanese alga cells cells in this species differ from those of G. r紅 ely produced 8-celled colonies ， and these multicoccum. multicoccum. In G. discoideum ， there are had two cell arrangements ， one of which was usua l1 y two pyrenoids per ce l1， and the cells characteristic of G. multicoccum ， as de- are are pyriform (Prescott 1942 ，Ettl 1983). By scribed by Pocock (1 955) (Figs. 4，5) ， while contrast ， G. multicoccum has two to 12 the other resembled the 訂 rangement of G. pyrenoids pyrenoids in mature vegetative cells ，which pectorale (Fig. 3; Nozaki 1984 ， 1988). are are almost spherical or elongate (Pocock Nozaki and Kuroiwa (1 991) reported both of 1955 ， Nozaki and Kuroiwa 199 1). these g.・・ ce l1 ed colony arrangements in Accordingly ， our Japanese collection can be Nepalese strains of G. multicoccum ，and our assigned assigned to G. multicoccum. rbcL gene tree demonstrated a close relation- June June 2006 Joumal of Japanese Botany Vo l. 81 No. 3 145

G. G. octonarium NIES-851 (USA)

G. G. quadr αtum NIES-653 (ltah 訂 i ，Nepal) viridistellatum G. viridistellatum NIES-289 (Okinawa ，Japan) 991 991 G. viridistellatum NIES-857 (Kathmandu ，Nepal) 97 97 L G. viridistellatum NIES-654 (Kanagawa ，Japan) 盟問問一間 G. G. multicoccum NIES-I038 (Texas ，USA) G. G. multicoccum UTEX 783 (Califomia ，USA) G. multicoccum Asa. Goni. 84 (Fukuoka ，Japan) G. G. multicoccum UTEX 2580 (Kathmandu ，Nepal) 100 G. pectorale Kita.Goni.3 (Hokkaido ，Japan) 100 G. pectorale Kanek04 (Okinawa ，Japan) G.pector αle NIES-569 (Okayama ，Japan) Pα ndorina colem α niae NIES-572 (Okayama ，Japan) Pandorina Pandorina morum UTEX 880 (Califomia ，USA) 0.005 0.005 ト一一寸

Fig. Fig. 13. Neighbor-joining (NJ) tree based on 1128 bp of the rbcL genes from 12 strains of five five Gonium (G.) species and two strains of Pandorina (Table 1). Branch lengths are propotional propotional to Kimura (1 980) distances ， which are indicated by the scale bar below the tree. tree. Numbers above or below the branches represent 50 % or more bootstrap values based based on 1000 replications of the NJ or maximum parsimony analyses ，respectively.

ship ship between our Japanese strain of G. Laboratory of Plant Systematics of multicoccum multicoccum and the Nepalese strain (UTEX Department of Biological Science ，Graduate 2580) that Nozaki and Kuroiwa (1991) stud- School of Botany ，University of Tokyo for ied ied (Fig. 13). Furthermore ， the sequence of their kind supports. We are also grateful to the the rbcL gene [including two inte 町 upted Ms. S. Tsuda of University of Tokyo for her group 1 introns (1972 bp)] of our Japanese providing soil materials. This study was sup- strain strain exactly matched that of the Nepalese ported in part by a grant from the Institute strain strain (UTEX 2580; Nozaki et al. 2002). for Fermentation ，Osaka (to H. N.). This indicates a very recent radiation of these these two strains of G. multicoccum ，and References there there is no genetic differentiation between Akiyama M. ， H. Hirose ， Yamagishi T. and Hirano M. them. However ， there were differences in the 1977. Class Chlorophyceae. In. Hirose H. and ITS2 sequences between the two strains (Fig. Yamagishi T. (eds.) ， Ill ustrations of the Japanese Fresh-water Fresh-water Algae ，pp. 275-760. Uchida Rokakuho 12). 12). Publishing ，Tokyo (i n Japanese). Coleman Coleman A. W. ， Suarez A. and Goff L. J. 1994. We are grateful to D r. M. Kato of National Molecular delineation of species and syngens in Science Museum ， and D r. Y. Kita of volvocacean green alge (Chlorophyta). J. Phyco l. University of Tokyo for their helpful sugges- 30: 80-90. tions. tions. We want to thank all the members of Ettl H. 1983. Chlorophyta 1. Phytomonadina In. Ettl 146 146 植物研究雑誌第81 巻第3号 平成18 年6月

H. ，Gerloff J. and Mollenhauer D. (eds.) ，Suβwass- ferred from cladistic analysis based on mo 叩hologi- erflora erflora von 民1itteleuropa ， Bd. 9. xiv + 807 pp. cal data. J. Phyco l. 30: 353-365. Gustav Gustav Fischer Verlag ，Stuttgart. 一一一，一一一， Sano R.， Uchida H. and Watanabe M. M. Felsenstein Felsenstein J. 1985. Confidence limits on phylogenies: 1995. Phylogenetic relationships within the colo- an an approach using the bootstrap. Evolution 39: nial Volvocales (Chlorophyta) inferred from rbcL 783-791. 783-791. gene sequence data. J. Phyco l. 31: 970-979. Kasai Kasai F. ，Kawachi M. ， Erata M. and Watanabe M. M. 一一and Kuroiwa T. 199 1. Morphology and sexual (eds.). (eds.). 2004. NIES-Collection. List of Strains. reproduction of Gonium multicoccum (Volvocals ， Microalgae Microalgae and Protozoa. 7th ed. 257 pp. National Chlorophyta) from Nepa l. Phycologia 30: 381- Institute Institute for Environmental Studies ， Tsukuba. 393. Kato Kato S. 1982. Laboratory culture and mo 叩hology of 一一一， Misawa K.， Kajita T. ， Kato M. ， Nohara S. and Colacium vesiculosum E 担rb. (Euglenophyceae). Watanabe M. M. 2000. Origin and evolution of the Jpn. Jpn. J. Phyco l. 30: 63-67 (in Japanese with English colonial Volvocales (Chlorophyceae) as inferred abstract). abstract). from multiple ，chloroplast gene sequences. Mo l. Kimura M. 1980. A simple method for estimating evo- Phylogene t. Evo l. 17: 256-268. lutionary lutionary rates of base substitutions through com- 一一一， Song L.， Liu Y.， Hiroki M. and Watanabe M. M. parative parative studies of sequences. nucleotide J. Mo l. 1998. Taxonomic re-examination of a Chinese Evo l. 16: 111-120. strain labeled ‘Eudorina sp.' (Volvocaceae ， Kusumoto M. ，Sonoda S. ， Kajino M. and Hamamatu Chlorophyta) based on morphological and DNA N. N. 1978. Gonium pectorale Muller isolated from sequence data. Phyco l. Res. 46， supplemen t: 63- paddy paddy field soil collected from various localities in 70. Japan. Japan. Jpn. J. Phyco l. 26: 19-26 (in Japanese with 一一一， Takahara M. ，Nakazawa A.， Ki ta Y.， Yamada T. English English abstract). Takano H. ， Kawano S. and Kato M. 2002. Mai J. C. and Coleman A. W. 1997. The intemal tran- Evolution of rbcL group IA introns and intron open scribed scribed spacer 2 exhibits a common secondary reading frames within the colonial Volvocales structure structure in green algae and flowering plants. 1. (Chlorophyceae). Mo l. Phylog. Evo l. 23: 326-338. Mo l. Evo l. 44: 258-27 1. Pocock M. A. 1955. Studies in North American Nakazawa A. ， Kri enitz L. and Nozaki H. 200 1. Volvocales. 1. The genus Gonium. Madrono. 13: Taxonomy of the unicellular green algal genus 49-80. Vitreochlamys Vitreochlamys (Volvocales) ， based on comparative Prescott G. W. 1942. The algae of Louisiana ， with des- mo 叩hology of cultured materia l. Eu r. J. Phyco l. criptions of some new forms and notes on distribl ト 36: 36: 113-128. tion. Trans. Am. Microsc. Soc. 61: 109-119. Nozaki Nozaki H. 1984. Newly found facets in the asexual and Pringsheim E. G. 1946. Pure Cultures of Algae. 119 sexual sexual reproduction of Gonium pectorale pp. Cambridge University Press ， Cambridge. (Chlorophyta ，Volvocales). J. Phyco l. 32: 130- Saito N. and Nei M. 1987. The neighbor サoining 133. 133. method: a new method for reconstructing 一一一 1988. Colonial Volvocales (Chlorophyta) from phylogenetic trees. Mo l. Bio l. Evo l. 4: 406 -4 25. Kathmandu ，Nepa l. In. Watanabe M. and Malla S. Starr R. C. and Zeikus J. A. 1993. UTEX - the Culture B. B. (eds.) ， Cryptogams of the Himalayas Vo l. 1， pp. Collection of Algae at the University of Texas at 39 -4 6 ，National Science Museum ， Tsukuba. Austin. J. Phyco l. 29 ，supplement: 1-106. 一一一 1989. Mo 中 hological variation and reproduction Stein J. R. 1958. A morphologic and genetic study of in in Gonium viridistellatum (Volvocales ， Gonium pectorale. Am. J. Bo t. 45: 664-672. Chlorophyta). Chlorophyta). Phycologia 28: 77-88. Swofford D. L. 2003. PAUP*. Phylogenetic Analysis 一一一 1993. Asexual and sexual reproduction in Using Parisomy (*and other methods). Version Gonium quadratum (Chlorophyta) with a discus- 4.0b 01 Sinauer Associates ，Sunderland ，Massachu- sion sion of phylogenetic relationships within the setts. Goniaceae. Goniaceae. J. Phyco l. 29: 369-376. Thompson J. D. ， Gibson T. J. ， Plewniak F. ， 一一一 2003. Flagellated green algae. In. Wehr J. D. and Jeanmougin F. and Higgins D. G. 1997. The Sheath Sheath R. G. (eds.) ， Freshwater Algae of North ClustalX windows interface: flexible strategies for America. ，Academic Press ，Amsterdam ，Boston ， multiple sequence alignment aided by quality London ，New Y ork ，Oxford ，Paris ， San Diego ， analysis tools. Nucleic Acids Res. 25: 4876 -4 882.

Singapore ，Sydney ，Tokyo ， pp. 225-252. Wakasugi T. ， Nagai T. ，Kapoor M. ， Sugita M. ，It oM 吋 一一- and It o 乱1. 1994. Phylogenetic relationships It o S. ， Tsudzuki J. ，Nakashima K.， Tsudzuki T. ， within within the colonial Volvocales (Chlorophyta) in- Suzuki Y.， Hamada A.， Ohta T. ， Inamura A. ， June June 2006 Journal of Japanese Botany Vo l. 81 No. 3 147

Y oshinaga K. and Sugiura M. 1997. Complete nu- Yamada): 379-384. cleotide cleotide sequence of the chloroplast genome from Yoshinaga K.， Ohta T. ， Suzuki Y. and Sugiura M. the the green alga Chlorella vulgaris: the existence of 1988. Chlorella chloroplast DNA sequence con- genes genes possib1y involved in chloroplast division. taining a gene for the large subunit of ribulose-l ，5- Proc. Proc. Nat l. Acad. Sci. U.S. A. 94: 5967-5972. bisphosphate carboxylase/oxygenase and a part of Watanabe M. 1977. A preliminary study of Gonium a possible gene for the bata' subunit of RNA viridistellatum viridistellatum sp. nov. (Chlorophyta ，Volvoca- polymerase. Plant Mo l. Bio l. 10: 245-250. ceae). ceae). Bull. Jap. Soc. Phyco l. 25 (supp l.， Mem. Iss.

山白敏寛ぺ仲田崇志b ，宮地和幸a ，野崎久義b • 日本新産 Gonium multicoccum (緑藻綱，オオヒゲ マワリ目)の形態と分子系統 ル産の本種と最も近縁であることが判明した. 福岡県朝倉郡筑前町の水田土壌より Gonium を (a 東邦大学理学部生物学科， 分離・培養し，光学顕微鏡による形態観察を行っ *現所属:東京大学大学院理学系研究科 た結果，これまでに日本より報告のない Gonium 生物科学専攻 multicoccum と同定された.葉緑体 rbcL 遺伝子を b東京大学大学院理学系研究科生物科学専攻) 用いた分子系統解析を実施した結果，本藻はネパー