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Page 1 植物研究雜誌 J. Jpn. Bot. 80: 197-207 (2005) Two Species Of 植物研究雑誌 J. J. Jpn. Bo t. 80: 80: 197-207(2005) Two Species of Chlorogonium (Volvocales ，Chlorophyceae) from Japan Takashi Takashi NAKADA a， Atsusi NAKAZAWA b and Hisayoshi NOZAKI a aDepartment aDepartment of Biological Sciences ， Graduate School of Science ，University of Tokyo ， Hongo 7-3-1 ，Bunkyo-ku ，Tokyo ，113 ・0033 JAPAN; E 司 mail: nozaki@biol ふ u-tokyo.ac.jp bRIKEN (The Institute of Physical and Chemical Research) ， Hirosawa 2-1 ，Wako ，Saitama ， 351-0198 JAPAN (Received on December 7，2004) Strains Strains from two species of Chlorogonium were isolated from Japanese soil samples ， and and were examined with light and electron microscopy. A molecular phylogeny based on rbcL rbcL sequences was also constructed for these strains. One of the two species ，Chloro- gonium gonium elongatum (P. A. Dang.) France ， has not been previously studied in culture from Japan. Japan. The other species was identified as Chlorogonium kasakii Nozaki ， which has not been been previously recorded from Japan. Key words: Chlorogonium ，Chlorophyceae ，culture strain ，distribution ，taxonomy. Chlorogonium Ehrenb. (Haematococca- light microscope. These species can be relia- ceae ，Volvocales ， Chlorophyceae) is a uni- bly distinguished using an electron micro- cellular cellular genus characterized by its spindle- scope and examination of pyrenoid stability shaped shaped vegetative cells (e. g. ，Smith 1950). under photoheterotrophic culture condition Ettl Ettl (1 980) distinguished this genus from (Nozaki et al. 1998). Therefore ， the records Chlamydomonas Ehrenb. by its first trans- of C. euchlorum and C. elongatum from verse verse cell division without protoplasmic ro- Japan 訂 'e to be re 四 assessed using newly iso- tation. tation. Approximately 20 species have been lated culture strains ， that allow reliable and recognized recognized in Chlorogonium (Ettl 1983 ， reproducible identification of the species. Nozaki et al. 1998). Nozaki et al. (1 998) We isolated strains of two species of studied studied 23 worldwide strains of Chlorogo- Chlorogonium from soil samples collected in nium and recognized six species using light Chiba ， Kanagawa and Hokkaido ， Japan. and electron microscopy and by comparison They were identified as C. elongatum and of of gene sequences of the large subunit of C. kasakii ，respectively ， using light and elec- RubisCO (rbcL). tron microscopic and molecular phylogenetic Eight Eight species of Chlorogonium have been cnteria. recorded recorded from Japan (Nozaki 2000). Three of of the eight species were studied by N ozaki Materials and Methods et et a1. (1 998) using Japanese strains. How- Soil samples used in this study were col- ever ， the other five species ，in cI uding C. lected from four localities in J apan. SkCl- 2 euchlorum Ehrenb. and C. elongatum (P. A. was isolated from a paddy field at Ootake ， Dang.) Dang.) France ，have not been investigated in Narita-shi ，Chiba ，35 0 48'34"N 140 0 16'40"E ， such such detai l. Vegetative cells of C. euchlo ・ on 23 July 2003. Chlogo-1 /l 998-3-7 was rum ， C. elongatum and C. kasakii Nozaki are isolated from Tanasawa-paddy fields of similar similar to each other when viewed under a Tokyo University of Agriculture ，Shimo 幽 -197- 198 198 植物研究雑誌第80 巻第4号 平成 17 年8月 2 Figs. Figs. 1 ，2. Vegetative cells of two species of Chlorogonium Ehrenb. ， shown at the same magnifica- tion. tion. Fig. 1. C. elongatum (P. A. Dang.) France. Fig. 2. C. kasakii Noz 誌 I. 2 kawairi ，Atsugi-shi ，Kanagawa ， on 7 March about 130-200μmol.m- .s 一1 provided by cool 1998. 1998. SkCl-5 was isolated from bottom of white fluorescent lamps. In order to deter- Sakataga-ike Sakataga-ike Pond (pH 7.1 ， 22.1 OC) ，Ootake ， mine whether the pyrenoids are stable or un- Narita-shi ，Chiba ，35 049'6"N 140 016'26"E ， stable (Nozaki et al. 1994 ，1995) ， the cells on on 23 July 2003. Kh Cl-3 was isolated from were grown photoheterotrophically in AF 目 6 a paddy field at Nakanosawa ，Kitahiroshima- medium supplemented with major organic shi ，Ishikari Subprefecture ，Hokkaido ，42 0 compounds (modified acetate medium: 400 58 58 '51 "N 141 035'4 "E ，on 27 March 2004. mg/l each of sodium acetate.3HzO ，glucose ， Clonal Clonal cultures were established using the bacto yeast extract ， and bacto tryptone) as pipette-washing pipette-washing method (Pringsheim 1946) described by Nozaki et al. (1 995). Light mi- from from Petri dishes in which a small amount of croscopy was carried out using an Olympus the the dried soil samples had been rewetted BX60 microscope equipped with Nomarski with with ion-exchanged or distilled wate r. The interference optics. Two representative cultures cultures were grown in screw-cap tubes strains (SkCl-2 and SkCl-5) were examined (18 (18 x 150 mm) containing 9-10 ml of AF-6 by transmission electron microscopy (TEM). medium (Kato 1982) modified by the elimi- The methods for TEM have been described nation nation of CaC03 and by the addition of 400 elsewhere (Nozaki et al. 1994). mg/l mg/l MES (Kasai et al. 2004). They were For phylogenetic analysis ， 1128 base pairs kept kept at 20 oC ， with alternating periods of 14 of the coding region of rbcL genes from four h light and 10 h dark ， at a light intensity of new strains of Chlorogonium (SkCl ・2，SkCl- August 2005 August Joumal of Japanese Botany Vo l. 80 No. 4 199 5 ，Chlogo- 1/ 1998-3 田 7 ，and Kh Cl-3) were sponded to positions 31-1158 of the sequenced as described previously (Nozaki Chlorella vulgaris Beり • rbcL gene (Waka- et et al. 1997). The region sequenced co 町 e- sugi et al. 1997). These sequence data were Table Table 1. List of strains and/or species of Chlorogonium (Cg.) ，Chlamydomonas (Cd.) and their related green algae used used in this study. Strain designation and source ， database accession number ， and origin of rbcL sequence 訂 C glven glven GenBank Species Species Strain designation Locality access lO n Reference****** number 11、‘.，ノ、‘.，，，、‘，，，，、‘.，，，、‘，，ノ、‘.，，，、、.，ノ、‘.，ノ、、.，，，、‘，，ノ、‘.， Cg. Cg. euchlorum CCAP* 12/2C Amiens ， France ABOI0224 フ CCAP 12/3 Berlin ，Germany AJ001882 -111111111111111111211111111111 CCAP 12/6 Unknown ABOI0225 UTEX** 1639 Nebraska ，U. S.A. ABOI0226 UTEX 2010 Germany ABOI0227 UTEX 2011 Germany ABOI0228 SAG*** 12-2d Cape Flats ， S. Africa ABOI0229 Cg. Cg. capillatum NIES****-692 Fukushima ， Japan ABOI0230 CCAP 12/2A Berlin ，Germany ABOI0231 CCAP 12/2B Cape Flats ， S. Africa AB010232 CCAP 12/5 Unknown ABOI0233 J 、、， UTEX 201 Czech/Slovakia ABOI0234 ノ、、.，E UTEX 1643 Massachusetts ，U.S. A. ABOI0235 J 、、.， UTEX 2160 Germany ABO 10236 J 、‘.，ノ、‘.， SAG 12-2e Prague ，Czech Republic AB010237 SAG 47.84 Unknown AB010238 r 、‘.，ノ、‘.，ノ、‘.， SAG 4.93 Massachusetts ，U.S. A. AB010239 今 Cg. Cg. elong αtum IAM* 料** C-293 Caldbeck ， England AJ001881 L1111 UTEX 2571 Texas ， U.S.A. ABOI0240 J 、、.，ノ、、.，，，、‘，，，、、，， UTEX 2572 Texas ， U.S.A. ABOI0241 SkCI-2 SkCI-2 Chiba ， Japan AB206329 3司 司3titi-- Chlogo 闇 111998-3-7 Kanagawa ， Japan AB206330 NIES-123 Hokkaido ， Japan ABOI0242 J 、、.，，、‘.， Cg. βlsiforme NIES-439 Ibaraki ， Japan AB010243 Cg. Cg. neglectum CCAP 12/8 Cumbria ， England AB010244 r 司 、‘.，，，、、.，ノ、‘.，ノ、、.，，，、、.，，，、、，， Cg. Cg. kasakii SkCI-5 Chiba ， Japan AB206331 3 今 Kh CI-3 Hokkaido ， Japan AB206332 3A Cd. Cd. pulsatilla CCAP 11/ 44 Tvarminne ， Finland AB007323 『ヴ臼今，， Cd. Cd. tetragama NIES-446 Ibaraki ，J apan AJO01880 Dunaliella Dunaliella parva UTEX 1983 Unknown AJO01877 B 今ゐ戸、 Dysmorphococcus globosus SAG 20-1 TN ，U.S. A. AJ001885 r 、‘.，，，、‘.，ノ H aematococcus lacustris NIES-144 Hokkaido ， Japan AB084336 JζJ Characiochloris Characiochloris sasae NIES-567 Kanagawa ，J apan AB084338 *Culture *Culture Collection of Algae and Protozoa (Thompson et al. 1988). **Culture **Culture Collection of Algae at the University of Texas at Austin (Starr and Zeikus 1993). ***Sammlung von Algenkulturen at the University of Gottingen (Schlosser 1994). ****Microbial ****Microbial Culture Collection at the National Institute for Environmental Studies (Kasai et al. 2004). *****IAM Culture Collection at the University of Tokyo (Yokota et al. 2004). 料 ****1) Nozaki et al. (1 998) ， 2) Hepperle et al. (1998) ， 3) present study ， 4) pub1ished on1y in database ， 5) Nozaki et et al. (2002). 200 植物研究雑誌第80 巻第4号 平成17 年8月 unambiguously unambiguously aligned with those of 11 Results other other Chlorogonium strains (see Nozaki Observations et et al. 1998) and six species from related Chlorogonium elongatum (P. A. Dang.) genera genera (Table 1) ， possibly belonging to France (Figs. 1， 3-8) group group C of Nozaki et al. (2003; see also Cells fusiform or broadly fusiform. Nozaki Nozaki et al. 1998 ， Proschold et al. 2001 ， Anterior end of the cell blunt or narrowed. Buchheim et al. 2002). No insertions or dele- Posterior end acute and somewhat attenuate tions tions were required for the alignmen t. When (Figs. 1，3 ， 4). Numerous contractile vacu- two or more sequences were identical ， they oles (-10) distributed throughout the surface were were treated as a single operational taxo- of the protoplast (Figs. 3 ，4). Nucleus located nomic unit (OTU). From this alignment ，a in the center of the cell (Fig. 3). Chloroplast distance distance matrix was calculated by applying parietal ，covering almost the whole periphery the the two-parameter method (Kimura 1980) in of the protoplast ， with usually two large CLUSTAL X (Thompson et al. 1997). A pyrenoids positioned longitudinally (Figs. 1， phylogenetic phylogenetic tree was constructed with the 3) ， but sometimes one or three to four neighbor-joining neighbor-joining (NJ) algorithm (Saitou and pyrenoids. Pyrenoids spherical (Fig. 3) ，sta- Nei Nei 1987) ， also using CLUSTAL X ， and the ble under photoheterotrophic growth condi- robustness robustness of the resulting lineages was tions (Fig. 5). Tubular thylakoid lamellae tested tested by a bootstrap analysis (Felsenstein penetrating centripetally into the pyrenoid 1985) 1985) with 1000 replications.
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