植物研究雑誌 J. J. Jpn. Bo t. 80: 80: 197-207(2005)

Two Species of Chlorogonium (Volvocales ，) 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 ，.

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、 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. From this matrix (Fig. 7). Stigma single ， ovoid to ellip- alignment ， a maximum parsimony (MP) soidal ，positioned in the anterior one quarter analysis ， and a bootstrap analysis with 1000 to one third of the cell (Fig. 4) ， and com- replications ，was performed using PHYLIP posed of a single layer of globules (Fig. 8). 3.6a3 3.6a3 (Felsenstein 2002). For the phylo- Flagella one quarter to one third of the cell in genetic genetic analyses ， Characiochloris sα sae length ，extending laterally from the anterior Nozaki Nozaki was designated as the outgroup ， end of the protoplast (Fig. 1). Cells 28-51 because because Ch αraciochloris Pascher represents 阿n long and 7-21 開n wide. Ratio of cell the the most basal branch within group C length to width 2-5. The first cell division in (Nozaki (Nozaki et al. 2003) or the DunaliellalLobo- asexual reproduction without preceding rota- characium characium lineage (Buchheim et al. 2002). tion of protoplas t. During the successive cell divisions ， the most anterior daughter protoplast protoplast connected with the parental flagellar flagellar base (Fig. 6).

Figs. Figs. 3-8. Chlorogonium elongatum (P. A. Dang.) France (SkCl-2). Figs. 3-6. Nomarski interference micros- copy copy of the vegetative cells ， shown at the same magnification. Figs. 3，4 ， 6-8. Cells grown photoautotrophically photoautotrophically (3 -4 days old in AF-6 medium). Fig 3. Optical section of a cell. Fig. 4. Surface view of of the cell in Fig. 3. Fig. 5. Cell grown photoheterotrophically (24-h culture in the modified acetate me- dium) ， showing stable pyrenoids (Note that the cell is slightly compressed by the microscopic preparation). Fig. Fig. 6. Eight-celled stage of asexuaI reproduction. Note the plasma strand connecting the anterior daughter protoplast protoplast and the flagellar base of the parental cell. Figs. 7 ，8. Transmission electron microscopy of cells. Fig. Fig. 7. Pyrenoid with penetrating thylakoid lamellae. Fig. 8. Stigma with a single layer of globules. Abbreviations: Abbreviations: b，flagellar base; v，contractile vacuole; p ，pyrenoid; n ，nucleus; e ，stigma; ps ， plasma strand; c，chloroplas t. August August 2005 Joumal of Japanese Botany Vo l. 80 No. 4 201 202 植物研究雑誌第80 巻第4号 平成17 年8月

Figs.9-14. Figs.9-14. Chlorogonium kasakii Nozaki (SkCI-5). Figs. 9-12. Nomarski interference microscopy ofthe vege- tative tative cells ，shown at the same magnification. Figs. 9，10 ， 12-14. Cells grown photoautotrophically (3 -4

days days old in AF 国 6 medium). Fig. 9. Optical section of a cell. Note the polygonal pyrenoids. Fig. 10. Surface view view of the cell in Fig. 9. Fig. 11. Cell grown photoheterotrophically (24-h culture in the modified acetate medium) ， showing stable pyrenoids. Fig. 12. Eight-celled stage of asexual reproduction. Note the plasma strand strand connecting the anterior daughter protoplast and the flagellar base of the parental cell. Figs. 13 ， 14. Transmission Transmission electron microscopy of cells. Fig. 13. Pyrenoid without pene 住ating thylakoid lamellae. Fig. 14. 14. Stigma with three layers of globules. Abbreviations: f，flagellum; for other abbreviations ， see Figs. 3- 8. 8. August August 2005 Journal of Japanese Botany Vo l. 80 No. 4 203

Chlorogonium kasakii Nozaki (Figs. 2，9- strains of Chlorogonium kasakii (CCAP 14) 14) 12/8 ， SkCl-5 ， and Kh Cl-3) and Chloro- Cells Cells fusiform to narrowly fusiform. Both gonium neglectum Pascher. The three strains anterior anterior and posterior ends of the cell acute ， of C. kasakii formed a robust monophyletic but but anterior end slightly broader (Figs. 2，9 ， group in clade 2， where C. kasakii SkCl-5 10). 10). Numerous contractile vacuoles (1 0 or and Kh Cl-3 from Japan were sister groups more) more) distributed throughout the surface of (with 71 % bootstrap value in the NJ analy- the the protoplast (Fig. 10). Nucleus located in sis). Clade 3 contained all the other Chloro- the the center of the cell (Fig. 9). Chloroplast pa- gonium species (C. elongatum ， C. capillatum rietal ， covering almost the whole periphery Nozaki & al. and C. euchlorum) and of of the protoplast ， with usually two large Chlamydomonas pulsatilla Wollenw. This pyrenoids ，positioned longitudinally (Figs. 2， clade was supported in only the NJ analysis ， 9) ， but sometimes three to four pyrenoids. with 64 % bootstrap suppor t. In this clade ， Pyrenoids Pyrenoids polygonal in shape (Fig. 9) ，stable C. elongatum ， C. capillatum and C. under under photohetero 甘ophic growth conditions euchlorum constituted a well-supported (Fig. (Fig. 11). Thylakoid lamellae not penetrating monophy letic group (with a 99 % bootstrap into into the pyrenoid matrix ， but surrounding it value in the NJ analysis and 98 % in the MP just just inside the associated starch granules analysis) ， and C. elongatum and C. capilla- (Fig. (Fig. 13). Stigma single ，ovoid ，positioned in tum were sister groups (with bootstrap values the the anterior one fifth to one quarter of the of 86 % in the NJ analysis and 66 % in the cell cell (Fig. 9) ， and composed of three layers of MP analysis). Both C. capillatum and C. globules globules (Fig. 14). Flagella one third or euchlorum formed robust monophyletic sometimes sometimes one half of the cell in length ，ex- groups (with 100 % bootstrap values in all tending tending laterally from the anterior end of the analyses except for a bootstrap value of 99 % protoplast protoplast (Figs. 2 ，9). Cells 26-59μm long for C. capillatum in the MP analysis) ， but and and 3-13μm wide. Ratio of cell length to C. elongatum formed a less robust clade (94 width width 4-8. The first cell division in asexual % bootstrap values in the NJ analysis and 58 reproduction reproduction without preceding rotation of % in the MP analysis) within which two protoplas t. During the successive cell divi- Japanese strains (SkCl-2 and Chlogo- 1/ 1998 ・・ sions ， the most anterior daughter protoplast 3-7) had identical sequences. connected connected with the parental f1 agellar base (Fig. (Fig. 12). Discussion Nozaki Nozaki et al. (1 998) re-examined five spe-

Phylogenetic Phylogenetic analyses cies of Chlorogonium ，including C. elonga 閏 A distance tree is shown in Fig. 15. Three tum and C. kasakii ，on the basis of the

clades clades containing Chlorogonium species comparative mo 中hology of cultivated mate 国 were were resolved (clades 1-3 in Fig. 15). Clade rials. They demonstrated that TEM of 1 contained Chlorogonium μ 'iforme Matv. ， pyrenoids and stigmata and pyrenoid stabili- Dysmorphococcus globosus H. C. Bold & R. ties under photoheterotrophic conditions C. C. Starr ， and Haematococcus lacustris (Gir.- were diagnostic at the species leve l. In the Chantr.) Chantr.) Rostaf. ， but this clade had weak Japanese strains isolated for this study ，these support support from bootstrap analyses (57 % in the characteristics (Figs. 5，7 ，8 ，11 ，13 ， 14) are NJ analysis and 63 % in the MP analysis). essentially the same as those of C. elongatum Clade Clade 2 was strongly supported by bootstrap and C. kasakii ， as described in Nozaki et al. analyses analyses (93 % in the NJ analysis and 80 % (1 998). The rbcL gene phylogeny of the in in the MP analysis) and consisted of three strains in this study supports the morphologi- 204 204 植物研究雑誌 第80 巻 第4号 平成17 年8月

Cg. 丘lsif01 'lll e NIES'123 1 Q |ω Dysm01phococcus Dysm01phococcus globosus SAG 20'1 I 2- ICD

・Ha θ'matococcus lacustds NIES'144 J -¥.

Cg. Cg. kasakii CCAP 12/8 (England) 可 100 100 71 Cg. kasakiiSkCl-5 (Chiba ， Japan) Clade Clade 2 g. Cg. kasakiiKhCl ・3 (Hokk 田do ， Japan) I Cg. Cg. neglectum NIES-439 j Dunaliella Dunaliella parva UTEX 1983

Cg. Cg. elongatum UTEX 2571 ， 2572 (Texas ，U.S .A.)

Cg. Cg. elongatum IAM C293 (England)

g. Cg. elongatum I~~?l 合 (C凶 a ， Japan) ， しChlogo ・111998 ・3-7 (Kanagawa ， Japan) 。一 100 rCg. capillatum NIES-692 ωaoω 而両 LíI~ ~~~':11~ .J-.._ rCCAP 12/2 A， 12/2B ， UTEX 201 ，1643 ，2160 ， 100 100 L Cg. 却 ，illatum L削 12-2e ，4.93 ， 47.84 Cg. Cg. capillatum CCAP 12/5

100rCg. 100rCg. euchlol'um CCAP 12/2C ，12/3 ， SAG 12 ・2d 100 100 98 ICg. eUChlOl'Um CCAP 12/6 ， UTEX 2010 ， 2011 100 100 Cg. θ uchlol'um UTEX 1639

Chlamydomonas Chlamydomonas pulsat J1 1a CCAP 11 ・44 Chlamydomonas Chlamydomonas tetragama NIES'442 0.01 0.01 substitutiO l1 s/site Cha l' aciochlo l'.I s sasae NIES'567

Fig. Fig. 15. Neighbor-joining tree based on 1128 base pairs of nucleotide sequences in the coding regions of the the rbcL genes from 26 strains of six Chlorogonium (Cg.) species ， as well as from six other related green algal algal OTUs. Chlorogonium strains with identical sequences were treated as a single OTU. Branch lengths lengths are proportional to Kimura (1980) distances ，which are indicated by the scale bar below the tree. Numbers above branches indicate the bootstrap values from neighbor joini 時 analysis ， whereas bootstrap values values from most parsimonious analysis are shown below branches. Both bootstrap analyses are based on 1，000 replicates. Only bootstrap values > 50 % are shown. cal cal identification of the strains (Fig. 15). elong αtum (Figs. 3-5) and C. kasakii (Figs. Chlorogonium euchlorum has been re- 9-11) are well consistent with these ranges. corded corded from Japan without TEM observa- However ， according to Ak iyama et al. tions tions and culture studies (Akiyama et al. (1977) ， the celllength of Japanese materials 1977). 1977). When grown photoauto 住ophically ， C. of C. euchlorum is 60-80μm. euchlorum ， C. elongatum and C. kasakii ap- The Japanese strains of C. elongatum are pe 訂 very similar to each other viewed with more closely related to each other when a light microscope (Nozaki et al. 1998) ，al- compared to other strains collected from though though the cell sizes are different between England and the United States (Fig. 15). The these these species. Vegetative cells of C. kasakii present two Japanese strains of C. kα sakii are are are 26-70μm long ， whereas those of C. more closely related to each other than they euchlorum and C. elongatum measure less are to the other strain collected from England than than 60 阿n (Nozaki et al. 1998). The cell (Fig. 15). These phylogenetic relationships sizes sizes of the present Japanese materials of C. likely reflect diversification of Chloro- August August 2005 Journal of Japanese Botany Vo l. 80 No. 4 205 gonium species. However ， only UK ， penetrating thylakoid lamellae; stigma American and Japanese strains of C. composed of a single layer of globules …・・

elongatum elongatum have been studied and sequenced ・・ H ・H ・. C. elongatum (P. A. Dang.) France to to date (Hepperle et al. 1998 ，Nozaki et al. 4. Pyrenoids ne 訂 ly polygonal in shape ，with- 1998; 1998; Fig. 15) ， although this species has out penetrating thylakoid lamellae; stigma been reported to be cosmopolitan (Ettl composed of multiple layers of globules

1983). 1983). Chlorogonium kasakii has been previ 国 -… C. kas ・akii Nozaki ously ously reported only from England (Nozaki 5. Stigma absent; flagella about the cell et et al. 1998). Further cultured strains of length ....・... C. ovatum Skvortsov & Noda Chlorogonium species in Asia are required to 5. Stigma generally present; flagella about a

identify identify the origin and map the detailed half of the cell length …...・ H ・.....・ H ・H ・H ・... 6 radiations radiations of each species of Chlorogonium. 6. Stigma positioned in the middle part of the cell cell .… C. ellipsoideum Skvortsov & Noda Key to species of Chlorogonium Ehrenb. 6. Stigma positioned in the anterior part of

in in Japan the cell ， if present ...・ H ・.....・ H ・.....・ H ・H ・H ・... 7 There has been no comprehensive key to 7. Number of contractile vacuoles generally species species of Chlorogonium in Japan. Nozaki et 2，3 or 4 . l. al. (1998) gave a key to 20 worldwide spe- .. C. latilanceolatum Skvortsov & Noda cies cies of Chlorogonium ， but C. ovatum 7. Number of contractile vacuoles 5 or more Skvortsov Skvortsov & Noda ， C. ellipsoideum .. 8 Skvortsov Skvortsov & Noda and C. latilanceolatum 8. Cells with narrowed or acute posterior

Skvortsov Skvortsov & Noda ，which were recorded and/or anterior ends under both photo 田 from Japan (Skvortzov and Noda 1967) ， autotrophic and photoheterotrophic growth

were excluded. Nozaki (2000) distinguished conditions .…....・ H ・.. C. euchlorum Ehrenb.

these these three species from other Chlorogonium 8. Anterior and posterior ends of cells gener 回 species species in that they lack pyrenoids. However ， ally blunt under photohetero 住ophic growth as as the absence of pyrenoids was examined conditions …… C. capillatum Nozaki & a l. under under only photoheterotrophic conditions (Skvortzov (Skvortzov and Noda 1967) ， they possibly We are grateful to the staff of Sakataga- have unstable pyrenoids. A revised key to ike Park for the facilities for our research at

species species of Chlorogonium in Japan is pre 国 the par k. This study was supported in part by sented sented here with addition of C. kasakii and a grant from the Institute for Fermentation re-assessment re-assessment of C. ovatum ， C. ellipsoideum (to H. N.). and C. latilanceolatum.

1. 1. Pyrenoids present ，stable …...・ H ・.....・ H ・.... 2 References 1. 1. Pyrenoids completely absent or unstable Akiyama M. ， Hirose H. ， Yamagishi T. and Hirano M. …5 1977. Class Chlorophyceae. In. Hirose H. and 2. 2. Number of contractile vacuoles 3 . Yamagishi T. (eds.) ，lllustrations of the Japanese Fresh-Water Fresh-Water Algae. pp. 275-760. Uchida Roka-

...・ H ・.....・ H ・...・ H ・...・ H ・...・ H ・.C. fusiforme Matv. kuho Pu blishing ，Tokyo (in Japanese). 2. 2. N umber of contractile vacuoles 5 or more Buchheim M. A. ，Buchheim J. A.， Carlson T. and

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仲田崇志a，中沢 敦b ，野崎久義a .日本産ヤリミ ドリ属(オオヒゲマワリ目，緑藻綱) 2 種につい て ドリ (新称; C. elongatum) および， 日本未記録 日本の水田土壌よりヤリミドリ属(新称; のカサキヤリミドリ(新称; c. kasakii) と同定さ Chlorogonium) Chlorogonium) 2 種が分離され，光学および電子 れた. 顕微鏡を用いた詳細な観察と分子系統に基づき日 (a 東京大学大学院理学系研究科生物科学専攻，

本産の培養株を用いた研究のないフタツメヤリミ b独立行政法人理化学研究所)