J1ふか冒志位一叫物取研け内九拘か不仁hEHr-三百士、心 aa町噌JJA←引〆{司・ん、-泊JF『 Rediscovery of the Wild-Extinct Species Nitellopsis obtusa (Charales) in Lake Kawaguchi，Ja pan Syou KATOa• ，*Sumio HIGUCHIb，Y oichi KONDO ，c Satoshi KITANOd，Hi sayoshi NOZAKIe and Jiro TANAKAa aDepartment of Ocean Sciences，Fa culty of Marine Science，T okyo University of Marine Science and Technology， Konan 4-5-7，Mi nato却，Tokyo，1 088477JAPAN; ・ *Present address: Department of Biological Sciences，G raduate School of Science， University of Tokyo，H ongo 7-3-1，B unkyo-ku，T okyo，1 13・0033JAPAN E-mai1: [email protected] bAmori Office，N agano Environment Conservation Research Institute， Komemura 1978，A mori，N agano，38 0-0944 JAPAN; CN吋iri-koMuseum，N 吋iri287-5，Sh inano-machi，N agano 389-1303，J APAN; Iizuna Office，N agano Environment Conservation Research Institute， d Kitago 2054-120，Na gano-shi，N agano，3 81-0075 JAPAN; eDep紅tmentof Biological Sciences，Gr aduate School of Science，Un iversity of Tokyo， Hongo 7-3-1，B unkyo-ku，T okyo，1 130033JAPAN ・ (Received on September 29，2 004) The Charales are especially endangered in Jap anese inland waters. Of the 74 charalean green algae taxa known from Jap an，a bout half eon the Jap anese red list of 紅 threatened or endangered species. Although the charalean species Nitellopsis obtusa (Desvaux) Groves once grew in four Japanese lakes，fi eld surveys in 1992-1993 sug- gested that白isspecies was extinct in the wild in Japan. However，du ring our 2003 field survey，N. obtusa was collected from natural populations growing in Lake Kawaguchi. The thalli had giant bract -cells and sts(asexual reproductive organs)，w hich ethe di- 紅 訂 agnostic characters of N. obtusa. One-yearoldcultured plants developed oogonia. In ad- 国 dition，a p hylogeny developed by using sequences from the gene encoding the large subunit of the enzyme rubisco (rbcL) demonstrated that the alga collected from Lake Kawaguchi，a l aboratory-maintained sample of N. obtusa from Lake Nojiri (Japan) and aG erman N. obtusa specimen formed ar obust clade within the Charales. Key words: Charales，N itellopsis，r bcL gene，r ediscovery，w ild-extinct species. As the biodiversity of freshwater habitats ean taxa had been dramatically reduced in Jap an shrinks because of water pollution (Noz紘iet al. 1995). The Japanese red list following the economic boom of the 1970s， now includes five extinct，o ne wild-extinct， the charalean green algae seem to be espe- and 24 threatened taxa from the charalean cially endangered. Before the 1970s，t axo- flora in Jap an (Environmental Agency of nomic studies recorded 74 taxa in four Japan 2000). genera (Chara，L amprothamnium，N itellop- Nitellopsis obtusα(Desvaux) Groves is the sis，a nd Nitella) from Japan (lmahori 1954; largest charalean species found in Europe Kasaki 1964; Imahori and Kasaki 1977). and Asia between 180N and 650N (Wood However，fi eld surveys of Japanese lakes in 1965). In Japan，th is dioecious species was 1993-1994 found that the number of charal- previously recorded from four lakes: Lake -84- Apri12005 Journal of Japanese Botany Vol. 80 No. 2 85 Ashinoko (Kanagawa Pref.)，L ake Kawa- medium for Charales (SWC-l; Sakayama guchi (Yamanashi Pref.)，L ake Nojiri (Naga- et al. 2004). The cultures were maintained no Pref.)，a nd Lake Yamanaka (Yamanashi under controlled laboratory conditions at 20 oc Pref.) (Kasaki 1964). However，a fter field with a 14: 10-h lightdark cycle and ca. “ surveys in 1992-1993，N ozaki et al. (1994) 140μmol m-2 S-1 illumination provided by concluded that N. obtusa was extinct in natu- f1uorescent lamps. The thalli collected from ral habitats in Jap an. However cultures of N. site 12 (Fig. 1) were also grown in a1 2-L obtusa previously collected from Lake Nojiri tank containing SWC-l and were maintained have been maintained as experimental mate- at room temperature (15-300C) with a1 4: rial in the laboratory of Osaka Medical 10占 light-darkcycle and ca. 500μmol m-2 University (Nozaki et al. 1994). Therefore， S-1 fluorescent lamp illumination. in Jap an N. obtusa is listed as an“extinct The thalli were observed with aU FX- IIA species in the wild" (Environmental Agency microscope (Nikon，T okyo，J apan) and a of Japan 2000). MS5 stereo microscope (Leica Micro- During our 2003 field surveys，li ving sam- systems，T okyo，Ja pan). Taxonomic identifi- ples of N. obtusa were collected from natural cations were based on Kasaki (1956)，W ood populations growing in Lake Kawaguchi. (1964， 1965)， and Imahori and Kasaki Here we report field data，m orphology，a nd (1977). the gene sequence of the large subunit of rubisco (rbcL) for the N. obtusa obtained from Lake Kawaguchi. 9 Materials and Methods Field surveys of charalean species were made at various sites around Lake Kawa- guchi (Minamitsuru-gun，Y amanashi Pref， 4 Japan) in June and October 2003 (Fig. 1). Thalli were collected using ah andmade an- chor (Imahori 1954)，w hich was thrown into the water from the lakeshore or piers and Fig. 1. Map of Lake Kawaguchi，Y amanashi Pref.， then dragged along the bottom. Japan. Collection sites enumbered 1-15. 訂 Uni-charalean cultures (STAR strain origi- Collections were made in June 2003 at sites 1-3， nating from site 12; Fig. 1) were established 5，6 ，8 ，1 1-13，a nd 15，a nd in October 2003 at by inoculating p紅tof aN . obtusa thallus into sites 1，4 ，5 ，7 ，9 ，1 0，1 2，a nd 14. Nitellopsis a9 00-ml glass jar containing as oil-water- obtusa was collected at sites 5a nd 12. Table 1. Cornparison of the charalean taxa found in Lake Kawaguchi from three surveys in different decades Kasaki (1964) Nozaki et al. (1995) the present study Chara braunii Gmelin C. braunii C. braunii C. globularis Thuillier var. globularis C. globularis var. globularis C. globularis var. globularis C. corallina Willdenow Nitella flexilis Agardh v訂.flexilis N. flexilis var. flexilis N. gracilens Morioka N. hyalina Agardh Nitellopsis obtusa (Desvaux) Groves Nitellopsis obtusa 86 植物研究雑誌第80巻第2号 平成17年4月 Table 2. Species and culture strains used for the rbcL gene phylogeny strain designation and origin of rbcL gene GenBank Spesies col1ection information sequence data accession number Chara globularis Thuillier F124C，G ermany McCourt et a .1(1999) AF097163 C. connivens Salzmann ex Braun X214，Is rael McCourt et a .1(1999) AF097162 仁 vulgarisLinnaeus X152，D enmark McCourt et a .1(1999) AF097166 C. rusbyana Howe X066，A rgentina McCourt et a .1(1999) AF097168 Lamprothamnium papulosum F137，Fr ance McCourt et a .1(1999) AF097170 (Wal1roth) Groves Nitellopsis obtusa (Desvaux) F131，G ermany McCourt et a .1(1999) U27530 Groves STAR，L ake Kawaguchi， the present study AB195319 Yamanashi，Ja p an，O ct. 2003 NOJIRI，L ake N吋iri， the present study AB195320 Nagano， Japan，A ug. 1974 Lychnothαmnus barbatus Ger，G ermany McCourt et a .1(1999) AF097172 お(1eyen)Leonhardi Nitella flexilis (Linnaeus) Agardh SOlO， Japan Sakayama et al. (2002) AB076056 N. praelonga Braun P/CR7，Co sta Rica McCourt et a .1(1999) AF097173 N. pulchella Al1en S011，Ja pan Sakayama et a .1(2002) AB076057 N.β4町G的 (Roxburghex S037，Ja pan Sakayama et al. (2002) AB076059 Bruzelius) Agardh N. gracilens Morioka KINU，Ja pan Sakayama et a .1(2002) AB076063 N. translucens (Persoon) Agardh F108，Fr ance McCourt et al. (1999) AF097745 Tolypella. nidifica (M剖ler)Braun F138，Fr ance McCourt et a .1(1999) U27531 Coleochaete nitellarum Jost Nishiyama and Kato (1999) AB013662 C. orbicularis Pringsheim Manhart (1994) L13477 Zygnema peliosporum Wittrock Mccourt and Karol (1995) U38701 DNA sequencing of the rbcL gene of the (Jukes and Cantor 1969) in PAUP* 4.0b10. N. obtusa from Lake Kawaguchi (STA R A phylogenetic tree was then constructed by strain) and from Lake Nojiri (NOJIRI strain; a neighbor-joining (NJ) algorithm (Saitou No zaki et a1. 1994) was performed as de- and Nei 1987)，a gain using PAUP* 4.0b10. scribed by Sakayama et a .1(2002). For the The robustness of the phylogeny was tested phylogenetic analysis，a d ata matrix contain- by ab ootstrap analysis with 1000 replica- ing 1194 bp of unambiguously aligned rbcL tions. In these phylogenetic analyses， gene sequences from three samples of N. Coleochaete nitellarum Jost，C . orbicularis obtusa，13 other charalean species，a nd three Pringsheim， and Zygnema peliosporum other charophycean species (Table 2) was Wittrock were designated as the outgroup subjected to unweighted maximum parsi- because they belong to the Charophyceae mony (MP) analysis using PAUP* 4.0b10 sensu Mattox and Stewart (1984)，w hile re- (Swofford 2002). A bootstrap analysis cent molecular phylogenetic studies give rea- (Felsenstein 1985) was carried out based on sonably high bootstrap support for the 1000 replications of the general heuristic monophyly of the Characeae within the search (full heuristic type with the甘eebisec Charophyceae (McCourt et a1. 1999，2000). 四 tion-reconnection branch-swapping algo- rithm). From the same alignment used in the Results MPa nalysis，a d istance matrix was calcu- Field collections and cultures - Five lated by applying the Jukes-Cantor method charalean taxa including Nitellopsis obtusa April2005 Journal of Japanese Botany Vo .l80 No. 2 87 were collected for this study (Table 1). The thickened nodal cells was collected at site 5. collection sites 'eshown in Fig 1. In June After several months，cu ltures of this sample 紅 2003，a p iece of aN . obtusa thallus with demonstrated the typical vegetative morphol- Fig.2. Mo中hologyof Nitellopsis obtusa Groves specimens collected at site 12 in Lake Kawaguchi. a-d. Thalli field-collected in October 2003. a. Habit. b. Apical portion of thallus. Arrows or arrowheads indicate bract- cells or branchlet segments，r espectively. c. Stars (asexual reproductive organs). d. Thickened nodal cells. e-g. Oogonia developing in plants cultured in a1 2-L tank for about one ye e. Oogonia formed as solitary 紅. or geminate at branchlet nodes. f. Oogonia. g. Apical portion of immature oogonium showing coronula com- posed of five cells arranged in one tier. 88 植物研究雑誌 第80巻 第2号 平成17年4月 ogy of N. obtusa (not shown). 1n October by Kasaki (1956) and Wood (1964，1 965): 2003，th alli with the typical vegetative mor- thalli were 15-40c m high; axes were up to phology of N. obtusa were collected at site 1m mi n diameter; and intemode cells were 12. N0 N. obtusa was found at site 5a t that 1-1.5 times as long as branchlet cells (Figs. t1me. 2a，b ). Whorls consisted of five to six branchlets，u p to 6c m long (Figs. 2a，b) . Morphology -The morphology of the N. Bract-cells were 1-2， similar to the obtusa thalli collected at site 12 of Lake branchlet -segments，an d were directed some- Kawaguchi agreed with descriptions given what toward the axis; they measured 1.2-1.7 の『 Lamprothamnium populosum dω @ω『 。 4Nitellopsis obtusa from Lake Kawaguchi 胤 lIopsisobtusa fr吋 e陶jiri 月。Z 一 Nitella furcata =oω 。 Tolypella nidifica Coleochaete nitellarum Coleochaete orbicularis Zygnema peliosporum 一ーー 0.01substitutions/site Fig. 3. Distance tree based on aligned nucleotide sequences for 1194 bp of the rbcL gene for 16 charalean taxa and three outgroup taxa. The tree was derived using the neighbor- joining method (Saitou and Nei 1987). This method is based on the Jukes-Cantor dis- tance (Jukes and Cantor 1969)，w hich is indicated by the scale bar below the tree. Numbers above or below the branches ebootstrap values (50 % or more) based on 訂 1000 replications of the NJ or MPa nalyses. April2005 Journal of Japanese Botany Vol. 80 No. 2 89 cml ong (Fig. 2b). White stars were ca. 3m m molecular phylogenetic analysis demon- in diameter (Fig. 2c). Nodal cells were thick- strated that the specimens from Lake ened and ca. 3m min diameter (Fig. 2d). Kawaguchi are very closely related to N. Oogonia developed on thalli cultured for obtusa specimens originating from Lake approximately one year in a1 2-L tank. The Nojiri and Germany (Fig. 3). Nitellopsis oogonia，w hich formed as solitary or gemi- obtusa habitats in Lake Kawaguchi are，ho w- nate at the lowest 1-2 branchlet-nodes， ever，m uch reduced from those reported by measured 1000-1140阿nlong and 870-1050 Kasaki (1964). He reported that N. obtusa 阿nwide (Figs. 2e，f ). The coronula were was found at several sites circling the pe- composed of five cells arranged in one tier， rimeter of Lake Kawaguchi (Fig. 1). In con- measuring 40阿nhigh and 120阿nwide at 佐ast，we collected N. obtusa at only two of the base (Fig. 2g). Antheridia were not ob- the 15 sites we surveyed around the lake. served. Furthermore，N. obtusa populations may be sporadic or very small，as the alga was not Molecular phylogenetic analyses-The rbcL present at the same site in both June and gene sequences of two Jap anese plants of N. October 2003. Therefore，p opulations of N. obtusa (from Lake Kawaguchi and Lake obtusa growing in Lake Kawaguchi should Nojiri) were identical. They differed from be conserved as soon as possible. the sequence of aG erman N. obtusa speci- The rediscovery of N. obtusa in Lake men (McCourt et al. 1996) by only one base Kawaguchi may be explained by one of two pmr. possibilities. First，ei ther oospores (zygotes) The NJ tree of the rbcL gene sequences is or stsof N. obtusa embedded in the lake 訂 shown in Fig. 3. Branches that resolved with bottom may have germinated. According to bootstrap values of 50 % or more by the NJ Kasaki (1964)，N . obtusa plants collected and/or MPa nalyses are shown. The phylo- from Lake Kawaguchi and Lake Ashinoko genetic relationships among the six genera of were female，wh ereas those from Lake Nojiri the Characeae resolved in this study were es- were male. The thalli we collected from sentially the same as those found by Lake Kawaguchi produced only female or- Sakayama et al. (2002). Four genera of the gans (Figs. 2e-g). Nitellopsis obtusa can also Chareae (Chara，L amprothamnium，L ychno- reproduce asexually by stars. Therefore，b ur- thamnus and Nitellopsis) were resolved as a ied stars may have given rise to the vegeta- monophyletic group，w hereas two genera of tive thalli，af ter the survey of Nozaki et al. the Nitelleae (Nitella and Tolypella) repre- (1994). However，t he stars do not have a sented a p aphyleticgroup. Within the thick cell wall (Wo od 1965) and thus may 征 Chareae，th e samples of N. obtusa from Lake not be viable after ac ertain time. Second，th e Kawaguchi， Lake Nojiri， and Germany rediscovery may result from differences in formed ar obust clade with high bootstrap collection methods. Nozaki et al. (1994) sur- values (100 %) using both the MPa nd NJ veyed Lake Kawaguchi only once in 1992， methods (Fig. 3). whereas we surveyed the lake on three days in June and October 2003 for the present Discussion study. Our two collections of viable Nitellopsis The charalean taxa found in this survey obtusa thalli from Lake Kawaguchi demon- are different from those collected from Lake strate that natural populations of the algae Kawaguchi by Kasaki (1964) and No zaki are extan .tThe species was identified based et al. (1995) (Table 1). Although Kasaki on its morphology (Fig. 2). In addition，a (1964) and Nozaki et al. (1995) collected 90 植物研究雑誌第80巻第2号 平成17年4月 Nitella flexilis Agardh var. flexilis，w e did Uchida Rokakuho publishing，T okyo (in Japanese). not find this species. N. gracilens Morioka Jukes T. H. and Cantor C. R. 1969. Evolution of pro- tein molecules. In: Munro H. N. (edよMammalian has not been col1ected from Lake Kawaguchi Protein Metabolism. pp. 21-132. Academic Press， since Kasaki's survey (1964). In contrast， New York. Chara corallina Willdenow and N. hyalina Kasaki H. 1956. On the newly found genus Nitellopsis Agardh were newly collected in the present from Japan. (Notes on the Charophyta-flora in study (Table 1). The different taxa repre- Japan-3). J. Jpn. Bot. 31: 97-101 (in Japanese). 一一ー 1964.The Charophyta from the lakes of Japan. J. sented in the various surveys may indicate Hattori. Bot. Lab. 27: 215-314. recent environmental changes in Lake Mattox K. R. and Stewart K. D. 1984. Classification of Kawaguchi and suggests an eed for detaHed the green algae: ac oncept based on comparative resurveys of current Jap anese charalean flora cytology. In: Irvine D. E. G. and John D. M. (Edsよ in other lakes (Nozaki et al. 1995). Systematics of the Green Algae. pp. 29-72. Since Kasaki (1956) first reported Academic Press，L ondon. McCourt R. M.，Ka rol K. G.，Ca sanova M. T. and Feist Nitellopsis obtusa in Japan，t axonomic iden- M. 1999. Monophyly of genera and species of tifications of Jap anese species have been Characeae based on rbcL sequences，w ith special based solely on morphological attributes reference to Australian and European Lychnotham- (e. g.，K asaki 1964; Nozaki et al. 1994). Our nus barbatus (Characeae，C harophyceae). Aust. J. study used rbcL gene sequences to demon- Bot. 47: 361-369. 一， 一一一， Guerlesquin M. and Feist M. 1996. S佐atethat N. obtusa specimens originating Phylogeny of extant genera in the family Chara- from Lake Kawaguchi，L ake No jiri，a nd ceae (Charales，C harophyceae) based on rbcL se- Germany every closely related to one an- 紅 quences and morphology. Am. J. Bot. 83: 125- other，a dding support to the previous mor- 131. phological identification of Jap anese N. 一一，一一一， Helm-Bychowski J. B. K. M.，G rajewska obtusa collections. A.，Wo jciechowski M. F. and Hoshaw R. W. 2000. Phylogeny of the conjugating green algae (Zygnemophyceae) based on rbcL sequences. J. We egrateful to Dr. Atsushi Kobayashi， 訂 Phycol. 36: 747-758. Mr. Sozo Hayasaka，M r. Yo hei Tsukamoto Nozaki H.，K asaki H.，S ano S. and Watanabe M. M. and Ms. Tomoyo Ishimaru of Tokyo Univer- 1994. Extinction of the natural populations and sity of Pisheries for their kind help in the possibility of the revival of Japanese Nitellopsis obtusa (Characeae). Proc. Jap. Soc. Pl. Taxon. 10: field surveys. Thanks are also due to Dr. 45-50 (in Japanese with English abstract). Hidetoshi Sakayama，N a tional Institute for 一一一， Watanabe M. M.，K asaki H.，S ano S.，K ato M. Environmental Studies，f or his helpful ad- and Omori Y. 1995. Current state of the Charales vise. This study was in part supported by a (Charophyta) in Japanese lakes. Jpn. J. Phycol. 43: Grant-in-Aid for Scientific Research (No. 213-218 (in Japanese with English abstract). 15651102 to H.N.) from the Japan Society Saitou N. and Nei M. 1987. The neighbor-joining method: an ew method for reconstructing phylo- for the Promotion of Science. genetic trees. Mol. Biol. Evol. 4: 406-425. Sakayama H.，H ara Y. and Nozaki N. 2004. Taxono- References mic re-examination of six species of Nitella Environment Agency of Jap an (edふ2000.Threatened (Charales，C harophyta) from Asia，a nd phylo- wildlife of Japan-Red data book 2nd ed.-vo1.9 genetic relationship within the genus based on rbcL Bryophytes，A lgae，L ichens，Fu ngi. 429 pp. Japan and atpB gene sequences. Phycologia 43: 91-104. Wildlife Research Center，T okyo (in Japanese). 一一一， Nozaki H. and Hara Y. 2002. Taxonomic re- Felsenstein J. 1985. Confidence limits on phylogenies: examination of Nitella (Charales， Charophyta) an approach using bootstrap. Evolution 38: 16-24 from Jap an，b ased on microscopical studies of Imahori K. and Kasaki H. 1977. Class Charophyceae. oospore wall omamentation and rbcL gene se- In: Hirose H. and Yamagishi T. (edsよI1lustrations quences. Phycologia 41: 397-408. of the Japanese Fresh-Water Algae. pp. 761-829. Swofford D. L. 2002. PAUP* 4.0: phylogenetic analy- Apri12005 Joumal of Japanese Botany Vol. 80 No. 2 91 sis using psimony，version 4.0blO. Computer Weinheim. 訂 program distributed by Sinauer Associates，I nc.， 一一一and Imahori K. 1964. Iconograph of the Fitchburg. Characeae. In: Wood R. D. and Imahori K. 但(e也d吋s..) Wood R. D. 1965. Monograph of the Characeae. In: A Revision of the Characeae.vol. 2. 395 pls，x v + 紅 Wood R. D. and Imahori K. (edsよARevision of 7p p. J. Cramer，W einheim. the Characeae. vol. 1. 904 pp. J. Cramer. 加藤将，*，樋口澄男 ，近藤洋一 ，北野聡 ， a b c d 野崎久義 ，田中次郎 .本邦野生絶滅種ホシツリ e d モ(シヤジクモ目)の再発見 後1年藻体から雌器(生卵器)が発達した.さら 日本産の車軸藻類はこれまでに4属74分類群が に今回の河口湖産の rbcL遺伝子の塩基配列は， 報告されているが，近年の環境汚染による水質の 野尻湖産及びドイツ産のものとほぼ一致し，本藻 汚染等が原因で，全国で激減しており，レッドデー 体がホシツリモであることのより確かな確証を得 タブックにも多数の絶滅危慎種・絶滅種が記載さ た.現在の河口湖における生育域は非常に減少し れている.ホシツリモ (Nitellopsisobtusa)は，日 ており，緊急な保護対策が必要と考えられる. 本では4湖沼(芦ノ湖，河口湖，野尻湖，山中湖) (a東京海洋大学海洋科学部海洋環境学科， に生育していたが野生絶滅種とされている.今回， *現所属:東京大学大学院理学系研究科 山梨県河口湖において2003年6月， 10月に車軸藻 生物科学専攻 類の調査を行ったところ，ホシツリモと思われる 長野県環境保全研究所安茂里庁舎， b 藻体が発見された.本藻体はホシツリモに特有な 野尻湖ナウマン象博物館， c 長大な萄と，無性生殖器官である星状体が確認で 長野県環境保全研究所飯綱庁舎， d き，加崎 (1956)の記載と一致した.また，培養 東京大学大学院理学系研究科生物科学専攻) e