植物研究雑誌 J. J. Jpn. Bo t. 76: 76: 129-136(2001)

New Species of and ( ， )

Nobutaka HANAGATA

Research Research Center for Advanced Science and Technology ， The University of Tokyo Komaba Komaba 4-6- 1，Meguro-ku ，Tokyo ，153-8904 JAPAN (Received (Received on August 11 ， 2000)

Two green algae were collected from tree bark and coastal water. One isolated from tree tree bark is solitary and unicellular ， and has network ribs on the cell wal l. This alga is morphologically morphologically similar to Coelastrella vacuolata (Shihira & Kr auss) Hegewald & Hanagata ， but differs from it in the shape of cells and visible omamentation on the cell wall wall in light microscopy. The other isolated from coastal water is in colony ， and has a meridional meridional ribs on the cell wall. Th e gross morphology of the second alga resembles that of of Coelastrella multistriata (Trenkwalder) var. corcontica Kalina & Pu nとochaiova ， but differs differs from it in habita t. The phylogenetic trees constructed with sequence data of 18S rRNA gene showed 由at the second alga is distant from Co. multistriata var. corcontica. This This alga is placed in the subgenus Acutodesmus of the genus Scenedesmus ， and forms monophyletic monophyletic relationship together with Scenedesmus rubescens (Dangeard) Kessler & al. al. and S. dissociatus (Verses & Trainor) Hegewald & Hanagata. Therefore ， these two algae algae are described as new species ，Coelastrella saipanensis Hanagata and Scenedesmus littoralis littoralis Hanagata ，respectively.

Key words: Bark inhabiting ，Coelastrella saipanensis ，new species ， Scenedesmus littorl αlis

Introduction Scenedesmus komarekii on bar k. The gross The genus Scenedesmus is common fresh- mo 中hology of the alga is close to that of water water planktonic algae and characterized by Chlorella ， but the cell wall OIτlamentation 印刷 the the formation of coenobia of 2- ，4- ，8- ，16- ， vealed by electron microscopy showed that or or rarely 32-cells 紅 ranged in one or two the alga is a member of Scenedesmus. Kalina rows. rows. The occurrence ， a町 angement and and Pun とochaiova (1987) re-examined sev- length length of the spines on the surface of the eral species of Chlorella using elecron mi- cells cells have been used as taxonomical criteria croscope ， and excluded C h. ルsca var. for for the classification of Scenedesmus for vacuolata and Ch. かsca v紅 . rubescens from

more than one hundred years. However ，re 司 Chlorella on the basis of the cell wall struc- cently ， the cell wall ultrastructure under elec- ture. They assigned these two algae to 住on microscopy is used as the most Graesiella vacuolata and Halochlorella important important criteria at species rank of rubescens ，respectively ，and placed them in Scenedesmus (Hegewald et al. 1990 ，Hege- the subfamily Scotiellocystoideae ， the family wald and Schnef 1991). Chlorellaceae. However ， Kessler et al. Hanagata et al. (1996) found single-celled (1997) later transferred these algae into

-129- 130 130 植物研究雑誌第76 巻第3号 平成13 年6月

Scenedesmus Scenedesmus based on the phylogenetic Primers for the amplification and PCR con- study. study. Hanagata (1998) also transferred sev- ditions were the same as those shown in eral eral single -c elled algae known as the mem- Nakayama et al. (1 996). Double strand PCR bers bers of the subfamily Scotiellocystoideae product was directly sequenced using a DNA into into Scenedesmus on the basis of the se- autosequencer SQ-5500 (Hitachi Co. ，Ltd. ， quence quence analyses of 18S rRNA gene. Japan) according to manufacture's instruc- Recently ，Hegewald and Hanagata (2000) re- tions. classified classified the family Scenedesmaceae on the The sequences determined in this study basis basis of morphology and phy logenetic data ， were manually aligned with other previously and transferred these single-celled Scenedes- known 18S rDNA sequences. No ambigui- mus into the genus CoelastreU α. ties in alignment occurred. The sequences

Many specimens of coccoid green algae included in this study 訂 'e as follows have have been collected from 佐ee bark ，soil ， (with accession numbers): Chlamydomonas freshwater freshwater and coastal water in this study. reinhardtii Dangeard (M32703) ， Coela- They were cultured and examined with light streU αmultistriata (Trenkwalder) Kalina & and electron microscopy. Of these ， the cell Pun とochafova var. multistriata (ABOI2846) ， walls walls of two algae isolated from tree b担・ k C. multistriata var. corcontica Kalina & and and coastal water ，respectively ， have p訂 ticu- Pun とochafuva (AB037082) ， C. oocystiformis lar lar ornamentation patterns which 紅 e the (Lund) Hegewald & Hanagata (AB012848 characteristic characteristic features of the subfamily as oocystiformis) ， C. terrestris Scotiellocystoideae. Scotiellocystoideae. In this paper ， these two (Reisigl) Hegewald & Hanagata (AB012847 algae algae are described as new taxa Coelastrella as Scotiellopsis terrestris) ， C. vacuolata saip α nensis Hanagata and Scenedesmus (Shihira & Kr auss) Hegewald & Hanagata littoralis littoralis Hanagata ，respectively ， and their (X56104 as Scenedesmus vacuolatus) ， systematic systematic positions are discussed on the Dunaliella salina (Dunal) Teodoresco basis basis of the results obtained by light and (M84320) ， pupukensis (Kalina electron electron microscopic observations as well as et Pun とochafova) Hegewald & Hanagata phylogenetic phylogenetic study. (X91267 as Scenedesmus pupukensis) ， Scenedesmus (Lagerheim) Materials Materials and Methods Chodat (AB037088) ， S. acutus 乱1eyen Coelastrella Coelastrella saipanensis was collected (X56103 as S. obliquus) ， S. bacillaris

from the bark of Cocos nucifera ，on 8 Gutwinski (X91266 as S. producto ・capita- Octorber ，1993 ， in Saipan ， North Mariana tus) ， S. dissociatus (Verses & Trainor) Islands. Islands. Scenedesmus littoralis was isolated Hegewald & Hanagata (AB037084 as ftom seawater collected in the coastal 紅側 Dac η lococcus dissociatus) ， S. distendus faced faced to the Tokyo Bay of Chiba Prefecture ， (Holtmann) Hegewald & Hanagata J apan. The algae examined in this study were (AB037093 as S. pectinatus var. distendus) ， cultured cultured in Bold's basal medium (1949). The S. hindakii Hegewald & Hanagata methods of unialgal culture and scanning (AB037098 as apiculata) ， S. electron electron microscopy were described in a pre- obtusus Meyen (AB037091) ， S. reginae vious vious paper (Hanagata et al. 1996). (Holtmann) Hegewald & Hanagata All All information on extraction procedure of (AB037096 as S. wisconsinensis var. regi- genomic DNA was provided previously nae) and S. rubescens (Dangeard) Kessler & (Hanagata (Hanagata 1998). To obtain almost complete al. (X74002 as S. rubescens).

18S rDNA ， polymerase chain reaction (PCR) Sequences were analyzed with the maxi 帽 protocols protocols (Saiki et al. 1988) were used. mum parsimony (MP) and distance matrix June June 2001 Journal of Japanese Botany Vo l. 76 No. 3 131 methods. methods. The maximum parsimony analysis neighbor-joining methods were estimated was implemented with the PAUP computer using the bootstrap method (Felsenstein program program (version 3. 1. 1; Swofford 1993) 1985). using using a heuristic search under the unweight- ed ed scheme. A random addition of sequence Descriptions with with 10 replicates and a branch-swapping Coelastrella saipanensis Hanagata ， sp. algorithm algorithm (TBR ， or tree bisection-recon- nov. [Figs. 1-5 ，9， 10]

nection) nection) was used. The same sequence align 幽 Alga unicelluaris et solitaria. Cellulae ment data were converted to a distance sphaericae ，4- 14μm diam. Paries cellulae in matrix matrix by means of the two-p 紅 ameter microscopio optico omamentum reticulatio- method (Kimura 1980) using CLUSTAL W nis supra superficiei habens. Chloroplastum (Thompson et al. 1994). The distance matrix parietale ，tabuloeforme ，cum uno pyrenoide. was converted to a phylogenetic 位ee using Pyrenoides sphaerica vel ovata ， et granis

the the neighbor 聞 joining (NJ) algorithm (Saitou amyli circumcincta. Cellula interdum grana and and Nei 1987) of CLUSTAL W. Support for oleosa et vacuolas habens. Reproductio intemal intemal branches in both the trees con- asexualis per 2-16 autosporas magnitudinis structed structed with the maximum parsimony and aequalis effecta. Autosporae sphaericae ， 3-7

5μm

Figs. Figs. 1-5. Coelastrella saipanensis Hanagata ，sp. nov. 1-3. Vegetative cells. 4. Autospores in spo ・ rangium. rangium. 5. Autospores discharged from parent cel l. 132 132 植物研究雑誌第76 巻第3号 平成 13 年6月

開n， per aperturam parietis sporangii Scenedesmus littoralis Hanagata ，sp. nov. liberatae. liberatae. Substantia carotenoidis secunda [Figs. 6-8 ，11 ， 12] praesens. praesens. Alga colonialis 2-4 -8 cellulis formans. Holotype: Holotype: Figure 1. Cellulae sphaericae ，3.5-12 阿n diam ， vel Type locality: Saipan ， North Mariana ovatae ， 3-8 x 3.5-10μm. Paries cellulae or- Islands. Islands. namentum meridianum supra superficiei

Habita t: Specimens examined were iso- habens. Chloroplastum p紅 ietale ，cum uno lated lated from the samples attached on the bark pyrenoide. Pyrenoides sphaerica vel ovata ，et of of Cocos nucifera. granis amyli circumcincta. Cellula saepe Type culture: Saipan ， North Mariana grana oleosa habens. Cellulae maturae Islands ，80ctober ， 1993. Strain D9-1 depos- vacuolas numerosas habentes. Reproductio ited ited in the Culture Collection at Research asexualis per 2-16 autosporas. Autosporae Center Center for Advanced Science and ovatae ， semisphaericae ， per aperturam Technology Technology (RCAST) ，The University of parietis sporangii liberatae. Substantia Tokyo. Tokyo. carotenoidis secunda praesens.

5f1 m

Figs. Figs. 6-8. Scenedesmus littoralis Hanagata ，sp. nov. 6 ，7. Four-celled colonies. 8. Eight-celled col- ony. ony. June June 2001 Journal of Japanese Botany Vo l. 76 No. 3 133

Holotype: Holotype: Figure 6 Phylogenetic positions Type locality: Yawata-kaigan ，Ichihara ， The phylogenetic tree obtained from NJ Chiba Chiba Prefecture ， J apan. analysis is shown in Fig. 13. MP analysis

Habita t: Specimen examined was isolated resulted in fifteen equally p紅 simonious reso 由 from from the coastal wate r. lutions (l ength = 286 steps ， consistency Type culture: Yawata-kaigan ， Ichihara ， index = 0.914). A strict consensus tree is pre- Chiba Chiba Prefecture ，6 Apri11997. Strain CI de- sented in Fig. 14. Coelastrella saipanensis posited posited in the Culture Collection at Research (AB055800) is placed in a monophyletic Center Center for Advanced Science & Technology lineage of the genus Coelastrella. Two varie- (RCAST) ，The University of Tokyo. ties of Co. multistriata form a monophyletic Scenedesmus Scenedesmus littoralis is able to grow in relationship to a group consisting of Co. the the salinity up to 2 % N aCl ， although the terrestris and Co. oocystiformis. Coelastrella maximum growth rate is obtained in the cul- saipanensis forms a sister relationship to ture ture medium without added NaC l. these combined. Coelastrella vacuolat αfirst，

Figs. Figs. 9， 10. Scanning electron micrographs of Coelastrella saipanensis Hanagata ， sp. nov. Figs. 11 ， 12. Scanning Scanning electron micrographs of Scenedesmus littoralis Hanagata ， sp. nov. 134 134 植物研究雑誌 第76 巻 第3号 平成13 年6月

Subgenus Subgenus Genus

58 、乙!j S ，dissociatus S. dissociatus 14 14 与円程 13 13 S. rubescens 'S. rubescens H9hhra 以2 'S. littoralis 'S. littoralis 匂町内奈良叫 'Scenedesmus 'Scenedesmus acuminatus 'S. acuminatus 遠足句 z.J 9z.J S. distendus 'S. distendus 2SHhh .S. .S. acutus 'S. acutus 76 Js L S. reginae reglnae 町内向号待両選h 95 日 95 r S. hindakii 'S. hindakii I. S. o btusus S.obtusus S. S. bacillaris 伝 bacillaris S C. C. vacuolata C. vacuolata 円。ぇ 611 ・C. multistriata var. multistiata C. multistriata var. multistiata bhH3Rb 92 凶L. C. multist 巾旬var. corcontica C. multistriata var. corcontica I rC. terrestris C. terrestris 69 'J9 8L C. 00 のIstiformis C.oo のIstiformis ...... C. saipanens お 'C. saipanensis Neodesmus pupukensis 'N eodesmus pupukensis Chlamydomonas reinhardtii Chlamydomonas reinhardtii Dunariella Dunariella salina salina 'Dunariella

Figs. Figs. 13 ， 14. Phylogenetic trees inferred from 18S rDNA sequences. The numbers on the branches indicate bootstrap bootstrap values (1000 replicates). 13. A distance tree constructed with the neighbor-joining method. 14. A strict consensus tree generated by heuristic search. diverges diverges in this lineage. Scenedesmus (1 981) ， but differs from it in the absence of littoralis littoralis (AB055801) is placed in the lineage pol 訂 thickenings. of of the subgenus Acutodesmus in the genus The characteristic features of Scenedesmus Scenedesmus. Scenedesmus. Scenedesmus littoralis forms a littoralis are the presence of meridional ribs monophyletic monophyletic group together with S. on the cell wall and the formation of rubescens rubescens and S. dissociatus. coenobial colony. The ornamentation pattern of of cell wall in this species is sim i1 ar to that of Discussion Discussion Co. oocystiformis ， Co. terrestris and Co. The size of cells ， the shape of chloroplast multistriata v紅 . multistriata ， but differs and and the cell wall ornamentation of from them in the formation of colony. Coelastrella Coelastrella saipanensis resemble those of Coelastrella compac ωSk 吋a (1959) also has Co. Co. vacuolatl α. The new species ，however ， meridion a1 ribs and forms a sim i1 ar colony ， differs differs from Co. vacuolata in the shape of but the cells of this species 紅 e larger than S. cells cells and ornamentation pattern on the sur- littoralis. In addition ， one of the cell poles of face face of the cells. In Co. vacuolata ，cells 紅 e Co. compacta is provided with a papilla ， often often broadly ellipsoidal in shape and the while S. littoralis does not have such a struc- cell cell wall is smooth when viewed by light mi- ture. The morphological features including croscopy croscopy (Kalina and Pun ゐcharova 1987). the shape and the size of cells ， and the shape On the other ，hand in Co. saipanensis the of chloroplast of S. littoralis basically resem- cells cells are always spheroidal in shape and the bles those of Co. multistriata var. corcontica network network of ribs on their surface can be ob- which has meridional ribs on the cell wall served served even by light microscopy. The cell and forms colonies. The difference between wall wall ornamentation of Co. saipanensis is S. littoralis and Co. multistri α ta var. also also similar to that of Scotiellopsis reticulata corcontica is their habitats ， the former inhab- as as observed by Pun とochaf ova and Kalina its coastal while water the latter inhabits peat June 2001 2001 June Journa1 of Japanese Botany Vo l. 76 No. 3 135 pools pools in the subalpine region (Kalina and 1 express my gratitude to Dr. Eberhard Pun とochafova 1987). The phylogenetic trees Hegewald ， Research Center Julich ， and Dr. in in this study show that S. littoralis is dis- Mitsuo Chihara ， N atural History Museum tantly tantly related to Co. multistriat αvar.， and Institute ，Chiba ， for helpful suggestion corcontica ， although these two species 紅 C and comments. morphologically morphologically similar to one another. Scenedesmus Scenedesmus littoralis is phylogenetically References close close to S. rubescens and S. dissociatus. Bold H. 1949. The morphology of Chlamydomonas Scenedesmus Scenedesmus rubescens is also halotolerant chlamydogama sp. nov.. Bull. Torrey Bo t. Club (Kessler (Kessler 1982) ， but the species differs from 70: 101-108. S. S. littolaris in the following morphological Dangeard P. 1965. Sur quelques algues vertes marines nouvelles nouvelles observees en culture. Botaniste 49: 5-4 5. features; features; the shape and size of cells ， the cell Felsenstein J. 1985. Confidence limits on phylogenies: wall wall omamentation ， and the shape of an approach using bootstrap. Evolution 39: 783- chloroplas t. The solitary cells of S. rubescens 79 1. are are sometimes connected polarly by append- Hanagata N. 1998. Phylogeny of the subfamily ages ages to short chains (Kalina and Pun とochaf- Scotiellocystoideae (Chlorophyceae ， Chlorophyta) and and related taxa inferred from 18S ribosomal RNA ， 訂 ova ova 1987). In addition this alga has pol gene sequence data. J. Phyco l. 34: 1049-1054. thickenings thickenings and often develops appendages ， 一一一， Karube 1. and Chihara M. 1996. Bark-inhabiting composed of single microfibrils. It was first green algae in Japan (1). Scenedesmus komarekii described described as R αlochlorella rubescens by and Coelastrella multistriata var. multistriata Dangeared Dangeared (1 965) ， and later transferred into (Scotiellocystoideae ， Chlorellaceae ， Chlorophy- ceae). ceae). J. Jpn. Bo t. 71: 87-97. Chlorella Chlorella by Kessler et al. (1 968). Kessler et Hegewald E. 1982. Taxonomisch-morphologische l. al. (1997) ，however ，re-transferred this spe- Untersuchungen von Scenedesmus Isolaten aus cies cies into Scenedesmus on the basis of Stammsammlungen. Archiv fur Hydrobiologie ， phylogenetic phylogenetic study as described above. Supp l. 60， Algological Studies 29: 375 -4 06. Scenedesmus Scenedesmus littolaris is also distinguishable 一一一 and Hanag 剖 a， N. 2000. Phylogenetic studies on from from S. dissociatus by gross morphology. Scenedesmaceae. Algological Studies. 100: 29 -4 9. 一一一， Hindak F. and Schnepf E. 1990. Studies on the Scenedesmus Scenedesmus dissociatus was isolated from genus genus Scenedesmus Meyen (Chlorophyta ， farmland farmland soil and originally described as ) from Sourthern India ， with special Dac η lococcus dissociatus (Verses and reference to the cell wall ultrastructure. Nova Trainor Trainor 1966). This species is spindle-shaped Hedwigia ，Beiheft 99: 1-75. with with bristles often connecting the cells to 一一- and Schnepf E. 1991. Scenedesmus abundans chains. chains. The halotolerance of the species has (Kirchn.) Chod. ， an older name for Chlorellafusca Shih. Shih. et Kr auss. Arch. Protistenkd. 139: 133-176. not not been verified. The phylogenetic analysis Kalina T. and Pun とochaiova M. 1987. Taxonomy of revealed revealed that S. littoralis is a member of the the subfamily Scotiellocystoideae Fott 1976 subgenus subgenus Acutodesmus. One of the morpho- (Chlorellaceae ，Chlorophyceae). Ar chiv fur Hydro- logical logical features of Acutodesmus is the shape biologie ，Supp l. 73 ，Algological Studies 45: 473- of of cells which are spindle-like with acute cell 52 1. poles poles (Hegewald and Hanagata 2000). While Kessler E. ，Czygan F. ， Fott B. and Novakova M. 1968. Uber Halochlorella rubescens Dangeard. Arch. the the cells of S. littoralis are spheroidal or Protistenkd. 110: 42 6-4 68. ovoidal ， and do not share the mo 中hological 一一一， Schafer M. ，Hummer c.， Kloboucek A. and Huss feature feature with those of Acutodesmus. v. A. R. 1997. Physiological ，biochemical ， and molecular molecular characters for the taxonomy of the sub- Scenedesmus Scenedesmus littoralis seems to be mo 中ho ・ logically logically an exceptional taxon in the genera of Scenedesmus (Chlorococcales ，Chloro- phyta). phyta). Bo t. Acta 110: 24 4- 250. subgenus subgenus Acutodesm 払s'. Ki mura M. 1980. A simple method for estimating rate 136 136 植物研究雑誌第76 巻第3号 平成13 年6月

of of base substitutions through comparative studies Algological Studies 27: 119-147. of of nucleotide sequences. J. Mo l. Evo l. 16: 111- Saiki R. K.， Gelfand ， D. H. ，Stoffel S. ， Scharf S. J. ， 120. 120. Higuchi R.， Horn G. T. ， Mullis K. B. and Erlich H. Komarek J. and Ludvik J. 197 1. Die Zellwandstruktur A. 1988. Primer-directed enzymatic amplification als als taxonomisches Merkmal in der Gattung of DNA with thermostable DNA polymerase. Scenedesmus Scenedesmus 1. Die Ultrastrukturelemente. Archiv Science 239: 487 -4 91. fur fur Hydrobiologie ，Supp l. 39 ，Algological Studies Saitou N. and Nei M. 1987. The neighbor-joining 5: 5: 301-333. method: a new method for reconstructing 一一一 and 一一一. 1972. Die Zellwandstruktur a1 s phylogenetic trees. Mo l. Bio l. Evo l. 4: 40 6-4 25. taxonomisches taxonomisches Merkmal in der Gattung Swofford D. L. 1993. PAUP: Phylogenetic Analysis Scenedesmus Scenedesmus 2. Taxonomische Auswertung der Using Parsimony ， version 3. 1.1. Laboratory of untersuchten untersuchten Arten. Ar chiv fur Hydrobiologie ， Molecular Systematics ， Smithonian Institute ， Supp l. 41 ，Algological Studies 6: 11 -4 7. Washington. Nakayama T. ，Watanabe S. ， Mitsui K. ， Uchida H. and Skuja H. 1959. Gloeococcus bav αricus n. sp. und Inouye Inouye 1. 1996. The phylogenetic relationship be- Coelastrella compacta n. sp. Protoplasma 50: 493- tween tween the Chlamydomon a1 es and Chlorococcales 497. inferred inferred from 18S rDNA sequence data. Phyco l. Thompson J. D. ， Higgins D. G. and Gibson T. J. 1994.

Res. Res. 44: 47-55. CLUSTAL W: improving the sensitive of progres 司 Pelicaric Pelicaric S. 1971. Verschiedene Typen der sive multiple sequence alignment through sequence Ultrastruktur Ultrastruktur der Zellwand bei drei Arten der weighting ，positions-specific gap penalties and Gattung Gattung Scenedesmus Meyen. Archiv fur weight matrix choice. Nuc l. Acids Res. 22: 4673- Hydrobiologie ，Supp l. 39 ，Algological Studies 4: 4680. ‘ 137-145. 137-145. Verses P. A. and Trainor F. R. 1966. Dactylococcus Pun とocharova M. and Kalina T. 198 1. Taxonomy of dissociatus ，a new species from a Connecticut the the genus Scotiellopsis Vinatzer (Chlorococcales ， cornfield soi l. Phycologia 6: 79-82. Chlorophyta). Chlorophyta). Archiv fur Hydrobiologie ，Supp l. 60，

花方信孝: Coelastrella 属及びScenedesmus 属(緑 藻綱)の 2 新種 北マリアナ諸島サイパン島の樹木皮および東京 Hanagata とした.沿岸海水より単離した藻は 2--4- 湾内房地区の沿岸海水より単離した鞭毛をもたな 8 細胞のコロニーを形成し細胞表面には子午線

い 2 種の緑藻について分類学的な研究を行った. 状の隆起を有する.形態は Co. multistriata v低 樹皮から単離した藻は単細胞で，細胞は球形であ corcontica に類似するが 食塩を含む培地でも増 り，細胞壁表面には子午線状の隆起を有する. 殖できる点で異なる. この藻は分子系統上は Coelastrella Coelastrella vacuolata に類似するが，細胞壁表面 Scenedesmus 属 Acutodesmus 亜属に位置するが， の隆起が光学顕微鏡でも観察できる点で異なる. この亜属に類似の形態的特徴をもっ既知種がない この藻は， 18S rDNA 領域の塩基配列の解析では ので新種と認め， Scenedesmus littoralis Hanagata Coelastrella 属に位置するが近縁の既知種がない とした. ことから新種と認め， Coelastrella saipanensis (東京大学先端科学技術研究センター)