植物研究雑誌 J. J. Jpn. Bot. 74: 74: 204-211 (1 999) Coenocystis Coenocystis inconstans ， a New Species of Bark-Inhabiting Green AIgae (Chlorophyceae ， Chlorophyta) a Nobutaka HANAGATA and Mitsuo CHIHARA b aResearch aResearch Center for Advanced Science and Technology ，University of Tokyo ， 4-6-1 4-6-1 Komaba ，Meguro-ku ，Tokyo ， 153-8904 JAPAN; bNatural bNatural History Museum and Institute ，Chiba ， 955-2 955-2 Aoba-cho ，Chuo-ku ，Chiba ， 26-8682 JAPAN (Received (Received on November 11 ， 1998) An alga with mucilaginous matrix around the cell wall was isolated from the bark bark of Ulmus campestris var. vulgaris collected from Christchurch ，New Zealand. The cells when young are ellipsoidal to narrowly ellipsoidal in shape ， and ovoid or spheroidal spheroidal when mature. The chloroplast of young cells is 甘ough-like or cup-shaped with with smooth margin ， while that of mature cells is often divided into a few lobes. The unbranched unbranched thylakoid lamellae pene 位ate the pyrenoid in almost parallel a町 angemen t. Many starch grains are often located around the pyrenoid. The cells are capable of producing producing 2-4 -(8) autospores. These characteristic features agree fundamentally with those those of the genus Coenocystis Korsikov ， the family Radiococcaceae ， but any de- scribed species species scribed of this genus do not share the gross morphology with our alga. The alga alga is ，therefore ， recognized as a new species named Coenocystis inconstans Hanagata Hanagata & Chihara Key words: Chlorophyceae ，Coenocystis ，new species Introduction habit freshwate r. In the course of our stud- One of the significant features which ies of bark-inhabiting green algae ，many characterize characterize the family Radiococcaceae Fott specimens were collected and their tax- et et Komarek is the presence of mucilage onomy has been studied by light and elec- around cells. Komarek and Fott (1 983) di- tron microscopy (Hanagata et al. 1996a ， vided the family into four subfamilies ， 1996b ， 1997). We encountered an alga with mainly on the basis of the features of the a mucilaginous matrix around the cells surrounding surrounding mucilage: the Radiococcoideae ， among the bark samples collected from New the the Disporoideae ， the Dictyochlorelloideae Zealand and the alga was recognized as a and the Palmodictyoideae. new species of Coenocystis on the basis of The genus Coenocystis Korsikov (1 953) ， morphological features. In this paper the with C. planctonia Korsikov as the type ， is description of the new species ， Coenocystis a member of the Radiococcoideae. Komarek inconstans Hanagata & Chihara ，and its phy- and Fott (1983) listed seven species and two logenetic position inferred from 18S rDNA varieties varieties in this genus ，most of which in 司 sequence data are presented. -204- August 1999 Journal of Japanese Botany Vo l. 74 No. 4 205 Materials Materials and Methods reticulata Tschermak- Woess (Z47207) ， Coenocystis Coenocystis inconstans was isolated from Fusochloris perforata (Lee & Bold) Floyd ， the the bark of Ulmus campestris var. vulgaris S. Watanabe & Deason (M62999 as collected collected by one of us ，M. Chihara from Characium performatum) ， Gloeotilopsis Christchurch ，New Zealand ，on 31 Decem- planctonia Iyenger & Philipose (Z28970) ， ber ， 1995. The alga examined in this study Microthamnion kuetzinganum Nageli was cultured in Bold's basal medium (Bold (Z28974) ，Mychonastes zofingiensis (Donz) 1949). 1949). The methods of obtaining and grow- Kalina & Pun とocharova (X73996 as Chlo- ing ing the unialgal culture ，and the procedure rella zofingiensis) ，Myrmecia biatorellae of of transmission electron microscopy were Boye-Petersen (Z28971) ，Nanochlorum obtained obtained in a previous paper (Hanagata et al. eucaryotum Wilhelm et al. (X06425) ， 1996a). 1996a). The strain described here is main- Scenedesmus fuscus (Shihira & Krauss) tained tained in the Environmental Biotechnology Hegewald (X73995 as Chlorella fusc α) ， Laboratory ， Research Center for Advanced Tetraselmis striata Butcher (X70802) ，and Science Science and Technology at the University of Trebouxia impressa Ahmadjian (Z21551). Tokyo. The data set excluded the ambiguously Total Total DNA was extracted in TE buffer aligned regions and was 1754 base pairs in (1 0 mmol/L Tris-HC1 ，1 mmol/L EDTA ，pH total length. 8.0) 8.0) using a mortar and pestle in the pres- Sequences were analyzed with both the ence of glass microfibre filter (Whatman maximum parsimony and the distance ma- GF/B). The polymerase chain reaction trix methods. The maximum parsimony (PCR) protocols (Saiki et al. 1988) was fol- analysis was implemented with the PAUP lowed for the amplification of 18S rDNA computer program (version 3.1.1; Swofford region. region. PCR amplification was conducted 1993) using a heuristic search under the with with 30 cycles of 94 0 C for 1 min ，55 0 C for unweighted scheme. A random addition of 1 min ，and 72 0 C for 2 min. The PCR prod- sequence with 10 replicates and a branch:- uct uct was directly sequenced using a DNA swapping algorithm (TBR) was used. For autosequencer ABI 377 (Applied the distance matrix method ，Kimura' s two- Biosystems ， Foster City ，CA ，USA) with the parameter method (Kimura 1980) was used dye termination method. Primers used were for the calculation of the distance matrix and reported reported by Nakayama et al. (1 996). Acces- neighbor-joining method (Saitou and Nei sion sion number of Coenocystis inconstans is 1987) to construct the tree. These proce- AB017435. dures were conducted with CLUST AL W The determined sequence was manually (Thompson et al. 1994) and PHYLIP aligned aligned with other previously known ones. (Felsenstein 1993). Supporting for internal The sequences included in this study are as branchs in both the trees constructed with follows follows (accession numbers): Chlorell α the maximum parsimony and neighbor-join- ellipsoidea ellipsoidea Gerneck (X63520) ， Chlorella ing methods were estimated by the bootstrap kessleri kessleri Fott et N ovakova SAG 211-11 g analysis (Felsenstein 1985). (X56105) ， Chlorella mirabilis Andreeva (X74000) ，Chlorella saccharophil α( Kr uger) Description Migula (X63505 and X73991) ， Chlorella Coenocystis inconstans Hanagata & sorokiniana sorokiniana Shihira & Krauss SAG 211-8k Chihara ， sp. nov. (X62441) ， Chlorella vulgaris Beijerinck Alga unicelluaris ，solitaria vel colonialis SAG 211 ・11 b (X 13688) ，Choricystis minor in matrice gelatinosa. Cellulae juveniles (Sk 吋a) Fott (X89012) ， Dictyochloropsis ellipsoideae vel anguste ellipsoideae ， rectae 206 206 植物研究雑誌 第74 巻 第4号 平成11 年8月 vel vel curvatae ，2.5-4.0 x3.5-7.5μm. Cellulae numerosis circumcincta. Matrix pyrenoidis maturae maturae ellipsoideae vel ovatae ， 3.5-6.5 x per lamellas parallelas invasa. Reproductio 5.0-9.5μm ， vel sphaericae ，3.5-8.0μm in asexualis per 2-4-(8) autosporas effecta. diametro. diametro. Chloroplastus unus ，parietalis ， Autosporae ellipsoideae vel anguste cupulatus ， saepe margine grandi lobus. ellipsoideae ， 2.0-4.0 x 3.0-6.0μm. Sub- Pyrenoides singla ， granis amylaceis stantia carotenoide secundaria carens. -〆~'. :\t~!~号、. _，..・，_，.川 :.~:!f，.-;"::.":rr.. dト~~. .;mさ均:;::~~，、 E~戸己完3 ・ :はさ:;;:\'刊行:\;~ Wg~;~\ . ぶがが ，:j ， '~\"-句-凡..: ...... 九、，，_ .'!o';-旨γアア':_.i-_'~'.!~::、V沖μ白':J :':/.:.仰川品川h片日ワ糾1ル以刈h可叫4占::.~H削h肝納約J向均H切r!1噌川山ι代~:'町~::~'町:::~'刊;汗 σ科刊む小叩机判判円N爪川川刈可仇川吋吋吋れNη川勾:苫芯Z苫芯口r巳匂:.;_:， 古 . 耳切:~t_::-:::~i!!i':~:!:-:~:i:;:~~:;I_~~~':~・ 1 '<i'品':!~::::バ，'~・:'，:-:::_:::'，:::・- - ， 苅議将司、、心¥-.-，，.，'，'，、. 'i;'::.~;:~:V__ 3 4 5μm Figs. Figs. 1-9. Coenocystis inconstans ，sp. nov. 1. Young vegetative cell. 2-4. Mature vegetative cells. cells. 5. Aggregation of cells surrounded by mucilaginous substance. 6 ，7. Colony embed- ded in common mucilaginous matrix. 8， 9. Autospores in parent cells. August August 1999 Journal of Japanese Botany Vo l. 74 No. 4 207 Holotype: Holotype: Figure 14. The alga is unicellular and solitary or in Type locality: Christchurch ，New colony embedded in a common gelatinous Zealand. Zealand. matrix. The cells are surrounded by a mu- Habitat: Habitat: Specimens examined were iso- cilaginous substance ，whose matrix is not lated lated from the samples attached on the bark stratified. Young cells are ellipsoidal or nar- of of Ulmus campestris va r. vulgaris. rowly ellipsoidal ，sometimes ovoidal and Type culture: Christchurch ，New Zealand ， curved ， 2.5-4.0 x 3.5-7.5μm in size ， 31 31 December 1995. Strain K4-3 is deposited with rounded ends. Mature cells are ellipsoi- in in the Culture Collection of Environmental dal to ovoid ， 3.5-6.5 x 5.0-9.5μm in Biotechnology Biotechnology Laboratory at Research Cen- size ， or spheroidal ，3.5-8.0μm in diameter. ter ter for Advanced Science and Technology ， The cell wall is composed of a single layer ， The University of Tokyo. 0.04-0.12μm thick and smooth on the sur- Figs. Figs. 10-14. Coenocystis inconstans sp. nov. (1 0-12. Photomicrographs. 13 ， 14. Transmission electron micrographs). micrographs). 10. India ink preparation to show the presence of mucilaginous substance around cells. 1. 11. Mature e11i psoidal cells. 12. Mature spheroidal cells. 13. Young vegetative cells. 14. Mature veg- etative etative cell. 208 208 植物研究雑誌 第74 巻 第4号 平成11 年8月 face. face. The chloroplast is parietal and cup- observed. shaped ，often with deeply divided lobes. The chloroplast chloroplast consists of thylakoid lamellae Phylogenetic position that that are arranged in several almost parallel Maximum parsimony analysis of the 18S rows. rows. Several electron-dense granules ， rDNA sequences resulted in the