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Oltmannsiellopsis, a new Genus of Marine Flagellate (Dunaliellaceae, Chlorophyceae)
Arch. Protistenkd. 132 (1986): 313-324 VEB Gustav Fischer Verlag Jena
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan
Oltmannsiellopsis, a new Genus of Marine Flagellate (Dunaliellaceae, Chlorophyceae)1) By MITSUO CHIHARA, ISAO INOUYE and NAOHIRO TAKAHATA With 34 Figures
Summary Three species of Oltmannsiellopsis CHIHARA et IXOUYE gen. nov., viz., O. viridis (HARGRAVES et STEELE) CHIHARA et INOUYE, O. geminata INOUYE et CHIHARA sp. nov. and O. unicellularis INOUYE et CHIHARA sp. nov. were studied using laboratory culture and electron microscopy. The new genus is characterized by having I) unicellular and colonial members, 2) quadriflagellate cells, 3) no cell wall, 4) longitudinal cell division of a cell while swimming. These features are considered sufficient to separate the new genus from Oltmannsiella ZIMMERMANN (1930). ZIMMERMANN'S genus is placed in the Chlamydomonadaceae, while ours is referred to the Dunaliellaceae. The three species of Oltmannsiellopsis are distinguished from one another as follows: O. viridis forms a four-celled colony, O. gemiuata a two-celled colony and O. unicellularis is one· celled.
Introduction Oltmann8iella ZIMMERMANN (1930) is a marine colonial green flagellate. Since the genus was established with the single species, O. lineata, in the Mediterranean Sea, no other species was added until 1980, when HARGRAVES and STEELE described a second species from Narragansett Bay, Rhode Island, on the east coast of North America. It was named O. virich8 (incorrectlJ spelled O. virida by the authors) and distinguished from the type primarily in having four flagella. For the last several years, we have collected and examined many marine flagellates which possibly cause red tide in Japanese coastal water. In the course of the study, we have often encountered two colonial green flagellates at several localities along the Pacific coast of Japan. One of them is undoubtedly identical with O. riridi8 while the other is apparently distinct from both O. lineata and O. 'ciridi8 in forming a two-celled colony. Examination with the electron microscope of these two colonial algae show that they are similar to an unicellular flagellate collected from Harima-nada in the Inland Sea of Japan with respect to cellular details and method of asexual reproduction. Moreover, these algae differ in the features from Oltmann81·ella. We therefore establish a new genus to accommodate them.
Material and Methods Specimens examined in this study are listed in Table 1 with their source and collection data. All of these specimens were cultured in GPM medium (LOEBLICH 1975) or ESM medium (OKAICHI 1) The authors dedicate the paper to Prof. ALFRED RIETH on the occasion of his 75th birthday.
314
M. CHIHARA et al.
Table 1. Sources and collection data of Oltmannsiellopsis strains used in this study. Specimed examined
Sources and collection data
Isolators
Oltmannsiellopsis
viridi8
Mie University Culture Collection
HIDEo I WASARI
Oltmann8iellop8is
viridi8
Oct. 1979, Kesen-numa, Miyagi Prefecture
ISAO INOUYE
Oltmannsiellopsi8
geminata
Apr. 1981, Setta, Shizuoka Prefecture
ISAO INOUYE
Oltmannsiellopsis
geminata
May 1982, Itsu-ura, Ibraki Prefecture
TAKEO HORIGUCHI
Oltmannsiellopsis
unicellularis Microbial Culture Collection of the National Institute for Environmental Studies
SYOICHIRO SUDA
et al. 1982) and exposed to 2,000 Ix provided with white fluorescent tubes and subjected to 12 h/12 h light/dark regime at 20°C. For scanning electron microscopy cells were fixed in 4 % osmium tetroxide for 2 h, dehydrated with graded ethanol series, subjected to critical point drying and sputtered with gold. Observations were made by JEOL T-20 scanning electron microscope. Materials for thin sections were prepared as follows. Cells collected by gentle centrifuging or retained by membrane filters of 3.0 pm pore size were fixed in 2.5 % glutaraldehyde in 0.2 M sodium cocadylate buffer (pH 8.0) containing 0.25 M sucrose for 1 h, rinsed in the same buffer for three times (5 min each) and postfixed in 2 % osmium tetroxide for 2 h. Cells were embedded in SPURR'S low viscosity resin (SPURR 1969) after dehydration in a graded ethanol series. Sections were cut with diamond knives and strained in 2 % uranyl acetate followed by lead citrate (REYNOLD 1963). Observations were made using JEOL JEM 100 CXIl transmission electron microscope.
Description and Discussion Oltmannsiellopsis
CHIHARA
et
INOUYE
gen. nov.
Plant a unicellularis et colonialis; colonia plerumque bicellularis vel quadricellularis ; cellulae coloniae in parallelis seriatim dispostitae; cellula ovoidea vel ellipsoidea, ali quando paulo complana, pariete cellulae carens, unum chloroplastum et quadriflagellas habens, flagella flagelliformis, sine pilis et squamis in pagina, e depressie apicale calderi-forme oriunda; chloroplastus cupulatus, unum stignum et unum pyrenoidem habens; pyrenoides per thylakoides invasus, per granes multa amyloidea circumcinctus; reproductio asexualis per cellulam mobilem per bipartitionem longitudinalem protoplasti effecta; reproductio sexualis ignota. Typus generis. Oltmannsiellopsis viridis (HARGRAVES et STEELE) CHIHARA et INOUYE.
Key to species 1. 1. 2. 2.
Plant colonial Plant unicellular. . . . . . . . . . Colony usually consisting of four cells Colony usually consisting of two cells
Oltmannsiellopsis viridis (HARGRAVES et
2
O. unicellulari8 O. viridi8 . . O. geminata STEELE) CHIHARA
et
INOUYE
comb. nov.
Basionym. Oltmannsiella viridis HARGRAVES et STEELE (as O. vin·da). Phycologia 19: 97, figs. 1-8. 1980. Our specimens from the Japanese coastal waters agree very well with the description of Oltmannsiella viridis by HARGRAVES and STEELE (1980). The alga as founn in Japan is colonial, usually consisting of four cells joined laterally (Figs. 1, 2, 5
315
Oltmannsiellopsis, a new Genus of Marine Flagellat e
1
2
3
4
Figs . 1 and 2. Side and polar views of Oltmannsiellopsis viridis (HARGRAVES et STEELE) CHIHARA et I XOUYE. A colony is consisting of four cells joined laterally. Each cell of the colony has four fl age ll a emerging to sides of the tran sverse axis of the colony. The eyespot is situated in the pos· terior h alf of the cell. 1 and 2 : X 4, 300. Fig. 3. Side view of Oltmannsiellopsis geminata INOUYE et CHIHARA. A colony is symmetrical and cordate. Cells consisting the colony join to each other facing their ventral side. Each cell is asymmetrical, has four flagell a em erging from a depression located in the subapical portion of coherent s ide (ventral side) of the cell. The eyespot is situated in the anterior half of the cell. X 3,300. Fig. 4. Side view of Oltmannsiellopsis unicellularis INOUYE et CHIHARA, unicellular species of the genus . Cell is symmetrical and has four flagella emerging in the same way as in O. viridis and O. geminata. The eyespot is situa t ed in the anterior half of the cell. X 3,200.
316
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et al.
Figs. 5- 10. Light microscopy of the three species of Oltmannsiellopsis. Side and polar vie w of O. viridis (5 and 6), O. geminata (7 and 8) and O. unicellularis (9 and 10). 5 and 6: X 2,000; 7: X 1,800; 8: X 2,200; 9: X 1,700; 10: X 2,100. Figs. 11-14. Scanning electron microscopy. 11. A symmetrical cell of O. viridis with four flagella. 12. An asymmetrical cell of O. geminata with foul' flagella emerging from subapical portion of the cell. 13 a nd 14. Flagellar insertion of O. geminata (13) and O. vi1'idis (14). 11: X 3,700; 12: X 6,000; 13: X 8,000; 14: X 15.000.
Figs. 15- 20. Transmission electron microscopy of Oltmannsiellopsis viridis. 15. Longitudinal section of the cell, showing nucleus (N). pyrenoid (P) surrounded by numerous starch grains (S). dictyosome (D). refractive bodies (Rb). flagella (F) and other cell structures. 16. Detail of pyrenoid. Th e m atrix is penetrated by single thylakoids. 17. The eyespot composed of single layered globules. 18. A microbody (Mb) located beneath the nucleus and just external to the pyrenoid region of
Oltmannsiellopsis, a new Genus of Marine Flagellate
317
the chloroplast. 19. Detail of the anterior r egion of the cell. Two basal bodies are crossing to each other. 20. Transverse section of the basal body apparatuses, showing counterclockwise orientation of two internal basal bodies and three of cruciate microtubular fl agellar roots. 15: X 8,000; 16 : X 9,500; 17: X 30,000; 18: X 19,000 ; 19: X 28,000; 20: X 16,000.
318
M.
CHIHARA
et al.
and 6). The cells are somewhat ovoid or almost cylindrical, occasionally bent longitudinally, with almost rounded apices and rounded end (Figs. 1 and 5). In polar view, the cells appear square with rounded corners (Figs. 2 and 6). They measure 10-20 flm long and 5-10 flm wide and lack a cell wall (Fig. 15). Each cell possesses a single chloroplast (Fig. 15) and four flagella of the whiplash type, which arise from an apical caldera-shaped depression (Figs. 11, 14 and 20). The chloroplast is cup-shaped, has two to four lobes and contains a single pyrenoid in its posterior region (Fig. 15) and an eyes pot composed of single layered osmiophilic globules (Fig. 17). The pyrenoid matrix is penetrated by single thylakoids and surrounded by many starch grains (Fig. 16). In the external pair of cells of a colony, the eyes pot is situated laterally, while in the internal pair of cells it faces the inside (Fig. 1). Nucleus and some other organelles such as mitochondria, dictyosomes and basal body apparatuses are situated in a pocket surrounded by chloroplast lobes (Fig. 15). An elongated microbody is present between the nucleus and the pyrenoid region of the chloroplast (Fig. 18). With the light microscope, numerous refractive grains are observed distributed in the peripheral region of the cell and around the nucleus (Figs. 5 and 15). These may be muciferous bodies containing closely packed fibrous material which are discharged from the cell when cells are treated with fixatives (Fig. 15). Four flagellar basal bodies are arranged as follows. Two internal basal bodies have a counterclockwise orientation, crossing each other at 45-90 degrees (Fig. 19), while one external body is parallel to each internal basal body (Fig. 20). This arrangement results in a characteristic flagellar orientation, viz., a pair of flagella emerge on each side of the transverse axis of the colony (Figs. 2 and 6). The microtubular flagellar root system is of the so-called cruciate type (Fig. 20). Asexual reproduction takes place by longitudinal bipartition of a cell while swimming, and the sequence is as follows. First, the colony splits into two swimming colonies, each consisting of two cells (Figs. 21 and 28A). Second, each cell of the two-celled colony then divides longitudinally to reconstitute the four-celled colony (Figs. 22-24 and 28A). Each of the two internal daughter cells produces an eyespot de novo facing the inside of the colony (Figs. 23, 24 and 28A). During longitudinal bipartition, each daughter cell receives a pair of flagella from the parent and later produces a new pair de novo (Fig. 28A). The process of the asexual reproduction described above in addition to the absence of a cell wall is in contrast to the features known in Oltmannsiella. According to the description and the illustrations given by ZIMMERMANN (1930), in that genus the cells are bounded by walls and asexual reproduction takes place in the non-motile state. The first cell division is longitudinal and it occurs within the mother cell wall. The daughter cells then rotate 90 degrees. The second longitudinal bipartition takes place almost synchronously in the two cells, resulting in four laterally coherent daughter cdls with their long axis perpendicular to the long axis of the mother cell wall. An autocolony is thus produced. This method of reproduction is fundamentally identical with that of Chalmydomonas (ETTL 1976) and Chloromonas (PASCHER 1943; ETTL 1980) in the Chlamydomonadaceae, whereas the method of the asexual reproduction seen in O. viridis is basically identical with that of Dunaliella (LEVAIN and MARANO-LE BARON 1973; EYDEN 1975; CHIHAltA, INOlTYE and TAKAHATA, unpublished observations) in the Dunaliellaceae. It is thus appropriate to remove O. viridis from Oltmannsiella and place it in a new genus of the Dunaliellaceae, which we name Oltmannsiellopsis. Type locality: Narragansett Bay, Rhode Island, U.S.A. Habitat: Coastal waters, often occurring in great abundance in brackish waters in embayments in summer.
319
Oltmannsiellopsis, a new Genus of Marine Flagellate
21
24
23
Figs. 21-27. Sequence of asexual reproduction of O. viridis (21-24) and O. geminata (25-27). Four·celled colony splits into two·celled colony (21). Each cell reproduces by longitudinal bipartition (22-24), resulting in a four-celled colony. Two-celled colony splits into single cells (25-26) and each of them divides by longitudinal bipartition after the chloroplast duplicates (27). Arrow heads indicate eyespots produced de novo. 21-24: X 1,750; 25-27: X 2,300.
Geographic distribution: Known at present from the type locality and in Japan (Teshio, Hokkaido; Kesen-numa, Miyagi Pref.; Itsu-ura, Ibaraki Pref.; Ito, Ko-ura and Heta, Shizuoka Pref.; Awaji-shima, Hyogo Pref.; Aoshima, Miyazaki Pref.: Nagasaki-hana, Kagoshima Pref.). Probably widely distributed in the temperate regions.
Oltmannsiellopsis geminata INOUYE et
CHIHARA
sp. nov.
Plant a colonialis; colonia plerumque bicellularis; cellula semicordata a let ere visus, 10.5-15.0 flm longa, 5.0-9.0 flm lata, elliptica vel obovata a let ere dorsale visus. Plants colonial, usually consisting of two cells, cordate when viewed from broad side; cells somewhat depressed, semicordate when viewed from broad side, 10.5-15.0 flm long, 5.0-9.0 flm wide, broader anteriorly, narrower posteriorly, with somewhat rounded apex and rounded end, elliptical or obovata when viewed from narrow side,
320
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CHIHARA
et al.
A
B Fig. 28. Schematic drawings showing asexual reproduction of O. viridis (A) and O. geminata (B).
with somewhat rounded apex and rounded end; chloroplast markedly asymmetrical, restricted to the lateral and basal parts of the cell, lying close to the dorsal lateral wall; flagella arising from the bottom of a caldera-shaped depression near the ventral shoulder of the cell. Holotype: Figure 3. Type locality: Heta, Shizuoka Pref., Japan. Habitat: Coastal waters. Geographical distribution: Type locality and Itsu-ura, Ibaraki Pref.
Oltmanllsiellop8i8, a new Genus of Marme Flagellate
321
Figs. 29-30. Transmission electron microscopy of Oltmannsiellop8'is geminata. Two longitudinal sections of the cell cut through different planes, one through lateral (29) and the other through dorsiventral plane (30). Major cell organelles are seen in both micrographs. Arrow head in Fig. 30 shows a caldera-shaped depression located in the subapical portion of the ventral side of the cell. Abbreviations: bb - basal bodies; F - flagellum; M - microbody; Mb - muciferous body N - nucleus; P - pyrenoid; Rb - refractive body. 29: X 9,700; 30: X 10,000.
Oltmannsiellopsis geminata has fundamentally the same cell structure as O. viridis (most of major cell organelles indistinguishable from those of O. viridis can be seen in Fig. 29). It differs ,from that species, however, in forming two-celled rather than four-celled colonies (Figs. 7 and 8) and in having markedly asymmetrical cells. While the colony is symmetrical, each cell shows dorsiventral asymmetry (Figs. 12 and 30). For convenience of description, the coherent side of each cell is designated ventral, while the free side is designated dorsal. A caldera-shaped depression is located subapically on the ventral side of the cell (Figs. 2, 12 and 30), and from there four flagella arise in the same way as in O. viridis (Fig. 13). The chloroplast, which is dorsilaterally stiuated (Fig. 30), possesses an eyespot in the anterior half of the dorsal side (Fig. 7). A pyrenoid is situated somewhat toward the dorsal side in the basal portion of the
322
M.
CHIHARA
et al.
Figs. 31-34. Transmission electron microscopy of Oltmannsiellopsis unicellularis. Longitudinal (31) and transverse (32) section of the cell. Longitudinal (33) and transverse (34) section of the anterior region of the cell showing similar appearance to that of O. viridi8. 31: X 12,000; 32: X 6,000; 33: X 25,000; 34: x 17,000.
Oltmannsiellopsis, a new Genus of Marine Flagellate
323
chloroplast (Figs. 29 and 30). Asexual reproduction is similar to that in O. viridis. The first sign of cell division observable with the light microscope is the separation of the colony into two cells (Figs. 25-26 and 28B). Separated cells tend to become symmetrical externally and internally (Fig. 26). The chloroplast becomes cup-shaped and divides entirely before longitudinal cell cleavage proceeds (Figs. 27 and 28B). Resultant cells usually remain loosely attached to form a daughter colony. These processes occur while the cell is swimming, as in O. viridis.
Oltmannsiellopsis unicellularis INOUYE et CHIHARA sp. nov. Plant a unicellularis; cellula aliquantum complana, obovata a latere lato visus, 9.0-14.0,um longa, 7.0-10.0,um lata, elliptic a a latere angusto visus. Plants unicellular, somewhat compressed, obovate when viewed from broad side, 9.0-14.0,um long, 7.0-10,um wide, elliptical when viewed from narrow side; chloroplast cup-shaped; pyrenoid posteriorly; eyespot in the anterior half of the cell; cells forming gelatinous mass in culture. Holotype: Figure 4. Type locality: Harima-nada, Inland Sea of Japan. Habitat: Coastal waters. Geographic distribution: Known from the type locality. Although O. um'cellulan's (Figs. 4, 9-10 and 31-34) is very different in appearance from the colonial members of the genus, it shares many characteristics with them, viz., it lacks a cell wall, possesses four flagella arranged in exactly the same pattern as in O. viridis and O. geminata (Figs. 9-10 and 31-32) and reproduces by longitudinal bipartition while swimming. Most cellular structures are fundamentally indistinguishable from those of the colonial species (Figs. 31-24). An interesting feature unique to O. unicellularis, however, is the development in culture of a gelatinous mass in which the cells swim. Recently, a very similar alga was found in coastal waters of Natal, South Africa (PIENAAR 1985). PIENAAR's alga is unicellular, lacks a cell wall, prossesses four flagella arranged in the same way as in three species described in this paper and develops a gelatinous mass in culture (PIENAAR 1985 and pers. comm.). These features coincide with those of O. unicellularis, suggesting that PIENAAR'S alga is It member of the genus Oltmannsiellopsis, although the method of cell division has not been determined. PIENAAR, however, reported the presence of diamond-shaped body scales above the cell membrane. We examined O. unicellularis carefully using materials fixed in various ways, but could not found any trace of scales. It seems likely, therefore, that PIENAAR'S alga is specifically different from O. unicellularis, but these two algae must be studied in more detail before a decision can be made.
Acknowledgements We should like to express our thanks to Professor HIDEO IWASAKI, Mie University, Dr. TAKEO HORIGUCHI, University of Natal, Pietermaritzburg, South Africa and Dr. MAKOTO WATANABE and Mr. SYOICHIRO SUDA, National Institute for Environmental Studies, for providing culture strains. We are also grateful to Dr. PAUL C. SILVA, University of California, Berkeley, U.S.A., for his reading of the manuscript.
Literature ETTL, H. (1976): Die Gattung Chlamydomonas EHRENBERG. Nova Hedwigia 49: 1-1122. - (1980): GrundriB der all~emeinen Algologie. Jena.
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M. CHIHARA et al., Oltmannsiellopsis, a new Genus of Marine Flagellate
EYDEX, B. (1975): Light and electron microscope study of Dunaliella primolecta BUTCHER (Volvocida). J. Protozool. 2.2: 316--344. HARGRAYES, P. E., and STEELE, R. L. (1980): Morphology and ecology of Oltmannsiella virida, sp. nov. (Chlorophyceae: Volvocales). Phycologia 19: 96-102. I,EVAIX, N., and MARAKO-LE BARON, F. (1973): Etude des effets d'un insecticide organochlore, Ie lindane, sur la croissance et la structure de deux algues unicellularis, l'Euglena gracilis Z. et la Dunaliella bioculata. C. R. Acad. Sci. Paris 2.76: 37-40. LOEBLICH, A. R. III. (1975): A seawater medium for dinoflagellates and the nutrition of Cachonina niei. J. Phycol. 11: 80-86. OKAICHI, T., NISHIO, S., and IMATOMI, Y. (1982): Collection and mass culture. In: Jpn. Soc. Sci. Fish. (ed.): Toxic phytoplankton - Occurrence, mode of action and toxin. KoseishaKoseikaku, Tokyo: 23-34. P ASCHER, A. (1943): Beitrage zur Morphologie der ungeschlechtlichen und geschlechtlichen Vermehrung der Gattung Chlamydomonas. Beih. Bot. Centralbl. 62./A: 197-220. PIEKAAR, R. N. (1985): An unusual scale bearing flagellate resembling the genus Oltmannsiella (Chlorophyceae). Abstract. Second International Phycological Congress, Copenhagen 4-10 August, 1985: 126. REYKOLDS, E. C. (1963): The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell BioI. 17: 208--212. SPURR, A. R. (1969): A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruc. Res. 2.6: 31-42. ZIMMERMANN, W. (1930): Neue und wenig bekannte Kleinalgen von Neapel. I-V. Zeitschr. Bot. 23: 419-428. Authors' address: Dr. MITSUO CHIHARA, Dr. ISAo INOUYE and Mr. NAOHIRO TAKAHATA, Institute of Biological Sciences, University of Tsubuka, Sakura-mura, Ibaraki-ken, 305 Japan.
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan
Oltmannsiellopsis, a new Genus of Marine Flagellate (Dunaliellaceae, Chlorophyceae)1) By MITSUO CHIHARA, ISAO INOUYE and NAOHIRO TAKAHATA With 34 Figures
Summary Three species of Oltmannsiellopsis CHIHARA et IXOUYE gen. nov., viz., O. viridis (HARGRAVES et STEELE) CHIHARA et INOUYE, O. geminata INOUYE et CHIHARA sp. nov. and O. unicellularis INOUYE et CHIHARA sp. nov. were studied using laboratory culture and electron microscopy. The new genus is characterized by having I) unicellular and colonial members, 2) quadriflagellate cells, 3) no cell wall, 4) longitudinal cell division of a cell while swimming. These features are considered sufficient to separate the new genus from Oltmannsiella ZIMMERMANN (1930). ZIMMERMANN'S genus is placed in the Chlamydomonadaceae, while ours is referred to the Dunaliellaceae. The three species of Oltmannsiellopsis are distinguished from one another as follows: O. viridis forms a four-celled colony, O. gemiuata a two-celled colony and O. unicellularis is one· celled.
Introduction Oltmann8iella ZIMMERMANN (1930) is a marine colonial green flagellate. Since the genus was established with the single species, O. lineata, in the Mediterranean Sea, no other species was added until 1980, when HARGRAVES and STEELE described a second species from Narragansett Bay, Rhode Island, on the east coast of North America. It was named O. virich8 (incorrectlJ spelled O. virida by the authors) and distinguished from the type primarily in having four flagella. For the last several years, we have collected and examined many marine flagellates which possibly cause red tide in Japanese coastal water. In the course of the study, we have often encountered two colonial green flagellates at several localities along the Pacific coast of Japan. One of them is undoubtedly identical with O. riridi8 while the other is apparently distinct from both O. lineata and O. 'ciridi8 in forming a two-celled colony. Examination with the electron microscope of these two colonial algae show that they are similar to an unicellular flagellate collected from Harima-nada in the Inland Sea of Japan with respect to cellular details and method of asexual reproduction. Moreover, these algae differ in the features from Oltmann81·ella. We therefore establish a new genus to accommodate them.
Material and Methods Specimens examined in this study are listed in Table 1 with their source and collection data. All of these specimens were cultured in GPM medium (LOEBLICH 1975) or ESM medium (OKAICHI 1) The authors dedicate the paper to Prof. ALFRED RIETH on the occasion of his 75th birthday.
314
M. CHIHARA et al.
Table 1. Sources and collection data of Oltmannsiellopsis strains used in this study. Specimed examined
Sources and collection data
Isolators
Oltmannsiellopsis
viridi8
Mie University Culture Collection
HIDEo I WASARI
Oltmann8iellop8is
viridi8
Oct. 1979, Kesen-numa, Miyagi Prefecture
ISAO INOUYE
Oltmannsiellopsi8
geminata
Apr. 1981, Setta, Shizuoka Prefecture
ISAO INOUYE
Oltmannsiellopsis
geminata
May 1982, Itsu-ura, Ibraki Prefecture
TAKEO HORIGUCHI
Oltmannsiellopsis
unicellularis Microbial Culture Collection of the National Institute for Environmental Studies
SYOICHIRO SUDA
et al. 1982) and exposed to 2,000 Ix provided with white fluorescent tubes and subjected to 12 h/12 h light/dark regime at 20°C. For scanning electron microscopy cells were fixed in 4 % osmium tetroxide for 2 h, dehydrated with graded ethanol series, subjected to critical point drying and sputtered with gold. Observations were made by JEOL T-20 scanning electron microscope. Materials for thin sections were prepared as follows. Cells collected by gentle centrifuging or retained by membrane filters of 3.0 pm pore size were fixed in 2.5 % glutaraldehyde in 0.2 M sodium cocadylate buffer (pH 8.0) containing 0.25 M sucrose for 1 h, rinsed in the same buffer for three times (5 min each) and postfixed in 2 % osmium tetroxide for 2 h. Cells were embedded in SPURR'S low viscosity resin (SPURR 1969) after dehydration in a graded ethanol series. Sections were cut with diamond knives and strained in 2 % uranyl acetate followed by lead citrate (REYNOLD 1963). Observations were made using JEOL JEM 100 CXIl transmission electron microscope.
Description and Discussion Oltmannsiellopsis
CHIHARA
et
INOUYE
gen. nov.
Plant a unicellularis et colonialis; colonia plerumque bicellularis vel quadricellularis ; cellulae coloniae in parallelis seriatim dispostitae; cellula ovoidea vel ellipsoidea, ali quando paulo complana, pariete cellulae carens, unum chloroplastum et quadriflagellas habens, flagella flagelliformis, sine pilis et squamis in pagina, e depressie apicale calderi-forme oriunda; chloroplastus cupulatus, unum stignum et unum pyrenoidem habens; pyrenoides per thylakoides invasus, per granes multa amyloidea circumcinctus; reproductio asexualis per cellulam mobilem per bipartitionem longitudinalem protoplasti effecta; reproductio sexualis ignota. Typus generis. Oltmannsiellopsis viridis (HARGRAVES et STEELE) CHIHARA et INOUYE.
Key to species 1. 1. 2. 2.
Plant colonial Plant unicellular. . . . . . . . . . Colony usually consisting of four cells Colony usually consisting of two cells
Oltmannsiellopsis viridis (HARGRAVES et
2
O. unicellulari8 O. viridi8 . . O. geminata STEELE) CHIHARA
et
INOUYE
comb. nov.
Basionym. Oltmannsiella viridis HARGRAVES et STEELE (as O. vin·da). Phycologia 19: 97, figs. 1-8. 1980. Our specimens from the Japanese coastal waters agree very well with the description of Oltmannsiella viridis by HARGRAVES and STEELE (1980). The alga as founn in Japan is colonial, usually consisting of four cells joined laterally (Figs. 1, 2, 5
315
Oltmannsiellopsis, a new Genus of Marine Flagellat e
1
2
3
4
Figs . 1 and 2. Side and polar views of Oltmannsiellopsis viridis (HARGRAVES et STEELE) CHIHARA et I XOUYE. A colony is consisting of four cells joined laterally. Each cell of the colony has four fl age ll a emerging to sides of the tran sverse axis of the colony. The eyespot is situated in the pos· terior h alf of the cell. 1 and 2 : X 4, 300. Fig. 3. Side view of Oltmannsiellopsis geminata INOUYE et CHIHARA. A colony is symmetrical and cordate. Cells consisting the colony join to each other facing their ventral side. Each cell is asymmetrical, has four flagell a em erging from a depression located in the subapical portion of coherent s ide (ventral side) of the cell. The eyespot is situated in the anterior half of the cell. X 3,300. Fig. 4. Side view of Oltmannsiellopsis unicellularis INOUYE et CHIHARA, unicellular species of the genus . Cell is symmetrical and has four flagella emerging in the same way as in O. viridis and O. geminata. The eyespot is situa t ed in the anterior half of the cell. X 3,200.
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Figs. 5- 10. Light microscopy of the three species of Oltmannsiellopsis. Side and polar vie w of O. viridis (5 and 6), O. geminata (7 and 8) and O. unicellularis (9 and 10). 5 and 6: X 2,000; 7: X 1,800; 8: X 2,200; 9: X 1,700; 10: X 2,100. Figs. 11-14. Scanning electron microscopy. 11. A symmetrical cell of O. viridis with four flagella. 12. An asymmetrical cell of O. geminata with foul' flagella emerging from subapical portion of the cell. 13 a nd 14. Flagellar insertion of O. geminata (13) and O. vi1'idis (14). 11: X 3,700; 12: X 6,000; 13: X 8,000; 14: X 15.000.
Figs. 15- 20. Transmission electron microscopy of Oltmannsiellopsis viridis. 15. Longitudinal section of the cell, showing nucleus (N). pyrenoid (P) surrounded by numerous starch grains (S). dictyosome (D). refractive bodies (Rb). flagella (F) and other cell structures. 16. Detail of pyrenoid. Th e m atrix is penetrated by single thylakoids. 17. The eyespot composed of single layered globules. 18. A microbody (Mb) located beneath the nucleus and just external to the pyrenoid region of
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the chloroplast. 19. Detail of the anterior r egion of the cell. Two basal bodies are crossing to each other. 20. Transverse section of the basal body apparatuses, showing counterclockwise orientation of two internal basal bodies and three of cruciate microtubular fl agellar roots. 15: X 8,000; 16 : X 9,500; 17: X 30,000; 18: X 19,000 ; 19: X 28,000; 20: X 16,000.
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and 6). The cells are somewhat ovoid or almost cylindrical, occasionally bent longitudinally, with almost rounded apices and rounded end (Figs. 1 and 5). In polar view, the cells appear square with rounded corners (Figs. 2 and 6). They measure 10-20 flm long and 5-10 flm wide and lack a cell wall (Fig. 15). Each cell possesses a single chloroplast (Fig. 15) and four flagella of the whiplash type, which arise from an apical caldera-shaped depression (Figs. 11, 14 and 20). The chloroplast is cup-shaped, has two to four lobes and contains a single pyrenoid in its posterior region (Fig. 15) and an eyes pot composed of single layered osmiophilic globules (Fig. 17). The pyrenoid matrix is penetrated by single thylakoids and surrounded by many starch grains (Fig. 16). In the external pair of cells of a colony, the eyes pot is situated laterally, while in the internal pair of cells it faces the inside (Fig. 1). Nucleus and some other organelles such as mitochondria, dictyosomes and basal body apparatuses are situated in a pocket surrounded by chloroplast lobes (Fig. 15). An elongated microbody is present between the nucleus and the pyrenoid region of the chloroplast (Fig. 18). With the light microscope, numerous refractive grains are observed distributed in the peripheral region of the cell and around the nucleus (Figs. 5 and 15). These may be muciferous bodies containing closely packed fibrous material which are discharged from the cell when cells are treated with fixatives (Fig. 15). Four flagellar basal bodies are arranged as follows. Two internal basal bodies have a counterclockwise orientation, crossing each other at 45-90 degrees (Fig. 19), while one external body is parallel to each internal basal body (Fig. 20). This arrangement results in a characteristic flagellar orientation, viz., a pair of flagella emerge on each side of the transverse axis of the colony (Figs. 2 and 6). The microtubular flagellar root system is of the so-called cruciate type (Fig. 20). Asexual reproduction takes place by longitudinal bipartition of a cell while swimming, and the sequence is as follows. First, the colony splits into two swimming colonies, each consisting of two cells (Figs. 21 and 28A). Second, each cell of the two-celled colony then divides longitudinally to reconstitute the four-celled colony (Figs. 22-24 and 28A). Each of the two internal daughter cells produces an eyespot de novo facing the inside of the colony (Figs. 23, 24 and 28A). During longitudinal bipartition, each daughter cell receives a pair of flagella from the parent and later produces a new pair de novo (Fig. 28A). The process of the asexual reproduction described above in addition to the absence of a cell wall is in contrast to the features known in Oltmannsiella. According to the description and the illustrations given by ZIMMERMANN (1930), in that genus the cells are bounded by walls and asexual reproduction takes place in the non-motile state. The first cell division is longitudinal and it occurs within the mother cell wall. The daughter cells then rotate 90 degrees. The second longitudinal bipartition takes place almost synchronously in the two cells, resulting in four laterally coherent daughter cdls with their long axis perpendicular to the long axis of the mother cell wall. An autocolony is thus produced. This method of reproduction is fundamentally identical with that of Chalmydomonas (ETTL 1976) and Chloromonas (PASCHER 1943; ETTL 1980) in the Chlamydomonadaceae, whereas the method of the asexual reproduction seen in O. viridis is basically identical with that of Dunaliella (LEVAIN and MARANO-LE BARON 1973; EYDEN 1975; CHIHAltA, INOlTYE and TAKAHATA, unpublished observations) in the Dunaliellaceae. It is thus appropriate to remove O. viridis from Oltmannsiella and place it in a new genus of the Dunaliellaceae, which we name Oltmannsiellopsis. Type locality: Narragansett Bay, Rhode Island, U.S.A. Habitat: Coastal waters, often occurring in great abundance in brackish waters in embayments in summer.
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24
23
Figs. 21-27. Sequence of asexual reproduction of O. viridis (21-24) and O. geminata (25-27). Four·celled colony splits into two·celled colony (21). Each cell reproduces by longitudinal bipartition (22-24), resulting in a four-celled colony. Two-celled colony splits into single cells (25-26) and each of them divides by longitudinal bipartition after the chloroplast duplicates (27). Arrow heads indicate eyespots produced de novo. 21-24: X 1,750; 25-27: X 2,300.
Geographic distribution: Known at present from the type locality and in Japan (Teshio, Hokkaido; Kesen-numa, Miyagi Pref.; Itsu-ura, Ibaraki Pref.; Ito, Ko-ura and Heta, Shizuoka Pref.; Awaji-shima, Hyogo Pref.; Aoshima, Miyazaki Pref.: Nagasaki-hana, Kagoshima Pref.). Probably widely distributed in the temperate regions.
Oltmannsiellopsis geminata INOUYE et
CHIHARA
sp. nov.
Plant a colonialis; colonia plerumque bicellularis; cellula semicordata a let ere visus, 10.5-15.0 flm longa, 5.0-9.0 flm lata, elliptica vel obovata a let ere dorsale visus. Plants colonial, usually consisting of two cells, cordate when viewed from broad side; cells somewhat depressed, semicordate when viewed from broad side, 10.5-15.0 flm long, 5.0-9.0 flm wide, broader anteriorly, narrower posteriorly, with somewhat rounded apex and rounded end, elliptical or obovata when viewed from narrow side,
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A
B Fig. 28. Schematic drawings showing asexual reproduction of O. viridis (A) and O. geminata (B).
with somewhat rounded apex and rounded end; chloroplast markedly asymmetrical, restricted to the lateral and basal parts of the cell, lying close to the dorsal lateral wall; flagella arising from the bottom of a caldera-shaped depression near the ventral shoulder of the cell. Holotype: Figure 3. Type locality: Heta, Shizuoka Pref., Japan. Habitat: Coastal waters. Geographical distribution: Type locality and Itsu-ura, Ibaraki Pref.
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Figs. 29-30. Transmission electron microscopy of Oltmannsiellop8'is geminata. Two longitudinal sections of the cell cut through different planes, one through lateral (29) and the other through dorsiventral plane (30). Major cell organelles are seen in both micrographs. Arrow head in Fig. 30 shows a caldera-shaped depression located in the subapical portion of the ventral side of the cell. Abbreviations: bb - basal bodies; F - flagellum; M - microbody; Mb - muciferous body N - nucleus; P - pyrenoid; Rb - refractive body. 29: X 9,700; 30: X 10,000.
Oltmannsiellopsis geminata has fundamentally the same cell structure as O. viridis (most of major cell organelles indistinguishable from those of O. viridis can be seen in Fig. 29). It differs ,from that species, however, in forming two-celled rather than four-celled colonies (Figs. 7 and 8) and in having markedly asymmetrical cells. While the colony is symmetrical, each cell shows dorsiventral asymmetry (Figs. 12 and 30). For convenience of description, the coherent side of each cell is designated ventral, while the free side is designated dorsal. A caldera-shaped depression is located subapically on the ventral side of the cell (Figs. 2, 12 and 30), and from there four flagella arise in the same way as in O. viridis (Fig. 13). The chloroplast, which is dorsilaterally stiuated (Fig. 30), possesses an eyespot in the anterior half of the dorsal side (Fig. 7). A pyrenoid is situated somewhat toward the dorsal side in the basal portion of the
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Figs. 31-34. Transmission electron microscopy of Oltmannsiellopsis unicellularis. Longitudinal (31) and transverse (32) section of the cell. Longitudinal (33) and transverse (34) section of the anterior region of the cell showing similar appearance to that of O. viridi8. 31: X 12,000; 32: X 6,000; 33: X 25,000; 34: x 17,000.
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chloroplast (Figs. 29 and 30). Asexual reproduction is similar to that in O. viridis. The first sign of cell division observable with the light microscope is the separation of the colony into two cells (Figs. 25-26 and 28B). Separated cells tend to become symmetrical externally and internally (Fig. 26). The chloroplast becomes cup-shaped and divides entirely before longitudinal cell cleavage proceeds (Figs. 27 and 28B). Resultant cells usually remain loosely attached to form a daughter colony. These processes occur while the cell is swimming, as in O. viridis.
Oltmannsiellopsis unicellularis INOUYE et CHIHARA sp. nov. Plant a unicellularis; cellula aliquantum complana, obovata a latere lato visus, 9.0-14.0,um longa, 7.0-10.0,um lata, elliptic a a latere angusto visus. Plants unicellular, somewhat compressed, obovate when viewed from broad side, 9.0-14.0,um long, 7.0-10,um wide, elliptical when viewed from narrow side; chloroplast cup-shaped; pyrenoid posteriorly; eyespot in the anterior half of the cell; cells forming gelatinous mass in culture. Holotype: Figure 4. Type locality: Harima-nada, Inland Sea of Japan. Habitat: Coastal waters. Geographic distribution: Known from the type locality. Although O. um'cellulan's (Figs. 4, 9-10 and 31-34) is very different in appearance from the colonial members of the genus, it shares many characteristics with them, viz., it lacks a cell wall, possesses four flagella arranged in exactly the same pattern as in O. viridis and O. geminata (Figs. 9-10 and 31-32) and reproduces by longitudinal bipartition while swimming. Most cellular structures are fundamentally indistinguishable from those of the colonial species (Figs. 31-24). An interesting feature unique to O. unicellularis, however, is the development in culture of a gelatinous mass in which the cells swim. Recently, a very similar alga was found in coastal waters of Natal, South Africa (PIENAAR 1985). PIENAAR's alga is unicellular, lacks a cell wall, prossesses four flagella arranged in the same way as in three species described in this paper and develops a gelatinous mass in culture (PIENAAR 1985 and pers. comm.). These features coincide with those of O. unicellularis, suggesting that PIENAAR'S alga is It member of the genus Oltmannsiellopsis, although the method of cell division has not been determined. PIENAAR, however, reported the presence of diamond-shaped body scales above the cell membrane. We examined O. unicellularis carefully using materials fixed in various ways, but could not found any trace of scales. It seems likely, therefore, that PIENAAR'S alga is specifically different from O. unicellularis, but these two algae must be studied in more detail before a decision can be made.
Acknowledgements We should like to express our thanks to Professor HIDEO IWASAKI, Mie University, Dr. TAKEO HORIGUCHI, University of Natal, Pietermaritzburg, South Africa and Dr. MAKOTO WATANABE and Mr. SYOICHIRO SUDA, National Institute for Environmental Studies, for providing culture strains. We are also grateful to Dr. PAUL C. SILVA, University of California, Berkeley, U.S.A., for his reading of the manuscript.
Literature ETTL, H. (1976): Die Gattung Chlamydomonas EHRENBERG. Nova Hedwigia 49: 1-1122. - (1980): GrundriB der all~emeinen Algologie. Jena.
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EYDEX, B. (1975): Light and electron microscope study of Dunaliella primolecta BUTCHER (Volvocida). J. Protozool. 2.2: 316--344. HARGRAYES, P. E., and STEELE, R. L. (1980): Morphology and ecology of Oltmannsiella virida, sp. nov. (Chlorophyceae: Volvocales). Phycologia 19: 96-102. I,EVAIX, N., and MARAKO-LE BARON, F. (1973): Etude des effets d'un insecticide organochlore, Ie lindane, sur la croissance et la structure de deux algues unicellularis, l'Euglena gracilis Z. et la Dunaliella bioculata. C. R. Acad. Sci. Paris 2.76: 37-40. LOEBLICH, A. R. III. (1975): A seawater medium for dinoflagellates and the nutrition of Cachonina niei. J. Phycol. 11: 80-86. OKAICHI, T., NISHIO, S., and IMATOMI, Y. (1982): Collection and mass culture. In: Jpn. Soc. Sci. Fish. (ed.): Toxic phytoplankton - Occurrence, mode of action and toxin. KoseishaKoseikaku, Tokyo: 23-34. P ASCHER, A. (1943): Beitrage zur Morphologie der ungeschlechtlichen und geschlechtlichen Vermehrung der Gattung Chlamydomonas. Beih. Bot. Centralbl. 62./A: 197-220. PIEKAAR, R. N. (1985): An unusual scale bearing flagellate resembling the genus Oltmannsiella (Chlorophyceae). Abstract. Second International Phycological Congress, Copenhagen 4-10 August, 1985: 126. REYKOLDS, E. C. (1963): The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell BioI. 17: 208--212. SPURR, A. R. (1969): A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruc. Res. 2.6: 31-42. ZIMMERMANN, W. (1930): Neue und wenig bekannte Kleinalgen von Neapel. I-V. Zeitschr. Bot. 23: 419-428. Authors' address: Dr. MITSUO CHIHARA, Dr. ISAo INOUYE and Mr. NAOHIRO TAKAHATA, Institute of Biological Sciences, University of Tsubuka, Sakura-mura, Ibaraki-ken, 305 Japan.