Shoichiro Suda

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 16S rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204T), Planktothrix rubescens (type strain CCAP 1459/22T), Planktothrix pseudagardhii sp. nov. (type strain T1-8-4T), Planktothrix mougeotii (type strain TR1-5T), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207T), Tychonema bourrellyi (type strain CCAP 1459/11BT) and Limnothrix redekei (type strain NIVA CYA 277/1T).

A proposal for the unification of five species of the cyanobacterial genus Microcystis Kutzing ex Lemmermann 1907 under the rules of the Bacteriological Code

Genomic DNA homologies were examined from six Microcystis (cyanobacteria) strains, including five different species, Microcystis aeruginosa, Microcystis ichthyoblabe, Microcystis novacekii, Microcystis viridis and Microcystis wesenbergii. All DNA-DNA reassociation values between two strains of M. aeruginosa and the other four species exceeded 70%, which is considered high enough for them to be classified within the same bacterial species. It is proposed to unify these five species into M. aeruginosa under the Rules of the Bacteriological Code and NIES843T (= IAM M-247T) is proposed as the type strain. Two other species, Microcystis flos-aquae and Microcystis pseudofilamentosa, should be regarded as morphological variations of this unified M. aeruginosa. The current taxonomy of cyanobacteria depends too much upon morphological characteristics and must be reviewed by means of bacteriological methods as well as traditional botanical methods.

LEPIDODINIUM VIRIDE GEN. ET SP. NOV. (GYMNODINAIALES, DINOPHYTA), A GREEN DINOFLAGELLATE WITH A CHLOROPHYLL A- AND B-CONTAINING ENDOSYMBIONT1,2

A further description of the green dinoflagellate, strain Y‐100, with a chlorophyll a‐ and b‐containing endosymbiont is given with special emphasis on the morphology of the host cell. The host dinoflagellate cell is unarmored and has a gymnodinoid overall appearance. The theca or amphiesma basically consists of the outer membrane and flattened thecal vesicles in which no thecal plates are developed. Unusual hand basket‐shaped scales cover the entire cell surface together with a layer of mucilaginous material. These findings suggest that the organism is a new member of the Gymnodiniaceae (Gymnodiniales). We propose the name Lepidodinium viride gen. et sp. nov. The ultrasturcture of the host cell is typical of the dinoflagellates; however, the organism has 1) an unusual cytoplasmic projection that may be a homologue of the peduncle, 2) a single membrane‐bounded body containing membranous sheets, closely situated next to the endosymbiont, and 3) an electron opaque network‐forming appendage surrounding the transverse flagellum. Name of these features have been found in other dinoflagellates.

A green dinoflagellate with chlorophylls A and B: morphology, fine structure of the chloroplast and chlorophyll composition

A green‐colored marine unicell has been grown in unialgal culture and its morphology, chloroplast fine structure, and chlorophyll composition investigated. The organism is typical of dinoflagellates in its shape, flagellation, nucleus, mitochondria, and trichocysts. It is similar to Gymnodinium but possesses fine body scales. Chloroplasts and two kinds of vesicles bounded by double membranes, but no organelles obviously identifiable as nuclei or mitochondria, are associated in ribosome‐dense cytoplasm separated by a double membrane from the dinophycean cytoplasm. The chloroplasts are unlike any previously reported for dinoflagellates. Each is enclosed by an envelope consisting of a double membrane. Chloroplast lamellae consist of three appressed thylakoids. Interlamellar pyrenoids are present. Pigment analysis reveals chlorophylls a and b but not chlorophyll c. It seems likely that the organism is an undescribed dinoflagellate containing an endosymbiont with chlorophylls a and b and that the reduction of the endosymbiont nucleus and mitochondria has permitted a more initmate symbiosis.

Genetic diversity and distribution of the ciguatera-causing dinoflagellate Gambierdiscus spp. (Dinophyceae) in coastal areas of Japan.

Background The marine epiphytic dinoflagellate genus Gambierdiscus produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet. Methods/Principal Finding A total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006–2011. The SSU rDNA, the LSU rDNA D8–D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities. Conclusions/Significance This study revealed a surprising diversity of Japanese Gambierdiscus and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8–D10 as genetic markers are recommended for further use.

Phylogeography of Ostreopsis along West Pacific Coast, with Special Reference to a Novel Clade from Japan

Background A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. Methods/Principal Finding In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8–D10 region of the nuclear large subunit rDNA (D8–D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1–5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8–D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8–D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. Conclusions/Significance This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8–D10 marker will be suitable for these purposes.

REVISION OF THE MASTOPHOROIDEAE (CORALLINALES, RHODOPHYTA) AND POLYPHYLY IN NONGENICULATE SPECIES WIDELY DISTRIBUTED ON PACIFIC CORAL REEFS(1).

The subfamily Mastophoroideae (Corallinaceae, Rhodophyta) is characterized by species possessing nongeniculate, uniporate tetrasporangial conceptacles without apical plugs, the presence of cell fusions, and the absence of secondary pit connections. However, molecular phylogenetic studies not including the type genus Mastophora indicated that the Mastophoroideae was polyphyletic. Our molecular phylogenetic analysis of the subfamily including the type genus using DNA sequences of SSU rDNA and plastid‐encoded gene of PSII reaction center protein D1 (psbA) revealed that Mastophora formed a robust clade only with Metamastophora. The other mastophoroid genera were divided into six lineages within the family Corallinaceae. Five supported lineages—(i) Pneophyllum; (ii) Hydrolithon gardineri (Foslie) Verheij et Prud’homme, Hydrolithon onkodes (Heydr.) Penrose et Woelk., and Hydrolithon pachydermum (Foslie) J. C. Bailey, J. E. Gabel et Freshwater; (iii) Hydrolithon reinboldii (Weber Bosse et Foslie) Foslie; (iv) Spongites; and (v) Neogoniolithon—were clearly distinguished by the combination of characters including the presence or absence of palisade cells and trichocytes in large, tightly packed horizontal fields and features of tetrasporangial and spermatangial conceptacles. Therefore, we amend the Mastophoroideae to be limited to Mastophora and Metamastophora with a thin thallus with basal filaments comprised of palisade cells, tetrasporangial conceptacles formed by filaments peripheral to fertile areas, and spermatangia derived only from the floor of male conceptacles. This emendation supports Setchell’s (1943) original definition of the Mastophoroideae as having thin thalli. We also propose the establishment of three new subfamilies, Hydrolithoideae subfam. nov. including Hydrolithon, Porolithoideae subfam. nov. including the resurrected genus Porolithon, and Neogoniolithoideae subfam. nov. including Neogoniolithon. Taxonomic revisions of Pneophyllum and Spongites were not made because we did not examine their type species.

Characterization of morphospecies and strains of the genus Microcystis (Cyanobacteria) for a reconsideration of species classification

For characterization of Microcystis species and strains, cell size, growth temperature optimum, salinity tolerance, dark chemoheterotrophy, photoheterotrophy, guanine + cytosine content in DNA, total fatty acid composition and restriction fragment length polymorphism of a polymerase chain reaction product (PCR‐RFLP) of the cpcBA intergenic spacer and flanking region were examined using 24 strains of Microcystis isolated from various lakes and ponds in Japan. From the results obtained it was observed that Microcystis spp. displayed low phenotypic diversity. Cell diameters of these strains were overlapping and there was no clear correlation with morphospecies. Slight differences in growth temperature optimum and salinity tolerance were observed among all strains. No strains showed either chemoheterotrophy or photoheterotrophy. The fatty acids present were the same in different strains although the amounts were different. All the strains had a similar G + C content ranging from 39 to 43 mol%. The phonogram constructed from the PCR‐RFLP analysis showed that the species assignment for Microcystis species by morphology did not correspond with the genetic background.

Taxonomic characterization of a marine Nannochloropsis species, N. oceanica sp. nov. (Eustigmatophyceae)

Abstract Ten strains of Nannochloropsis species in the Marine Biotechnology Institute Culture Collection (MBIC), Japan, were classified as N. granulata (one strain), N. salina (one strain), N. gaditana (three strains) and N. oceanica Suda & Miyashita sp. nov. (five strains) by determining nearly complete 18S rDNA sequences. The 18S rDNA sequences of strains belonging to the same species were all identical. Rubisco (rbcL) gene sequences of the 10 strains were also determined. The substitution frequencies among the rbcL genes in these four species were more than three times higher than those among 18S rDNA, especially between N. gaditana and N. salina, where 16 substitutions in the rbcL genes were found, in contrast to one substitution in 18S rDNA. These data suggest strongly that rbcL gene sequences provide better resolution than 18S rDNA sequences for classification of Nannochloropsis species. Morphology was characterized by light microscopy (LM) of exponentially growing cells. Cells of N. gaditana and N. salina were cylindrical to oval. The majority of cells of other species were globose. Cells of N. granulata had many refractile granules and other granular inclusions, which exhibited vigorous Brownian movement. Few N. oculata had refractile granules. Some N. oceanica had refractile granules and granular inclusions that were nonmotile. LM observation of old cells and electron microscopy of cells of all growth stages revealed no morphological traits that distinguish between these species.

Phylogeny and ultrastructure of Nephroselmis and Pseudoscourfieldia (Chlorophyta), including the description of Nephroselmis anterostigmatica sp. nov. and a proposal for the Nephroselmidales ord. nov

T. Nakayama, S. Suda, M. Kawachi, and I. Inouye. 2007. Phylogeny and ultrastructure of Nephroselmis and Pseudoscourfieldia (Chlorophyta), including the description of Nephroselmis anterostigmatica sp. nov. and a proposal for the Nephroselmidales ord. nov. Phycologia 46: 680–697. DOI: 10.2216/04-25.1 A new species of Nephroselmis, Nephroselmis anterostigmatica, is described from coastal waters of the Palau Islands and the southern part of Japan. The long flagellum was stretched out at angle to the cell and showed a characteristic sigmoid line. A cup-shaped yellowish green chloroplast with two narrow sinuses included a pyrenoid with tubular invaginations of thylakoids. In contrast to other species of Nephroselmis, the eyespot was located at the anterior side of the cell. The transitional region of the flagellum possessed a single transitional plate and a stellate structure. The cell body was covered by two types of square scales (windmill/Maltese cross scales absent), small stellate scales and two types of large spiny scales. The flagellar surface was covered by inner square and outer rod-shaped scales, and ornamented with T- and Pl-hairs. Tip hairs are absent. Nephroselmis anterostigmatica is superficially similar to Nephroselmis pyriformis (N. Carter) Ettl, but a detailed comparison of morphological characters, such as chloroplast shape, position of eyespot, composition of hair scales, presence or absence of the two types of small square scales and the large spiny scales, reveals clear differences between the two species. The molecular phylogenetic analysis on the basis of small subunit ribosomal DNA (SSU rDNA) sequences showed that N. anterostigmatica was not closely related to N. pyriformis, but was allied to Nephroselmis astigmatica Inouye & Pienaar. Comparisons of the hair scales and SSU rDNA sequences confirm that the identity of strain CCMP717 is N. pyriformis, and Pseudoscourfieldia marina (Throndsen) Manton has flagella ornamented with a different type of hair scales. The morphological evolution within the genus Nephroselmis is discussed and the order Nephroselmidales is proposed.