Satoshi Shimada

Molecular phylogenetic analyses of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva

In order to elucidate the species composition of free‐floating Ulva that cause green tide in several bays in Japan, and to clarify the generic status of Ulva and Enteromorpha (Ulvales, Ulvophyceae), the nuclear encoded internal transcribed spacer (ITS) region including the 5.8S gene and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/ oxgenase (rbcL) gene sequences for 15 species were determined. Both ITS and rbcL analyses indicate that free‐floating Ulva samples are divided into four different lineages that correspond to Ulva lactuca Linnaeus, U. pertusa Kjellman, U. armoricana Dion etal. and U. fasciata Delile. These four species are distinguished by cell morphology including the arrangement of cells, the shape and size of cells and the position of chloroplasts. Molecular data also indicated that Ulva and Enteromorpha are not separated as respective monophyletic groups within a large monophyletic clade and congeneric as shown by previous molecular studies using the ITS sequences alone. This strongly suggests that these genera are congeneric and Enteromorpha should be reduced to the synonym of Ulva.

Phylogeography of the genus Ulva (Ulvophyceae, Chlorophyta), with special reference to the Japanese freshwater and brackish taxa

The nuclear-encoded ITS and associated 5.8S rDNA regions were sequenced for 72 specimens of Ulva collected from 44 rivers across Japan, including U. prolifera Müller from the Shimanto River, Kochi Prefecture, as well as 26 samples originally identified as U. linza L. from 20 coastal marine areas. Sequence data revealed that the samples fall into six distinct clades: the U. flexuosa Wulfen clade (2 samples), the Ulva linza-procera-prolifera (LPP) complex clade (75 samples), Ulva sp. 1 clade (3 samples), Ulva sp. 2 clade (7 samples), Ulva sp. 3 clade (4 samples) and Ulva sp. 4 clade (7 samples). The LPP complex contained a mixture of 26 samples collected from seashores and 49 samples obtained from rivers, including U. prolifera from the Shimanto River, and GenBank data for U. linza and U. procera Ahlner. The samples of the LPP complex differed by only 0–7 substitutions (0–1.149%). Subsequent phylogeographic analyses of the LPP complex based on the 5S rDNA spacer region revealed the presence of two further groupings: a group including 22 strictly marine littoral U. linza samples and a U. prolifera group composed of a mixture of 4 marine samples and all 49 river samples. The monophyly of all river samples indicates that adaptation to low salinity might have occurred only once in the evolutionary history of the LPP complex.

GALEIDINIIUM RUGATUM GEN. ET SP. NOV. (DINOPHYCEAE), A NEW COCCOID DINOFLAGELLATE WITH A DIATOM ENDOSYMBIONT1

A new sand‐dwelling dinoflagellate from Palau, Galeidinium rugatum Tamura et Horiguchi gen. et sp. nov., is described. The life cycle of this new alga consists of a dominant nonmotile phase and a brief motile phase. The motile cell transforms itself directly into the nonmotile cell after swimming for a short period, and cell division takes place in the nonmotile phase. The nonmotile cell possesses a dome‐like cell covering, which is wrinkled and equipped with a transverse groove on the surface. The cell has 10–20 chloroplasts and a distinct eyespot. The motile cell is Gymnodinium‐like in shape. The dinoflagellate possesses an endosymbiotic alga to which the chloroplasts belong and which is separated from the host (dinoflagellate) cytoplasm by a unit membrane. The endosymbiont cytoplasm also possesses its own eukaryotic nucleus and mitochondria. The eyespot is surrounded by triple membranes and is located in the host cytoplasm. Photosynthetic pigment analysis, using HPLC, revealed that G. rugatum possesses fucoxanthin as the principal accessory pigment instead of peridinin. The rbcL tree showed that G. rugatum is monophyletic with Durinskia baltica (Levander) Carty et Cox and Kryptoperidinium foliaceum (Stein) Lindemann and that this clade is closely related to the pennate diatom, Cylindrotheca sp. The endosymbiont of G. rugatum is therefore shown to be a diatom. Phylogenetic analysis based on small subunit rDNA sequences demonstrated that G. rugatum, D. baltica, and K. foliaceum, all of which are known to harbor an endosymbiont of diatom origin, are closely related.

A study of sexual and asexual populations of Scytosiphon lomentaria (Scytosiphonaceae, Phaeophyceae) in Hokkaido, northern Japan, using molecular markers

The presence or absence of gamete fusion in the dioecious and isogamous brown alga Scytosiphon lomentaria (Scytosiphonaceae, Phaeophyceae) was investigated for thalli collected from three localities, Oshoro, Asari and Muroran in Hokkaido, northern Japan. Both sexual and asexual thalli were found in each locality. These sexual (35 samples) and asexual thalli (15 samples) were used to establish unialgal cultures. ITS2 sequences were determined in all cultures, the rbcL-spacer-S in 14, and cox3 in 25 cultures. Thirteen haplotypes (A1–4, B, C1–4, D, E1–3) were found in ITS2 sequences (241–252u2009bp). Ten haplotypes (A1–4, B, C1–4 and D) were found in sexual samples: haplotypes A and B were found in Oshoro, C and D in Asari, and C in Muroran. Haplotypes E1–3 were found in asexual samples. Sequence divergence values (including gap information) were less than 4.03% among sexual samples (A, B, C and D types), 0.38–0.77% among asexual samples (E types) and 4.39–5.70% between sexual and asexual samples. In the mitochondrial cox3 gene region (543u2009bp), six haplotypes (K1–3, L, M and N) were obtained. Cox3-K types were found in samples of ITS2-A types, cox3-L type in ITS2-B type, cox3-M type in ITS2-C and D types, and cox3-N type in ITS2-E types. For cox3 sequences, nucleotide differences were 0.18–4.42% among sexual samples (K, L and M types), but 8.66–10.31% between sexual and asexual samples (N type). In partial rbcL (174u2009bp)-spacer (188u2009bp)-partial rbcS (90u2009bp) sequences, six haplotypes (R–W) were found although there were 1–4u2009bp nucleotide differences among these haplotypes. R, U and W types were found in sexual samples, and S, T and V types in asexual samples. Results of the sequence analyses suggest that our presumed asexual thalli are genetically different from sexual thalli and may be derived from populations that lack sexual reproduction.

A new green‐tide‐forming alga, Ulva ohnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) from Japan

Ulva ohnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) is described from southern and western Japan and is characterized by the following combination of features: (i) the large, fragile, easily torn thalli, which are 30–55 μm thick in the upper and middle regions and often have microscopic marginal teeth; (ii) the production of zoids in the upper marginal region; (iii) a regular alternation of dioecious gametophytes and a sporophyte; (iv) the production of free‐floating thalli from torn‐off attached thalli, which reproduce vegetatively by fragmentation and form green tides in summer to autumn; (v) disorderly arranged cells that are polygonal or quadrangular in the upper and middle regions; and (vi) the chloroplast covering the outer face of cell, with 1–3 pyrenoids. Ulva ohnoi differs from U. armohcana Dion et al., U. fasciata Delile, U. reticulata Forsskal, U. scandinavica Eliding and U. spiulosaOkamura et Segawa, which all possess microscopic marginal serrations, in thallus shape, cell shape or life history pattern. It is also distinguished from morphologically similar species by sequences of the nuclear encoded internal transcribed spacers and the 5.8S ribosomal RNA gene and the plastid encoded large subunit of ribulose‐l,5‐bisphosphate carboxylase/oxgenase gene. Furthermore, crossing tests demonstrate that there is a reproductive boundary between U. ohnoi and the most closely related species, U. fasciata and U. reticulata.

Reinstatement of Grateloupia subpectinata (Rhodophyta, Halymeniaceae) based on morphology and rbcL sequences

Morphological observations and molecular analyses of the north‐western Pacific species of the red algal genus Grateloupia (Halymeniaceae) indicate the presence of an entity, which is somewhat similar in gross morphology to G. asiatica Kawaguchi et Wang but is distinguished from the latter species by some morphological features. These include: (i) a somewhat fleshy texture; (ii) wider and much thicker (4.5–10 mm wide and up to 1300 μm thick) axes, of which an inner cortex consists of more (6–9) cells; (iii) generally longer (up to 17 cm), marginal and surface proliferations that are clearly constricted (terete) at bases; and (iv) much elongated, oblong auxiliary cells. Phylogenetic analysis using the ribulose‐l,5‐bisphosphate carboxylase/ oxygenase (rbcL) gene of G. asiatica and the alga in question shows them to be distantly related and strongly supports the differentiation of these two entities at the species level. Judging from the literature, this entity is actually Grateloupia subpectinata Holmes, which has been placed into synonymy under G. asiatica [as G. filicina (Lamouroux) C. Agardh] or G. prolongata J. Agardh in previous reports, and therefore the Holmes name is reinstated.

The new genus Yonagunia Kawaguchi et Masuda (Halymeniaceae, Rhodophyta), based on Y. Tenuifolia Kawaguchi et Masuda sp. nov. from southern Japan and including Y. formosana (Okamura) Kawaguchi et Masuda comb. nov. from southeast Asia

Yonagunia Kawaguchi et Masuda, gen. nov. (Halymeniaceae, Rhodophyta) is proposed to accommodate a new species, Yonagunia tenuifolia Kawaguchi et Masuda and the species currently known as Prionitis formosana (Okamura) Kawaguchi et Nguyen. Based on auxiliary cell ampullar features, Yonagunia is included in the group of genera with the simplest type of ampulla (the Grateloupia type) that comprises Dermocorynus, Grateloupia, Kintokiocolax, Phyllymenia, and Zymurgia. However, Yonagunia differs from these genera in the behavior of cells in the ampullar filaments immediately after diploidization, most cells of the primary and secondary filaments simultaneously dividing to form grape‐like clusters of small globular cells that subsequently elongate and produce involucral filaments to laxly surround the maturing carposporophyte. Yonagunia is resolved by our rbcL gene sequence analyses as one of five monophyletic clades within the Halymeniaceae (an Aeodes/Pachymenia, a Polyopes, a Carpopertis/Cryptonemia/Halymenia, a Yonagunia, and a Grateloupia clade) that is positioned as sister to the Grateloupia clade. Carpogonial branch apparatuses are identified as a potential taxonomic significance on the same level as auxiliary cell ampullae.

Asexual life history by quadriflagellate swarmers of Ulva spinulosa (Ulvales, Ulvophyceae)

This is the first report of a Ulva species reproducing asexually solely by quadriflagellate swarmers. Ulva spinulosa Okamura et Segawa specimens were collected from Ukibuchi on the Pacific coast of Kochi Prefecture, southern Japan. Quadriflagellate swarmers were released from these specimens. The swarmers showed negative phototaxis before settlement. Thalli cultured from these swarmers also released quadriflagellate swarmers in culture. Microspectrophotometric studies demonstrated equivalent DNA in nuclei of vegetative cells in thalli of U. spinulosa and in sporo‐phytes of the other Ulva species with sexual life history (U. fasciata Delile). Furthermore, the quadriflagellate swarmers of U. spinulosa had the same DNA value, demonstrating that the quadriflagellate swarmers are produced without meiosis.

Taxonomic Notes on Marine Algae from Malaysia. II. Seven Species of Rhodophyceae

Abstract Six species of marine algae are reported from Malaysia for the first time and their characteristic features are described: Hildenbrandia rubra (Sommerfelt) Meneghini (Hildenbrandiales, Hildenbrandiaceae); Grateloupia filicina (Lamouroux) C. Agardh (Cryptonemiales, Halymeniaceae); Taenioma perpusillum (J. Agardh) J. Agardh, Taenioma dotyi Hollenberg and Zellera tawallina Martens (Ceramiales, Delesseriaceae), and Amansia rhodantha (Harvey) J. Agardh (Ceramiales, Rhodomelaceae). The occurrence of Taenioma dotyi in Malaysian waters is the first report outside the Hawaiian Islands. Amansia rhodantha is newly recorded for the Pacific Ocean. The presence of Gelidiella pannosa (Feldmann) Feldmann et G. Hamel (Gelidiales, Gelidiaceae) in Malaysia is confirmed.

Two new species of Gelidium (Rhodophyta, Gelidiales), Gelidium tenuifolium and Gelidium koshikianum, from Japan

Two new marine red algae, Gelidium tenuifolium sp. nov. and Gelidium koshikianum sp. nov. (Gelidiales, Gelidiaceae) are described from Japan. Gelidium tenuifolium with large‐sized thalli (up to 30 cm tall) is distinguished from other species with such thalIi by the production of wide, flattened and thin branches (up to 2 mm wide and 60–80 urn thick), the presence of an apical depression and simple determinate branches. Gelidium koshikianum with middle‐sized thalli (5–8 cm tall) is distinguished from other species with such thalli by having wide axes (up to 2.5 mm wide) and short (2.0–3.2 mm), unbranched, second‐ and third‐order branches issuing at short intervals (0.8–1.4 mm). In phylogenetic analyses of rbcL sequences, four Gelidium species that are chiefly distributed in Japan including G. tenuifolium were clustered together with 99% bootstrap value (Japanese Gelidium‐complex clade). Gelidium linoides Kützing came to the position of the sister group to G. tenuifolium with 99% bootstrap value. There were four substitutions (0.3% divergence) between G. linoides and G. tenuifolium sequences. Gelidium koshikianum and Gelidium allanii Chapman were clustered together with 100% bootstrap value and they came to the position of the sister group to the Japanese GeIidium‐complex clade with 83% bootstrap value. There were six substitutions (0.4% divergence) between G. koshikianum and G. allanii sequences.