Takeaki Hanyuda

PHYLOGENETIC RELATIONSHIPS WITHIN CLADOPHORALES (ULVOPHYCEAE, CHLOROPHYTA) INFERRED FROM 18S rRNA GENE SEQUENCES, WITH SPECIAL REFERENCE TO AEGAGROPILA LINNAEI 1

The phylogenetic position of a freshwater green alga, Aegagropila linnaei (Cladophorales, Ulvophyceae), was investigated using nuclear 18S rRNA gene sequences. This alga has usually been called Cladophora aegagropila (L.) Rabenhorst or Cladophora sauteri (Nees ex Kütz.) Kütz. Based on morphology, it was formerly classified into the section Aegagropila or into the subgenus Aegagropila, together with several marine species of the genus Cladophora. This classification is not supported by the present phylogenetic analyses in which two very distinct Cladophorales clades are recognized. Aegagropila linnaei groups together in a well‐supported clade with Cladophora sp., Pithophora sp., Chaetomorpha okamurae, Arnoldiella conchophila, Wittrockiella lyallii, and Cladophora conchopheria. Aegagropila linnaei and its closely related species share some ultrastructural and biochemical characteristics, like pyrenoid structure, carotenoid composition, and cell wall composition. Freshwater species, included in the analysis, were located in two distantly related lineages, indicating that adaptation from a marine to a freshwater habitat has happened at least twice independently in the Cladophorales.

MOLECULAR PHYLOGENY OF DISCOSPORANGIUM MESARTHROCARPUM (PHAEOPHYCEAE) WITH A REINSTATEMENT OF THE ORDER DISCOSPORANGIALES1

A molecular phylogenetic analysis of the little‐studied filamentous brown alga Discosporangium mesarthrocarpum (Meneghini) Hauck using rbcL and partial 18S rDNA sequences revealed that the species forms a monophyletic clade with Choristocarpus tenellus (Kütz.) Zanardini that is sister to all other brown algae. Although D. mesarthrocarpum has unique disk‐shaped plurilocular reproductive organs, D. mesarthrocarpum and C. tenellus share the following basic morphological features, which are considered to be plesiomorphic characters in the brown algae: (1) apical (and diffuse) growth; (2) uniseriate, subdichotomously branched filaments; (3) multiple chloroplasts per cell without pyrenoids; and (4) lack of heterotrichy and of phaeophycean hairs. The rbcL DNA sequence of an Australian D. mesarthrocarpum specimen showed considerable deviation from Mediterranean and Macaronesian specimens. Therefore, the presence of a second species in the genus is suggested; however, the taxonomic treatment of this putative species is not pursued in the present report. Regarding the higher‐ranking systematic position of D. mesarthrocarpum, reinstatement of Discosporangiaceae and Discosporangiales is proposed, and the inclusion of Choristocarpaceae in the order is also suggested. Under short‐day and long‐day culture conditions at 15°C–25°C, Mediterranean D. mesarthrocarpum exhibited a direct type of life history, with a succession of uniseriate filamentous thalli bearing characteristic disk‐shaped plurilocular zoidangia, but thalli did not survive at 10°C and below.

Reinstatement of Ectocarpus crouaniorum Thuret in Le Jolis as a third common species of Ectocarpus (Ectocarpales, Phaeophyceae) in Western Europe, and its phenology at Roscoff, Brittany

Based on morphological characters, cross‐fertility and molecular systematics, two species are currently recognized in the ubiquitous temperate brown algal genus Ectocarpus: the type species E. siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey. We studied diversity, cross‐fertility and ecology of Ectocarpus in megatidal areas in northwest France (Western Europe) and propose to reinstate a third species, E. crouaniorum Thuret in Le Jolis. Genotyping of 67 individuals from five localities, including the type locality of E. crouaniorum, using internal transcribed spacer 1 (ITS1) length as a marker, showed that the three species co‐occurred whenever the habitat was suitable. Our survey also revealed a single putative field hybrid between E. crouaniorum and E. siliculosus, and a single individual of a further Ectocarpus genotype. In laboratory experiments, E. crouaniorum was crossed with E. siliculosus and E. fasciculatus. In 12 of 13 crosses, the zygotes did not develop (postzygotic sterility); in one experiment a viable hybrid was produced after crossing a female E. crouaniorum with a male E. siliculosus, but this hybrid was unable to form meiospores. Phylogenetic analysis of five molecular markers from the nuclear, mitochondrial and plastid genomes (in total 1818 bp) confirmed genetic separation of the three species. Ecologically, E. crouaniorum was confined to high intertidal pools and run‐offs, where the gametophyte was common from spring to summer. Another characteristic was that it usually occurred as an epiphyte of up to 12 cm in length on erect thalli of Scytosiphon lomentaria. Sporophytes of E. crouaniorum were found all year long; they were <3 cm in size or microscopic and were epilithic in the same habitat. The presence of a third species of Ectocarpus in Western Europe suggests that species diversity in this genus is larger than recognized during the last 40 years.

BIOGEOGRAPHY AND TAXONOMY OF BATRACHOSPERMUM HELMINTHOSUM (BATRACHOSPERMALES, RHODOPHYTA) IN JAPAN INFERRED FROM RBCL GENE SEQUENCES1

Twenty‐seven individuals of Batrachospermum helminthosum Bory were collected from various sites in Japan, from temperate Iwate Prefecture to subtropical Okinawa Prefecture. The chloroplast‐encoded rbcL gene was sequenced from each sample. Phylogenetic analyses were performed using these and previously published sequences of 12 samples from North America. There were five haplotypes among the Japanese samples. Haplotype 1 was collected throughout a large geographical area corresponding to most of the sampling range in this study and was represented by most individuals (21 individuals). The other haplotypes were represented by one to three individuals, respectively. These distributions of haplotypes suggest the occurrence of one large metapopulation and/or recent colonization over a wide geographical area in Japan. Phylogenetic analysis showed two well‐supported clades within B. helminthosum, one clade containing four haplotypes (1–4) from Japan and the other clade comprising five North American haplotypes. Such differences between Japanese and North American haplotypes might be a result of continental drift and subsequent isolation in the Mesozoic era. The position of haplotype 5 from Okinawa in Japan is poorly resolved, but it is not closely related to the other four Japanese haplotypes. It is suggested that several taxonomic characteristics (sexuality, point of origin of carpogonium‐bearing branches, and trichogynes with or without basal knobs or branching) is not fixed genetically but is affected by environmental conditions. Based on the results of the present study, B. coerulescens Sirodot and B. elegans Sirodot should be placed in synonymy with B. helminthosum.

Carotenoid compositions of Cladophora balls (Aegagropila linnaei) and some members of the Cladophorales (Ulvophyceae, Chlorophyta): Their taxonomic and evolutionary implication

Photosynthetic pigments were analyzed by HPLC for 27 samples of the Cladophorales (Ulvophyceae, Chlorophyta). The carotenoid compositions of the examined algae were classified into three types based on the final compound of biosynthesis of the α‐carotene branch: lutein type, characterized by containing lutein as a major carotenoid and lacking loroxanthin and siphonaxanthin; loroxanthin type, characterized by containing loroxanthin and lacking siphonaxanthin; and siphonaxanthin type, characterized by containing siphonaxanthin. We constructed molecular phylogenetic tree of the species examined in the present study using 18S rRNA gene sequences and mapped the carotenoid types of the species onto the tree. The molecular phylogenetic analysis divided the Cladophorales into two major clades, clade 1 and Aegagropila‐clade (clade 2), and divided clade 1 into subclade 1‐1 and subclade 1‐2. All the examined species positioned in the Aegagropila‐clade and those of the subclade 1‐1 belonged to the loroxanthin type, whereas both lutein type and siphonaxanthin type appeared only in the subclade 1‐2. The clades delineated by molecular phylogenetic analysis were congruent with distribution of the carotenoid types, indicating that the carotenoid types are of taxonomic significance in the Cladophorales. Considering the distribution pattern of these carotenoid types and minimum state changes in the Cladophorales, we concluded that the loroxanthin type was the primitive (plesiomorphic) state and the siphonaxanthin type and lutein type appeared in the subclade 1‐2 as advanced (apomorphic) state within this order and suggested that the cladophoralean siphonaxanthin type would have been secondarily acquired.

Variability in the rbcL Introns of Caulerpalean Algae (Chlorophyta, Ulvophyceae)

rbcL gene have been reported to date. Four new cases from Caulerpales, Ulvophyceae are described here. In the genus Caulerpa, the presence of an intron was unstable even in the infraspecific taxa. Based on comprehensive comparisons of the inserted positions, lengths of introns and so on, the presence of at least three kinds of introns, which probably have independent origins, was suggested in Caulerpales.

Four new species of Padina (Dictyotales, Phaeophyceae) from the western Pacific Ocean, and reinstatement of Padina japonica

Ni-Ni-Win, Hanyuda T., Arai S., Uchimura M., Prathep A., Draisma S.G.A., Soe-Htun and Kawai H. 2010. Four new species of Padina (Dictyotales, Phaeophyceae) from the western Pacific Ocean, and reinstatement of Padina japonica. Phycologia 49: 136–153. DOI: 10.2216/09-54.1 Molecular phylogenetic analyses of Padina species collected from the western Pacific Ocean using rbcL and mitochondrial cox3 genes revealed the occurrence of four genetically distinctive clades of unknown Padina species: clade A ( = Padina okinawaensis sp. nov.) from Ryukyu Islands (Japan), Hawaii, Indonesia and Thailand, clade B ( = Padina undulata sp. nov.), clade C ( = Padina terricolor sp. nov.) and clade D ( = Padina fasciata sp. nov.) from Ryukyu Islands (Japan). Morphologically, these new taxa are all bistratose species, and different from any known species in the following morphological features: P. okinawaensis sp. nov., reniform or circular thallus of entire margin with inconspicuous hair lines on the superior surface of the thallus, and reproductive organs (oogonia and tetrasporangia) in small groups or discontinuous sori under thin flakes of calcium on the inferior surface; P. undulata sp. nov., circular or semicircular thallus with undulate margin, conspicuous hair lines on both surfaces of the thallus, oogonial and tetrasporangial sori in continuous or discontinuous lines covered with a persistent indusium, and cylindrical oogonia; P. terricolor sp. nov., grayish brown on the inferior surface of the thallus, and oogonial and tetrasporangial sori in continuous lines covered with a persistent indusium; P. fasciata sp. nov., broad white stripes on both surfaces of the thallus due to unique calcification, and oogonial and tetrasporangial sori in continuous lines, which are entirely embedded in a gelatinous layer, on the inferior surface of the thallus. In addition, the independence of Padina japonica from Padina sanctae-crucis is suggested on the basis of molecular and morphological evidence.

Three new records of Padina in Japan based on morphological and molecular markers

Three species of the brown algal genus Padina (Dictyotales), Padina melemele I.A. Abbott et Magruder, Padina moffittiana I.A. Abbott et Huisman, and Padina thivyae Doty et Newhouse, which were originally described from Hawaii, are newly reported from some of the offshore islands of Southern Japan: P. melemele collected from Okinawa (Ryukyu Islands) and Chichi‐jima (Ogasawara Islands), P. moffittiana from Okinawa I. and P. thivyae from Tanega‐shima. Identifications are based on morphological comparisons with the original descriptions and herbarium specimens of these species identified by I. A. Abbott. Some minor differences included tetrasporangial sori that were formed only on one surface of the thallus in Japanese P. melemele and P. thivyae, whereas in the Hawaiian specimens they were sometimes formed on both surfaces. Japanese P. moffittiana had entire margins, whereas the Hawaiian specimens generally had fimbriate margins. Molecular analyses using rbcL and mitochondrial cox3 genes as molecular markers confirmed the identification of P. melemele and P. moffittiana. Unfortunately the record of P. thivyae could not be confirmed because we were unable to extract DNA from this species.

MORPHOLOGY AND MOLECULAR PHYLOGENY OF AUREOPHYCUS ALEUTICUS GEN. ET SP. NOV. (LAMINARIALES, PHAEOPHYCEAE) FROM THE ALEUTIAN ISLANDS(1).

A previously unknown species of kelp was collected on Kagamil Island, Aleutian Islands. The species can be easily distinguished from any known laminarialean alga: the erect sporophytic thallus is composed of a thin lanceolate blade attaining ∼2 m in height and ∼0.50 m in width, without midrib, and the edge of the blade at the transition zone is thickened to form a V‐shape; the stipe is solid and flattened, slightly translucent, attaining ∼1 m in length; the holdfast is semidiscoidal and up to 0.15 m in diameter. Anatomically, the blade has the typical trumpet‐shaped hyphae characteristic of the Chordaceae and derived foliose laminarialean species (i.e., Alariaceae/Laminariaceae/Lessoniaceae). No hair pits or mucilaginous structures were observed on the blade or stipe. No fertile sporophytes were collected, but abundant juvenile sporophytes were observed in the field. In the molecular phylogenetic analyses using chloroplast rbcL gene, nuclear ITS1‐5.8S‐ITS2 rDNA, and mitochondria nad6 DNA sequences, the new species (Aureophycus aleuticus gen. et sp. nov.) showed a closer relationship with Alariaceae of conventional taxonomy, or the “Group 1” clade of Lane et al. (2006) including Alaria and related taxa than with other groups, although the species was not clearly included in the group. Aureophycus may be a key species in elucidating the evolution of the Alariaceae within the Laminariales. Because of the lack of information on reproductive organs and insufficient resolution of the molecular analyses, we refrain from assigning the new species to a family, but we place the new species in a new genus in the Laminariales.

Species Diversity and Seasonal Changes of Dominant Ulva Species (Ulvales, Ulvophyceae) in Mikawa Bay, Japan, Deduced from ITS2 rDNA Region Sequences

Frequent occurrences of green tides caused by Ulva species (Ulvales, Ulvophyceae) associated with eutrophication along enclosed coasts are currently causing environmental problems in coastal ecosystems. In addition, increasing intercontinental introductions of coastal marine organisms, including Ulva, are also a serious issue. However, due to the considerable morphological plasticity of this genus, the taxonomy of Ulva species based on morphological studies is problematic. Therefore, in order to elucidate the species diversity and seasonal changes of the dominant Ulva species in Mikawa Bay, central Honshu, Japan, we made seasonal collections of Ulva species at seven localities, and identified the dominant species using the ITS2 rDNA region sequences. We identified the following nine taxa as common Ulva species in the area: 1) Ulva pertusa Kjellman; 2) U. ohnoi Hiraoka et Shimada; 3) U. linza L.; 4) U. californica Wille; 5) U. flexuosa Wulfen; 6) U. fasciata Delile; 7) U. compressa L.; 8) U. armoricana Dion et al.; 9) U. scandinavica Bliding. Among the species, U. pertusa was most common and dominant from spring to summer, and U. ohnoi from autumn to winter. Ulva californica and U. scandinavica have not been reported before from Japan.