Masanori Hiraoka

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.

The induction of reproductive cell formation of Ulva pertusa Kjellman (Ulvales, Ulvophyceae)

Synchronous zooid formation in Ulva pertusa Kjellman was induced in excised disks maintained in sterilized seawater at 20°C, 12:12 h L:D cycle and fluorescent light at 100 μmol photons m 2s 1. Zooids were reieased from mature disk tissue on the morning of the second or the third day after excision. The degree of zooid formation was found to be dependent on disk size and the region of the mother thalius from which the disk tissue was excised. Zooid formation was induced in more than 90% of small disks (0.9 mm in diameter) which were taken from the margins of the Ulva thalli. When disks were incubated together with a perforated mother thalius, the disks remained sterile. The presence of maturation inhibitors in vegetative thalli is suggested.

Culture and hybridization experiments on an ulva clade including the Qingdao strain blooming in the yellow sea.

In the summer of 2008, immediately prior to the Beijing Olympics, a massive green tide of the genus Ulva covered the Qingdao coast of the Yellow Sea in China. Based on molecular analyses using the nuclear encoded rDNA internal transcribed spacer (ITS) region, the Qingdao strains dominating the green tide were reported to be included in a single phylogenetic clade, currently regarded as a single species. On the other hand, our detailed phylogenetic analyses of the clade, using a higher resolution DNA marker, suggested that two genetically separate entities could be included within the clade. However, speciation within the Ulva clade has not yet been examined. We examined the occurrence of an intricate speciation within the clade, including the Qingdao strains, via combined studies of culture, hybridization and phylogenetic analysis. The two entities separated by our phylogenetic analyses of the clade were simply distinguished as U. linza and U. prolifera morphologically by the absence or presence of branches in cultured thalli. The inclusion of sexual strains and several asexual strains were found in each taxon. Hybridizations among the sexual strains also supported the separation by a partial gamete incompatibility. The sexually reproducing Qingdao strains crossed with U. prolifera without any reproductive boundary, but a complete reproductive isolation to U. linza occurred by gamete incompatibility. The results demonstrate that the U. prolifera group includes two types of sexual strains distinguishable by crossing affinity to U. linza. Species identification within the Ulva clade requires high resolution DNA markers and/or hybridization experiments and is not possible by reliance on the ITS markers alone.

Low genetic variability of Sargassum muticum (Phaeophyceae) revealed by a global analysis of native and introduced populations.

Sargassum muticum (Yendo) Fensholt is one of the most well‐known invasive species in the world. There have, however, been few genetic investigations on both its introduced and native populations. There are also some questions about the taxonomic status of this species. This study is the first to assess the genetic diversity of S. muticum on a global scale, by utilizing one marker each from the extranuclear genomes, namely, plastidial RUBISCO and mitochondrial TrnW_I spacers, as well as the nuclear internal transcribed spacer 2 (ITS2). Based on the markers investigated, both the invasive as well as the native populations of this species appeared very homogenous, when compared with other invasive and brown macroalgae. No variation in ITS2 and RUBISCO spacer was revealed in S. muticum populations, including those from its native ranges in Asia and the introduced ranges in Europe and North America. Two TrnW_I spacer haplotypes with a fixed two‐nucleotide difference were found between the populations of eastern Japan and the other 15 populations examined. This study confirms that there is no cryptic diversity in the introduced range of this species. All the materials collected globally are indeed S. muticum. Results depicting the distribution range of the two TrnW_I spacer haplotypes also support the earlier suggestion that the source of the introduced S. muticum populations is most likely western and central Japan (Seto Inland Sea), where the germlings of S. muticum were likely to have been transported with the Pacific oysters previously introduced for farming in Canada, UK, and France in earlier years.

Different life histories of Enteromorpha prolifera (Ulvales, Chlorophyta) from four rivers on Shikoku Island, Japan

Abstract Populations of Enteromorpha prolifera collected from four rivers on Shikoku Island in Japan had three different types of life history. One type was the typical sexual life history of Enteromorpha, which is isomorphic, with biflagellate anisogametes and quadriflagellate meiospores. The other two types were asexual life histories involving biflagellate or quadriflagellate zoids. Both biflagellate and quadriflagellate asexual zoids were intermediate in size between gametes and meiospores in the sexual life history, and both were negatively phototactic. Microspectrophotometric studies demonstrated similar amounts of DNA in the nuclei of vegetative cells in thalli generated asexually from biflagellate or quadriflagellate zoids, and in vegetative cells of sporophytes (2C) in the sexual life history. Furthermore, both types of asexual zoids had the same DNA value (2C), demonstrating that the asexual zoids are produced without meiosis. The three strains with different life histories were determined to be conspecific, on the basis of morphological similarities and low sequence divergence of the nuclear ribosomal DNA internal transcribed spacer (ITS1, ITS2 and 5.8S). However, there was no correlation between ITS sequence and life history. One river contained only E. prolifera reproducing via a sexual life history. In the other three rivers, where a mixture of life histories was found, individuals with different life histories were indistinguishable on the basis of morphology and ITS sequence.

New species of freshwater Ulva, Ulva limnetica (Ulvales, Ulvophyceae) from the Ryukyu Islands, Japan

Ulva limnetica Ichihara et Shimada, sp. nov. (Ulvales, Ulvophyceae) is described from the Ryukyu Islands, Japan, and is characterized by thalli that are: (i) branched, tubular, fragile and wrinkled; (ii) up to 80 cm in height and up to 2 cm in diameter; (iii) light to yellowish green in color; and (iv) having an asexual reproduction by means of quadriflagellate swarmers. Rhizoidal cells bear tubular extensions on the outside of the cell layer in the stipe. Ulva limnetica is distinguished from species with similar thalli by chloroplast disposition, branching pattern, number of pyrenoids per cell and gross morphology. It is also distinguished by sequences of the nuclear‐encoded 18S ribosomal RNA gene, internal transcribed spacer 2 region and the plastid‐encoded large subunit of ribulose‐1,5‐bisphosphate carboxylase/oxgenase gene (rbcL). Ulva limnetica was clustered with other Ulva species in an early diverging lineage within the genus.

Microwave-assisted hydrothermal extraction of sulfated polysaccharides from Ulva spp. and Monostroma latissimum.

Microwave-assisted hydrothermal extraction was applied for production of sulfated polysaccharides from Ulva spp. and Monostroma latissimum. The maximum ulvan yields attained 40.4±3.2% (Ulva meridionalis) and 36.5±3.1% (Ulva ohnoi) within 4min of come-up time and 10min of extraction time at 160°C, respectively. The rhamnan sulfate yield from M. latissimum further attained 53.1±7.2% at 140°C. The sulfated polysaccharides were easily recovered from the extract by simple ethanol precipitation. In addition, molecular weights and viscosity of the extracted polysaccharides could be controlled by varying the extraction temperature. Dielectric measurement revealed that ionic conduction was the important parameter that affect the microwave susceptibility of algae-water mixture. The sulfated polysaccharides extracts are expected as potential feedstock for medical and food applications.

Hydrolysis of green-tide forming Ulva spp. by microwave irradiation with polyoxometalate clusters

Effectiveness of microwave irradiation was demonstrated for hydrolysis of Ulva spp. with addition of polyoxometalate (POM) clusters. Aqueous solution of POMs (2–50 mM) at 140 °C yielded 349–435 mg g−1 of neutral sugars (predominantly constituted of rhamnose) and uronic acids from Ulva spp. under an electromagnetic field. POMs were also capable of producing significantly larger amounts of reducing sugars than hydrochloric and sulfuric acids by 1.67–6.27 times with only very low amount of furfural derivatives (≤0.1%). Phosphotungstic acid (PW) was found to be the most suitable POM for hydrolysis of Ulva spp. Microwave irradiation showed higher production of sugars than external conduction heating using an induction oven by 5–10% (neutral sugars) and 24–33% (uronic acids) at the same thermal history. In addition, PW was recycled at least three times by diethylether extraction without changing its activity.

Functional group dependent dielectric properties of sulfated hydrocolloids extracted from green macroalgal biomass

Dielectric properties of aqueous solutions of sulfated hydrocolloids (ulvan and rhamnan sulfate) extracted from green macroalgal biomass were studied in a frequency range of 100 MHz-10 GHz. Counterion exchange of native hydrocolloids (mixture of Na(+), Mg(2+) and Ca(2+)) to H(+)-form showed significant increase in loss factor due to ionic conduction. On the other hand, desulfations decreased their loss factors. The results suggested that ionic conduction of H(+) has significant contribution to loss factors. Additionally, H(+)-form hydrocolloids showed significant improvement in hydration, which might also affect the dielectric property of the solution by reducing the amount of free water. The viscosity, however, did not show apparent relevance with the dielectric property.