Different Growth and Sporulation Responses to Temperature Gradient among Obligate Apomictic Strains of Ulva prolifera.

The green macroalga Ulva prolifera has a number of variants, some of which are asexual (independent from sexual variants). Although it has been harvested for food, the yield is decreasing. To meet market demand, developing elite cultivars is required. The present study investigated the genetic stability of asexual variants, genotype (hsp90 gene sequences) and phenotype variations across a temperature gradient (10-30 °C) in an apomictic population. Asexual variants were collected from six localities in Japan and were isolated as an unialgal strain. The hsp90 gene sequences of six strains were different and each strain included multiple distinct alleles, suggesting that the strains were diploid and heterozygous. The responses of growth and sporulation versus temperature differed among strains. Differences in thermosensitivity among strains could be interpreted as the result of evolution and processes of adaptation to site-specific environmental conditions. Although carbon content did not differ among strains and cultivation temperatures, nitrogen content tended to increase at higher temperatures and there were differences among strains. A wide variety of asexual variants stably reproducing clonally would be advantageous in selecting elite cultivars for long-term cultivation. Using asexual variants as available resources for elite cultivars provides potential support for increasing the productivity of U. prolifera.

Massive Ulva Green Tides Caused by Inhibition of Biomass Allocation to Sporulation.

The green seaweed Ulva spp. constitute major primary producers in marine coastal ecosystems. Some Ulva populations have declined in response to ocean warming, whereas others cause massive blooms as a floating form of large thalli mostly composed of uniform somatic cells even under high temperature conditions-a phenomenon called "green tide". Such differences in population responses can be attributed to the fate of cells between alternative courses, somatic cell division (vegetative growth), and sporic cell division (spore production). In the present review, I attempt to link natural population dynamics to the findings of physiological in vitro research. Consequently, it is elucidated that the inhibition of biomass allocation to sporulation is an important key property for Ulva to cause a huge green tide.

Systematics, distribution, and sexual compatibility of six Scytosiphon species (Scytosiphonaceae, Phaeophyceae) from Japan and the description of four new species.

The brown alga Scytosiphon lomentaria (Scytosiphonaceae, Ectocarpales) has been reported from cold and warm waters worldwide. Although previous molecular phylogenetic studies and crossing experiments have shown that it is a complex of multiple distinct species, the taxonomic position and accurate distribution of each species have remained largely unclear. For the S. lomentaria complex from Japan, our cox1 and rbcL phylogenetic analyses detected six species. In addition to five previously detected species (species Ia-Va), one species (species VI) was newly found in the subtropical area, Okinawa Island. Species VI was recovered as a sister to species Ia-Va in rbcL. Based on the morphological and phylogenetic data, we concluded that species Ia is S. lomentaria, species IIIa is S. promiscuus, and the other four species are newly described: S. shibazakiorum for species IIa, S. tosaensis for species IVa, S. arcanus for species Va, and S. subtropicus for species VI. The cox1-based analysis also showed that S. lomentaria, S. shibazakiorum, and S. promiscuus have worldwide distributions, while the other three species were not found outside of Japan. In Japan, except for S. subtropicus, distributions of these species overlapped. Our crossing experiments showed that gametic incompatibility (pre-zygotic barrier) was less developed between the allopatric species (i.e., S. subtropicus and others) compared with the sympatric species. It may suggest that pre-zygotic barriers have evolved among the sympatric species due to reinforcement.

Probing rapid carbon fixation in fast-growing seaweed Ulva meridionalis using stable isotope 13C-labelling.

The high growth rate of Ulva seaweeds makes it a potential algal biomass resource. In particular, Ulva meridionalis grows up to fourfold a day. Here, we demonstrated strong carbon fixation by U. meridionalis using 13C stable isotope labelling and traced the 13C flux through sugar metabolites with isotope-ratio mass spectrometry (IR-MS), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), 13C-nuclear magnetic resonance spectrometry (13C-NMR), and gas chromatography-mass spectrometry (GC-MS). U. meridionalis was first cultured in 13C-labelled enriched artificial seawater for 0-12 h, and the algae were collected every 4 h. U. meridionalis grew 1.8-fold (dry weight), and the 13C ratio reached 40% in 12 h, whereas 13C incorporation hardly occurred under darkness. At the beginning of the light period, 13C was incorporated into nucleic diphosphate (NDP) sugars in 4 h, and 13C labelled peaks were identified using FT-ICR-MS spectra. Using semiquantitative 13C-NMR measurements and GC-MS, 13C was detected in starch and matrix polysaccharides after the formation of NDP sugars. Moreover, the 14:10 light:dark regime resulted into 85% of 13C labelling was achieved after 72 h of cultivation. The rapid 13C uptake by U. meridionalis shows its strong carbon fixation capacity as a promising seaweed biomass feedstock.

Fourfold daily growth rate in multicellular marine alga Ulva meridionalis.

Microalgae with high growth rates have been considered as promising organisms to replace fossil resources with contemporary primary production as a renewable source. However, their microscopic size makes it hard to be harvested for industrial applications. In this regard, multicellular macroalgae are more suitable for harvesting. Here, we show that Ulva meridionalis has the highest growth rate ever reported for a multicellular autotrophic plant. Contrasted to the known bloom-forming species U. prolifera growing at an approximately two-fold growth rate per day in optimum conditions, U. meridionalis grows at a daily rate of over fourfold. The high growth ability of this multicellular alga would provide the most effective method for CO2 fixation and biomass production.

Asexual thalli originated from sporophytic thalli via apomeiosis in the green seaweed Ulva.

Apomixis is an asexual reproduction system without fertilization, which is an important proliferation strategy for plants and algae. Here, we report on the apomeiosis in the green seaweed Ulva prolifera, which has sexual and obligate asexual populations. Genomic PCR of mating type (MT)-locus genes revealed asexual thalli carrying both MT genomes. Observation of the chromosomes during the formation of each type of reproductive cell revealed that cells in asexual thalli performed apomeiosis without chromosome reduction. Moreover, genotyping revealed that laboratory-cultured sporophytic thalli produced not only each type of gametophyte but also diploid thalli carrying the mt- and mt+ genome (mt± thallus strains). The mt± thallus strain released diploid biflagellate zoids, with ultrastructure and behavior similar to mt+ gametes. Additionally, a transcriptomic analysis revealed that some meiosis-related genes (Mei2L and RAD1) were highly expressed in the quadriflagellate zoosporoids. Our results strongly suggest that asexual thalli originally evolved via apomeiosis in sporophytic thalli.

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae, Chlorophyta): evidence for ongoing speciation.

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm-temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata-ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS-based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.

Mitochondrial and chloroplast genome sequences of Ulva ohnoi, a green-tide-forming macroalga in the Southern coastal regions of Japan.

Ulva is a green macroalga often causing a macroalgal bloom, 'green tide'. Ulva ohnoi is a major species composing the green tide of the southern coastal regions of Japan. Here, we sequenced the complete mitochondrial and chloroplast genomes of the authentic strain of U. ohnoi. The mitochondrial and chloroplast genomes were of 65,326 bp and 103,313 bp, respectively, and the gene content was highly conserved in the Ulva species. The phylogenetic analyses using mitochondrial or chloroplast proteins represented the same topology with high supporting values. These results show that mitochondrial and chloroplast genomes can be used as reliable phylogenetic markers.

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.

Novel distribution pattern between coexisting sexual and obligate asexual variants of the true estuarine macroalga Ulva prolifera.

Where sexual and asexual forms coexist within a species, the asexuals are often found to be prevalent in marginal habitats. This asexual distribution pattern has received evolutionary attention linked to the paradox of sex. In many marine species, there is a distributional trend of asexual modes being more common in lower salinity waters regarded as the ecogeographic marginal, being explained by negative effects of low salinities on sexual reproductive success. However, the distribution pattern of estuarine species recently adapted to low salinity waters has remained unknown. The brackish macroalga Ulva prolifera being a major benthic component of estuarine ecosystems includes a sexual variant and obligate asexual variants by means of motile spores. We examined the sexual-asexual distribution pattern of this alga along a salinity gradient in river estuaries. Surprisingly, opposite to the distributional trend of marine organisms, the results clearly showed the persistent predominance of sexuals in the lower salinity reaches than the asexuals. In addition, a frequent alternating of dioecious gametophytes and sporophytes in the sexual population was observed, suggesting the sexual reproductive process would be robustly performed in the low salinity waters. Considering U. prolifera had evolved from the ancestral marine species to become a true estuarine species of which the core habitat is the low salinity reaches, in a broad sense its sexual-asexual distribution pattern would be involved in asexual marginal occupations of the species range previously reported in other organisms. Based on the frozen niche variation model, we can give a concise explanation for the evolutionary process of this pattern; multiple asexuals with frozen genotypic variation had arisen from sexual ancestors undergoing low salinity adaptation and share the estuarine habitat with the sexuals at present.

Effects of acidic functional groups on dielectric properties of sodium alginates and carrageenans in water.

This study investigated the dielectric properties of sodium alginates and carrageenans in water at frequencies between 100 MHz and 20 GHz in regard to water-hydrocolloid interactions via acidic functional groups. Both sodium alginates and carrageenans showed conduction loss at lower frequencies and dielectric loss at higher frequencies. Reduction and desulfation of sodium alginates and carrageenans, which decreased the numbers of acidic functional groups, decreased their conduction loss. In addition, H(+)-form carrageenans showed the highest ionic conduction. Correlational analysis of dielectric properties and related physical parameters showed that the loss tangent (tanδ) of the hydrocolloid solution was determined by the conductivity of the aqueous solution. Especially at pH below 2, strong H(+) conduction was associated with high tanδ probably due to the Grotthuss mechanism. The molecular dynamics of free water and H(+), viscosity conditions were also suggested to be associated with dielectric property of water-hydrocolloid system.

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.

Warming off southwestern Japan linked to distributional shifts of subtidal canopy-forming seaweeds.

To assess distributional shifts of species in response to recent warming, historical distribution records are the most requisite information. The surface seawater temperature (SST) of Kochi Prefecture, southwestern Japan on the western North Pacific, has significantly risen, being warmed by the Kuroshio Current. Past distributional records of subtidal canopy-forming seaweeds (Laminariales and Fucales) exist at about 10-year intervals from the 1970s, along with detailed SST datasets at several sites along Kochi's >700 km coastline. In order to provide a clear picture of distributional shifts of coastal marine organisms in response to warming SST, we observed the present distribution of seaweeds and analyzed the SST datasets to estimate spatiotemporal SST trends in this coastal region. We present a large increase of 0.3°C/decade in the annual mean SST of this area over the past 40 years. Furthermore, a comparison of the previous and present distributions clearly showed the contraction of temperate species' distributional ranges and expansion of tropical species' distributional ranges in the seaweeds. Although the main temperate kelp Ecklonia (Laminariales) had expanded their distribution during periods of cooler SST, they subsequently declined as the SST warmed. Notably, the warmest SST of the 1997-98 El Niño Southern Oscillation event was the most likely cause of a widespread destruction of the kelp populations; no recovery was found even in the present survey at the formerly habitable sites where warm SSTs have been maintained. Temperate Sargassum spp. (Fucales) that dominated widely in the 1970s also declined in accordance with recent warming SSTs. In contrast, the tropical species, S. ilicifolium, has gradually expanded its distribution to become the most conspicuously dominant among the present observations. Thermal gradients, mainly driven by the warming Kuroshio Current, are presented as an explanation for the successive changes in both temperate and tropical species' distributions.

Effects of Gametophytes of Ecklonia Kurome on the Levels of Glucose and Triacylglycerol in db/db, Prediabetic C57BL/6J and IFN-γ KO Mice.

We have studied edible algae that have the potential to down-regulate blood glucose. In Japan, Ecklonia species have been believed to improve the circulation of blood. In this study, we used leptin receptor deficient type 2 diabetes model mice (db/db) and prediabetic C57BL/6J mice. We also focused on the role of IFN-γ in the control of blood levels of triacylglycerol and glucose, because it is reportedly engaged in the regulation of energy consumption together with leptin. We report that gametophytes of Ecklonia kurome down-regulate the blood level of glucose and serum level of triacylglycerol in db/db. We also report that gametophytes of Ecklonia kurome down-regulate the level of glucose but not the level of triacylglycerol in prediabetic C57BL/6J mice induced by a high fat diet. They increased the level of triacylglycerol compared to that of control group in C57BL/6J, but not in IFN-γ KO mice. Gametophytes of Ecklonia kurome were administered orally to prediabetic C57BL/6J and IFN-γ KO mice and oral glucose tolerance tests were performed to evaluate the effects of algae. During the administration of the normal diet, we found a higher level of blood glucose in a glucose tolerance test of IFN-γ KO mice compared with that of C57BL/6J. Although a high fat diet induced a higher level of blood glucose compared with a normal diet group in a glucose tolerance test of C57BL/6J mice, this effect of high fat diet was not observed clearly at first but appeared three hours after glucose administration in IFN-γ KO mice. Gametophytes of Ecklonia kurome down-regulated the level of blood glucose in both C57BL/6J and IFN-γ KO mice, when administered a normal diet after making them prediabetic. These results suggest that Ecklonia kurome are effective to down-regulate the blood glucose and IFN-γ is involved in the regulation of blood glucose and triacylglycerol.

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.

A vanadium-dependent bromoperoxidase in the marine red alga Kappaphycus alvarezii (Doty) Doty displays clear substrate specificity.

Bromoperoxidase activity was initially detected in marine macroalgae belonging to the Solieriaceae family (Gigartinales, Rhodophyta), including Solieria robusta (Greville) Kylin, Eucheuma serra J. Agardh and Kappaphycus alvarezii (Doty) Doty, which are important industrial sources of the polysaccharide carrageenan. Notably, the purification of bromoperoxidase was difficult because due to the coexistence of viscoid polysaccharides. The activity of the partially purified enzyme was dependent on the vanadate ion, and displayed a distinct substrate spectrum from that of previously reported vanadium-dependent bromoperoxidases of marine macroalgae. The enzyme was specific for Br- and I- ions and inactive toward F- and Cl-. The K(m) values for Br- and H2O2 were 2.5x10(-3) M and 8.5x10(-5) M, respectively. The halogenated product, dibromoacetaldehyde, that accumulated in K. alvarezii was additionally determined.