Kensuke Ichihara

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.

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.

Comparing the low‐salinity tolerance of Ulva species distributed in different environments

The green macroalgal genus Ulva (Ulvales, Ulvophyceae, Chlorophyta) is distributed worldwide from marine to freshwater environments. Comparative analyses of hyposalinity tolerance among marine, brackish, and freshwater Ulva species were performed by fluorescein diacetate viability counts. The subtidal marine species Ulva sp., collected from a depth of 30 m, showed the poorest tolerance to low salinity. This species died in 5 practical salinity units (PSU) artificial seawater or freshwater within 1 day. Its closely related species U. linza L. (an intertidal species) and U. prolifera Müller (a brackish species) showed varying tolerances to low salinity. After 7 days of freshwater exposure, the viability of U. linza L. decreased to approximately 20%, while U. prolifera Müller showed nearly 100% viability. The freshwater species U. limnetica Ichihara et Shimada, not yet found in coastal areas, was highly viable in seawater.

Systematics of Rhizoclonium-like algae (Cladophorales, Chlorophyta) from Japanese brackish waters, based on molecular phylogenetic and morphological analyses

Ichihara K., Shimada S. and Miyaji K. 2013. Systematics of Rhizoclonium-like algae (Cladophorales, Chlorophyta) from Japanese brackish waters, based on molecular phylogenetic and morphological analyses. Phycologia 52: 398–410. DOI: 10.2216/12–102.1 Analyses of partial small subunit (SSU) and large subunit (LSU) rRNA genes of Rhizoclonium-like filamentous green algae, collected from brackish waters in Japan, revealed the existence of five genetically distinct species. Four species (Rhizoclonium spp. 1–4) were closely related to Rhizoclonium hieroglyphicum. The phylogenetic position of the Rhizoclonium-like sp. 5 remained largely unresolved, although LSU analyses suggested a relationship with Chaetomorpha ligustica. Rhizoclonium spp. 1 and 4 were identified as Rhizoclonium riparium and Rhizoclonium lubricum, respectively. Rhizoclonium spp. 2 and 3 were morphologically distinguishable from other Rhizoclonium species and minute Chaetomorpha species. Rhizoclonium sp. 2 exhibited a characteristically small L/D (cell length/cell diameter) ratio and large number of nuclei (3–7 per cell), while Rhizoclonium sp. 3 was characterized by unique spindle or band-like, densely arranged chloroplasts; therefore, we described them as new species, Rhizoclonium breve and Rhizoclonium confertum. Rhizoclonium-like sp. 5 was characterized by perforated chloroplasts and polypyramidal pyrenoids and was clearly distinct from known Rhizoclonium or Chaetomorpha species. Based on the phylogenetic position and morphological features of Rhizoclonium-like sp. 5, we described it as a new species of Chaetomorpha, Chaetomorpha tokyoensis.

cDNA cloning of a lectin‐like gene preferentially expressed in freshwater from the macroalga Ulva limnetica (Ulvales, Chlorophyta)

The macroalgal species Ulva limnetica Ichihara et Shimada was investigated to understand the molecular mechanism of its tolerance or adaptation to freshwater. We detected a 19 kDa protein by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, which accumulated in greater amounts in freshwater conditions compared with marine conditions. The band was excised and the partial amino acid sequence was determined by Edoman degradation. Based on the sequences, we isolated the corresponding cDNA by the rapid amplification of cDNA ends (RACE) technique. The constructed, full‐length cDNA was 1272 bp in length, consisting of 198 bp 5′‐non‐coding region, an open reading frame of 840 bp (279 amino acids), 233 bp 3′‐non‐coding region and poly (A) tail. The protein encoded by the cDNA showed 30% identity and 45% similarity to lectin isolated from Ulva pertusa Kjellman, and we named this gene ULL (encoding Ulva limnetica lectin‐like protein). Northern blot analysis demonstrated that the expression level of the ULL in the freshwater‐cultured sample was higher than in the seawater‐cultured sample.

ISOLATION AND TEMPORAL EXPRESSION ANALYSIS OF FRESHWATER-INDUCED GENES IN ULVA LIMNETICA (ULVALES, CHLOROPHYTA)(1).

The macroalga Ulva limnetica K. Ichihara et S. Shimada is the only known Ulva species to be distributed exclusively in freshwater and is restricted to freshwater bodies in the Ryuku archipelago. Molecular phylogenetic analysis suggests that U. limnetica originally evolved from marine forms of Ulva. The mechanisms of adaptation to freshwater in Ulva spp. are poorly understood. In this study, we isolated genes potentially involved in adaptation or tolerance to freshwater conditions in U. limnetica, using suppression subtractive hybridization between mRNAs of samples cultured in freshwater and seawater conditions. A total of 219 genes, up‐regulated by the exposure of the macroalga to freshwater, were isolated. Reverse transcription–PCR (RT–PCR) revealed 39 clones, including malate dehydrogenase, soluble starch synthase, triosephosphate isomerase, plastid ribosomal protein, DnaJ‐like protein, and chloroplast ascorbate peroxidase (APX), which were specifically or preferentially expressed in freshwater conditions. These 39 clones were also analyzed for their temporal transcriptional response to freshwater conditions. A large majority of these up‐regulated genes showed a transient peak of expression after 1–4 h, followed in the next 24 h by a decrease to a stable level (over the 7 d of the experiment). After the initial response peak, the level of expression either remained higher than in the control (long‐term response) or returned to a level similar to pretreatment level. A few genes showed a more delayed response (i.e., after several days) to freshwater exposure. Finally, we discussed the possible contributions of the freshwater‐induced genes in the acquisition of freshwater adaptation or tolerance of U. limnetica.

Taxonomic reinvestigation of Petalonia (Phaeophyceae, Ectocarpales) in southeast of Honshu, Japan, with a description of Petalonia tenuis sp. nov.

Abstract: We conducted a phylogenetic and morphological reinvestigation of Petalonia species from the Pacific coast southeast of Honshu, Japan. Phylogenetic analyses based on plastid rbcL and the nuclear ITS regions resulted in three genetically distinct species: P. binghamiae, P. fascia, and Petalonia sp. Petalonia sp. was characterized by the medullary structure showing intermediate characteristics between those of P. binghamiae and P. fascia as well as by the thinner thallus. In addition, the habitat and the seasonality of Petalonia sp. were slightly different from those of the two species. Sexual fusion of plurizoids was observed on Petalonia sp., and the morphology of the prostrate sporophytes agreed with the previous reports of P. binghamiae and P. fascia. According to the characteristics, Petalonia sp. is described as a new species, P. tenuis sp. nov.

Taxonomic reassessment of Ulva prolifera (Ulvophyceae, Chlorophyta) based on specimens from the type locality and Yellow Sea green tides

Abstract: Since 2008, the green seaweed Ulva prolifera has caused the worlds largest green tide in the Yellow Sea, China. It has subsequently attracted considerable research interest. However, species identification is an essential step for advancing this research. Based on phylogenetic analyses using molecular sequences such as internal transcribed spacer (ITS) or ribulose bisphosphate carboxylase large chain (rbcL), specimens of U. prolifera collected worldwide were separated into a European clade and the Ulva linza–procera–prolifera (LPP) complex clade that included the Chinese bloom-forming strains and Japanese brackish strains. This has resulted in considerable controversy as to the identity of U. prolifera and the bloom-forming species in the Yellow Sea. To resolve this issue, we examined populations of U. prolifera from the type locality at Lolland Island, Denmark, and globally significant sites including sites from Japan and China using morphological, developmental, molecular and crossing studies. We found that almost all the Danish strains agree with the description of the type specimen and were included in the LPP clade. They had a branched morphology in culture and an obligate asexual life history with quadriflagellate zoosporoids. We conclude that this taxon in the LPP clade is true U. prolifera. Based on culture morphology, mating compatibility and the 5S rDNA spacer sequences, the Chinese bloom-forming strains were distinct, and the new subspecies U. prolifera subsp. qingdaoensis subsp. nov. is described. Strains of the European clade showing gamete incompatibility with the sexual members of the LPP clade were assigned to the species Ulva splitiana.