Hidetoshi Sakayama

Taxonomic re-examination of Nitella (Charales, Charophyceae) from Japan, based on microscopical studies of oospore wall ornamentation and rbcL gene sequences

Abstract Nine taxa of Nitella (Charales, Charophyceae) from Japan, including five Japanese or east Asian endemics, were examined to improve understanding of their taxonomic status. The approaches used were light and scanning electron microscopy (SEM) of the oospores and sequencing of the gene (rbcL) encoding the large subunit of Rubisco. The species delineated were N. pulchella (= N. dualis f. pulchella), N. furcala, N. inversa (= N. furcata f. inversa), N. gracilens (= N. furcata f. gracilens), N. pseudoflabellata, N. hyalina, N. spieiformis (= N. gracilis f. spiciformis), N. moriokae (= N. rigida f. moriokae) and N. axillaris (= N. translucens var. axillaris). SEM observations showed that the oospore morphology of four taxa – N. inversa, N. gracilens, N. spiciformis and N. axillaris – was distinctly different from that of the species (N. furcata, N. furcata, N. gracilis and N. translucens, respectively) to which R.D. Wood assigned them as infraspecific taxa. Furthermore, the rbcL data showed that N. gracilens was separated phylogenetically from N. furcata and N. axillaris from N. translucens. This is the first integration of SEM oospore morphology and molecular phylogenetics in charalean taxonomy, demonstrating the efficiency of both approaches to address problems at lower taxonomic levels.

Diversification of a Transcription Factor Family Led to the Evolution of Antagonistically Acting Genetic Regulators of Root Hair Growth

Summary Streptophytes colonized the land some time before 470 million years ago [1, 2, 3]. The colonization coincided with an increase in morphological and cellular diversity [4, 5, 6, 7]. This increase in diversity is correlated with a proliferation in transcription factors encoded in genomes [8, 9, 10]. This suggests that gene duplication and subsequent diversification of function was instrumental in the generation of land plant diversity. Here, we investigate the diversification of the streptophyte-specific Lotus japonicus ROOTHAIRLESS LIKE (LRL) transcription factor (TF) [11, 12] subfamily of basic loop helix (bHLH) proteins by comparing gene function in early divergent and derived land plant species. We report that the single Marchantia polymorpha LRL gene acts as a general growth regulator required for rhizoid development, a function that has been partially conserved throughout multicellular streptophytes. In contrast, the five relatively derived Arabidopsis thaliana LRL genes comprise two antagonistically acting groups of differentially expressed genes. The diversification of LRL genes accompanied the evolution of an antagonistic regulatory element controlling root hair development.

Taxonomic re-examination of six species of Nitella (Charales, Charophyceae) from Asia, and phylogenetic relationships within the genus based onrbcL andatpB gene sequences

Abstract Six taxa of Nitella (Charales, Charophyceae), collected from Asia, were investigated using light and scanning electron microscopy (SEM) for the oospores, and sequencing of the gene encoding the large subunit of Rubisco (rbcL), in order to improve our understanding of their taxonomic status. Our SEM observations demonstrated that the oospore morphology of four taxa belonging to the subgenus Tieffa1lenia [N. megaspora (J. Groves) Sakayama, comb. nov. (= N. pseudoflabellata f. megaspora), N. tumulosa (= N. furcata f. tumulosa), N. gracillima (= N. gracilis f. gracillima) and N. axilliformis (= N. translucens f. axilliformis)] is distinctly different from that of the species N. pseudoflabellata, N. furcata, N. gracilis and N. translucens, respectively, to which R.D. Wood assigned them as infraspecific taxa. Our rbcL sequence data showed that N. megaspora is separated phylogenetically from N. pseudoflabellata, N. tumulosa from N. furcata and N. axilliformis from N. translucens. In addition, to re-examine the taxonomic system of Nitella proposed by R.D. Wood, who treated oospore wall ornamentations as diagnostic at the infraspecific level, we carried out molecular phylogenetic analyses using the combined sequence data set for the gene encoding the beta subunit of ATP synthase (atpB) and the rbcL gene of these six species, as well as eleven species of Nitella studied previously. The combined sequence data resolved fiverobust clades within the subgenus Tieffallenia that were characterized by differences in oospore wall ornamentation. However, the species and sectional diagnoses of R.D. Wood, such as the form and cell number of dactyls in vegetative thalli, were variable within the clades. Therefore, R.D. Woods taxonomic system appears unnatural, at least within the subgenus Tieffallenia.

Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae

RAB5 GTPases are important regulators of endosomal membrane traffic in yeast, plants, and animals. A specific subgroup of this family, the ARA6 group, has been described in land plants including bryophytes, lycophytes, and flowering plants. Here, we report on the isolation of an ARA6 homologue in a green alga. CaARA6 (CaRABF1) from Chara australis, a member of the Characeae that is a close relative of land plants, encodes a polypeptide of 237 aa with a calculated molecular mass of 25.4kDa, which is highly similar to ARA6 members from Arabidopsis thaliana and other land plants and has GTPase activity. When expressed in Nicotiana benthamiana leaf epidermal cells, fluorescently tagged CaARA6 labelled organelles with diameters between 0.2 and 1.2 µm, which co-localized with fluorescently tagged AtARA6 known to be present on multivesicular endosomes. Mutations in the membrane-anchoring and GTP-binding sites altered the localization of CaARA6 comparable to that of A. thaliana ARA6 (RABF1). In characean internodal cells, confocal immunofluorescence and immunogold electron microscopy with antibodies against AtARA6 and CaARA6 revealed ARA6 epitopes not only at multivesicular endosomes but also at the plasma membrane, including convoluted domains (charasomes), and at the trans-Golgi network. Our findings demonstrate that ARA6-like proteins have a more ancient origin than previously thought. They indicate further that ARA6-like proteins could have different functions in spite of the high similarity between characean algae and flowering plants.

Characterization of the Heterotrimeric G-Protein Complex and Its Regulator from the Green Alga Chara braunii Expands the Evolutionary Breadth of Plant G-Protein Signaling

The presence of a complete repertoire of heterotrimeric G-protein complex components and its regulator in a green alga confirms that the origin of this signaling mechanism in plants is more ancient than previously thought and is not related to the colonization of land by plants. The lack of heterotrimeric G-protein homologs in the sequenced genomes of green algae has led to the hypothesis that, in plants, this signaling mechanism coevolved with the embryophytic life cycle and the acquisition of terrestrial habitat. Given the large evolutionary gap that exists between the chlorophyte green algae and most basal land plants, the bryophytes, we evaluated the presence of this signaling complex in a charophyte green alga, Chara braunii, proposed to be the closest living relative of land plants. The C. braunii genome encodes for the entire G-protein complex, the Gα, Gβ, and Gγ subunits, and the REGULATOR OF G-PROTEIN SIGNALING (RGS) protein. The biochemical properties of these proteins and their cross-species functionality show that they are functional homologs of canonical G-proteins. The subunit-specific interactions between CbGα and CbGβ, CbGβ and CbGγ, and CbGα and CbRGS are also conserved, establishing the existence of functional G-protein complex-based signaling mechanisms in green algae.

Morphological variation and intraspecific phylogeny of the ubiquitous species Chara braunii (Charales, Charophyceae) in Japan

S. Kato, H. Sakayama, S. Sano, F. Kasai, M.M. Watanabe, J. Tanaka and H. Nozaki. 2008. Morphological variation and intraspecific phylogeny of the ubiquitous species Chara braunii (Charales, Charophyceae) in Japan. Phycologia 47: 191–202. DOI: 10.2216/07-27.1 Chara braunii is a charalean species that grows in a range of aquatic habitats from shallow water in paddy fields to the bottom of deep lakes. More than 30 infraspecific taxa have been proposed within this species because of its morphological variability. In order to reexamine the infraspecific taxa and habitat preference of C. braunii, we carried out molecular phylogenetic analyses and quantitative measurements of morphological characters used for infraspecific taxa, based on samples collected from various habitats and localities in Japan. Sequence data from the gene encoding the large subunit of Rubisco (rbcL) and the intergenic spacer regions between the beta subunit of the ATP synthase and rbcL genes demonstrated two robust clades (groups A and B). Samples of group A were collected mainly from shallow aquatic environments (< 15 cm in depth) such as paddy fields, while those of group B were mainly composed of samples found in deep aquatic environments (> 1 m in depth) such as lakes or ponds. Groups A and B therefore seem to represent closely related entities that have recently differentiated and might adapt to two different aquatic environments. However, the morphological measurements suggested these groups can be delineated by neither of the traditional diagnoses that were different between infraspecific taxa of Japanese plants of C. braunii.

Phylogenetic analyses of Nitella subgenus Tieffallenia (Charales, Charophyceae) using nuclear ribosomal DNA internal transcribed spacer sequences

Abstract Nucleotide sequences from the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal RNA (rRNA) gene were obtained for 13 Nitella species belonging to the subgenus Tieffallenia in order to test the chloroplast gene phylogeny previously used for taxonomic studies. The phylogenetic relationships of the 13 Nitella species based on nuclear DNA markers were congruent with those in the chloroplast gene phylogeny using the combined sequence data set for the genes encoding the beta subunit of ATP synthase (atpB) and the large subunit of Rubisco (rbcL). Therefore, the chloroplast gene phylogeny at the species level within the subgenus Tieffallenia seems to be unaffected by the maternal inheritance of chloroplast genes and can be used to obtain a natural taxonomy at the species level. This may result from the monoecious sexual reproduction that occurs in all the Nitella species examined. Phylogenetic analyses based on the concatenated chloroplast atpB and rbcL and nuclear 5.8S rRNA and ITS sequences generally gave higher resolution at or above species level within the subgenus Tieffallenia than the ITS–5.8S rRNA phylogeny or the rbcL–alpB analyses.

Morphology, molecular phylogeny and taxonomy ofNitella comptonii (Charales, Characeae)

H. Sakayama, S. Arai, H. Nozaki, F. Kasai and M.M. Watanabe. 2006. Morphology, molecular phylogeny and taxonomy of Nitella comptonii (Charales, Characeae).Phycologia 45: 417–421. DOI: 10.2216/05-31.1 The taxonomic status of Nitella comptonii (Charales, Characeae) is not well understood because this species has not been recorded since the early 20th century, and detailed examinations of fully mature individuals have not been performed. Recently, we collected fully mature N. comptonii from the Okinawa Islands in Japan. These exhibited a distinctive fertile spherical head in the axils, and their vegetative and reproductive structures were consistent with those in the original description of the species. In 1965 in his ‘A revision of the Characeae’, R. D. Wood assigned N. comptonii as a subspecies of N. pseudoflabellata. In our study, scanning electron microscopic analysis of N. comptonii oospores revealed that external and internal oospore morphology of N. comptonii was clearly different from that of N. pseudoflabellata. In addition, multiple DNA phylogeny using the concatenated sequences of the chloroplastrbcL,atpB, andpsaB genes, and nuclear 5.8S ribosomal RNA gene and internal transcribed spacer regions, clearly separated N. comptonii from N. pseudoflabellata. Therefore, N. comptonii should be classified as a separate species.

Occurrence of the endangered species Nitellopsis obtusa (Charales, Charophyceae) in western Japan and the genetic differences within and among Japanese populations

Nitellopsis obtusa (Charales, Charophyceae) are widely distributed from Europe to Asia; however, this species has been recorded in only the five lakes in central Honshu in Japan. This species was thought to be extinct in Japan, but was rediscovered in limited areas of Lake Kawaguchi in central Honshu. More recently, we discovered more Japanese populations of N. obtusa in Lake Biwa in western Honshu, and it became clear that the species had a broader distribution in Japan than originally believed. In addition, although only male or female thalli have been collected at each lake, both male and female thalli were found from Lake Biwa. This is the first report of a potentially sexual population of N. obtusa in Japan. The DNA sequences of three chloroplast DNA markers, including both coding and non‐coding regions, were identical in all specimens from Lake Kawaguchi and Lake Nojiri (Central Honshu), and differed from those of Lake Biwa and German specimens. Although Japanese and German specimens were genetically similar, Japanese specimens displayed considerable genetic diversity according to locality.

AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS 1

Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat‐based dimorphism of chloroplast DNA (cpDNA). Here, we examined the “shallow” and “deep” subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima’s D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both “shallow” and “deep” haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat‐based dimorphism of cpDNA from field‐collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes.