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.

NBRP databases: databases of biological resources in Japan.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.

Recombination, cryptic clades and neutral molecular divergence of the microcystin synthetase (mcy) genes of toxic cyanobacterium Microcystis aeruginosa.

BACKGROUND: The water-bloom-forming cyanobacterium Microcystis aeruginosa is a known producer of various kinds of toxic and bioactive chemicals. Of these, hepatotoxic cyclic heptapeptides microcystins have been studied most intensively due to increasing concerns for human health risks and environmental damage. More than 70 variants of microcystins are known, and a single microcystin synthetase (mcy) gene cluster consisting of 10 genes (mcyA to mcyJ) has been identified to be responsible for the production of all known variants of microcystins. Our previous multilocus sequence typing (MLST) analysis of the seven housekeeping genes indicated that microcystin-producing strains of M. aeruginosa are classified into two phylogenetic groups. RESULTS: To investigate whether the mcy genes are genetically structured similarly as in MLST analysis of the housekeeping genes and to identify the evolutionary forces responsible for the genetic divergence of these genes, we used 118 mcy-positive isolates to perform phylogenetic and population genetic analyses of mcy genes based on three mcy loci within the mcy gene cluster (mcyD, mcyG, and mcyJ), none of which is involved in the production of different microcystin variants. Both individual phylogenetic analysis and multilocus genealogical analysis of the mcy genes divided our isolates into two clades, consistent with the MLST phylogeny based on seven housekeeping loci. No shared characteristics within each clade are known, and microcystin analyses did not identify any compositional trend specific to each clade. Statistical analyses for recombination indicated that recombination among the mcy genes is much more frequent within clades than between, suggesting that recombination has been an important force maintaining the cryptic divergence of mcy genes. On the other hand, a series of statistical tests provided no strong evidence for selection to explain the deep divergence of the mcy genes. Furthermore, analysis of molecular variance (AMOVA) indicated a low level of geographic structuring in the genetic diversity of mcy. CONCLUSION: Our phylogenetic analyses suggest that the mcy genes of M. aeruginosa are subdivided into two cryptic clades, consistent with the phylogeny determined by MLST. Population genetic analyses suggest that these two clades have primarily been maintained as a result of homology-dependent recombination and neutral genetic drift.

Fine-scale spatial and temporal genetic differentiation of water bloom-forming cyanobacterium Microcystis aeruginosa: revealed by multilocus sequence typing.

Accumulating evidence suggests that physical isolation can potentially make a significant contribution to microbial population structure and incipient speciation. However, the effect of geographic factors on population structure has not been explicitly studied for ubiquitous cyanobacteria such as a water bloom-forming Microcystis aeruginosa. To investigate whether a fine-scale geographic structure is developed within M. aeruginosa, 96 isolates from five Japanese lakes separated by < 55 km were analysed using multilocus sequence typing. The results of multilocus sequence typing analyses indicated that M. aeruginosa was not phylogeographically structured, although a high level of genetic differentiation was observed among locations (FST = 0.372). Most surprisingly, the highest levels of genetic differentiation were observed between populations at the same location at different times of the same year, and between those separated by only 3 km on the same day. The isolation-by-distance pattern was not supported, but not completely ruled out. Taken together, our results suggest that geographic factors, if present, have far less impact on the fine-scale spatial genetic diversity of M. aeruginosa than local genetic drift or, possibly, selection.

TAXONOMIC REEXAMINATION OF CHARA GLOBULARIS (CHARALES, CHAROPHYCEAE) FROM JAPAN BASED ON OOSPORE MORPHOLOGY AND rbcL GENE SEQUENCES, AND THE DESCRIPTION OF C. LEPTOSPORA SP. NOV.(1).

Chara globularis Thuillier (=f. globularis sensu R. D. Wood) is a widespread species of the genus and inhabits fresh- and brackish-water environments. In an attempt to reexamine the taxonomic status of C. globularis collected from Japan, we reassessed vegetative and oospore morphology of Japanese material and herbarium specimens originating from Europe (including the type specimen) and conducted molecular phylogenetic analyses based on rbcL gene sequences. Although the other vegetative morphologies were consistent with the description of C. globularis f. globularis sensu R. D. Wood, we identified two types of branchlets within the Japanese materials: one has elongate end segments (EL type), and the other has short end segments (SH type) corresponding to the type material. Moreover, the oospore wall of the EL type was different from that in the SH type. The oospores of the EL type were dark brown to reddish brown and had a spongy pattern with the pusticular elevations on the fossa wall, whereas the fossa wall of the SH type was black with a granulate to papillate or fine pusticular pattern. In addition, our sequence data demonstrated that these two types are separated phylogenetically from each other. Therefore, we describe the EL type as a new species, C. leptospora sp. nov.

Isolation and absolute configuration determination of aliphatic sulfates as the Daphnia kairomones inducing morphological defense of a phytoplankton-part 2.

4,8-Dimethylnonyl sulfate (1) and 3-methyl-4E-decenyl sulfate (2) were isolated from Daphnia pulex as the Daphnia kairomones that induced morphological defense of a freshwater phytoplankton Scenedesmus gutwinskii var. heterospina (NIES-802). The absolute configuration at C4 of 1 was determined by Ohrui's method applied to alcohol 3. The absolute stereochemistry at C3 of 2 was determined by (1)H-NMR analysis of the (R)-1NMA ester of alcohol 11.

Isolation of new aliphatic sulfates and sulfamate as the Daphnia Kairomones inducing morphological change of a phytoplankton Scenedesmus gutwinskii.

New aliphatic sulfates and sulfamates were isolated from Daphnia pulex as the Daphnia kairomones that induced morphological defense of a freshwater phytoplankton Scenedesmus gutwinskii var. heterospina (NIES-802). Their structures were determined by spectroscopic and synthetic studies.

Multilocus sequence typing (MLST) reveals high genetic diversity and clonal population structure of the toxic cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa is one of the most prevalent bloom-forming cyanobacteria and has been the cause of increasing public health concern due to the production of hepatotoxins (microcystins). To investigate the genetic diversity, clonality and evolutionary genetic background with regard to the toxicity of M. aeruginosa, a multilocus sequence typing (MLST) scheme was developed, based on seven selected housekeeping loci (ftsZ, glnA, gltX, gyrB, pgi, recA and tpi). Analysis of a collection of 164 isolates from Japan and other countries identified 79 unique sequence types (STs), revealing a high level of genetic diversity (H=0.951). Although recombination between loci was indicated to be substantial by Shimodaira-Hasegawa (SH) tests, multilocus linkage disequilibrium analyses indicated that recombination between strains probably occurs at some frequency but not to the extent at which alleles are associated randomly, suggesting that the population structure of M. aeruginosa is clonal. Analysis of subsets of strains also indicated that the clonal population structure is maintained even in a local population. Phylogenetic analysis based on the concatenated sequences of seven MLST loci demonstrated that microcystin-producing genotypes are not monophyletic, providing further evidence for the gain and loss of toxicity during the intraspecific diversification of M. aeruginosa. However, toxic strains are genetically distinct from non-toxic strains in MLST allelic profiles, and it was also shown that non-toxic strains harbouring toxin genes fall into a single monophyletic clade, except for one case. These results suggest that the toxicity of M. aeruginosa is relatively stable in the short term, and therefore can be unequivocally characterized by MLST. The MLST scheme established here will be of great help for future detailed population genetic studies of M. aeruginosa.

Glycosylation of bisphenol A by freshwater microalgae.

The endocrine disruptor bisphenol A (BPA, 4,4'-isopropylidenediphenol) is used to manufacture polycarbonate plastic and epoxy resin linings of food and beverage cans, and the residues from these products are then sometimes discharged into rivers and lakes in waste leachates. However, the fate of BPA in the environment has not yet been thoroughly elucidated. Considering the effect of BPA on aquatic organisms, it is important that we estimate the concentration of BPA and its metabolites in the aquatic environment, but there are few data on the metabolites of BPA. Here, we focused on freshwater microalgae as organisms that contribute to the biodegradation or biotransformation of BPA in aquatic environments. When we added BPA to cultures of eight species of freshwater microalgae, a reduction in the concentration of BPA in the culture medium was observed in all cultures. BPA was metabolized to BPA glycosides by Pseudokirchneriella subcapitata, Scenedesmus acutus, Scenedesmus quadricauda, and Coelastrum reticulatum, and these metabolites were then released into the culture medium. The metabolite from P. subcapitata, S. acutus, and C. reticulatum was identified by FAB-MS and (1)H-NMR as bisphenol A-mono-O-beta-d-glucopyranoside (BPAGlc), and another metabolite, from S. quadricauda, was identified as bisphenol A-mono-O-beta-d-galactopyranoside (BPAGal). These results demonstrate that freshwater microalgae that inhabit universal environments can metabolize BPA to its glycosides. Because BPA glycosides accumulate in plants and algae, and may be digested to BPA by beta-glycosidase in animal intestines, more attention should be given to levels of BPA glycosides in the environment to estimate the ecological impact of discharged BPA.

Complete genomic structure of the bloom-forming toxic cyanobacterium Microcystis aeruginosa NIES-843.

The nucleotide sequence of the complete genome of a cyanobacterium, Microcystis aeruginosa NIES-843, was determined. The genome of M. aeruginosa is a single, circular chromosome of 5,842,795 base pairs (bp) in length, with an average GC content of 42.3%. The chromosome comprises 6312 putative protein-encoding genes, two sets of rRNA genes, 42 tRNA genes representing 41 tRNA species, and genes for tmRNA, the B subunit of RNase P, SRP RNA, and 6Sa RNA. Forty-five percent of the putative protein-encoding sequences showed sequence similarity to genes of known function, 32% were similar to hypothetical genes, and the remaining 23% had no apparent similarity to reported genes. A total of 688 kb of the genome, equivalent to 11.8% of the entire genome, were composed of both insertion sequences and miniature inverted-repeat transposable elements. This is indicative of a plasticity of the M. aeruginosa genome, through a mechanism that involves homologous recombination mediated by repetitive DNA elements. In addition to known gene clusters related to the synthesis of microcystin and cyanopeptolin, novel gene clusters that may be involved in the synthesis and modification of toxic small polypeptides were identified. Compared with other cyanobacteria, a relatively small number of genes for two component systems and a large number of genes for restriction-modification systems were notable characteristics of the M. aeruginosa genome.

UV protection in the photosymbiotic ascidian Didemnum molle inhabiting different depths.

Didemnum molle is a colonial ascidian that harbors the prokaryotic photosymbiont Prochloron in its cloacal cavity. Colonies occur over a relatively wide bathymetric range (approximately 0-30 m), and colony color is widely variable, partly depending on depth. Colonies in shallow sites are bright white, with densely distributed spicules, and often with brown or dark gray pigmentation, while colonies in deeper sites are less pigmented, with sparsely distributed spicules. Didemnum molle colonies contain mycosporine-like amino acids (MAAs) as UV-absorbing substances. These include mycosporine-glycine, shinorine, and porphyra-334. Among colonies from 5-, 10-, 15-, and 20-m depths, the concentration of total MAAs was significantly high at 10 m and low at 20 m. Colonies at 10 m need to maintain low spicule densities to have enough photosynthetically active radiation (PAR) to maintain the photosymbionts, and they probably concentrate MAAs to block UV radiation without attenuating PAR. Because high levels of PAR cause photoinhibition of photosynthesis, spicules and pigment cells would be more effective for photoprotection in shallow water. Colonies of D. molle may adjust the light conditions for photosymbionts by combining MAAs, spicules, and pigment cells in varying amounts.

Isolation and absolute configuration determination of aliphatic sulfates as the Daphnia kairomones inducing morphological defense of a phytoplankton.

2,6-Dimethylheptyl sulfate (1) and 6-methyloctyl sulfate (3) were isolated from Daphnia pulex as the Daphnia kairomones that induced morphological defense of a freshwater phytoplankton Scenedesmus gutwinskii var. heterospina (NIES-802). The absolute stereochemistry at C2 of 1 was determined by (1)H-NMR analysis of the (R)-MTPA ester of alcohol 2. The absolute configuration at C6 of 3 was determined by Ohrui's method applied to alcohol 4.

Morphology, genetic diversity, temperature tolerance and toxicity of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) strains from Thailand and Japan.

Cylindrospermopsis raciborskii is a planktonic, nostocalean cyanobacterium, which produces an alkaloid heptatoxin, cylindrospermopsin. We performed morphological observations, 16S rDNA sequence analysis, PCR fingerprint analysis of short tandemly repeated repetitive (STRR) sequences, temperature tolerances and toxin analysis to characterize 24 strains of this toxic cyanobacterium isolated from Thailand and Japan. All strains shared common morphological traits characteristic of C. raciborskii and showed high 16S rDNA sequence similarity, forming a defined cluster together with the reference strains from Australia. In particular, some of the Thai strains shared 99.9% to 100% similarity with the Australian strains. Various combinations of STRR primers revealed different and unique DNA band patterns among strains of C. raciborskii. The phylogenetic tree revealed two main clusters of C. raciborskii strains, the Thai/Japan-Shinobazugaike cluster (cluster I) and the Japan-Gonoike cluster (cluster II). Cluster I was further divided into two subclusters, A (only Thai strains) and B (one Thai strain and the Japan-Shinobazugaike strains). Thus, the results from 16S rDNA and STRR analyses showed no clear geographical distinction between Japanese and Thai strains and between Thai and Australian strains. Thai strains were separated into adaptive and non-adaptive groups to low temperature (15 and 17.5 degrees C) and Japanese strains were composed of only low-temperature-adaptive ones. The toxin cylindrospermopsin was detected in some strains of cluster I-A and in one strain of cluster II. We conclude that C. raciborskii is a species that has recently begun to invade, and a species with different physiological strains or ecotypes in temperature tolerance; the toxin is synthesized without any relation to phylogenetic or genetic clusters and to geography.