Molecular phylogenetics of the fresh water sleepers Odontobutis (Gobiiformes: Odontobutidae) and its implications on biogeography of freshwater ichthyofauna of East Asia.

The genus Odontobutis is a group of freshwater fishes endemic to East Asia. Phylogenetic relationships among the Odontobutis species have never been fully tested due to incomplete taxon sampling and that molecular data have not been collected in many Odontobutis species. In the present study, we sampled 51 specimens from all known eight Odontobutis species with two outgroups (Perccottus glenii and Neodontobutis hainanensis). We collected sequence data of 4434 single-copy nuclear coding loci using gene capture and Illumina sequencing. A robust phylogeny of the Odontobutis with many individuals for each species was built, supporting the current taxonomy that all extant Odontobutis species are valid. The two species from Japan (O. hikimius + O. obscurus) formed an independent clade sister to the "continental odontobutids", whereas the species from southern China (O. sinensis + O. haifengensis) separated from the rest species of the genus. Surprisingly species from the lower reaches of the Yangtze River (O. potamophilus) was more closely related to species from the Korean Peninsula and northeastern China than to the middle reaches of the Yangtze River, such that their relationship was ((O. sinensis + O. haifengensis)(O. platycephala + (O. yaluensis + (O. potamophilus + O. interruptus)))). Divergence time among the Odontobutis was estimated using 100 most clock-like loci and three fossil calibration points. The crown group of the Odontobutis was estimated at 9.0 Ma during the late Miocene (5.6-12.7 Ma, 95% HPDs). Ancestral range of the genus was reconstructed using Reconstruct Ancestral States in Phylogenies (RASP) and BioGeoBEARS. The result suggested that the common ancestor of modern Odontobutis probably was distributed in Japan, southern China or the Korean Peninsula. A series of geographical events in East Asia since the late Miocene, such as the opening of the Japan/East Sea, rapid uplift of the Tibetan Plateau and climate change in the northern region of the Yellow River might account for diversification and current distribution pattern of the Odontobutis.

A simple SNP genotyping method reveals extreme invasions of non-native haplotypes in pale chub Opsariichthys platypus, a common cyprinid fish in Japan.

Biological invasion by non-native subspecies or populations is one of the most serious threats to ecosystems, because these species might be easily established in the introduced area and can negatively affect native populations through competition and hybridization. Pale chub Opsariichthys platypus, one of the most common fish in East Asia, exhibits clear genetic differentiation among regional populations; however, introgression and subsequent loss of genetic integrity have been occurring throughout Japan due to the artificial introduction of non-native conspecifics. In this study, we developed a simple SNP genotyping method to discriminate between native and non-native mitochondrial DNA (mtDNA) haplotypes in pale chub using real-time PCR assay. We then investigated the distribution patterns of non-native pale chub in Tokai region, located in the center of Honshu Island, Japan and developed a predictive model of the occurrence of non-natives to reveal the factors influencing their invasion. The specificity and accuracy of the genotyping method were confirmed by using samples whose haplotypes were determined previously. Extensive occurrence of non-native haplotypes in Tokai region was detected by this method. In addition, our models suggested that the presence of non-natives varied greatly depending on the river system, and was positively influenced by the impounded water areas. Our method could accurately distinguish between native and non-native haplotypes of pale chub in Japan and suggested key environmental factors associated with the presence of non-natives. This approach can greatly reduce experimental costs be a great contribution for quantitative investigation.

Biogeographical role of the Kuroshio Current in the amphibious mudskipper Periophthalmus modestus indicated by mitochondrial DNA data.

Quaternary climatic cycles have influenced marine organisms' spatial distribution and population dynamics. This study aimed to elucidate the evolutionary influences of contemporary and glacial physical barriers on the population structure, demography and colonization history of the mudskipper (Periophthalmus modestus) based on a mitochondrial gene segment (ND5) from 131 individual fish sampled in the northwestern Pacific Ocean. The current Kuroshio Current and the glacial exposure of the Taiwan Strait appeared to have restricted migration among the South China Sea, coastal East China and Japan. However, genetic homogeneity (Nm>1) also suggested contemporary larval transportation by sea circulation between the East China Sea and the South China Sea or historical dispersal along the glacial exposed shoreline among China, Japan and the Ryukyu Islands. Evolutionary signals of the strengthened East Asian Summer Monsoon in the mid-Pleistocene and regional difference in intertidal primary productions were indicated by a late-Pleistocene population expansion of P. modestus with a higher effective population size in the South China Sea than in the East China Sea. Furthermore, a potential colonization origin from the South China Sea was consistently inferred by different clues, including the populations' coalescence times, the ancestral haplotype distribution, the number of private haplotypes and species/genetic diversity.

Phylogeny, hybridization, and life history evolution of Rhinogobius gobies in Japan, inferred from multiple nuclear gene sequences.

Rhinogobius fishes (Gobiidae) are distributed widely in East and Southeast Asia, and represent the most species-rich group of freshwater gobies with diversified life histories (i.e., amphidromous, fluvial, and lentic). To reveal their phylogenetic relationships and life history evolution patterns, we sequenced six nuclear and three mitochondrial DNA (mtDNA) loci from 18 species, mainly from the mainland of Japan and the Ryukyu Archipelago. Our phylogenetic tree based on nuclear genes resolved three major clades, including several distinct subclades. The mtDNA and nuclear DNA phylogenies showed large discordance, which strongly suggested mitochondrial introgression through large-scale interspecific hybridization in these regions. On the basis of the molecular dating using geological data as calibration points, the hybridization occurred in the early to middle Pleistocene. Reconstruction of the ancestral states of life history traits based on nuclear DNA phylogeny suggests that the evolutionary change from amphidromous to freshwater life, accompanied by egg size change, occurred independently in at least three lineages. One of these lineages showed two life history alterations, i.e., from amphidromous (small egg) to fluvial (large egg) to lentic (small egg). Although more inclusive analysis using species outside Japan should be further conducted, the present results suggest the importance of the life history evolution associated with high adaptability to freshwater environments in the remarkable species diversification in this group. Such life history divergences may have contributed to the development of reproductive isolation.

Population divergence of Biwia zezera (Cyprinidae: Gobioninae) and the discovery of a cryptic species, based on mitochondrial and nuclear DNA sequence analyses.

Mitochondrial and nuclear DNA analyses were used to examine the native distribution range and population structure of Biwia zezera (Cyprinidae: Gobioninae) based on specimens from the species' presumed distribution range. We found two greatly differentiated groups, with 8.6% uncorrected sequence differences in the mtDNA cytochrome b gene; one group was distributed exclusively in the Yodo River system (excluding Lake Biwa and rivers flowing into the lake). This differentiation was supported by results from three nuclear DNA loci, and it was concluded that the Yodo population is an undescribed cryptic species. Based on mtDNA haplotype distribution and endemicity, B. zezera was determined to be indigenous to the Ise Bay area (Nobi Plain), Lake Biwa basin (excluding the Yodo River outlet and its tributaries), the Sanyo region, and northern Kyushu. The population in the Ise Bay area was the most differentiated from the others, while that in Kyushu was the second-most differentiated. It was confirmed that an endemic population closely related to the Lake Biwa population is naturally distributed in the Sanyo region.

Intraspecific variation in the mitochondrial genome among local populations of Medaka Oryzias latipes.

The draft genome data of Medaka Oryzias latipes shows that it has distinct intraspecific genetic variation. To survey the genetic variations contributing to environmental adaptation, we focused on the mitochondrial DNA (mtDNA). The complete mtDNA sequences of Medaka were compared among 8 local population stocks and 4 inbred strains established from genetically divergent groups. Inbred strain HSOK, derived from the Eastern Korean group of Medaka, has a mitochondrial gene order that was distinct from other Medaka groups. Phylogenetic trees based on the mitochondrial genome sequences indicated that the mitogenome from the Shanghai stock (China) and HSOK strain were highly diverged from Japanese Medaka, and that the Japanese Medaka mitogenome was diverged into two groups; this result was fully consistent with those of the previous study using mtDNA-encode gene sequences. Among tRNA genes, the most divergent was the tRNA(Thr) gene as reported in humans previously. The number of tandemly repeated 11 nucleotide units in the Medaka mtDNA control region (CR) varied greatly among local populations. The number of repeats was more variable in the Northern Japanese group (10-34) than in the Southern group (7-12), while two other Oryzias species, inhabiting tropical regions, had no repeats. A comprehensive comparison between the number of repeat units and meteorological data indicated that the number of repeats correlated to the index data of a cold environment and seasonal climatic change. In cold (5 degrees C) acclimated fish, the mRNA levels varied among mitochondria coding genes. mRNA of the cytochrome oxidase subunit I gene in some local stocks was induced by cold temperature and seemed to be correlated with the number of repeated sequences in the CR. This study revealed that the repeated sequences in the mtDNA CR might function for mtDNA gene expression and that the number of tandem repeats in Medaka mtDNA is likely related to adaptation to a harsh habitat.

DNA sequences identify numerous cryptic species of the vertebrate: a lesson from the gobioid fish Schindleria.

Schindleria (Gobioidei, Schindleriidae), believed to include one of the smallest and youngest reproducing vertebrates, is broadly distributed in the Indo-Pacific Oceans, inhabiting coral reef lagoons. They are all characterized by a reduced larval-like form, such as a slender translucent and scaleless body. The three nominal species recognized in the genus to date have been distinguished by only combination of dorsal and anal fin-ray counts, and the existence of some undescribed species has been suggested in Schindleria; thus a total picture of species composition of the genus is poorly known. Towards the disclosure of diversity of Schindleria, a molecular phylogenetic analysis using partial mitochondrial 16S rRNA sequences was conducted for specimens from the Ryukyu and Ogasawara Islands, Japan. This analysis showed clearly that as many as 21 genetically distinguishable species occurred within the geographical areas. The degree of species crypticness of "S. praematura" [15.0=15 (new cryptic species + known species)/1 (known species)] is higher than the values of well-known animal examples, such as the pan-mesopelagic bristlemouth fish Cyclothone alba (5.0) and the South American skipper butterfly Astraptes fulgerator (10.0). This discovery of many cryptic species in Schindleria suggests that the use of DNA sequences is necessary for species identification of such morphologically conserved taxa. Because molecular analyses should increase the number of hitherto unnamed and pseudonymous species, especially in tropical areas, it is proposed that DNA-based designation is necessary for such taxa in order to compile the full "species lists", although there is presently no consensus for the inclusion of DNA sequencing data in the formal descriptions of new species.

Genetic differentiation of the gobies Gymnogobius castaneus and G. taranetzi in the region surrounding the sea of Japan as inferred from a mitochondrial gene genealogy.

The phylogenetic relationships between gobies of the genus Gymnogobius were analyzed using mitochondrial cytochrome b gene sequences, focusing on the species currently classified as G. taranetzi and G. castaneus that occur in Japan, South Korea, and Russia. Gobies of the two species collected at 12 localities in Japan, South Korea, and Russia formed a monophyletic clade (called the "castaneus species complex" here) with G. breunigii as the sister clade. Within the species complex, six lineages were recognized: (L1) G. castaneus from the Akigawa River, Tokyo, Japan; (L2) G. castaneus from Yuza, Yamagata, Japan; (L3) G. taranetzi from Russia and South Korea; (L4) G. castaneus from the Tonegawa River, Chiba, Japan; (L5a) G. taranetzi from Shimane, Japan; and (L5b) G. castaneus + G. taranetzi from the Japan Sea coast of northern Japan. The two local lineages of G. castaneus (L1 and L2) are highly divergent from the others. The Japanese populations of G. taranetzi have diverged from the continental G. taranetzi populations, while one mitochondrial lineage (L5b) is shared with G. castaneus of northeast Japan. Therefore, the current species G. taranetzi and G. castaneus as defined morphologically are polyphyletic, necessitating a taxonomic revision. The genetic differentiation of isolated local lineages and the evolution of taranetzi-and castaneus-type gobies have likely occurred repeatedly in brackish/freshwater habitats around the Sea of Japan. We discussed the time of divergence for these gobies based on a tree with the molecular clock assumption.

Major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial DNA sequences.

A recent preliminary study using complete mitochondrial DNA sequences from 48 species of teleosts has suggested that higher teleostean phylogenies should be reinvestigated on the basis of more intensive taxonomic sampling. As a second step towards the resolution of higher teleostean phylogenies, which have been described as the "(unresolved) bush at the top of the tree," we reanalyzed their relationships using mitogenomic data from 100 purposefully chosen species that fully represented all of the higher teleostean orders, except for the Batrachoidiformes. Unweighted and weighted maximum parsimony analyses were conducted with the data set that comprised concatenated nucleotide sequences from 12 protein-coding genes (excluding 3rd codon positions) and 21 transfer RNA (tRNA) genes (stem regions only) from each species. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high statistical values. All major, comprehensive groups above ordinal level as currently defined in higher teleosts (with the exception of the Neoteleostei and several monotypic groups), such as the Eurypterygii, Ctenosquamata, Acanthomorpha, Paracanthopterygii, Acanthopterygii, and Percomorpha, appeared to be nonmonophyletic in the present tree. Such incongruities largely resulted from differences in the placement and/or limits of the orders Ateleopodiformes, Lampridiformes, Polymixiiformes, Ophidiiformes, Lophiiformes, Beryciformes, Stephanoberyciformes, and Zeiformes, long-standing problematic taxa in systematic ichthyology. Of these, the resulting phylogenetic positions of the Ophidiiformes and Lophiiformes were totally unexpected, because, although they have consistently been considered relatively primitive groups within higher teleosts (Paracanthopterygii), they were confidently placed within a crown group of teleosts, herein called the Percomorpha. It should be noted that many unexpected, but highly supported relationships were found within the Percomorpha, being highly promising for the next investigative step towards resolution of this remarkably diversified group of teleosts.