Cobitis takenoi (hereinafter, Tango loach), an endangered species of tetraploid spined loach, is known to inhabit only a limited area of the Noda River in Kyoto Prefecture, Japan; it coinhabits this area with the tetraploid congener Cobitis sp. BIWAE type A (hereinafter, Ohshima loach) without interbreeding. This study used field surveys to reveal the spatial distribution and habitat selectivity of Tango loach and its interspecific relationships with Ohshima loach during the non-breeding season. Tango loach seemed to prefer a muddy bottom and be more concentrated in specific areas than Ohshima loach.

Abstract Background Intestinal atresia (IA) is a congenital gut obstruction caused by the absence of gut opening. Genetic factors are assumed to be critical for the development of IA, in addition to accidental vascular insufficiency or mechanical strangulation. However, the molecular mechanism underlying IA remains poorly understood. Results In this study, to better understand such a mechanism, we isolated a mutant ofOryzias latipes(the Japanese rice fish known as medaka) generated by N-ethyl-N-nitrosourea mutagenesis, in which IA develops during embryogenesis. Positional cloning identified a nonsense mutation in themyosin phosphatase target subunit 1(mypt1) gene. Consistent with known Mypt1 function, the active form of myosin regulatory light chain (MRLC), which is essential for actomyosin contraction, and F-actin were ectopically accumulated in the intestinal epithelium of mutant embryos, whereas cell motility, proliferation and cell death were not substantially affected. Corresponding to the accumulation site of F-actin/active MRLC, the intestinal epithelium architecture was disordered. Importantly, blebbistatin, a non-muscle myosin inhibitor, attenuated the development of IA in the mutant. Conclusions Cytoskeletal contraction governed bymypt1regulates the integrity of the embryonic intestinal epithelium. This study provides new insight into our understanding of the mechanism of IA development in humans. Bullet Points Medakamypt1mutants display intestinal atresia. The level of phosphorylated myosin regulatory light chain was higher inmypt1mutant embryos than inwild-typeembryos. The levels of F-actin appeared elevated in the intestinal epithelium ofmypt1mutants. Blebbistatin, an inhibitor of non-muscle myosin II, rescued intestinal atresia inmypt1mutant embryos.

In the Naka-ikemi Wetland, a cryptic species of the dojo loach,Misgurnussp. Type I (called Type I loach herein), inhabits along with the common nominal species,Misgurnus anguillicaudatus(called Type II loach herein). In this study, the distribution patterns of two loaches were investigated by environmental DNA (eDNA) analysis and previously suggested habitat segregation between two sympatric loaches was discussed. Type I loach tended to have a limited distribution, mainly inhabiting the northern part of the wetland; Type II loach tended to be widely distributed across the central to southern parts of the wetland. Both loaches occurred sympatrically in some areas throughout the sampling periods, indicating that they did not completely segregate their habitats.

The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.

An endangered tetraploid spined loach species,Cobitis takenoi(Cypriniformes: Cobitidae; hereafter calledTangoloach) is known to inhabit only a single river in Kyoto Prefecture, Japan. SinceTangoloach was discovered recently, in 2010, and only described in 2016, its morphology, ecology, and genetics are not well studied. Another tetraploid spined loach speciesCobitissp. BIWAE type A (hereafter, calledOhshimaloach) inhabits the same river. The two loaches are reported as morphologically distinguishable from each other. Although the habitats of the two species in the river are segregated (Ohshimaloach andTangoloach inhabit the upper and lower reaches, respectively), they overlap to a small degree in the boundary area. Recently, some individuals with morphological characteristics that are intermediate between the two species were found in the overlap zone. It was suspected that hybrids between the two species were produced since breeding seasons of the two species overlapped. To investigate whether the two species produce hybrids, we performed mitochondrial and nuclear DNA analyses on the unidentifiable individuals. Eight individuals unidentifiable to the species level collected in the river between 2017 and 2018 were examined and compared with theTangoandOhshimaloach species. Using mitochondrial DNA (mtDNA) cytochromebanalysis, we found that six individuals had mtDNA types identical toTangoloach and two individuals had mtDNA types identical toOhshimaloach. Furthermore, sequencing analysis of nuclear recombination activating gene 1 (RAG-1) revealed that each species had species-specific alleles. The phylogenetic analysis indicated that alleles inTangoloach were divided into two clusters and those fromOhshimaloach formed a single cluster. There were no discrepancies in the combination between mtDNA and nuclear DNA species types within each specimen. DNA fingerprinting analysis (AFLP) showed that the species-unidentifiable individuals exhibited distinctly segregated genetic groups corresponding withTangoandOhshimaloaches. In summary, no hybrids were detected from among any unidentifiable individual examined in this study. New conventional genetic method for discriminating the two sympatric loach species developed here can be effective tool for the conservation of theTangoloach since there was no strict diagnostic morphological character between them.

Medaka (comprising two species Oryzias latipes and Oryzias sakaizumii) is a small freshwater fish that is globally used as an experimental model organism. Wild Japanese medaka populations have been declining and their genetic diversity has been affected by extensive artificial introduction of non-native populations and 'himedaka', an orange-red variety derived from O. latipes, which is a commercially popular in Japan. To detect artificially caused genetic introgression in wild medaka populations, we developed a novel and effective nuclear markers. Sequencing analysis of a single-copy nuclear DNA (scnDNA) belonging to linkage group 21 (LG21b) was performed to identify specific haplotype in O. sakaizumii, nine genetically localized groups of O. latipes, and himedaka populations. A total of 28 haplotypes were identified from the 16 tested wild populations. Of the 28 haplotypes, 27 were considered specific to O. sakaizumii and to each genetically localized group of O. latipes; thus, these haplotypes could be used as diagnostic markers to detect artificially caused genetic introgression. Eight haplotypes were confirmed in himedaka, and four were considered specific to himedaka. Our tests involved direct sequencing of the 3 '-sequence of the LG21b region as a diagnostic marker, which effectively detected artificially caused genetic introgression in wild populations. Analysis of the actual level of genetic introgression will be improved by incorporating the new scnDNA marker in combination with conventional DNA markers of medaka.

A mitochondrial cytochrome b genotype of Oryzias latipes, mitotype B15, is widely and discontinuously distributed in various localities in Japan. To re-examine the origin of mitotype B15 detected in various localities, we conducted a sequence analysis of the NADH dehydrogenase subunit 2 (ND2) gene region. Twenty-eight haplotypes were defined among mitotype B15 fish from 23 localities throughout Japan. Most of the haplotypes detected in each area are unique to the location and genetically closely related to each other, indicating that they are native haplotypes. Our results suggest that ND2 analysis is important for identifying native populations of O. latipes.

This paper proposes a novel environmental monitoring strategy, incremental environmental monitoring, that enables scientists to reveal the ecology of wild animals in the field. We applied this strategy to the habitat of endangered freshwater fish. Specifically, we designed and implemented a network-based system using distributed sensors to continuously monitor and record the habitat of endangered fish. Moreover, we developed a set of analytical tools to exploit a variety of sensor data, including environmental time-series data such as amount of dissolved oxygen, as well as underwater video capturing the interaction of fish and their environment. We also describe the current state of monitoring the behavior and habitat of endangered fish and discuss solutions for making such environmental monitoring more efficient in the field.

To determine the actual state of hybridization between two Japanese medaka species (Oryzias latipes and Oryzias sakaizumii) in their natural environment, we used nuclear DNA markers at 10 loci to analyze 215 individuals from eight wild populations in the middle reaches of the Yura River basin in Japan, where the two species are sympatric. Despite large genetic differentiation between the two species, reproductive isolation between them could not be confirmed. We also discussed the formation of the current distribution patterns of the two species and their hybridization zone in the Yura River basin.

Information about species distribution is crucial to ecological studies. Environmental DNA (eDNA) analysis has recently been used to estimate the distribution of aquatic organisms. Several analytical methods including metabarcoding and species-specific PCR are being used for eDNA analysis. However, when only a few species are targeted, metabarcoding is not cost-effective because of the wasted consumption of read due to amplification of non-target species DNA. On the other hand, speciesspecific PCR requires tests to be repeated multiple times resulting in consuming more DNA templates, and experimental consumables. Here we propose a methodological framework for simultaneously detecting a few species using real-time multiplex PCR. We developed the species-specific primer-probe sets for two species of Japanese medaka (Oryzias latipes and o. sakaizumii), and we used them in the real-time multiplex PCR. In aquarium experiment, even when the species abundances were biased, both species were simultaneously detected in all samples. In a field survey, eDNA analysis and capture survey produced consistent results in all sampling sites, including sites with low fish densities. eDNA analysis using real-time multiplex PCR can be easily applied to other aquatic organisms, enabling a more cost-effective distribution survey of multiple target organisms.

Although it has been reported that populations of the Japanese dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) belong to two distinct mitochondrial (mt)DNA (Type I and Type II), the taxonomic status of the species remains unresolved. To address this question, nuclear DNA and morphological analyses were performed on M. anguillicaudatus population in the Nakaikemi Wetland, where Type I and Type II lineages are sympatric. Results suggest the existence of a cryptic species (Type I) within the Japanese dojo loach.

We revealed the range and current status of genetic disturbances in wild medaka populations (Oryzias latipes species complex) using two DNA markers (cytb gene and b-marker). Genetic disturbances were detected in many wild populations throughout Japan and were primarily caused by artificial introduction of the commercial medaka variety, himedaka. We identified native medaka populations without introgressions, which may be significant conservation targets. To conserve the native genetic diversity of the medaka species complex, further introduction of himedaka should be prevented by educating the public about the current status and risks of introducing non-native medaka varieties into the wild.

In wild populations of the Japanese endemic freshwater fish Minami-medaka (Oryzias latipes), genetic disturbances caused by crossing with artificially introduced exotic populations, especially an orange-red commercial variety of medaka commonly called himedaka, have been reported throughout Japan. Minami-medaka populations in the Kyushu-Ryukyu Islands region include many genetically distinct groups, indicating that the region is important for the conservation of genetic diversity in Minami-medaka populations. In this study, genetic analyses using two DNA markers (one for a mitochondrial gene, the other for a nuclear gene) were conducted to reveal the current status of genetic disturbance in wild Minami-medaka populations in the Kyushu-Ryukyu Islands region. In 125 individuals from 18 populations analyzed, one individual, from the northern Kyushu Islands, was found to carry alleles originating from the himedaka strain. Based on the results of all available surveys, including those of the present study, a total of six introgressed populations are currently known in the region. All other populations are considered unaffected. The native genetic diversity of Minami-medaka populations in the Kyushu-Ryukyu Islands region is comparatively well preserved; results of surveys throughout the rest of the range of the species indicate that almost half of all Minami-medaka populations have been introgressed by non-native genes. Conservation measures and regular genetic monitoring are recommended to preserve the genetic diversity of this species.

Genetic disturbance in wild populations of medaka (Oryzias latipes complex) has been mainly caused by the introduction of the orange-red commercial variety medaka (himedaka) in Japan. To examine whether survival, reproduction, and species recognition would be influenced by this difference in body coloration, we conducted three laboratory experiments (predatory pressure, mate choice, schooling behavior) using wild type medaka and himedaka. In the predation experiment using dark chub (Candidia temminckii) as a predator, himedaka were predated upon more often than wild type medaka. However, individuals did not choose mates or select schooling groups based on himedaka or wild type medaka phenotypes. The results indicate that himedaka receive higher predation pressure but are able to easily mate with wild type medaka in a natural environment. To conserve the genetic diversity of wild medaka populations, we need to control the risk of genetic disturbance caused by himedaka. J. Exp. Zool. 323A: 349-358, 2015. (c) 2015 Wiley Periodicals, Inc.

The oriental weather loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inhabits the waters of East Asia including Japanese Islands. The Japanese population of M anguillicaudatus includes two major mitochondrial DNA (mtDNA) clades, but their evolutionary origin is unknown. In this study, we conducted phylogeographic analyses of M anguillicaudatus that were based on mtDNA cytochrome b sequences to clarify the evolutionary origin of the two distinct mtDNA clades. This newly obtained data were integrated with the mtDNA sequence data obtained in previous studies and reanalysed. The results showed that one major clade originated because of mtDNA introgression from a loach of the genus Cobitis. The geographic range of the populations carrying non-introgressed mtDNA tended to be limited to the peripheral areas of the Japanese Islands, whereas the range of the populations carrying introgressed mtDNA was spread over wide regions of the Japanese Islands. These distribution and divergence patterns suggested that M anguillicaudatus populations carrying introgressed mtDNA have spread and replaced the range of populations carrying non-introgressed mtDNA.

Genetic disturbances in wild populations of Medaka (Oryzias latipes) have resulted from the introduction of populations originating from the commercial orange-red strain (himedaka). The "himedaka" phenotype, caused by a defect in melanin deposition in skin cells, is conferred by a mutated recessive allele (b allele) on the slc45a2 locus (linkage group 12). To examine genetic introgression from "himedaka" to wild Medaka populations, a new DNA marker (b-marker) was constructed that detects fish with the b allele according to a length polymorphism of the promoter region between the wildtype (B) and b alleles. Among 169 fish from 45 wild populations in the Yamato River, Nara Prefecture, all specimens with the orange-red phenotype (from 7 populations) had a homozygous b/b genotype. Although most of the wildtype fish had a homozygous B/B genotype, 12 wildtype fish from 9 populations (3 of which consisted of fish with the orange-red phenotype) had the heterozygous genotype (B/b). These results indicated a high level of cryptic genetic introgression from "himedaka" to wild Medaka populations.

The Yoshino diversion (Yoshino Bunsui) was established to supply irrigation water from the Yoshino River to the Yamato plain. In order to examine fish introductions through the canals of the Yoshino Bunsui, the genetic structure of 17 populations of dark chub Zacco temminckii collected from the Yamato and Yoshino rivers were analyzed and their mtDNA ND2 sequences studied. A total of 16 haplotypes were detected in 261 individuals, 6 haplotypes being present in both rivers. In the Yamato River, the shared haplotypes were found to be associated with the locations of inflows from the Yoshino Bunsui. On the other hand, the majority of haplotypes found in individuals from the Yamato River were not found in those from the Yoshino River. This study confirmed the secondary introduction of dark chub from the Yoshino River into the Yamato River via the Yoshino Bunsui.

Genetic disturbance in wild populations of Medaka (Oryzias latipes) has resulted from the introduction of populations originating from commercial strains. To clarify the genetic composition of orange-red type and wild type commercial strains of O. latipes ('himedaka' and 'kuromedaka', respectively), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing analyses of the mitochondrial cytochrome b gene were conducted for Medaka populations cultured in 2 fish farms and others purchased from 21 aquarium fish shops. All 'himedaka' populations showed a high frequency of the B27 mitotype, an original mitotype of 'himedaka', and a low frequency of the B1a mitotype, an introduced mitotype from wild populations in Okayama or Nara Prefectures. 'Kuromedaka' populations, originating from cultured populations, were characterized primarily by the B27 and B1a mitotypes, being a result of hybridization between wild populations and 'himedaka'. All 'kuromedaka' populations originating from wild populations had their own native mitotypes, although some also had the B1a mitotype, which was inferred as being of 'himedaka' origin.

During the development of the vertebrate nervous system, mitosis of neural progenitor cells takes place near the lumen, the apical side of the neural tube, through a characteristic movement of nuclei known as interkinetic nuclear migration (INM). Furthermore, during the proliferative period, neural progenitor cells exhibit planar cell divisions to produce equivalent daughter cells. Here, we examine the potential role of extracellular signals in INM and planar divisions using the medaka mutant tacobo (tab). This tab mutant shows pleiotropic phenotypes, including neurogenesis, and positional cloning identified tab as laminin gamma 1 (lamc1), providing a unique framework to study the role of extracelluar signals in neurogenesis. In tab mutant neural tubes, a number of nuclei exhibit abnormal patterns of migration leading to basally mislocalized mitosis. Furthermore, the orientation of cell division near the apical surface is randomized. Probably because of these defects, neurogenesis is accelerated in the tab neural tube. Detailed analyses demonstrate that extracellular signals mediated by the FAK pathway regulate INM and planar divisions in the neuroepithelium, possibly through interaction with the intracellular dynein-motor system.

メダカは、本来北海道を除く日本各地に生息しているメダカ科の淡水魚で、かつては水田の用水路などに多く生息していたが、近年では都市化やオオクチバスなどの外来魚の影響により分布域が縮小し続けている。そのため現在では、環境省RLにおいて絶滅危惧II類に指定されている。また本種の国内集団は、大きく遺伝的に異なる北日本集団と南日本集団、さらに南日本集団は様々な地方型に分けられる。本研究では、奈良県大和川水系におけるヒメダカからの遺伝子移入の実態の解明を目的とし、奈良県大和川水系に生息するメダカ集団についてmtDNAチトクロームb領域を対象とした遺伝子解析を行ったので報告する。

日本産シマドジョウ属魚類の分類は歴史的に混乱しており未だに決着を見ていないが、形態学的分類および核型・アロザイム・DNA分析等による遺伝学的研究結果を総合すると、従来、シマドジョウ、ヤマトシマドジョウ（またはタイリクシマドジョウ）、スジシマドジョウ、イシドジョウとよばれてきた4グループに区分でき、それぞれのグループが複数の独立種を含んでいることが明らかになっている。斉藤では、池田と整合させるため、従来のヤマトシマドジョウとスジシマドジョウのグループをタイリクシマドジョウ群とまとめ、それぞれをヤマトシマドジョウ亜群、スジシマドジョウ亜群に区分している。しかし、遺伝学的研究からヤマトシマドジョウのグループはシマドジョウとスジシマドジョウのグループの個体間の交雑を起源とする異質4倍体で、このグループの名称の根拠となったヨーロッパ産のタイリクシマドジョウとは全く異なるものであること、さらにmtDNA分析によりスジシマドジョウのグループとタイリクシマドジョウが近縁関係にないことが明らかになっていることから、タイリクシマドジョウ群として両者をまとめることは不適当である。本報告では、英語表記を基本として、それぞれの和名、学名をシマドジョウ種群、スジシマドジョウ種群、ヤマトシマドジョウ種群、イシドジョウ種群とよぶ。

The distribution patterns of seven Cobitis species are analysed, namely, C. takatsuensis, C. biwae, the yamato complex, the Kyushu form of C. striata (small race), C. striata (middle race), the Hakata form of C. striata (middle race), and the Onga form of C. striata (middle race) in the northern Kyushu Island, Japan. C. takatsuensis is distributed in the upstream areas of some rivers in the northeastern part of the island. The yamato complex is widely distributed in the northern part of the island. The other five species are distributed in five areas separated by mountains. Although the yamato complex and the four striata complex species are often distributed in the same river system, the former inhabit upstream areas, while the latter inhabit the downstream reaches. The results of this study reveal that habitat fragmentation by mountains and the difference in longitudinal distribution lead to this complicated distribution pattern.

We have isolated and characterized a ventralized mutant in medaka (the Japanese killifish; Oryzias latipes), which turned out to have a mutation in the chordin gene. The mutant exhibits ventralization of the body axis, malformation of axial bones, over-bifurcation of yolk sac blood vessels, and laterality defects in internal organs. The mutant exhibits variability of phenotypes, depending on the culture temperature, from embryos with a slightly ventralized phenotype to those without any head and trunk structures. Taking advantages of these variable and severe phenotypes, we analyzed the role of Chordin-dependent tissues such as the notochord and Kupffer's vesicle (KV) in the establishment of left-right axis in fish. The results demonstrate that, in the absence of the notochord and KV, the medaka lateral plate mesoderm autonomously and bilaterally expresses spaw gene in a default state.

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1(hdf) is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1(hdf) mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1(hdf) mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the

The Japanese rosy bitterling (Rhodeus ocellatus kurumeus) is a cyprinid subspecies endemic to Japan. It is now on the verge of extinction due to hybridization with its subspecies R. o. ocellatus, introduced from mainland China. In this study, we examined 13 populations of R. ocellatus from Nara Prefecture, Japan, using morphological analysis and molecular analyses of mtDNA and microsatellites. One of the populations in Nara Park (Stn. 9) closely matched the morphological characteristics of R. o. kurumeus, while other populations more closely resembled R. o. ocellatus. The former population possessed mitotypes of R. o. kurumeus only, while the latter possessed those of both subspecies or those of R. o. ocellatus only. Notably, mitotypes of R. o. kurumeus were widely observed in the Nara Basin. In addition to the presence of mitotypes reported in an Osaka population of R. o. kurumeus, the low genetic diversity of the population at Stn. 9 was comparable to that of the Osaka R. o. kurumeus population. In a principal component analysis of microsatellites data, three clusters were recognized; 1. R. o. kurumeus (including the population at Stn. 9), 2. R. o. ocellatus and 3. their probable hybrids. These results suggest that R. o. kurumeus was once distributed widely in the middle reaches of the Yamato River, but the remaining populations have hybridized with R. o. ocellatus, except for the population at Stn. 9. Low genetic diversity in the Nara Park population is considered a result of a population bottleneck in a small isolated habitat.

The biogeography of freshwater fishes in Japan was reviewed in terms of achievements and perspectives. In the last three decades, biogeographic studies have changed from earlier descriptions of the freshwater fish fauna, based on the Linnean classification system, to phylogenetic approaches using various molecular markers. Especially, the phylogeographic approach, which explores the formation of geographic distribution patterns of genealogical lineages within species, has become predominant. Analyses of genuine freshwater fishes have disclosed their speciation and dispersal patterns throughout temperate East Asia since the Neogene, along with the formation of the Japanese Archipelago. In particular, molecular clocks of mitochondrial DNA have played an important role in examinations of biogeographic relationships between the Japanese Archipelago and Chinese continent/Korean Peninsula, and vicariance by Fossa Magna in central Honshu Island. Patterns of range expansion through the sea and landlocking in coldtemperature euryhaline fishes have indicated their speciation and distribution dynamics under the fluctuating climatic conditions of the Plio-Pleistocene. Likewise, phylogeographic implications of unusual biological entities arising from interspecific hybridization or gynogenesis have been discussed. Nevertheless, despite the emphases given to some groups, the present knowledge of phylogeographic patterns of Japanese freshwater fishes is for the most part still insufficient for quantitative analyses of the overall history of the freshwater fish fauna and geographic regions of Japan. Improved research techniques and methodologies for the integration of findings from multiple taxa and/or genes are essential. Further, evolutionary formation of distributional ranges should be considered together with ecological biogeography, including the processes of local adaptation, interspecific interaction and extinction. Modern day disturbances of freshwater fish distributions, including fish transportation, are rapidly leading to artificial distribution patterns and extinctions. Exhaustive phylogeographic analyses should be necessary as a primary requirement for conserving freshwater fish biodiversity in Japan.

The "striata complex," a group of spined loaches included in the genus Cobitis and characterized by a striped coloration pattern on the lateral midline, is distributed in rivers in northeastern Asia to western Japan. The complex comprises 2 continental species (Cobitis tetralineata and Cobitis lutheri) and 3 Japanese races of species rank (large race, middle race, and small race), the small race further comprising 6 local forms of subspecific rank (Tokai form, Biwa form, Yodo form, Sanyo form, San-in form, and Kyushu form). Previous karyological studies have revealed that the large race is an allotetraploid, the others being diploid. In this study, mitochondrial (mt) DNA analyses were conducted for 30 diploid populations of the Cobitis striata complex from Japan and Korea to examine: (1) their phylogenetic relationships and the position of the complex among the major lineages of Cobitis; and (2) the genetic relationships among the Japanese and Korean populations. The results, based on cytochrome b sequences (724 base pairs) analyzed with those of the main lineage of European and Japanese Cobitis, indicated that the striata complex should be considered as a monophyletic group, which evolved in northeastern Asia. Initially considered as a subspecies of Cobitis taenia, widely distributed from Europe to Asia, the striata complex does not have a sister-relationship with the former. Although the Korean species C tetralineata was formerly believed to be closely related to the middle race in Japan, and a second continental species, C lutheri, closely related to the Kyushu or San-in forms of the small race in Japan, the trees resulting from the present study revealed that the two Korean species were clustered with each other and separated from all Japanese races.

The original population of the Ikehara Reservoir consisted of a subspecies of the largemouth bass, Micropterus salmoides salmoides. In 1988, another subspecies, M. s. floridanus, originating from Florida was transplanted there. In 1996/97 a genetic study was made on the population in regard to their mitochondrial (mt) DNA with the result that M. s. floridanus was detected from 56.8% of fish. We re-examined the population in 2003 and found that fish having the mtDNA originating from M. s. floridanus accounted for a significantly higher percentage than before (86.7%). Moreover, fish having the mtDNA originating from M. s. floridanus were found from other lakes and ponds in the Kinki District.

The distribution range of the Japanese spined loach Cobitis biwae is in the middle stretches of rivers in most of Honshu and Shikoku Islands. In Kyushu Island, its close relative, the yamato complex sensu Saitoh et al.(2000), is distributed, but C. biwae is thought to be absent there. Comparison of the morphological characters and erythrocytic size revealed that the specimens of Cobitis collected from the Oita River system in Kyushu Island are the tetraploid form of C. biwae. Subsequent mitochondrial DNA analyses also supported this finding. Taking the connection pattern of drainages during glacial periods into consideration, it is suggested that C. biwae from the Oita R. s. is native.

The Japanese spined loach Cobitis biwae includes the tetraploid form, which has 96 chromosomes, as well as the diploid form, which has 48 chromosomes. In the present study, we analyzed the mitochondrial DNA (mtDNA) to examine the genetic relationships among 82 populations of diploid-tetraploid complexes of C. biwae. Restriction fragment length polymorphism (RFLP) analysis of the ND1 region on 202 individuals revealed that C. biwae contains three genetically divergent major groups corresponding to geographical proximities (the Eastern, Western, and Kochi groups). Phylogenetic analyses (neighbor-joining, NJ and maximum-parsimony, MP) of a part of the cytochrome b gene sequence (748 bp) in 31 individuals supported the three major groups recognized by RFLP, and indicated considerable genetic differentiation between the Western group and the other two groups (average, 15.2%). The genetic relationship and distribution pattern of the three major groups hypothesized two major dispersions of C. biwae during the middle Miocene: first, the ancestor of the Eastern and Kochi groups had spread from the West through wide regions of present-day Honshu and Shikoku Islands, and following that period, the ancestor of the Western group with a different mtDNA composition, probably resulting from the mtDNA introgression from C. takatsuensis, moved into western Japan and pushed the former north eastward. All tetraploid form populations were included in the Western group and treated as a monophyletic cluster with low genetic divergence. It is notable that two diploid populations geographically adjacent to the tetraploid range were genetically closely related to the tetraploid forms. This result suggests that these diploid populations were directly related to the maternal origin of the tetraploid form.

The Japanese allotetraploid spined loach of the genus Cobitis "yamato complex" sensu SAITOH et al. (2000), distributed in Western Japan, originated from hybridization between C. biwae on the maternal side and C. striata (Kyushu form) on the paternal side. Mitochondrial (mt) and nuclear DNA were analyzed in order to determine the genetic relationships among 15 populations spanning the entire range of the yamato complex. PCR-RFLP analysis of the ND1 mtDNA gene indicated that the yamato complex contains two divergent types of mtDNA: type A, consisting of one haplotype observed only in the Fukagawa River and type B consisting of 12 haplotypes found in the entire area. Phylogenetic analysis based on the cytochrome b mtDNA gene corroborated RFLP analysis, and indicated that type A was closely related to a different species, C. biwae (Kochi group) and C. striata (large race), rather than type B. The results of RAPD analysis on the Fukagawa River individuals, where types A and B sympatrically existed suggested that no reproductive isolation occurs between them. The existence of two distinct mtDNA types within the yamato complex suggest either multiple maternal origin at the speciation (tetraploidization) time or mtDNA introgression from other species afterwards.

A comparison of allozyme variation, restriction fragment length polymorphisms in the mitochondrial DNA and partial sequences of the ND II gene (496 bp) was made among two lacustrine populations of the piscivorous chub (Opsariichthys uncirostris uncirostris) in Japan, four fluvial populations in Korea, one lacustrine population in Russia and one specimen from the Amur River (O. u. amurensis). All analyses indicate that the Japanese populations of piscivorous chub are separable from the Asian mainland populations of Korea and Russia. The latter populations were further divided into the Korean and Russian fish. Although opinions are divided on the phylogenetic position of the population in Lake Mikata, Japan, which shows unique morphological traits intermediate between those of the population in Lake Biwa and the mainland populations, the current analysis indicates a closer relationship to the population in Lake Biwa.

Population structures of the delicate loach, Niwaella delicata. were inferred from morphology and restriction fragment length polymorphism (RFLP) analysis of part of the mitochondrial DNA (mtDNA) of 25 populations, representing the species range in central Honshu Island. The existence of two types of morphological variation corresponding to regional distributions, the "Pacific slope type". and "Sea of Japan slope type." has been known in N. delicata. Our morphological reexamination of the two types revealed some discrepancies in their distribution pattern. Therefore, we reclassified two new color types corresponded to their distribution areas as "gathered spots type (G type)" and "scattered spots type (S type)," respectively. The: present classification of G and S types is closely related to the mtDNA divergence pattern. The current analysis also indicated that each G and S type population was further divided into two genetic groups, corresponding to geographic proximity. In spite of marked morphological differentiation, the genetic diversity between G and S type populations (1.153%) was comparable only to that reported for intraspecific levels in most freshwater fishes. Moreover, in the population of which the color patterns of all fish were characterized to the S type, mtDNA haplotypes corresponding to G and S types were sympatrically detected. This result indicates secondary contact between the two type populations and the possibility that they are not reproductively isolated.

The largemouth bass Micropterus salmoides consists of two subspecies of the northern subspecies M. s. salmoides and the Florida subspecies M. s. floridanus. It has been widely regarded that largemouth bass, introduced and widespread in Japan, is the northern subspecies. In 1988, the Florida subspecies was also introduced into the Ikehara Reservoir (Nara Prefecture), where northern subspecies were already present. To assess the influence of this introduction, the genetic structure of the largemouth bass population in Ikehara Reservoir and thirteen other locations throughout Japan were examined and compared by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for mitochondrial DNA (mtDNA). Analysis of the ND1 region of mtDNA using sixteen restriction enzymes revealed 10 haplotypes (1-10) that form two distinct clusters in the UPGMA tree. One cluster consisted of two haplotypes (1 and 2) commonly detected in most populations throughout Japan, and the other cluster consisted of eight haplotypes (3-10) detected only in Ikehara and its connected Reservoirs. The reported rapid assay to identify mtDNA from two subspecies using D-loop region clearly revealed that eight haplotypes (3-10) were from the Florida subspecies.

Partial sequences of the cytochrome b gene (740 bp) and restriction fragment length polymorphisms in the mitochondrial DNA were used to examine inter-and intraspecific relationships among nine species of bagrid catfishes (family Bagridae) in Japan and Korea. Several opinions have been expressed regarding the kinship among Japanese and Korean bagrid catfishes, based on external morphological similarities. Almost all of them, however, were rejected by the current data sets. For instance, it has been considered that the Korean species, Pseudobagrus brevicorpus and P. fulvidraco, were closely-related to P. ichikawai and P. nudiceps, respectively, found in Japan. Resulting trees indicated that P. ichikawai branched off separately from all of the remaining Pseudobagrus species. Similarly, P. nudiceps and P. fulvidraco were represented by distantly separated branches. The intraspecific genetic divergence of bagrid catfishes was relatively smalt, even among geographically distant populations.