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Aphyocypris chinensis is a small cyprinid that is widely distributed in lowland areas of continental China, the Korean Peninsula, and the northwestern part of Kyushu, Japan. However, this species is severely threatened in Japan due to human impacts; thus, several facilities and citizen groups implemented captive breeding and reintroduction/reinforcement programs using several dozen founder fish collected from Tanushimaru, the location with the only known natural population, in the 1980s and 1990s. To determine the phylogenetic position and genetic authenticity of Japanese populations of A. chinensis, we conducted mtDNA and genome-wide SNP analyses using a total of 254 specimens from 31 wild and captive samples, including specimens of Chinese and Korean A. chinensis and the close relative A. kikuchii from Taiwan. The mtDNA divergence and phylogeny indicated that the haplotypes found exclusively in Japan were differentiated from the Chinese/Korean haplotypes (uncorrected p = 2.6% in the cytochrome b gene) to the same extent as they were from A. kikuchii haplotypes (p = 2.9%). Results from mtDNA sequences and 47–359 SNPs, obtained using the MIG-seq method with different parameter sets, revealed that the initial captive populations and an extinct wild population in Ukiha, adjacent to Tanushimaru, were genuine Japanese populations, whereas all extant captive and wild populations in Japan are hybrids between Japanese A. chinensis and A. kikuchii, or A. kikuchii itself. The details of the captive breeding and exchange programs, as well as evidence for the aquarium trade of A. kikuchii since the 1990s, strongly suggest that the mixture of A. kikuchii into Japanese populations occurred within the first several years of the establishment of captive populations in 1994. The present case of the highly probable extinction of genuine Japanese populations of A. chinensis emphasizes the importance of confirmation and management of the genetic authenticity of conservation-targeted species.

Hemigrammocypris neglecta is a small-sized cyprinid fish that is endemic to Japan and the sole member of the genus Hemigrammocypris. We examined the osteological features of the species, and its relationships with related genera are discussed. The present data suggest that H. neglecta is most likely the sister taxon of Metzia lineata. This relationship is supported by one possible synapomorphy: the presence of a postepiphysial fontanelle, which is absent in all other Xenocypridinae.

We re-describe and illustrate the syntypes of Candidia barbata. The syntypes differ from the original description of Candidia pingtungensis, their only congener, in several counts: lateral line scales, scales between the dorsal-fin origin and occipital, scales between the lateral line and dorsal-fin origin, scales between the lateral line and anal-fin origin, barbels, gill rakers, and pharyngeal teeth. On the basis of the original description of C. barbata, we also tentatively proposed a revision of the type locality of this species.

We evaluated inter- and intraspecific variations of the intestinal coiling patterns and intestinal length of the oily bitterlings, Tanakia limbata, Tanakia koreensis, Tanakia somjinensis, and Tanakia signifer. All the oily bitterling species shared a unique intestinal coiling pattern, expressed as an oval shaped loop. The ratio of the intestinal length to standard length varied among species, viz., both T. koreensis and T. signifer have significantly longer intestines than T. limbata and T. somjinensis. The intestinal length may be a useful diagnostic character between two sibling species, T. koreensis and T. somjinensis, although we observed intraspecific variation in the intestinal length of T. limbata and T. koreensis among collection sites.

The lakeweed chub Ischikauia steenackeri is a medium-sized, herbivorous fish and the sole extant member of the genus Ischikauia, which is endemic to Lake Biwa and the Yodo River drainage, Japan. In order to clarify its systematic position, the skeletal anatomy of I. steenackeri is described and its relationships with related genera are discussed. The present data suggest the monophyly of Ischikauia and seven cultrine genera (Culter, Chanodichthys, Megalobrama, Sinibrama, Hemiculter, Toxabramis, and Anabarilius) based on a unique character, the metapterygoid elongated dorsally. Additionally, our data suggest that Ischikauia is closely related to Culter, Chanodichthys, Megalobrama, and Sinibrama. This relationship is supported by three synapomorphies that are common to them: a narrow third infraorbital, dorsal extension of the third supraneural, and a large quadrate foramen.

Ichthyoxenus amurensis (Crustacea: Isopoda: Cymothoidae) was found in the body cavity of the Amur bitterling, Rhodeus sericeus (Cypriniformes: Cyprinidae), from Primorsky, Russia, in August 2003. A total of 13 individuals of I. amurensis were obtained from nine of 29 fish specimens (prevalence = 31.0%). Rhodeus sericeus is a new host for I. amurensis. In the body cavity of R. sericeus, I. amurensis was found within a thin-walled membranous sac, and the intestines of the host were malformed as a result of infection. There was no significant difference in the standard length of infected and uninfected R. sericeus.

In order to elucidate phylogenetic relationships and intraspecific variations and to infer the evolutionary process of loaches of the genus Lefua, we analyzed nucleotide sequences of the mitochondrial D-loop region of 100 specimens obtained from 97 localities in Japan and Korea. The genus Lefua includes three described species, L. nikkonis, L. echigonia, and L. costata and an undescribed species, Lefua sp. Our results showed that each species of Lefua formed a monophyletic group, indicating clearly that Lefua species can be genetically distinguished from one another. Lefua nikkonis was the most closely related to L. costata, while L. sp. was the most closely related to L. echigonia. Specimens of L. sp. were grouped into two intraspecific populations and specimens of L. echigonia were grouped into six populations. These populations were well separated geographically from one another by mountain ranges and highlands. We estimated the evolutionary time for splitting of the species and intraspecific populations, and speculated on the evolutionary process of the genus Lefua. Species of Lefua are severely threatened. Fundamental genetic information is indispensable for conservation. We presented genetic background in order to protect these threatened loaches.

The sex ratio of bitterling hybrids (subfamily: Acheilognathinae) is often likely to be biased toward males. Artificial hybridization was carried out in 10 species of bitterlings (three genera) in order to elucidate the masculinization mechanism of hybrids. Tanakia himantegus never produced viable F1 hybrids with other species, while hybrids of most other species were viable. In terms of sex ratio and fertility, hybrids were clearly divided into two groups: congeneric Tanakia hybrids and others. Both male and female congeneric Tanakia hybrids were fertile. The sex ratio was nearly 1:1 in all groups of Tanakia hybrids. Except for the congeneric Tanakia hybrids, sterile males appeared predominantly in groups of hybrids in which females were very rare but remained fertile. Sterile intersexes were also observed in five hybrid groups: T. lanceolata (female) x Acheilognathus cyanostigma (male), Rhodeus uyekii (female) x T. lanceolata (male), A. rhombeus (female) x T. lanceolata (male), A. rhombeus (female) x T. limbata (male), and A. tabira tabira (female) x A. cyanostigma (male). In the development of male-predominant hybrids, although hybrid and control (parental species) hatching and survival rates do not differ, no females appeared in hybrids, contrary to the controls. Taking the female heterogametic sex-determining system (ZW) and the phylogenetic relationship of bitterlings into consideration, the masculinization mechanism of hybrids in bitterlings can be explained by the interaction of two sex chromosomes, derived from each parental species. The basic genetic sex in bitterlings is male (ZZ) and the derivative is female (ZW). When parental species are related, the sex phenotype of hybrids coincides with the genetic sex. However, when the parental species differ, the sex phenotype of the ZW genotype is reversed to become male by an abnormal interaction between the Z and W chromosomes. The rare appearance of females and intersexes in male-predominant hybrids might be due to complete or partial functional expression of the W chromosome.

The phylogenetic relationships among threePseudorasbora fishes (Cyprinidae, Sarcocheiichthyinae) occurring in Japan (P. parva, P. pumila pumila andP. pumila subsp. sensu Nakamura [1963]) were inferred from nucleotide sequences of the mitochondrial 16S rRNA gene. The sequences. of 1240 bp, were determined and compared for 22 specimens from 2–8 populations for each taxon, with a singlePungtungia herzi specimen as an outgroup. A total of 171 sites (13.8%) were variable among the specimens, but only 0–2 sites within each population. The phylogenetic relationships estimated by neighbor-joining, maximum-parsimony and maximum-likelihood methods confirmed a sister relationship between the twoP. pumila subspecies, with a high level of confidence. However, their genetic distinction from each other (4.1±0.4SD % sequence difference on average) was at a level similar to that between them andP. parva (5.9±0.5%). The geographic distribution of the twoP. pumila subspecies, which are separated by the Fossa Magna region, suggests that the genetic divergence of the two subspecies originated from a vicariant process separating the freshwater ichthyofaunas of eastern and western Honshu.Pseudorasbora parva populations were divided into two genetic groups (1.8±0.2% sequence difference), one group comprising continental and part of the Japanese populations, and the other the remaining Japanese populations. This suggests that at least two genetically divergent lineages had been originally distributed in Japan, but a strong possibility remains that the present situation has resulted from artificial transplantation.

The larval development of the Japanese flounder,Paralichthys olivaceus, was surveyed using two types of morphometric analyses, modified allometry and polar coordinate analysis by principal component analysis (PCA). In the former, centroid size was used as a growth index instead of total length (TL), such enabling the determination of more detailed changes in each character than ordinary allometry based upon TL. Polar coordinate analysis disclosed two remarkable inflexions during the larval development ofP. olivaceus. Postlarvae ofP. olivaceus were found to undergo four developmental phases. From the point of view of metamorphosis, the phases were named drifting larva, premetamorphic larva, metamorphic larva and postmetamorphic larva, respectively. These phases were also tested by other characters related to flounder metamorphosis.

The evolutionary history of eight-barbel loaches of the genus Lefua contains important phylogenetic information that will aid in resolution of the faunal formations and evolutionary histories of Japanese and East Asian freshwater fishes. Our sequencing of the mitochondrial D-loop region in a large number of samples allowed construction of the most comprehensive phylogeny of these loaches to date; we demonstrated monophyly of five Lefua species and identified populations of Lufua. sp. and Lefua echigonia. Loaches inhabiting the Tokai region in Japan were morphologically and ecologically indistinguishable from Lefua sp. However, they were included in the L. echigonia lineage. We determined a novel phylogeny by sequencing the nuclear ribosomal S7 subunit and showed that nuclear DNA phylogeny essentially matched the mitochondrial DNA phylogeny. Loaches from the Tokai region were part of the L. echigonia lineage, indicating parallel evolution between Tokai loaches and Lefua sp. in western Japan. We presented the most robust phylogeny to date using concatenated mitochondrial and nuclear sequences. The wealth of molecular information allowed us to speculate on evolutionary processes in the genus Lefua.

Two types of the dark chub,Zacco temmincki, collected from 10 river systems in Japan were genetically characterized at 27 protein coding loci using starch-gel electrophoresis. They were fixed for different alleles at 13 loci. No hybrid individuals were observed, even in specimens collected in stations where both types appear sympatrically, indicating that each type of the dark chub represents a distinct species.

Three nominal species are known in East Asian balitorid loaches of the genus Lefua, i.e. L. echigonia, L. nikkonis, and L. costata. Lefua echigonia, with large morphological variations was recently separated into two groups, L. echigonia including the holotype and L. sp., based on morphological and ecological traits. We performed protein and DNA analyses to elucidate phylogenetic relationships among loaches of the genus Lefua and to settle the taxonomic status of L. sp. We also investigated intraspecific variations in L. echigonia s. str. to shed light on the process of formation of freshwater fish fauna in Japan. Protein analyses using two-dimensional gel electrophoresis showed that genetic distances between L. sp. and L. echigonia s. str. and between L. sp. and L. nikkonis were as large as that between L. echigonia s. str. and L. nikkonis. DNA analyses of the mitochondrial D-loop region showed that L. sp. and L. echigonia s. str. were monophyletic, respectively, while neither L. nikkonis nor L. costata was monophyletic and these species formed together a clade. The results supported the specific status of L. sp. and proposed reevaluation of the taxonomic status of L. nikkonis and L. costata. DNA analyses also showed that L. sp. was more closely related to L. echigonia s. str. than to the L. nikkonis-L. costata complex, and four local populations were distinguished in L. echigonia s. str. Distribution patterns of the four local populations of L. echigonia s. str. in Japan were approximately congruent with those of the medaka, Oryzias latipes, suggesting that differentiation in the two distantly related fishes have a common historical background.

It is almost a miracle that T. Nakabo, K. Nakayama, N. Muto, and M. Miyazawa (2011) have discovered an extant population of Oncorhynchus kawamurae, or Kunimasu as the standard Japanese name, from Lake Saiko, Yamanashi Prefecture, Japan, from outside of its original distribution. Kunimasu had been believed to be extinct for more than 70 years in the type locality (and only known locality), Lake Tazawa, Akita Prefecture, owing to the introduction of strongly acidic water to the lake. This great discovery is certainly epoch-making in the study of the conservation of threatened freshwater fishes in Japan. The extant population seems to be safe so far under the conditions of local fishery activity in Lake Saiko. Nakabo et al. (2011) basically succeeded in clarifying both the morphological distinction and genetic isolation of Kunimasu from those of another sympatrically occurring kokanee, Oncorhynchus nerka, or Himemasu, which was also introduced to Lake Saiko from Hokkaido before World War II. Those facts consequently confirm the specific (not subspecific) status (sensu Mayr 1942, 1969) of Kunimasu as O. kawamurae, referring to some ecological peculiarity, i.e., deep swimming/spawning layer and spawning in winter or spring. However, its basic biology remains too obscure, so the information to certify them as O. kawamurae and subsequent protection for this valuable extant population is not yet sufficient. Major problems are as follows. (1) Genetic property: The small number of the specimens examined (n = 9) is not sufficient to show their full population genetic traits. Nakabo et al. (2011) successfully rejected large-scale hybridization between Kunimasu and Himemasu in Lake Saiko. The presumed differences in spawning season and sites may have worked as reproductive isolation mechanisms. However, their data cannot rule out the possibility that they have partially hybridized and some gene introgression from Himemasu to Kunimasu (and vice versa) has occurred. A huge number of Himemasu had already been introduced even to Lake Tazawa for a period of 30 years (Sugiyama 2000). Only morphologically typical specimens might have been examined by Nakabo et al. (2011). Information on the founder effect during the introduction process is lacking for both Kunimasu and Himemasu in Lake Saiko. Taking into consideration the repeated artificial transplantation of Himemasu among several lakes, the original geographic variation of Himemasu should be clarified, and the Kunimasu population in Lake Saiko should be compared with it by using adequate molecular markers. Verifiable information on the presence/ absence or extent of gene introgression is essential when reintroduction or conservation/benign introduction of this ‘‘extinct in the wild’’ species is considered. It has not been concluded yet whether the Kunimasu population in Lake Saiko is genetically pure. Genetic comparison between the current and original Kunimasu specimens would be the only direct test to certify the ‘‘rediscovery.’’ DNA analysis for old formalin-fixed specimens is generally difficult or impossible (Sugiyama 2000). However, recently developed next-generation sequencers might have the power to pick up and sequence short DNA fragments even from such old specimens. K. Hosoya Nature Conservation Committee of the Ichthyological Society of Japan, Tokyo, Japan

Transparent-scaled variant (TSV) of the rosy bitterling Rhodeus ocellatus ocellatus (Kner) was observed on both morphology and heredity. Compared with the normal-scaled type (NST), TSV is characterized by the blackish coloration in both eyes and peritoneum, and the luminescent one over the whole body. Histologically, the density of guanophores containing reflecting platelets was conspicuously low, especially in scale, iris, choroid and peritoneum, while the increase in the number of goblet cells (mucous cells) was recognized all over the dermal/epidermal regions. The heredity of TSV was recessive and supposed to be controlled by a single pair of genes unrelated to sex, judging from the result of crossbreeding experiments between NST and TSV. In growth and reproduction, no difference was seen between these two phenotypes. Transparent-scaled variant of the rosy bitterling can be competent for a genetic marker in experimental and developmental biology.

The skeletal system of Zacco pachycephalus, sensu Jordan & Evermann (1903), is described and compared with that of other related barilines to clarify its systematic position. Osteological characters fall generally into two categories in terms of apomorphic features. The first category is exemplified by autapomorphies such as lower jaw bone shape and the number of branchiostegal rays and hypurals. The latter characters are highly unusual among Cyprinidae. The second category consists of synapomorphies linking Z. pachycephalus and Opsariichthys uncirostris. Specialization of jaw bones for a piscivorous habit is also notable. The quadratepterygoid fenestra of Z. pachycephalus and O. uncirostris is considered derived. We conclude that Zacco pachycephalus should be transferred from Zacco to Opsariichthys based on the present osteological analysis.

This study was carried out to shed light on confused subfamilial groupings in the Cyprinidae from the biochemical viewpoint at the molecular level, specifically by using two-dimensional gel electrophoresis of liver proteins. Six pairs of cypriniform fishes, which are different from one another at familial, subfamilial, generic, specific, subspecific, and individual levels, were compared. The genetic distances between pairs of fishes increased as taxonomic ranks of the pairs became higher, confirming the reliable usefulness of this technique. Four species representing the different subfamilies, Cyprininae, Gobioninae, Acheilognathinae, and Leuciscinae, were compared to give new insight into relationships at the subfamilial level. Cyprinus carpio (Cyprininae) and Pseudogobio esocinus esocinus (Gobioninae) gave the smallest genetic distance and the largest values were obtained between either one of the above species and Acheilognathus melanogaster (Acheilognathinae), suggesting that the former two subfamilies compose the most closely related group that is in turn distantly related to Acheilognathinae. Tribolodon hakonensis (Leuciscinae) had almost equal genetic distances to the three other species.

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.