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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.

A new nemacheilid loach, Lefua tokaiensis sp. nov., is described from the small mountain streams in the Tokai region, central Honshu, Japan. Lefua tokaiensis is distinguished from all other species of Lefua by the following combination of characters: eyes located dorsally on the head, presence of a narrow conspicuous longitudinal mark between the base of the outer rostral barbel and the eye, absence of a rhomboid or triangular dark blotches on the middle of the caudal fin base, absence of a black longitudinal stripe on both body sides in mature males, absence of dusky cross bars on the dorsal area of the body, absence of a dusky bar beside the dorsal fin base, typical presence of small dark spots on the dorsal and caudal fins, presence of the obscure dark band in skin of the caudal fin base, caudal fin slightly squared off at the apex, and modally four hypurals.

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.

We investigated the cephalic lateral line canal system of the golden venus chub, Hemigrammocypris rasborella. The cephalic lateral line canal system consists of the infraorbital canal (IOC), the preopercular canal (POC), the mandibular canal (MC), the supraorbital canal (SOC), the temporal canal (TC), and the supratemporal canal (STC), and is characterized by the following pedomorphic features: disjunction of IOC and SOC, of TC and POC, and of POC and MC. We also discuss the phylogenetic significance of the cephalic lateral line canal system of H. rasborella.

メダカは，遺伝的特徴によって北日本集団と南日本集団に大きく二分される。両集団は，京都府由良川水系において側所的に分布しているにもかかわらず，それぞれ純系を維持している。このことは，両集団間に生殖的隔離が成立していることを示唆している。そこで，両集団の生態的分化の程度を探る一環として，京都府由良川水系の南北集団を用いて，通常時と刺激を与えた時の群れ行動について比較実験を行った。通常時では，南日本集団は北日本集団より小さな魚群半径を形成した。しかし，4尾の場合では魚群半径にばらつきが見られた。刺激を与えた時では，南日本集団は一度分散した後，密集し，静止した。一方，北日本集団は活発に動いていたが，冬期に南日本集団と同様に静止することを確認した。以上の結果から，南北集団間で群れ行動が異なることが確かめられ，そのことから地域性を無視した放流は生態的地域固有性を失わせるものと危惧された。

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.

The habitat of Aphyocypris chinensis in Jeollabuk-do, Korea was surveyed in March and July 2007 to obtain basic information on its habitat preference. The data obtained are expected to contribute to the conservation of the endangered Japanese population of the present species. Twenty species including A. chinensis, belonging to nine fish families, were caught at four stations using a wide hand net. Among these, Carassius spp., Macropodus ocellatus and Monopterus albus were found in habitats similar to that of A. chinensis. The habitats in Korea were similar to a nearly traditional irrigation canal, which are identical to those in Japan. From this survey, we confirm that a traditional style of farming provides the most suitable habitat for A. chinensis.

The hypothesis that the Ajime-loach, Niwaella delicata, is guided to groundwater seepages by a positive thermotaxis in autumn, was tested by a field investigation and aquarium-based experiments. A total of 763 individuals of N. delicata were captured from October to November in a groundwater trap in the Yasu River, Shiga Prefecture. Niwaella delicata began to be captured as the temperature of the surface water fell to 15.8° ± 1.1°C (mean ± SD) and that of the groundwater to 15.5° ± 0.9°C. Groundwater was often warmer than surface water at night or occasionally all day, and the difference in temperature reached a maximum of 1.3°C at the night on 5 November. For the diel pattern of captures, nocturnal capture was higher than diurnal capture when the groundwater was warmer at night and colder during the daytime, whereas both diurnal and nocturnal captures were high when the groundwater was always warmer than the surface water. The aquarium-based experiments showed that N. delicata choose warmer water, ranging from 18.4° to 22.2°C, just before the capture period in the Yasu River, and are sensitive to differences in water temperature of 1.3° ± 0.1°C. Although the present results broadly support the hypothesis, a part of the results indicates that water temperature gradients may not be the only factor involved in the groundwater selection of N. delicata.

To elucidate the phylogenetic position of Lefua loaches from Aichi and Shizuoka Prefectures of Honshu Island, Japan, we determined their nucleotide sequences for the mitochondrial D-loop region and compared these to sequences from four other Lefua species: L. costata, L. nikkonis, L. echigonia, and L. sp. Loaches identified as L. sp. on the basis of morphology comprised a clade (the L. sp. Tokai population) that grouped together with L. echigonia; hence, the processes involved in evolution within the genus Lefua were unclear. We performed randomly amplified polymorphic DNA (RAPD) analyses to obtain genetic information on nuclear DNA. The RAPD patterns of the L. sp. Tokai population differed from those of the local L. echigonia and L. sp. populations. The L. sp. Tokai population was similar to L. echigonia with regard to mitochondrial DNA but differed from L. echigonia and L. sp. with respect to nuclear DNA; this indicated that the evolutionary background of the L. sp. Tokai population was unique. We suggest that introgression of mitochondria occurred from L. echigonia to the L. sp. Tokai population, and speculate on the process of evolution of the latter population of Lefua. As with six L. echigonia populations and two L. sp. populations, we regard the L. sp. Tokai population as an evolutionary significant unit (ESU) that qualifies for protection as an endangered loach.

An anomalous individual with an adipose fin-like projection was discovered during seedling production of Biwa rock catfish, Silurus lithophilus (Teleostei: Siluridae). The external shape of the projection resembled an adipose fin, but soft rays were clearly observed within it. The projection was proximally supported by a series of pterygiophores and by 14 soft rays. The projection can be explained as: I, homologous with a primitive adipose fin, which occurred as a result of localized ontogenetic reversion; II, an extra fin that occurred as a result of abnormal expression of a regeneration mechanism; III, a remnant of a dorsal fin and/or a second dorsal fin; and IV, an inversion phenomenon of the polarity in the formation process of the anal fin.

The early development of the endangered freshwater goby, Rhinogobius sp. BI (ogasawara-yoshinobori in Japanese), was described in the course of a serial rearing experiment over generations as ex situ preservation. The eggs, measuring 2.0 mm in long diameter and 0.7 mm in short diameter, were elliptical with a colorless transparent chorion, a slightly yellowish yolk, and some oil globules. Hatching occurred naturally at 6 to 7 days after spawning at 24.0°C. Newly hatched larvae, measuring 3.2–3.4 mm in total length (TL), had opened mouth and a globular yolk sac. The yolk was completely absorbed at 3.5 mm TL (5 days after hatching). Notochord flexion initiated at 5.7 mm TL (18 days) and finished at

The cephalic lateral line systems of seven Far Eastern Phoxinus species (P. phoxinus, P. kumgangensis, P. semotilus, P. lagowskii, P. oxycephalus, P. perenurus, and P. czekanowskii) were investigated. In this genus, the infraorbital canal is not connected with either the supraorbital canal or the preoperculomandibular canal. Phoxinus phoxinus is unique for having an underdeveloped condition, such as canal formation or remaining as uncovered. A unified supraorbital canal was observed in all species, but the infraorbital canal of both P. perenurus and P. czekanowskii was not unified into a single canal throughout their development. Unification between both sides of the supratemporal canal occurred in larger individuals of P. lagowskii, P. oxycephalus, and P. czekanowskii. The preoperculomandibular canal of P. kumgangensis, large P. lagowskii, and large P. oxycephalus was unified. The pore number of each part of the canal system also varied depending on the species. Intraspecific variations were observed between Korean and Japanese specimens of P. lagowskii in the unification of the supratemporal canal and the preoperculomandibular canal, and the number of pores of the supratemporal canal. It was inferred that the specific characteristic patterns of their cephalic lateral line systems reflected the following two factors: different environmental requirement for their microhabitat and different maximum body size.

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.

1988 年にオオクチバスフロリダ半島産亜種が放流された池原貯水池では，1996, 1997 年の遺伝学的調査においてフロリダ半島産亜種由来のミトコンドリア DNA（mtDNA）をもつ個体の割合は 56.8% であった。今回（2003 年）再調査を行った結果，本亜種由来の mtDNA をもつ個体は全体の 86.7% となり，前回調査より有意な増加がみとめられた。また，近畿地方の池原貯水池とは別水系の湖沼（津風呂湖，宝ヶ池，深泥池）からも本亜種由来の mtDNA をもつ個体が検出された。

The relationships between environmental variables, distribution of freshwater fishes and their diet were investigated at 40 sites on irrigation ditches for rice fields in central Japan. Multivariate analyses showed that fish species richness and diversity in ditches was high when fishes were able to easily invade the rice fields. Natural stream beds not covered by concrete had greater total number and biomass of fishes in irrigation ditches. The connection between ditches and rice fields was of secondary importance. Canonical correlation analysis indicated that the two principal fish community variables, abundance and diversity, were respectively represented by the presence of the minnow, Gnathopogon elongatus elongatus, and the absence of the loach, Misgurnus anguillicaudatus. Most fishes in the irrigation ditches predominantly preyed upon aquatic insects such as ephemeropteran nymphs, chironomid larvae and trichopteran larvae, but aerial insects and benthic algae were also fed upon by a few species. Small fishes (

Two biological types of Japanese dark chub, so-called types A and B of Zacco temminckii, were taxonomically inspected. A comparison of types A and B with the lectotypes of Leuciscus sieboldii and L. temminckii in Siebold's collection revealed that type A is identical to L. sieboldii, whereas type B matches L. temminckii. Hence, Zacco sieboldii and Z. temminckii were redescribed on the basis of the lectotype and additional specimens from Japan. Zacco sieboldii is distinguishable from Z. temminckii by having a narrower band on the anterior portion of both body sides, nine branched rays of anal fin (10 in Z. temminckii), lateral line scales not less than 53 (not more than 52 in Z. temminckii), and scales above lateral line not less than 13 (not more than 11 in Z. temmincki). A key to the species of Japanese Zacco is also provided.

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.

Artificially induced triploid male Rhodeus ocellatus ocellatus showed typical nuptial colorations, irrespective of spermiation. In milt from triploids, abnormal spermatozoa (malformation of the head and mitochondrion, excessive formation of the head, mitochondrion and flagellum, and no flagellum) occurred at 78°4% frequency. Spermatozoa with multiflagella were most common, often with a saccate-like organ. Many triploid spermatozoa moved actively as long as those of diploids (10·92±0·91 min=mean±S.D., P>0·05), but did not advance like diploids, spinning around until movement ceased. The sperm density in triploids was < 2% of that from diploids. In triploid testes, deformed and variously sized spermatids were often observed, and normal spermatids and spermatozoa were seldom recognized. The DNA content of triploid spermatozoa varied greatly, compared with that of diploids. Peak of sperm DNA content differed slightly between two triploid samples with two peaks at 1·5 n and 1·9 n (P

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.

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.

Japanese freshwater fishes, including lampreys, comprise 15 orders, 35 families, and 96 genera, with 211 species and subspecies. Most belong to the families Cyprinidae (29% of species and subspecies), Gobiidae (21%), Salmonidae (10%), and Cobitidae (8%). Cyprinids and cobitids presumably originated from east Asia, gobiids from southeast Asia, and cottids and salmonids from the north Pacific. Japanese freshwater fishes include 88 endemic species and subspecies, of which three have been extirpated. Fishes introduced into natural rivers and lakes for inland commercial fisheries and sport fishing, and by accident, include many exotic species, of which 23 now inhabit natural freshwaters. These often have destroyed the local fish fauna by predation, and caused genetic pollution by hybridization with local strains. Destruction of freshwater environments by land development also poses a threat to Japanese freshwater fish communities. In addition Japanese freshwater systems have been markedly altered by development of rice paddy fields which have caused some species to decline but others to flourish, and changed the distribution patterns of fishes between upstream and downstream areas. To conserve endangered species and declining communities of Japanese freshwater fishes, we need to clarify the characteristics of their original habitats and the effects of developing paddy fields, from both the ecological and historical points of view.

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.