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Overuse of natural resources by humans is a major threat to biodiversity. Overuse often involves species of economic or esthetic value, and fish are a typical example for a group that is exploited both for economic reasons (for human consumption) and for esthetic reasons (e.g. by aquarists). Pseudorhodeus tanago (Tanaka, 1909) (formerly known as Tanakia tanago) is a small colorful but legally protected (fishing, keeping and transfer are banned) bitterling fish distributed around Tokyo, Japan. Whereas it is critically endangered and more and more habitat loss has occurred, at least four stocks have been newly found during the last decade. To explore whether emergence of these newly found habitats is a consequence of incomplete survey, we genotyped mitochondrial cytochrome b sequence of P. tanago from 17 localities and an illegal home aquarium. Populations known by the past extensive survey (13 localities) showed geographically structured population genetic characteristics. Population-specific haplotypes were common indicating past divergence and bottleneck events. Four (north, {center + west}, south_1, south_2) or five (north, center, west, south_1, south_2) geographic groups were detectable as for these known localities. On the other hand, newly found stocks were polymorphic and showed identical haplotypes from distant known localities. If we assume historical basis of distribution and genetic characteristics of these newly found stocks, it must be a series of unlikely geological events and haplotype sorting. We discuss potential issues posed by these questionable stocks.

Genetic diversity at a major histocompatibility complex (MHC) class II B gene was examined for two wild and three captive populations of the endangered Tokyo bitterling Tanakia tanago. A specific primer set was first developed to amplify the MHC II B exon 2 locus. Using single strand conformation polymorphism (SSCP) and sequencing analysis, 16 DAB3 alleles were detected with 56 nucleotide substitutions in the 276-bp region. In the putative antigen-binding sites of exon 2, the rate of nonsynonymous substitutions was significantly higher than that of synonymous substitutions (dN/dS = 2.79), indicating positive selection on the retention of polymorphism. The population from the Handa Natural Habitat Conservation Area and that from the Tone River system exhibited low variation (one and three alleles, respectively), whereas the captive population that originated from a mix of three distinct populations had the highest amounts of variation (14 alleles). The levels of heterozygosity at the MHC varied considerably among populations and showed significant correlations with those at putative neutral microsatellite markers, suggesting that genetic drift following a bottleneck has affected MHC variability in some populations. Comparisons between endangered and non-endangered fish species in previous reports and the present results indicate that the number of MHC alleles per population is on average 70% lower in endangered species than non-endangered species. Considering the functional consequence of this locus, attention should be paid to captive and wild endangered fish populations in terms of further loss of MHC alleles.

The maintenance of genetic diversity is one of the chief concerns in the captive breeding of endangered species. Using microsatellite and mtDNA markers, we examined the effects of two key variables (parental number and duration of breeding period) on effective population size (N(e) ) and genetic diversity of offspring in an experimental breeding program for the endangered Tokyo bitterling, Tanakia tanago. Average heterozygosity and number of alleles of offspring estimated from microsatellite data increased with parental number in a breeding aquarium, and exhibited higher values for a long breeding period treatment (9 weeks) compared with a short breeding period (3 weeks). Haplotype diversity in mtDNA of offspring decreased with the reduction in parental number, and this tendency was greater for the short breeding period treatment. Genetic estimates of N(e) obtained with two single-sample estimation methods were consistently higher for the long breeding period treatment with the same number of parental fish. Average N(e) /N ratios were ranged from 0.5 to 1.4, and were high especially in the long breeding period with small and medium parental number treatments. Our results suggest that the spawning intervals of females and alternative mating behaviors of males influence the effective size and genetic diversity of offspring in bitterling. To maintain the genetic diversity of captive T. tanago, we recommend that captive breeding programs should be conducted for a sufficiently long period with an optimal level of parental density, as well as using an adequate number of parents.

The Tokyo bitterling Tanakia tanago (Cyprinidae) was once found throughout the Kanto Plain, central Japan, but most of their habitats have been lost due to human activities such as urbanization and improvement of paddy fields. Subsequently, conservation efforts, including captive breeding and reintroduction, have been ongoing. However, the genetic relationships among populations of this species including captive and remnant wild populations have been uncertain and thus management units for this species have been unidentified. We examined the population differentiation among 12 populations, including four wild and eight captive populations, and their relative genetic diversities to assist in conservation management decisions. Phylogeographic analyses based on partial mitochondrial cytochrome b gene sequences and microsatellite polymorphisms revealed four geographically associated genetic groups in the populations. Northern Tochigi populations have diverged from other populations (0.77% of d A ), likely stemming from allopatric fragmentation following a change in the route of the Naka River, which occurred during the middle of the Pleistocene epoch. Microsatellite analysis has revealed that the genetic diversity of each population is generally low, and that most of the populations have experienced genetic bottlenecks. For future in- and ex-situ conservation programs to succeed, the population structure and genetic variability of remnant populations need to be taken into consideration.

The Japanese bitterling Tanakia tanago is an endangered cyprinid species; thus, captive breeding programs are being conducted in various facilities as ex situ conservation. To examine the genetic diversity in one wild and three reared populations, and its changes during the process of captive breeding, sequences of the mitochondrial cytochrome b gene and control region were determined. The wild population, collected in 1993, was monomorphic. Although the reared population that originated from the wild population was almost monomorphic, a rare haplotype, distinct from all others by a relatively large sequence divergence, was also observed. In the other reared populations, some degree of genetic diversity had been maintained. A reared hybrid population, which originated from a mixture of three distinct populations, showed the greatest genetic diversity. These results suggest considerable genetic diversity within and among populations of T. tanago in the past. Although a loss of genetic diversity was observed in some year-classes of reared populations, there was no tendency for genetic diversity to decrease as a result of captive breeding, probably because offspring were obtained from multi-year-class parents in the captive breeding program. Accordingly, this breeding method should be appropriate for conserving the genetic diversity of T. tanago.

The physical habitat characteristics associated with spatial distribution patterns of the freshwater mussel Pronodularia japanensis, which is used for oviposition by the Tokyo bitterling Tanakia tanago, were investigated in a small stream within a Tokyo bitterling protected area. The distribution of the mussels was found to be in an under-dispersed, non-random spatial pattern. Mussel occurrence correlated negatively with sediment softness, and positively with flow velocity, while mussel abundance was associated negatively with sediment softness and positively with sediment type (particle size). Furthermore, mussels were scarce in riverbed areas with a lack of sediment. These correlations suggest that the population dynamics of mussels and Tokyo bitterling may be influenced by changes in stream sediment conditions. To conserve the symbiosis between Tokyo bitterlings and mussels, a suitable benthic environment is required.

From June 2000 to September 2001, we investigated the presence of eggs spawned in Margaritifera laevis and the seasonal changes in the gonads of Tanakia tanago. Eggs were observed from mid-March to mid-September. In females with a shrunken ovipositor, as the GSI gradually increased, most ovaries were in the prespawning phase (Oct-Mar). As the GSI increased further, most ovaries were in the early spawning phase (Mar-Jun). As the GSI gradually deceased, ovaries in the late spawning phase appeared (Jun-Sep). When the GSI was very low, most ovaries were in the postspawning phase (Sep-Oct). In males, when the GSI was low, most testes were in the early prespawning phase from Oct-Dec. As the GSI gradually increased, most testes were in the late prespawning phase (Dec-Jan). As the GSI increased further, testes were in the early spawning phase (Jan-Jun). As the GSI gradually decreased, amost testes were in the late spawning phase (Jun-Sep). When the GSI was very low, most testes were in the postspawning phase (Sep-Oct). These results indicate that T. tanago has a distinct annual reproductive cycle and is a spring-autumn spawner. Based on the relationship between reproductive activity and environmental factors, the spawning season of T. tanago appears to be initiated by increasing temperature and / or longer days in spring and to be terminated by shorter days in autumn.

In order to study the phylogenetic relations between rhodeine species a number of interspecific and intergeneric crossings have been performed. This communication deals with the intergeneric hybrids obtained from the combinations Acheilognathus lanceolata ♂ × Acanthorhodeus atremius ♀ and Tanakia tanago ♂ × Acheilognathus rhombea ♀.From the first combination adult hybrids have been obtained which were intermediate between both parental species and showed a male phenotype. The second combination produced larvae, but a deficient yolk supply prevented them from reaching the free-swimming stage.Experimental evidence is presented for the occurrence of a dominant genetic factor in A. rhombea males, which controls the duration of embryonic development in this and other bitterling species.

The development of eggs and larvae and minute tubercles on the skin surface in larvae ofTanakia tanago were observed. The eggs began to hatch approximately 52 hours after insemination and the larvae reached free-swimming stage 19 days after hatching at water temperature of 22±1°C. The egg and larval development and minute tubercles on the skin surface in larvae of this species were similar to those ofAcheilognathus lanceolata andA. limbata. However,T. tanago was distinguishable in egg and larval development fromA. lanceolata andA. limbata by the following characters: the perivitelline space was narrower, embryonic and larval development was faster, and minute tubercles on the skin surface of the anteriormost parts of the yolk sac, and of the body and head were hemispheric in shape. From these characters,T. tanago is considered to be more specialized thanA. lanceolata andA. limbata.

The hybrids between female Acheilognathus lanceolatus and Rhodeus amarus, and male Acheilognathus himantegus. Can. J. Zool. 40:255-259. . 1962d. Hybridization experiments in acheilognathine fishes (Cyprinidae, Teleostei). Intergeneric hybrids between Rhodeus spinalis, Rhodeus ocellatus from Korea and Japan, and Acheilognathus tabira. Zool. Anz. 169 (9-10):373-384. . 1962e. Hybridization experiments in acheilognathine fishes (Cyprinidae, Teleostei). The interspecific hybrids between Acheilognathus lanceolatus and Acheilognathus tabira. Can. J. Zool. 40:421-424. 1964. Hybridization experiments in acheilognathine fishes (Cyprinidae, Teleostei). Hybrids between female Paracheilognathus rhombeus, and male Rhodeus ocellatus, Tanakia tanago, Acheilognathus tabira, A. limbatus, and Rhodeus spinalis. Copeia 1964(1): 150-155.