Morphological discrimination of two genetic groups of a Japanese salamander, Hynobius naevius (Amphibia, Caudata)

Hynobius naevius, distributed on western Honshu, Shikoku, and Kyushu Islands of Japan, includes two genetically distinct groups (Groups A and B) that have never been delimited morphologically. Using specimens from the entire species range, we investigated the possibility of distinguishing these groups morphologically. Multivariate analyses of morphometric characters resulted in recognition of two groups that corresponded well to the two genetic groups. One (Group A) was characterized by larger body, compressed tail, shallower vomerine tooth series, bluish- or reddish-purple ground color, and pale-white lateral markings. In contrast, another (Group B) was characterized by smaller body, cylindrical tail, longer vomerine tooth series, reddish-brown ground color, and white lateral markings. Group A was composed of populations from the Chugoku District of Honshu and northern Kyushu, and could not be divided into subgroups, while Group B encompassed populations from the Chubu and Kinki Districts of Honshu, Shikoku and Kyushu, and was subdivided into three local subgroups that are geographically separated by marine straits. Morphometric differentiation in Group A is presumed to have been less affected by genetic factors than by other factors, such as ecological relationships with other, coexisting species. Differentiation in Group B is assumed to have been enhanced not only by genetic but also by climatological factors.     

Mitochondrial DNA phylogeography of the red fox (Vulpes vulpes) in northern Japan

Mitochondrial DNA variation in the cytochrome b (cyt b) gene and the control region was examined in the red fox Vulpes vulpes from Japan, with special focus on the population divergence between Hokkaido and northern Honshu. Resultant haplotypes from Hokkaido were subdivided into two distinct groups (I and II), with an average genetic distance of 0.027 for cyt b. Divergence time is roughly estimated to be 1-2 million years ago, given that the conventional divergence rate of the mammalian cyt b gene is 2% per million years. Notably, Group II was only found in Hokkaido, whereas Group I comprised haplotypes from Honshu, Kyushu (Japan), eastern Russia, and Europe, as indicated by a comparison of our own data to the literature. On the other hand, judging from constructed trees, Group I haplotypes from Hokkaido appeared to differ from those from other parts of Japan, i.e., Honshu and Kyushu. This implies that Blakiston's Line, which demarcates the boundary between Hokkaido and Honshu, has been an effective barrier and has allowed the structuring of genetic variation in maternal lineages. Thus, these results suggest that the Hokkaido population, which is sometimes referred to as the distinct subspecies V. v. schrencki, has its own genetic background with multiple migration events and differs from the parapatric subspecies V. v. japonica found in Honshu and Kyushu.     

Two Genetically Distinct Lineages of the Sika Deer, Cervus nippon, in Japanese Islands: Comparison of Mitochondrial D-Loop Region Sequences

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705–824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.     

Intraspecific variation of cuticular hydrocarbon composition in Formica japonica Motschoulsky (Hymenoptera: Formicidae)

Cuticular hydrocarbons and morphological features were compared among 80 Formica japonica colonies collected in Japan. Although a few morphological differences were found in workers among the colonies, four different types of cuticular hydrocarbon composition were observed. This was supported by a principal component analysis. We further compared the cuticular hydrocarbons among a total of approximately 400 F. japonica colonies, and categorized the hydrocarbon components into four types based on the result of discriminant analyses for the first 80 colonies. Type 1 was observed in colonies mainly collected in southern Honshu, Shikoku, and Kyushu. Types 2, 3, and 4 were from colonies with primary collections in Southern Honshu, central and Pacific coast northern Honshu, and the Sea of Japan coasts of northern Honshu and Hokkaido, respectively. The occurrence of four distinct types of CHC composition suggests that the colonies that produce them are separate species.     

Genetic differentiations of Hynobius naevius (Amphibia,Hynobiidae) as revealed by allozyme analysis

A survey of genetic variation of lotic breeding salamander Hynobius naevius by starch gel electrophoresis using 26 populations from southwestern Japan revealed the presence of two genetically distinct groups (GROUP-A and B) which overlap distributional ranges in some parts of Kyushu. Degree of genetic differentiation between the two groups was larger in Kyushu, including sympatric area, than in allopatric areas. We surmise that the two groups first differentiated allopatrically and overlapped their distributions secondarily. The first genetic differentiation is considered to have occurred in the Pliocene as a result of isolation between northern (GROUP-A) and southern (GROUP-B) ancestral populations by the formation of the Seto Inland Sea, largely separating the present distributions of the two groups. Genetic diversities within the GROUP-A seem to have become complicated by isolations, disappearances of local populations, and subsequent range expansion of distributions in Chugoku and northern half of Kyushu regions. Within the GROUP-B, diversities first formed by isolation by distance have been retained and accelerated secondarily by the formations of the Kii and Bungo straits. Sympatric populations of the two groups in northern half of Kyushu seem to have overlapped their ranges by niche segregation.     

Intraspecific Genetic Diversity of Undaria pinnatifida in Japan, based on the Mitochondrial cox3 Gene and the ITS1 of nrDNA

The intraspecific genetic diversity of the kelp Undaria pinnatifida (Harvey) Suringar (Laminariales, Phaeophyceae) was investigated using DNA sequences of the mitochondrial cytochrome oxidase subunit 3 (cox3) gene and internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA in plants collected from 21 localities along the Japanese coast between 2001 and 2003. Morphological variation was also examined and compared with the genetic diversity. Cox3 analyses of 106 plants revealed 9 haplotypes (I–IX) that differed from each other by 1–7 bp (all synonymous substitutions). Haplotype I was distributed in Hokkaido and the northern Pacific coast of Honshu, while haplotype III was found along the Sea of Japan coast of Honshu. Other types were found along the central and southern coast of Honshu. ITS1 analyses of 42 plants revealed 0–1.7% nucleotide differences, but plants from the Sea of Japan coast and northern Japan had similar sequences. The lower genetic differentiation along the Sea of Japan and northern coasts might be due to the recent establishment (after the middle of the last glacial period) of the Sea of Japan flora. The cox3 haplotype of cultivated plants was found in natural populations occurring close to cultivation sites (Naruto, Tokushima Pref., and Hokutan, Hyogo Pref.). This suggests that cultivated plants possibly escaped and spread or crossed with plants of natural populations. Morphological analyses of variation in 10 characters were conducted using 66 plants. The results showed no significant local variation owing to the wide variation in each population and did not support any forma previously described. No correlations between the morphological characters and cox3 haplotypes were detected.     

Two genetically distinct lineages of the sika deer, Cervus nippon, in Japanese islands: comparison of mitochondrial D-loop region sequences

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705-824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.     

Comments on cephalometrical configurations of the Japanese

Cephalometrical configurations of Japanese people were investigated on two hundred and sixteen groups living on the four main islands of Japan: Hokkaido, Honshu, Shikoku, and Kyushu. The Japanese in Honshu were divided geographically into a narrow-featured eastern major group and a broad-featured western major group. A physical characteristic of Japanese living in the western mid-Honshu was the lengthened feature of their cephalic measurement, an influence of a long-headed group of Korean people; Japanese residents in eastern mid-Honshu, however, showed the result of mixing with a short-headed group of Korean people. Japanese people living in Hokkaido, the northernmost island, and in the Ryukyu Islands in the South of Japan, presented no important finding which suggests their influence on the physical characteristics of the Japanese. Ongoing studies indicating physical relationships between Kyushu or Shikoku residents and Honshu residents, are as yet insufficient.     

Phylogeography of Japanese population of Phelotrupes auratus (Coleoptera, Geotrupidae) inferred from mitochondrial DNA sequences

A partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene (745 bp) was determined for 57 specimens of a geotrupid beetle (Phelotrupes auratus) from throughout the Japanese archipelago. Of the 57 beetles examined, 42 haplotypes were identified. Phylogenetic trees inferred using maximum parsimony, neighbor joining, and Bayesian inference methods were highly congruent. Reconstructed phylogenetic relationships indicated that P. auratus from the Japanese archipelago was separated into two distinct lineages: Group A, which consisted of 35 haplotypes from Honshu, Shikoku, and Hokkaido Islands, and Group B, which consisted of seven haplotypes from Kyushu and Yakushima Islands. In addition, two sublineages were also recognized within Group A: Subgroup A-1, which consisted of 11 haplotypes from eastern Honshu and Hokkaido, and Subgroup A-2, which consisted of 10 haplotypes from western Honshu and Shikoku. Average genetic distances within Group A were positively correlated to geographic distance between sampling localities. Phylogenetic relationships among haplotypes did not correspond to subspecies classification.     

Geographic variability in expression of the sex-linked AAT-1 gene of the bell-ring frog, Buergeria buergeri

Cytological observation and artificial crossing experiments were used to examine the geographic differences in the sex-determining mechanism and mode of inheritance of the sex-linked AAT-1 gene in the bell-ring frog, Buergeria buergeri. The AAT-1 phenotypes were also examined by allozyme analysis using field-caught females and males collected from 19 populations from the Honshu, Shikoku, and Kyushu islands of Japan, in order to comprehensively elucidate the geographic variability in the expression of the sex-linked AAT-1 gene of B. buergeri. The results showed that the Aomori population of B. buergeri from the northern end of Honshu was female heterogametic in sex determination, that chromosome No. VII was a sex chromosome of the ZZ/ZW type, and that the sex-linked AAT-1 gene was expressed on both the Z and W chromosomes. This mode of AAT-1 expression in the Aomori population was different from that in the Hiroshima population from western Honshu, in which the AAT-1 gene was expressed on the Z chromosome but not on the W chromosome. The results also showed that there was no differentiation among populations in the expression of the AAT-1 genes on the Z chromosome, whereas two populations, the Hiroshima and Aomori frogs, exhibited distinct modes of expression of the AAT-1 gene on the W chromosome. These two modes of expression may be widely distributed in western and eastern Japan, and coexist in the central part of Honshu.     

Spatial framework of nine distinct local populations of the Japanese dormouse Glirulus japonicus based on matrilineal cytochrome b and patrilineal SRY gene sequences

We previously revealed the presence of six genetically distinct matrilineal populations of the Japanese dormouse Glirulus japonicus in the distribution range of Honshu, Shikoku, and Kyushu islands. In this study, we extended this analysis using mitochondrial cytochrome b gene sequences (n = 96) and Y-chromosome-specific SRY gene sequences (n = 22) from individuals collected from Honshu, Shikoku, Kyushu, and Oki Dogo I. The cytochrome b sequence data allowed us to define precise geographic ranges of the six previously known and three newly found distinct matrilineal lineages: northeastern Honshu (I), east-central Honshu (II), west-central Honshu and the Kii Peninsula (III), the western part of Honshu (IV), Shikoku (V), westernmost Honshu and Kyushu (VI), the northern part of central Honshu (VII), the southern part of central Honshu (VIII), and Oki Dogo I. (IX). Our inference of geographic borders suggests that regions of lower and higher altitudes in the mountain systems played important roles in driving the hosting and separation of lineages, respectively. Six matrilineal lineages (I, II, V, VI, VIII, and XI) were shown to possess their own SRY haplotypes, while lineages III and IV shared one haplotype. These data together with our previous observation of nuclear ribosomal RNA gene variation indicate advanced populational subdivision in this species. It is thus evident that each of the populations, including those living at high latitudes and in limited geographic spaces, have survived for several million years. A specific ability to tolerate cold may have permitted G. japonicus to preserve anciently diverged lineages in each locality.     

Reinstatement of Myelophycus caespitosus (Scytosiphonaceae,Phaeophyceae) from Japan

Reinstatement of Myelophycus caespitosus Kjellman (Ectocarpales s.l., Phaeophyceae) described from western Japan is proposed based on the comparisons of DNA sequences of Japanese Myelophycus specimens, the type specimen of Chordaria simplex and lectotypified specimen of Myelophycus caespitosus. In the genetic analyses using mitochondrial cox1 and cox3, chloroplast atpB, psbA and rbcL DNA sequences, the specimens morphologically referable to M. simplex formed two distinct clades (clade‐1 and clade‐2) supported by high statistical values. Clade‐1 was distributed on the western coast of Japan (Honshu, Shikoku and Kyushu) from the Kii Peninsula to Tsushima and the Pacific coast of Aomori, northeastern Honshu, and clade‐2 on the Pacific coast of central Honshu from Sanriku to the Kii Peninsula. Based on the cox3 DNA sequences and the location of the type locality at Shimoda, Izu Peninsula, clade‐2 was concluded to correspond to true M. simplex. There were no recognizable differences in the representative morphological features (height and diameter of sporophytes and gametophytes, number of cells comprising cortical, subcortical and medullary layers, thickness of plurilocular gametangia, or length and diameter of unilocular zoidangia) between the specimens included in the two taxa. Analyses of the specimens of the two taxa, including sympatric populations on the Kii Peninsula, using a nuclear genetic marker ocm3 did not suggest any genetic exchanges between the two taxa. On the other hand, cox3 gene sequence of the voucher specimen of Myelophycus caespitosus collected from Goto, Kyushu, western Japan housed in the UPS herbarium was included in clade‐1. In conclusion, we propose the reinstatement of M. caespitosus and to lectotypify the specimen in UPS. Korean Myelophycus specimens reported from Cheju Island and Wando were considered to belong to M. caespitosus based on the reported rbcL sequences.     

Differential geographic patterns of mitochondrial DNA variation in two sympatric species of Japanese wood mice, Apodemus speciosus and A. argenteus

We examined the gene sequences of mitochondrial cytochrome b (cyt b) in two Japanese wood mouse species, Apodemus speciosus (n = 89) and A. argenteus (n = 46), which are distributed on the four main islands of Japan (Hokkaido, Honshu, Shikoku, and Kyushu) and on the small islands surrounding them. Apodemus speciosus, the larger of the two species, showed substantial genetic variation, with a maximum of 3% sequence divergence, and remarkable phylogenetic subdivision with two major clades. One clade represents haplotypes from a central region, including Honshu, Shikoku, Kyushu, and their adjacent islands; the other clade includes haplotypes from Hokkaido and the peripheral islands, forming four subclades: a) Hokkaido, b) Sado Island, c) Satsunan Islands, and d) the Izu Islands. Sequence divergence among the four subclades was 1.0 to 1.5%, implying that A. speciosus colonized these geographic regions 0.2 to 0.3 million years ago, assuming a substitution rate of 2.4% per million years. The population on the Izu Islands has preserved haplotypes that are distinct from those in any other region, providing good evidence for the natural colonization of the volcanic islands of the Izu Islands. The cyt b sequence variation had no relation to the karyotypic dimorphism for the eastern (2n = 48) and western (2n = 46) geographic groups, between which a strict border exists at central Honshu. On the other hand, Apodemus argenteus, the smaller of the two species, showed a similar level of sequence divergence (maximum of 3%) but no substantial geographic differentiation: populations in Hokkaido, Sado, and Yakushima shared similar haplotypes with each of the central populations, suggesting that genetic exchanges occurred among the localities in the last 0.15 million years. The apparent genetic structure of the mitochondrial DNA found in the A. speciosus population might be caused solely by long-term existence in insular regions, presumably due to ecological superiority relative to A. argenteus.     

Phylogenetic relationships and biogeography of the Japanese clawed salamander, Onychodactylus japonicus (Amphibia: Caudata: Hynobiidae), and its congener inferred from the mitochondrial cytochrome b gene

Using the mitochondrial cytochrome b gene, we investigated phylogenetic relationships between and within the Japanese clawed salamander, Onychodactylus japonicus, and its close continental relative O. fischeri. Monophyly of O. japonicus was well supported, and O. japonicus was clearly distinguished from O. fischeri. However, O. fischeri comprises genetically distinct populations from Russia, NE China, and Korea that do not form a monophyletic group. Within O. japonicus, four major clades were clearly recognized: Clade I from northern Tohoku district, Clade II from southern Tohoku district and the Tsukuba Mountains, Clade III from southwestern Honshu, and Clade IV from Kinki and Chugoku districts in Honshu and from Shikoku. Although genetic distances among these clades were large (5.5-9.6%), relationships among the clades were unresolved. All clades except Clade I contained two or three distinct subclades. In several localities in Kinki and Chugoku, Clades III and IV were sympatric. The estimated divergence times and available geohistorical data suggest that O. japonicus began to differentiate in the Upper Late Miocene and that the pattern of genetic differentiation of this species has been affected strongly by climate changes and geohistorical events such as volcanic activity and mountain formation. Our results suggest that both O. fischeri and O. japonicus comprise multiple cryptic species.     

Genetic differentiation among eight color types of the freshwater goby,Rhinogobius brunneus,from western Japan

Genetic differentiation among eight color types of the freshwater goby,Rhinogobius brunneus, from the western part of Japan was investigated by using electrophoretic methods. Four sympatric types (Cross-band, Dark, Cobalt and Large-Dark (A) types) did not share alleles at between one and six loci out of 12 loci tested. No hybrid specimens were found among these types. The average genetic distances among these four types ranged from 0.13 to 0.72, which fall within the range of values among congeneric species of fishes. The average genetic distances among the other four types, Large-Dark (B), Orange, Shinji-Lake and Boso types, were only 0.01 to 0.03, and fall within the range of values among conspecific populations. These results suggest that the former four types are clearly discrete species and the latter four types may be considered as intraspecific variations of a fifth species.     

Multiple origins of Locusta migratoria (Orthoptera: Acrididae) in the Japanese Archipelago and the presence of two major clades in the world: evidence from a molecular approach

Phylogenetic relationships among migratory locust (Locusta migratoria) populations in different climatic regions were analysed by sequencing four mitochondrial DNA regions, with special reference to the origin of Japanese populations. The populations are clearly separated into two clades: one consists of individuals from temperate and cold‐temperate areas of Japan and the Chinese continent, and the other comprises those from subtropical islands of Japan, Hainan Island in China, Timol Leste, Australia, Ethiopia, France, and some individuals from Tsushima Island and Honshu of Japan. The divergence time between the two clades is estimated to be 0.86–1.89 Mya. The phylogenetic analysis revealed that Japanese L. migratoria populations were composed of individuals of six different origins: (1) Hokkaido populations possibly from the Russian continent; (2) Honshu–Kyushu populations from the Chinese continent; (3) Southwest Island populations from Hainan Island or adjacent areas; (4) Ogasawara populations that might have originated from Micronesia; (5) part of the Tsushima population that originated from somewhere in the Asian tropics; and (6) a possible relict population of ancient southern haplotypes that exists in western areas of northern Honshu. The Tsugaru Straits and Tokara Straits have acted as effective geographical barriers, as in other organisms, isolating locust populations for a few thousand years. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 570–581.     

Molecular phylogeny of the genus Apatrobus (Coleoptera: Carabidae: Patrobinae) in western Japan

We performed a molecular phylogenetic analysis of the ground beetles Apatrobus (Carabidae), endemic to Japan, using the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear 28S rRNA (28S) genes. We focused on the species divergence in Kyushu, Shikoku and western Honshu and used 15 of 19 species and three populations with undetermined species in the DNA analysis. The gene trees showed that, of the Apatrobus species studied, A. hayachinensis Nakane from northern Honshu was not included in the monophyletic group of the other Apatrobus species and likely to be of a different genus. Divergence time estimation suggested that Apatrobus species excluding A. hayachinensis diverged 5.2 million years ago and the subsequent divergence of species occurred during the Pliocene and Pleistocene. In each of the main islands, Kyushu, Shikoku and Honshu, two or more distinct lineages occurred and all species had restricted distribution areas, suggesting that ancient dispersal and vicariance among the three main islands resulted in the nested biogeographical pattern of species distribution.     

Onset of cryptic vicariance in the Japanese dormouse Glirulus japonicus (Mammalia, Rodentia) in the Late Tertiary, inferred from mitochondrial and nuclear DNA analysis

The sequences of the mitochondrial cytochrome b gene and restriction site variation in the spacer region of the nuclear ribosomal RNA gene [rDNA-restriction fragment length polymorphism (RFLP)] were analysed to determine the phylogeographic structure of the Japanese dormouse ( Glirulus japonicus ), which is threatened by deforestation and has been designated an endangered species in Japan. The phylogenetic tree of cytochrome b grouped G. japonicus into six geographical populations: north-eastern Honshu (I), central Honshu (II), west-central Honshu/Kii Peninsula (III), western Honshu (IV), Shikoku (V), and westernmost Honshu/Kyushu (VI); the genetic distances among these groups suggest divergence in the Late Tertiary. The lineage of group VI was located at the basal position in the phylogenetic tree, followed by the radiation of the other lineages. An rDNA-RFLP analysis of 15 restriction sites roughly supported such genetic isolation; groups I, II, III, IV, V and VI have five, two, one, one, one and four unique restriction sites, respectively, revealing four geographic groups as cryptic species: I, II, III + IV + V and VI. Our results reveal the ancient divergences of the local population, which has a complicated evolutionary history, and should be useful in developing a framework for the conservation of this species.     

Low genetic diversities in isolated populations of the Asian black bear (<Emphasis Type="Italic">Ursus thibetanus</Emphasis>) in Japan,in comparison with large stable populations

Populations of the Asian black bear (Ursus thibetanus) are relatively large and continuous in central Honshu, the main island of Japan, but they are isolated in western Honshu. To clarify the degree of genetic isolation of the populations in western Honshu, we compared the genetic diversities of four populations in western Honshu with that of one of the continuous populations of central Honshu. Three of the four western Honshu populations were isolated and the other was continuous with the central Honshu populations on a geographical distribution basis. The genotypes at 10 microsatellite loci of the sampled individuals were determined and the genetic structures of the populations examined. Genetic diversities were significantly lower in the isolated populations than in the continuous populations. The continuous population in central Honshu had high levels of genetic diversity, comparable to those in populations of the American black bear (Ursus americanus) and the brown bear (Ursus arctos). The genetic distances between the two continuous populations were smallest, even though their geographic distance was largest (>200 km) among all the pairs of neighboring populations examined. Low genetic diversity within the isolated populations suggested genetic drift due to the small population size; the genetic differentiation among the populations indicated low rates of gene flow among them.     

Low genetic variation and inbreeding depression in small isolated populations of the Japanese rosy bitterling, Rhodeus ocellatus kurumeus

The Japanese rosy bitterling, Rhodeus ocellatus kurumeus, has been affected not only by the invasion of another subspecies, R. o. ocellatus, from China, but also by habitat fragmentation. In this study, the effects of habitat fragmentation on the fitness of R. o. kurumeus were investigated. Owing to exclusion by R. o. ocellatus, R. o. kurumeus in Honshu and Shikoku has disappeared entirely, except for small populations in isolated man-made ponds in Osaka and Kagawa. In Kyushu it still occupies open water systems, into which R. o. ocellatus has only recently invaded. Meristic and genetic data show that the diversity of R. o. kurumeus is significantly lower in the isolated Osaka and Kagawa populations than the non-isolated Fukuoka population. The Osaka population is inferior to the Fukuoka population in terms of viability and growth. The viability of reciprocal inter-population hybrids between the Osaka and Fukuoka populations was, however, as high as that of the Fukuoka population. In addition to the high scores of band sharing index (BSI) in RAPD-PCR analysis, acceptance of transplanted scales among individuals, irrespective of natal pond, indicates that the Osaka population forms a highly inbred line. These results suggest that low genetic variation is associated with inbreeding depression in the small isolated Osaka populations. Consequently, the management of ponds, including the free movement of individuals, in addition to measures to prevent the invasion of R. o. ocellatus, is necessary for the conservation of R. o. kurumeus.