Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice

When the Y chromosome of the laboratory inbred mouse strain C57BL/6 (B6) is replaced by the Y of certain strains of Mus musculus domesticus, testis determination fails and all XY fetuses develop either as hermaphrodites or XY females (XY sex reversal). This suggests the presence of at least two alleles of Sry, the male-determining gene on the Y:M. m. domesticus and B6. The B6 Y chromosome is derived from the Japanese house mouse, M. m. molossinus and therefore carries a molossinus Sry allele. As a first step to determine how the molossinus Sry allele evolved, its distribution pattern was determined in wild mice. The cumulative data of 96 M. musculus samples obtained from 58 geographical locations in Europe, North Africa, and Asia show the molossinus Sry allele is restricted to Japan and the neighboring Asian mainland and confirm that Japanese M. m. molossinus mice were derived in part from a race of M. m. musculus from Korea or Manchuria. Sry polymorphisms, as illustrated by the molossinus Sry allele, can serve as molecular markers for studies on the evolution of wild M. musculus populations and can help determine the role sex determination plays in speciation.      

Hybrid origin of Japanese mice "Mus musculus molossinus": evidence from restriction analysis of mitochondrial DNA

The Japanese mouse, Mus musculus molossinus, has long been considered an independent subspecies of the house mouse. A survey of restriction- site haplotypes of mitochondrial DNA (mtDNA) showed that Japanese mice have two main maternal lineages. The most common haplotype is closely related to the mtDNA of the European subspecies M. m. musculus. The other common haplotype and two minor ones are closely related to each other and to the mtDNA of an Asiatic subspecies, M. m. castaneus. Two other rare variants are probably the result of recent contamination by European M. m. domesticus. The musculus type of mtDNA is found in the southern two-thirds of Japan, whereas the common castaneus type is found in the northern third and the minor variants are found sporadically throughout Japan. The castaneus mtDNA lineage had a few minor variants, whereas the musculus lineage was completely monomorphic. By contrast, the native population of M. m. castaneus and the Chinese and Korean musculus populations were highly polymorphic. These results suggest that M. m. molossinus is a hybrid between ancestral colonies, possibly very small, of M. m. musculus and M. m. castaneus, rather than an independent subspecies.      

Acquisition of two endogenous ecotropic murine leukemia viruses in distinct Asian wild mouse populations.

Wild mouse DNAs were analyzed for two types of endogenous ecotropic murine leukemia viruses (MuLVs), Akv and Fv-4r-associated MuLV. Endogenous Akv viruses were found only in northern Chinese mice, Korean mice, and Japanese (Mus musculus molossinus) mice. The Fv-4r gene, which is a truncated endogenous MuLV with ecotropic interference properties, was carried by Southeast Asian (M. m. castaneus) mice, Korean mice, and M. m. molossinus. Sequences related to Fv-4r MuLV env were found only in M. m. castaneus. These findings suggest that endogenous Akv viruses were acquired by northern Chinese mice and that the Fv-4r gene or its related endogenous MuLVs were acquired independently by M. m. castaneus. The Fv-4r gene appears to have been generated hundreds of thousands of years ago, before the amplification of the Fv-4r-related endogenous MuLVs in M. m. castaneus. The coexistence of Akv viruses and the Fv-4r gene in M. m. molossinus may be explained by the hybrid origin of M. m. molossinus in crosses between northern Chinese mice and M. m. castaneus, as described in other articles. The absence of the Fv-4r-related endogenous MuLVs in M. m. molossinus may indicate that the ancestral mice of this subspecies either were an ancient type of M. m. castaneus that had acquired the Fv-4r gene but had not yet acquired the Fv-4r-related endogenous MuLVs or were a rare fraction of a mixed population of M. m. castaneus and northern Chinese mice.     

Evolutionary and dispersal history of Eurasian house mice Mus musculus clarified by more extensive geographic sampling of mitochondrial DNA

We examined the sequence variation of mitochondrial DNA control region and cytochrome b gene of the house mouse (Mus musculus sensu lato) drawn from ca. 200 localities, with 286 new samples drawn primarily from previously unsampled portions of their Eurasian distribution and with the objective of further clarifying evolutionary episodes of this species before and after the onset of human-mediated long-distance dispersals. Phylogenetic analysis of the expanded data detected five equally distinct clades, with geographic ranges of northern Eurasia (musculus, MUS), India and Southeast Asia (castaneus, CAS), Nepal (unspecified, NEP), western Europe (domesticus, DOM) and Yemen (gentilulus). Our results confirm previous suggestions of Southwestern Asia as the likely place of origin of M. musculus and the region of Iran, Afghanistan, Pakistan, and northern India, specifically as the ancestral homeland of CAS. The divergence of the subspecies lineages and of internal sublineage differentiation within CAS were estimated to be 0.37–0.47 and 0.14–0.23 million years ago (mya), respectively, assuming a split of M. musculus and Mus spretus at 1.7 mya. Of the four CAS sublineages detected, only one extends to eastern parts of India, Southeast Asia, Indonesia, Philippines, South China, Northeast China, Primorye, Sakhalin and Japan, implying a dramatic range expansion of CAS out of its homeland during an evolutionary short time, perhaps associated with the spread of agricultural practices. Multiple and non-coincident eastward dispersal events of MUS sublineages to distant geographic areas, such as northern China, Russia and Korea, are inferred, with the possibility of several different routes.     

Ancient onset of geographical divergence,interpopulation genetic exchange,and natural selection on the Mc1r coat‐colour gene in the house mouse (Mus musculus)

We examined genetic variation in house mice from India and Pakistan, a predominant part of the predicted homeland of this species and also the territory of the subspecies Mus musculus castaneus (CAS), using a nuclear marker for seven tandemly arranged genes (Fanca–Spire2–Tcf25–Mc1r–Def8–Afg3l1–Dbndd1) and compared them with those previously determined for mice from other parts of Eurasia. Construction of a network with the concatenate sequences yielded three distinct clusters representing the three major subspecies groups: CAS, Mus musculus domesticus (DOM) and Mus musculus musculus (MUS). STRUCTURE analysis provided evidence for further subdivision of CAS into two main haplogroups within the Indian subcontinent. Single‐gene networks revealed not only gene‐specific architecture for subgrouping in CAS, but also allelic exchange among subspecies. These results suggest the earlier onset of allopatric divergence in the predicted homeland (the Middle East and Indian subcontinent) and subsequent intermittent admixing via gene flow across the CAS haplogroups and among the three subspecies groups. A comparison of the levels of nucleotide diversity among the gene regions revealed a less divergent state in the chromosome region containing Mc1r and its adjacent genes, indicative of a selective sweep, suggesting the involvement of natural selection in the Mc1r allelic variation. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 778–794.     

Diverse wild mouse origins of xenotropic, mink cell focus-forming, and two types of ecotropic proviral genes

We analyzed wild mouse DNAs for the number and type of proviral genes related to the env sequences of various murine leukemia viruses (MuLVs). Only Mus species closely related to laboratory mice carried these retroviral sequences, and the different subclasses of viral env genes tended to be restricted to specific taxonomic groups. Only Mus musculus molossinus carried proviral genes which cross-reacted with the inbred mouse ecotropic MuLV env gene. The ecotropic viral env sequence associated with the Fv-4 resistance gene was found in the Asian mice M. musculus molossinus and Mus musculus castaneus and in California mice from Lake Casitas (LC). Both M. musculus castaneus and LC mice carried many additional Fv-4 env-related proviruses, two of which are common to both mouse populations, which suggests that these mice share a recent common ancestry. Xenotropic and mink cell focus-forming (MCF) virus env sequences were more widely dispersed in wild mice than the ecotropic viral env genes, which suggests that nonecotropic MuLVs were integrated into the Mus germ line at an earlier date. Xenotropic MuLVs represented the major component of MuLV env-reactive genes in Asian and eastern European mice classified as M. musculus molossinus, M. musculus castaneus, and Mus musculus musculus, whereas Mus musculus domesticus from western Europe, the Mediterranean, and North America contained almost exclusively MCF virus env copies. M. musculus musculus mice from central Europe trapped near the M. musculus domesticus/M. musculus musculus hybrid zone carried multiple copies of both types of env genes. LC mice also carried both xenotropic and MCF viral env genes, which is consistent with the above conclusion that they represent natural hybrids of M. musculus domesticus and M. musculus castaneus.     

Origins of Laboratory Mice Deduced from Restriction Patterns of Mitochondrial DNA

To determine the origins of laboratory mice, the restriction patterns of mitochondrial DNAs (mtDNAs) from various strains were compared with those of relevant subspecies and/or races of mus musculus . In most strains and substrains of laboratory mice examined (50/55), the cleavage patterns were identical to those of the European subspecies M. m. domesticus . Those that varied include two sublines of NZB, the strain NZC, and the Japanese strain RR. The NZB and NZC patterns were identical to that of the European subspecies M. m. brevirostris , which itself has restriction patterns similar to M. m. domesticus . On the other hand, the RR pattern was identical to M. m. molossinus -like mice trapped in Western China and slightly different from Japanese M. m. molossinus . These findings suggest that the strains NZB and NZC stemmed from a European founder stock which differed from the ancestral stocks of other laboratory strains and that the ancestral mice of the RR strain had been transported from China to Japan. Therefore, most laboratory strains of mice are derived from the European subspecies M. m. domesticus while M. m. brevirostris and M. m. molossinus have made minor contributions. M. m. musculus does not appear to have made any contribution.     

The musculus-type Y Chromosome of the laboratory mouse is of Asian origin

Mus musculus domesticus, M.m. bactrianus, M. m. musculus, M.m. castaneus, and M.m. molossinus wild mice were investigated for polymorphisms of the Y Chromosome (Chr) genes Zinc finger-Y (Zfy) and Sex-determining region-Y (Sry). Zfy divided the Y Chrs of these mice into domesticus- (domesticus) and musculus-types (musculus, castaneus, molossinus). M.m. bactrianus specimens had both Y Chrs, possibly owing to the introgression of a musculus-type Y into this population. Sry identified a subpopulation of musculus-type Y chromosomes. This subpopulation, designated the molossinus-type, was found in M.m. molossinus, a M. musculus subspecies specimen from northern China (Changchun), and laboratory mice. The cumulative data suggest that M.m. musculus of northern China and Korea are subpopulation distinct from M.m. musculus of Europe and central China and that this subpopulation invaded Japan, giving rise to M.m. molossinus. Furthermore, the data suggest that the musculus-type Y of the laboratory mouse originated from this subpopulation, corroborating early historical record reporting that Chinese and Japanese mice that were imported into Europe for the pet trade contributed to the genome of the laboratory mouse.     

Origins of laboratory mice deduced from restriction patterns of mitochondrial DNA

To determine the origins of laboratory mice, the restriction patterns of mitochondrial DNAs (mtDNAs) from various strains were compared with those of relevant subspecies and/or races of Mus musculus. In most strains and substrains of laboratory mice examined (50/55), the cleavage patterns were identical to those of the European subspecies M. m. domesticus. Those that varied include two sublines of NZB, the strain NZC, and the Japanese strain RR. The NZB and NZC patterns were identical to that of the European subspecies M. m. brevirostris, which itself has restriction patterns similar to M. m. domesticus. On the other hand, the RR pattern was identical to M. m. molossinus-like mice trapped in Western China and slightly different from Japanese M. m. molossinus. These findings suggest that the strains NZB and NZC stemmed from a European founder stock which differed from the ancestral stocks of other laboratory strains and that the ancestral mice of the RR strain had been transported from China to Japan. Therefore, most laboratory strains of mice are derived from the European subspecies M. m. domesticus while M. m. brevirostris and M. m. molossinus have made minor contributions. M. m. musculus does not appear to have made any contribution.     

Different phylogeographic patterns in two Japanese Silpha species (Coleoptera: Silphidae) affected by climatic gradients and topography

To reveal differences in phylogeographic patterns of flightless insect species occurring in different regions of Japan, we studied the phylogeography and demographic history of Silpha beetles occurring in cool-temperate habitats of two major islands, Honshu and Hokkaido, using sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. Honshu has a more mountainous topography, and cool-temperate habitats occur discontinuously, whereas Hokkaido, located to the north of Honshu, has more continuous cool-temperate habitats. A species endemic to Honshu, S. longicornis occurs on Honshu, whereas S. perforata occurs on Hokkaido and the East Asian continent. Our results indicate that the ancestors of S. longicornis colonized Honshu via a south-west route c . 0.7 Mya and the species has highly divergent populations in isolated mountainous areas of Honshu, whereas S. perforata colonized Hokkaido via a northern route less than 90 000 years ago and has less divergent geographic populations. During the last glacial period, S. perforata was probably restricted to refugia in southern Hokkaido and later expanded into northern Hokkaido, whereas S. longicornis populations existed in many isolated refugia, probably because of the complex topography of Honshu. Thus, our study demonstrates that, even between closely related species, interactions among biology, latitudinal climatic gradients and topography can produce different phylogeographic patterns.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 452–467.     

Sry promoters from domesticus (Tirano) and C57BL/6 mice function similarly in embryos and adult animals.

Introduction of the Y chromosome from a Mus musculus domesticus (Tirano) subspecies into the Mus musculus musculus C57BL/6 (B6) inbred strain background results in sex reversal in XY offspring. It has been hypothesized that the domesticus testis-determining Y (Tdy) locus is misregulated in B6 genome, thereby impairing sex determination in B6.Y(Dom) animals. The identification of a gene in the sex-determining region on the Y chromosome (Sry) as the Tdy has provided a means to experimentally examine this hypothesis. We have generated several lines of B6 transgenic mice harboring a green fluorescent protein gene directed by a Sry promoter from the domesticus (Tirano) Y chromosome. Detailed analysis of the transgene expression was conducted in both fetal and adult tissues of the transgenic mice. The domesticus Sry promoter was capable of directing the expression of the green fluorescent protein gene in a pattern similar, if not identical, to that of the endogenous B6 Sry gene. These observations suggest that the domesticus Sry promoter is not involved in the postulated misregulation of the domesticus (Tirano) Sry gene in the B6 genomic background. These results are discussed with reference to a second hypothesis invoking incompatible protein interaction(s) as a mechanism of aberrant sex determination in B6.Y(Dom) animals.     

Genetic differentiation of house mice in the fauna of the former U.S.S.R.: results of cytogenetic studies

A cytogenic study of nearly 200 house mice (Mus musculus sensu stneto) and aboriginal mice (Mus hortulanus, Mus abbotti) of the subgenus Mus was carried out. Mice were sampled from most localities in the former U.S.S.R., from the western borders to the Far East, and it was shown that it is possible to use cytogenetic markers to classify the species and rare subspecies of the subgenus. Such markers included the characteristic morphology of the sex chromosomes and the pattern of distribution of the C-heterochromatin in the karyotype. Thus, the aboriginal mice, together with M. spretus , are characterized by a significant reduction in the size of the Y chromosome. In addition, the variant of the X chromosome (so called 'molossinus' lype) previously only observed in Japanese M. in. molossinus was found in all the Mus musculus sampled from the fauna of the former U.S.S.R. Another type, the so called 'domeslicus' is a plesiomorphic variant of the X chromosome which is normally found in M. domestuus. M. hortulanus, M. abbotti and possibly in M. spretus. The presence of the common variant X chromosome in the house mice of the various subspecies in the fauna of the former U.S.S.R., Mongolia (raddei) and Japan (molossinus) provides the basis for the integration of Asian house mice into the one species, At. musculus sensu stricto. The problems of morphology, ecology and systematics of the mouse fauna of the former U.S.S.R. are also discussed with special attention being paid to the studies of the so called 'wagnen' form.     

Differing populations of intracisternal A-particle genes in myeloma tumors and mouse subspecies.

Intracisternal A-particle genes form a family of endogenous retrovirus-like genetic elements that are transcribed in mouse plasmacytomas (myeloma tumors). Two types of A-particle genes that can be differentiated by a sequence of 0.5 kilobase found in one type but not the other have been identified. Quantitative Southern blot analysis was used to measure the populations of different A-particle genes in DNAs from BALB/c mice, the Japanese subspecies Mus musculus subsp. molossinus, and myeloma tumors. The majority of the genes (715 copies per haploid genome or 76%) were found to be nearly identical except for small changes in conserved restriction enzyme sites. The second type of A-particle gene was much less abundant with 90 copies representing approximately 10%. The A-particle RNA in MOPC104E and MOPC315 was found to be colinear with a small portion of this latter type, comprising only 2% of the endogenous intracisternal A-particle sequences. Myeloma tumor DNA was found to have a two- to fourfold increase in the number of these genes, suggesting that the intracellular viruses have been activated to produce a double-stranded complementary DNA which subsequently integrated into the tumor genome. Analysis of M. musculus subsp. molossinus DNA revealed similar but shifted populations of A-particle genes, when compared with BALB/c DNA, except for the absence of a prominent EcoRI-HindIII band at 3.9 kilobases. This latter band, representing approximately 15% of the A-particle genes in BALB/c DNA, was shown to be a deletion variant of the most abundant gene family.     

Microsatellite genetic variation of Asian populations of Dolly Varden char

Genetic variation at eight microsatellite loci was examined in 21 populations of Dolly Varden charrs, Salvelinus malma, representing five geographical regions (Kamchatka Peninsula, Sea of Okhotsk coast, Sea of Japan coast, Sakhalin Island, and Kuril islands). Hierarchical analysis of molecular variance showed that 11% (58% in terms of R-statistics) of the variation was distributed among of northern subspecies of Dolly Varden, Salvelinus malma malma and southern Asian Dolly Varden, S. m. krascheninnikovi while similar values were attributed to the among-regional level within northern Dolly Varden 9% (7%) and southern Asian Dolly Varden 11% (14%). Permutation-based tests indicated a mutational component to genetic differentiation based on allelic size variance and suggested that divergence of the two subspecies had occurred at least 3,000 generations ago. On large spatial scales (within the Asian range of the species), populations clustered according to their geographical location. On smaller scales (within regions and subregions) correlation between genetic and geographic distances was not significant. Northern Dolly Varden has higher allelic diversity and more private alleles than southern subspecies, this probably indicating differences in demographic history.     

Mosaic genealogy of the Mus musculus genome revealed by 21 nuclear genes from its three subspecies

Patterns of genetic variation provide insight into the evolutionary history of a species. Mouse (Mus musculus) is a good model for this purpose. Here we present the analysis of genealogies of the 21 nuclear loci and one mitochondrial DNA region in M. musculus based on our nucleotide sequences of nine inbred strains from three M. musculus subspecies (musculus, domesticus, and castaneus) and one M. spicilegus strain as an outgroup. The mitochondrial DNA gene genealogy of those strains confirmed the introgression pattern of one musculus strain. When all the nuclear DNA data were concatenated to produce a phylogenetic tree of nine strains, musculus and domesticus strains formed monophyletic clusters with each other, while the two castaneus strains were paraphyletic. When each DNA region was treated independently, the phylogenetic networks revealed an unnegligibly high level of subspecies admixture and the mosaic nature of their genome. Estimation of ancestral and derived population sizes and migration rates suggests the effects of ancestral polymorphism and gene flow on the pattern of genetic variation of the current subspecies. Gene genealogies of Fut4 and Dfy loci also suggested existence of the gene flow between M. musculus and M. spicilegus or other distant species.     

Phylogeography of a freshwater sculpin, Cottus nozawae, from the northeastern part of Honshu Island, Japan

 The fluvial sculpin, Cottus nozawae, is a coldwater-adapted fish distributed in Hokkaido Island and the northeastern part of Honshu Island (Tohoku District), Japan. Mitochondrial DNA (mtDNA) control region sequencing was used to investigate the geographic distribution of genetic variation and phylogeography of C. nozawae. Most populations possessed unique haplotypes, few being shared across river systems. Phylogenetic analysis of the sequences of the mtDNA control region and adjacent regions of C. nozawae revealed three distinct phylogenetic groups that differed by 3.05% to 3.11%, corresponding to distinct geographic regions, Hokkaido Island, northern Tohoku District, and Yamagata Prefecture (southwestern Tohoku District), respectively. The divergence times of three groups were estimated to be about 1.5 million years ago by applying a general rate for mtDNA, suggesting that the divergence among them might have occurred in the early Pleistocene. Divergence among the haplotypes within the group from the northern Tohoku District was also high (1.84%), no haplotypes being shared by local populations in different river systems in this region. Local populations from a single river system in this region comprise a distinct lineage that differed from other river systems. Such genetically divergent population structures among the different regions and river systems are considered to have resulted mainly from long-term isolation and restricted gene flow among river systems, probably promoted by the fluvial benthic life history and low dispersal ability of this species. Received: April 12, 2001 / Revised: December 1, 2001 / Accepted: December 19, 2001     

C57BL/6J-T-associated sex reversal in mice is caused by reduced expression of a Mus domesticus Sry allele

C57BL/6J-T-associated sex reversal (B6-TAS) in XY mice results in ovarian development and involves (1) hemizygosity for Tas, a gene located in the region of Chromosome 17 deleted in T(hp) and T(Orl), (2) homozygosity for one or more B6-derived autosomal genes, and (3) the presence of the AKR Y chromosome. Here we report results from experiments designed to investigate the Y chromosome component of this sex reversal. Testis development was restored in B6 T(Orl)/+ XY(AKR) mice carrying a Mus musculus Sry transgene. In addition, two functionally different classes of M. domesticus Sry alleles were identified among eight standard and two wild-derived inbred strains. One class, which includes AKR, did not initiate normal testis development in B6 T(Orl)/+ XY mice, whereas the other did. DNA sequence analysis of the Sry ORF and a 5' 800-bp segment divided these inbred strains into the same groups. Finally, we found that Sry is transcribed in B6 T(Orl)/+ XY(AKR) fetal gonads but at a reduced level. These results pinpoint Sry as the Y-linked component of B6-TAS. We hypothesize that the inability of specific M. domesticus Sry alleles to initiate normal testis development in B6 T(Orl)/+ XY(AKR) mice results from a biologically insufficient level of Sry expression, allowing the ovarian development pathway to proceed.     

Restriction fragment length variations and chromosome mapping of two mouse metallothionein genes,Mt-1 andMt-2

Restriction endonuclease fragment length variations (RFLVs) were found through the use of cDNA probes for metallothionein genes 1 (Mt-1) and 2 (Mt-2) in the mouse. RFLVs were detected in restriction patterns generated by BglII and XbaI in the Mt-1 gene and by PvuII in the Mt-2 gene. All laboratory strains carry the Mt-1a and Mt-2a alleles. Among strains of wild origin, some Western European subspecies (Mus mus domesticus and M. m. brevirostris) also carry the Mt-1a and Mt-2a alleles. In contrast, a European subspecies (M. m. musculus) and the great majority of subspecies from East Asian countries (M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai) carry the Mt-1b and Mt-2b alleles. A domesticus strain from Bulgaria and two castaneus strains from Thailand and Philippines carry the intermediate combination of Mt-1b and Mt-2a alleles. Using the RFLVs, we mapped the Mt-1 and Mt-2 genes on chromosome 8, and they appear to be very closely linked since no recombination was observed between them in any of the mice examined. Data from three-point cross tests showed that the recombination frequencies are 4.31% between Os and Mt, 15.52% between Mt and Prt-2, and 19.83% between Os and Prt-2. The gene order of Os-Mt-1,Mt-2-Prt-2 has been confirmed.     

Genetic variation and differentiation of the two river sculpins,Cottus nozawae andC. amblystomopsis,deduced from allozyme and restriction enzyme-digested mtDNA fragment length polymorphism analyses

Genetic differentiation of the two sibling species,Cottus nozawae andC. amblystomopsis, from the northern part of Japan (Hokkaido Island and the Tohoku District) was investigated using allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA. Although the two species are morphologically very similar, previously being thought to be a single species, they have different life-cycles;C. nozawae has a fluvial life-cycle with a small number of large-sized eggs, whereasC. amblystomopsis is an amphidromous species with a large number of small-sized eggs. Four populations ofC. amblystomopsis from Hokkaido Island and 24 populations ofC. nozawae (22 from Hokkaido Island and 2 from the Tohoku District) were sampled and examined Intrapopulational differentiation in the two species was measured by examining several indexes, including proportion of polymorphic loci (P), mean heterozygosity (H) and nucleotide diversity (π). All measurements were higher in theC. amblystomopsis populations, suggesting that intrapopulational variation inC. nozawae was less than inC. amblystomopsis and reflecting the difference in effective population sizes between them. Cluster analyses were performed using the UPGMA method, based on the data matrices of genetic distance (D) and the net nucleotide difference (δ) between populations. TheC. nozawae andC. amblystomopsis populations from Hokkaido Island composed a large cluster (Hokkaido group), while theC. nozawae populations from the Tohoku District composed a different cluster (Tohoku group). Bootstrap probabilities deduced from 1000 bootstrap replications for presence or absence of restriction sites showed that the mtDNA haplotypes detected within the Tohoku Group occurred in 99.9% of the bootstrap replicates outside the mtDNA haplotypes of the Hokkaido group, while those within the Hokkaido group occurred in 3.5–64.9% of bootstrap replicates. Consequently, the Hokkaido populations of the two species (Hokkaido group) were genetically close to each other, whileC. nozawae from the Tohoku District (Tohoku group) were distant from the Hokkaido group. These results suggest that the ancestral populations of the two species on Hokkaido Island shared the same gene pool, even after becoming geographically isolated from the ancestral population ofC. nozawae in the Tohoku District by the formation of the Tsugaru Straits.