Reproductive trait divergence and hybrid fertility patterns between chromosomal races of the house mouse in Tunisia: analysis of wild and laboratory-bred males and females

The divergence in reproductive features and hybrid fertility patterns between two chromosomal races (2 n  = 40, 40St, and 2 n  = 22, 22Rb) of the house mouse in Tunisia were re-assessed on a larger sample of wild and laboratory-bred individuals than studied hitherto. Results showed that litter sizes were significantly smaller in 40St than in 22Rb mice, contrary to previous analyses. This suggests that variation in litter size between the two chromosomal races is more likely related to selective and/or environmental factors acting locally than to interracial reproductive trait divergence. However, the significantly reduced litter size of F₁ hybrids compared with parental individuals was confirmed, and further highlighted a sex difference in hybrid infertility, as F₁ females produced fewer litters and of smaller size than males. Histological analyses of F₁ and backcrosses showed a breakdown of spermatogenesis in males and a significantly reduced primordial follicle pool in females. The degree of gametogenic dysfunction was not related to the level of chromosomal heterozygosity per se , but a significant effect of two Rb fusions on follicle number was observed in hybrid females. These results suggest that genetic incompatibilities contribute to primary gametogenic dysfunction in hybrids between the chromosomal races in Tunisia.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 407–416.     

Origin and evolution of the Robertsonian populations of the house mouse (Rodentia, Muridae) in Tunisia based on allozyme studies

The central coastal region of Tunisia harbours two chromosomal races of the house mouse (2 n  = 22, 22Rb; 2 n  = 40, 40Std), which are genetically differentiated and show a high level of reproductive isolation. This study presents an allozyme analysis of house mice from the inland city of Kairouan 75 km from the coastal region. Results showed that the 22Rb and 40Std mice from Kairouan shared the same high degree of variability, and were not genetically differentiated. This contrasts with the genetic data registered for the two chromosomal races from the coastal towns, in which a particularly low genic diversity was observed in the 22Rb populations. As the two races in Kairouan show the same low hybridization rates as the populations in the coastal region, these results argue in favour of a local differentiation of the Rb race in Tunisia, most likely originating in Kairouan. The data further suggest that the decrease in variability observed in the coastal 22Rb populations is related to a founder effect or a bottleneck following the introduction of mice from Kairouan into one of these localities, prior to their dispersion throughout the coastal region.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 515–521.     

Geographical pattern of genetic variation in the Robertsonian system of Mus musculus domesticus in central Italy

The Robertsonian (Rb) system of Mus musculus domesticus in central Italy consists of three Rb races (CD, 2 n  = 22; CB, 2 n  = 22; ACR, 2 n  = 24), and additional Rb populations with new metacentrics have recently been reported. The aim of the present paper is to provide insight into the process of house mouse raciation in this area. Here we present new data concerning the geographical distribution of mtDNA haplotypes (control region sequence) in the three Rb races and in the surrounding standard populations. The presence of exclusive clades in all the three Rb races supports the idea of a past fragmentation event and a subsequent period of isolation. However, comparison of mtDNA polymorphism between Rb and standard races, and the shape of the mismatch distribution within the races, does not reveal any clear sign of a strong reduction in population size, at this geographical scale, related to race formation.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 395–405.     

Mitochondrial DNA polymorphisms of the house mouse Mus musculus domesticus from Greece, focusing on the Robertsonian chromosomal system of north-west Peloponnese

This study focused on the sequence variation in a mitochondrial region in house mice Mus musculus domesticus from chromosomally variable populations in the north-west Peloponnese, Greece. The mitochondrial molecular variation revealed was among the highest found so far for M. m. domesticus populations. The haplotype distribution pattern was rather complex. There was no clear differentiation between the populations characterized by Robertsonian chromosomes and standard all-acrocentric populations. There is therefore no indication that the Robertsonian populations were formed during a prolonged period of geographical isolation.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 643–651.     

Is chromosomal speciation occurring in house mice in Tunisia?

Two chromosomal races of house mice are present in Tunisia, one represented by mice carrying the 40-acrocentric standard karyotype and the other by a Robertsonian race (2re = 22) homozygous for nine centric fusions (Rb). A comparative summary on allozyme divergence, geographical distribution and level of reproductive isolation in the Tunisian and European Rb races is presented, to which new data on mitochondrial DNA and morphological divergence are added. The Tunisian 22Rb race revealed unique features not matched by the European chromosomal races, such as a decrease in allozymic variability, a higher level of genetic and morphological differentiation and a mosaic geographical distribution. The mtDNA analysis argued in favour of a local origin of the chromosomal divergence suggesting that the higher level of differentiation between the Tunisian races resulted from the older age of the 22Rb race and/or from a severe botdeneck. The decrease in fertility of chromosomal hybrids between the Tunisian races was compatible with the limited genetic introgression between diem. Furthermore, data on the restricted distribution of hybrid populations suggested that premating reproductive barriers may be evolving. The Tunisian 22Rb race is thus an appropriate model to investigate a chromosomally-mediated speciation event.     

Analysis of molecular differentiation in a hybrid zone between chromosomally distinct races of the common shrew Sorex araneus (Insectivora: Soricidae) suggests their common ancestry

During postglacial colonization, populations that diverged in different refugia produced a patchwork of genomes, often delimited with sharp hybrid zones. The outcome of hybridization following the secondary contact of two genetically distinct populations is hard to predict. In this context, the present study investigated the genetic structure of the hybrid zone between the Drnholec and Białowieża chromosome races of the common shrew ( Sorex araneus ) in Poland using biparentally inherited (seven autosomal microsatellites) and uniparentally inherited (Y-linked microsatellite and mtDNA) molecular markers. On the basis of diagnostic chromosomes, the Drnholec and Białowieża races were classified to different karyotypic groups, which were believed to have independent glacial histories. It was found that genetic differentiation between the Drnholec and Białowieża races was weak and nonsignificant with respect to all molecular markers. However, these results are in contrast with the chromosomal structure of this hybrid zone. The very sharp frequency clines of the diagnostic chromosomes strongly suggest that gene flow between the Drnholec and Białowieża races was reduced. Nonsignificant correlations between genetic differentiation and both the presence of an environmental barrier and geographical distance reveal that only differences in karyotypes might be a reason for limited gene exchange between the races. It is assumed that a lack of molecular differences between the Drnholec and Białowieża races results from a shared ancestral variation.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 79–90.     

Chromosomal evolution of the house mouse, Mus musculus domesticus, in the Aeolian Archipelago (Sicily, Italy)

We describe the chromosomal evolution of the metacentric populations of the house mouse, Mus musculus domesticus , which constitute the Robertsonian System of Aeolian Islands (Sicily, Italy). Eighty-nine specimens from all the seven islands that form the Archipelago were cytogenetically examined. The analysis shows the presence of 4 Rb races with a large number of shared metacentric chromosomes: 2 n  = 36 on Panarea, 2 n  = 34 on Alicudi, 2 n  = 26 on Lipari and Stromboli, and a different 2 n  = 26 race on Vulcano. On Salina and Filicudi, the standard karyotype was found. Polymorphism was only found in a population on Panarea Island and this population shares no metacentrics with the other races. The distribution of metacentrics among the races and the comparison between the Aeolian metacentrics and those found in the 97 previously documented metacentric populations allows us to formulate a hypothesis of chromosomal evolution for the Aeolian Robertsonian system. Six of the twelve metacentric chromosomes found in the Aeolian Islands come from localities outside the archipelago. The evolutionary model highlights how the chromosomal races originated inside the Archipelago and involve several factors, such as formation in situ of metacentrics, zonal raciation and, whole arm reciprocal translocation. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 194–202.     

Microsatellite variability in wild populations of the house mouse is not influenced by differences in chromosomal recombination rates

Differences in recombination rates along the chromosomes can influence the evolution of neutral loci via hitchhiking effects. Generally, these effects should be stronger in regions of low recombination than in regions of high recombination. Detailed information on physical and genetic maps in the house mouse now allows an assessment of the correlation between neutral variability and recombination rates at given chromosomal regions. We chose 29 microsatellite loci from chromosomal regions which show differences in recombination rates and tested their variability in samples from five wild populations of Mus musculus musculus and M. m. domesticus . Our results provide no evidence for a correlation between microsatellite variability and recombination rates. This suggests that the high average mutation rate of microsatellites in mammals counterbalances the effects of long-range hitchhiking in the mouse genome.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 629–635.     

Non-metric morphological divergence in the western house mouse, Mus musculus domesticus, from the Barcelona chromosomal hybrid zone

The effect of hybridization on morphological variation was investigated in 120 western house mice, Mus musculus domesticus , from the hybrid zone between the Barcelona and standard chromosomal races. The incidence of 37 non-metric cranial traits was calculated for standard mice (2 n  = 40) and Barcelona-standard hybrids (2 n  = 27–39). Subsequent analyses were conducted on several karyological subgroups, established by grouping the animals according to either their diploid number or their degree of chromosomal heterozygosity. Results revealed no significant difference by sex, asymmetry, or geographical distance. Significant phenetic divergences were found between the karyotypes studied in relation to several variants. Differences were especially substantial between the standard race and hybrid mice, even with respect to those hybrids with karyotypes close to that of the standard race. Within the hybrids, the maximum divergence corresponded to the 28-chromosome homozygotes, chromosomally close to the Barcelona race, and to the heterozygotes with more than two fusions. Since differences in non-metric trait frequencies are generally considered a measure of genetic divergence, the results suggest the occurrence of a barrier to gene flow in the Barcelona hybrid zone. The decrease of genetic exchange between the chromosomally differentiated mice might be due to reduced fertility in hybrids, associated with chromosomal heterozygosity.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 313–322.     

Chromosomal variation in the house mouse

Although the standard karyotype of the western house mouse, Mus musculus domesticus , consists entirely of acrocentric chromosomes, there are 97 distinct 'populations' that are characterized by various combinations of metacentric chromosomes that have arisen by Robertsonian (Rb) fusions and whole-arm reciprocal translocations (WARTs). In this review we discuss the processes behind the origin and fixation of these rearrangements and then present a unified list of all known metacentric populations and evaluate their phylogenetic relationships. Eleven independent phylogeographical 'systems', each consisting of 2–25 metacentric populations, were identified in Scotland, Denmark, Northern Europe–Northern Switzerland, Southern Switzerland, Northern Italy, Croatia, Spain, Central–Southern Italy, Peloponnesus, Mainland Greece and Madeira. There are six isolated metacentric populations that do not belong to any of these systems. To generate phylogenies of the metacentric populations within each system, we determined those outcomes with the fewest steps regarding accumulation of metacentrics by Rb fusions, WARTs and zonal raciation and taking into account geographical proximity. These phylogenies should be viewed as working hypotheses that will be refined with further chromosomal and molecular data and improvements in methods of phylogenetic reconstruction. The list of metacentric populations and our phylogenies are also published electronically and can be accessed at http://www.studenec.ivb.cz/Projects/RobertsonianMice/ .  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 535–563.     

Phylogeography and species limits in the Gymnodactylus darwinii complex (Gekkonidae, Squamata): genetic structure coincides with river systems in the Brazilian Atlantic Forest

Phylogenetic analyses based on mtDNA cytochrome  b were performed in 42 lizards of the Gymnodactylus darwinii complex from three regions within Brazil's Atlantic Forest. Mainland regions and continental shelf islands in the south-eastern range and mainland areas from the north-east were sampled. The criteria of maximum parsimony (MP), maximum likelihood (ML) and Bayesian methods were explored, with the robustness for nodes assessed by bootstrapping (MP and ML) and posterior probabilities (Bayesian searches). By all methods, three distinctive phylogroups were recovered: a south-eastern clade (SE) and two clades from northern regions (NE₁ and NE₂). The pattern of genetic structure of the major clades coincided with the presence of river systems in the Atlantic Forest, and based on corrected genetic distances between those clades, divergence times were tentatively estimated using mtDNA rates calibrated for squamate reptiles. The putative role of Atlantic Forest rivers in generating differentiation is discussed. We present a hypothesis of species limits for G. darwinii , based on concordant lines of evidence including cytogenetic and mtDNA analyses. Two chromosome races (cytotype A, 2n = 38; and cytotype B, 2n = 40) had distributions concordant with clades SE and NE₁ + NE₂, respectively. These races are interpreted to be full species on the basis of a number of empirical criteria.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 13–26.     

Genetic and morphological divergence reveals local subdivision of perch (Perca fluviatilis L.)

The level of gene flow is an important factor influencing genetic differentiation between populations. Typically, geographic distance is considered to be the major factor limiting dispersal and should thus only influence the degree of genetic divergence at larger spatial scales. However, recent studies have revealed the possibility for small-scale genetic differentiation, suggesting that the spatial scale considered is pivotal for finding patterns of isolation by distance. To address this question, genetic and morphological differentiation were studied at two spatial scales (range 2–13 km and range 300 m to 2 km) in the perch ( Perca fluviatilis L.) from the east coast archipelago of Sweden, using seven microsatellite loci and geometric morphometrics. We found highly significant genetic differentiation between sampled locations at both scales. At the larger spatial scale, the distance per se was not affecting the level of divergence. At the small scale, however, we found subtle patterns of isolation by distance. In addition, we also found morphological divergence between locations, congruent with a spatial separation at a microgeographic scale, most likely due to phenotypic plasticity. The present study highlights the importance of geographical scale and indicates that there might be a disparity between the dispersal capacity of a species and the actual movement of genes. Thus, how we view the environment and possible barriers to dispersal might have great implications for our ability to fully understand the evolution of genetic differentiation, local adaptation, and, in the end, speciation.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 746–758.     

Studies on the Robertsonian chromosomal variation of Mus musculus domesticus (Rodentia, Muridae) in Greece

The karyotype of the house mouse, Mus musculus domesticus , was examined in 282 specimens from 44 localities, in an effort to gain better understanding of the Robertsonian (Rb) variation known to exist in Greece. We consider that an Rb system exists in Peloponnisos, southern Greece, distributed in an area that is substantially larger than previously known. It consists of at least three Rb races with 2 n  = 30, 2 n  = 24 and 2 n  = 28, respectively, the last being reported for the first time in this paper and carrying Rb(3.6), Rb(8.12), Rb(10.14), Rb(13.15), Rb(9.16) and Rb(11.17) in a homozygous state. Additional instances of variation in this Rb system include individuals with 2 n  = 31 and 32 of variable Rb constitution and hybrids between the Rb races with 2 n  = 30 and 2 n  = 24. In southern Peloponnisos, Rb(10.14) was found in either a homozygous or a heterozygous state (2 n  = 38 or 39). The relationships among the Rb populations of Peloponnisos are discussed and hypotheses for their evolution are proposed. Rb variation was also recorded in two new locations of eastern Sterea Ellas (2 n  = 28 and 29) and one in Ipiros, north-west Greece (2 n  = 38). These findings corroborate the existence of two separate Rb systems in those two areas. Moreover, among a number of islands surveyed, Rb variation was only found in Kythira island, with Rb(10.14) in a heterozygous state (2 n  = 39). Finally, the typical all-acrocentric karyotype (2 n  = 40) was found in 51 of the animals studied from 13 localities. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 503–513.     

Morphological variation in house mice from the Robertsonian polymorphism area of Barcelona

Morphometric variation in the Robertsonian polymorphism zone of Barcelona of Mus musculus domesticus was studied by geometric morphometrics. This system is characterized by populations of reduced diploid number (2 n  = 27–39) surrounded by standard populations (2 n  = 40). We investigated the morphological variation in mice from this area, as well as the effect of geographical distance and karyotype on this variation. We also investigated the degree of co-variation between the two functional units of the mandible to explore the origin of this system (primary intergradation or secondary contact). The size and shape of the cranium, mandible and scapula were analysed for 226 specimens grouped by population, chromosome number and structural heterozygosity. Size was estimated as the centroid size, and shape was estimated after Procrustes superimposition. No significant differences in size between populations or chromosomal groups were detected. Diploid number, structural heterozygosity and local geographical isolation contributed to the differentiation in shape. Morphological differentiation between standard mice and Robertsonian specimens was observed, suggesting genetic isolation between these groups. Co-variation between the ascending ramus and alveolar region of the mandible was quantified by the trace correlation between landmark subsets of these modules. The trace values showed an ascending trend, correlated with the distance from the centre of the polymorphism area, a pattern consistent with a primary intergradation scenario.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 555–570.     

Genetic cohesion of Eresus walckenaeri (Araneae, Eresidae) in the eastern Mediterranean

The eresid spider genus Eresus is morphologically and ecologically conservative. At least three species occur in Europe. However, deep genetic divergence among geographical samples within two species, E. cinnaberinus and E. sandaliatus , may suggest more cryptic species. In the present study we investigate the genetic cohesion of the third species, Eresus walckenaeri , throughout its eastern Mediterranean distribution range, relative to the E. cinnaberinus–E. sandaliatus species complex. Eresus walckenaeri specimens were monophyletic. Genetic discreteness of E. walckenaeri in a region of sympatry with its sister species in Greece provides evidence for species integrity of E. walckenaeri within the European Eresus species complex. Eresus walckenaeri exhibited high concordance between geographical location and mtDNA genealogy. Two major phylogeographical clades were found in the Greek–Turkish and Syrian–Israel parts of the investigated area, respectively (∼6.5% sequence divergence). Concordance between geography and genetic divergence was further observed between Aegean island samples and their corresponding Greek and Turkish mainland samples, suggesting regional subdivision with gradual but potentially high dispersal propensity. Monophyly and limited regional distribution indicate Mediterranean endemic origin.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 86 , 1–9.     

Probable origin of the Robertsonian phenomena in domestic mice in Tunisia

The Robertsonian phenomenon in house mice (Mus musculus domesticus) from Tunisia consists in the presence of only one 22-chromosome Robertsonian race (22Rb) carrying the maximum number of fusions observed until now. The 22Rb populations exclusively occupy urban centers in the Eastern-Central region of Tunisia where standard population with 40-all acrocentric chromosomes (40Std) occur in surrounding neighborhoods and rural environments. In addition to the habitat partition, allozyme and mitochondrial DNA analyses showed that the 22Rb populations were genetically differentiated from the 40Std ones. This differentiation mostly stemmed from an important decrease in genetic variability in the 22Rb populations from the Sahel towns. The extent of morphological, ecological and genetical divergence observed between these chromosomal races in Tunisia is in agreement with the predictions of the chromosomal speciation model of White which advocates that karyotypic differentiation between taxa can lead to their reproductive isolation and independent evolution. Such a process is verified if the Rb process in Tunisia results from local differentiation which is supported by both the genetic and morphological data. However, the hypothesis of an origin by introduction of these mice from another region of Tunisia or from another country cannot be totally dismissed. In this study, an allozymic analysis of mice (22Rb and 40Std) from the geographically distant city of Kairouan was performed. Results showed that 22Rb and 40Std mice from Kairouan shared the same high degree of variability, and were not genetically differentiated. This contrasts with the results registered in the two chromosomal races in the Sahel towns. Such data argue in favor of a local differentiation of the Robertsonian process in Tunisia and suggest that the decrease in variability of the structural nuclear genes in the Sahel 22Rb populations can be related to an introduction from Kairouan into a Sahel locality resulting in a founder effect or followed by a severe bottleneck prior to its dispersion throughout the Sahel region.     

Conflicting patterns of genetic and morphological variation in European Gentianella section Gentianella

Genetics in European Gentianella Moench sect. Gentianella were investigated using internal transcribed spacer (ITS) nrDNA, trn L-F cpDNA and Amplified Fragment Length Polymorphisms (AFLP) fingerprint data to analyse relationships within the section. Both a cladistic and a phenetic approach, as well as nonhierarchical analysis (PCA) and partitioning of molecular variance (AMOVA), were used. Sequence data did not resolve groups within the section. They reflected, however, geographical isolation between central European and eastern Carpathian ( G. lutescens and G. bulgarica ) as well as Caucasian samples ( G. caucasea ). AFLP data revealed a strong differentiation within G. germanica between populations of the Alps and north-western middle Europe, although there is hardly any morphological differentiation of taxonomic significance. Morphological similarities between G. aspera and G. pilosa were not reflected in genetics, and no support was found for G. aspera . Genetic similarities were not in line with morphology in G. lutescens and G. bulgarica . AFLP data only partly resolve relationships among taxa. High genetic similarities, as well as partitioning of molecular variance among taxa/populations of eastern middle Europe ( G. austriaca , G. bohemica , G. germanica , G. stiriaca ) and intermediate morphological characters in G. bohemica and G. stiriaca , might indicate reticulate evolution. The overall low sequence divergence within the section points to a very young origin of all taxa.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 175–187.     

The genomics of speciation: investigating the molecular correlates of X chromosome introgression across the hybrid zone between Mus domesticus and Mus musculus

Understanding the genetic details of reproductive isolation is a key goal in the study of speciation. Hybrid zones, geographical regions where two species meet and exchange genes, can provide insight into the genetic basis of reproductive isolation. This is especially true in species with mapped molecular markers because patterns of gene flow can be compared among different genomic regions. Even greater insight can be obtained in species with complete genome sequences because gene identity, gene number and other features of interest can be assessed for genomic regions with different patterns of introgression. Here, we review recent studies on the well-characterized hybrid zone between Mus domesticus and M. musculus , including a detailed survey of patterns of introgression for 13 markers on the X chromosome. We then compare levels of introgression for these 13 regions to a number of genomic attributes inferred from the complete sequence of the X chromosome, with two purposes. First, we identify candidate genes for reproductive isolation by finding genes that map to an X-linked region of reduced introgression and that are only expressed in the male germ line or that show high rates of protein evolution in comparison with rat. Second, we ask whether patterns of gene flow are correlated with recombination rate, gene density, base composition, CpG island density, mutation rate and the rate of protein evolution, as might be expected if many genes contribute to reproductive isolation. We identify seven candidate genes for reproductive isolation between M. domesticus and M. musculus , and our analyses reveal no general correlations between levels of introgression and other measured sequence characteristics. These results underline the utility of the house mouse as a model system for the study of speciation.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 523–534.     

Hybrids between Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) do not show endogenous postzygotic isolation

Studies of prezygotic and postzygotic isolation in Drosophila have shown in general that species in sympatry tend to evolve prezygotic barriers earlier than do species in allopatry. However, postzygotic barriers tend to evolve at the same evolutionary rate in both sympatric and allopatric species. In contrast to these observations, the grasshoppers Chorthippus parallelus parallelus and C. p. erythropus show complete hybrid male sterility but only limited prezygotic isolation after an estimated 0.5 millions years of divergence. Like their congeners, C. brunneus and C. jacobsi form a hybrid zone where their ranges meet in northern Spain. However, the hybrid zone is mosaic and bimodal and, in contrast to the high levels of postzygotic isolation between C. parallelus subspecies, these two species showed no significant reduction in hybrid fitness in F₁ or backcross generations relative to the parental generations. The level of prezygotic isolation in laboratory tests was comparable to that between C. parallelus subspecies. These results suggest that endogenous postzygotic isolation does not play an important role in the reproductive isolation between C. brunneus and C. jacobsi , or in determining the structure of the hybrid zone. Exogenous postzygotic isolation may be present and should be tested in future studies.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 195–203.     

Geographic allozyme variation in the Japanese clouded salamander, Hynobius nebulosus (Amphibia: Urodela)

Using starch gel electrophoresis, geographical enzyme variation was surveyed in 46 populations of the Japanese salamander Hynobius nebulosus , which occurs widely in western Japan. This species exhibits substantial local genetic differentiation and is more diverse in inland regions than on small islands. In several different analyses, two groups of populations, one from Kyushu Island to the westernmost part of Chugoku district, Honshu (western group), and another from Shikoku through Kinki to Chubu district, Honshu (eastern group), were consistently recognized. Most of the remaining populations were placed in two less clearly-defined groups: the montane group from the Chugoku Mountains and the Chugoku group from the coastal regions of Chugoku district. The divergence patterns of H. nebulosus are thought to be related to the geological history of its range. The populations near the boundary of the western group were found to share several alleles that were predominant on both sides of the boundary, indicating past secondary contact. Differentiation in the montane group did not follow the isolation by distance model, probably because several populations included were genetically mixed with lowland populations of the Chugoku group.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 311–330.