Strong selection against hybrids maintains a narrow contact zone between morphologically cryptic lineages in a rainforest lizard

Phenotypically cryptic lineages comprise an important yet understudied part of biodiversity; in particular, we have much to learn about how these lineages are formed and maintained. To better understand the evolutionary significance of such lineages, we studied a hybrid zone between two morphologically cryptic phylogeographic lineages in the rainforest lizard, Lampropholis coggeri. Analyzing a multilocus genetic dataset through cline inference, individual-based methods and population measures of disequilibrium and using simulations to explore our genetic results in context of theoretical expectations, we inferred the processes maintaining this hybrid zone. We find that these lineages meet in a hybrid zone that is narrow (≈400 m) relative to inferred dispersal rate. Further, the hybrid zone exhibits substantial genetic disequilibrium and sharply coincident and largely concordant clines. Based on our knowledge about the region's biogeography, the species' natural history, and our simulation results, we suggest that strong selection against hybrids structures this system. As all clines show a relatively narrow range of introgression, we posit that this hybrid zone might not yet be in equilibrium. Nonetheless, our results clearly show that phylogeographic lineages can evolve substantial reproductive isolation without concomitant morphological diversification, suggesting that such lineages can constitute a significant component of evolutionary diversity.     

Discordant patterns of introgression across a narrow hybrid zone between two cryptic lineages of an Iberian endemic newt

The study of natural hybrid zones can illuminate aspects of lineage divergence and speciation in morphologically cryptic taxa. We studied a hybrid zone between two highly divergent but morphologically similar lineages (south‐western and south‐eastern) of the Iberian endemic Bosca's newt (Lissotriton boscai) in SW Iberia with a multilocus dataset (microsatellites, nuclear and mitochondrial genes). STRUCTURE and NEWHYBRIDS analyses retrieved few admixed individuals, which classified as backcrosses involving parental individuals of the south‐western lineage. Our results show asymmetric introgression of mtDNA beyond the contact from this lineage into the south‐eastern lineage. Analysis of nongeographic introgression patterns revealed asymmetries in the direction of introgression, but except for mtDNA, we did not find evidence for nonconcordant introgression patterns across nuclear loci. Analysis of a 150‐km transect across the hybrid zone showed broadly coincident cline widths (ca. 3.2–27.9 km), and concordant cline centres across all markers, except for mtDNA that is displaced ca. 60 km northward. Results from ecological niche modelling show that the hybrid zone is in a climatically homogenous area with suitable habitat for the species, suggesting that contact between the two lineages is unlikely to occur further south as their distributions are currently separated by an extensive area of unfavourable habitat. Taken together, our findings suggest the genetic structure of this hybrid zone results from the interplay of historical (biogeographic) and population‐level processes. The narrowness and coincidence of genetic clines can be explained by weak selection against hybrids and reflect a degree of reproductive isolation that is consistent with cryptic speciation.     

Genetic analysis of a contact zone between two lineages of the ocellated lizard (Lacerta lepida Daudin 1802) in south‐eastern Iberia reveal a steep and narrow hybrid zone

Measuring the diffusion of genes between diverging taxa through zones of secondary contact is an essential step to understand the extent and nature of the reproductive isolation that has been achieved. Previous studies have shown that the ocellated lizard (Lacerta lepida Daudin, 1802) has endured repeated range fragmentation associated with the climatic oscillations of the Plio‐Pleistocene that promoted diversification of many different evolutionary units within the species. However, the oldest divergence within the group is estimated to have occurred much earlier, during the Miocene, around 9 Ma and corresponds to the split between the subspecies Lacerta lepida nevadensis Buchholz (1963) and Lacerta lepida lepida Daudin (1802). Although these two evolutionary units have documented genetic and morphological differentiation, most probably accumulated during periods of allopatry, little is known about patterns of gene flow between them. In this study, we performed a population genetic analysis of a putative area of secondary contact between these two taxa, using mtDNA and microsatellite data. We assessed levels of gene flow across the contact zone to clarify to what extent gene flow may be occurring. Hybridization between the subspecies was observed by the presence of genetically introgressed individuals. However, the overall coincidence of mitochondrial and multilocus nuclear clines and generally steep clines support the idea that this contact zone is acting as a barrier to gene flow. Taken together, these results suggest that L. l. lepida and L. l. nevadensis are in independent evolutionary trajectories and should be considered as two different species.     

Analysis of a secondary contact between divergent lineages of brown trout Salmo trutta L. from Duero basin using microsatellites and mtDNA RFLPs

The analysis of 12 microsatellite loci in 16 native populations of Salmo trutta from Duero basin evidenced a strong genetic differentiation in accordance with the existence of two divergent Atlantic mtDNA lineages, Atlantic (AT) and Duero (DU). These lineages were observed spatially segregated mainly in the Lower-course and in the inner part of the basin. Unlike previous isozyme information, microsatellite data suggested a more downstream location of the sharpest genetic divergence in Duero basin and a more complex structure in the inward area. Putative hybrid populations evidenced higher Hardy–Weinberg and gametic disequilibria than pure ones (Pisuerga, Lower-course), not explained by mixture due to differential immigration pattern across the basin. Hybridization indexes suggested a bimodal pattern of hybridization and a higher weight of Pisuerga region in the genetic composition of hybrid samples in accordance with mtDNA data. The results suggested a limited introgression between AT and DU lineages. Taking into account the time of divergence between both lineages, selection and/or genetic incompatibility appeared necessary to explain the genetic structure observed and the ancient location of DU lineage restricted to this area.     

Hybridization and asymmetric introgression across a narrow zone of contact between Neotoma fuscipes and N. macrotis (Rodentia: Cricetidae)

When lineages diverge in allopatry and come into secondary contact, we have a unique opportunity to examine the degree to which they have become reproductively isolated from one another and the mechanisms that contribute to rates of interspecific gene flow. If hybridization and introgression have occurred in the past or are ongoing, examining patterns of variation in differentially inherited genetic markers can provide insight into underlying mechanisms determining interspecific reproductive interactions. We investigated genetic variation in a recently discovered contact zone between two species of woodrats (Neotoma fuscipes and N. macrotis) in central coastal California. Previous studies have found evidence of historic hybridization between these species, but an active site of sympatry had yet to be discovered. Here, we describe the first known area of sympatry between the two species and present evidence of ongoing hybridization at this site. We intensively sampled throughout the narrow area of interspecific contact and continuously into the adjacent areas where each taxon existed largely in isolation of the other. We genotyped 851 individuals sampled in a single year at 15 nuclear microsatellite loci and sequenced a portion of the cytochrome b gene in a subset of these to examine patterns of introgression. We find that the area of sympatry is less than 1 km in length, but hybridization appeared to be fairly common, with 15% of the woodrats being of hybrid origin. At least some hybrids are fertile because introgression was evident. However, introgression appeared to be asymmetric with backcrosses toward N. macrotis being more common, a pattern we suspect is due in part to directional mate choice related to differential body size. This study adds to our growing understanding of the nature of species boundaries, especially between lineages that are far along the continuum towards speciation. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 162–172.     

Asymmetric and differential gene introgression at a contact zone between two highly divergent lineages of field voles (Microtus agrestis)

Secondary contact zones have the potential to shed light on the mode and rate at which reproductive isolation accumulates during allopatric speciation. We investigated the population genetics of a contact zone between two highly divergent lineages of field voles (Microtus agrestis) in the Swiss Jura mountains. To shed light on the processes underlying introgression, we used maternally, paternally, and bi-parentally inherited markers. Though the two lineages maintained a strong genetic structure, we found some hybrids and evidence of gene flow. The extent of introgression varied with the mode of inheritance, being highest for mtDNA and absent for the Y chromosome. In addition, introgression was asymmetric, occurring only from the Northern to the Southern lineage. Both patterns seem parsimoniously explained by neutral processes linked to differences in effective sizes and sex-biased dispersal rates. The lineage with lower effective population size was also the more introgressed, and the mode-of-inheritance effect correlated with the male-biased dispersal rate of microtine rodents. We cannot exclude, however, that Haldane's effect contributed to the latter, as we found a marginally significant deficit in males (the heterogametic sex) among hybrids. We propose a possible demographic scenario to account for the patterns documented, and empirical extensions to further investigate this contact zone.     

The past and future influence of geographic information systems on hybrid zone, phylogeographic and speciation research

Over the past two decades geographers have developed an increasingly sophisticated technology termed a geographic information system (GIS). A GIS has the ability to store, map and analyse spatial data. The powerful analytical capabilities of a GIS could serve to enhance our understanding of the spatial component of the evolutionary process. In particular, phylogeographers, hybrid zone and speciation researchers could benefit enormously from incorporating this sophisticated technology from the discipline of geography, as they have done so readily from other disciplines (e.g. genetics). Indeed, an increasing number of researchers in these fields are beginning to include GIS analyses into their research programmes. Some of this integration has taken the form of analysing the spatial relationship between populations and hybrid zones. Several other researchers have also begun to incorporate GIS into their work through the use of GIS-based niche models. These models estimate a multidimensional niche for a species using known geo-referenced populations and digital climate maps. Here, I review the recent integration of GIS and GIS-based predictive niche models into the above evolutionary sub-disciplines. I also describe evolutionary analyses that could be further enhanced through the implementation of GIS.     

Genetic panmixia within a narrow contact zone between chromosomally and ecologically distinct black fly sibling species (Diptera: Simuliidae)

Hybrid zones are windows into the speciation process, and their study can give clues into the maintenance and breakdown of species boundaries. Using both genetic and ecological tools, we investigate lineage diversification across a contact zone characterized by chromosome rearrangements. We show that black fly sibling species, Simulium arcticum sensu stricto (s.s.) and Simulium saxosum, lack genetic differentiation at both microsatellite and mtDNA loci in allopatry and sympatry, as well as exhibit high levels of gene flow and continuous chromosome variation in sympatry. Furthermore, hybrid frequencies at the contact zone are similar to those seen between races, rather than species. In contrast, S. arcticum s.s. and S. saxosum maintain ecological differences and distinct habitat associations ‐ the contact zone situated at the margin of suitable habitat for each sibling species. Moreover, gene flow occurs only in a narrow band along an ecological transition. Except for the contact zone, S. arcticum s.s. and S. saxosum hybrids do not occur elsewhere within the sibling species' ranges. Although S. arcticum s.s. and S. saxosum maintain the potential to interbreed freely, we conclude that habitat associations and, perhaps, chromosome systems prevent expansion of ranges and assimilation of lineages.     

Parallel pattern of differentiation at a genomic island shared between clinal and mosaic hybrid zones in a complex of cryptic seahorse lineages

Diverging semi‐isolated lineages either meet in narrow clinal hybrid zones, or have a mosaic distribution associated with environmental variation. Intrinsic reproductive isolation is often emphasized in the former and local adaptation in the latter, although both reduce gene flow between groups. Rarely are these two patterns of spatial distribution reported in the same study system. Here, we report that the long‐snouted seahorse Hippocampus guttulatus is subdivided into discrete panmictic entities by both types of hybrid zones. Along the European Atlantic coasts, a northern and a southern lineage meet in the southwest of France where they coexist in sympatry—i.e., in the same geographical zone—with little hybridization. In the Mediterranean Sea, two lineages have a mosaic distribution, associated with lagoon‐like and marine habitats. A fifth lineage was identified in the Black Sea. Genetic homogeneity over large spatial scales contrasts with isolation maintained in sympatry or close parapatry at a fine scale. A high variation in locus‐specific introgression rates provides additional evidence that partial reproductive isolation must be maintaining the divergence. We find that fixed differences between lagoon and marine populations in the Mediterranean Sea belong to the most differentiated SNPs between the two Atlantic lineages, against the genome‐wide pattern of structure that mostly follow geography. These parallel outlier SNPs cluster on a single chromosome‐wide island of differentiation. Since Atlantic lineages do not map to lagoon‐sea habitat variation, genetic parallelism at the genomic island suggests a shared genetic barrier contributes to reproductive isolation in contrasting contexts–i.e., spatial versus ecological. We discuss how a genomic hotspot of parallel differentiation could have evolved and become associated both with space and with a patchy environment in a single study system.     

Genetic structure, introgression, and a narrow hybrid zone between northern and California spotted owls (Strix occidentalis)

The northern spotted owl (Strix occidentalis caurina) is a threatened subspecies and the California spotted owl (Strix occidentalis occidentalis) is a subspecies of special concern in the western United States. Concern for their continued viability has arisen because of habitat loss caused by timber harvesting. The taxonomic status of the northern subspecies has been the subject of continuing controversy. We investigated the phylogeographical and population genetic structure of northern and California spotted owls with special reference to their region of contact. Mitochondrial DNA (mtDNA) control region sequences confirmed the existence of two well-differentiated lineages connected by a narrow hybrid zone in a region of low population density in north central California. Maximum-likelihood estimates indicated bidirectional gene flow between the lineages but limited introgression outside the region of contact. The lengths of both the mtDNA hybrid zone and the reduced density patch were similar and slightly exceeded estimates of natal dispersal distances. This suggests that the two subspecies were in secondary contact in a hybrid zone trapped by a population density trough. Consequently, the zone of interaction is expected to be geographically stable. We discovered a third, rare clade of haplotypes, which we interpreted to be a result of incomplete lineage sorting; those haplotypes result in a paraphyletic northern spotted owl with respect to the California spotted owl. A congeneric species, the barred owl (Strix varia), occasionally hybridizes with spotted owls; our results indicated an upper bound for the frequency of barred owl mtDNA haplotypes in northern spotted owl populations of 3%.     

Morphology, genetics, and ecology of pocket gophers (genus Geomys) in a narrow hybrid zone

A population of hybrid pocket gophers ( Geomys bursarius × G. lutescens ) exists 1–2 km west of Oakdale, Antelope County, Nebraska, U.S.A. The hybrids occur in soil that has characteristics intermediate between that occupied by G. lutescens (sand) and by G. bursarius (silt loam); the vegetation associations on the different soils are Sandhills Prairie and Tall-grass Prairie, respectively, with mixed prairie on the intermediate soils. Hybrids are identifiable on the basis of both qualitative and quantitative morphological characteristics, allozymes and karyology. Concordance between morphological, allozymic, karyotypic and ecological data sets is very high. Hybrids appear to reproduce normally and survive well; i.e. they suffer no obvious loss of fitness. Backcrossing to either parental type is apparently rare. The parental species each support obligate parasitic lice ( Geomydoecus : Trichodectidae) of different species; these species are not sister species. We suggest that hybrid zones resulting from primary and secondary contact may be distinguished by (1) concordance of clines in different character sets, (2) fossil and biogeographic data, and (3) parasite data. We conclude that this zone resulted from secondary contact, and that the zone is maintained either by selection against hybrids (less likely) or by hybrid superiority (more likely).     

Sperm divergence in a passerine contact zone: Indication of reinforcement at the gametic level

Postcopulatory sexual selection may promote evolutionary diversification in sperm form, but the contribution of between‐species divergence in sperm morphology to the origin of reproductive isolation and speciation remains little understood. To assess the possible role of sperm diversification in reproductive isolation, we studied sperm morphology in two closely related bird species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia), that hybridize in a secondary contact zone spanning Central and Eastern Europe. We found: (1) striking divergence between the species in total sperm length, accompanied by a difference in the length of the mitochondrial sperm component; (2) greater divergence between species in sperm morphology in sympatry than in allopatry, with evidence for character displacement in sperm head length detected in L. megarhynchos; (3) interspecific hybrids showing sperm with a length intermediate between the parental species, but no evidence for decreased sperm quality (the proportion of abnormal spermatozoa in ejaculates). Our results demonstrate that divergence in sperm morphology between the two nightingale species does not result in intrinsic postzygotic isolation, but may contribute to postcopulatory prezygotic isolation. This isolation could be strengthened in sympatry by reinforcement.     

Secondary contact between Lycaeides idas and L. melissa in the Rocky Mountains: extensive admixture and a patchy hybrid zone

Studies of hybridization have increased our understanding of the nature of species boundaries, the process of speciation, and the effects of hybridization on the evolution of populations and species. In the present study we use genetic and morphological data to determine the outcome and consequences of secondary contact and hybridization between the butterfly species Lycaeides idas and L. melissa in the Rocky Mountains. Admixture proportions estimated from structure and geographical cline analysis indicate L. idas and L. melissa have hybridized extensively in the Rocky Mountains and that reproductive isolation was insufficient to prevent introgression for much of the genome. Geographical patterns of admixture suggest that hybridization between L. idas and L. melissa has led to the formation of a hybrid zone. The hybrid zone is relatively wide, given estimates of dispersal for Lycaeides butterflies, and does not show strong evidence of cline concordance among characters. We believe the structure of the Lycaeides hybrid zone might be best explained by the patchy distribution of Lycaeides, local extinction and colonization of habitat patches, environmental variation and weak overall selection against hybrids. We found no evidence that hybridization in the Rocky Mountains has resulted in the formation of independent hybrid species, in contrast to the outcome of hybridization between L. idas and L. melissa in the Sierra Nevada. Finally, our results suggest that differences in male morphology between L. idas and L. melissa might contribute to isolation, or perhaps even that selection has favoured the spread of L. melissa male genitalia alleles.     

Secondary contact followed by gene flow between divergent mitochondrial lineages of a widespread Neotropical songbird (Zonotrichia capensis)

Understanding how genetic and phenotypic differences that arise in geographically isolated populations influence the outcome of secondary contact advances our knowledge of speciation. In the present study, we investigate the secondary contact between divergent lineages of a widespread Neotropical songbird, the Rufous‐collared sparrow (Zonotrichia capensis). Zonotrichia capensis is morphologically and behaviourally diverse, and shows a pattern of lineage diversification produced by a Pleistocene expansion and colonization of South America from a probable Central American origin. Consistent with previous results, we find three lineages throughout the species range, showing between 1.5% and 2.5% divergence in mitochondrial control region sequences. These lineages come into secondary contact in the Dominican Republic, La Paz (Bolivia), and North‐eastern Argentina. We use DNA microsatellite data to study a broad secondary contact zone in North‐eastern Argentina, finding that Bayesian clustering analyses do not assign individuals to their respective mitochondrial lineages. Overall, we did not observe nuclear genetic discontinuities in the study area. We conclude that, if genetic, morphological, and/or cultural differences accumulated among lineages during isolation, they were insufficient to prevent gene flow after secondary contact. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 863–868.     

A hybrid zone between two subspecies of Chorthippus parallelus. X-chromosome variation through a contact zone

Two grasshopper subspecies, Chorthippus parallelus parallelus (Cpp) and Chorthippus parallelus erythropus (Cpe), meet along the Pyrenees where they hybridize and produce a hybrid zone. A contact zone located in the Col de Portalet has been analyzed for the distribution of chromosome markers on the sex (X) chromosome in pure and hybrid populations. C-banding allowed us to distinguish both pure subspecific forms and recombinant forms, and to demonstrate their different frequencies through the contact zone. Interestingly, the distal C-band (P) on the X chromosome that characterizes Cpp individuals occurs at very high frequency through the zone and then drops sharply, close to where pure Cpe populations are found. A novel interstitial C-band (H) has been found, probably derived from that characterizing Cpe individuals (E). This marker band is only present in hybrid populations. These data are discussed in the light of the dynamics of the hybrid zone.     

Morphological and mitochondrial DNA analyses indicate the presence of a hybrid zone between two species of leaf beetle (Coleoptera; Chrysomelidae) in Southern Spain

The present study reports a case where the survey of morphological and mitochondrial DNA variation among populations of a species complex of leaf beetle, the Gonioctena variabilis complex, has lead to the identification of a hybrid zone between two species of the complex in Southern Spain. The complex is divided into four species distributed around the western Mediterranean region. The four species, G. variabilis, Gonioctena aegrota, Gonioctena gobanzi, and Gonioctena pseudogobanzi, are traditionally determined by differences in the morphology of the male genitalia (aedeagus). To gain insight into the history of the speciation process within this species complex, we sampled populations in Portugal, Spain, Southern France, and Northern Italy. We sequenced a portion of the mitochondrial control region of each individual collected. A haplotype network of these sequences was found to comprise four distinct groups of sequence types, separated by a relatively large number of mutations. Moreover, in most of the samples for which morphological and molecular variation is available, there is a one‐to‐one correspondence between haplotype group, defined by mitochondrial sequence variation, and morphological groups defined on the basis of the aedeagus, showing evidence of four historically independent evolutionary units. This supports the use of the aedeagus morphology as a taxonomically informative trait in this species complex and a recent taxonomic revision upgrading four formerly subspecies, corresponding to the evolutionary units identified in the present study, to species status. However, some of the individuals from our samples in Southern Spain, morphologically identified as G. aegrota, were found to possess mitochondrial sequences typical of G. pseudogobanzi. The opposite case was also found. This suggests the presence of a zone of contact and hybridization between G. aegrota and G. pseudogobanzi. The location of this hybrid zone appears to be unusual. We identify historical scenarios that may explain our observations. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94 , 105–114.     

Genetic population structure of two cryptic Gammarus fossarum types across a contact zone

Previous studies have revealed inconsistent results about the taxonomic status of European Gammarus fossarum forms. The variability in morphology and hybridization ability has not shown clear geographic patterns, whereas on a genetical basis two Central European G. fossarum forms have been proposed. In the present study the genetic structure of G. fossarum populations was investigated across a natural contact zone. For the first time direct comparisons of allotopic versus syntopic populations were feasible. 24 Populations of G. fossarum plus 4 populations of G. pulex (as outgroup) were sampled along 2 transects across the contact zone. The genotypes of about 60 individuals per population were revealed by allozyme electrophoresis at 6 polymorphic loci. Parallel step ‘clines’ in allele frequencies indicate secondary contact of the two G. fossarum forms. The level of genetic variation between the forms was comparable to an interspecific level (G. pulex versus G. fossarum). The pronounced genetic bipartition in syntopic G. fossarum populations and the lack of potential F1-hybrids further corroborated the taxonomic status of the G. fossarum types. The geographic patterns of the genetic differentiation between and within the G. fossarum types, the allelic diversities and the genotypic disequilibria revealed recent introgression at the analysed loci to be insignificant. In addition it is shown that the population structure of the western G. fossarum type is out of equilibrium, which suggest a recent colonization of this type probably by displacement of the other type.     

Stable coexistence of incompatible Wolbachia along a narrow contact zone in mosquito field populations

In arthropods, the intracellular bacteria Wolbachia often induce cytoplasmic incompatibility (CI) between sperm and egg, which causes conditional embryonic death and promotes the spatial spread of Wolbachia infections into host populations. The ability of Wolbachia to spread in natural populations through CI has attracted attention for using these bacteria in vector‐borne disease control. The dynamics of incompatible Wolbachia infections have been deeply investigated theoretically, whereas in natural populations, there are only few examples described, especially among incompatible infected hosts. Here, we have surveyed the distribution of two molecular Wolbachia strains (wPip11 and wPip31) infecting the mosquito Culex pipiens in Tunisia. We delineated a clear spatial structure of both infections, with a sharp contact zone separating their distribution areas. Crossing experiments with isofemale lines from different localities showed three crossing types: wPip11‐infected males always sterilize wPip31‐infected females; however, while most wPip31‐infected males were compatible with wPip11‐infected females, a few completely sterilize them. The wPip11 strain was thus expected to spread, but temporal dynamics over 7 years of monitoring shows the stability of the contact zone. We examined which factors may contribute to the observed stability, both theoretically and empirically. Population cage experiments, field samples and modelling did not support significant impacts of local adaptation or assortative mating on the stability of wPip infection structure. By contrast, low dispersal probability and metapopulation dynamics in the host Cx. pipiens probably play major roles. This study highlights the need of understanding CI dynamics in natural populations to design effective and sustainable Wolbachia‐based control strategies.     

Gene flow across secondary contact zones of the Emys orbicularis complex in the Western Mediterranean and evidence for extinction and re‐introduction of pond turtles on Corsica and Sardinia (Testudines: Emydidae)

European pond turtles represent a phylogeographically deeply structured complex of distinct taxa. Here, we use mitochondrial DNA sequences (cytochrome b gene) and eight polymorphic microsatellite loci to investigate genetic differentiation and gene flow of Sicilian, Corsican and Sardinian pond turtles and of subspecies involved in two secondary contact zones in the Pyrenean region and Southern Italy. Mitochondrial and microsatellite differentiation is largely concordant in populations from the core regions of the distribution ranges of the studied taxa. Both marker systems provide no evidence for gene flow between Sicilian pond turtles (Emys trinacris) and Southern Italian subspecies of E. orbicularis. By contrast, in the contact zones limited gene flow occurs between distinct subspecies of E. orbicularis. Although the Southern Italian contact zone is significantly older than the Pyrenean contact zone of Holocene age, patterns of asymmetric introgression are similar. Introgressive hybridization leads to the exchange of mitochondria, but microsatellite data indicate only a few individuals with mixed ancestry. This suggests that incipient isolating mechanisms maintain largely discrete nuclear genomic gene pools. Furthermore, this implies that Southern Italy acted as a hotspot rather than as a melting pot of genetic diversity during the last glacial. Pond turtles from Corsica and Sardinia are not differentiated from continental populations of the subspecies E. o. galloitalica, neither in the mitochondrial nor in the quickly evolving microsatellite markers. As the fossil record argues for a continuous presence of pond turtles on both islands since the Middle Pleistocene, this suggests that the native island populations became extinct and the extant turtles were later introduced by prehistoric settlers. The lack of genetic differentiation of pond turtles from Corsica and Sardinia supports the view that the subspecies described from these islands are not valid.