High genomic diversity in the bank vole at the northern apex of a range expansion: The role of multiple colonizations and end-glacial refugia.

The history of repeated northern glacial cycling and southern climatic stability has long dominated explanations for how genetic diversity is distributed within temperate species in Eurasia and North America. However, growing evidence indicates the importance of cryptic refugia for northern colonization dynamics. An important geographic region to assess this is Fennoscandia, where recolonization at the end of the last glaciation was restricted to specific routes and temporal windows. We used genomic data to analyse genetic diversity and colonization history of the bank vole (Myodes glareolus) throughout Europe (>800 samples) with Fennoscandia as the northern apex. We inferred that bank voles colonized Fennoscandia multiple times by two different routes; with three separate colonizations via a southern land-bridge route deriving from a "Carpathian" glacial refugium and one via a north-eastern route from an "Eastern" glacial refugium near the Ural Mountains. Clustering of genome-wide SNPs revealed high diversity in Fennoscandia, with eight genomic clusters: three of Carpathian origin and five Eastern. Time estimates revealed that the first of the Carpathian colonizations occurred before the Younger Dryas (YD), meaning that the first colonists survived the YD in Fennoscandia. Results also indicated that introgression between bank and northern red-backed voles (Myodes rutilus) took place in Fennoscandia just after end-glacial colonization. Therefore, multiple colonizations from the same and different cryptic refugia, temporal and spatial separations and interspecific introgression have shaped bank vole genetic variability in Fennoscandia. Together, these processes drive high genetic diversity at the apex of the northern expansion in this emerging model species.

Genomics of end-Pleistocene population replacement in a small mammal.

Current species distributions at high latitudes are the product of expansion from glacial refugia into previously uninhabitable areas at the end of the last glaciation. The traditional view of postglacial colonization is that southern populations expanded their ranges into unoccupied northern territories. Recent findings on mitochondrial DNA (mtDNA) of British small mammals have challenged this simple colonization scenario by demonstrating a more complex genetic turnover in Britain during the Pleistocene-Holocene transition where one mtDNA clade of each species was replaced by another mtDNA clade of the same species. Here, we provide evidence from one of those small mammals, the bank vole (Clethrionomys glareolus), that the replacement was genome-wide. Using more than 10 000 autosomal SNPs we found that similar to mtDNA, bank vole genomes in Britain form two (north and south) clusters which admix. Therefore, the genome of the original postglacial colonists (the northern cluster) was probably replaced by another wave of migration from a different continental European population (the southern cluster), and we gained support for this by modelling with approximate Bayesian computation. This finding emphasizes the importance of analysis of genome-wide diversity within species under changing climate in creating opportunities for sophisticated testing of population history scenarios.

Phylogenetic evidence for a new species of Barbus in the Danube River basin.

Three species of small-sized rheophilic Barbus fishes are endemic to and widely distributed throughout the mountain regions in the Danube River basin. In Hungary, barbels referred to as B. petenyi occur in streams in the foothills of the Carpathians near the borders with Slovakia, Ukraine and Romania. However, up to now, no genetic investigations were carried out on rheophilic barbels in this region. This study aims to clarify the taxonomic identity and distribution of the rheophilic barbels in the Hungarian plain based on molecular and morphological analyses. Two mitochondrial genes (cytochrome b, ATPase 6/8) and one nuclear gene (beta-actin intron 2) were sequenced and several morphometric and meristic characters were recorded. Phylogenetic and morphological analyses revealed that there are four genetically distinct lineages among the rheophilic barbels in the Carpathian Basin. The results demonstrated that North-Hungarian Barbus populations belong to B. carpathicus and that B. petenyi presumably does not occur in Hungary. As expected, B. balcanicus was only recorded in samples from the Balkans analyzed for reference. A distinct species, new to science, was discovered to be present in Sebes-Körös River (Crisul Repede) in eastern Hungary and western Romania and is formally described here as B. biharicus Antal, László, Kotlík - sp. nov.

Mapping 3' transcript ends in the bank vole (Clethrionomys glareolus) mitochondrial genome with RNA-Seq.

BACKGROUND: Although posttranscriptional modification of mitochondrial (mt) transcripts plays key roles in completion of the coding information and in the expression of mtDNA-encoded genes, there is little experimental evidence on the polyadenylation status and the location of mt gene poly(A) sites for non-human mammals. RESULTS: Poly(A)-enriched RNA-Seq reads collected for two wild-caught bank voles (Clethrionomys glareolus) were mapped to the complete mitochondrial genome of that species. Transcript polyadenylation was detected as unmapped adenine residues at the ends of the mapped reads. Where the tRNA punctuation model applied, there was the expected polyadenylation, except for the nad5 transcript, whose polyadenylated 3' end is at an intergenic sequence/cytochrome b boundary. As in human, two pairs of bank vole genes, nad4l/nad4 and atp8/atp6, are expressed from bicistronic transcripts. TAA stop codons of four bank vole protein-coding genes (nad1, atp6, cox3 and nad4) are incompletely encoded in the DNA and are completed by polyadenylation. This is three genes (nad2, nad3 and cob) less than in human. The bank vole nad2 gene encodes a full stop codon (TAA in one vole and TAG in the other), which is followed by a 2 bp UTR and the gene conforms to the tRNA punctuation model. In contrast, the annotations of the reference mouse and some other rodent mt genomes in GenBank include complete TAG stop codons in both nad1 and nad2, which overlap downstream trnI and trnW, respectively. Thus the RNA-Seq data of bank voles provides a model for stop codons of mt-encoded genes in mammals comparable to humans, but at odds with some of the interpretation based purely on genomic data in mouse and other rodents. CONCLUSIONS: This work demonstrates how RNA-Seq data were useful to recover mtDNA transcriptome data in a non-model rodent and to shed more light on mammalian mtDNA transcriptome and post-transcriptional modification. Even though gene content and organisation of mtDNA are strongly conserved among mammals, annotations that neglect the transcriptome may be prone to errors in relation to the stop codons.

Mitogenomic phylogenetics of the bank vole Clethrionomys glareolus, a model system for studying end-glacial colonization of Europe.

We have revisited the mtDNA phylogeny of the bank vole Clethrionomys glareolus based on Sanger and next-generation Illumina sequencing of 32 complete mitochondrial genomes. The bank vole is a key study species for understanding the response of European fauna to the climate change following the Last Glacial Maximum (LGM) and one of the most convincing examples of a woodland mammal surviving in cryptic northern glacial refugia in Europe. The genomes sequenced included multiple representatives of each of the eight bank vole clades previously described based on cytochrome b (cob) sequences. All clades with the exception of the Basque - likely a misidentified pseudogene clade - were highly supported in all phylogenetic analyses and the relationships between the clades were resolved with high confidence. Our data extend the distribution of the Carpathian clade, the marker of a northern glacial refugium in the Carpathian Mountains, to include Britain and Fennoscandia (but not adjacent areas of continental Europe). The Carpathian sub-clade that colonized Britain and Fennoscandia had a somewhat different history from the sub-clade currently found in or close to the Carpathians and may have derived from a more north-westerly refugial area. The two bank vole populations that colonized Britain at the end of the last glaciation are for the first time linked with particular continental clades, the first colonists with the Carpathian clade and the second colonists with the western clade originating in a more southerly refugium in the vicinity of the Alps. We however found no evidence that a functional divergence of proteins encoded in the mitochondrial genome promoted the partial genetic replacement of the first colonists by the second colonists detected previously in southern Britain. We did identify one codon site that changed more often and more radically in the tree than expected and where the observed amino acid change may affect the reductase activity of the cytochrome bc1 complex, but the change was not specific to a particular clade. We also found an excess of radical changes to the primary protein structure for geographically restricted clades from southern Italy and Norway, respectively, possibly related to stronger selective pressure at the latitudinal extremes of the bank vole distribution. However, overall, we find little evidence of pervasive effects of deviation from neutrality on bank vole mtDNA phylogeography.

Speciation history and widespread introgression in the European short-call tree frogs (Hyla arborea sensu lato, H. intermedia and H. sarda).

European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form six genetically differentiated but morphologically cryptic taxa, H. arborea sensu stricto, H. orientalis and H. molleri from across Europe to western Asia (together referred to as H. arborea sensu lato), two putative taxa within H. intermedia (Northern and Southern) from the Italian Peninsula and Sicily, and H. sarda from Sardinia and Corsica. Here, we assess species limits and phylogenetic relationships within these 'short-call tree frogs' based on mitochondrial DNA and nuclear protein-coding markers. The mitochondrial and nuclear genes show partly incongruent phylogeographic patterns, which point to a complex history of gene flow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogs and to infer the species tree, we used coalescent-based approaches. The monophyly of H. arborea sensu lato is supported by the mtDNA as well as by the all-gene species tree. The Northern and Southern lineages of H. intermedia have been connected by nuclear gene flow (despite their deep mtDNA divergence) and should be treated as conspecific. On the contrary, the parapatric taxa within H. arborea sensu lato should be considered distinct species (H. arborea, H. orientalis, H. molleri) based on the coalescent analysis, although signs of hybridization were detected between them (H. arborea×H. orientalis; H. arborea×H. molleri). A mitochondrial capture upon secondary contact appears to explain the close mtDNA relationship between the geographically remote Iberian H. molleri and H. orientalis from around the Black Sea. Introgressive hybridization occurred also between the Balkan H. arborea and northern Italian H. intermedia, and between the Minor Asiatic H. orientalis and Arabian H. felix arabica (the latter belonging to a different acoustic group/clade). Our results shed light on the species limits in the European short-call tree frogs and show that introgression played an important role in the evolutionary history of the short-call tree frogs and occurred even between taxa supported as distinct species.

Adaptive phylogeography: functional divergence between haemoglobins derived from different glacial refugia in the bank vole.

Over the years, researchers have used presumptively neutral molecular variation to infer the origins of current species' distributions in northern latitudes (especially Europe). However, several reported examples of genic and chromosomal replacements suggest that end-glacial colonizations of particular northern areas may have involved genetic input from different source populations at different times, coupled with competition and selection. We investigate the functional consequences of differences between two bank vole (Clethrionomys glareolus) haemoglobins deriving from different glacial refugia, one of which partially replaced the other in Britain during end-glacial climate warming. This allows us to examine their adaptive divergence and hence a possible role of selection in the replacement. We determine the amino acid substitution Ser52Cys in the major expressed ß-globin gene as the allelic difference. We use structural modelling to reveal that the protein environment renders the 52Cys thiol a highly reactive functional group and we show its reactivity in vitro. We demonstrate that possessing the reactive thiol in haemoglobin increases the resistance of bank vole erythrocytes to oxidative stress. Our study thus provides striking evidence for physiological differences between products of genic variants that spread at the expense of one another during colonization of an area from different glacial refugia.

Genomic signatures of climate adaptation in bank voles.

Evidence for divergent selection and adaptive variation across the landscape can provide insight into a species' ability to adapt to different environments. However, despite recent advances in genomics, it remains difficult to detect the footprints of climate-mediated selection in natural populations. Here, we analysed ddRAD sequencing data (21,892 SNPs) in conjunction with geographic climate variation to search for signatures of adaptive differentiation in twelve populations of the bank vole (Clethrionomys glareolus) distributed across Europe. To identify the loci subject to selection associated with climate variation, we applied multiple genotype-environment association methods, two univariate and one multivariate, and controlled for the effect of population structure. In total, we identified 213 candidate loci for adaptation, 74 of which were located within genes. In particular, we identified signatures of selection in candidate genes with functions related to lipid metabolism and the immune system. Using the results of redundancy analysis, we demonstrated that population history and climate have joint effects on the genetic variation in the pan-European metapopulation. Furthermore, by examining only candidate loci, we found that annual mean temperature is an important factor shaping adaptive genetic variation in the bank vole. By combining landscape genomic approaches, our study sheds light on genome-wide adaptive differentiation and the spatial distribution of variants underlying adaptive variation influenced by local climate in bank voles.

Local adaptation and future climate vulnerability in a wild rodent.

As climate change continues, species pushed outside their physiological tolerance limits must adapt or face extinction. When change is rapid, adaptation will largely harness ancestral variation, making the availability and characteristics of that variation of critical importance. Here, we used whole-genome sequencing and genetic-environment association analyses to identify adaptive variation and its significance in the context of future climates in a small Palearctic mammal, the bank vole (Clethrionomys glareolus). We found that peripheral populations of bank vole in Britain are already at the extreme bounds of potential genetic adaptation and may require an influx of adaptive variation in order to respond. Analyses of adaptive loci suggest regional differences in climate variables select for variants that influence patterns of population adaptive resilience, including genes associated with antioxidant defense, and support a pattern of thermal/hypoxic cross-adaptation. Our findings indicate that understanding potential shifts in genomic composition in response to climate change may be key to predicting species' fate under future climates.

A mitochondrial DNA phylogeny of the endangered vipers of the Vipera ursinii complex.

The last two populations of the Hungarian meadow viper Vipera ursinii rakosiensis were thought to persist in the steppe fragments of Hungary until meadow vipers were discovered in central Romania (Transylvania), suggesting a possible existence of remnant populations elsewhere. We assessed the phylogenetic position of the Transylvanian vipers using 2030 bp of mitochondrial DNA sequence. We showed that they were closely related to the Hungarian vipers, while those from northeastern Romania (Moldavia) and Danube Delta belonged to the subspecies Vipera ursinii moldavica. Montane subspecies from Europe (Vipera ursinii ursinii and Vipera ursinii macrops) formed a sister clade to the two lowland subspecies. Vipera renardi formed a sister clade to V. ursinii, with populations from the Greater Caucasus (Vipera renardi lotievi) and Tien Shan (Vipera renardi tienshanica) as the sister group to Vipera renardi renardi, and Vipera renardi eriwanensis from the Lesser Caucasus as the most basal taxon in the species. Our results illustrate that the divergence between the lowland and montane populations occurred separately in each species and several times in V. renardi. We demonstrated that the recently discovered Transylvanian population is the third surviving population of V. u. rakosiensis and the only known population outside of Hungary.

Genic distribution modelling predicts adaptation of the bank vole to climate change.

The most likely pathway for many species to survive future climate change is by pre-existing trait variation providing a fitness advantage under the new climate. Here we evaluate the potential role of haemoglobin (Hb) variation in bank voles under future climate change. We model gene-climate relationships for two functionally distinct Hb types, HbS and HbF, which have a north-south distribution in Britain presenting an unusually tractable system linking genetic variation in physiology to geographical and temporal variation in climate. Projections to future climatic conditions suggest a change in relative climatic suitability that would result in HbS being displaced by HbF in northern Britain. This would facilitate local adaptation to future climate-without Hb displacement, populations in northern Britain would likely be suboptimally adapted because their Hb would not match local climatic conditions. Our study shows how pre-existing physiological differences can influence the adaptive capacity of species to climate change.

Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction.

Orthohantaviruses are rodent-borne emerging viruses that may cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses-Puumala virus (PUUV), responsible for a mild to moderate form of hemorrhagic fever with renal syndrome in humans, Tula virus (TULV) with low pathogenicity, and non-pathogenic Prospect Hill virus (PHV)-with human and rodent host cell lines. Besides the fact that cell susceptibility to virus infection was shown to depend on the cell type and virus strain, the three orthohantaviruses were able to infect Vero E6 and HuH7 human cells, but only the former secreted infectious particles. In cells derived from PUUV reservoir, the bank vole (Myodes glareolus), PUUV achieved a complete viral cycle, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting differences in host specificity. A search for mature virions by electron microscopy (EM) revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. We described differential interactions of orthohantaviruses with cellular factors, as supported by the cellular distribution of viral nucleocapsid protein with cell compartments, and proteomics identification of cellular partners. Our results also showed that interferon (IFN) dependent gene expression was regulated in a cell and virus species dependent manner. Overall, our study highlighted the complexity of the host-virus relationship and demonstrated that orthohantaviruses are restricted at different levels of the viral cycle. In addition, the study opens new avenues to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how it depends on tissue type and host species.

Niche differentiation in a postglacial colonizer, the bank vole Clethrionomys glareolus.

Species-level environmental niche modeling has been crucial in efforts to understand how species respond to climate variation and change. However, species often exhibit local adaptation and intraspecific niche differences that may be important to consider in predicting responses to climate. Here, we explore whether phylogeographic lineages of the bank vole originating from different glacial refugia (Carpathian, Western, Eastern, and Southern) show niche differentiation, which would suggest a role for local adaptation in biogeography of this widespread Eurasian small mammal. We first model the environmental requirements for the bank vole using species-wide occurrences (210 filtered records) and then model each lineage separately to examine niche overlap and test for niche differentiation in geographic and environmental space. We then use the models to estimate past [Last Glacial Maximum (LGM) and mid-Holocene] habitat suitability to compare with previously hypothesized glacial refugia for this species. Environmental niches are statistically significantly different from each other for all pairs of lineages in geographic and environmental space, and these differences cannot be explained by habitat availability within their respective ranges. Together with the inability of most of the lineages to correctly predict the distributions of other lineages, these results support intraspecific ecological differentiation in the bank vole. Model projections of habitat suitability during the LGM support glacial survival of the bank vole in the Mediterranean region and in central and western Europe. Niche differences between lineages and the resulting spatial segregation of habitat suitability suggest ecological differentiation has played a role in determining the present phylogeographic patterns in the bank vole. Our study illustrates that models pooling lineages within a species may obscure the potential for different responses to climate change among populations.

A dynamic history of admixture from Mediterranean and Carpathian glacial refugia drives genomic diversity in the bank vole.

Understanding the historical contributions of differing glacial refugia is key to evaluating the roles of microevolutionary forces, such as isolation, introgression, and selection in shaping genomic diversity in present-day populations. In Europe, where both Mediterranean and extra-Mediterranean (e.g., Carpathian) refugia of the bank vole (Clethrionomys glareolus) have been identified, mtDNA indicates that extra-Mediterranean refugia were the main source of colonization across the species range, while Mediterranean peninsulas harbor isolated, endemic lineages. Here, we critically evaluate this hypothesis using previously generated genomic data (>6,000 SNPs) for over 800 voles, focusing on genomic contributions to bank voles in central Europe, a key geographic area in considering range-wide colonization. The results provide clear evidence that both extra-Mediterranean (Carpathian) and Mediterranean (Spanish, Calabrian, and Balkan) refugia contributed to the ancestry and genomic diversity of bank vole populations across Europe. Few strong barriers to dispersal and frequent admixture events in central Europe have led to a prominent mid-latitude peak in genomic diversity. Although the genomic contribution of the centrally located Carpathian refugium predominates, populations in different parts of Europe have admixed origins from Mediterranean (28%-47%) and the Carpathian (53%-72%) sources. We suggest that the admixture from Mediterranean refugia may have provisioned adaptive southern alleles to more northern populations, facilitating the end-glacial spread of the admixed populations and contributing to increased bank vole diversity in central Europe. This study adds critical details to the complex end-glacial colonization history of this well-studied organism and underscores the importance of genomic data in phylogeographic interpretation.

Relics of the Europe's warm past: phylogeography of the Aesculapian snake.

Understanding how species responded to past climate change can provide information about how they may respond to the current global warming. Here we show how a European reptile species responded to the last natural global warming event at the Pleistocene-Holocene transition that led to the Holocene climatic optimum approximately 5000-8000 years ago. The Aesculapian snake, Zamenis longissimus, is a thermophilous species whose present-day distribution in the southern half of Europe is a remnant of much wider range during the Holocene climatic optimum when populations occurred as far north as Denmark. These northern populations went extinct as the climate cooled, and presently the species is extinct from all central Europe, except few relic populations in locally suitable microhabitats in Germany and the Czech Republic. Our phylogenetic and demographic analyses identified two major clades that expanded from their respective western and eastern refugia after the last glacial maximum (18,000-23,000 years ago) and contributed approximately equally to the present range. Snakes from the relic northern populations carried the Eastern clade, showing that it was primarily the snakes from the eastern, probably Balkan, refugium that occupied the central and northern Europe during the Holocene climatic optimum. Two small, deep-branching clades were identified in near the Black Sea and in Greece. These clades provide evidence for two additional refugia, which did not successfully contribute to the colonization of Europe. If, as our results suggest, some populations responded to the mid-Holocene global warming by shifting their ranges further north than other populations of the same species, knowing what populations were able to expand in different species may provide information about what populations will be important for the species' ability to cope with the current global warming.

Phylogeography of the Middle Eastern tree frogs (Hyla, Hylidae, Amphibia) as inferred from nuclear and mitochondrial DNA variation, with a description of a new species.

Evolutionary relationships of the tree frogs from the Middle East and the demographic histories of their populations were studied using a combination of mitochondrial and nuclear genes. Hyla savignyi and neighboring populations of H. orientalis (former eastern populations of H. arborea) were the main focus taxa. Within H. savignyi, a deep phylogenetic divergence dated about 8.4Ma was discovered. Southern populations from Yemen, Jordan, southern Syria and extreme north-eastern Israel are hereby described as a new species, H. felixarabica sp. nov. Our study points to a biogeographic connection of the south-western Arabian Peninsula and southern Levant and to the importance of the Dead Sea Rift as a historical barrier geographically separating the new species from H. savignyi. Major genetic breaks revealed within species (H. felixarabica: Yemen vs. Jordan-Syria; H. savignyi sensu stricto: Levant vs. Turkey-Iran) are probably connected to climate changes during the Plio-Pleistocene boundary, while the finer phylogeographic structuring probably resulted from the Quaternary climate oscillations. The Cypriote population of H. savignyi originated from southern Anatolia relatively recently. Hyla orientalis from the southern Black Sea region seems to be genetically quite uniform, although two phylogeographic units with western Turkish and Caucasus-Caspian affinities might be detected. Hyla savignyi and H. orientalis carry signals of population expansions dated to the middle to late Pleistocene, while populations of H. felixarabica seem to have rather been constant in size, which might indicate more stable climatic conditions in the southern regions during the Quaternary.

Nuclear and mitochondrial DNA sequence data reveal the evolutionary history of Barbus (Cyprinidae) in the ancient lake systems of the Balkans.

Freshwater fauna of ancient lakes frequently contain endemic taxa thought to have originated during the long existence of these lakes, yet uncertainties remain as to whether they represent distinct genetic lineages with respect to more widespread relatives and to the relative roles of isolation and dispersal in their evolution. Phylogenetic analyses of sequence variation at nuclear and mitochondrial genes were used to examine these issues for the freshwater fish genus Barbus in two European ancient lake systems on the Balkan Peninsula. The nuclear and mitochondrial data yielded concordant phylogeographic patterns though incomplete sorting of nuclear haplotypes between some mitochondrial clades was detected. The distributions of two currently recognized species investigated here do not match the distributions of evolutionary lineages revealed by phylogenetic analyses. The Prespa barbel, Barbus prespensis, is not endemic to the lakes Prespa as previously thought but is instead found to be widespread in the south-eastern Adriatic Sea basin, with a distribution largely corresponding to the basin of the now extinct Lake Maliq historically connected with Lake Prespa. On the other hand, a cryptic phylogenetic subdivision in a widespread species, B. rebeli, was discovered to be more distant from B. rebeli than from other Barbus species and to be endemic to the system of connected lakes Ohrid and Shkodra. The division coincides with the hydrogeographical boundary delimiting distributions of other freshwater fishes, and we suggest that this newly discovered evolutionary lineage represents a distinct species. These findings support the emerging pattern that endemic taxa have evolved not through isolation of individual lakes, but in systems of currently and historically interconnected lakes and their wider basins.

The Celtic fringe of Britain: insights from small mammal phylogeography.

Recent genetic studies have challenged the traditional view that the ancestors of British Celtic people spread from central Europe during the Iron Age and have suggested a much earlier origin for them as part of the human recolonization of Britain at the end of the last glaciation. Here we propose that small mammals provide an analogue to help resolve this controversy. Previous studies have shown that common shrews (Sorex araneus) with particular chromosomal characteristics and water voles (Arvicola terrestris) of a specific mitochondrial (mt) DNA lineage have peripheral western/northern distributions with striking similarities to that of Celtic people. We show that mtDNA lineages of three other small mammal species (bank vole Myodes glareolus, field vole Microtus agrestis and pygmy shrew Sorex minutus) also form a 'Celtic fringe'. We argue that these small mammals most reasonably colonized Britain in a two-phase process following the last glacial maximum (LGM), with climatically driven partial replacement of the first colonists by the second colonists, leaving a peripheral geographical distribution for the first colonists. We suggest that these natural Celtic fringes provide insight into the same phenomenon in humans and support its origin in processes following the end of the LGM.

Divergence with gene flow between Ponto-Caspian refugia in an anadromous cyprinid Rutilus frisii revealed by multiple gene phylogeography.

The Black and Caspian Seas have experienced alternating periods of isolation and interconnection over many Milankovitch climate oscillations and most recently became separated when the meltwater overflow from the Caspian Sea ceased at the end of the last glaciation. Climate-induced habitat changes have indisputably had profound impacts on distribution and demography of aquatic species, yet uncertainties remain about the relative roles of isolation and dispersal in the response of species shared between the Black and Caspian Sea basins. We examined these issues using phylogeographical analysis of an anadromous cyprinid fish Rutilus frisii. Bayesian coalescence analyses of sequence variation at two nuclear and one mitochondrial genes suggest that the Black and Caspian Seas supported separate populations of R. frisii during the last glaciation. Parameter estimates from the fitted isolation-with-migration model showed that their separation was not complete, however, and that the two populations continued to exchange genes in both directions. These analyses also suggested that majority of migrations occurred during the Pleistocene, showing that the variation shared between the Black and Caspian Seas is the result of ancient dispersal along the temporary natural connections between the basins, rather than of incomplete lineage sorting or recent human-mediated dispersal. Gene flow between the refugial populations was therefore an important source of genetic variation, and we suggest that it facilitated the evolutionary response of the populations to changing climate.

Around or across the Carpathians: colonization model of the Danube basin inferred from genetic diversification of stone loach (Barbatula barbatula) populations.

Despite increasing information about postglacial recolonization of European freshwater systems, very little is known about pre-Pleistocene history. We used data on the recent distribution and phylogenetic relationships of stone loach mitochondrial lineages to reconstruct the initial colonization pattern of the Danube river system, one of the most important refuges for European freshwater ichthyofauna. Fine-scale phylogeography of the Danubian populations revealed five highly divergent lineages of pre-Pleistocene age and suggested the multiple origin of the Danubian stone loach. The mean sequence divergence among lineages extended from 7.0% to 13.4%, which is the highest intraspecific divergence observed so far within this river system. Based on the phylogeographical patterns, we propose the following hypothesis to relate the evolution and dispersal of the studied species with the evolution of the Danube river system and the Carpathian Mountains: (i) during the warmer period in the Miocene, the areas surrounding the uplifting Alps and Carpathians served as mountainous refuges for cold-water adapted fish and promoted the diversification of its populations, and (ii) from these refuges, colonization of the emerging Danube river system may have taken place following the retreat of the Central Paratethys. Co-existence of highly divergent mtDNA lineages in a single river system shows that range shifts in response to climatic changes during the Quaternary did not cause extensive genetic homogenization in the stone loach populations. However, the wide distribution of some mtDNA lineages indicates that the Pleistocene glaciations promoted the dispersal and mixing of populations through the lowlands.