Endemic species may have complex histories: within-refugium phylogeography of an endangered Iberian vole.

Glacial refugia protected and promoted biodiversity during the Pleistocene, not only at a broader scale, but also for many endemics that contracted and expanded their ranges within refugial areas. Understanding the evolutionary history of refugial endemics is especially important in the case of endangered species to recognize the origins of their genetic structure and thus produce better informed conservation practices. The Iberian Peninsula is an important European glacial refugium, rich in endemics of conservation concern, including small mammals, such as the Cabrera vole (Microtus cabrerae). This near-threatened rodent is characterized by an unusual suite of genetic, life history and ecological traits, being restricted to isolated geographic nuclei in fast-disappearing Mediterranean subhumid herbaceous habitats. To reconstruct the evolutionary history of the Cabrera vole, we studied sequence variation at mitochondrial, autosomal and sex-linked loci, using invasive and noninvasive samples. Despite low overall mitochondrial and nuclear nucleotide diversities, we observed two main well-supported mitochondrial lineages, west and east. Phylogeographic modelling in the context of the Cabrera vole's detailed fossil record supports a demographic scenario of isolation of two populations during the Last Glacial Maximum from a single focus in the southern part of the Iberian Peninsula. In addition, our data suggest subsequent divergence within the east, and secondary contact and introgression of the expanding western population, during the late Holocene. This work emphasizes that refugial endemics may have a phylogeographic history as rich as that of more widespread species, and conservation of such endemics includes the preservation of that genetic legacy.

A voyage to Terra Australis: human-mediated dispersal of cats.

BACKGROUND: Cats have been transported as human commensals worldwide giving rise to many feral populations. In Australia, feral cats have caused decline and extinction of native mammals, but their time of introduction and origin is unclear. Here, we investigate hypotheses of cat arrival pre- or post-European settlement, and the potential for admixture between cats of different invasion events. We analyse the genetic structure and diversity of feral cats from six locations on mainland Australia, seven Australian islands and samples from Southeast Asia and Europe using microsatellite and mitochondrial DNA data. RESULTS: Our results based on phylogeographic model selection are consistent with a European origin of cats in Australia. We find genetic distinctiveness of Australian mainland samples compared with Dirk Hartog Island, Flinders Island, Tasman Island and Cocos (Keeling) Island samples, and genetic similarities between some of the island populations. Historical records suggest that introduction of cats to these islands occurred at the time of European exploration and/or in connection with the pearling, whaling and sealing trades early in the 19th century. On-going influx of domestic cats into the feral cat population is apparently causing the Australian mainland populations to be genetically differentiated from those island populations, which likely are remnants of the historically introduced cat genotypes. CONCLUSION: A mainly European origin of feral cats in Australia, with possible secondary introductions from Asia following the initial establishment of cats in Australia is reasonable. The islands surrounding Australia may represent founding populations and are of particular interest. The results of the study provide an important timeframe for the impact of feral cats on native species in Australia.

Relationship between wild greylag and European domestic geese based on mitochondrial DNA.

The origins of the European domestic goose are uncertain. The available information comes from archaeological findings and historical literature, but genetic evidence has hitherto been scarce. The domestic goose in Europe is derived from the greylag goose (Anser anser), but it is not known where the initial domestication took place and which of the two subspecies of greylag goose was ancestral. We aimed to determine the amount and geographical distribution of genetic diversity in modern populations of greylag geese as well as in different breeds of the domestic goose to make inferences about goose domestication. We studied DNA sequence variation in the mitochondrial control region of greylag geese from multiple populations across Europe and western Asia as well as specimens of domestic geese representing 18 modern breeds and individuals not belonging to any recognised breed. Our results show notable differences in genetic diversity between different greylag goose populations and the presence of six mitochondrial haplogroups which show a degree of geographical partitioning. The genetic diversity of the domestic goose is low, with 84% of sampled individuals having one of two major closely related haplotypes, suggesting that modern European domestic geese may derive from a narrow genetic base. The site of domestication remains unresolved, but domestic geese in Turkey were unusually diverse, indicating the importance of further sampling in the vicinity of the eastern Mediterranean and the Near East. There appears to be past or ongoing hybridisation between greylags and domestic geese in particular areas, consistent with field observations.

Of mice and the 'Age of Discovery': the complex history of colonization of the Azorean archipelago by the house mouse (Mus musculus) as revealed by mitochondrial DNA variation.

Humans have introduced many species onto remote oceanic islands. The house mouse (Mus musculus) is a human commensal and has consequently been transported to oceanic islands around the globe as an accidental stowaway. The history of these introductions can tell us not only about the mice themselves but also about the people that transported them. Following a phylogeographic approach, we used mitochondrial D-loop sequence variation (within an 849- to 864-bp fragment) to study house mouse colonization of the Azores. A total of 239 sequences were obtained from all nine islands, and interpretation was helped by previously published Iberian sequences and 66 newly generated Spanish sequences. A Bayesian analysis revealed presence in the Azores of most of the D-loop clades previously described in the domesticus subspecies of the house mouse, suggesting a complex colonization history of the archipelago as a whole from multiple geographical origins, but much less heterogeneity (often single colonization?) within islands. The expected historical link with mainland Portugal was reflected in the pattern of D-loop variation of some of the islands but not all. A more unexpected association with a distant North European source area was also detected in three islands, possibly reflecting human contact with the Azores prior to the 15th century discovery by Portuguese mariners. Widening the scope to colonization of the Macaronesian islands as a whole, human linkages between the Azores, Madeira, the Canaries, Portugal and Spain were revealed through the sharing of mouse sequences between these areas. From these and other data, we suggest mouse studies may help resolve historical uncertainties relating to the 'Age of Discovery'.

Relaxed functional constraints on triplicate α-globin gene in the bank vole suggest a different evolutionary history from other rodents.

Gene duplication plays an important role in the origin of evolutionary novelties, but the mechanisms responsible for the retention and functional divergence of the duplicated copy are not fully understood. The α-globin genes provide an example of a gene family with different numbers of gene duplicates among rodents. Whereas Rattus and Peromyscus each have three adult α-globin genes (HBA-T1, HBA-T2 and HBA-T3), Mus has only two copies. High rates of amino acid evolution in the independently derived HBA-T3 genes of Peromyscus and Rattus have been attributed to positive selection. Using RACE PCR, reverse transcription-PCR (RT-PCR) and RNA-seq, we show that another rodent, the bank vole Clethrionomys glareolus, possesses three transcriptionally active α-globin genes. The bank vole HBA-T3 gene is distinguished from each HBA-T1 and HBA-T2 by 20 amino acids and is transcribed 23- and 4-fold lower than HBA-T1 and HBA-T2, respectively. Polypeptides corresponding to all three genes are detected by electrophoresis, demonstrating that the translated products of HBA-T3 are present in adult erythrocytes. Patterns of codon substitution and the presence of low-frequency null alleles suggest a postduplication relaxation of purifying selection on bank vole HBA-T3.

Genetic identification of Iberian rodent species using both mitochondrial and nuclear loci: application to noninvasive sampling.

Species identification through noninvasive sampling is increasingly used in animal conservation genetics, given that it obviates the need to handle free-living individuals. Noninvasive sampling is particularly valuable for elusive and small species such as rodents. Although rodents are not usually assumed to be the most obvious target for conservation, of the 21 species or near-species present in Iberia, three are considered endangered and declining, while several others are poorly studied. Here, we develop a genetic tool for identifying all rodent species in Iberia by noninvasive genetic sampling. To achieve this purpose, we selected one mitochondrial gene [cytochrome b (cyt-b)] and one nuclear gene [interphotoreceptor retinoid-binding protein (IRBP)], which we first sequenced using tissue samples. Both genes allow for the phylogenetic distinction of all species except the sibling species Microtus lusitanicus and Microtus duodecimcostatus. Overall, cyt-b showed higher resolution than IRBP, revealing a clear barcoding gap. To allow these markers to be applied to noninvasive samples, we selected a short highly diagnostic fragment from each gene, which we used to obtain sequences from faeces and bones from owl pellets. Amplification success for the cyt-b and IRBP fragment was 85% and 43% in faecal and 88% and 64% in owl-pellet DNA extractions, respectively. The method allows the unambiguous identification of the great majority of Iberian rodent species from noninvasive samples, with application in studies of distribution, spatial ecology and population dynamics, and for conservation.

Origin of the chromosomal radiation of Madeiran house mice: a microsatellite analysis of metacentric chromosomes.

Chromosome races of Mus musculus domesticus are characterised by particular sets of metacentric chromosomes formed by Robertsonian fusions and whole-arm reciprocal translocations. The Atlantic island of Madeira is inhabited by six chromosome races of house mice with 6-9 pairs of metacentric chromosomes. Three of these races are characterised by the metacentric 3.8 also found elsewhere in the distribution of M. m. domesticus, including Denmark and Spain. We investigated the possibility that metacentric 3.8 was introduced to Madeira during the initial colonisation, as this could have 'seeded' the cascade of chromosomal mutation that is the basis of the extraordinary chromosomal radiation observed on the island. Variation at 24 microsatellite loci mapping to three different chromosomal regions (proximal, interstitial and distal) of mouse chromosomes 3 and 8 was investigated in 179 mice from Madeira, Denmark, Portugal, Spain, Italy and Scotland. Analyses of microsatellite loci closely linked to the centromeres of these chromosomes ('proximal loci') do not support a common evolutionary origin of metacentric 3.8 among Madeiran, Danish and Spanish mouse populations. Our results suggest that Madeiran mice are genetically more similar to standard karyotype mice from Portugal than to metacentric mice from elsewhere. There is expected to be an interruption to gene flow between hybridising metacentric races on Madeira, particularly in the chromosomal regions close to the rearrangement breakpoints. Consistent with this, relating to differentiation involving chromosomes 3 and 8 on Madeira, we found greater genetic structure among races for proximal than interstitial or distal loci.

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia.

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered 'cryptic species'. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex-linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent-based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.

Fellow travellers: a concordance of colonization patterns between mice and men in the North Atlantic region.

BACKGROUND: House mice (Mus musculus) are commensals of humans and therefore their phylogeography can reflect human colonization and settlement patterns. Previous studies have linked the distribution of house mouse mitochondrial (mt) DNA clades to areas formerly occupied by the Norwegian Vikings in Norway and the British Isles. Norwegian Viking activity also extended further westwards in the North Atlantic with the settlement of Iceland, short-lived colonies in Greenland and a fleeting colony in Newfoundland in 1000 AD. Here we investigate whether house mouse mtDNA sequences reflect human history in these other regions as well. RESULTS: House mice samples from Iceland, whether from archaeological Viking Age material or from modern-day specimens, had an identical mtDNA haplotype to the clade previously linked with Norwegian Vikings. From mtDNA and microsatellite data, the modern-day Icelandic mice also share the low genetic diversity shown by their human hosts on Iceland. Viking Age mice from Greenland had an mtDNA haplotype deriving from the Icelandic haplotype, but the modern-day Greenlandic mice belong to an entirely different mtDNA clade. We found no genetic association between modern Newfoundland mice and the Icelandic/ancient Greenlandic mice (no ancient Newfoundland mice were available). The modern day Icelandic and Newfoundland mice belong to the subspecies M. m. domesticus, the Greenlandic mice to M. m. musculus. CONCLUSIONS: In the North Atlantic region, human settlement history over a thousand years is reflected remarkably by the mtDNA phylogeny of house mice. In Iceland, the mtDNA data show the arrival and continuity of the house mouse population to the present day, while in Greenland the data suggest the arrival, subsequent extinction and recolonization of house mice--in both places mirroring the history of the European human host populations. If house mice arrived in Newfoundland with the Viking settlers at all, then, like the humans, their presence was also fleeting and left no genetic trace. The continuity of mtDNA haplotype in Iceland over 1000 years illustrates that mtDNA can retain the signature of the ancestral house mouse founders. We also show that, in terms of genetic variability, house mouse populations may also track their host human populations.

Colonization of Ireland: revisiting 'the pygmy shrew syndrome' using mitochondrial, Y chromosomal and microsatellite markers.

There is great uncertainty about how Ireland attained its current fauna and flora. Long-distance human-mediated colonization from southwestern Europe has been seen as a possible way that Ireland obtained many of its species; however, Britain has (surprisingly) been neglected as a source area for Ireland. The pygmy shrew has long been considered an illustrative model species, such that the uncertainty of the Irish colonization process has been dubbed 'the pygmy shrew syndrome'. Here, we used new genetic data consisting of 218 cytochrome (cyt) b sequences, 153 control region sequences, 17 Y-intron sequences and 335 microsatellite multilocus genotypes to distinguish between four possible hypotheses for the colonization of the British Isles, formulated in the context of previously published data. Cyt b sequences from western Europe were basal to those found in Ireland, but also to those found in the periphery of Britain and several offshore islands. Although the central cyt b haplotype in Ireland was found in northern Spain, we argue that it most likely occurred in Britain also, from where the pygmy shrew colonized Ireland as a human introduction during the Holocene. Y-intron and microsatellite data are consistent with this hypothesis, and the biological traits and distributional data of pygmy shrews argue against long-distance colonization from Spain. The compact starburst of the Irish cyt b expansion and the low genetic diversity across all markers strongly suggests a recent colonization. This detailed molecular study of the pygmy shrew provides a new perspective on an old colonization question.

Properties of a hybrid zone between highly distinct chromosomal races of the house mouse (Mus musculus domesticus) in Northern Italy, and comparisons with other hybrid zones.

Here we provide the first detailed description of the hybrid zone between the Cremona chromosomal race of house mouse (ICRE; 2n = 22) and the standard all-telocentric race (40ST; 2n = 40), with full karyotypes of 106 individuals from 17 localities along a transect between the 2 races to the west of Lake Garda in Northern Italy. The ICRE race is characterised by 9 pairs of metacentric chromosomes in a homozygous state and we use the metacentric frequency data along the transect to fit tanh metacentric clines. The clines are narrow (5-8 km, standardised width) suggesting low hybrid fitness. However, the lack of occurrence of ICRE × 40ST F(1) hybrids and presence of other hybrid types suggests that the F(1) hybrids initially produced in this hybrid zone were at least partially fertile, despite having 9 meiotic trivalent configurations. We apply the same cline-fitting methodology to 3 previously studied hybrid zones between metacentric races and the 40ST race. Taken together with published clinal data on 4 further metacentric-40ST hybrid zones, we are able to make objective generalisations on the characteristics of such zones in the house mouse. Zones involving 22-chromosome races are narrower, on average, than other metacentric-40ST hybrid zones and do not show a tendency towards the generation of new races as found with zones where the metacentric race has a higher 2n. It appears that metacentric-40ST zones are unlikely to be sites of speciation (even when a 22-chromosome race is involved), although a mosaic structure to the hybrid zone may enhance this possibility. We make a comparison between metacentric-40ST zones and contacts between 2 metacentric races, for a comprehensive perspective of chromosomal hybrid zones in the house mouse.

Natural hybridization between extremely divergent chromosomal races of the common shrew (Sorex araneus, Soricidae, Soricomorpha): hybrid zone in Siberia.

Chromosomal races of the common shrew differ in sets of metacentric chromosomes and on contact may produce hybrids with extraordinarily complex configurations at meiosis I that are associated with reduced fertility. There is an expectation that these may be some of the most extreme tension zones available for study and therefore are of interest as potential sites for reproductive isolation. Here, we analyse one of these zones, between the Novosibirsk race (characterized by metacentrics go, hn, ik, jl, mp and qr) and the Tomsk race (metacentrics gk, hi, jl and mn and acrocentrics o, p, q and r), which form hybrids with a chain-of-nine (CIX) and a chain-of-three (CIII) configuration at meiosis I. At the Novosibirsk-Tomsk hybrid zone, the CIX chromosomes form clines of 8.53 km standardized width on average, whereas the cline for the CIII chromosomes was 52.83 km wide. The difference in these cline widths fits with the difference in meiotic errors expected with the CIX and CIII configuration, and we produce estimates of selection against hybrids with these types of configurations, which we relate to dispersal and age of the hybrid zone. The hybrid zone is located at the isocline at 200 m altitude above sea level; this relationship between the races and altitude is suggested at both coarse and fine scales. This indicates adaptive differences between the races that may in turn have been promoted by the chromosome differences. Thus, the extreme chromosomal divergence between the Novosibirsk and Tomsk may be associated with genic differentiation, but it is still striking that, despite the large chromosomal differences, reproductive isolation between the Novosibirsk and Tomsk races has not occurred.

Natural hybridization between extremely divergent chromosomal races of the common shrew (Sorex araneus, Soricidae, Soricomorpha): hybrid zone in European Russia.

The Moscow and Seliger chromosomal races of the common shrew differ by Robertsonian fusions and possibly whole-arm reciprocal translocations (WARTs) such that their F1 hybrids produce a chain-of-eleven configuration at meiosis I and are expected to suffer substantial infertility. Of numerous hybrid zones that have been described in the common shrew, those between the Moscow and Seliger races involve the greatest chromosomal difference. We collected 211 individuals from this zone to generate a total dataset of 298 individuals from 187 unique global positioning system (GPS) locations within the vicinity of interracial contact. We used a geographic information system (GIS) to map the location of the hybrid zone, which follows a direct route between two lakes, as would be anticipated from tension zone theory. Even within the central area of the hybrid zone, there is a much higher frequency of pure race individuals than hybrid, making this a clear example of a bimodal zone in the sense of Jiggins & Mallet (2000). The zone runs through good habitat for common shrews, but nevertheless it is very narrow (standard cline widths: 3-4 km), as would be anticipated from low hybrid fitness. There is clear potential for an interruption to gene flow and build-up of reproductive isolation. As found in some other hybrid zones, there is a high frequency of novel genetic variants, in this case, new chromosomal rearrangements. Here, we report a de novo Robertsonian fission and a de novo reciprocal translocation, both for the first time in the common shrew. There is an extraordinarily high frequency of de novo mutations recorded in F1 hybrids in the zone and we discuss how chromosomal instability may be associated with such hybrids. The occurrence of a de novo Robertsonian fission is of considerable significance because it provides missing evidence that fissions are the basis of the novel acrocentric forms found and apparently selected for in certain common shrew hybrid zones.

New metacentric population of the house mouse (Mus musculus domesticus) found in Valchiavenna, Northern Italy.

Although the standard karyotype of the western house mouse (Mus musculus domesticus) consists entirely of telocentric chromosomes, there are over 100 populations across western Europe and North Africa characterized by different sets of metacentrics formed by Robertsonian fusions and whole-arm reciprocal translocations. Here we report the discovery of a new metacentric population from Valchiavenna, northern Italy, that we have named the 'Lower Valchiavenna population' (abbreviated as ILVC). This metacentric population is found in villages and on farms over a 10-kilometer stretch comprising the southern half of Valchiavenna. ILVC is characterized by the metacentrics 1.18, 2.4, 3.8, 5.15, 6.7, 9.14, 10.12, 11.13 and 16.17, and appears to be closely related to the Chiasso population (CHCH), which possesses the same set of metacentrics except 1.18. We discuss the evolutionary origin of ILVC in relation to human occupation of the region. We also suggest that the geographical position of ILVC between 2 other metacentric populations with entirely different sets of metacentrics (Chiavenna, ICHI, and lower Valtellina, ILVA) may provide 2 additional chromosomal hybrid zones for the study of speciation.

Molecular insights into the colonization and chromosomal diversification of Madeiran house mice.

The colonization history of Madeiran house mice was investigated by analysing the complete mitochondrial (mt) D-loop sequences of 156 mice from the island of Madeira and mainland Portugal, extending on previous studies. The numbers of mtDNA haplotypes from Madeira and mainland Portugal were substantially increased (17 and 14 new haplotypes respectively), and phylogenetic analysis confirmed the previously reported link between the Madeiran archipelago and northern Europe. Sequence analysis revealed the presence of four mtDNA lineages in mainland Portugal, of which one was particularly common and widespread (termed the 'Portugal Main Clade'). There was no support for population bottlenecks during the formation of the six Robertsonian chromosome races on the island of Madeira, and D-loop sequence variation was not found to be structured according to karyotype. The colonization time of the Madeiran archipelago by Mus musculus domesticus was approached using two molecular dating methods (mismatch distribution and Bayesian skyline plot). Time estimates based on D-loop sequence variation at mainland sites (including previously published data from France and Turkey) were evaluated in the context of the zooarchaeological record of M. m. domesticus. A range of values for mutation rate (mu) and number of mouse generations per year was considered in these analyses because of the uncertainty surrounding these two parameters. The colonization of Portugal and Madeira by house mice is discussed in the context of the best-supported parameter values. In keeping with recent studies, our results suggest that mutation rate estimates based on interspecific divergence lead to gross overestimates concerning the timing of recent within-species events.

Ecomorphometric variation and sexual dimorphism in the common shrew (Sorex araneus).

We investigated the evolution of the biomechanics of the mandible in island and mainland populations of the common shrew on the west coast of Scotland. We predicted that climatic differences between populations should cause differences in prey composition leading to changes in the mechanical potential (MP) of the mandible. In females, MP was correlated with climate, with greater MP in warmer and drier habitats. In males, MP was significantly greater than in females but there was no relationship between male MP and climate. This led to increased sexual dimorphism in colder and wetter climates. The same pattern was found after a phylogenetic least squares analysis was conducted to account for shared phylogenetic history. We discuss possible reasons for this pattern, including male-male combat and the greater necessity of females to feed as efficiently as possible to meet their extremely high energy requirements during lactation.

Mandible asymmetry and genetic diversity in island populations of the common shrew, Sorex araneus.

Mandibles from 13 island and six mainland populations of common shrews from the west coast of Scotland were subjected to geometric morphometric analysis in order to investigate the relationship between genetic diversity and fluctuating asymmetry. Although population mean shape fluctuating asymmetry (FA) and size FA were significantly inversely correlated with population genetic diversity this result was substantially due to one island. Sanda, the smallest island with by far the lowest genetic diversity, also had the highest FA. When Sanda was removed from the analysis, the relationship was not significant. There was no relationship between genetic diversity and FA at the individual level, whether measured as mean locus heterozygosity or d(2). In general, if genetic variation affects FA at all, the effect is weak and may only be of biological interest in very small populations.

Patterns of genic diversity and structure in a species undergoing rapid chromosomal radiation: an allozyme analysis of house mice from the Madeira archipelago.

The chromosomal radiation of the house mouse in the island of Madeira most likely involved a human-mediated colonization event followed by within-island geographical isolation and recurrent episodes of genetic drift. The genetic signature of such processes was assessed by an allozyme analysis of the chromosomal races from Madeira. No trace of a decrease in diversity was observed suggesting the possibility of large founder or bottleneck sizes, multiple introductions and/or a high post-colonization expansion rate. The Madeira populations were more closely related to those of Portugal than to other continental regions, in agreement with the documented human colonization of the island. Such a Portuguese origin contrasts with a study indicating a north European source of the mitochondrial haplotypes present in the Madeira mice. This apparent discrepancy may be resolved if not one but two colonization events took place, an initial north European introduction followed by a later one from Portugal. Asymmetrical reproduction between these mice would have resulted in a maternal north European signature with a nuclear Portuguese genome. The extensive chromosomal divergence of the races in Madeira is expected to contribute to their genic divergence. However, there was no significant correlation between chromosomal and allozyme distances. This low apparent chromosomal impact on genic differentiation may be related to the short time since the onset of karyotypic divergence, as the strength of the chromosomal barrier will become significant only at later stages.

Identification of all pachytene bivalents in the common shrew using DAPI-staining of synaptonemal complex spreads.

A major problem in studies of synaptonemal complexes (SC) is the difficulty in distinguishing individual chromosomes. This problem can be solved combining SC immunostaining with FISH of chromosome-specific sequences. However, this procedure is expensive, time-consuming and applicable only to a very limited number of species. In this paper we show how a combination of SC immunostaining and DAPI staining can allow identification of all chromosome arms in surface-spreads of the SC of the common shrew (Sorex araneus L.). Enhancement of brightness and contrast of the images with photo editing software allowed us to reveal clear DAPI-positive and negative bands with relative sizes and positions similar to DAPI landmarks on mitotic metaphase chromosomes. Using FISH with DNA probes prepared from chromosome arms m and n we demonstrated correct recognition of the chromosomes mp and hn on the basis of their DAPI pattern. We show that the approach we describe here may be applied to other species and can provide an important tool for identification of individual bivalents in pachytene surface-spreads.

Beyond the Mediterranean peninsulas: evidence of central European glacial refugia for a temperate forest mammal species, the bank vole (Clethrionomys glareolus).

This study details the phylogeographic pattern of the bank vole, Clethrionomys glareolus, a European rodent species strongly associated with forest habitat. We used sequences of 1011 base pairs of the mitochondrial DNA cytochrome b gene from 207 bank voles collected in 62 localities spread throughout its distribution area. Our results reveal the presence of three Mediterranean (Spanish, Italian and Balkan) and three continental (western, eastern and 'Ural') phylogroups. The endemic Mediterranean phylogroups did not contribute to the post-glacial recolonization of much of the Palaearctic range of species. Instead, the major part of this region was apparently recolonized by bank voles that survived in glacial refugia in central Europe. Moreover, our phylogeographic analyses also reveal differentiated populations of bank voles in the Ural mountains and elsewhere, which carry the mitochondrial DNA of another related vole species, the ruddy vole (Clethrionomys rutilus). In conclusion, this study demonstrates a complex phylogeographic history for a forest species in Europe which is sufficiently adaptable that, facing climate change, survives in relict southern and northern habitats. The high level of genetic diversity characterizing vole populations from parts of central Europe also highlights the importance of such regions as a source of intraspecific genetic biodiversity.