Nuclear and mitochondrial phylogeography of the European fire-bellied toads Bombina bombina and Bombina variegata supports their independent histories

Exact location and number of glacial refugia still remain unclear for many European cold‐blooded terrestrial vertebrates. We performed a fine‐scaled multilocus phylogeographic analysis of two Bombina species combining mitochondrial variation of 950 toads from 385 sites and nuclear genes (Rag‐1, Ncx‐1) from a subset of samples to reconstruct their colonization and contemporary variation patterns. We identified the lowlands northwest of the Black Sea and the Carpathians to be important refugial areas for B. bombina and B. variegata, respectively. This result emphasizes the importance of Central European refugia for ectothermic terrestrial species, far north of the Mediterranean areas regarded as exclusive glacial refugia for the animals. Additional refugia for B. variegata have been located in the southern Apennines and Balkans. In contrast, no evidence for the importance of other east European plains as refugial regions has been found. The distribution of mtDNA and Ncx‐1 variation suggests the presence of local refugia near the Black Sea for B. bombina; however, coalescent simulations did not allow to distinguish whether one or two refugia were present in the region. Strong genetic drift apparently accompanied postglacial expansions reducing diversity in the colonization areas. Extended sampling, coupled with the multilocus isolation with migration analysis, revealed a limited and geographically restricted gene flow from the Balkan to Carpathian populations of B. variegata. However, despite proximity of inferred B. bombina and B. variegata refugia, gene exchange between them was not detected.     

Mitochondrial DNA variation in the hybridizing fire-bellied toads, Bombina bombina and B. variegata

Using five restriction enzymes, geographical variation of mitochondrial DNA (mtDNA) in Bombina bombina and B. variegata was studied in samples from 20 locations. Each restriction enzyme produced a species-specific fragment pattern. B. bombina haplotypes A and B were closely related to each other. In contrast, haplotypes A and B of B. variegata formed two distinct lineages. A very distinctive haplotype (C) was found in the Carpathian Mountains, whereas two other haplotypes, D and E (differing by a single AvaI site), were present in western Europe and the Balkans, respectively. Populations polymorphic for haplotypes D and E occurred in the central Balkans where the haplotypes could replace each other clinally. mtDNA sequence divergence between B. bombina and B. variegata was estimated as 6.0-8.1% and 4.7-5.2% between type C and types D/E of B. variegata. The latter divergence is contrary to allozyme and morphological data that place the western and Carpathian B. v. variegata together (Nei's D = 0.07) and separate them from the Balkan subspecies B. v. scabra (Nei's D = 0.18). Broad interspecific correlation among morphology, allozymes and mtDNA types in European fire-bellied toads argues that, despite continuous hybridization (interrupted perhaps during Pleistocene glacial maxima), little or no mtDNA introgression between the species has occurred outside the narrow hybrid zones that separate these parapatric species.     

Hybridization between the fire-bellied toads Bombina bombina and Bombina variegata in the karst regions of Slovakia and Hungary: morphological and allozyme evidence

Geographic variability and genetic interactions in the contact zone between the fire-bellied toads, Bombina bombina and B. variegata, were studied using analysis of morphological and genetic variation in sixteen samples from the Slovak Karst and Aggtelek Karst regions. Genotype frequencies at four marker loci (Ldh-1, Mdh-1, Adk, Hem) demonstrate the existence of a hybrid zone with highly variable population structures. While some samples appear to represent panmictic hybrid populations, other samples are very heterogeneous. Pure individuals of both species occurred together with hybrids at one site. Habitat segregation among genetically differentiated demes probably causes this heterogeneity of population structures. Increased frequencies of the allele Ldh-1^M, which is present in low proportion in B. bombina south of the contact zone, were found in some hybrid populations.     

Phylogeography and demography of Guianan harlequin toads (Atelopus): diversification within a refuge

We investigated the genetic structure of populations of Guianan harlequin toads (genus Atelopus) and their evolutionary affinities to extra-Guianan congeners. Phylogenetic analysis of mitochondrial cytochrome b (cyt b) and NADH dehydrogenase subunit 2 (ND2) gene sequences produced well-supported clades largely corresponding to the four recognized taxa in the Guianas (Atelopus spumarius hoogmoedi, Atelopus spumarius barbotini, Atelopus franciscus, and Atelopus flavescens). Our findings suggest that the Guianan A. spumarius represent distinct evolutionary lineages that merit distinction from Amazonian conspecifics, and that the status of A. flavescens and A. franciscus is somewhat less clear. Approximately 69% of the observed genetic variation is accounted for by differences between these four recognized taxa. Coalescent-based estimates of gene flow between taxa suggest that these lineages are largely isolated from one another. Negligible rates of migration between populations and significant divergence within such close proximity suggests that although the region inhabited by these taxa is almost entirely undisturbed, significant habitat heterogeneity exists as to have produced a remarkable diversification of Atelopus within the eastern Guiana Shield. These results contradict the commonly held view of the Guiana Shield as a 'refuge' whose stability during late Tertiary and Quaternary climatic fluctuations served as a biotic reservoir. Instead, we provide evidence that climatic fluctuations during this time had a diversifying effect within the Guianan region.     

Phylogeography of the endangered Cathaya argyrophylla (Pinaceae) inferred from sequence variation of mitochondrial and nuclear DNA

Cathaya argyrophylla is an endangered conifer restricted to subtropical mountains of China. To study phylogeographical pattern and demographic history of C. argyrophylla, species-wide genetic variation was investigated using sequences of maternally inherited mtDNA and biparentally inherited nuclear DNA. Of 15 populations sampled from all four distinct regions, only three mitotypes were detected at two loci, without single region having a mixed composition (G(ST) = 1). Average nucleotide diversity (theta(ws) = 0.0024; pi(s) = 0.0029) across eight nuclear loci is significantly lower than those found for other conifers (theta(ws) = 0.003 approximately 0.015; pi(s) = 0.002 approximately 0.012) based on estimates of multiple loci. Because of its highest diversity among the eight nuclear loci and evolving neutrally, one locus (2009) was further used for phylogeographical studies and eight haplotypes resulting from 12 polymorphic sites were obtained from 98 individuals. All the four distinct regions had at least four haplotypes, with the Dalou region (DL) having the highest diversity and the Bamian region (BM) the lowest, paralleling the result of the eight nuclear loci. An AMOVA revealed significant proportion of diversity attributable to differences among regions (13.4%) and among populations within regions (8.9%). F(ST) analysis also indicated significantly high differentiation among populations (F(ST) = 0.22) and between regions (F(ST) = 0.12-0.38). Non-overlapping distribution of mitotypes and high genetic differentiation among the distinct geographical groups suggest the existence of at least four separate glacial refugia. Based on network and mismatch distribution analyses, we do not find evidence of long distance dispersal and population expansion in C. argyrophylla. Ex situ conservation and artificial crossing are recommended for the management of this endangered species.     

Mapping Bombina mitochondrial genomes: the conundrum of Carpathian Bombina variegata (Anura: Discoglossidae)

Complete mitochondrial genomes (mtDNA) of five individuals representing two haplotypes of Bombina bombina and three of Bombina variegata were compared using restriction site maps. Phylogenetic analyses reveal three ancient mitochondrial lineages: (1) two very similar haplotypes A and B of B. bombina ; (2) almost identical haplotypes D and E of B. variegata ; and (3) haplotype C of B. variegata . Haplotype C is as different from haplotypes D/E as from A/B. These data are strikingly discordant with relationships based on morphology and allozymes. Haplotypes C and D/E represent a pre-Pleistocene mitochondrial divergence within B. variegata , nearly coincident with speciation between B. variegata and B. bombina . Geographical partitioning of the two divergent B. variegata mitochondrial lineages indicates repeated localization of the lineages in separate glacial refugia during the Pleistocene. That nuclear genes do not show a similar divergence, but rather indicate relatively free genetic exchange between populations with divergent mtDNAs, suggests that males dispersed much more widely than females during expansions from glacial refugia. Comparison of Bombina mtDNA maps with a restriction site map of Xenopus laevis mtDNA revealed 16 homologous sites; 12 of these may be nearly invariant across primitive anuran mtDNAs. Two distinct regions of heteroplasmy, representing two regions with variable numbers of sequence repeats [length variable (LV) regions], were characterized. Comparison with the Xenopus map places LV1, present in all five haplotypes, near the 5'-end of the control region, and LV2 present only in B. variegata , near the 3'-end. Although phylogenetic analyses did not group the two major B. variegata lineages together, presence of LV2 in both lineages supports placement of both within B. variegata .     

Phylogeography of the North American red fox: vicariance in Pleistocene forest refugia

Fossil, archaeological, and morphometric data suggest that indigenous red foxes in North America were derived from vicariance in two disjunct refugia during the last glaciation: one in Beringia and one in the contiguous USA. To test this hypothesis, we conducted a phylogeographical analysis of the North American red fox within its presettlement range. We sequenced portions of the mitochondrial cytochrome b (354 bp) gene and D-loop (342 bp) from 220 historical red fox specimens. Phylogenetic analysis of the cytochrome b gene produced two clades that diverged c . 400 000 years before present ( bp ): a Holarctic and a Nearctic clade. D-loop analyses of the Nearctic clade indicated three distinct subclades (≥ 99% Bayesian posterior probability); two that were more recently derived (rho estimate c . 20 000 bp ) and were restricted to the southwestern mountains and the eastern portion of North America, and one that was older (rho estimate c . 45 000 bp ) and more widespread in North America. Populations that migrated north from the southern refugium following deglaciation were derived from the colonization of North America during or prior to the Illinoian glaciation (300 000–130 000 bp ), whereas populations that migrated south from the northern refugium represent a more recent colonization event during the Wisconsin glaciation (100 000–10 000 bp ). Our findings indicate that Nearctic clade red foxes are phylogenetically distinct from their Holarctic counterparts, and reflect long-term isolation in two disjunct forest refugia during the Pleistocene. The montane lineage, which includes endangered populations, may be ecologically and evolutionarily distinct.     

Phylogeography of shy and white-capped albatrosses inferred from mitochondrial DNA sequences: implications for population history and taxonomy

The evolutionary relationship between shy (Thalassarche cauta) and white-capped (T. steadi) albatrosses was examined using mitochondrial control region sequences. Results were interpreted in the context of a recent and contentious taxonomic revision that recommended full species status for shy and white-capped albatrosses. Low sequence divergence between shy and white-capped albatrosses (1.8%) and between their close relatives, Salvin's and Chatham albatrosses (2.9%), was observed. Much higher sequence divergence was found between the shy/white-capped pair and the Salvin's/Chatham pair (7.0%). Phylogenetic analyses confirmed the separation of the shy/white-capped pair from the Salvin's/Chatham pair but did not provide species-level resolution. Phylogeographic analyses, including a nested clade analysis, FST estimates and an analysis of molecular variance, indicated unambiguous genetic structuring between shy and white-capped albatrosses, thus confirming the demographic isolation of the species, but showed little to no structuring within each species. The geographical distribution of mtDNA haplotypes and other evidence suggest that shy albatrosses arose through range expansion by white-capped albatrosses.     

Phylogeography of field voles (Microtus agrestis) in Eurasia inferred from mitochondrial DNA sequences

In a distribution-wide phylogeographic survey of the field vole (Microtus agrestis), 75 specimens from 56 localities across Eurasia were examined for DNA sequence variation along the whole 1140 base pair (bp) mitochondrial (mt) cytochrome b gene. The species is subdivided into three main mtDNA phylogeographic groups - western, eastern and southern - with largely allopatric distributions. The western phylogeographical group is found in west and central Europe and spread most probably from a glacial refugium in the Carpathians. The eastern group covers a large range from Lithuania to central Asia, and probably originated from a southeast European source (e.g. the southern Urals or the Caucasus). The southern group occupies an area from Portugal to Hungary, with division into two distinct mtDNA sublineages that presumably derive from separate glacial refugia in the Iberian Peninsula. Molecular clock estimates suggest that the western and eastern field vole populations separated during the last glaciation, whereas the southern population dates back 0.5-0.9 Myr. High levels of mtDNA variation indicate relatively large population sizes and subdivisions within phylogeographic groups during the last glaciation. We report a possible new suture zone in east Europe.     

Phylogeography of the barbel (Barbus barbus) assessed by mitochondrial DNA variation

Using the phylogeographic framework, we assessed the DNA sequence variation at the mitochondrial cytochrome b gene across the distribution range of the barbel Barbus barbus, a widely distributed European cyprinid. Reciprocal monophyly of non-Mediterranean European and Balkan/Anatolian populations is taken as evidence for a long-term barrier to gene flow, and interpreted as a consequence of survival of the species in two separate refugia during several later glacial cycles. Lack of profound genealogical divergence across Europe from western France to the northwestern Black Sea basin is consistent with recent colonization of this area from a single glacial refuge, which was probably located in the Danube River basin. This may have occurred in two steps: into the Western European river basins during the last interglacial, and throughout the Central European river basins after the last glacial. The populations from the Balkans and Anatolia apparently did not contribute mitochondrial DNA to the post-Pleistocene colonization of non-Mediterranean Europe. Lack of detectable variation within the Balkans/Anatolia is attributed mainly to recent expansion throughout these regions, facilitated by the freshwater conditions and seashore regression in the Black Sea during the Late Pleistocene and Early Holocene.     

Phylogeography and counter-intuitive inferences in island biogeography: evidence from morphometric markers in the mobile butterfly Maniola jurtina (Linnaeus) (Lepidoptera, Nymphalidae)

Distribution of mobile organisms on near-continent islands is mainly shaped by factors operating over ecological rather geological time. However, the phylogeography of single species has the potential to expose historical factors at work. In the present study, West Mediterranean populations of the butterfly Maniola jurtina are studied using geometric morphometrics. The distribution of the two well established lineages ( Maniola jurtina jurtina in the Atlanto–Mediterranean area and Maniola jurtina janira in the Central–Eastern-Mediterranean area) on 12 islands and the adjoining continents are compared. The south-western lineage unexpectedly occurs on islands close to shores occupied by the eastern lineage. We have modelled the distribution of the lineages using three different hypotheses: (1) a contemporary isolation model, which predicts lineage occupancy of islands is linked to relative distances from neighbouring continental areas; (2) a refugial hypothesis, which predicts one lineage to be the ancestral one for the whole region studied, and then successively replaced over part of it; (3) a changing geography hypothesis, which predicts the two lineages to have evolved in their currently occupied areas, continuously sourcing islands subsequent to the Würm maximum glaciation. Of the three models, the refugial hypothesis is most highly correlated with the observed pattern, suggesting that Mediterranean islands may function as refugia during cold periods, much as the three mainland peninsulas of Iberia, Italy and Greece are known to have done. Thereafter, hybridization on the nearest and smallest islands has occurred, with the entire process supporting the notion of the joint influence of factors in ecological and geological time.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 677–692.     

Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data

In order to contribute to the debate about southern glacial refugia used by temperate species and more northern refugia used by boreal or cold-temperate species, we examined the phylogeography of a widespread snake species (Vipera berus) inhabiting Europe up to the Arctic Circle. The analysis of the mitochondrial DNA (mtDNA) sequence variation in 1043 bp of the cytochrome b gene and in 918 bp of the noncoding control region was performed with phylogenetic approaches. Our results suggest that both the duplicated control region and cytochrome b evolve at a similar rate in this species. Phylogenetic analysis showed that V. berus is divided into three major mitochondrial lineages, probably resulting from an Italian, a Balkan and a Northern (from France to Russia) refugial area in Eastern Europe, near the Carpathian Mountains. In addition, the Northern clade presents an important substructure, suggesting two sequential colonization events in Europe. First, the continent was colonized from the three main refugial areas mentioned above during the Lower-Mid Pleistocene. Second, recolonization of most of Europe most likely originated from several refugia located outside of the Mediterranean peninsulas (Carpathian region, east of the Carpathians, France and possibly Hungary) during the Mid-Late Pleistocene, while populations within the Italian and Balkan Peninsulas fluctuated only slightly in distribution range, with larger lowland populations during glacial times and with refugial mountain populations during interglacials, as in the present time. The phylogeographical structure revealed in our study suggests complex recolonization dynamics of the European continent by V. berus, characterized by latitudinal as well as altitudinal range shifts, driven by both climatic changes and competition with related species.     

Genetic diversity and phylogeography of the Apennine yellow-bellied toad Bombina pachypus, with implications for conservation

Genetic variation was investigated in 17 populations of the Italian endemic Apennine yellow-bellied toad using both mitochondrial (598 bp of the cytochrome b gene) and nuclear (21 allozyme loci) markers. Populations from central Calabria (southern Italy) showed the highest levels of intrapopulation genetic variation, whereas samples located north of this region were nearly lacking in variation. This appears to be a typical pattern of 'southern richness and northern purity', usually attributed to the prolonged population stability within southern refugia coupled with the loss of variation during postglacial northward expansion. However, the overall pattern of genetic variation observed has a strong geographical component, suggesting two Calabrian plains, Catanzaro and Crati-Sibari, as historical barriers to dispersal separating three population groups. These findings cannot be explained by the prolonged stability of southern populations alone, and suggest that the southern richness has been at least in part shaped by allopatric differentiation within the refugial range, followed by intermixing of previously differentiated lineages. From a conservation standpoint, Calabria is the major genetic diversity reservoir for this species, thus deserving particular conservation efforts. Furthermore, although the low intrapopulation genetic variation outside Calabria appears to be of clear historical origin, evidence of a current reduction of gene flow suggests that human disturbance has also played a part, particularly in the anthropogenic impacted Volturno river drainage basin.     

Reproductive biology of the fire-bellied toads, Bombina bombina and B. variegata (Anura: Discoglossidae): egg size, clutch size and larval period length differences

Clutch size, egg size, larval size and duration of larval period were estimated for samples of Bombina bombina and B. variegata from several populations. Bombina variegata lays fewer eggs per batch (mean 17.4) than B. bombina (mean 32.5). The volume of B. variegata eggs is 2.4 times greater and the dry weight 2.3 times greater than that of B. bombina. As a consequence, the volume of B. variegata hatchlings and their dry weight are respectively 1.9 times and 2.5 times greater than that of B. bombina. Analysis of variance showed that variation of egg size in B. variegata is significantly greater than in B. bombina. When reared under laboratory conditions freshly metamorphosed individuals of both species showed no difference in body size or weight. Although growth rates are equal, tadpoles of B. variegata reached metamorphosis 14 days earlier than B. bombina tadpoles because of differences in size of hatchlings. Differences in reproductive parameters found for the two European Bombina species can be interpreted as adaptations to contrasting environments in which they breed.     

Fat and glycogen utilization in the larynx muscles of fire-bellied toads (Bombina bombina L.) during calling activity

Summary Both fat and glycogen are present in abundance in the larynx muscles of anurans. To clarify their role, the Musculus dilatator laryngis of the male fire-bellied toad, Bombina bombina was studied.In untreated males, the mean fat content of this larynx muscle was 14%; the muscle contained glycogen amounting to 57% of that measured in the liver tissue of the same animal. After thirteen days of continual calling activity induced by administration of a gonadotropin, the fat content fell to 6%, the glycogen to 34%. The fat content was essentially unchanged (13%) by four hours of electrical stimulation of the muscle; the glycogen content, however, had fallen to 42% after this treatment. Neither component was altered in amount by castration, the fat content being about 13% and that of glycogen, 52%. Nor did treatment with gonadotropic hormone reduce either the fat content (13%) or the amount of glycogen (59%).From these results it was concluded that fats represent a direct source of energy for the larynx muscles, which is used up gradually over long periods of calling. The glycogen in these muscles, on the other hand, is a short-term store sufficient to supply energy for only a few hours of calling activity.Supported by the Deutsche Forschungsgemeinschaft.     

Pleistocene evolutionary history of the Clouded Apollo (Parnassius mnemosyne): genetic signatures of climate cycles and a 'time-dependent' mitochondrial substitution rate

Genetic data are currently providing a large amount of new information on past distribution of species and are contributing to a new vision of Pleistocene ice ages. Nonetheless, an increasing number of studies on the 'time dependency' of mutation rates suggest that date assessments for evolutionary events of the Pleistocene might be overestimated. We analysed mitochondrial (mt) DNA (COI) sequence variation in 225 Parnassius mnemosyne individuals sampled across central and eastern Europe in order to assess (i) the existence of genetic signatures of Pleistocene climate shifts; and (ii) the timescale of demographic and evolutionary events. Our analyses reveal a phylogeographical pattern markedly influenced by the Pleistocene/Holocene climate shifts. Eastern Alpine and Balkan populations display comparatively high mtDNA diversity, suggesting multiple glacial refugia. On the other hand, three widely distributed and spatially segregated lineages occupy most of northern and eastern Europe, indicating postglacial recolonization from different refugial areas. We show that a conventional 'phylogenetic' substitution rate cannot account for the present distribution of genetic variation in this species, and we combine phylogeographical pattern and palaeoecological information in order to determine a suitable intraspecific rate through a Bayesian coalescent approach. We argue that our calibrated 'time-dependent' rate (0.096 substitutions/ million years), offers the most convincing time frame for the evolutionary events inferred from sequence data. When scaled by the new rate, estimates of divergence between Balkan and Alpine lineages point to c. 19 000 years before present (last glacial maximum), and parameters of demographic expansion for northern lineages are consistent with postglacial warming (5-11 000 years before present).     

A long‐standing Pleistocene refugium in southern Africa and a mosaic of refugia in East Africa: insights from mtDNA and the common eland antelope

Aim Previous genetic studies of African savanna ungulates have indicated Pleistocene refugial areas in East and southern Africa, and recent palynological, palaeovegetation and fossil studies have suggested the presence of a long‐standing refugium in the south and a mosaic of refugia in the east. Phylogeographic analysis of the common eland antelope, Taurotragus oryx (Bovidae), was used to assess these hypotheses and the existence of genetic signatures of Pleistocene climate change. Location The sub‐Saharan savanna biome of East and southern Africa. Methods Mitochondrial DNA control‐region fragments (414 bp) from 122 individuals of common eland were analysed to elucidate the phylogeography, genetic diversity, spatial population structuring, historical migration and demographic history of the species. The phylogeographic split among major genetic lineages was dated using Bayesian coalescent‐based methods and a calibrated fossil root of 1.6 Ma for the split between the common eland and the giant eland, Taurotragus derbianus. Results Two major phylogeographic lineages comprising East and southern African localities, respectively, were separated by a net nucleotide distance of 4.7%. A third intermediate lineage comprised only three haplotypes, from Zimbabwe in southern Africa. The estimated mutation rate of 0.097 Myr^?1 revealed a more recent common ancestor for the eastern lineage (0.21 Ma; 0.07–0.37) than for the southern lineage (0.35 Ma; 0.10–0.62). Compared with the latter, the eastern lineage showed pronounced geographic structuring, lower overall nucleotide diversity, higher population differentiation, and isolation‐by‐distance among populations. Main conclusions The data support the hypothesis of Pleistocene refugia occurring in East and southern Africa. In agreement with palynological, palaeovegetation and fossil studies, our data strongly support the presence of a longer‐standing population in the south and a mosaic of Pleistocene refugia in the east, verifying the efficacy of genetic tools in addressing such questions. The more recent origin of the common eland inhabiting East Africa could result from colonization following extinction from the region. Only two other dated African ungulate phylogenies have been published, applying different methods, and the similarity of dates obtained from the three distinct approaches indicates a significant event c. 200 ka, which left a strong genetic signature across a range of ungulate taxa.     

Late Pleistocene origin of the entire circumarctic range of the arctic‐alpine plant Kalmia procumbens

The circumarctic ranges of arctic‐alpine plants are thought to have been established in the late Pliocene/early Pleistocene, when the modern arctic tundra was formed in response to climate cooling. Previous findings of range‐wide genetic structure in arctic‐alpine plants have been thought to support this hypothesis, but few studies have explicitly addressed the temporal framework of the genetic structure. Here, we estimated the demographic history of the genetic structure in the circumarctic Kalmia procumbens using sequences of multiple nuclear loci and examined whether its genetic structure reflects prolonged isolation throughout the Pleistocene. Both Bayesian clustering and phylogenetic analyses revealed genetic distinction between alpine and arctic regions, whereas detailed groupings were somewhat discordant between the analyses. By assuming a population grouping based on the phylogenetic analyses, which likely reflects a deeper intraspecific divergence, we conducted model‐based analyses and demonstrated that the intraspecific genetic divergence in K. procumbens likely originated during the last glacial period. Thus, there is no need to postulate range separation throughout the Pleistocene to explain the current genetic structure in this species. This study demonstrates that range‐wide genetic structure in arctic‐alpine plants does not necessarily result from the late Pliocene/early Pleistocene origin of their circumarctic ranges and emphasizes the importance of a temporal framework of the current genetic structure for understanding the biogeographic history of the arctic flora.     

Phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum: contrasting results from mitochondrial and microsatellite data

Phylogeographical studies are typically based on haplotype data, occasionally on nuclear markers such as microsatellites, but rarely combine both. This is unfortunate because the use of markers with contrasting modes of inheritance and rates of evolution might provide a more accurate and comprehensive understanding of a species' history. Here we present a detailed study of the phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum , using 1098 bp of the mitochondrial ND2 gene from 45 localities from across its Palaearctic range to infer population history. In addition, we re-analysed a large microsatellite data set available for this species and compared the results of both markers to infer population relationships and the historical processes influencing them. We show that mtDNA, the most popular marker in phylogeography studies, yielded a misleading result, and would have led us to conclude erroneously that a single expansion had taken place in Europe. Only by combining the mitochondrial and microsatellite data sets are we able to reconstruct the species' history and show two colonization events in Europe, one before the Last Glacial Maximum (LGM) and one after it. Combining markers also revealed the importance of Asia Minor as an ancient refugium for this species and a source population for the expansion of the greater horseshoe bat into Europe before the LGM.