Factors related to the occurrence of hybrids between brown hares Lepus europaeus and mountain hares L. timidus in Sweden

Hybridization occurs among many species, and may have implications for conservation as well as for evolution. Interspecific gene flow between brown hares Lepus europaeus and mountain hares L. timidus has been documented in Sweden and in continental Europe, and has probably to some extent occurred throughout history in sympatric areas. What local factors or ecological relationships that correlate with or trigger hybridization between these species has however been unclear. We studied spatial distribution of hybrids between brown hares and mountain hares in Sweden in relation to characteristics of the sampled localities (hunting grounds). In a sample of 70 brown hares collected from 39 populations in south‐central Sweden during 2003–2005, 11 (16%) showed introgressed mtDNA from mountain hares. Among the brown hares from their northern range, i.e. in general the most recent establishments, the corresponding figure was 75% (9/12). The frequency of samples with hybrid ancestry increased significantly with latitude, altitude and hilliness, and were higher (p<0.1) in recently established populations and/or where the proportion of arable land was low. Several site‐specific parameters were correlated, e.g. latitude as expected to hilliness, and no parameter explained the occurrence of hybrids exclusively. Instead, the appearance of mountain hare mtDNA among brown hares was associated with a conglomerate of parameters reflecting landscapes atypical for the brown hare, e.g. forest dominated and steep areas where the species quite recently was established. We suggest that these abiotic factors mirror the main aspect influencing hybridization frequency, namely the density or relative frequency of the two species. In atypical brown hare landscapes with recent establishment, mountain hares are probably relatively more common. When one species dominate in numbers, or when both species display low densities, increased frequency of hybridization is expected due to low availability of conspecific partners, a phenomenon referred to as Hubbs’ principle.     

High mtDNA haplotype diversity among introduced Swedish brown haresLepus europaeus

The brown hareLepus europaeus Pallas, 1778 occurs naturally in central Eurasia, but has been introduced to parts of northern Europe, South- and North America, Australia and New Zealand. Brown hares were introduced to Sweden from central Europe for hunting purposes during the 19th century. We investigated how the human--mediated brown hare colonisation of Sweden is reflected in the amount of genetic variation present by assessing variation and composition of mitochondrial DNA (mtDNA) lineages among Swedish brown hares. MtDNA from a total of 40 brown hare specimens from 15 localities were analysed for Restriction Fragment Length Polymorphisms. The haplotype diversity is surprisingly high (0.893 ± 0.002) when compared to the mtDNA diversity among brown hares on the European continent as well as to other mammalian species. Admixture of haplotypes from different source populations combined with a reduced effect of random genetic drift and a relaxed selection pressure due to rapid population growth after introduction are mechanisms that are likely to account for the observed high mtDNA haplotype diversity.     

The hidden history of the snowshoe hare,Lepus americanus: extensive mitochondrial DNA introgression inferred from multilocus genetic variation

Hybridization drives the evolutionary trajectory of many species or local populations, and assessing the geographic extent and genetic impact of interspecific gene flow may provide invaluable clues to understand population divergence or the adaptive relevance of admixture. In North America, hares (Lepus spp.) are key species for ecosystem dynamics and their evolutionary history may have been affected by hybridization. Here we reconstructed the speciation history of the three most widespread hares in North America – the snowshoe hare (Lepus americanus), the white‐tailed jackrabbit (L. townsendii) and the black‐tailed jackrabbit (L. californicus) – by analysing sequence variation at eight nuclear markers and one mitochondrial DNA (mtDNA) locus (6240 bp; 94 specimens). A multilocus–multispecies coalescent‐based phylogeny suggests that L. americanus diverged ~2.7 Ma and that L. californicus and L. townsendii split more recently (~1.2 Ma). Within L. americanus, a deep history of cryptic divergence (~2.0 Ma) was inferred, which coincides with major speciation events in other North American species. While the isolation‐with‐migration model suggested that nuclear gene flow was generally rare or absent among species or major genetic groups, coalescent simulations of mtDNA divergence revealed historical mtDNA introgression from L. californicus into the Pacific Northwest populations of L. americanus. This finding marks a history of past reticulation between these species, which may have affected other parts of the genome and influence the adaptive potential of hares during climate change.     

The rise and fall of the mountain hare (Lepus timidus) during Pleistocene glaciations: expansion and retreat with hybridization in the Iberian Peninsula

The climatic fluctuations during glaciations have affected differently arctic and temperate species. In the northern hemisphere, cooling periods induced the expansion of many arctic species to the south, while temperate species were forced to retract in southern refugia. Consequently, in some areas the alternation of these species set the conditions for competition and eventually hybridization. Hares in the Iberian Peninsula appear to illustrate this phenomenon. Populations of Iberian hare (Lepus granatensis), brown hare (Lepus europaeus) and broom hare (Lepus castroviejoi) in Northern Iberia harbour mitochondrial haplotypes from the mountain hare (Lepus timidus), a mainly boreal and arctic species presently absent from the peninsula. To understand the history of this past introgression we analysed sequence variation and geographical distribution of mitochondrial control region and cytochrome b haplotypes of L. timidus origin found in 378 specimens of these four species. Among 124 L. timidus from the Northern Palaearctic and the Alps we found substantial nucleotide diversity (2.3%) but little differentiation between populations. Based on the mismatch distribution of the L. timidus sequences, this could result from an expansion at a time of temperature decrease favourable to this arctic species. The nucleotide diversity of L. timidus mtDNA found in Iberian L. granatensis, L. europaeus and L. castroviejoi (183, 70 and 1 specimens, respectively) was of the same order as that in L. timidus over its range (1.9%), suggesting repeated introgression of multiple lineages. The structure of the coalescent of L. granatensis sequences indicates that hybridization with L. timidus was followed by expansion of the introgressed haplotypes, as expected during a replacement with competition, and occurred when temperatures started to rise, favouring the temperate species. Whether a similar scenario explains the introgression into Iberian L. europaeus remains unclear but it is possible that it hybridized with already introgressed L. granatensis.     

On shortcomings of using mtDNA sequence divergence for the systematics of hares (genus Lepus): An example from cape hares

Despite the well-known fact that evolutionary patterns of single genes or sequences are not necessarily paralleled by organismic evolution, using mitochondrial sequence divergence for inferring phylogenetic relationships among hare species is not uncommon. Earlier studies reported interspecific introgression in the genus Lepus that may slur systematic conclusions drawn exclusively from mtDNA data. We examined pairwise divergence in partial mtHV-1 sequences and nuclear gene pools based on microsatellite and allozyme loci separately in a South and North African population of cape hares, Lepus capensis, and did not find significant correspondence on the individual level within either population. Also, the former population had a significantly higher level of mtHV-1 sequence divergence compared to the latter, but levels of individual nuclear gene pool divergence did not differ significantly between the two populations. Hence, the absence of correspondence between mtHV-1 sequence divergence and nuclear gene pool divergence among individuals within a population might be independent of the population-specific level of differentiation among individuals. Our results complement earlier findings in hares, and strongly recommend to include nuclear gene pool evidence for systematic inferences within this genus, even under the absence of mitochondrial introgression.     

Polymorphism of mtDNA regions in brown hare (Lepus europaeus) populations from Vojvodina (Serbia and Montenegro)

It is well known that there is heterogeneity of mitochondrial DNA (mtDNA) within and among natural populations of same species. The polymorphism level of particular regions of mtDNA gives valuable results in detection of population genetic structure. The aim of this paper was to detect polymorphism of three mtDNA regions: cytochrome oxidase I (COI), Control region, and 12S/16S rRNA, by the mtDNA RFLP-PCR method, in three Lepus europaeus populations from Vojvodina province (Serbia and Montenegro). Polymorphism was detected within the two regions, COI and Control region, while 12S/16S rRNA region was monomorphic in all 77 individuals. Eight haplotypes were detected in the brown hare population in Vojvodina, and three were unique for the Srem brown hare population.     

The diet of the mountain hare (Lepus timidus hibernicus) on coastal grassland

Across most of their range in Europe, mountain hares are usually restricted to upland areas with poor food quality. In these areas they generally feed on browse species such as heather or twigs and barks of trees. On lowland areas in Europe, with better food quality, the mountain hare is replaced by the brown hare ( Lepus europaeus ) which feeds predominantly on greasses. This khas led some authors to conclude that mountain hares are primarily adapted for browsing. In the absence of brown hares in Ireland, mountain hares are found on a wide variety of habitats including grassland. On grassland, their diet consists almost exclusively of grasses, up to 94% of their annual diet, which is more than has been reported for brown hares on similar habitat. Based on this evidence, and other work, it is proposed that the mountain hare in primarily a grazing animal and competitive exclusion by brown hares may underlie much of their present distribution in Europe.     

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.     

The recent expansion of the brown hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis>) in Sweden with possible implications to the mountain hare (<Emphasis Type="Italic">L. timidus</Emphasis>)

The brown hare (Lepus europaeus) expanded its Swedish distribution since the 1980s northwards and locally to new areas within its former range. Of 115 brown hare populations within the former range reported in a hunter enquiry, those established after 1980 were situated higher above the sea level than older ones and higher than neighbouring (<50 km) older populations. Reports on increased use of forest habitats by brown hares were equally frequent among recent and older populations, suggesting a process promoted solely by less harsh winters. Supposed hare hybrids were more often reported from hunting grounds with recent brown hare establishment, i.e. where the species expands in time and in space. In a 27-year dataset on brown hare observations, the recent increased use of forest habitats was supported in that maximum distances to agricultural land for brown hare sightings were higher in mild winters, whereas the proportions of the annual observations made during winter were lower. In 40-year bag records from two Swedish counties, the dynamics of the mountain hare (Lepus timidus) responded positively to snow parameters, whereas brown hares responded negatively. We suggest that the state of mountain hare populations primarily depends on winter conditions and predation pressure, whereas possible effects of hybridization are unclear. If winter conditions remain as in the last 15 years, mountain hare numbers are not likely to increase in southern Sweden, whereas the brown hare may expand even further. In either case, hybrids will occur in sympatric areas in frequencies probably related to the density of the respective true species.     

Mitochondrial Cytochrome b Gene Sequence Diversity in the Korean Hare, Lepus coreanus Thomas (Mammalia, Lagomorpha)

Partial sequences of the mitochondrial cytochrome b gene of the Korean hare (Lepus coreanus) were analyzed to determine the degree of genetic diversity. Nine haplotypes were observed, and the maximum Tamura-Nei nucleotide distance among them was 2.8%, indicating that genetic diversity of L. coreanus is moderate. In order to clarify the Korean hare's taxonomic status and relationship with the Manchurian hare (L. mandshuricus) and the Chinese hare (L. sinensis), these nine haplotypes of the Korean hare were compared with 13 haplotypes from five other species of eastern Asian Lepus including L. mandshuricus and L. sinensis. The Korean hare was distinct in its cytochrome b gene, and it is confirmed that L. coreanus is a valid species, as noted by Jones and Johnson (1965, Univ. Kansas Publ. (Mus. Nat. Hist.) 16:357). Further analyses of mtDNA cytochrome b gene with additional specimens of L. coreanus from North Korea and other species of Lepus from eastern Asia are needed to clarify the taxonomic status of the divergent mtDNA clades of L. mandshuricus and L. sinensis.     

The genomic legacy from the extinct Lepus timidus to the three hare species of Iberia: contrast between mtDNA, sex chromosomes and autosomes

Extensive interspecific genetic introgression is often reported, and appraising its genomic impact can serve to determine whether it results from selection on specific loci or from demographic processes affecting the whole genome. The three species of hares present in the Iberian Peninsula harbour high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species now extinct in the region. This could result from the invasive replacement of L. timidus by the temperate species during deglaciation but should then have left traces in the nuclear genome. We typed single nucleotide polymorphisms (SNPs) discovered by sequencing 10 autosomal loci, two X-linked and one Y-linked in species-wide samples of the four taxa. Based on lineage-diagnostic SNPs, we detected no trace of L. timidus sex chromosomes in Iberia. From the frequencies of inferred haplotypes, autosomal introgression into L. granatensis appeared mostly sporadic but always widespread instead of restricted to the north as mtDNA. Autosomal introgression into Iberian L. europaeus , inhabiting the Pyrenean foothills, was hardly detectable, despite quasi-fixation of L. timidus mtDNA. L. castroviejoi , endemic to the Cantabrian Mountains and fixed for L. timidus mtDNA, showed little traces of autosomal introgression. The absence of sex-chromosome introgression presumably resulted from X-linked hybrid male unfitness. The contrasting patterns between the autosomes and mtDNA could reflect general gender asymmetric processes such as frequency-dependent female assortative mating, lower mtDNA migration and higher male dispersal, but adaptive mtDNA introgression cannot be dismissed. Additionally, we document reciprocal introgression between L. europaeus and both L. granatensis in Iberia and L. timidus outside Iberia.     

The distribution of mountain hares Lepus timidus in Europe: a challenge from brown hares L. europaeus?

1. Throughout the most recent glacial period (Weichsel), the mountain hare Lepus timidus had a continuous distribution in the tundra habitat south of the ice‐rim. When the ice retreated, mountain hares colonized deglaciated land, and spread over northern Europe. 2. Since the Weichsel, the mountain hare's distribution in Europe has been gradually reduced and at present comprises Ireland and the Scottish Highlands, high altitudes in the Alps, isolated forests in eastern Poland, most of Fennoscandia and from the Baltic countries eastwards through Russia. Declines during the last century have been observed in Sweden and Russia. 3. This review defines and evaluates causes for this gradual reduction and fragmentation of the mountain hare's distribution, with special focus on interactions with brown hares Lepus europaeus. The relative importance of diseases, predation, cultivation and interactions with other herbivores than brown hares are discussed. 4. A plausible cause of the possible permanent disappearance of mountain hares in Europe appears to be exclusion by interspecific competition and hybridization with, and/or epidemic diseases mediated by, the congeneric brown hare.     

European hare (Lepus europaeus) invasion ecology: implication for the conservation of the endemic Irish hare (Lepus timidus hibernicus)

European hare Lepus europaeus populations have undergone recent declines but the species has successfully naturalised in many countries outside its native range. It was introduced to Ireland during the mid-late nineteenth century for field sport and is now well established in Northern Ireland. The native Irish hare Lepus timidus hibernicus is an endemic subspecies of mountain hare L. timidus and has attracted major conservation concern following a long-term population decline during the twentieth century and is one of the highest priority species for conservation action in Ireland. Little is known about the European hare in Ireland or whether it poses a significant threat to the native mountain hare subspecies by compromising its ecological security or genetic integrity. We review the invasion ecology of the European hare and examine evidence for interspecific competition with the mountain hare for habitat space and food resources, interspecific hybridisation, disease and parasite transmission and possible impacts of climate change. We also examine the impact that introduced hares can have on native non-lagomorph species. We conclude that the European hare is an emerging and significant threat to the conservation status of the native Irish hare. Invasive mammal species have been successfully eradicated from Ireland before and immediate action is often the only opportunity for cost-effective eradication. An urgent call is issued for further research whilst the need for a European hare invasive Species Action Plan (iSAP) and Eradication strategy are discussed.     

Phylogenetic analysis of mtCR-1 sequences of Tunisian and Egyptian hares (Lepus sp. or spp., Lagomorpha) with different coat colours

North African hares are currently considered belonging to cape hares (Lepus capensis), except for an isolated occurrence of L. victoriae in NW Algeria. However, the few existing molecular data are not unequivocal. Here, we study sequence variation (415 bp) in the hypervariable domain-1 of the mitochondrial (mt) control region, of hares with different coat colour from north-central Tunisia and NW Egypt, to test Petter's [(1959): Eléments d’une révision des Lièvres africains du sous-genre Lepus. Mammalia 23, 41–67] hypothesis that North African hares belong to L. capensis. Seven Tunisian and one Egyptian haplotypes were revealed from 28 hares and compared phylogenetically to 245 haplotypes of various Lepus species downloaded from GenBank. Neighbour joining (NJ) and principal coordinate (PCO) analyses based on a Tamura-Nei 93 distance matrix, as well as maximum parsimony (MP) analysis concordantly grouped all currently obtained haplotypes together into one monophyletic clade, and revealed relatively close relationships to the clades of African scrub hares (L. saxatilis) and brown hares (L. europaeus). The three distinguished coat colour types of Tunisian hares were paralleled only to a small extent by sequence differentiation. Haplotypes of L. capensis from the nominal Cape province of South Africa, North Africa, and China clustered into different major clades, respectively, with Chinese L. capensis haplotypes forming only a subclade within a major clade that encompassed predominantly “mountain/arctic hare-type sequences” in addition to sequences of several other palaearctic and nearctic species. One further Chinese L. capensis haplotype clustered into the L. comus clade. These results indicated occurrence of introgression and/or shared ancestral polymorphism. Such an evolutionary scenario implies using nucelar markers in addition to mtDNA for phylogenetic inferences in the genus Lepus; nevertheless, mtDNA is still useful for inferring phylogenetic history and biogeography of hares.     

Ancient introgression of Lepus timidus mtDNA into L. granatensis and L. europaeus in the Iberian Peninsula

A 587 bp fragment of cytochrome b sequences from 90 individuals of 15 hare (Lepus) species and two outgroups were phylogenetically analysed and compared to an analysis derived from 474 bp sequences of the nuclear transferrin gene. Mountain hare (Lepus timidus) type mtDNA was observed in L. granatensis and L. europaeus from the Iberian Peninsula, far away from the extant distributional range of L. timidus. In addition to these two hare species, other hare species may also contain mtDNA from L. timidus. This species may have introgressed with other species of Lepus that occur within its present range, or where fossils indicate its historical presence during glacial periods. L. timidus mtDNA is common in the northern part of the L. granatensis range. Finally, we reassessed the phylogenetic relationships of the five European hare species based on both mitochondrial and nuclear DNA sequences.     

Recurrent introgression of mitochondrial DNA among hares (Lepus spp.) revealed by species-tree inference and coalescent simulations

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.     

Molecular Approaches Revealing Prehistoric, Historic, or Recent Translocations and Introductions of Hares (Genus Lepus) by Humans

Although only of medium size, and thus of little nutritional value compared to big game such as mammoths and ungulates, hares (Lepus spp.) probably have always been a food source for humans, as documented in archaeological finds. Nowadays, hares, particularly such species as the brown hare (L. europaeus), are among the most important game species in many European countries. For hunting, perhaps religious reasons, and in connection with certain myths, hares have been and are still being intentionally translocated. Ancient translocations by humans can be inferred from the presence of hares on islands that had no mainland connections, at least during the Pleistocene, the major evolutionary period of the genus Lepus. We review some of the literature on anthropogenic translocations of hares. We focus on three examples [the brown hare (L. europaeus), the Corsican hare (L. corsicanus), and the Sardinian hare (L. capensis)], where some molecular data could be used to trace the translocation routes and possible origins of introduced hare populations. Certain molecular marker systems, such as sequences of the hypervariable part I (HV-1) of the mitochondrial control region, show high variability in hare species and are thus promising for tracing both recent and ancient origins of translocated hares. Some other molecular marker systems as well as caveats connected with the use of such marker systems in the genus Lepus are also discussed.     

Influence of management regime and population history on genetic diversity and population structure of brown hares (Lepus europaeus) in an Italian province

In many areas, the management of overexploited populations of brown hare (Lepus europaeus) is based on annual restocking. While in some cases exotic hares are introduced, in some others hares are captured locally within protected areas and subsequently released into hunting grounds. We evaluated the genetic effects of this management regime in an Italian province where the brown hare population has recovered in the last few decades, by sequencing the hypervariable domain 1 of the mitochondrial control region and by genotyping eight autosomal microsatellites in hares sampled in both hunting and non-hunting areas. Both nuclear (H _e?=?0.68 and H _o?=?0.65) and mitochondrial variability (h?=?0.853 and π?=?0.012) were in line with other European populations. When comparing our data with mitochondrial sequences retrieved from GenBank, out of the 21 detected haplotypes, 14 were private to our study area. While 4.6 % of the individuals were found to carry haplotypes attributable to past introductions, 41.5 % grouped within a well-supported lineage, previously identified with a presumed native Italian taxon, L. e. meridiei. Despite the detectable geographic partitioning of mitochondrial haplotypes across the province, no genetic structure resulted from microsatellites analysis, indicating that no reproductive barriers exist among hares carrying different mitochondrial lineages. In conclusion, the local management seems to have contributed to the recovery of the species and to a full admixture of nuclear genes in the province. However, neither the extensive translocations nor the possible introductions of exotic heads seem to have completely undermined the local mitochondrial lineages.     

Phylogeography of the brown hare (Lepus europaeus) in Europe: a legacy of south‐eastern Mediterranean refugia?

Aim We analysed the population genetics of the brown hare (Lepus europaeus) in order to test the hypothesis that this species migrated into central Europe from a number of late glacial refugia, including some in Asia Minor. Location Thirty‐three localities in Greece, Bulgaria, Italy, Croatia, Serbia, Poland, Switzerland, Austria, France, Germany, the Netherlands, Spain, the United Kingdom, Turkey and Israel. Methods In total, 926 brown hares were analysed for mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism (RFLP) performed on polymerase chain reaction‐amplified products spanning cytochrome b (cyt b)/control region (CR), cytochrome oxidase I (COI) and 12S–16S rRNA. In addition, sequence analysis of the mtDNA CR‐I region was performed on 69 individuals, and the data were compared with 137 mtDNA CR‐I sequences retrieved from GenBank. Results The 112 haplotypes detected were partitioned into five phylogeographically well‐defined major haplogroups, namely the ‘south‐eastern European type haplogroup’ (SEEh), ‘Anatolian/Middle Eastern type haplogroup’ (AMh), ‘European type haplogroup, subgroup A’ (EUh‐A), ‘European type haplogroup, subgroup B’ (EUh‐B) and ‘Intermediate haplogroup’ (INTERh). Sequence data retrieved from GenBank were consistent with the haplogroups determined in this study. In Bulgaria and north‐eastern Greece numerous haplotypes of all five haplogroups were present, forming a large overlap zone. Main conclusions The mtDNA results allow us to infer post‐glacial colonization of large parts of Europe from a late glacial/early Holocene source population in the central or south‐central Balkans. The presence of Anatolian/Middle Eastern haplotypes in the large overlap zone in Bulgaria and north‐eastern Greece reveals gene flow from Anatolia to Europe across the late Pleistocene Bosporus land‐bridge. Although various restocking operations could be partly responsible for the presence of unexpected haplotypes in certain areas, we nevertheless trace a strong phylogeographic signal throughout all regions under study. Throughout Europe, mtDNA results indicate that brown hares are not separated into discernable phyletic groups.