Purifying selection shaping the evolution of the Toll-like receptor 2 TIR domain in brown hares (Lepus europaeus) from Europe and the Middle East.

Toll-like receptors (TLRs) are transmembrane proteins of the innate immune system, composed of the ectodomain involved in pathogen recognition and the intracellular Toll/interleukin-1 receptor (TIR) domain important for downstream signal transduction. Here, we analyze the genetic variability of TIR nucleotide and amino-acid sequences of the TLR2 gene in 243 brown hares from Europe and the Middle East and tested for the presence of selection signals and spatial structuring. TLR2 TIR domain sequences were PCR amplified and sequenced, while genotyping was performed by phasing. Genetic diversity indices were calculated in DnaSP and Arlequin, while presence of selection signals was tested using MEGA and the Datamonkey web server. The presence of spatial patterns in TIR sequence distribution was tested by spatial Principal Component Analysis (sPCA) in adegenet. A total of 13 haplotypes were revealed with haplotype diversity of 0.424, and nucleotide diversity (π) of 0.00138. Two spatial clusters were revealed: "Anatolia/Middle East" and "Europe". In Anatolia the two most prevalent amino-acid variants, A and B (the latter being the most ancestral) were maintained at similar frequencies; but in Europe a shift in genotype frequencies was observed as well as a higher number of nonsynonymous substitutions giving rise to novel amino-acid protein variants originating from the evolutionarily younger protein variant. Molecular diversity (haplotype and nucleotide diversity) indices were significantly higher in the "Anatolia/Middle East" cluster. A signal of purifying selection was detected acting on the TIR sequences.

Genetic analysis of 12 X-STR loci in the Serbian population from Vojvodina Province.

The analysis of 12 X-STR loci included in the Investigator® Argus X-12 kit was performed on a sample of 325 unrelated persons from Vojvodina Province, Republic of Serbia. No significant differences were observed in the allele frequencies in males and females. Heterozygosity values among the studied loci ranged from 67.62 to 94.28%. All loci in female individuals were consistent with the Hardy-Weinberg equilibrium test. The combined power of discrimination values in male and female individuals was 0.9999999994 and 0.999999999999999, respectively. The combined mean exclusion chance was 0.999998 in deficiency cases, 0.9999999977 in normal trio cases, and 0.9999994 in duo cases. Loci DXS10135 and DXS10101 were found to be most polymorphic. The haplotype diversity was found to be greater than 0.993 for all linkage groups. The exact test for pairwise linkage disequilibrium for the 12 loci in the male samples showed significant linkage disequilibrium for the DXS10103-DXS10101 and DXS10134-DXS10146 pairs of loci. The results from the current study confirmed that the panel of 12 X-STR loci is highly polymorphic and informative and can be implemented as a powerful tool in deficient paternity testing and kinship analysis, as well as a useful complement tool of autosomal short tandem repeats (STRs) in forensic investigation. Population differentiation analyses indicated significant differences in genetic structure between the Serbian population and the geographically and ethno-linguistically distant populations, while genetic homogeneity was present in populations with similar geographic origin.

Different associations of apoE gene polymorphism with metabolic syndrome in the Vojvodina Province (Serbia).

The metabolic syndrome (MetS) is a polygenic multifactorial metabolic disorder with strong socioeconomic influence. MetS has became a worldwide epidemic, that directly increases the risk of cardiovascular diseases and type 2 diabetes mellitus. The human apoE gene, coding Apolipoprotein E, has three common polymorphisms in human population: e2, e3 and e4, which are proved to be associated with impaired lipid metabolism. The contribution of apoE polymorphism to MetS disorders has not been investigated previously in Vojvodina Province, region with the highest number of obese people in Serbia. The aim of this study was to evaluate apoE gene polymorphism in relation to MetS disorders. The healthy control group of 30 individuals and 63 MetS patients were examined for apoE variants in relation to biochemical and anthropometric parameters. The genotypes were determined by PCR-RFLP. Regarding all parameters, significantly higher values were detected in MetS group compared to control. The MetS group of patients had significantly higher frequency of e4 allele. In addition, positive relation was revealed between e4 allele presence and all measured parameters. It was found that the e4 allele was related with a significantly increased OR of MetS disorders according to the International Diabetes Federation definition. These results suggested that e4 allele may act as a one of determinants for development of metabolic syndrome.

Dose-dependent survival of K562 cells subjected to irradiation, time course of endogenous enzyme activity and protective effect of applied superoxide dismutase.

PURPOSE: Irradiation-generated reactive species are proven to affect the cell survival and antioxidant enzyme levels. Radioresistance is a phenomenon which includes many cell mechanisms and signaling pathways. Superoxide dismutase (SOD) acts in and outside of cells after irradiation. The aim of this study was to determine LD50 (lethal dose for 50% of K562 cells), to monitor the effect of a chosen dose and exogenously applied superoxide dismutase (ExSOD) on the cell number and the activity of SOD, glutathione peroxidase (GSH-Px) and catalase (CAT). METHODS: The survival of irradiated (20-32.5 Gy) K562 cells was determined using the trypan-blue exclusion. Besides irradiated and non-irradiated cells (controls), another two groups of cells were treated with SOD (10-6 M) which then served as SOD-treated controls or were irradiated (30 Gy) one hour later. The number of cells and the activity of SOD, GSH-Px and CAT (using kinetic methods) were monitored after 1, 24, 48, 72 and 96 hrs in unirradiated, irradiated, SOD-treated and SOD-treated/irradiated experimental groups. RESULTS: K562 cells showed dose-dependent survival in the chosen range of doses. A dose of 30 Gy induced 50% cell mortality and increased the activity of all three investigated enzymes after 24 hrs. Pretreatment with SOD preserved the survival of irradiated cells and increased SOD, GSH-Px and CAT activity. ExSOD induced an increase of the activity of all examined enzymes. CONCLUSION: A balanced enhancement in endogenous antioxidative activity may be the cause of the increased radioresistance of K562 SOD-pretreated cells.

Radiobiological effects of multiple vs. single low-dose pre-irradiation on the HT29 cell line.

AIM OF THE STUDY: Aim of the study was to compare radiobiological effects of multiple vs. single low-dose pre-irradiation on the HT29 cell line. This regime is designed to be as similar as possible to fractionated tumour radiotherapy treatment, and to provide data on radiobiological effects on human tumour cells. MATERIAL AND METHODS: The cell line used in the study was HT29 (human colorectal adenocarcinoma, American Type Culture Collection HTB-38™). Also, for comparison, the MRC5 cell line (human foetal lung fibroblasts, American Type Culture Collection CCL 171) was used. Four-day treatment in a 4 × 2 Gy regime was performed. Cell viability was evaluated by tetrazolium colorimetric MTT assay. RESULTS: Multiple low-dose pre-irradiation induced a stronger radioadaptive response compared to single low-dose application in the HT29 cell line. Multiple pre-irradiation with 0.03 Gy and 0.05 Gy caused radioadaptive effects, while in both single and multiple low-dose pre-irradiation regimes 0.07 Gy led to radiosensitivity. Radiobiological effects induced in the HT29 cell line by low-dose pre-irradiation were evidently weak during the treatment time, because a single low-dose applied only on the first day gave no radioadaptive effects. In the MRC5 cell line different effects were registered, since radioadaptive response has not been observed after multiple or single pre-irradiation. CONCLUSIONS: The obtained data are interesting, especially for the possible application of low-dose pre-irradiation in radiotherapy.

The Extraordinary Diversity of Merodon avidus Complex (Diptera: Syrphidae)-Adding New Areas, New Species and a New Molecular Marker.

In this paper, the Merodon avidus (Diptera, Syrphidae) species complex was revised, whereupon we discovered and described four new species for science: Merodon atroavidus Vujic, Radenkovic et Likov sp. nov., M. magnus Vujic, Kocis Tubic et Acanski sp. nov., M. nigroscutum Vujic, Radenkovic et Likov sp. nov. and M. pseudomoenium Vujic, Kocis Tubic et Acanski sp. nov. An integrative taxonomy approach was used to delimit species boundaries. Two molecular markers (the mitochondrial COI gene and nuclear 28S rRNA gene-newly analysed marker for the complex) and geometric morphometry of the wing shape, together with morphological data and distribution, successfully separated all species from the complex. The morphological variability of the analysed species is described and discussed and an illustrated diagnostic key for typical morpho-forms of species from the M. avidus complex is presented. A distribution map of all investigated species from the complex is provided. The level of endemicity of the M. avidus complex was discussed.

Revisions of the clavipes and pruni species groups of the genus Merodon Meigen, 1803 (Diptera, Syrphidae).

This study focuses on the avidus-nigritarsis lineage within the genus Merodon, exploring morphological, genetic, and distributional aspects of two related assemblies within this lineage: the clavipes and pruni species groups. An integrative taxonomic approach was followed to ensure comprehensive species identification and validation, using adult morphology, wing geometric morphometrics, and genetic analysis of the mtDNA COI gene. In the clavipes group, seven species were identified, including three new species: M.aenigmaticus Vujic, Radenkovic & Likov, sp. nov., M.latens Vujic, Radenkovic & Likov, sp. nov., and M.rufofemoris Vujic, Radenkovic & Likov, sp. nov. In the pruni group, our revision revealed a new species, M.aequalis Vujic, Radenkovic & Likov, sp. nov., and the revalidation of Merodonobscurus Gil Collado, 1929, stat. rev. Merodonpallidus Macquart, 1842 is redescribed. Diagnoses, identification keys to species, and distribution maps are provided, and neotypes for Syrphusclavipes Fabricius, 1781 and Merodonquadrinotatus (Sack, 1931) are designated. Additionally, the following new synonyms are proposed: M.clavipesalbus syn. nov., M.clavipesater syn. nov., M.clavipesniger syn. nov., and M.splendens syn. nov. are junior synonyms of M.clavipes; and M.veloxarmeniacus syn. nov. and M.veloxanathema syn. nov. are junior synonyms of M.velox.

Monitoring of species' genetic diversity in Europe varies greatly and overlooks potential climate change impacts.

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species' joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union's Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity.

Three new species of the Merodon ruficornis group (Diptera: Syrphidae) discovered at the edge of its range.

Further revision of material from the Merodon ruficornis group (Diptera: Syrphidae) revealed three new species, M. acutus Vuji et Radenkovi sp. nov. from Russia, M. fulvitarsis Vuji et Radenkovi sp. nov. from Iran, and M. trispinus Vuji et Radenkovi sp. nov. from Turkey. We provide an upgraded and illustrated identification key for the species of the group, as well as distribution maps for the new species. Moreover, we describe the female of M. portschinskyi Stackelberg, 1924. These new descriptions extend the range of the M. ruficornis group in almost all directions and bolster the very high level of endemism previously observed within this group. Of the 21 taxa (including the three new species described herein), 15 are narrow-range endemics.

Pan-European phylogeography of the European roe deer (Capreolus capreolus).

To provide the most comprehensive picture of species phylogeny and phylogeography of European roe deer (Capreolus capreolus), we analyzed mtDNA control region (610 bp) of 1469 samples of roe deer from Central and Eastern Europe and included into the analyses additional 1541 mtDNA sequences from GenBank from other regions of the continent. We detected two mtDNA lineages of the species: European and Siberian (an introgression of C. pygargus mtDNA into C. capreolus). The Siberian lineage was most frequent in the eastern part of the continent and declined toward Central Europe. The European lineage contained three clades (Central, Eastern, and Western) composed of several haplogroups, many of which were separated in space. The Western clade appeared to have a discontinuous range from Portugal to Russia. Most of the haplogroups in the Central and the Eastern clades were under expansion during the Weichselian glacial period before the Last Glacial Maximum (LGM), while the expansion time of the Western clade overlapped with the Eemian interglacial. The high genetic diversity of extant roe deer is the result of their survival during the LGM probably in a large, contiguous range spanning from the Iberian Peninsula to the Caucasus Mts and in two northern refugia.

Location and Species Matters: Variable Influence of the Environment on the Gene Flow of Imperiled, Native and Invasive Cottontails.

The environment plays an important role in the movement of individuals and their associated genes among populations, which facilitates gene flow. Gene flow can help maintain the genetic diversity both within and between populations and counter the negative impact of genetic drift, which can decrease the fitness of individuals. Sympatric species can have different habitat preferences, and thus can exhibit different patterns of genetic variability and population structure. The specialist-generalist variation hypothesis (SGVH) predicts that specialists will have lower genetic diversity, lower effective population sizes (Ne), and less gene flow among populations. In this study, we used spatially explicit, individual-based comparative approaches to test SGVH predictions in two sympatric cottontail species and identify environmental variables that influence their gene flow. New England cottontail (Sylvilagus transitionalis) is the only native cottontail in the Northeast US, an early successional habitat specialist, and a species of conservation concern. Eastern cottontail (S. floridanus) is an invasive species in the Northeast US and a habitat generalist. We characterized each species' genomic variation by developing double-digest Restriction-site Associated DNA sequence single nucleotide polymorphism markers, quantified their habitat with Geographic Information System environmental variables, and conducted our analyses at multiple scales. Surprisingly, both species had similar levels of genetic diversity and eastern cottontail's Ne was only higher than New England cottontail in one of three subregions. At a regional level, the population clusters of New England cottontail were more distinct than eastern cottontail, but the subregional levels showed more geographic areas of restricted gene flow for eastern cottontail than New England cottontail. In general, the environmental variables had the predicted effect on each species' gene flow. However, the most important environmental variable varied by subregion and species, which shows that location and species matter. Our results provide partial support for the SGVH and the identification of environmental variables that facilitate or impede gene flow can be used to help inform management decisions to conserve New England cottontail.

Population genetic structure of wolves in the northwestern Dinaric-Balkan region.

The Balkan Peninsula and the Dinaric Mountains possess extraordinary biodiversity and support one of the largest and most diverse wolf (Canis lupus) populations in Europe. Results obtained with diverse genetic markers show west-east substructure, also seen in various other species, despite the absence of obvious barriers to movement. However, the spatial extent of the genetic clusters remains unresolved, and our aim was to combine fine-scale sampling with population and spatial genetic analyses to improve resolution of wolf genetic clusters. We analyzed 16 autosomal microsatellites from 255 wolves sampled in Slovenia, Croatia, Bosnia and Herzegovina (BIH), and Serbia and documented three genetic clusters. These comprised (1) Slovenia and the regions of Gorski kotar and Lika in Croatia, (2) the region of Dalmatia in southern Croatia and BIH, and (3) Serbia. When we mapped the clusters geographically, we observed west-east genetic structure across the study area, together with some specific structure in BIH-Dalmatia. We observed that cluster 1 had a smaller effective population size, consistent with earlier reports of population recovery since the 1980s. Our results provide foundation for future genomic studies that would further resolve the observed west-east population structure and its evolutionary history in wolves and other taxa in the region and identify focal areas for habitat conservation. They also have immediate importance for conservation planning for the wolves in one of the most important parts of the species' European range.

Dispersal ability predicts spatial genetic structure in native mammals persisting across an urbanization gradient.

As the rate of urbanization continues to increase globally, a growing body of research is emerging that investigates how urbanization shapes the movement-and consequent gene flow-of species in cities. Of particular interest are native species that persist in cities, either as small relict populations or as larger populations of synanthropic species that thrive alongside humans in new urban environments. In this study, we used genomic sequence data (SNPs) and spatially explicit individual-based analyses to directly compare the genetic structure and patterns of gene flow in two small mammals with different dispersal abilities that occupy the same urbanized landscape to evaluate how mobility impacts genetic connectivity. We collected 215 white-footed mice (Peromyscus leucopus) and 380 big brown bats (Eptesicus fuscus) across an urban-to-rural gradient within the Providence, Rhode Island (U.S.A.) metropolitan area (population =1,600,000 people). We found that mice and bats exhibit clear differences in their spatial genetic structure that are consistent with their dispersal abilities, with urbanization having a stronger effect on Peromyscus mice. There were sharp breaks in the genetic structure of mice within the Providence urban core, as well as reduced rates of migration and an increase in inbreeding with more urbanization. In contrast, bats showed very weak genetic structuring across the entire study area, suggesting a near-panmictic gene pool likely due to the ability to disperse by flight. Genetic diversity remained stable for both species across the study region. Mice also exhibited a stronger reduction in gene flow between island and mainland populations than bats. This study represents one of the first to directly compare multiple species within the same urban-to-rural landscape gradient, an important gap to fill for urban ecology and evolution. Moreover, here we document the impacts of dispersal capacity on connectivity for native species that have persisted as the urban landscape matrix expands.

Revision of the Merodon serrulatus group (Diptera, Syrphidae).

The phytophagous hoverfly genus Merodon Meigen, 1803 (Diptera, Syrphidae), which comprises more than 160 species distributed in Palaearctic and Afrotropical regions, can be differentiated into multiple groups of species that harbor high levels of hidden diversity. In this work, the serrulatus species group of Merodon is revised, providing an illustrated key to species, a detailed discussion on the taxonomic characters and a morphological diagnosis, including also the first data about the preimaginal morphology of this species group. The study includes characteristics of the 13 species of the M. serrulatus group, along with the available distributional data. Moreover, descriptions are provided for seven new species, namely M. defectus Vujic, Likov & Radenkovic sp. nov., M. disjunctus Vujic, Likov & Radenkovic sp. nov., M. medium Vujic, Likov & Radenkovic sp. nov., M. nigrocapillatus Vujic, Likov & Radenkovic sp. nov., M. nigropunctum Vujic, Likov & Radenkovic sp. nov., M. opacus Vujic, Likov & Radenkovic sp. nov., and M. trianguloculus Vujic, Likov & Radenkovic sp. nov. In addition, the taxa M. serrulatus (Wiedemann in Meigen, 1822), M. bequaerti Hurkmans, 1993, M. hirsutus Sack, 1913, M. kawamurae Matsumura, 1916, M. sacki (Paramonov, 1936) and M. sophron Hurkmans, 1993 are redefined and redescribed. Following a detailed study of the type material sourced from different entomological collections, the status of all available taxa related to M. serrulatus is revised and a new synonymy is proposed: M. tener Sack, 1913 syn. nov. (junior synonym of M. serrulatus). The identity of M. trizonus (Szilády, 1940) could not be assessed as the type specimens are lost. Thus, the name M. trizonus is considered as nomen dubium. The monophyly and composition of this species group are assessed through Maximum Parsimony and Maximum Likelihood analyses of the mitochondrial COI and nuclear 28S rRNA gene sequences.

The VEGF gene polymorphism in glioblastoma may be a new prognostic marker of overall survival.

PURPOSE: Glioblastoma (GBM) is the most aggressive primary brain tumor. Vascular endothelial growth factor (VEGF) gene polymorphisms and overexpression are involved in high-grade malignant gliomas. The aim of this study was to assess the distribution of +405C>G VEGF gene polymorphism in patients diagnosed by glioblastoma and to test its association with the overall survival (OS). METHODS: Patients diagnosed for glioblastoma were randomly selected, and follow-up was conducted for a minimum of 36 months. Tissue paraffin embedded GBM samples were subjected for the VEGF polymorphism detection. The associations of the observed genotypes and clinical data were evaluated. RESULTS: The most frequent single nucleotide polymorphism (SNP) variant was G (72.58%). The GG genotype was proved to have statistically significant longer OS and patient status (alive/dead) compared to CC and CG genotypes (p=0.022 and 0.005, respectively). CONCLUSION: Our results indicate that +405C>G VEGF gene polymorphism may be used as prognostic genetic marker of OS in GBM patients.

Positive selection and precipitation effects on the mitochondrial NADH dehydrogenase subunit 6 gene in brown hares (Lepus europaeus) under a phylogeographic perspective.

Previous studies in hares and jackrabbits have indicated that positive selection has shaped the genetic diversity of mitochondrial genes involved in oxidative phosphorylation, which may affect cellular energy production and cause regional adaptation to different environmental (climatic) pressures. In the present study, we sequenced the NADH dehydrogenase subunit 6 (MT-ND6) gene of 267 brown hares (L. europaeus) from Europe and Asia Minor and tested for positive selection and adaptations acting on amino acid sequences (protein variants). Molecular diversity indices and spatial clustering were assessed by DnaSP, Network, and Geneland, while the presence of selection signals was tested by codeml in PAML, and by using the Datamonkey Adaptive Evolution web server. The SPSS software was used to run multinomial regression models to test for possible effects of climate parameters on the currently obtained protein variants. Fifty-eight haplotypes were revealed with a haplotype diversity of 0.817, coding for 17 different protein variants. The MT-ND6 phylogeographic pattern as determined by the nucleotide sequences followed the earlier found model based on the neutrally evolving D-loop sequences, and reflected the earlier found phylogeographic Late Pleistocene scenario. Based on several selection tests, only one codon position consistently proved to be under positive selection. It did occur exclusively in the evolutionarily younger hares from Europe and it gave rise to several protein variants from the southeastern and south-central Balkans. The occurrence of several of those variants was significantly favored under certain precipitation conditions, as proved by our multinomial regression models. Possibly, the great altitudinal variation in the Balkans may have lead to bigger changes in precipitation across that region and this may have imposed an evolutionarily novel selective pressure on the protein variants and could have led to regional adaptation.

Close relatives of Mediterranean endemo-relict hoverflies (Diptera, Syrphidae) in South Africa: Morphological and molecular evidence in the Merodon melanocerus subgroup.

An ongoing study of the genus Merodon Meigen, 1803 in the Republic of South Africa (RSA) has revealed the existence of new species related to M. melanocerus Bezzi, 1915. The M. melanocerus subgroup belongs to the Afrotropical lineage of the M. desuturinus group. Revision of all available material from museums and detailed analyses of newly -collected specimens from our own expeditions to RSA resulted in delimitation of five species: M. capensis Hurkmans sp. n., M. commutabilis Radenkovic et Vujic sp. n., M. drakonis Vujic et Radenkovic sp. n., M. flavocerus Hurkmans sp. n. and M. melanocerus. In addition to classical morphological characters, sequences of the mitochondrial COI gene are provided for four related taxa. Results of molecular phylogenetic analyses supports monophyly of the M. desuturinus group and confirmed delimitation between species. Links between Palaearctic and Afrotropical faunas of this group, as well as possible evolutionary paths, are discussed. Based on phylogenetic analyses, four lineages (putative subgenera) have been recognized within the genus Merodon; besides the three previously established ones, albifrons+desuturinus, aureus (sensu lato) and avidus-nigritarsis, one new lineage named natans is distinguished.

Large-scale mitochondrial DNA analysis reveals new light on the phylogeography of Central and Eastern-European Brown hare (Lepus europaeus Pallas, 1778).

European brown hare, Lepus europaeus, from Central and Eastern European countries (Hungary, Poland, Serbia, Lithuania, Romania, Georgia and Italy) were sampled, and phylogenetic analyses were carried out on two datasets: 1.) 137 sequences (358 bp) of control region mtDNA; and 2.) 105 sequences of a concatenated fragment (916 bp), including the cytochrome b, tRNA-Thr, tRNA-Pro and control region mitochondrial DNA. Our sequences were aligned with additional brown hare sequences from GenBank. A total of 52 and 51 haplotypes were detected within the two datasets, respectively, and assigned to two previously described major lineages: Anatolian/Middle Eastern (AME) and European (EUR). Furthermore, the European lineage was divided into two subclades including South Eastern European (SEE) and Central European (CE). Sympatric distribution of the lineages of the brown hare in South-Eastern and Eastern Europe revealed contact zones there. BAPS analysis assigned sequences from L. europaeus to five genetic clusters, whereas CE individuals were assigned to only one cluster, and AME and SEE sequences were each assigned to two clusters. Our findings uncover numerous novel haplotypes of Anatolian/Middle Eastern brown hare outside their main range, as evidence for the combined influence of Late Pleistocene climatic fluctuations and anthropogenic activities in shaping the phylogeographic structure of the species. Our results support the hypothesis of a postglacial brown hare expansion from Anatolia and the Balkan Peninsula to Central and Eastern Europe, and suggest some slight introgression of individual haplotypes from L. timidus to L. europaeus.