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The European roe deer (Capreolus capreolus) is one of the most numerous and widespread ungulate species in Europe, which has complicated the assessment of its genetic diversity on a range-wide scale. In this study, we present the mitochondrial DNA control region (mtDNA CR) genetic diversity and population structure of roe deer in Europe based on the analyses of 3010 samples, which were described as European roe deer individuals. Our analyses revealed two main diversity hotspots, namely Eastern and Central Europe. We proposed that these hotspots result from the Siberian roe deer (C.pygargus) mtDNA introgression and the secondary contact of mtDNA clades, respectively. Significantly lower values of genetic diversity (nucleotide and haplotype diversity) were recorded in the peripheral areas of the species’ range, including the southernmost parts of the Last Glacial Maximum (LGM) refugial areas. Roe deer population in Europe consists of 2–3 genetic groups according to SAMOVA, and 15–16 clusters identified by GENELAND. The main driver of roe deer population structure in the eastern parts of the continent has been introgression of mtDNA of C. pygargus. Spatial genetic analyses revealed a complex structure of roe deer on a pan-European scale, which presumably results from post-glacial recolonization of the continent from various parts of a large LGM refugial area by different roe deer mtDNA clades and haplogroups. © 2022, The Author(s) under exclusive licence to Deutsche Gesellschaft für Säugetierkunde.

To provide the most comprehensive picture of species phylogeny and phylogeography of European roe deer (

Wild boar populations are strongly increasing across Europe. Although details of population dynamics and reproductive potential are known in this species, our knowledge about the social structure and a possible impact of hunting on this structure is poorly understood. Here we investigated the grouping behaviour of yearling wild boar females under enclosure housed conditions. All females were at the same age and older females (i.e., potential group leaders in a family group) were not present. The wild boar females were purchased at the age of six months from four different breeding sites. After a habituation period of ˜8 weeks the females were released randomly into two large enclosures, but ensuring that animals of different origin and body mass were evenly distributed. We analysed data from 111 female wild boars kept in two enclosures (enclosure east (EE): 72 females on ˜33 ha and enclosure west (EW): 39 females on ˜22 ha). Observations were carried out between March and August the year after birth (at the age of yearlings). The yearling females did not reproduce in the observation year. Data were analysed using social network analysis (SNA). We observed that the females formed stable groups during the study period. Group size did not differ significantly between the enclosures (group sizes between 16 and 20 individuals in the EE and between 11 and 17 individuals in the EW). Interestingly, the higher number of individuals lead to more instead of larger groups in the EE. Further, the group compositions were not based on the relatedness between the animals, as females from different breeding sites formed stable groups together. Rather, common habituation (familiarity) had a stronger effect on subsequent group formation. Within the groups we did not observe a linear hierarchy, although the groups were stable over time. Two females showed high betweenness values (i.e., an individual was often part of the shortest pathways in a network) compared with the average, indicating that females show differences in their connectivity within the social network of a population. Individuals with a high betweenness are very likely to rapidly spread diseases in a population and might play a key role in health management strategies.

Background Animal mitochondria play a central role in energy production in the cells through the oxidative phosphorylation (OXPHOS) pathway. Recent studies of selection on different mitochondrial OXPHOS genes have revealed the adaptive implications of amino acid changes in these subunits. In hares, climatic variation and/or introgression were suggested to be at the origin of such adaptation. Here we looked for evidence of positive selection in three mitochondrial OXPHOS genes, using tests of selection, protein structure modelling and effects of amino acid substitutions on the protein function and stability. We also used statistical models to test for climate and introgression effects on sites under positive selection. Results Our results revealed seven sites under positive selection in ND4 and three sites in Cytb. However, no sites under positive selection were observed in the COX1 gene. All three subunits presented a high number of codons under negative selection. Sites under positive selection were mapped on the tridimensional structure of the predicted models for the respective mitochondrial subunit. Of the ten amino acid replacements inferred to have evolved under positive selection for both subunits, six were located in the transmembrane domain. On the other hand, three codons were identified as sites lining proton translocation channels. Furthermore, four codons were identified as destabilizing with a significant variation of Δ vibrational entropy energy between wild and mutant type. Moreover, our PROVEAN analysis suggested that among all positively selected sites two fixed amino acid replacements altered the protein functioning. Our statistical models indicated significant effects of climate on the presence of ND4 and Cytb protein variants, but no effect by trans-specific mitochondrial DNA introgression, which is not uncommon in a number of hare species. Conclusions Positive selection was observed in several codons in two OXPHOS genes. We found that substitutions in the positively selected codons have structural and functional impacts on the encoded proteins. Our results are concordantly suggesting that adaptations have strongly affected the evolution of mtDNA of hare species with potential effects on the protein function. Environmental/climatic changes appear to be a major trigger of this adaptation, whereas trans-specific introgressive hybridization seems to play no major role for the occurrence of protein variants.

Hybridization may often be an important source of adaptive variation, but the extent and long-term impacts of introgression have seldom been evaluated in the phylogenetic context of a radiation. Hares (Lepus) represent a widespread mammalian radiation of 32 extant species characterized by striking ecological adaptations and recurrent admixture. To understand the relevance of introgressive hybridization during the diversification ofLepus, we analyzed whole exome sequences (61.7 Mb) from 15 species of hares (1- 4 individuals per species), spanning the global distribution of the genus, and two outgroups. We used a coalescent framework to infer species relationships and divergence times, despite extensive genealogical discordance. We found high levels of allele sharing among species and show that this reflects extensive incomplete lineage sorting and temporally layered hybridization. Our results revealed recurrent introgression at all stages along theLepusradiation, including recent gene flow between extant species since the last glacial maximum, but also pervasive ancient introgression occurring since near the origin of the hare lineages. We show that ancient hybridization between northern hemisphere species has resulted in shared variation of potential adaptive relevance to highly seasonal environments, including genes involved in circadian rhythm regulation, pigmentation, and thermoregulation. Our results illustrate how the genetic legacy of ancestral hybridization may persist across a radiation, leaving a long-lasting signature of shared genetic variation that may contribute to adaptation within and among species.

In natural populations, allelic diversity of the major histocompatibility complex (MHC) is commonly interpreted as resulting from positive selection in varying spatiotemporal pathogenic landscapes. Composite pathogenic landscape data are, however, rarely available. We studied the spatial distribution of allelic diversity at two MHC class II loci (DQA, DQB) in hares, Lepus capensis, along a steep ecological gradient in North Africa and tested the role of climatic parameters for the spatial distribution of DQA and DQB proteins. Climatic parameters were considered to reflect to some extent pathogenic landscape variation. We investigated historical and contemporary forces that have shaped the variability at both genes, and tested for differential selective pressure across the ecological gradient by comparing allelic variation at MHC and neutral loci. We found positive selection on both MHC loci and significantly decreasing diversity from North to South Tunisia. Our multinomial linear models revealed significant effects of geographical positions that were correlated with mean annual temperature and precipitation on the occurrence of protein variants, but no effects of co-occurring DQA or DQB proteins, respectively. Diversifying selection, recombination, adaptation to local pathogenic landscapes (supposedly reflected by climate parameters) and neutral demographic processes have shaped the observed MHC diversity and differentiation patterns.

Gedeon, Csongor Istvan/0000-0003-4027-2409; Velickovic, Nevena/0000-0003-0797-269X WOS:000522708700002 PubMed: 32236892 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 (pi) 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. [1/2018] We thank all local hunters, Prof. Philip U. Alkon, PhD (Israel), Orsolya Vincze, PhD (Romania) and Sim Broekhuizen, PhD (The Netherlands) for helping collecting the samples. Partial financial support for this work was provided by Wildlife Research-Franz Suchentrunk (Project No. 1/2018).

North African hares are currently included in cape hares, Lepus capensis sensu lato, a taxon that may be considered a superspecies or a complex of closely related species. The existing molecular data, however, are not unequivocal, with mtDNA control region sequences suggesting a separate species status and nuclear loci (allozymes, microsatellites) revealing conspecificity of L. capensis and L. europaeus. Here, we study sequence variation in the intron 6 (468 bp) of the transferrin nuclear gene, of 105 hares with different coat colour from different regions in Tunisia with respect to genetic diversity and differentiation, as well as their phylogenetic status. Forty-six haplotypes (alleles) were revealed and compared phylogenetically to all available TF haplotypes of various Lepus species retrieved from GenBank. Maximum Likelihood, neighbor joining and median joining network analyses concordantly grouped all currently obtained haplotypes together with haplotypes belonging to six different Chinese hare species and the African scrub hare L. saxatilis. Moreover, two Tunisian haploypes were shared with L. capensis, L timidus, L. sinensis, L. yarkandensis, and L. hainanus from China. These results indicated the evolutionary complexity of the genus Lepus with the mixing of nuclear gene haplotypes resulting from introgressive hybridization or/and shared ancestral polymorphism. We report the presence of shared ancestral polymorphism between North African and Chinese hares. This has not been detected earlier in the mtDNA sequences of the same individuals. Genetic diversity of the TF sequences from the Tunisian populations was relatively high compared to other hare populations. However, genetic differentiation and gene flow analyses (AMOVA, FST, Nm) indicated little divergence with the absence of geographically meaningful phylogroups and lack of clustering with coat colour types. These results confirm the presence of a single hare species in Tunisia, but a sound inference on its phylogenetic position would require additional nuclear markers and numerous geographically meaningful samples from Africa and Eurasia.

There is increasing evidence that animal personalities are linked to different life history strategies. However, studies examining whether these effects differ under varying environmental conditions are rare. Here, we investigated how animal personality affects reproductive success in a pulsed resource consumer, the wild boar. We determined the exploratory behaviour of 57 female wild boars in nine novel object tests and additionally assessed their aggressiveness. Exploration behaviour (i.e. approach latency and investigation duration) and aggressiveness were repeatable within individuals and both mapped on a single principal component yielding an individual personality score. Afterwards the females were kept together with 28 males under seminatural conditions in two large breeding enclosures from 2011 to 2014. Over winter 2013/2014 we applied high versus medium feeding regimes to the two enclosures. Our results show that adult body mass and reproductive success were affected by juvenile body mass and thus already determined early in life, which may point to a silver spoon effect in the wild boar. Whether a female reproduced or not, as well as the litter size shortly after birth, was only affected by female body mass. The postweaning litter size (i.e. at the time of independence at the age of about 6 months), however, was additionally affected by the personality score in interaction with food availability. Under high food availability less aggressive and explorative individuals raised more juveniles to independence. We conclude that lower aggressiveness and reduced exploratory tendency of the mother lead to lower juvenile mortality and hence have a positive impact on postweaning litter size. Under slightly decreased food availability, however, this effect vanished. As the impact of personality on reproductive success differed between changing environmental conditions, our results support the hypothesis that different personality phenotypes are evolutionarily maintained by varying selection pressures in heterogeneous environments.

Gedeon, Csongor Istvan/0000-0003-4027-2409; Stefanovic, Milomir/0000-0001-6294-3782 WOS: 000513587800001 PubMed: 31703111 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. Ministry of Education, Science and Technological Development, Republic of Serbia [43002]; Provincial Secretariat for Science and Technological Development [0601 78/37-316]; Wildlife Research - Franz Suchentrunk [1/2017] This work was financially supported by the Ministry of Education, Science and Technological Development, Republic of Serbia Grant No. 43002, project title "Biosensing technology and global system for long-term research and integrated management of ecosystems"; and the Provincial Secretariat for Science and Technological Development Grant No. 0601 78/37-316, project title "Evaluation of ecological networks in AP Vojvodina as support for nature conservation". Partial financial support was provided by Wildlife Research - Franz Suchentrunk (project No. 1/2017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Background The aim of the study was to use hybrid populations as well as island populations of the European brown hare (Lepus europaeus) to explore the effect of evolutionary events, such as the post-deglaciation translocations, spontaneous and human-mediated, local adaptation and the genetic drift in the shaping of the phylogeographic patterns of the species. For this purpose, we used molecular markers, both nuclear and mitochondrial, that are indicative for local adaptation as well as neutral markers to elucidate the patterns of population differentiation based on geographic isolation and the clade of origin. To broaden our analysis, we included data from our previous studies concerning mainland populations, to explore the genetic differentiation in the base of the geographic origin (mainland/island) of the populations. Results Our results suggest that local adaptation shapes the differentiation in both genomes, favoring specific alleles in nuclear genes (e.g. DQA) or haplotypes in mtDNA (e.g. Control Region, CR). mtDNA variation was found to be in a higher level and was able to give a phylogeographic signal for the populations. Furthermore, the degree of variation was influenced not only by the geographic origin, but also by the clade of origin, since specific island populations of Anatolian origin showed a greater degree of variation compared to specific mainland populations of the European clade. Concerning the hybrid population, we confirmed the existence of both clades in the territory and we provided a possible explanation for the lack of introgression between the clades. Conclusion Our results indicate that the Quaternary’s climatic oscillations played a major role in the shaping of the phylogeographic patterns of the species, by isolating populations in the distinct refugia, where they adapted and differentiate in allopatry, leading to genome incompatibilities observed nowadays. Electronic supplementary material The online version of this article (10.1186/s12862-019-1354-y) contains supplementary material, which is available to authorized users.

WOS: 000458165800012, We determined allelic variation at eleven microsatellite loci to study the effect of the Bosphorus, the Sea of Marmara, and the Dardanelles on the population genetic architecture of brown hares, Lepus europaeus, from Anatolia and Turkish Thrace in the southeastern most Balkans. The latter region was connected with Anatolia during periods of the late Pleistocene and the early Holocene (until ca. 8000 y BP) enabling natural gene flow. Following a recent mitochondrial (mt) DNA-based phylogeographic model that suggested unidirectional ancient gene flow from Anatolia to the SE Balkans, we expected lower genetic diversity in hares from Turkish Thrace than Anatolia. The Turkish Thrace population might have been established only relatively recently as natural northwestward expansion of a long existing Anatolian population. Alternatively, it might have received late Pleistocene or Holocene gene flow by hares from eastern Europe from north of the Black Sea, even after the formation of the marine barrier. This might have produced a notable genetic distinction between the hares from either side of the Bosphorus, the Sea of Marmara, and the Dardanelles. We found significantly lower allelic richness in hares from Turkish Thrace than Anatolia, very little genetic admixture in the Turkish Thrace population, and only one allele at low frequency in Turkish Thrace that was not observed in Anatolian hares. On the contrary, many alleles of Anatolian hares were not recovered in hares from Turkish Thrace. Nevertheless, genetic divergence (Fst, Cavalli-Sforza & Edwards distances, AMOVA) between the hares from either side of the current marine barrier was low (Fst values, Kirikkale University Scientific Research Projects Coordination Unit [BAP-2008/18, BAP-2012/32, BAP-2016/61], This work was supported by the Kirikkale University Scientific Research Projects Coordination Unit (project numbers: BAP-2008/18, BAP-2012/32, BAP-2016/61).

Toll-like receptors (TLRs) are a major group of proteins that recognize molecular components of infectious agents, known as pathogen associated molecular patterns (PAMPs). The structure of these genes is similar and characterized by the presence of an ectodomain, a signal transmembrane segment and a highly conserved cytoplasmic domain. The latter domain is homologous to the human interleukin-1 receptor (IL1R) and human IL-18 receptor (IL-18R) and designated TIR domain. The latter domain of the TLR genes was suggested to be very conservative and its evolution is driven by purifying selection. Variability and evolution of the TIR sequences of TLR2 gene were studied in three hare populations from Tunisia with different ecological characteristics (NT-North Tunisia with Mediterranean, CT-Central Tunisia with semi-arid, and ST-South Tunisia with arid climate). Sequencing of a 372bp fragment of TIR2 revealed 25 alleles among 110 hares. Twenty variable nucleotide positions were detected, of which 7 were non-synonymous. The highest variability was observed in CT, with 16 polymorphic positions. In ST, only 4 polymorphic nucleotide positions were detected with all diversity values lower than those recorded for the other two populations. By using several approaches, no positive selection was detected. However, evidence of purifying selection was found at two positions. The logistic models of the most common TIR2 protein variant that we run to examine whether its occurrence was affected by climatic variation independent of the geographic sample location suggested only a longitudinal effect. Finally, the mapping of the non-synonymous mutations to the inferred tertiary protein structure showed that they were all localized in the different loop regions. Among all non-synonymous substitutions, three were suggested to be deleterious as evidenced by PROVEAN analysis. The observed patterns of variability characterized by low genetic diversity in ST might suggest that the TIR region was more affected, than other markers, by genetic drift or/and that these patterns were shaped by different selective pressures under different ecological conditions. Notably, this low diversity was not detected by other (putatively neutral) microsatellite markers analysed in the course of other studies. But low diversity was also found for two MHC class II adaptive immune genes. As expected from functionally important regions, the evolution of the TIR2 domain is mainly driven by purifying selection. However, the occurrence of deleterious non-synonymous substitutions might highlight the flexible evolution of the TIR genes and/or their interactions with other proteins.

In Bulgaria, chamois (Rupicapra rupicapra balcanica) are protected by law and it has been recommended to assess their genetic diversity, level of inbreeding, and extent of introgression by Alpine chamois (R. r. rupicapra) that were released in the last century in the Rhodope Mountains. Chamois (n = 81) collected between 2009 and 2013 at the “Izvora State Hunting Reserve” (ISHR, western Rhodopes, a major source for chamois translocations in Bulgaria; census size: 200–250) were screened for allelic variability at 12 microsatellite loci and compared to chamois (R. r. rupicapra, n = 135) from six populations in the Austrian Alps. Indices of genetic variability were significantly lower for the ISHR population than for the Austrian populations. A significant bottleneck signal was observed for the ISHR population, but no distinct inbreeding signal due to non-random mating. F-statistics, absolute genetic distances, Bayesian STRUCTURE and assignment analyses, and a factorial correspondence analysis concordantly indicated distinct differentiation between the ISHR and Austrian chamois. STRUCTURE identified some few ISHR chamois as possibly introgressed by Alpine chamois. ONeSAMP revealed effective population size estimates of Ne ≈ 50 for the ISHR population. A reduction of shooting quotas, a reinforcement of poaching control, and exchanging chamois with other Bulgarian populations should increase its Ne to prevent further loss of genetic variability. Screening for introgression should be extended to other Bulgarian populations, particularly to areas where Alpine chamois were released in the past; and it should accompany all translocations, to prevent possible dissemination of introgressed chamois.

In lagomorphs, lymphocyte subset distributions and the importance of CD4(+) T cell levels has so far only been considered in the frame of rabbit disease models. In this study, the first assessment of CD4(+) T lymphocytes in peripheral blood cells in brown hares (Lepus europaeus L., 1758), a further leporid species using a cross-reactive rabbit anti-CD4 antibody in flow cytometry, is presented. In addition, the entire coding region of the hare CD4 gene (1380 bp) coding for a polypeptide of 459 amino acids has been sequenced. Using generalized least squares fitting by maximum likelihood (GLS) test, significantly (p=0.0095) higher CD4(+) T cell frequencies in males than in females and significantly (p=0.0001) higher frequencies for leverets (younger than 2 months of age) than for subadult and adult (older than 7 months of age) individuals were detected. No significant age influence, however, was found for subadult and adult hares. The study is particularly meant to provide a first step in establishing a toolbox for the assessment of the immune response in this leporid species.

International audience; Genetic introgression from a resident species into an invading close relative can result from repeated hybridisation along the invasion front and/or allele surfing on the expansion wave. Cases where the phenomenon is massive and systematic, such as for hares (genus Lepus) in Iberia, would be best explained by recurrent hybridisation but this is difficult to prove because the donor populations are generally extinct. In the Pyrenean foothills, Lepus europaeus presumably replaced Lepus granatensis recently and the present species border is parallel to the direction of invasion, so that populations of L. granatensis in the contact zone represent proxies of existing variation before the invasion. Among three pairs of populations sampled across this border, we find less differentiation of mitochondrial DNA (mtDNA) across than along it, as predicted under recurrent hybridisation at the invasion front. Using autosomal microsatellite loci and X-and Y-linked diagnostic loci, we show that admixture across the border is quasi-absent, making it unlikely that lack of interspecific mtDNA differentiation results from ongoing gene flow. Furthermore, we find that the local species ranges are climatically contrasted, making it also unlikely that ongoing ecologydriven movement of the contact account for mtDNA introgression. The lack of mtDNA differentiation across the boundary is mostly due to sharing of mtDNA from a boreal species currently extinct in Iberia (Lepus timidus) whose mitochondria have thus remained in place since the last deglaciation despite successive invasions by two other species. Home-loving mitochondria thus witness past species distribution rather than ongoing exchanges across stabilised contact zones.

Background Recent studies of selection on mitochondrial (mt) OXPHOS genes suggest adaptation due mainly to environmental variation. In this context, Tunisian hares that display several external phenotypes with phylogenetically rather homogenous gene pool and shallow population structure provide a good precondition to detect positive selection on mt genes related to environmental/climatic variation, specifically ambient temperature and precipitation. Results We used codon-based methods along with population genetic data to test for positive selection on ATPase synthase 6 (ATP6) and NADH dehydrogenase 2 (ND2) of cape hares (Lepus capensis) collected along a steep ecological gradient in Tunisia. We found significantly higher differentiation at the ATP6 locus across Tunisia, with sub-humid Mediterranean, semi-arid, and arid Sahara climate than for fourteen unlinked supposedly neutrally evolving nuclear microsatellites and mt control region sequences. This suggested positive selection on ATP6 sequences, which was confirmed by several codon-based tests for one sequence site that together with a second site translated into four different amino acids. Positive selection on ND2 sequences was also confirmed by several codon-based tests. The corresponding frequencies of the two most prevalent variants at each locus varied significantly across climate regions, and our logistic general linear models of occurrence of those proteins indicated significant effects of mean annual temperature for ATP6 and mean minimum temperature of the coldest month of the year for ND2, independent of geographical location, annual precipitation, and the respective co-occurring protein at the second locus. Moreover, presence of the ancestral ATP6 protein, as inferred from phylogenetic networks, was positively affected by the simultaneous presence of the derived ND2 protein and vice versa, independent of temperature, precipitation, or geographic location. Finally, we obtained a significant coevolution signal for the ancestral ATP6 and derived ND2 sequences and vice versa. Conclusions positive selection was strongly suggested by the population genetic approach and the codon-based tests in both mtDNA genes. Moreover, the two most prevalent proteins at the ATP6 locus were distributed at significantly varying frequencies across the study area with a significant effect of mean annual temperature on the occurrence of the ATP6 proteins independent of geographical coordinates and the co-occuring ND2 protein variant. For ND2, occurrence of the two most frequent protein variants was significantly influenced by the mean minimum temperature of the coldest month, independent of the co-occurring ATP6 protein variant and geographical coordinates. This strongly suggests direct involvement of ambient temperature in the adaptation of the studied mtOXPHOS genes. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0896-0) contains supplementary material, which is available to authorized users.

Recently, many new (extant) mammal species have been named, mostly by raising subspecies to species rank. This is primarily a consequence of the phylogenetic species concept (PSC) that has become very popular over the last few decades. We highlight several cases of splitting and argue that much of this taxonomic inflation is artificial due to shortcomings of the PSC and unjustified reliance on insufficient morphological and/or genetic data. We particularly discourage species splitting based on gene trees inferred from mitochondrial DNA only and phenetic analyses aimed at diagnosability. Uncritical acceptance of new species creates an unnecessary burden on the conservation of biodiversity.

The European ground squirrel (Spermophilus citellus) is endangered and in decline. Populations are increasingly fragmented, and only a coordinated conservation effort at the European level may guarantee its long-term survival. To obtain a general population genetic picture on a larger geographic scale, we screened 117 individuals from seven local populations in Hungary, Romania, and Austria for allelic variation at eleven microsatellite loci. We found a high (23.4%) proportion of private alleles, and a moderate to somewhat elevated level (15.27%) of partitioning of genetic diversity among populations, compared to that found in many other terrestrial mammals. Genetic variability was significantly higher than in earlier studied Czech populations that are considered genetically depleted, but significantly lower than in undisturbed populations of S. suslicus and S. brunneus, that are similar to the European ground squirrel in their ecological requirements, reproductive biology, and social organization. Genetic diversity was also lower than in most presumably “undisturbed” populations of other Sciurid species. This, together with the observed level and pattern of genetic differentiation among populations, such as no significant increase of genetic differentiation with geographic distance and similar variance of genetic differentiation between populations independent of geographic distance, indicated the prevalence of relatively strong drift effects for all populations. A Bayesian STRUCTURE analysis and a factorial correspondence analysis concordantly revealed a fairly complex genetic composition of local populations, but no major geographic trend in the pattern of the genetic composition. Overall, the results suggest disintegration of local colonies that might earlier have been more connected genetically. The STRUCTURE analysis also suggested anthropogenic translocations among single Hungarian populations. Our data on genetic diversity and its distribution do not object to such conservation measures. Translocation of individuals particularly from nearby populations may increase the chances of survival of small and isolated populations and counteract inbreeding at low densities.

We analysed 128 mountain hares Lepus timidus varronis from eastern Switzerland with respect to genetic variability, differentiation and phylogeography. The molecular markers chosen were 443 bp of the mitochondrial control region and 13 microsatellite loci (12 of which were polymorphic). Among the 113 successfully sequenced hares, five yielded introgressed brown hare Lepus europaeus haplotypes, making our study one of few to show introgression of mitochondrial brown hare alleles into mountain hare gene pools rather than the other way around. Overall haplotype and nucleotide diversities were 0.91 and 0.0081, and observed and expected heterozygosities were 0.40 and 0.54. Our Swiss sample did not show unequivocal signals of substructuring and probably represents a (nearly) pan-mictic population. We also analysed the 20 haplotypes we found phylogeographically in a global framework by adding 143 published sequences from throughout the species' distribution range. The resulting haplotype network lacked an overall geographical structure, but instead consisted of many geographically meaningful subclusters that were scattered throughout the network, including different groups of Russian, Scandinavian or Alpine sequences. This pattern is in line with earlier findings and expectations for arctic species and is indicative of a continuous population across the European continent during the last ice age. Unexpectedly, our Swiss haplotypes all clustered together, suggesting that most of them originated in situ after the isolation of the Alpine population in the late Pleistocene.

The link between adaptive genetic variation, individual fitness and wildlife population dynamics is fundamental to the study of ecology and evolutionary biology. In this study, a Bayesian modelling approach was employed to examine whether individual variability at two major histocompatibility complex (MHC) class II loci (DQA and DRB) and eight neutral microsatellite loci explained variation in female reproductive success for wild populations of European brown hare (Lepus europaeus). We examined two aspects of reproduction: the ability to reproduce (sterility) and the number of offspring produced (fecundity). Samples were collected from eastern Austria, experiencing a sub-continental climatic regime, and from Belgium with a more Atlantic-influenced climate. As expected, reproductive success (both sterility and fecundity) was significantly influenced by age regardless of sampling locality. For Belgium, there was also a significant effect of DQA heterozygosity in determining whether females were able to reproduce (95% highest posterior density interval of the regression parameter [−3.64, −0.52]), but no corresponding effect was found for Austria. In neither region was reproduction significantly associated with heterozygosity at the DRB locus. DQA heterozygotes from both regions also showed a clear tendency, but not significantly so, to produce a larger number of offspring. Predictive simulations showed that, in Belgium, sub-populations of homozygotes will have higher rates of sterile individuals and lower average offspring numbers than heterozygotes. No similar effect is predicted for Austria. The mechanism for the spatial MHC effect is likely to be connected to mate choice for increased heterozygosity or to the linkage of certain MHC alleles with lethal recessives at other loci.

Both the Cyt b gene of mtDNA and Y chromosome markers were studied in a relatively large sample of brown hares ( L. europaeus ) from Europe and Anatolia (Turkey and Israel), together with other seven Lepus species, in order to enable comparative analysis of possible sex-specific gene flow. Furthermore, Y chromosome markers were compared with data from biparentally inherited markers in an attempt to understand whether or not their pattern of distribution was congruent with that of allozymes or whether they rather matched mtDNA phylogenies, with which they share uniparental inheritance. Consistent with the general observation, levels of interspecific genetic variability were very low for the Y chromosome markers compared with mtDNA. Moreover, lack of interspecific variation for the Y-DNA studied within Lepus genus rendered these markers improper for any further phylogenetic analysis. With the highest nucleotide diversity in Anatolia compared with Europe, both marker systems confirmed an unbroken species history in Anatolia, corroborated the hypothesis of continuous gene flow from Anatolia's neighbouring regions, and supported the idea of a quick postglacial colonization followed by expansion of the species in large parts of Europe. Phylogenetic analysis under mtDNA revealed the existence of four different haplogroups with a well defined distribution across Europe and Anatolia. Both genetic systems supported the deep separation of Anatolian and European lineages of L. europaeus . Nevertheless, Anatolian Y-DNA lineages extended across a longer geographic distance in south-eastern Europe than Anatolian mtDNA haplotypes, probably as a result of higher female philopatry that makes mtDNA introgression more difficult in brown hares.

As a consequence of persecution and habitat fragmentation, wildcats (Felis silvestris silvestris) in Western Europe have experienced a severe reduction in population numbers and sizes. The remaining wildcat populations are considered to be endangered by losses of genetic variability and by hybridisation with free-ranging domestic cats. To investigate genetic diversity within and among wild and domestic cat populations in Germany and to estimate the extent of gene flow between both forms, we analysed a total of 266 individuals. PCR-amplification and sequencing of 322 base pairs of a highly variable part of the mitochondrial control region (HV1) of 244 specimens resulted in 41 haplotypes with 31 polymorphic sites. Additionally, eight microsatellite loci were examined for those 244 cats. Moreover, a total of 46 wildcats and 22 domestic cats could be genotyped for 13 polymorphic out of 31 enzyme loci. Genetic variability in both groups was generally high. Variability in domestic cat populations was higher than in wildcat populations. Almost no differentiation between domestic cat populations could be found (FST for microsatellites=3%). In contrast, wildcat populations differed significantly from one another (FST for microsatellites=9.55%) Within the smaller wildcat populations, a reduction of genetic diversity was detectable with regard to the nuclear DNA. Wildcat and domestic cat mitochondrial haplotypes were separated, suggesting a very low level of maternal gene flow between both forms. In microsatellites and to a somewhat lesser extent in allozymes, wildcats and domestic cats showed distinct differentiation, suggesting an only low extent of past hybridisation in certain populations. The microsatellite data set indicated a significantly reduced effective population size (bottleneck) in the recent past for one German wildcat population.

Mitochondrial DNA mutations create variation in the efficiency of the oxidative phosphorylation pathway and therefore cellular energy production. Mildly deleterious mutations may reduce the performance of sperm cells in particular, due to their high energy requirements and low number of mitochondria, yet have little or no effect on the viability of somatic cells or ova. Mutations will be maintained in the population, despite the fitness cost for males, because mtDNA is passed down the female line. We looked for this so-called mother's curse effect in our captive colony of European brown hares. Significantly reduced male reproductive success was detected for a divergent haplotype that could be traced back to hares imported from a remote population. Median reproductive success for these hares was 0.17 compared to 0.49 for the indigenous haplotypes (Wilcoxon rank-sum, P = 0.002). No difference was detected for female reproductive success, nor were we able to find a nuclear DNA component to variation in male fertility. Our data are strong evidence for a mother's curse effect persisting despite multiple crossings over seven generations. These data raise important issues relating to the reproductive fitness of small or intermixing populations and have particular implications for the management of populations for conservation.

Conventionally, Lepus capensis is considered to range across large parts of Africa, the Middle East, Central and Far East Asia. However, a recent morphological study restricts cape hares tentatively to a small range in the Western Cape Region of South Africa and groups all other L. capensis-type hares from South Africa into a new species: L. centralis. Here, we studied molecular relationships among L. capensis-type hares from South Africa. Phenotypically and morphologically the individuals matched either the newly described L. capensis or L. centralis. We examined 66 hares for allelic variation at 13 microsatellite loci and for sequence variation of the hypervariable domain 1 of the mitochondrial control region. All tree-generating analyses of the currently obtained sequences and all South African cape hare sequences downloaded from GenBank revealed monophyly when compared to sequences of various other Lepus species. A network analysis indicated close evolutionary relationships between hares of the “L. capensis-phenotype” and the “L. centralis-phenotype” (according to Palacios et al. 2008) from the southwest of the Western Cape, relative to their pronounced evolutionary divergence from all other more central, northern, and north-eastern L. capensis-type hares. F-statistics, a Bayesian admixture STRUCTURE model, as well as a principal coordinate analysis of microsatellite data indicated close genetic relationships among all South African L. capensis-type hares studied presently. A coalescence model-based migration analysis for microsatellite alleles indicated gene flow between most of the considered subspecies of cape hare, including L. capensis capensis and L. capensis centralis, theoretically sufficient to balance stochastic drift effects. Concordantly, AMOVA models revealed only little effects of partitioning microsatellite variation into the two suggested morpho-species “L. capensis” and “L. centralis”. Under an “Interbreeding Species Concept” (e.g. a strict or relaxed Biological Species Concept), the current molecular data demonstrate conspecificity of the two proposed morpho-species “L. capensis” and “L. centralis”. Based on the present molecular data the differentiation of subspecies of cape hares from southern Africa is discussed.

Variability and phylogenetic relationships of sequences of the hypervariable domain I (HVI) of the mitochondrial DNA was studied in 46 brown hares (Lepus europaeus) from Anatolia, to test the hypotheses that (i) hares from several islands off the Anatolian coast and from Cyprus are phylogenetically close to mainland Anatolian hares, (ii) Anatolian hare sequence variability is higher than that of typical European brown hares, and to iii) infer possible Anatolian source populations of hares from some islands in the eastern Mediterranean. Neighbor joining and Maximum Parsimony analyses revealed reciprocal monophyly for sequences from Anatolia, the considered eastern Mediterranean islands off the Anatolian coast, Cyprus, and those sequences published earlier form NE Greece that were supposed to originate from earlier immigration via the late-Pleistocene/early-Holocene land bridge that connected SE Europe and W Anatolia (Kasapidis et al., 2005. Mol. Phylogenet. Evol. 34, 55–66). A high sequence idiosyncrasy was found among the Anatolian samples. Almost all approaches to compare variability between Anatolian and the downloaded European sequence data indicated higher sequence diversity in Anatolia, in accordance with earlier findings for allozyme loci. Network and principal coordinate analyses of the Anatolian sequences and those from the islands off the Anatolian coast as well as the Anatolian-type NE Greek sequences suggested high mitochondrial gene exchange among local populations in Anatolia with little effect of possible geographic barriers, and did not provide clues for tracing possible origins of island populations.

Empirical data sets of Artiodactyla (Antilocapridae, Bovidae, Cervidae, Suidae), Carnivora (Mustelidae) and Rodentia (Sciuridae, Cricetidae, Arvicolidae, Muridae), obtained by horizontal starch el electrophoresis of 15–34 isoenzyme sstems, were used to calculate genetic distances and to construct phylogenetic trees by the following methods: Nei's D (corrected for small sample sizes) - UPGMA, FITCH, KITSCH (out of Felsenstein's PHYLIP-package); Rogers-distance - distance-Wanger tree; maximum likelihood approach (cavalli-Sforza-Edwards); maximum parsimony method (wagner); Hennigian cladogram. The results were re-examined using the statisticar methods of jackknife and bootstrap. The following problems became apparent and were studied in more detail: inconstancy of molecular evolutionary rate among taxa, non-uniformity of evolutionary rate among isoenzymes, possible convergence of alloenzymes, different evolutionary histories of taxa (radiations/bottlenecks), methodological influences sample sizes / rare alleles, comparability of data sets). The results show, that many branches of the various phylogenetic trees are fairly constant. The ambiguous position of the remaining OTU's is due to insufficient evidence in the primary data rather than to theroperties of cluster algorithms. However, since these problematic cases are also uncertain in phylogenies based on morphological characters and palaeontological results, even an increased data set may not lead to a cyear decision unless additional taxa of crucial importance are examined. Molecular evolutionary rate among taxa seems to be accelerated in some cases, possibly due to random fixation of different alleles during bottlenecks, when a highly polymorpic ancestral form underwent a series of adaptive radiations. Isoenzymes can be divided into groups with different evolutionary rates. Thus, data sets are only comparable with respect to genetic variability and differentiation, when they contain a similar amount of representatives of each of these categories.

We surveyed the genetic diversity of the expressed major histocompatibility complex class II DQA locus in natural populations of European brown hares, Lepus europaeus, from Austria and Belgium (267 individuals in total). Based on cDNA sequences, we designed hare-specific primers to amplify the highly variable second exon of the DQA gene. Using cloningsequencing methodology and capillary electrophoresis single-strand conformation polymorphism, we found ten alleles of the DQA exon 2 locus across these two European regions, of which eight are described for the first time. To search for signals of selection and recombination in the evolution of the DQA gene within the leporids, we augmented our sample with orthologous DQA alleles from the European rabbit, Oryctolagus cuniculus, in order to carry out a species level, species pairwise comparison. We found evidence of recombination in the history of the DQA sequences in leporids with some recombinant alleles bridging the species divide. In both species, selection on peptide binding site codons can be detected, though stronger for the rabbit. This result suggests that there may be a differential selection pressure in the deeper evolutionary history of these two species due to differences in several demographic and ecological traits likely subjecting them to differential selection by parasites. Finally, evolutionary relationships show a widespread and statistically significant intermingling of alleles from the two species. The many macroparasites shared between hares and rabbits may explain this pattern of trans-species polymorphism.

During the Pleistocene, the severe climatic changes induced major shifts in species distributions, forcing them to retract, expand, displace and/or fragment their ranges (Hewitt, 1996). In Europe, these continuous oscillations led to the production of greater subspecific and specific diversity in the southern peninsulas since they were the major ice age refugial areas (Hewitt, 1999). Hares (genus Lepus) seem to perfectly demonstrate these phenomena. Despite earlier controversies, the latest taxonomic view accepts five species of genus Lepus occurring naturally in Europe: L. europaeus, L. timidus, L. granatensis, L. castroviejoi, and L. corsicanus (Mitchell-Jones et al., 1999). Of these species, the latter two have allopatric and restricted ranges, L. castroviejoi in the Cantabrian Mountains of the Iberian Peninsula and L. corsicanus in the Apennines from central and southern Italy and also in Sicily (Fig. 1). These two southern European endemisms occupy highly specialized patches of scarce habitat and thus the establishment of suitable conservation mechanisms is a major concern (e.g., Temple and Terry, 2007). Several molecular studies have tried to clarify the evolutionary relationships between the European hare species (e.g., Perez-Suarez et al., 1994; Thulin et al., 1997a; Pierpaoli et al., 1999; Suchentrunk et al., 1999; Alves et al., 2000; Estonba et al., 2006; Ben Slimen et al., 2005). However

Phylogenetic relationships and systematics of hares and jackrabbits (genus Lepus, Lagomorpha) are notoriously difficult. One intriguing issue is the relationship between cape hares (Lepus capensis L., 1758) and brown hares (Lepus europaeus Pallas, 1778). According to Petter, F., [1959. Elements d'une revision des lievres africains du sous-genre Lepus. Mammalia 23, 41–67; 1961. Elements d'une revision des lievres europeens et asiatique du sous-genre Lepus. Z. Saugetierkd. 26, 30–40], these two phenotypically different taxa are conspecific. On the contrary, recent arguments were put forward to split African capensis into several species. Eleven variable microsatellite loci were used to determine the spatial genetic structure of African and South Israeli hares, currently considered cape hares, and European, Anatolian, and North Israeli hares, currently considered brown hares. Patterns of genetic differentiation of 294 hares collected at 18 locations from both species ranges were examined for accordance to the hypothesis of conspecificity or to the concept of two distinct species, the cape hare and the brown hare. Genetic variation was high (14.7 alleles/locus) and the distribution of alleles and genotypes was not homogeneous across the locations. Results from FST, RST, and GST′-statistics including various AMOVA models, comparisons of private alleles with corresponding relative genetic differentiation between neighbouring populations, as well as absolute genetic differentiation patterns were by and large congruent. They all indicated that the degree of differentiation between populations belonging to the two different taxa was not higher than within the taxa. Partitioning of genetic diversity was congruent with a isolation-by-distance model, and this also suggested that the differentiation was due to geographic distances rather than the occurrence of two species-specific gene pools. Homoplasy likely had no or only a marginal effect on our results, as indicated by the standardized genetic distance GST′ and the presence of differently fixed alleles between the South African cape hare and all European brown hare populations at the highly polymorphic Sol08 locus. The results are concordant with earlier multilocus allozyme and mtDNA RFLP data, but contrast with mtDNA sequence data published earlier, that suggest distinctly separate phylogenetic units in Africa, the Near East and Europe. Nevertheless, as concluded from the microsatellite data brown hares and African cape hares might be connected via the Near East by gene flow along a network of intergrading populations that have regionally distinct gene pools.

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.

WOS: 000393615100014, The wild boar (Sus scrofa L.) is distributed across most parts of Turkey, a major biogeographic crossroads connecting Southwest Asia and Southeast Europe. However, no information on genetic diversity and population structure of this species in Turkey is available. In this study, we report on mtDNA sequence variability and phylogenetic relationships among wild boars with variable external phenotypes from both its European (Turkish Thrace) and Asian (Anatolia) distributions in Turkey. Phylogenetic analyses of mtDNA D-loop sequences (413 bp) of 53 newly sequenced wild boars from different localities in Turkey and 432 wild boar sequences from various geographic origins downloaded from GenBank were performed to particularly compare the phylogeographic position of wild boars from the European part of the Turkish range with that of specimens from Anatolia and to explore a possible phylogeographic substructuring in Anatolia. Relatively high genetic diversity was found in the Turkish samples, with a total of 17 haplotypes. Phylogenetic analyses revealed partitioning of the currently found Turkish haplotypes into two haplogroups, which were, however, only partially concordant with the geographic origins of samples (central and southwestern Anatolia vs. Turkish Thrace and northeastern and southeastern Anatolia). A median-joining network grouped most Turkish haplotypes with those previously reported from the Near East, whereas the remaining two haplotypes were included in the European 1 haplogroup. The combined phylogenetic analysis of the currently obtained sequences and all sequences retrieved from GenBank supported the earlier findings of four major haplogroups. The present study will serve as a baseline for more comprehensive studies to understand phylogenetic relationships of wild boars in Turkey and the Near East., Kirikkale University Scientific Research Projects Coordination Unit [BAP-2014/35], We are very grateful to Prof Dr Nevena Velickovic (Department of Biology and Ecology, University of Novi Sad, Serbia) for her advice and valuable comments. We thank all those who helped us in collecting samples from Turkey: Ahmet Yasar Yildiz (1st Region Directorate of the Turkish Forestry and Water Affairs Ministry, Istanbul), Neslihan Akdemir (Kirikkale Post of the Turkish Forestry and Water Affairs Ministry), and several local hunters and hunting associations. This study was financially supported by the Kirikkale University Scientific Research Projects Coordination Unit (project number: BAP-2014/35). The Turkish Nature Protection and National Parks Directorate (Turkish Forestry and Water Affairs Ministry, Ankara) issued a research permit (permit number: 221163). We thank the editors Prof Dr Nusret Ayyildiz, Prof Dr Haluk Kefelioglu, and Prof Dr Mustafa Sozen, and three anonymous referees for their valuable comments and constructive suggestions on the manuscript.

In East Africa, cape hares (Lepus capensis) and savanna hares (L. victoriae) look much alike where their ranges overlap. Earlier studies suggested discrimination between the two species by several skull traits, but did not present morphometric statisitics. Our present discriminant analysis based on seven metric variables of the occipital bone related to skull length (condylobasal length) resulted in a high (95.1%) overall probability of correct separation of the two species. While all cape hares were classified correctly, correct classification was a bit lower in savanna hares (88.2%). A principal components analysis of the same variables confirmed the shape difference for the two species. Both region and sex-specific variation in the shape of the studied occipital bone complex was found in savanna hares but not in cape hares. The somewhat reduced level of correct identification of savanna hares might be due to a tendency for higher shape variation. Application of our discriminant analysis to other regions in East Africa is discussed.

To prevent the decline of the brown hare Lepus europaeus, especially as a result of hunting pressure, restocking programmes, using hares from elsewhere, were carried out in Greece up to 2001. Using diagnostic RFLP mitochondrial DNA markers we traced the origins of released captive-bred brown hares. We provide evidence that released animals survived long enough to have at least one reproductive cycle and to transmit their genome. If, in the long-term, introgressed foreign genes survive, forming new genotypes with indigenous genes, this would demonstrate that they are successful in terms of competition. As nuclear gene pools are not markedly divergent between Greek and other European hares foreign nuclear genes should not be a serious handicap. Hence, in certain situations release programmes may be appropriate.

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): Elements d’une revision des Lievres 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.

Fluctuating asymmetry (FA), used as an indicator of developmental stability, has long been hypothesized to be negatively correlated with genetic variability as a consequence of more variable organisms being better suited to buffer developmental pathways against environmental stress. However, it is still a matter of debate if this is due to metabolic properties of enzymes encoded by certain key loci or rather to overall genomic heterozygosity. Previous analyses suggest that there might be a general difference between homeo- and poikilotherms in that only the latter tend to exhibit the negative correlation predicted by theory. In the present study, we addressed these questions by analysing roe deer (Capreolus capreolus) from five German populations with regard to FA in metric and non-metric skull and mandible traits as well as variability at eight microsatellite loci. Genetic variability was quantified by heterozygosity and mean d2 parameters, and although the latter did not show any relationship with FA, we found for the first time a statistically significant negative correlation of microsatellite heterozygosity and non-metric FA among populations. Because microsatellites are non-coding markers, this may be interpreted as evidence for the role of overall genomic heterozygosity in determining developmental stability. To test if the threshold character of non-metric traits is responsible for the metric vs non-metric difference we also carried out calculations where we treated our metric traits as threshold values. This, however, did not yield significant correlations between FA and genetic variability either.

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.

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.