The secret of Pianosa island: an Italian native population of European brown hare (<Emphasis Type="Italic">Lepus europaeus meridiei</Emphasis> Hilzheimer, 1906)

Given its relevance as a game species, the brown hare (Lepus europaeus Pallas, 1778) is one of the most managed and translocated mammals in Europe. In Italy, the species shows a genepool consisting of a mix of native and exotic lineages, due to translocations and introductions for hunting purposes. Some authors argued that the introduction of exotic brown hares could have caused the extinction of an endemic subspecies, L. e. meridiei Hilzheimer 1906, once present in central and northern Italy. Here we genetically characterized for the first time the brown hare population living in Pianosa island (part of the Tuscan Archipelago National Park) using 13 STR loci and a fragment of the mtDNA control region. All individuals analyzed share a unique haplotype, the L. europaeus haplotype Leu2, recognized as the ancestral mitochondrial lineage corresponding to the subspecies L. e. meridiei. Furthermore, considering autosomal markers, Pianosa brown hare population and current Italian peninsular population are genetically distinct. The discovery of this ancient population in a protected area, isolated and not affected by recent translocation/restocking events, has a great relevance in conservation and confirms the current presence of the endemic subspecies L. e. meridiei in Italy.     

The occurrence of mountain hare mitochondrial DNA in wild brown hares

If interspecific hybrids are fertile and backcross to either parental species, transmission of mitochondrial DNA over the species barrier can occur. To investigate if such transmission has occurred between the brown hare Lepus europeus Pall and the mountain hare L. timidus L. in Scandinavia, an analysis of genetic variation in mitochondrial DNA from 36 hares, collected from 15 localities, was performed. Sequence divergence of mtDNA between species was estimated at 8 ± 1% (SD). Intraspecific mtDNA sequence divergence varied between 0.09 and 0.38% in brown hares and 0.10 and 1.44% in mountain hares. In six out of 18 brown hares examined, two different haplotypes of mountain hare origin were detected, demonstrating a transmission of mtDNA haplotypes from mountain hares to brown hares. The results indicate that interspecific hybridization between the two species occurs in wild populations.     

草兔线粒体基因组全序列及其系统发育关系分析

草兔(Lepus capensis)在我国数量多,分布广,但人们对草兔的系统地理学、草兔与其他兔类的系统发育关系了解不多,其亚种水平的分类也长期存在争议。本研究通过LA-PCR 技术对草兔线粒体基因组全序列进行PCR 扩增、序列测定和分析,并基于线粒体基因组12 个蛋白质编码基因10 776 bp的核苷酸序列,采用贝叶斯法(BI)和最大似然法(ML)构建哺乳纲(Mammalia) 灵长总目(Euarchontoglires)5 个目12 种动物的系统发育关系,结果支持兔形目(Lagomorpha)的单系起源,其中草兔与欧洲野兔(L. europaeus)亲缘关系最近,两者互为姐妹群,进而与家兔(Oryctolagus cuniculus)构成姐妹群关系。此外,基于线粒体细胞色素b 基因全序列,构建31 个兔类个体的系统发育关系,结果显示,草兔与雪兔(L. timidus),云南兔(L. comus) 与高原兔(L.oiostolus),东北兔(L. mandschuricus)与塔里木兔(L. yarkandensis) 有较近的亲缘关系,而在草兔内部,中国的草兔与南非草兔亲缘关系较远,采自江苏盐城的草兔与来自山东、陕西、四川的草兔聚为一支,具有明显较近的亲缘关系。该研究为更好地探讨草兔系统发育关系提供了进一步的分子生物学资料.     

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

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

High mtDNA haplotype diversity among introduced Swedish brown haresLepus europaeus

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

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

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

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

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

Molecular phylogeny of Japanese Leporidae,the Amami rabbit Pentalagus furnessi,the Japanese hare Lepus brachyurus,and the mountain hare Lepus timidus,inferred from mitochondrial DNA sequences

We determined mitochondrial 12S ribosomal RNA (rRNA) and cytochrome b (cyt b) gene sequences in three leporid species of Japan, the Amami rabbit Pentalagus furnessi from the Ryukyu Islands, the Japanese hare Lepus brachyurus from Honshu, and a Japanese form of the mountain hare Lepus timidus ainu from Hokkaido. We compared the sequences with those of other taxa of leporids available in databases. Phylogenetic trees of the 12S rRNA gene sequences indicated that the lineage of P. furnessi diversified during the generic radiation of the leporids at an ancient time, which was estimated to have been the middle Miocene. Cyt-b gene trees revealed that the lineage of L. brachyurus branched off at an early stage in the speciation of Lepus, probably at the beginning of the Pliocene. The cyt b sequences of L. t. ainu were somewhat distinct from those of continental conspecific populations; this lineage divergence is likely to have occurred during the middle or late Pleistocene. The results show that the three regions of the Japanese archipelago, Ryukyu, Honshu-Shikoku-Kyushu, and Hokkaido, now preserve their own leporid taxa, each with a different extent of genetic endemicity. It is possible that the zoogeographic traits of the Japanese leporids are a consequence of the evolutionary dynamics of leporids in East Asia, in that the radiation centers of leporids are likely to have shifted from tropical, through temperate, to arctic zones.     

The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus

Climatic oscillations during the glaciations forced dramatic changes in species distributions, such that some presently temperate regions were alternately occupied by temperate and arctic species. These species could have met and hybridized during climatic transitions. This phenomenon happened for three hare species present in Iberia (Lepus granatensis, Lepus europaeus and Lepus castroviejoi), which display high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species presently extinct in Iberia. Here, we extend our previous geographical survey to determine whether the distribution of this mtDNA lineage extends beyond the northern half of the Iberian Peninsula, where it is found at high frequencies. We also review the taxonomy, distribution and molecular phylogeny of the genus Lepus. The phylogenetic inference reveals the presence of L. timidus-like mtDNA in several other hare species in Asia and North America, suggesting that the mitochondrial introgression observed in Iberia might be generalized. Comparison with the available nuclear gene phylogenies suggests that introgression could have happened repeatedly, possibly during different climatic transitions. We discuss demographic and adaptive scenarios that could account for the repetition in time and space of this spectacular phenomenon and suggest ways to improve our understanding of its determinants and consequences. Such high levels of introgressive hybridization should discourage attempts to revise hare taxonomy based solely on mtDNA.     

Hares in Corsica: high prevalence of <Emphasis Type="Italic">Lepus corsicanus</Emphasis> and hybridization with introduced <Emphasis Type="Italic">L. europaeus</Emphasis> and <Emphasis Type="Italic">L. granatensis</Emphasis>

The Italian hare, Lepus corsicanus, was first described in Corsica more than 100 years ago, but the knowledge on the status of the species in this island remains scarce. Moreover, frequent introductions of thousands of individuals from other hare species, namely Lepus europaeus and Lepus granatensis, into Corsica are known to have occurred and an updated assessment of the prevalence of L. corsicanus in Corsica is therefore of utmost importance. Here, to estimate the relative prevalence of the hare species present in Corsica, we conducted a molecular analysis on 67 samples collected by hunters between 2002 and 2007 in 36 Corsican communes. Sequencing of portions of the nuclear gene transferrin and of the control region of the mitochondrial DNA allowed classifying most of the collected samples as belonging to L. corsicanus (70.1%). Of the sampled Corsican communes, 86.1% contained this species, while only in 11.1%, L. europaeus was present. Three of the analyzed specimens showed an inconsistent molecular assignment between markers suggesting a hybrid origin: L. corsicanus × L. europaeus, L. corsicanus × L. granatensis, and L. europaeus × L. granatensis. The first two cases of hybridization had never been described in nature, even in studies focusing on hares from Italy where L. corsicanus and L. europaeus are often sympatric. These results stress the real risk of corrosion of the native gene pool of L. corsicanus via hybridization with introduced species. We highlight the need of urgently rethinking the management plan of hare populations in Corsica.     

Evidence of autumn reproduction in female European hares (Lepus europaeus) from southern Europe

Information on reproductive biology of the European hare (Lepus europaeus) in different environmental and landscape conditions comprises part of fundamental knowledge regarding species’ adaptive responses as well as many aspects of its biology. Most of the studies conducted on European hare reproduction are confined to midlatitude and northern populations, whereas no data exist on the indigenous southern populations. Here, we present information on reproductive characteristics of European hares inhabiting Mediterranean ecosystems on the island of Crete, Greece for two successive hunting seasons. Although the annual reproductive cycle of the species is well known, with an autumn sexual inactivity, the duration of this period is subjected to fluctuations in different years and for different areas. According to our data, hare populations of Crete present an autumn–early winter reproductive activity with high proportions of pregnant females observed in all the months of the study. Furthermore, the estimated mean litter size (1.54 SE ± 0.07) while signed to the lowest values ever observed for European hares is similar to values obtained in continuous breeding species of the same genus, Lepus granatensis, Lepus corsicanus, Lepus (capensis) mediterraneus, and Lepus capensis also inhabiting warm climates. In conclusion, our results suggest that Cretan European hare populations exhibit a reproductively active period during autumn–early winter where proportions of pregnant females and litter size give a strong indication of a continuous reproduction throughout the year.     

Sexing European rabbits (Oryctolagus cuniculus), European brown hares (Lepus europaeus) and mountain hares (Lepus timidus) with ZFX and ZFY loci

We describe a new method for the sex determination of tissue originating from Oryctolagus cuniculus (European rabbit), Lepus europaeus (European brown hare) and Lepus timidus (mountain hare) based on PCR-RFLP analysis of point mutations that differentiate the ZFX and ZFY gene sequences. Among several applications, this PCR-RFLP method could be used to investigate gender ratio and evaluate the population dynamics of these species using samples collected when sex cannot be identified.     

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

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

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

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

A single multiplex of twelve microsatellite markers for the simultaneous study of the brown hare (Lepus europaeus) and the mountain hare (Lepus timidus)

The management of hunted species is challenging, as it must conciliate the conservation of species and their sustainable exploitation. Nongenetic tools are widely used in this context but they may present limitations notably when species can hybridize or when large‐scale spatial monitoring is required to establish optimal management actions. This is why genetic tools have been more and more integrated in wildlife management practices. However, the markers proposed are often amplified in small multiplexes when larger ones could allow to better cope with the small quantities of DNA obtained with noninvasive sampling methods. Here, we propose a unique multiplex of 12 autosomal microsatellite markers for the study of two hare species that exist in sympatry in some areas in Europe and are hunted notably in France: the brown hare Lepus europaeus and the mountain hare L. timidus. We tested 17 markers previously used in these two species or other lagomorph species, from which 12 were included in this single multiplex. Diversity was between 4 and 30 alleles per locus totaling 126 alleles, and we showed that these markers possess appropriate genetic resolution for individual and species identification for the populations under study. This multiplex panel represents the largest number of microsatellites amplified in one reaction proposed for these two hare species and provides a cost‐effective and valuable tool for further hybridization studies and the management of hares.     

Effect of fox hunting with small hound packs on spatial behaviour of brown hares

Control of red fox Vulpes vulpes populations is a fundamental game management tool used by hunters interested in increasing prey populations. In Italy, a popular method to control fox populations is hunting with small hound packs. The effects of this hunting technique on non-target species such as the brown hare Lepus europaeus, are unknown. In this study, we analysed for the first time the effects of fox hunting with hound packs on brown hares tagged with VHF collars. Our results showed that hunting with four trained hounds did not significantly modify the spatial behaviour of the brown hare.     

Invasion from the cold past: extensive introgression of mountain hare (Lepus timidus) mitochondrial DNA into three other hare species in northern Iberia

Mitochondrial DNA introgression from Lepus timidus into Lepus granatensis and Lepus europaeus was recently reported in Iberia, although L. timidus presumably retreated from this region at the end of the last ice age. Here we assess the extent of this ancient mtDNA introgression by RFLP analysis of 695 specimens representing the three hare species present in Iberia. The introgressed L. timidus lineage was found in 23 of the 37 populations sampled. It is almost fixed in L. europaeus across its Iberian range in the Pyrenean foothills, and in L. granatensis, which occupies the rest of the peninsula, it is predominant in the north and gradually disappears further south. We also found it in Lepus castroviejoi, a species endemic to Cantabria. Multiple hybridizations and, potentially, a selective advantage for the L. timidus lineage can explain the remarkable taxonomic and geographical range of this mitochondrial introgression.     

Temporal dynamics of European brown hare syndrome infection in Northern Italian brown hares (Lepus europaeus)

The progressive decline in the hare population across Europe has been associated with the occurrence of European brown hare syndrome (EBHS), a highly contagious disease considered endemic in all European countries. This study aimed to evaluate the in-field temporal dynamics of European brown hare syndrome virus (EBHSV) infection in wild European brown hares (Lepus europaeus) and to test the influence of population density on EBHS seroprevalence. A total of 512 blood samples were collected from free ranging hares captured for restocking in seven different areas of the province of Brescia (Northern Italy) during seven consecutive years (2006–2013) and tested using a competitive ELISA. A generalized linear mixed model estimated the yearly effects of population density on EBHS prevalence. Of the 512 tested, 344 (67.2 %) tested positive for EBHSV antibodies, with the annual seroprevalence ranging from 94.3 to 35.8 %. The prevalence was 3.303 times higher in areas with a density of over 15 hares/km² and declined over the years. The results indicate the ongoing transmission of the virus in the tested brown hare population. Since the eradication of EBHS in a wild population is not feasible, a strategy aimed at promoting the endemic stability of the virus through density-dependent mechanisms could be applied; however, this seems more difficult in practice than in theory and would most likely require a very high density of brown hares.     

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

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