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.     

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

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

The 24-hour cycle of the mountain hare Lepus timidus Linnaeus, 1758

The behavioral decisions of wild animals are influenced by the often conflicting needs to both feed efficiently and to avoid predators. Information regarding how lagomorphs such as the mountain hare (Lepus timidus) balance these factors throughout the day-night cycle is largely lacking despite its importance to management.To better understand the type and proportion of various behaviors the mountain hare engages in over the course of a 24-hour period, I monitored the behavior of six mountain hares under controlled conditions from early to mid-winter. The mountain hares spend the majority of their time resting (mean ± standard deviation: 32.4 ± 14.4%) and under canopy (22.4 ± 18.7%), and a smaller proportion of their time feeding (16.2 ± 6.1%). Activity peaks over the course of the 24-hour cycle reflect natural behavior patterns that emphasize the reduction of predation risk. The sunrise phase is characterized by the search for a suitable resting place; the day phase by resting and hiding. The sunset phase is characterized by the search for food, and the night phase by an equal mix of food intake and movement through the home range. These results can help land managers predict how factors such as increasing tourist activity (perceived by the hare as a safety threat) will influence the behavior of free-ranging mountain hares.     

Cladogenesis of the European brown hare (Lepus europaeus Pallas, 1778)

A substantial portion of today’s biodiversity is attributed to the climatic oscillations of the Pleistocene Ice Ages. Gradual but dramatic climate changes were accompanied by expansion, contraction, and isolation of populations, promoting the accumulation of genome differences and adaptations in refugial populations and resulting in allopatric differentiation in a variety of taxa. In the present study, partial mitochondrial DNA sequences of the widely distributed European brown hare (Lepus europaeus) were analyzed to test whether the species’ present genetic structure is the result of postglacial re-colonization of Europe from Asia Minor (clade A) and the Balkans (clade B) only, as suggested previously, or if additional refugia are likely. Analyses indicated the presence of an additional refugium (Italy, clade I). The genealogic network of Italian hares displayed the tree-like structure expected from refugial populations, whereas central European brown hare haplotypes revealed a clear star-phylogeny indicative of past-bottleneck population growth. This population size expansion, which was confirmed by mismatch analysis, was estimated to have occurred ∼50–55 thousand years ago (kya). The divergence of clade A* from the remaining matrilines is estimated at 239 kya, whereas the divergence of the ancestors of clades B* and I from A* occurred about 128 kya. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Preliminary studies on cryopreservation of hare (Lepus europaeus Pallas, 1778) semen

In the last decades, a significant decrease in hare population has been observed; for this reason, the aim of the study was to check if hare semen could be preserved in liquid nitrogen, with an extender used for rabbit semen. The results should provide a basis for creating a gene bank of the species. Ten ejaculates (volume above 0.4 ml, percentage of motile spermatozoa above 75%, spermatozoa concentration above 250 x 10(6) ml), obtained with electroejaculation method from four males, were frozen in an extender of the following composition: Tris (3.028 g), citric acid (1.675 g), glucose (1.25 g), dimethylsulphoxide (DMSO) (4.5%, v/v), egg yolk (17%, v/v) and distilled water to 100.00 ml. The motility of post-thawing spermatozoa was 40.50+/-7.97%, percentage of spermatozoa with normal acrosomes 76.10+/-3.69% and percentage of live spermatozoa 35.05+/-4.21%. Based on the properties of freezing-thawing semen, the hare semen can be successfully preserved in extender used for rabbit semen.     

A study of seasonally delayed puberty in the male hare, Lepus Europaeus.

The brown hare, Lepus europaeus, has a mating season which extends from January to September. Adult males exhibit pronounced seasonal changes in the reproductive tract which are associated with changes in LH secretion. Maximum plasma levels of immunoreactive LH occur between March and June and minimal levels in the autumn non-mating period from September to December; this seasonal cycle in gonadotrophin output is reflected by the appropriate changes in the secretion of testosterone from the testes and in the activity of the accessory sex glands. Juvenile animals reach puberty only during the adult mating season, and the age of puberty thus varies with the date of birth. Males born before May reach puberty and become fertile at 3 months of age, while those born from May to July grow to a mature body size during the autumn non-mating season but puberty is delayed for several months. Since some animals experiencing delayed puberty were found to have elevated plasma levels of LH and testosterone, it is concluded that puberty is not completely suppresed by the environmental effects of the autumn, but that the developmental process is prolonged, resulting in the juveniles being synchronized with the adults in their reproductive activity.     

Weights and growth rates in the Mountain hare Lepus timidus scoticus

Mountain hares were weighed during live trapping on a study area near Dufftown, Banffshire, from June 1958 to August 1966. Hares shot or killed by other means on the study area and elsewhere were also weighed. Females were heavier than males throughout the year and this difference became apparent in juveniles by August of their year of birth. Male hares lost weight during the breeding season (January to June) but regained it in late summer. Young hares gained weight initially at about 14 g per day, then at about 6 g per day to adult weight. Small juveniles, or those born late in the season, tended to become small adults, large or early juveniles to become large adults. Small hares moulted less completely andjbegan to breed later in the season than large hares. The effects of disease, starvation, severe weather and injury on hare weights were considered. Female weights were greater when the population was small, due to more late pregnancies or more embryos per female. Weight could not be used to distinguish between adult and juvenile hares above 2.1 kg, nor between young and older adults.     

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.     

Artificial insemination in the European hare (Lepus europaeus syriacus)

A simple technique for artificial insemination in hares was performed and used routinely in mass breeding of hares in captivity. Sperm was collected directly from the epididymides of males. Ovulation was induced by intravenous injection of human chorionic gonadotropin. Female hares were successfully inseminated throughout the year. Pregnancy occurred in 70% of inseminated females; the range in litter size was 1--3 offspring with a mean of 1.4. It was concluded that artificial insemination greatly simplified the breeding of hares in captivity.     

Preliminary results on plasma progesterone levels during pregnancy and superfetation in the hare, Lepus Europaeus.

Peripheral plasma progesterone levels were studied in pregnant hares. A rise occurred at the beginning of pregnancy, followed by a plateau from Days 10 to 35, and then a drop during the days just before parturition. No significant differences were noted between progesterone levels in pregnancies initiated at the pre-partum oestrus and those from other oestrous periods.     

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.     

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.     

Spatial patterns of genetic diversity across European subspecies of the mountain hare, Lepus timidus L

Fossil evidence shows that populations of species that currently inhabit arctic and boreal regions were not isolated in refugia during glacial periods, but instead maintained populations across large areas of central Europe. These species commonly display little reduction in genetic diversity in northern areas of their range, in contrast to many temperate species. The mountain hare currently inhabits both temperate and arctic-boreal regions. We used nuclear microsatellite and mtDNA sequence data to examine population structure and alternate phylogeographic hypotheses for the mountain hare, that is, temperate type (lower genetic diversity in northern areas) and arctic-boreal type (high northern genetic diversity). Both data sets revealed concordant patterns. Highest allelic richness, expected heterozygosity and mtDNA haplotype diversity were identified in the most northerly subspecies, indicating that this species more closely maps to phylogeographic patterns observed in arctic-boreal rather than temperate species. With regard to population structure, the Alpine and Fennoscandian subspecies were most genetically similar (F(ST) approximately 0.1). These subspecies also clustered together on the mtDNA tree and were assigned with highest likelihood to a common Bayesian cluster. This is consistent with fossil evidence for intermediate populations in the central European plain, persisting well into the postglacial period. In contrast, the geographically close Scottish and Irish populations occupied separate Bayesian clusters, distinct clades on the mtDNA maximum likelihood tree and were genetically divergent from each other (F(ST) > 0.4) indicating the influence of genetic drift, long isolation (possibly dating from the late glacial era) and/or separate postglacial colonisation routes.     

Diet of the Iberian hare (Lepus granatensis) in a mountain ecosystem

The diet of the Iberian hare (Lepus granatensis) was studied through microhistological pellet analysis in two areas from a mountain ecosystem in Central Portugal. Fecal pellets were collected monthly in 24 plots spatially distributed throughout the two study areas. For each period, a sample of 15 to 20 pellets was milled and 400 epidermal fragments were identified, by comparison with a reference collection. A wide range of plant species was observed in hare’s diet. Grasses represent the basis of the Iberian hare diet, with frequencies always higher than 50% in both study areas (annual average = 69.98%). Most of the 35 species of grasses assembled for the reference collection (91.43%) were identified in the pellets. Nevertheless, only six of these were consumed in proportions greater than 5%, being Anthoxanthum odoratum, Secale cereale and Agrostis spp. the species ingested in higher frequencies. The rate of grasses consumption reached 80.69% in winter but decreased in summer to around 55%. In this season, a concurrent rise in the ingestion of other plant groups, like herbs and shrubs, and of plant inflorescences was observed. This work provides the first results on the Iberian hare’s diet on mountain ecosystems, and suggests that the Iberian hare diet in a mountain ecosystem is similar to the observed in L. europaeus and L. timidus.