The genetic integrity and evolutionary persistence of declining wildcat populations are threatened by crossbreeding with widespread free-living domestic cats. Here we use allelic variation at 12 microsatellite loci to describe genetic variation in 336 cats sampled from nine European countries. Cats were identified as European wildcats (Felis silvestris silvestris), Sardinian wildcats (F. s. libyca) and domestic cats (F. s. catus), according to phenotypic traits, geographical locations and independently of any genetic information. Genetic variability was significantly partitioned among taxonomic groups (FST = 0.11; RST = 0.41; P < 0.001) and sampling locations (FST = 0.07; RST = 0.06; P < 0.001), suggesting that wild and domestic cats are subdivided into distinct gene pools in Europe. Multivariate and Bayesian clustering of individual genotypes also showed evidence of distinct cat groups, congruent with current taxonomy, and suggesting geographical population structuring. Admixture analyses identified cryptic hybrids among wildcats in Portugal, Italy and Bulgaria, and evidenced instances of extensive hybridization between wild and domestic cats sampled in Hungary. Cats in Hungary include a composite assemblage of variable phenotypes and genotypes, which, as previously documented in Scotland, might originate from long lasting hybridization and introgression. A number of historical, demographic and ecological conditions can lead to extensive crossbreeding between wild and domestic cats, thus threatening the genetic integrity of wildcat populations in Europe.

The taxonomic status of the Italian hare Lepus corsicanus has been uncertain since its first description by W. E. de Winton in 1898 (de Winton WE. Annual Magazine of Natural History, London, 1898, 1, 149-158). The distribution range of this species has shrunk severely over the last few decades owing to overhunting and restocking with nonindigenous brown hares (L. europaeus) in central and southern Italy and Sicily. Recently, scanty populations of Italian hares were rediscovered, and samples for morphological and molecular analyses were collected. Nucleotide sequences of the mitochondrial DNA (mtDNA) control region and cytochrome b indicate that L. corsicanus is a phylogenetically distinct species, which can be identified by concordant morphological and mtDNA traits. It seems to be reproductively isolated and apparently does not hybridize with sympatric brown hares. Phylogenetic analyses suggest that Italian and brown hares are not closely related sister taxa, but belong to distinct evolutionary lineages that dispersed in western Europe in different periods during the early Pleistocene. The Italian hare probably differentiated in isolated refuges in southern Italy during the last glaciation. Comparative analyses of mismatch distributions suggest that hares have had different demographic histories during the Pleistocene, which resulted in phylogeographical structuring in Italian hares, but not in brown and mountain (L. timidus) hares. The Italian hare is an endangered endemism and needs urgent conservation efforts.

Methods recently developed to infer population structure and admixture mostly use individual genotypes described by unlinked neutral markers. However, Hardy-Weinberg and linkage disequilibria among independent markers decline rapidly with admixture time, and the admixture signals could be lost in a few generations. In this study, we aimed to describe genetic admixture in 182 European wild and domestic cats (Felis silvestris), which hybridize sporadically in Italy and extensively in Hungary. Cats were genotyped at 27 microsatellites, including 21 linked loci mapping on five distinct feline linkage groups. Genotypes were analysed with structure 2.1, a Bayesian procedure designed to model admixture linkage disequilibrium, which promises to assess efficiently older admixture events using tightly linked markers. Results showed that domestic and wild cats sampled in Italy were split into two distinct clusters with average proportions of membership Q > 0.90, congruent with prior morphological identifications. In contrast, free-living cats sampled in Hungary were assigned partly to the domestic and the wild cat clusters, with Q < 0.50. Admixture analyses of individual genotypes identified, respectively, 5/61 (8%), and 16-20/65 (25-31%) hybrids among the Italian wildcats and Hungarian free-living cats. Similar results were obtained in the past using unlinked loci, although the new linked markers identified additional admixed wildcats in Italy. Linkage analyses confirm that hybridization is limited in Italian, but widespread in Hungarian wildcats, a population that is threatened by cross-breeding with free-ranging domestic cats. The total panel of 27 loci performed better than the linked loci alone in the identification of domestic and known hybrid cats, suggesting that a large number of linked plus unlinked markers can improve the results of admixture analyses. Inferred recombination events led to identify the population of origin of chromosomal segments, suggesting that admixture mapping experiments can be designed also in wild populations.

Crossbreeding with free-ranging domestic cats is supposed to threaten the genetic integrity of wildcat populations in Europe, although the diagnostic markers to identify "pure" or "admixed" wildcats have never been clearly defined. Here we use mitochondrial (mt) DNA sequences and allelic variation at 12 microsatellite loci to genotype 128 wild and domestic cats sampled in Italy which were preclassified into three separate groups: European wildcats (Felis silvestris silvestris), Sardinian wildcats (Felis silvestris libyca), and domestic cats (Felis silvestris catus), according to their coat color patterns, collection localities, and other phenotypical traits, independently of any genetic information. For comparison, we included some captive-reared hybrids of European wild and domestic cats. Genetic variability was significantly partitioned among the three groups (mtDNA estimate of F(ST) = 0.36; microsatellite estimate of R(ST) = 0.30; P < 0.001), suggesting that morphological diversity reflects the existence of distinct gene pools. Multivariate ordination of individual genotypes and clustering of interindividual genetic distances also showed evidence of distinct cat groups, partially congruent with the morphological classification. Cluster analysis, however, did not enable hybrid cats to be identified from genetic information alone, nor were all individuals assigned to their populations. In contrast, a Bayesian admixture analysis simultaneously assigned the European wildcats, the Sardinian wildcats, and the domestic cats to different clusters, independent of any prior information, and pointed out the admixed gene composition of the hybrids, which were assigned to more than one cluster. Only one putative Sardinian wildcat was assigned to the domestic cat cluster, and one presumed European wildcat showed mixed (hybrid) ancestry in the domestic cat gene pool. Mitochondrial DNA sequences indicated that three additional presumed European wildcats might have hybrid ancestry. These four cats were sampled from the same area in the northernmost edge of the European wildcat distribution in the Italian Apennines. Admixture analyses suggest that wild and domestic cats in Italy are distinct, reproductively isolated gene pools and that introgression of domestic alleles into the wild-living population is very limited and geographically localized.

The entire mitochondrial cytochrome b (cyt b) gene was compared for 11 species of the artiodactyl family Cervidae, representing all living subfamilies, i.e. the antlered Cervinae (Cervus elaphus, C. nippon, Dama dama), Muntiacinae (Muntiacus reevesi), and Odocoileinae (Odocoileus hemionus, Mazama sp., Capreolus capreolus, C. pygargus, Rangifer tarandus, Alces alces); and the antlerless Hydropotinae (Hydropotes inermis). Phylogenetic analyses using Tragulidae, Antilocapridae, Gira¤dae and Bovidae as outgroups provide evidence for three multifurcating principal clades within the monophyletic family Cervidae. First, Cervinae and Muntiacus are joined in a moderately-to-strongly supported clade of Eurasian species. Second, Old World Odocoileinae (Capreolus and Hydropotes) associate with the Holarctic Alces. Third, New World Odocoileinae (Mazama and Odocoileus) cluster with the Holarctic Rangifer. The combination of mitochondrial cyt b and nuclear k-casein sequences increases the robustness of these three clades. The Odocoileini + Rangiferini clade is unambiguously supported by a unique derived cranial feature, the expansion of the vomer which divides the choana. Contrasting with current taxonomy, Hydropotes is not the sister group of all the antlered deers, but it is nested within the Odocoileinae. Therefore, Hydropotes lost the antlers secondarily. Thus, the mitochondrial cyt b phylogeny splits Cervidae according to plesiometacarpal (Cervinae + Muntiacinae) versus telemetacarpal (Odocoileinae + Hydropotinae) conditions, and suggests paraphyly of antlered deer.