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.
