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.

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 European wildcat is an elusive felid that is declining across its range. Sicily hosts a distinctive insular wildcat population, the conservation of which requires much better ecological knowledge than is currently available, particularly population density. We simultaneously used two noninvasive methods (camera‐trapping and scat‐collection) to estimate the population density of wildcats on the Etna volcano. We conducted genetic analyses to identify individuals and to detect potential hybridization with the domestic cat. We analyzed individual capture‐histories from camera‐trapping and scat‐collection using the spatially explicit capture‐recapture (SECR) model. Furthermore, we applied the random encounter model (REM), which does not require individual identification, to the camera‐trapping data. We identified 14 wildcats from 70 photographic detections (6.48 detections/100 trap‐days) obtained from 1080 camera‐trapping days over 4 months, and we estimated to have identified all the individuals living in the study area (10.9 km−2). On the contrary, we identified 10 wildcats from 14 out of 39 scats collected from 391 km of transects walked. The estimated densities (individuals km−2 ± se) were 0.32 ± 0.1 (SECR camera‐trapping), 1.36 ± 0.73 (SECR scat‐collection) and 0.39 ± 0.03 (REM). The population density estimates obtained from SECR camera‐trapping and REM overlapped, although we recommend care when applying the latter. The SECR scat‐collection gave the highest population density (and less precise) estimates because of the low number of capture and recaptures; however, the population size estimated with this method matched the number of individuals photographed. The population density of the wildcat in Etna falls in the medium‐high range of those reported in literature, highlighting the role of this ecosystem for the long‐term conservation of the wildcat in Sicily. Camera‐trapping is confirmed as a useful tool to assess the wildcat population density and, in this case, was complemented by the genetic analysis that confirmed individual identity.

The increasing focus on infections in domestic cats (Felis catus) has raised questions about lungworm distribution in wild hosts. To enhance knowledge of the occurrence of lungworms in enzootic regions of central Italy, we examined the carcasses of 16 European wildcats (Felis silvestris silvestris). Adult nematodes, feces, respiratory flushings, and pulmonary tissues were collected at necropsy and then microscopically and genetically analyzed. Fourteen wildcats had single or mixed lungworm species. Aelurostrongylus abstrusus was the most common parasite retrieved, followed by Troglostrongylus brevior. In addition, three specimens of Angiostrongylus chabaudi were found in the pulmonary arteries of one wildcat. Histologically, the most common lesions were a mild-to-severe chronic catarrhal bronchitis and a chronic interstitial pneumonia with smooth muscle hypertrophy, associated with T. brevior and A. abstrusus, respectively. These results demonstrate that the European wildcats may harbor several species of lungworms that may impair their health and welfare. Also, F. s. silvestris is a potential reservoir for respiratory nematodes in domestic cats.

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Severe climatic changes during the Pleistocene shaped the distributions of temperate-adapted species. These species survived glaciations in classical southern refuges with more temperate climates, as well as in western and eastern peripheral Alpine temperate areas. We hypothesized that the European wildcat (Felis silvestris silvestris) populations currently distributed in Italy differentiated in, and expanded from two distinct glacial refuges, located in the southern Apennines and at the periphery of the eastern Alps. This hypothesis was tested by genotyping 235 presumed European wildcats using a panel of 35 domestic cat-derived microsatellites. To provide support and controls for the analyses, 17 know wildcat x domestic cat hybrids and 17 Sardinian wildcats (F. s. libyca) were included. Results of Bayesian clustering and landscape genetic analyses showed that European wildcats in Italy are genetically subdivided into three well-defined clusters corresponding to populations sampled in: (1) the eastern Alps, (2) the peninsular Apennines, and (3) the island of Sicily. Furthermore, the peninsular cluster is split into two subpopulations distributed on the eastern (Apennine mountains and hills) and western (Maremma hills and lowlands) sides of the Apennine ridge. Simulations indicated Alpine, peninsular, and Sicilian wildcats were isolated during the Last Glacial Maximum. Population subdivision in the peninsula cluster of central Italy arose as consequence of a more recent expansions of historically or ecologically distinct European wildcat subpopulations associated with distinct the Continental or Mediterranean habitats. This study identifies previously unknown European wildcat conservation units and supports a deep phylogeographical history for Italian wildcats.

Populations of Felis silvestris, the European, Asian and African wildcats, and the domestic cat, are characterized by a variable, genetically controlled, coat-colour and markings system. Samples of the three Italian cats, the European, the Sardinian (belonging to the African group) and the domestic striped tabby were studied for composition and occurrence of coat-colour and markings patterns, as well as for their association with craniometric and splanchnometric categories. Intergroup differences in the coat-colour and markings system were consistent with known metric differences. It was thus possible to distinguish, objectively, the three cats from each other by means of coat patterns, even more clearly than with metric procedures. Distances between taxa, calculated through the relative frequencies of the coat patterns, were fully consistent with those measured from allele frequencies for polymorphic loci. This confirmed the polytypical species status of Felis silvestris.