Genetic distinction of wildcat (Felis silvestris) populations in Europe,and hybridization with domestic cats in Hungary

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.     

Molecular analysis of hybridisation between wild and domestic cats (Felis silvestris) in Portugal: implications for conservation

The endangered European wildcat (Felis silvestris silvestris) is represented, today, by fragmented and declining populations whose genetic integrity is considered to be seriously threatened by crossbreeding with widespread free-ranging domestic cats. Extensive and recent hybridisation has been described in Hungary and Scotland, in contrast with rare introgression of domestic alleles in Italy and Germany. In Portugal, the wildcat is now listed as VULNERABLE in the Red Book of Portuguese Vertebrates. Nevertheless, genetic diversity of populations and the eventual interbreeding with domestic cats remain poorly studied. We surveyed genetic variation at 12 autosomal microsatellites for 34 wild and 64 domestic cats collected across Portugal. Wild and domestic cats were significantly differentiated both at allele frequencies and sizes (F _ST=0.11, R _ST = 0.18, P < 0.001). Population structure and admixture analyses performed using Bayesian approaches also showed evidence of two discrete groups clustering wild and domestic populations. Results did not show significant genetic divergence among Northern, Central and Southern wildcats. Six morphologically identified wildcats were significantly assigned to the domestic cluster, revealing some discrepancy between phenotypic and genetic identifications. We detected four hybrids (approximately 14%) using a consensus analysis of different Bayesian model-based software. These hybrids were identified throughout all sampled areas, suggesting that hybridisation is of major concern for the appropriate implementation of wildcat conservation strategies in Portugal.     

Detecting introgressive hybridization between free-ranging domestic dogs and wild wolves (Canis lupus) by admixture linkage disequilibrium analysis

Occasional crossbreeding between free-ranging domestic dogs and wild wolves (Canis lupus) has been detected in some European countries by mitochondrial DNA sequencing and genotyping unlinked microsatellite loci. Maternal and unlinked genomic markers, however, might underestimate the extent of introgressive hybridization, and their impacts on the preservation of wild wolf gene pools. In this study, we genotyped 220 presumed Italian wolves, 85 dogs and 7 known hybrids at 16 microsatellites belonging to four different linkage groups (plus four unlinked microsatellites). Population clustering and individual assignments were performed using a Bayesian procedure implemented in structure 2.1, which models the gametic disequilibrium arising between linked loci during admixtures, aiming to trace hybridization events further back in time and infer the population of origin of chromosomal blocks. Results indicate that (i) linkage disequilibrium was higher in wolves than in dogs; (ii) 11 out of 220 wolves (5.0%) were likely admixed, a proportion that is significantly higher than one admixed genotype in 107 wolves found previously in a study using unlinked markers; (iii) posterior maximum-likelihood estimates of the recombination parameter r revealed that introgression in Italian wolves is not recent, but could have continued for the last 70 (+/- 20) generations, corresponding to approximately 140-210 years. Bayesian clustering showed that, despite some admixture, wolf and dog gene pools remain sharply distinct (the average proportions of membership to wolf and dog clusters were Q(w) = 0.95 and Q(d) = 0.98, respectively), suggesting that hybridization was not frequent, and that introgression in nature is counteracted by behavioural or selective constraints.     

Hybridization between Felis silvestris silvestris and Felis silvestris catus in two contrasted environments in France

European wildcat (Felis silvestris silvestris) populations are fragmented throughout most of the whole range of the subspecies and may be threatened by hybridization with the domestic cat F.s. catus. The underlying ecological processes promoting hybridization remain largely unknown. In France, wildcats are mainly present in the northeast and signs of their presence in the Pyrenees have been recently provided. However, no studies have been carried out in the French Pyrenees to assess their exposure to hybridization. We compared two local populations of wildcats, one living in a continuous forest habitat in the French Pyrenees, the other living in a highly fragmented forest‐agricultural landscape in northeastern France to get insights into the variability of hybridization rates. Strong evidence of hybridization was detected in northeastern France and not in the Pyrenees. Close kin in the Pyrenees were not found in the same geographic location contrary to what was previously reported for females in the northeastern wildcat population. The two wildcat populations were significantly differentiated (F_ST = 0.072) to an extent close to what has been reported (F_ST = 0.103) between the Iberian population, from which the Pyrenean population may originate, and the German population, which is connected to the northeastern population. The genetic diversity of the Pyrenean wildcats was lower than that of northeastern wildcat populations in France and in other parts of Europe. The lower hybridization in the Pyrenees may result from the continuity of natural forest habitats. Further investigations should focus on linking landscape features to hybridization rates working on local populations.     

Detecting rare introgression of domestic dog genes into wild wolf (Canis lupus) populations by Bayesian admixture analyses of microsatellite variation

Hybridization with free-ranging dogs isthought to threat the genetic integrity ofwolves in Europe, although available mtDNA dataevidenced only sporadic cases of crossbreeding.Here we report results of population assignmentand genetic admixture analyses in 107wild-living Italian wolves, 95 dogs including30 different breeds and feral dogs, andcaptive-reared wolves of unknown or hybridorigins, which were genotyped at 18microsatellites. Two Italian wolves showedunusually dark coats (``black wolves'), and oneshowed a spur in both hindlegs (``fifth fingerwolf'), suggesting hybridization. Italianwolves showed significant deficit ofheterozygotes, positive F_IS values anddeviations from Hardy-Weinberg equilibrium.Genetic variability was significantlypartitioned between groups, suggesting thatwolves and dogs represent distinct gene pools.Multivariate ordination of individual genotypesand clustering of inter-individual geneticdistances split wolves and dogs into twodifferent clusters congruent with the priorphenotypic classification, but hybrids andwolves of unknown origin were not identifiedfrom genetic information alone. By contrast, aBayesian admixture analysis assigned all theItalian wolves and dogs to two differentclusters, independent of any prior phenotypicinformation, and simultaneously detected theadmixed gene composition of the hybrids, whichwere assigned to more than one cluster.Captive-reared wolves of unknown origin wereprevalently assigned to the Italian wolfpopulation. Admixture analyses showed that one``black wolf' had mixed ancestry in the dog genepool and could be a hybrid, while the other twowolves with unusual phenotypes were assigned tothe Italian wolf population.     

Genetic identification of wild and domestic cats (Felis silvestris) and their hybrids using Bayesian clustering methods

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.     

Genetic structure of wildcat (Felis silvestris) populations in Italy

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.     

Linking genetic diversity and temporal fluctuations in population abundance of the introduced feral cat (Felis silvestris catus) on the Kerguelen archipelago

Linking temporal variations of genetic diversity, including allelic richness and heterozygosity, and spatio-temporal fluctuations in population abundance has emerged as an important tool for understanding demographic and evolutionary processes in natural populations. This so-called genetic monitoring was conducted across 12 consecutive years (1996-2007) at three sites for the feral cat, introduced onto the Kerguelen archipelago fifty years ago. Temporal changes in allelic richness and heterozygosity at 18 microsatellite DNA loci were compared with temporal changes in the adult population abundance index, obtained by typical demographic monitoring. No association was found at the island spatial scale, but we observed an association between genetic diversity and adult population indices from year to year within each study site. More particularly, the magnitude of successive increases or decreases in the adult population abundance index appeared to be the major factor linking the trajectories of genetic diversity and adult population abundance indices. Natal dispersal and/or local recruitment, both facilitated by high juvenile survival when the adult population size is small, is proposed as the major demographic processes contributing to such an observed pattern. Finally, we suggested avoiding the use of the harmonic mean as an estimator of long-term population size to study the relationships between demographic fluctuations and heterozygosity in populations characterized by strong multiannual density fluctuations.     

Individual assignment using microsatellite DNA reveals unambiguous breed identification in the domestic dog

Modern individual clustering methods utilising hypervariable nuclear microsatellite DNA polymorphisms are being increasingly applied in the field of population genetics. This study explores the efficiency of the clustering methods in identifying the breeds of origin of 250 domestic dog (Canis familiaris) individuals based on 10 microsatellite loci. An allele sharing distance (DAS) matrix and the corresponding neighbour-joining tree of individuals revealed monophyletic assemblages that corresponded perfectly with the breeds of origin of the dogs. Individual assignment tests using a Bayesian statistical approach, an allele frequency based method, and a DCE genetic distance based method were all extremely powerful. Most strikingly, the Bayesian method provided 100% assignment success of individuals into their correct breeds of origin and 100% exclusion success of individuals from all alternate reference populations with a high level of statistical confidence (P < 0.0001). A Bayesian Markov Chain Monte Carlo clustering approach revealed clear distinction of individuals into groups according to their breeds of origin, with a near-zero level of 'genetic admixture' among breeds. The results demonstrate that an FST of 0.18, mean expected gene diversity of 0.6 across 10 loci, and approximately 50 individuals per reference population suffice to provide maximum individual assignment success in C. familiaris. This refutes the traditional view that DNA based dog breed identification is not feasible at the individual level of resolution.     

Food habits of the wildcat (Felis silvestris) in a peculiar habitat: the Mediterranean high mountain

The feeding spectrum of the wildcat Felis silvestris Schreber, 1777 was studied in two sites with different ecological characteristics, both situated in the same Mediterranean environment in the high mountain of the Sierra Nevada National Park, south-east Spain, where the rabbit Oryctolagus cuniculus is absent. Scat analysis ( n =101 faeces; n =402 prey items) showed that the diet is based on rodents, fundamentally wood mouse Apodemus sylvaticus , Mediterranean pine vole Microtus duodecimcostatus and south-western water vole Arvicola sapidus . Results showed strong differences between the two sites (χ²=74.04, d.f.=5, P <0.001), that is a predominance of voles in the mesic Chico river, whereas mice are predominant in the xeric Tejos ravine. Red-legged partridge Alectoris rufa and carrion also played an important role, especially in biomass terms. The overall diet differed essentially from that of the Mediterranean region, which surrounds the study area, since in these areas rabbits constitute the primary prey. However, the diet of the mountain wildcats is similar to that in the Eurosiberian floral region, despite its distance from the Sierra Nevada. In conclusion, the Iberian wildcat seems to behave as a facultative specialist, since it prefers rabbits whenever they are available, but rodents constitute most of its diet if rabbits are scarce or absent.     

Hybridization versus conservation: are domestic cats threatening the genetic integrity of wildcats (Felis silvestris silvestris) in Iberian Peninsula?

Cross-breeding between wild and free-ranging domestic species is one of the main conservation problems for some threatened species. The situation of wildcats (Felis silvestris silvestris) in Europe is a good example of this critical phenomenon. Extensive hybridization was described in Hungary and Scotland, contrasting with occasional interbreeding in Italy and Germany. First analyses in Portugal revealed a clear genetic differentiation between wild and domestic cats; however, four hybrids were detected. Here, we extended the approach to Iberian Peninsula using multivariate and Bayesian analyses of multilocus genotypes for 44 Portuguese wildcats, 31 Spanish wildcats and 109 domestic cats. Globally, wild and domestic cats were significantly differentiated (FST=0.20, p<0.001) and clustered into two discrete groups. Diverse clustering methods and assignment criteria identified an additional hybrid in Portugal, performing a total of five admixed individuals. The power of admixture analyses was assessed by simulating hybrid genotypes, which revealed that used microsatellites were able to detect 100, 91 and 85% of first-generation hybrids, second-generation genotypes and backcrosses, respectively. These findings suggest that the true proportion of admixture can be higher than the value estimated in this study and that the improvement of genetic tools for hybrids detection is crucial for wildcat conservation.     

European wildcat populations are subdivided into five main biogeographic groups: consequences of Pleistocene climate changes or recent anthropogenic fragmentation?

Extant populations of the European wildcat are fragmented across the continent, the likely consequence of recent extirpations due to habitat loss and over‐hunting. However, their underlying phylogeographic history has never been reconstructed. For testing the hypothesis that the European wildcat survived the Ice Age fragmented in Mediterranean refuges, we assayed the genetic variation at 31 microsatellites in 668 presumptive European wildcats sampled in 15 European countries. Moreover, to evaluate the extent of subspecies/population divergence and identify eventual wild × domestic cat hybrids, we genotyped 26 African wildcats from Sardinia and North Africa and 294 random‐bred domestic cats. Results of multivariate analyses and Bayesian clustering confirmed that the European wild and the domestic cats (plus the African wildcats) belong to two well‐differentiated clusters (average Ф_ST = 0.159, R_st  = 0.392, P > 0.001; Analysis of molecular variance [AMOVA]). We identified from c. 5% to 10% cryptic hybrids in southern and central European populations. In contrast, wild‐living cats in Hungary and Scotland showed deep signatures of genetic admixture and introgression with domestic cats. The European wildcats are subdivided into five main genetic clusters (average Ф_ST = 0.103, R_st  = 0.143, P > 0.001; AMOVA) corresponding to five biogeographic groups, respectively, distributed in the Iberian Peninsula, central Europe, central Germany, Italian Peninsula and the island of Sicily, and in north‐eastern Italy and northern Balkan regions (Dinaric Alps). Approximate Bayesian Computation simulations supported late Pleistocene–early Holocene population splittings (from c. 60 k to 10 k years ago), contemporary to the last Ice Age climatic changes. These results provide evidences for wildcat Mediterranean refuges in southwestern Europe, but the evolution history of eastern wildcat populations remains to be clarified. Historical genetic subdivisions suggest conservation strategies aimed at enhancing gene flow through the restoration of ecological corridors within each biogeographic units. Concomitantly, the risk of hybridization with free‐ranging domestic cats along corridor edges should be carefully monitored.     

Regionally high rates of hybridization and introgression in German wildcat populations (Felis silvestris, Carnivora, Felidae)

While the western populations of the wildcat ( Felis silvestris silvestris ) in Germany come into contact with wildcats in France and Switzerland, the eastern distribution area is geographically completely isolated and consists of scattered subpopulations. To investigate population structure, evolutionary relationships and degree of hybridization with domestic cats we analysed the mitochondrial control region of 86 cats in combination with 11 microsatellite loci of 149 cats. According to our microsatellite data, German wildcats are divided into two separate populations corresponding to the western and eastern distribution areas. We found no indication of a further subdivision of the eastern population. German wildcat populations are genetically distinct from domestic cats in the main, but we identified 18.4% of the whole wildcat sample as being of hybrid origin, corresponding to 4.2% of the eastern and 42.9% of the western wildcat population, and 2.7% of the domestic cat sample. The mitochondrial haplotypes form a network of three connected clusters and reveal a high level of genetic diversity, especially within the eastern population. Our findings are explained at best in terms of continuous introgression between domestic cats and wildcat populations and differing degrees of recent hybridization in the various populations. Future conservation efforts should focus on preserving the existing gene flow between the isolated distribution areas, but also on preventing the spread of hybrids and limiting the habitat alterations that lead to increased contact with domestic cats. In conclusion we discuss possible evolutionary reasons for the still traceable genetic integrity of the wildcat despite its long history of interbreeding.     

Craniological differentiation between European wildcats (Felis silvestris silvestris), African wildcats (F. s. lybica) and Asian wildcats (F. s. ornata): implications for their evolution and conservation

Intraspecific diversification of the wildcat (Felis silvestris), including the European wildcat (F. s. silvestris), the Asian wildcat (F. s. ornata) and the African wildcat (F. s. lybica), was examined based on 39 cranial morphology variables. The samples of free‐ranging cats originated from Britain, Europe, Central Asia and southern Africa, consisting of both nominal wildcat specimens (referred to henceforth as ‘wildcats’) and nominal non‐wildcat specimens (‘non‐wildcats’) based on museum labels. The skull morphology of ‘wildcats’ from Britain and Europe is clearly different from that of ‘wildcats’ of Central Asia and southern Africa. The latter are characterized especially by their proportionately larger cheek teeth. On the basis of principal component, discriminant function and canonical variate analyses, the skull morphology of British ‘non‐wildcats’ is less distinct than is that of British ‘wildcats’ from the skull morphologies of ‘wildcats’ of Central Asia and southern Africa. On the other hand, the skull morphology of southern African ‘non‐wildcats’ is as distinct from those of ‘wildcats’ of Britain and Europe as is that of southern African ‘wildcats’. We suggest that the evolution of the modern wildcat probably consisted of at least three different distribution expansions punctuated by two differentiation events: the exodus from Europe during the late Pleistocene, coinciding with the emergence of the steppe wildcat lineage (phenotype of Asian–African wildcat), followed by its rapid range expansion in the Old World. The second differentiation event was the emergence of the domestic cat followed by its subsequent colonization of the entire world with human assistance. Considering the recent evolutionary history of, and morphological divergence in, the wildcat, preventing hybridization between the European wildcat and the domestic cat is a high conservation priority. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 47–63.     

Tyrosinase mutations associated with Siamese and Burmese patterns in the domestic cat (Felis catus)

The Siamese cat has a highly recognized coat colour phenotype that expresses pigment at the extremities of the body, such as the ears, tail and paws. This temperature-sensitive colouration causes a 'mask' on the face and the phenotype is commonly referred to as 'pointed'. Burmese is an allelic variant that is less temperature-sensitive, producing more pigment throughout the torso than Siamese. Tyrosinase (TYR) mutations have been suspected to cause these phenotypes because mutations in TYR are associated with similar phenotypes in other species. Linkage and synteny mapping in the cat has indirectly supported TYR as the causative gene for these feline phenotypes. TYR mutations associated with Siamese and Burmese phenotypes are described herein. Over 200 cats were analysed, representing 12 breeds as well as randomly bred cats. The SNP associated with the Siamese phenotype is an exon 2 G > A transition changing glycine to arginine (G302R). The SNP associated with the Burmese phenotype is an exon 1 G > T transversion changing glycine to tryptophan (G227W). The G302R mutation segregated concordantly within a pedigree of Himalayan (pointed) Persians. All cats that had 'pointed' or the Burmese coat colour phenotype were homozygous for the corresponding mutations, respectively, suggesting that these phenotypes are a result of the identified mutations or unidentified mutations that are in linkage disequilibrium. Because the same mutations were identified in different breeds with similar phenotypes, the mutations are likely to be identical by descent rather than multiple mutation events occurring at the same site.     

Characterization of microsatellite loci for a barrenwort species (Epimedium diphyllum,Berberidaceae)

Microsatellites were identified and characterized from Epimedium diphyllum, a species of barrenworts, both attractive garden plants and valuable medicinal plants. Some Japanese species of Epimedium are threatened with extinction, and are listed in the Red Data Book of plants in Japan. Natural hybrid zones also have been reported among some taxa of Japanese Epimedium. We developed eight polymorphic microsatellite primers for population genetic analyses of E. diphyllum. The numbers of alleles per locus ranged from 10 to 20, with observed levels of heterozygosity between 0.85 and 1.00. These primer sets yielded amplification in the other three Japanese Epimedium. These markers will be valuable for conservation genetics, evolutionary biology, pharmacognostic study, and horticultural study of Epimedium.     

Development and characterization of polymorphic microsatellite loci in the endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri)

The Miami blue butterfly (Cyclargus thomasi bethunebakeri) is a state-endangered taxon in Florida and a candidate for federal listing. Here we report 12 polymorphic microsatellite loci appropriate for use in population and conservation studies. We genotyped 114 individuals sampled from a metapopulation in the lower Florida Keys over a 2-year period (2005-2006). These results show 4-14 alleles per locus, and ranges of observed and expected heterozygosities are 0.02679-0.79630 and 0.06154-0.69565, respectively. Large deviations from Hardy-Weinberg equilibrium (HWE) are observed across the whole sample set. When a single breeding population is analysed alone, seven of the loci are in HWE.