A methodological approach for non-invasive sampling for population size estimates in wild boars (Sus scrofa)

Composite microsatellite genotypes were determined at five loci from 35 tissue-sampled wild boars and used as reference genotypes to estimate both allelic drop-out rate and false allele rate in comparison to genotypes from scats and hair strands of the same animals. These rates allow to assess the genotyping reliability when only non-invasively collected material is available. Polymerase chain reaction (PCR) amplification from scats was often corrupted by inhibitors and worked poorly, whereas genotyping success in hair samples was high. Body region of hair origin had no influence on PCR suitability, whereas the type of hair had. We recommend the use of bristles. PCR conditions were optimized for single-hair (bristle) genotyping.     

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.     

Strong spatial segregation between wildcats and domestic cats may explain low hybridization rates on the Iberian Peninsula

The European wildcat (Felis silvestris silvestris) is an endangered felid impacted by genetic introgression with the domestic cat (Felis silvestris catus). The problem of hybridization has had different effects in different areas. In non-Mediterranean regions pure forms of wildcats became almost extinct, while in Mediterranean regions genetic introgression is a rare phenomenon. The study of the potential factors that prevent the gene flow in areas of lower hybridization may be key to wildcat conservation. We studied the population size and spatial segregation of wildcats and domestic cats in a typical Mediterranean area of ancient sympatry, where no evidence of hybridization had been detected by genetic studies. Camera trapping of wild-living cats and walking surveys of stray cats in villages were used for capture–recapture estimations of abundance and spatial segregation. Results showed (i) a low density of wildcats and no apparent presence of putative hybrids; (ii) a very low abundance of feral cats in spite of the widespread and large population sources of domestic cats inhabiting villages; (iii) strong spatial segregation between wildcats and domestic/feral cats; and (iv) no relationship between the size of the potential population sources and the abundance of feral cats. Hence, domestic cats were limited in their ability to become integrated into the local habitat of wildcats. Ecological barriers (habitat preferences, food limitations, intra-specific and intra-guild competition, predation) may explain the severe divergences of hybridization impact observed at a biogeographic level. This has a direct effect on key conservation strategies for wildcats (i.e., control of domestic cats).     

Genetic diversity and introgression in the Scottish wildcat

This paper describes a genetic analysis of wild-living cats in Scotland. Samples from 230 wild-living Scottish cats (including 13 museum skins) and 74 house cats from England and Scotland were surveyed for nine microsatellite loci. Pelage characteristics of the wild-living cats were recorded, and the cats were then grouped into five separate categories depending on the degree to which they conformed to the characteristics attributed to Felis silvestris Schreber, 1775. Allele frequency differences between the morphological groups are greater than those among the three house cat samples. Analysis of genetic distances suggests that more of the differences between individuals can be explained by pelage than geographical proximity, and that pelage and geographical location are not confounded. Ordination of the genetic distances suggests two main groups of wild-living cats, with intermediates, and one group is genetically very similar to the house cats, while the other group contains all cats taxonomically identified as wildcat based on morphology. A genetic mixture analysis gives similar results to the ordination, but also suggests that the genotypes of a substantial number of cats in the wildcat group are drawn from a gene pool with genotypes in approximately equilibrium proportions. We argue that this is evidence that these cats do not have very recent domestic ancestry. However, from the morphological data it is highly likely that this gene pool also contains a contribution from earlier introgression of domestic cat genes.     

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.     

Potential barriers to gene flow in the endangered European wildcat (Felis silvestris)

The European wildcat (Felis silvestris silvestris) is a focal species for conservation in many European countries. After a severe population decline during the 19th century, many populations became extinct or isolated. Within Germany, suitable wildcat habitat is assumed to be highly fragmented. We thus investigated fine-scale genetic structure of wildcat populations in Central Germany across two major potential barriers, the Rhine River with its valley and a major highway. We analyzed 260 hair and tissue samples collected between 2006 and 2011 in the Taunus and Hunsrück mountain ranges (3,500 km² study area). We identified 188 individuals by genotyping 14 microsatellite loci, and found significant genetic substructure in the study area. Both the Rhine River and the highway were identified as significant barrier to gene flow. While the long-term effect of the river has led to stronger genetic differentiation in the river compared to the highway, estimates of current gene flow and relatedness across barriers indicated a similar or even stronger barrier effect to ongoing wildcat dispersal of the highway. Despite these barrier effects, we found evidence for the presence of recent migration across both the river and the highway. Our study thus suggests that although wildcats have the capability of dispersal across major anthropogenic and natural landscape barriers, these structures still lead to an effective isolation of populations as reflected by genetic analysis. The results strengthen the need for currently ongoing national strategies of wildcat conservation aiming for large scale habitat connectivity.     

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.     

Development of SNP markers identifying European wildcats,domestic cats,and their admixed progeny

Introgression can be an important evolutionary force but it can also lead to species extinction and as such is a crucial issue for species conservation. However, introgression is difficult to detect, morphologically as well as genetically. Hybridization with domestic cats (Felis silvestris catus) is a major concern for the conservation of European wildcats (Felis s. silvestris). The available morphologic and genetic markers for the two Felis subspecies are not sufficient to reliably detect hybrids beyond first generation. Here we present a single nucleotide polymorphism (SNP) based approach that allows the identification of introgressed individuals. Using high‐throughput sequencing of reduced representation libraries we developed a diagnostic marker set containing 48 SNPs (Fst  > 0.8) which allows the identification of wildcats, domestic cats, their hybrids and backcrosses. This allows assessing introgression rate in natural wildcat populations and is key for a better understanding of hybridization processes.     

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.     

Large-scale genetic census of an elusive carnivore,the European wildcat (<Emphasis Type="Italic">Felis s. silvestris</Emphasis>)

The European wildcat, Felis silvestris silvestris, serves as a prominent target species for the reconnection of central European forest habitats. Monitoring of this species, however, appears difficult due to its elusive behaviour and the ease of confusion with domestic cats. Recently, evidence for multiple wildcat occurrences outside its known distribution has accumulated in several areas across Central Europe, questioning the validity of available distribution data for this species. Our aim was to assess the fine-scale distribution and genetic status of the wildcat in its central European distribution range. We compiled and analysed genetic samples from roadkills and hundreds of recent hair-trapping surveys and applied phylogenetic and genetic clustering methods to discriminate wild and domestic cats and identify population subdivision. 2220 individuals were confirmed as either wildcat (n = 1792) or domestic cat (n = 342), and the remaining 86 (3.9 %) were identified as hybrids between the two. Remarkably, genetic distinction of domestic cats, wildcats and their hybrids was only possible when taking into account the presence of two highly distinct genetic lineages of wildcats, with a suture zone in central Germany. 44 % of the individual wildcats where sampled outside the previously published distribution. Our analyses confirm a relatively continuous spatial presence of wildcats across large parts of the study area in contrast to previous analyses indicating a highly fragmented distribution. Our results suggest that wildcat conservation and management should take advantage of the higher than previously assumed dispersal potential of wildcats, which may use wildlife corridors very efficiently.     

A suite of genetic markers useful in assessing wildcat (Felis silvestris ssp.)-domestic cat (Felis silvestris catus) admixture

The wildcat (Felis silvestris ssp.) is a conservation concern largely due to introgressive hybridization with its congener F. s. catus, the common domestic cat. Because of a recent divergence and entirely overlapping ranges, hybridization is common and pervasive between these taxa threatening the genetic integrity of remaining wildcat populations. Identifying pure wildcats for inclusion in conservation programs using current morphological discriminants is difficult because of gross similarity between them and the domestic, critically hampering conservation efforts. Here, we present a vetted panel of microsatellite loci and mitochondrial polymorphisms informative for each of the 5 naturally evolved wildcat subspecies and the derived domestic cat. We also present reference genotypes for each assignment class. Together, these marker sets and corresponding reference genotypes allow for the development of a genetic rational for defining "units of conservation" within a phylogenetically based taxonomy of the entire F. silvestris species complex. We anticipate this marker panel will allow conservators to assess genetic integrity and quantify admixture in managed wildcat populations and to be a starting point for more in-depth analysis of hybridization.     

Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes,L. 1758) populations

We investigated the feasibility of using genetic techniques to census pine marten (Martes martes) populations by genotyping non-invasively collected samples (plucked hair and scats), with particular reference to the genetically depauperate Irish population. Novel real-time polymerase chain reaction methods were developed for species and sex identification, targeting short DNA sequences. Background genetic variation at 17 microsatellite loci was very low in the Irish population, with an average of 2.29 alleles per locus and expected heterozygosity of 0.35. Despite such low polymorphism, a panel of eight loci with a sibling probability of identity of 0.011 reliably identified individual pine marten and their gender, as determined by reference to genotypes of live trapped individuals. With high nuclear DNA amplification success rates (93.8%) and low genotyping error rates (1.8%), plucked hairs may represent a more reliable and cost-effective DNA source than scats for monitoring populations of this elusive carnivore, and similar taxa such as the sympatric stone marten Martes foina.

     

Genetic diversity and integrity of German wildcat (Felis silvestris) populations as revealed by microsatellites,allozymes, and mitochondrial DNA sequences

As a consequence of persecution and habitat fragmentation, wildcats (Felis silvestris silvestris) in Western Europe have experienced a severe reduction in population numbers and sizes. The remaining wildcat populations are considered to be endangered by losses of genetic variability and by hybridisation with free-ranging domestic cats. To investigate genetic diversity within and among wild and domestic cat populations in Germany and to estimate the extent of gene flow between both forms, we analysed a total of 266 individuals. PCR-amplification and sequencing of 322 base pairs of a highly variable part of the mitochondrial control region (HV1) of 244 specimens resulted in 41 haplotypes with 31 polymorphic sites. Additionally, eight microsatellite loci were examined for those 244 cats. Moreover, a total of 46 wildcats and 22 domestic cats could be genotyped for 13 polymorphic out of 31 enzyme loci. Genetic variability in both groups was generally high. Variability in domestic cat populations was higher than in wildcat populations. Almost no differentiation between domestic cat populations could be found (F_ST for microsatellites=3%). In contrast, wildcat populations differed significantly from one another (F_ST for microsatellites=9.55%) Within the smaller wildcat populations, a reduction of genetic diversity was detectable with regard to the nuclear DNA. Wildcat and domestic cat mitochondrial haplotypes were separated, suggesting a very low level of maternal gene flow between both forms. In microsatellites and to a somewhat lesser extent in allozymes, wildcats and domestic cats showed distinct differentiation, suggesting an only low extent of past hybridisation in certain populations. The microsatellite data set indicated a significantly reduced effective population size (bottleneck) in the recent past for one German wildcat population.     

Low rates of hybridization between European wildcats and domestic cats in a human‐dominated landscape

Hybridization between wild species and their domestic congeners is considered a major threat for wildlife conservation. Genetic integrity of the European wildcat, for instance, is a concern as they are outnumbered by domestic cats by several orders of magnitude throughout its range. We genotyped 1,071 individual wildcat samples obtained from hair traps and roadkills collected across the highly fragmented forests of western Central Europe, in Germany and Luxembourg, to assess domestic cat introgression in wildcats in human‐dominated landscapes. Analyses using a panel of 75 autosomal SNPs suggested a low hybridization rate, with 3.5% of wildcat individuals being categorized as F1, F2, or backcrosses to either parental taxon. We report that results based on a set of SNPs were more consistent than on a set of 14 microsatellite markers, showed higher accuracy to detect hybrids and their class in simulation analyses, and were less affected by underlying population structure. Our results strongly suggest that very high hybridization rates previously reported for Central Europe may be partly due to inadequate choice of markers and/or sampling design. Our study documents that an adequately selected SNP panel for hybrid detection may be used as an alternative to commonly applied microsatellite markers, including studies relying on noninvasively collected samples. In addition, our finding of overall low hybridization rates in Central European wildcats provides an example of successful wildlife coexistence in human‐dominated, fragmented landscapes.     

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.     

Spatial colonization by feral domestic catsFelis catus of former wildcatFelis silvestris silvestris home ranges

Presently, wildcatFelis silvestris silvestris Schreber, 1777 populations are fragmented and rapidly declining in most of Europe. Although habitat destruction possibly constitutes the most serious threat to wildcat survival, hybridisation with feral domestic cats is also a critical problem. However, the mechanisms that allow domestic cats to colonise former wild cat home ranges are yet unclear. The present paper describes the decrease of typical phenotypic wildcats and the increase of phenotypic domestic cats in a remote wild area of Portugal (Serra da Malcata). A field survey using box-traps and radio-tracking between 1998 and 2001 revealed that wildcats were widespread in the study area and no domestic cats were present. A second survey using camera traps between 2005 and 2007 revealed only one wildcat whereas four typical domestic phenotype individuals were photographed. The present study clearly emphasizes the need for urgent measures aimed at preserving wildcat populations. These measures should include a national census of the species and an extensive monitoring of genetic integrity of wildcat populations, followed by the elaboration of a wildcat conservation action plan.     

Morphological discriminants of Scottish wildcats (Felis silvestris), domestic cats (F. catus) and their hybrids

Skull characters of wildcats ( Felis silvestris ), classed as 'old', 'recent' or 'modern', depending on collection date, were compared with those of domestic cats ( F. catus ) and hybrids, using Fisher Linear Discriminant Functions, Principal Component Analyses and Canonical Variates. A key is derived for classification of individual (unknown) skulls. All analyses indicate that 'recent' and 'modern' wildcats were different from those collected earlier, and implied marked changes in the rate of hydridization during this century. We suggest that the older wildcat populations were a relatively 'pure' form but more recent populations contain a significant hybrid component. Although there may be a trend towards re-establishment of the earlier type, an alternative conclusion is that the 'pure' form of wildcat is effectively extinct in Scotland.     

Assessment of genotyping accuracy in a non-invasive DNA-based population survey of Asiatic black bears (Ursus thibetanus): lessons from a large-scale pilot study in Iwate prefecture,northern Japan

Non-invasive DNA genotyping using hair samples has become a common method in population surveys of Asiatic black bears (Ursus thibetanus) in Japan; however, the accuracy of the genotyping data has rarely been discussed in empirical studies. Therefore, we conducted a large-scale pilot study to examine genotyping accuracy and sought an efficient way of error-checking hair-trapping data. We collected 2,067 hair samples, successfully determined the genotypes of 1,245 samples, and identified 295 individuals. The genotyping data were further divided into 3 subsets of data according to the number of hairs used for DNA extraction in each sample (1–4, 5–9, and ≥10 hairs), and the error rates of allelic dropout and false alleles were estimated for each subset using a maximum likelihood method. The genotyping error rates in the samples with ≥10 hairs were found to be lower than those in the samples with 1–4 and 5–9 hairs. The presence of erroneous genotypes among the identified individuals was further checked using a post hoc goodness-of-fit test that determined the match between the expected and observed frequencies of individual homozygotes at 0–6 loci. The results indicated the presence of erroneous genotypes, possibly as a result of allelic dropout, in the samples. Therefore, for improved accuracy, it is recommended that samples containing ≥10 hairs should be used for genotyping and a post hoc goodness-of-fit test should be performed to exclude erroneous genotypes before proceeding with downstream analysis such as capture-mark-recapture estimation.     

Female in the inside,male in the outside: insights into the spatial organization of a European wildcat population

Hybridization between the European wildcat, Felis silvestris silvestris, and the domestic cat, Felis silvestris catus, has been found in several European countries with different landscape structures and in various proportions. In this study, we focus on a local population of European wildcats in forests fragmented by agricultural lands in northeastern France. Our aim is to better understand how the spatial organization of the wildcats in this particular type of environment might impact the proportion of hybridization. We combined radio-tracking and genetics through the use of microsatellite markers in order to assess both the spacing pattern and the level of hybridization of this wildcat population. Hybridization is rare in this wildcat population with only one putative hybrid (most likely backcrossed) detected out of 42 putative wildcats. We found that most females were concentrated inside the forest while males stood in the periphery or outside the forest. Furthermore, many males and females resulted related. Such a spacing pattern might limit contacts between male domestic cats and female wildcats and can be one of the causes that explain the low level of hybridization in the wildcat population in this environment. We could not exclude the possibility of hybrid presence in the neighboring domestic cat populations. Our results yield new insights on the influence that the landscape configuration and the spacing pattern can have on genetic flow between the populations of the two subspecies.     

Earliest “Domestic” Cats in China Identified as Leopard Cat (Prionailurus bengalensis)

The ancestor of all modern domestic cats is the wildcat, Felis silvestris lybica, with archaeological evidence indicating it was domesticated as early as 10,000 years ago in South-West Asia. A recent study, however, claims that cat domestication also occurred in China some 5,000 years ago and involved the same wildcat ancestor (F. silvestris). The application of geometric morphometric analyses to ancient small felid bones from China dating between 5,500 to 4,900 BP, instead reveal these and other remains to be that of the leopard cat (Prionailurus bengalensis). These data clearly indicate that the origins of a human-cat ‘domestic’ relationship in Neolithic China began independently from South-West Asia and involved a different wild felid species altogether. The leopard cat’s ‘domestic’ status, however, appears to have been short-lived—its apparent subsequent replacement shown by the fact that today all domestic cats in China are genetically related to F. silvestris.