Noninvasive genetic approaches continue to improve studies in molecular ecology, conservation genetics and related disciplines such as forensics and epidemiology. Noninvasive sampling allows genetic studies without disturbing or even seeing the target individuals. Although noninvasive genetic sampling has been used for wildlife studies since the 1990s, technological advances continue to make noninvasive approaches among the most used and rapidly advancing areas in genetics. Here, we review recent advances in noninvasive genetics and how they allow us to address important research and management questions thanks to improved techniques for DNA extraction, preservation, amplification and data analysis. We show that many advances come from the fields of forensics, human health and domestic animal health science, and suggest that molecular ecologists explore literature from these fields. Finally, we discuss how the combination of advances in each step of a noninvasive genetics study, along with fruitful areas for future research, will continually increase the power and role of noninvasive genetics in molecular ecology and conservation genetics.

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.

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 (F ST Z0.20, 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.

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, rst = 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, rst = 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.

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.