Microsatellite and Mitochondrial DNA Reveal Spatial Genetic Variation and Domestic Cat Admixture in Hungarian European Wildcats

The European wildcat (Felis silvestris) is threatened due to habitat loss and hybridisation with domestic cats (Felis catus), representing a significant conservation challenge in Hungary. We genotyped 118 field-collected cats from seven Hungarian sub-regions and 30 domestic cat references at 14 autosomal microsatellites, and sequenced a 669 bp mitochondrial ND5 fragment from the field samples. At K = 2, STRUCTURE assigned 84 field samples to the wildcat-associated cluster, 18 to the domestic-cat-associated cluster, and 16 (13.6%) to an intermediate membership interval. The combined non-wildcat proportion (28.8%) is not a hybridisation rate. Intermediate assignments fell to 10 at K = 3, demonstrating model dependence. Simulations recovered parental, F1 and F2 genotypes, but most second-generation and all third-generation backcrosses were assigned to a parental cluster. Genetic and geographic distances were significantly but weakly associated (Mantel r = 0.118, p = 0.005). Eight ND5 haplotypes were identified; dw4 predominated in the North Hungarian Mountains and w1 in Southern Transdanubia. These maternal patterns are compatible with historical lineage retention and/or introgression, but a single locus cannot date or distinguish these processes. The results support targeted genetic monitoring while highlighting the need for ancestry-informative SNP or genome-wide validation before management decisions are based on individual hybrid classifications.