Ancestral and contemporary genetic diversification of Toll-like receptor 2 in sympatric mustelid species from central Europe

The leucine-rich repeat (LRR) domain of Toll-like receptors (TLR) of the animal innate immune system recognises specific pathogen associated molecular patterns (PAMPs). It often exhibits elevated polymorphism driven by diversifying selection. We investigated variability and evolution of a 465bp fragment of the TLR2-LRR domain in six Mustelid species occurring sympatrically in eastern Austria. We specifically hypothesised higher diversity in the occasionally hybridising polecats, Mustela putorius , and steppe polecats, M. eversmannii , because of their potentially larger effective population size and possible trans-species polymorphism maintained by selection. However, among our studied species, polecats and steppe polecats exhibited the lowest allelic diversity, possibly reflecting their recent divergence. Observed heterozygosities were lower than expected heterozygosities across all studied species. Overall, five codon positions were identified as candidates for positive selection, with codon 149 supported by four independent methods, while two codons showed evidence of purifying selection. Structural modelling localized candidate positively selected sites in regions potentially relevant for function, including residues predicted to be involved in TLR1–TLR2 dimerization. Notably, several alleles were shared within and between Mustela and Martes , a pattern consistent with long-term ancestral polymorphism potentially maintained by pathogen-mediated selection. Overall, our results suggest that, for the studied species, the TLR2-LRR domain evolves under a dynamic interplay of purifying and diversifying selection, together with demographic processes.