Genetic structure of the red squirrel (Sciurus vulgaris) at the westernmost limit of its range

Peripheral populations are often more vulnerable to demographic instability and reduced genetic diversity due to historical habitat loss, fragmentation, and limited connectivity. The Eurasian red squirrel ( Sciurus vulgaris ) represents a notable case, having undergone local extinctions and population declines across Europe, including its extinction in Portugal in the sixteenth century. This was followed by recolonisation through both natural dispersal and human-mediated reintroductions in the late twentieth century. Despite this complex history, the genetic origins and structure of the contemporary Portuguese population remain poorly understood. In this study, we analysed the most comprehensive sampling of red squirrels in Portugal to date using two mitochondrial markers (cytb and D-Loop) to assess genetic diversity, phylogeographic structure, and potential sources of recolonisation. Portuguese red squirrels exhibit markedly low mitochondrial diversity relative to other European populations, consistent with historical bottlenecks, extinction, and founder effects during recent recolonisation. Our results corroborate the generally weak phylogeographic structure observed across Europe and reveal a largely panmictic population within Portugal, with haplotypes broadly distributed and not associated with geographic regions. Nonetheless, some degree of population differentiation concordant with isolation by distance is observed. Haplotype sharing with neighbouring Spain supports natural recolonisation via contiguous habitat, while shared haplotypes with France align with documented reintroductions. In contrast, the haplotype shared with Italy is more difficult to explain, possibly reflecting undocumented historical translocations or ancient refugial connectivity. Further analyses using fast-evolving markers or whole-genome data are necessary to fully resolve recolonisation dynamics, assess connectivity, and inform long-term conservation strategies for red squirrels in Portugal.