Staying with the pack: connectivity constraints in an isolated wolf subpopulation

European gray wolf ( Canis lupus ) populations experienced severe range fragmentation due to human persecution, habitat loss, and prey depletion. In Portugal, the Iberian wolf ( C. l. signatus ) subpopulation south of Douro River faces a high extinction risk driven by long-term demographic decline and limited gene flow. This study aimed to identify the main factors limiting functional connectivity within this endangered subpopulation. We tested two hypotheses: isolation by distance (IBD), where genetic differentiation is primarily driven by geographic distance; and isolation by resistance (IBR), which posits that genetic differentiation is influenced by landscape and ecological factors that impede movement. Resistance surfaces were derived from habitat suitability models incorporating trade-offs between ecological resources and human disturbance. To ensure robust inference, we assessed wolf-dog admixture using Bayesian clustering and assignment to parental and hybrid classes. We identified 30 of the 39 wolves estimated to occur in the region, and conducted an individual-based landscape genetics analysis, relating pairwise genetic distances to geographic and resistance distances (i.e., least-cost distances) using multiple regression. Both hypotheses significantly explained genetic distances between individuals. However, after accounting for population structuring, their contributions decreased substantially, likely reflecting the influence of social structure and kinship. We suggest much of the remaining variance is largely shaped by high human-induced mortality, which limits long-distance dispersal. Improving connectivity in this wolf subpopulation requires addressing social and demographic barriers alongside landscape-level constraints. Mitigating human-wolf conflict and reducing mortality rates is essential for enhancing functional connectivity and ensuring the long-term viability of this endangered subpopulation.