Wolf-dog hybridization: Population structure confounds genetic assignment and hybrid classification

Accurate hybrid detection is increasingly important for conservation, yet genetic assignment can be strongly biased when structured populations are analyzed as if they were homogeneous. This problem is especially relevant in recolonizing systems, where individuals from differentiated source populations meet and admix. We present and evaluate a monitoring-compatible framework that accounts for population structure before testing for hybridization, without relying on reference populations from outside the study area. Using four years of routine wolf monitoring in Czechia, a Central European contact zone of differentiated lineages, we show that ignoring population structure can inflate admixture and generate false signals of hybridization. Population-specific calibration combined with independent validation resolved ambiguous assignments and provided no evidence for ongoing wolf-dog introgression within the monitored wolf population. Our results demonstrate that population structure must be explicitly considered when interpreting hybridization signals and provide a practical framework for conservation monitoring in other structured wildlife populations.