Maldonado, Jesús E.

Abstract Concerns about declining gray fox (Urocyon cinereoargenteus) populations in the Midwest, United States, prompted a petition to list the prairie gray fox subspecies (Urocyon cinereoargenteus ocythous) under the US Endangered Species Act. Although previous studies identified a deep phylogenetic break between eastern and western lineages of the gray fox at the Great Plains Suture Zone, population structure within the Midwest remains poorly understood, complicating subspecies classification. To address this, we genotyped 339 individuals from the eastern lineage of the gray fox using single nucleotide polymorphisms (SNPs) generated from 2bRAD genomic sequencing to assess genetic diversity and structure. Bayesian clustering and principal component analysis supported three genetic clusters (K = 3), revealing a cryptic break within Missouri that subdivides the morphologically defined range of U. c. ocythous and a secondary break coinciding with the Mississippi River and the currently accepted subspecies boundary. However, low pairwise FST values and admixture indicate weak overall genetic differentiation with ongoing gene flow, whereas declining genetic diversity at northern latitudes is reflective of founder effects from post-glacial population expansion. Consistent with prior mitochondrial DNA (mtDNA) results, nuclear SNP data suggest that U. c. ocythous and Urocyon cinereoargenteus cinereoargenteus are unlikely to represent distinct evolutionary units. However, because 2bRAD detects primarily neutral variation, whole-genome sequencing will be needed to test for adaptive divergence that might persist despite gene flow. Our findings provide an important step toward a comprehensive understanding of population structure and refining subspecies boundaries within the eastern lineage of the gray fox.