Monitoring genetic parameters is important for setting effective conservation and management strategies, particularly for small, fragmented, and isolated populations. Small populations face increased rates of genetic drift and inbreeding, which increase extinction risk especially when gene flow is limited. Here, we applied a Genotyping-in-Thousands by sequencing (GT-seq) panel to inform recovery action for the northern Idaho ground squirrel (Urocitellus brunneus), listed as threatened under the U.S. Endangered Species Act. We evaluated genetic diversity, structure, connectivity, and effective population size to address species recovery goals. We delineated 15 conservation units: (1) three evolutionarily significant units that represent long-term population structure and evolutionary history, (2) nine management units that reflect current demographic connectivity and restrictions to gene flow, and (3) three adaptive units that capture adaptive differentiation across the species range. Effective population sizes per management unit were small overall (mean 38.16, range 2.3-220.9), indicating that recovery goals have not been reached. Our results suggest that connectivity within evolutionarily significant units should be maintained through the restoration of dispersal corridors. We recommend further sampling of subpopulations that harbor unique adaptive differentiation and that are geographically isolated to refine our understanding of adaptive variation. We recommend ongoing genetic monitoring with the same GT-seq marker panel to detect dispersal, assess effective population size, monitor the effects of inbreeding, and evaluate adaptive differentiation to monitor the effects of management action and environmental change. This study provides an illustration of how genetic and genomic tools can inform management and assess recovery goals for a threatened species.

Understanding the neutral (demographic) and adaptive processes leading to the differentiation of species and populations is a critical component of evolutionary and conservation biology. In this context, recently diverged taxa represent a unique opportunity to study the process of genetic differentiation. Northern and southern Idaho ground squirrels (Urocitellus brunneus – NIDGS, and U. endemicus - SIDGS, respectively) are a recently diverged pair of sister species that have undergone dramatic declines in the last 50 years and are currently found in metapopulations across restricted spatial areas with distinct environmental pressures. Here we genotyped single-nucleotide polymorphisms (SNPs) from buccal swabs with restriction site-associated DNA sequencing (RADseq). With these data we evaluated neutral genetic structure at both the inter- and intra-specific level, and identified putatively adaptive SNPs using population structure outlier and genotype-environment association (GEA) analyses. At the interspecific level, we found a clear separation between NIDGS and SIDGS, and evidence for adaptive differentiation relating to differences in hibernation. At the intraspecific level, we identified 3 Evolutionarily Significant Units for NIDGS and 2 for SIDGS plus multiple Management and Adaptive Units. Elevation appears to be the main driver of adaptive differentiation in NIDGS, while neutral variation patterns match and extend that identified in previous studies using microsatellite markers. For SIDGS, neutral substructure generally reflected the effect of natural geographic barriers, while adaptive variation reflected differences in land cover and temperature. These results clearly highlight the roles of neutral and adaptive processes for understanding species and population differentiation, which can have important conservation implications in threatened species.