Delineation of units below the species level is critical for prioritizing conservation actions for species at-risk. Genetic studies play an important role in characterizing patterns of population connectivity and diversity to inform the designation of conservation units, especially for populations that are geographically isolated. The northernmost range margin of Western Rattlesnakes (Crotalus oreganus) occurs in British Columbia, Canada, where it is federally classified as threatened and restricted to five geographic regions. In these areas, Western Rattlesnakes hibernate (den) communally, raising questions about connectivity within and between den complexes. At present, Western Rattlesnake conservation efforts are hindered by a complete lack of information on genetic structure and degree of isolation at multiple scales, from the den to the regional level. To fill this knowledge gap, we used Genotyping-in-Thousands by sequencing (GT-seq) to genotype an optimized panel of 362 single nucleotide polymorphisms (SNPs) from individual samples (n = 461) collected across the snake’s distribution in western Canada and neighboring Washington (USA). Hierarchical STRUCTURE analyses found evidence for population structure within and among the five geographic regions in BC, as well as in Washington. Within these regions, 11 genetically distinct complexes of dens were identified, with some regions having multiple complexes. No significant pattern of isolation-by-distance and generally low levels of migration were detected among den complexes across regions. Additionally, snakes within dens generally were more related than those among den complexes within a region, indicating limited movement. Overall, our results suggest that the single, recognized designatable unit for Western Rattlesnakes in Canada should be re-assessed to proactively focus conservation efforts on preserving total genetic variation detected range wide. More broadly, our study demonstrates a novel application of GT-seq for investigating patterns of diversity in wild populations at multiple scales to better inform conservation management.

GT-seq_308_488.vcf: vcf file containing genotypic data for 488 Western rattlesnakes at 308 SNPs generated via GT-seq

Metadata_488.xlsx: locality and sample information for 488 Western rattlesnakes in British Columbia (Canada) and Washington (USA)

Locus_info_362.csv: GT-seq genotyping input file