Evaluating genotyping-in-thousands by sequencing as a genetic monitoring tool for a climate sentinel mammal using non-invasive and archival samples.

Genetic tools for wildlife monitoring can provide valuable information on spatiotemporal population trends and connectivity, particularly in systems experiencing rapid environmental change. Multiplexed targeted amplicon sequencing techniques, such as genotyping-in-thousands by sequencing (GT-seq), can provide cost-effective approaches for collecting genetic data from low-quality and quantity DNA samples, making them potentially useful for long-term wildlife monitoring using non-invasive and archival samples. Here, we developed a GT-seq panel as a potential monitoring tool for the American pika () and evaluated its performance when applied to traditional, non-invasive, and archival samples, respectively. Specifically, we optimized a GT-seq panel (307 single nucleotide polymorphisms (SNPs)) that included neutral, sex-associated, and putatively adaptive SNPs using contemporary tissue samples ( = 77) from the Northern Rocky Mountains lineage of American pikas. The panel demonstrated high genotyping success (94.7%), low genotyping error (0.001%), and excellent performance identifying individuals, sex, relatedness, and population structure. We subsequently applied the GT-seq panel to archival tissue ( = 17) and contemporary fecal pellet samples ( = 129) collected within the Canadian Rocky Mountains to evaluate its effectiveness. Although the panel demonstrated high efficacy with archival tissue samples (90.5% genotyping success, 0.0% genotyping error), this was not the case for the fecal pellet samples (79.7% genotyping success, 28.4% genotyping error) likely due to the exceptionally low quality/quantity of recovered DNA using the approaches implemented. Overall, our study reinforced GT-seq as an effective tool using contemporary and archival tissue samples, providing future opportunities for temporal applications using historical specimens. Our results further highlight the need for additional optimization of sample and genetic data collection techniques prior to broader-scale implementation of a non-invasive genetic monitoring tool for American pikas.