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Historical DNA analysis of archival samples has added new dimensions to population genetic studies, enabling spatiotemporal approaches for reconstructing population history and informing conservation management. Here we tested the efficacy of Genotyping-in-Thousands by sequencing (GT-seq) for collecting targeted single nucleotide polymorphism genotypic data from archival scale samples, and applied this approach to a study of kokanee salmon ( Oncorhynchus nerka ) in Kluane National Park and Reserve (KNPR; Yukon, Canada) that underwent a severe 12-year population decline followed by a rapid rebound. We genotyped archival scales sampled pre-crash and contemporary fin clips collected post-crash, revealing high coverage (> 90% average genotyping across all individuals) and low genotyping error (< 0.01% within-libraries, 0.60% among-libraries) despite the relatively poor quality of recovered DNA. We observed slight decreases in expected heterozygosity, allelic diversity, and effective population size post-crash, but none were significant, suggesting genetic diversity was retained despite the severe demographic contraction. Genotypic data also revealed the genetic distinctiveness of a now extirpated population just outside of KNPR, revealing biodiversity loss at the northern edge of the species distribution. More broadly, we demonstrated GT-seq as a valuable tool for collecting genome-wide data from archival samples to address basic questions in ecology and evolution, and inform applied research in wildlife conservation and fisheries management.

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Genotyping-in-Thousands by sequencing of archival fish scales reveals maintenance of genetic variation following a severe demographic contraction in kokanee salmon