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Fig. S1 (a) Plot showing a decrease in average PCR success across all samples as target amplicon size increased. Mitochondrial amplicons ranged from 91 to 135 basepairs. (B) Logistic regression of PCR amplification success (1) or failure (0) as a function of fragment size for 322 PCR reactions. Chi square = 39.0657; df = 1; P = 0.0000.

Table S1 List of museum samples used in the study, including information on the drainage, museum, specimen accession number, collector, year collected, collection locality, the number of DNA extractions completed at the University of Colorado (CU) and the Australian Center for Ancient DNA (ACAD), and the number of DNA samples that yielded successful amplification of DNA.

Table S2 Mitochondrial DNA primers sequences used in the study.

Table S3 Modern cutthroat trout populations sampled across seven drainage basins in Colorado and New Mexico.

Table S4 The extant lineage, the diagnostic SNPs for each lineage (based on 430 bps), and the number of historic samples that have all of the diagnostic SNPs of each extant lineage.

Table S5 Number of ND2 mitochondrial haplotypes assigned to extant major lineages from fish that were sampled from 53 populations across major drainage systems.

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