Background: Biodiversity is facing global change at an unprecedented rate; understanding how species respond to this accelerated change is important to inform future environmental and wildlife-related policies. Serially collected specimens from natural history repositories can provide a unique window into how populations change over time and highlight further potential vulnerabilities in remaining populations. Changes in sampling abundance during field surveys suggest that salamanders from the genus Plethodon have experienced severe population declines over the past four decades. The causes of these declines remain unidentified, but factors such as habitat loss, climate change, acid rain, and pathogens are proposed as contributors. The population genetic consequences of these declines have not yet been assessed. Therefore, Plethodon serve as an ideal model to test the utility of historical DNA and serial sampling to identify shifts in genetic diversity at recent time scales in populations with documented declines. Here we estimate levels of genetic diversity in six Plethodon species through time using DNA from museum specimens and contemporary sampling. We quantify the impact of missing data, locus type, and sequencing depth on estimates of diversity. Results: Our estimates of genetic diversity are consistent with previously documented declines in P. cinereus at Indian Grave Gap, but genetic diversity increased for all other species and populations we characterized. Conclusions: Our study outlines important considerations for leveraging historical DNA in time series collections to quantify the genomic effects of localized population declines.

Until recently many historical museum specimens were largely inaccessible to genomic inquiry, but high-throughput sequencing (HTS) approaches have allowed researchers to successfully sequence genomic DNA from dried and fluid-preserved museum specimens. In addition to preserved specimens, many museums contain large series of allozyme supernatant samples but the amenability of these samples to HTS has not yet been assessed. Here, we compared the performance of a target-capture approach using alternative sources of genomic DNA from ten specimens of spring salamanders (Plethodontidae: Gyrinophilus porphyriticus) collected 1985–1990: allozyme supernatants, allozyme homogenate pellets, and formalin-fixed tissues. We designed capture probes based on double-digest restriction-site associated (RADseq) sequencing derived loci from seven of the specimens and assessed the success and consistency of capture and RADseq technical replicates. This study design enabled direct comparisons of data quality and potential biases among the different datasets for phylogenomic and population genomic analyses. We found that in phylogenetic analyses, all replicates for a given specimen clustered together, but in principal component space, RADseq replicates did not cluster with corresponding capture-based replicates. SNP calls were on average 18.3% different between technical replicates, but these discrepancies were primarily due to differences in heterozygous/homozygous SNP calls. We demonstrate that both allozyme supernatant and formalin-fixed samples can be successfully used for population genomic analyses and we discuss ways to identify and reduce biases associated with combining capture and RADseq data.