The utility of formalin-fixed tissues and allozyme supernatant for population genomics and considerations for combining capture- and RADseq-based SNP datasets

Abstract

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.

Understanding “Alexandrian” extinctions using genomic DNA from fluid-preserved museum specimens of Desmognathus salamanders

“…While our sample size is small and coverage limits our ability to test all hypotheses fully, there is an increased prevalence of homozygous SNPs unique to each of the uid-preserved specimens captured on the same day in the same population relative to the two recent specimens from widely separated populations in Florida and Georgia. Thus, we suspect at least some of the genetic divergence in the historical samples is artifactual (Ewart et al 2019;O'Connell et al 2021). In contrast, the peninsular populations are nonetheless supported as distinct by multiple SNPs in both the mitochondrial and nuclear data, shared by both historical specimens, called with high coverage and accuracy.…”

“…Our sampling of both populations and loci is too sparse to allow ne-scale population-genetic inference or estimation of phylogenetic networks showing hybridization between species, though both would be desirable in future studies due to their conservation relevance (Ewart et al 2019). We are also cautious regarding the potential for high rates of historical-sample-related base composition error confounding such estimates (see Hykin et al 2015;Oh et al 2015;O'Connell et al 2021). Thus, we do not phase alleles, model admixture, or estimate networks.…”