Sequence capture phylogenomics of historical ethanol-preserved museum specimens: Unlocking the rest of the vault

Natural history collections play a crucial role in biodiversity research, and museum specimens are increasingly being incorporated into modern genetics-based studies. Sequence capture methods have proven incredibly useful for phylogenomics, providing the additional ability to sequence historical museum specimens with highly degraded DNA, which until recently have been deemed less valuable for genetic work. The successful sequencing of ultraconserved elements (UCEs) from historical museum specimens has been demonstrated on multiple tissue types including dried bird skins, formalin-fixed squamates and pinned insects. However, no study has thoroughly demonstrated this approach for historical ethanol-preserved museum specimens. Alongside sequencing of "fresh" specimens preserved in >95% ethanol and stored at -80°C, we used extraction techniques specifically designed for degraded DNA coupled with sequence capture protocols to sequence UCEs from historical museum specimens preserved in 70%-80% ethanol and stored at room temperature, the standard for such ethanol-preserved museum collections. Across 35 fresh and 15 historical museum samples of the arachnid order Opiliones, an average of 345 UCE loci were included in phylogenomic matrices, with museum samples ranging from six to 495 loci. We successfully demonstrate the inclusion of historical ethanol-preserved museum specimens in modern sequence capture phylogenomic studies, show a high frequency of variant bases at the species and population levels, and from off-target reads successfully recover multiple loci traditionally sequenced in multilocus studies including mitochondrial loci and nuclear rRNA loci. The methods detailed in this study will allow researchers to potentially acquire genetic data from millions of ethanol-preserved museum specimens held in collections worldwide.