New DNA sequencing technologies are allowing researchers to explore the genomes of the millions of natural history specimens collected prior to the molecular era. Yet, we know little about how well specific next-generation sequencing (NGS) techniques work with the degraded DNA typically extracted from museum specimens. Here, we use one type of NGS approach, sequence capture of ultraconserved elements (UCEs), to collect data from bird museum specimens as old as 120 years. We targeted 5060 UCE loci in 27 western scrub-jays (Aphelocoma californica) representing three evolutionary lineages that could be species, and we collected an average of 3749 UCE loci containing 4460 single nucleotide polymorphisms (SNPs). Despite older specimens producing fewer and shorter loci in general, we collected thousands of markers from even the oldest specimens. More sequencing reads per individual helped to boost the number of UCE loci we recovered from older specimens, but more sequencing was not as successful at increasing the length of loci. We detected contamination in some samples and determined that contamination was more prevalent in older samples that were subject to less sequencing. For the phylogeny generated from concatenated UCE loci, contamination led to incorrect placement of some individuals. In contrast, a species tree constructed from SNPs called within UCE loci correctly placed individuals into three monophyletic groups, perhaps because of the stricter analytical procedures used for SNP calling. This study and other recent studies on the genomics of museum specimens have profound implications for natural history collections, where millions of older specimens should now be considered genomic resources.

1New DNA sequencing technologies are allowing researchers to explore the 1 2 genomes of the millions of natural history specimens collected prior to the 1 3 molecular era. Yet, we know little about how well specific next-generation

Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.

Genomic studies are revealing that divergence and speciation are marked by gene flow, but it is not clear whether gene flow has played a prominent role during the generation of biodiversity in species-rich regions of the world where vicariance is assumed to be the principal mode by which new species form. We revisit a well-studied organismal system in the Mexican Highlands, Aphelocoma jays, to test for gene flow among Mexican sierras. Prior results from mitochondrial DNA (mtDNA) largely conformed to the standard model of allopatric divergence, although there was also evidence for more obscure histories of gene flow in a small sample of nuclear markers. We tested for these 'hidden histories' using genomic markers known as ultraconserved elements (UCEs) in concert with phylogenies, clustering algorithms and newer introgression tests specifically designed to detect ancient gene flow (e.g. ABBA/BABA tests). Results based on 4303 UCE loci and 2500 informative SNPs are consistent with varying degrees of gene flow among highland areas. In some cases, gene flow has been extensive and recent (although perhaps not ongoing today), whereas in other cases there is only a trace signature of ancient gene flow among species that diverged as long as 5 million years ago. These results show how a species complex thought to be a model for vicariance can reveal a more reticulate history when a broader portion of the genome is queried. As more organisms are studied with genomic data, we predict that speciation-with-bouts-of-gene-flow will turn out to be a common mode of speciation.

Molecular studies have uncovered significant diversity in the Mexican Highlands, leading to the description of many new endemic species. DNA approaches to this kind of species discovery have included both mitochondrial DNA (mtDNA) sequencing and multilocus genomic methods. While these marker types have often been pitted against one another, there are benefits to deploying them together, as linked mtDNA data can provide the bridge between uncovering lineages through rigorous multilocus genomic analysis and identifying lineages through comparison to existing mtDNA databases. Here, we apply one class of multilocus genomic marker, ultraconserved elements (UCEs), and linked mtDNA data to a species complex of frogs (Sarcohyla bistincta, Hylidae) found in the Mexican Highlands. We generated data from 1,891 UCEs, which contained 1,742 informative SNPs for S. bistincta and closely related species and captured mitochondrial genomes for most samples. Genetic analyses based on both whole loci and SNPs agree there are six to seven distinct lineages within what is currently described as S. bistincta. Phylogenies from UCEs and mtDNA mostly agreed in their topologies, and the few differences suggested a more complex evolutionary history of the mtDNA marker. Our study demonstrates that the Mexican Highlands still hold substantial undescribed diversity, making their conservation a particularly urgent goal. The Trans-Mexican Volcanic Range stands out as a significant geographic feature in Sarcohyla and may have acted as a dispersal corridor for S. bistincta to spread to the north. Combining multilocus genomic data with linked mtDNA data is a useful approach for identifying potential new species and associating them with already described taxa, which will be especially important in groups with undescribed subadult phenotypes and cryptic species.