Efficient cross-species capture hybridization and next-generation sequencing of mitochondrial genomes from noninvasively sampled museum specimens.

The ability to uncover the phylogenetic history of recently extinct species and other species known only from archived museum material has rapidly improved due to the reduced cost and increased sequence capacity of next-generation sequencing technologies. One limitation of these approaches is the difficulty of isolating and sequencing large, orthologous DNA regions across multiple divergent species, which is exacerbated for museum specimens, where DNA quality varies greatly between samples and contamination levels are often high. Here we describe the use of cross-species DNA capture hybridization techniques and next-generation sequencing to selectively isolate and sequence partial to full-length mitochondrial DNA genomes from the degraded DNA of museum specimens, using probes generated from the DNA of a single extant species. We demonstrate our approach on specimens from an enigmatic gliding mammal, the Sunda colugo, which is widely distributed throughout Southeast Asia. We isolated DNA from 13 colugo specimens collected 47-170 years ago, and successfully captured and sequenced mitochondrial DNA from every specimen, frequently recovering fragments with 10%-13% sequence divergence from the capture probe sequence. Phylogenetic results reveal deep genetic divergence among colugos, both within and between the islands of Borneo and Java, as well as between the Malay Peninsula and different Sundaic islands. Our method is based on noninvasive sampling of minute amounts of soft tissue material from museum specimens, leaving the original specimen essentially undamaged. This approach represents a paradigm shift away from standard PCR-based approaches for accessing population genetic and phylogenomic information from poorly known and difficult-to-study species.