Laboratory methods
All laboratory work was performed at the Center for Conservation Genomics (CCG), Smithsonian Conservation Biology Institute, Washington, DC. We extracted DNA from liver and ear punch samples using a DNeasy Blood and Tissue Kit (Qiagen, Valencia CA) following the manufacturer’s protocol. We amplified whole mitogenomes in two fragments using long range PCR, fragmented the PCR products to an average length of 500 base pairs (bps) using a Qsonica Q800R sonicator (QSonica, Newtown, CT, USA), and prepared single-indexed DNA libraries for sequencing using a Kapa LTP Library Preparation kit (Kapa Biosystems, Wilmington, MA) following Hawkins et al. (2016). We pooled libraries equimolarly and sequenced on an Illumina MiSeq with 2 × 100 base pair (bp) reads (Illumina, Inc., San Diego, CA).
We used in-solution DNA hybridization to enrich genomic DNA for UCEs following Hawkins et al. (2016). We sheared DNA extracts and constructed indexed libraries as above. We quantified libraries using a Qubit fluorometer (Life Technologies) with a 1× dsDNA HS assay kit and multiplexed 4-8 samples equimolarly prior to enrichment. We used a NimbleGen SeqCap EZ kit (Roche, Basel, Switzerland) containing 54,689 unique 60-bp DNA probes representing 5,561 vertebrate UCE loci with an average of 4× tiling per base per locus to enrich multiplexed libraries following the manufacturer’s protocol. Post-enrichment libraries were amplified with 12–14 cycles of PCR using Kapa HiFi HotStart DNA polymerase (Kapa Biosystems, Wilmington, MA) following the manufacturer’s protocol.
Following visualization on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with High Sensitivity DNA kits, enriched libraries were quantified via qPCR using the Kapa Biosystems Illumina Library Quantification Kit (Kapa Biosystems, Wilmington, MA). Samples were pooled equimolarly and sequenced with 2 × 150 bp reads on Illumina HiSeq2000 (Semel Institute of Neurosciences, UCLA, & University of Copenhagen, Denmark) and MiSeq platforms (CCG).