ddRADSeq of Poecilimon orbelicus: muscle
We used Qiagen DNeasy kits (Qiagen, Hilden, Germany) to extract and purify genomic DNA from the hind femur of each individual. We processed genomic DNA into one genomic library (100 individuals/library) using the double-digestion restriction-site associated DNA sequencing procedure (ddRAD-seq) described in Peterson et al. (2012) (PLOS One, 7: e37135). In brief, we digested DNA with the restriction enzymes MseI and EcoRI (New England Biolabs, Ipswich, MA, USA) and ligated Illumina adaptors including unique 11-bp barcodes to the digested fragments of each individual. We pooled ligation products and size-selected them between 350-450 bp with a Pippin Prep machine (Sage Science, Beverly, MA, USA). We amplified the fragments by PCR with 12 cycles using the iProofTM High-Fidelity DNA Polymerase (BIO-RAD, Hercules, CA, USA) and sequenced the library in two single-read 150-bp lanes on an Illumina HiSeq2500 platform at The Centre for Applied Genomics (SickKids, Toronto, ON, Canada).