RAD-seq of Phyllodactylus benedettii
Genomic DNA was extracted from 161 individuals collected from nine sampling locations throughout Jalisco, Mexico (Fig. 1, Supplementary Table 1). Animal care protocols were approved by the Animal Care Committee at the Royal Ontario Museum (Number 2010-07; Toronto, Ontario, Canada). The ddRADseq libraries were prepared following the protocol of Peterson et al. (2012) and then submitted for paired-end 100 bp sequencing on two lanes of an Illumina Hi-Seq 2500 platform. Genomic DNA was digested using two restriction enzymes, SphI and MluCI, which recognize GCATGC and AATT sequences respectively. The DNA was then separated into seven pools of 43-48 samples, each with a unique index sequence which was ligated to one end of the DNA. At the other end of the indexed DNA, an inline-barcode was ligated. Size selection was performed for each pool using a Blue Pippin prep 2% dye-free gel cassette (V1; BDF2010, Sage Science) with the size set to narrow at 400 bp. The DNA was multiplexed and amplified after direct size selection. All library preparation and sequencing was performed by the University of Arizona Genetics Core (UAGC). Raw ddRADseq reads were subsequently assembled de novo using ipyrad v.0.6.11 (Eaton & Overcast, 2020). Raw reads were demultiplexed to individuals based on unique barcode sequences, allowing no mismatches in the barcodes.