Molecular methods
DNA was extracted using the DNEasy Blood & Tissue kit (Qiagen, Hilden,
Germany), quantified using a Qubit 2.0 fluorometer (Life Technologies
Inc., ON, Canada), and subjected to a double digest restriction
associated DNA (ddRAD) sequencing protocol (Peterson et al. ,
2012, Supplement M1). Samples were grouped into pools of 48 individuals
and cleaned with Speedbeads. Each pool was size-selected for fragments
of 300-400 bp in length using a Pippin Prep system (Sage Science,
Beverly, MA, USA). This range should yield approximately 38,000 ddRAD
loci, based on in silico digestion of the C. glareolusgenome sequence (GCA_001305785.1) using SimRAD (Lepais & Weir, 2014).
For each pool, we performed qPCR to determine the optimal number of PCR
cycles based on the onset of the saturation phase on amplification plots
(range: 11-14 cycles) (Gansauge & Meyer, 2013). Pools were then
amplified in four parallel reactions of 40 µl with primers that amplify
only fragments containing both P1 and P2 adapters. The resulting
libraries were sequenced in two separate runs on an Illumina NextSeq 500
with mid-output kits. We first sequenced the libraries with 75 bp
paired-end (PE) and then performed another sequencing run with 150 bp PE
sequencing.