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.