2.1 Sampling, extraction and PCR amplification of gDNA
A total of 276 birds (all adults, i.e. having already dispersed) spanning the entire known breeding distribution of Puffinus lheminieri and four populations of interest of P .bailloni were selected (Supplementary Material 1). Four individuals from the Pacific Ocean (taxon dichrous , by far the most widespread lineage in the Pacific (Onley & Scofield 2007); Fig. 1) were also used. Individuals were sexed using PCR amplification (with the 2250F and 2781R primers (Fridolfsson & Ellegren 1999)). The overall sex ratio was unbiased (120 females, 107 males; Pearson’s Chi² with Yates’ continuity correction, p=0.42; 49 individuals could not be sexed successfully, see Supplementary Material 1).
Total genomic DNA was extracted from blood samples (except for the population of the Bahamas, for which samples were derived from toepads collected on dead birds, lherminieri 1 and 2 on Figure 1) using NucleoSpin® Tissue XS Kit (Macherey & Nagel, Düren, Germany). Samples were incubated overnight in 4 mg of Proteinase K. Purified genomic DNA was eluted twice in 50 µL of TE buffer pre-heated at 70°C. DNA concentration was measured using Nanodrop spectrophotometry. Three mitochondrial markers (cox1 , cytb , and the mitochondrial Control Region, CR) and six nuclear markers (Beta-fibrinogen exon6 through 8, βfib ; Cold Shock Domain-containing E1 intron5,csde ; Interferon Regulatory Factor 2 intron2, irf2 ; PAX interacting protein 1 intron20, pax ; Recombination activating protein 1, rag1 ; and Tropomyosin 1 alpha exon7, tpm ) were targeted (primer sequences, PCR profile and conditions: Supplementary Material 2). These markers were previously shown to be polymorphic within and between petrel species (Gangloff et al. 2013, Silva et al. 2011).