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).