77 3.2.3 Molecular analyses Molecular analyses were used to investigate intergenerational connectivity between the RP and the GLS nesting areas. To do that, we tested for population differentiation at both the mitochondrial and nuclear levels (Moritz 1994; Fitzsimmons et al. 1997a). Tissue samples (n = 76) were collected from the RP (n = 38) and the GLS (n = 38). Samples were obtained from females encountered during nesting activities or from dead hatchlings found during nest excavations. All samples were stored in ethanol 96° until laboratory analyses. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN ®, Valencia, CA, USA), following the manufacturer's instructions. A fragment of the mitochondrial control region of approximately 800 bp was amplified (Table 3 SI Appendix) and sequenced using the primers LCM15382 and H950g (Abreu-Grobois et al. 2006). Sequence visualization, edition, and ClustalW alignment were performed with Geneious V7.1.9. Software (Biomatters). Genotyping was conducted using nine previously described polymorphic microsatellite loci for marine turtles: Cc1F01, Cc5F01, Cc7B07, Cc2G10 (Shamblin et al. 2007), Cc117, Cm84, Cm72 (FitzSimmons et al. 1995), Cc141, Cc7 (Bowen et al. 2005) (Table 4 SI Appendix). A substantial optimization step has been achieved to redefine the four PCR thermocycler conditions from original protocols (Table 5 SI Appendix). Allele sizes were assigned using the software Geneious Prime (Geneious Prime ® 2023.2.1). Peaks were visually assessed and bins manually checked. Only those samples that amplified for a minimum of seven microsatellites were retained for further microsatellite analysis. Then pairwise tests for population differentiation (FST) were conducted based on the mtDNA control region and the microsatellite loci using the program Arlequin 3.5 (Excoffier and Lischer 2010). Confidence Intervals (CI) were calculated for FST values by running pairwise analysis of molecular variance analyses with 10.000 permutations using the compute distance matrix with ‘conventional F-statistics’ option for mitochondrial data and ‘number of different alleles (FST-like)’ option for microsatellite data. Departure from the Hardy-Weinberg equilibrium was also tested using Arlequin.
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