Molecular laboratory work
DNA of 384 individuals of different species and Iberian localities was extracted using commercial extraction kit (EZNA® Tissue DNA Kit) according to manufacturer’s instructions. For all of them, a fragment of the mitochondrial gene COI was amplified using the universal primer pair LCO1490/HCO2198, common in DNA barcoding (see Folmer, Black, Hoeh, Lutz & Vrijenhoek 1994 for details on the primer sequences and PCR protocols). Sequencing was performed using Big-Dye (Perkin-Elmer) technology and an ABI3700 sequencer. We obtained good quality DNA sequences for 375 individuals (21 species), the DNA from the remaining 9 individuals could not be further used due to sequencing failures. Sequence chromatograms were assembled, inspected individually and edited using Sequencher 4.6 (Gene Codes Corp., Ann Arbor, MI, USA). The final alignment length after trimming was 658 bp. To rule out the presence of nuclear mitochondrial insertions (numts) we translated all the sequences into aminoacids using the software MacClade (Maddison & Maddison 2005). After translation we did not find any stop codons indicating the presence of numts (no stop codons can be present in the intronless mitochondrial COI gene). Moreover, there were additional evidences against the presence of numts. In no case the sequences of the same species were paraphyletic and intraspecific genetic divergence was lower than that expected if some of the sequences were numts (Bonal et al . 2018)