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)