Preparation of databases of DNA barcodes
Sequences of the 375 individuals collected during our field sampling
were queried in BOLD search engine (Ratnasingham & Hebert 2007) to
double-check the species identity in those specimens previously
determined according to morphology. We used the option All Barcode
Records in BOLD, as all our sequences were longer than 500bp and doing
so the query sequences are compared with a larger number of reference
barcodes. Besides, we mined BOLD searching for COI DNA barcodes of the
same species that we had sequenced in the Iberian Peninsula; in total we
could download 277 public sequences (available in January 2018), which
were used in further analyses (see Supplementary material section for
BOLD process IDs).
The final database pooling the sequences downloaded from BOLD with the
new Iberian sequences added up to 652 COI barcodes: 21 taxa from 136
localities in 14 European countries (Fig. 1). For the data downloaded
from BOLD, we considered two sequences to come from the same locality
when the name for that field site was identical in the database or when
the coordinates showed that the specimens had been collected within a
distance lower than 5 kilometres. We chose this threshold because, given
that these species of oak feeding moths are not migratory and have
limited dispersal abilities (Ruíz-Carbayo et al. 2017),
individuals within this range may belong to the same population.