DNA barcode geographical bias and intra-specific genetic
diversity
The number of Iberian haplotypes recorded in this study added up to 109,
from which 84 (77%) were new for BOLD. In all cases the morphological
identification of the caterpillars was coincident with the closest
species match in BOLD. However, in 3 species the intra-specific K2P
distance with respect any reference barcode exceeded 1% (Table S4),
what is over the maximum BOLD strict threshold for unequivocal species
assignment. After pooling together the DNA sequences available in BOLD
with those obtained from the new specimens sampled in Iberia, the total
number of barcodes scaled up to 652. The new sequences also increased
the number of Iberian localities with DNA barcodes, thereby allowing
more pairwise comparisons with non-Iberian Europe. The
species-by-species comparisons among the Europe-Europe, Europe-Italy,
Europe-Iberia and Iberia-Iberia contrasts (EUEU, EUIT, EUIB and IBIB,
respectively) showed that the inclusion of Iberian samples increased the
intra-specific genetic divergence (not considering spatial distance)
(Table 1). In all cases except one (Orthosia cruda ) the maximum
genetic divergence was higher in EUIB comparisons than in EUEU ones. In
the case of Italy the effect was not so strong, as the genetic
divergence in Europe-Europe and Europe-Italy contrasts (EUEU and EUIT
respectively) was very similar (Table 1). Only in some cases (e. g.Archips xylosteana ) the maximum genetic divergence was higher in
EUIT comparisons than in EUEU ones. The EUEU and IBIB contrasts
retrieved the same divergent results: maximum pairwise intra-specific
genetic divergence between Iberian populations was much higher, and
almost reached 2% in 7 out of 18 species (Catocala nymphagoga,
Peribatodes ilicaria, Ennomos quercaria, Tortricodes alternella,
Watsonalla uncinula, Colotois pennaria and Euproctis chrysorrhoea )
(Table 1). These differences are particularly striking, since the
maximum pairwise spatial distance between populations was lower in
Iberia compared to the rest of Europe (852.63 km vs. 2476.70 km).
The higher intra-specific genetic divergence in the pairwise comparisons
including Iberian populations (EUIB and IBIB) held true also when the
spatial distance was included as a covariate. In the analyses comparing
EUEU vs EUIB contrasts the distance between localities had a significant
positive effect on genetic divergence (Table 3; Fig. 3). However, the
type of contrast (EUEU or EUIB) had an independent and significant
effect too as, for any pairwise spatial distance, the average genetic
divergence was higher if one of the two populations compared was Iberian
(EUIB divergence higher than EUEU) (Table 3; Fig. 3). The same happened
in the analyses of EUEU vs IBIB over a maximum range of 1000 km. The
genetic divergence between Iberian populations (IBIB) was higher than in
the pairwise comparisons between European ones (EUEU) independently of
the effect of the geographical distance between localities (Table 3;
Fig. 4). In the analyses comparing EUEU vs EUIT contrasts, the distance
between localities had a significant positive effect on genetic
divergence too (Table 3; Fig. 3). The effect of including Italian
populations was not as high as in the case of Iberia, but the pairwise
genetic divergence was higher between EUIT contrasts than between EUEU
ones (Table 3; Fig. 3). As expected, the maximum sample size of each
pairwise comparison had a significant effect in all LMMs but, after
controlling for it, the type of contrast remained highly significant in
all cases.