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.