Study system and field sampling
The Heteroceran Lepidoptera (moths) whose caterpillars feed onQuercus spp. leaves were used as study model. There is a high
diversity of lepidopterans linked to the Quercus genus as host
plant. The most abundant oak herbivore families are noctuids,
tortricids, erebids, geometrids and nolids, which start feeding on the
new shoots in early April (Elkinton et al . 2016). The community
of caterpillars also changes during the season, with tortricids the
first to feed on the new shoots and geometrid species the last (Soria
1988). Herbivory damage on oak differs among years too, being able to
have outbreaks under specific conditions of temperature and climate
(Schroeder & Degen 2008). These insects are widely spread over Europe
and have been recorded feeding on different oak species (Soria 1988;
Führer 1998). The number of DNA barcodes available for the southern
European peninsulas (Balkans, Italy and Iberia) at public repositories
was very low compared with the rest of continental Europe (see below).
Thus, we carried out a field campaign in Spain to reduce such an
imbalance. We sampled 10 oak forests along a latitudinal gradient (Table
S1; Fig. 1) from April to June 2017, the period of maximum activity of
oak defoliating caterpillars in the Iberian Peninsula (Soria 1988). Oak
branches were shaken and the falling caterpillars collected on a white
cloth of a fixed surface placed beneath (see Ruiz-Carbayo, Bonal,
Espelta, Hernández & Pino 2017 for a detailed description of the
sampling methodology). The caterpillars collected at each oak were
placed within a plastic box and taken to the laboratory, where they were
housed individually in Petri dishes and fed with fresh oak leaves.
Caterpillars were identified to species level based on morphological
characters following guides and dichotomous keys (Gómez de Aizpurúa
2002; Gaytán, Canelo, González-Bornay, Pérez-Izquierdo & Bonal 2018).
In those few cases in which the caterpillar could not be identified it
was raised to the adult stage and then identified using guides (Fibiger
1997; Goater, Ronkay & Fibiger 2003; Sihvonen & Skou 2015). In total
21 species of 9 families were collected (Table S2). All specimens
(caterpillars and adults) were stored in 1.5 ml Eppendorf tubes filled
with 96% alcohol for further molecular analyses.