Plant sampling and herbivore bioassays
To analyse constitutive leaf GSL production, the 14 species ofCardamine were sampled at the flowering stage, from May until
August, following the natural phenology of the plants. The flowering
stage for sampling was chosen in order to avoid potential ontogenetic
effects on plant chemistry (Barton & Koricheva 2010), since most
species flower very rapidly and for long periods, sometimes throughout
the whole growing season. All plants (n = 10 per species) were collected
directly from the field in each species’ optimal habitat (Aeschimannet al. 2004).
For each plant species, we performed insect resistance bio-assays (N =
four insect species × 10 replicates = 40/plant spp.) with P.
brassicae, S. littoralis, B. brassicae and M. persicae .
To this end, individual plants that were visibly not damaged by
herbivores, and with a minimum distance of 10 m apart, were carefully
excavated, transplanted in cylindrical 20 cm diameter plastic pots by
adding common potting soil where needed (Ricoter AG, Aarberg,
Switzerland), and placed in a climate-controlled chamber (14:10 hrs and
23:15 °C day: night, and 55% relative humidity). For assays with
chewing herbivores, we used one 6-days old caterpillar per plant, while
for sap-feeding aphids we used one adult per plant. For each herbivore
species, we randomly choose two fully-expanded leaves per plant and
placed them in a Petri dish on a filter paper moisten with one drop of
distilled water. After five days of feeding, we estimated plant
resistance against the two different feeding guilds by calculating
larval gain weight for caterpillars using the formula: ln (final fresh
weight - initial fresh weight), and the number of progenies for aphids.
We specifically used detached leaves in order to avoid plant defence
induction due to feeding, since here the aim was to correlate the
measured chemical diversity of GSLs across different species (see below)
with insect resistance.