Study species, seed sampling and amplification
We focused on Biscutella didyma L., a winter annual Brassicaceae
with Mediterranean and Irano-Turanian distribution (Feinbrun-Dothan &
Danin 1998). It was selected because it is one of few species occurring
commonly across the entire rainfall gradient. As is typical for many
annuals in our study region, B. didyma has short dispersal
distances (Siewert & Tielbörger 2010) and low outcrossing rates (Boazet al. 1990), making gene-flow among manipulated plots unlikely.
Seeds of B. didyma were collected in spring 2012 from twenty
randomly selected individuals within each 10m × 25m plot, i.e. 100 per
rainfall treatment and site. From these seeds, one offspring per
individual was raised with regular watering for one generation in
Tübingen (Germany) to reduce potential parental effects. The growth
period (Nov 2012 – May 2013) approximated the natural vegetation season
and photoperiod, and greenhouse temperatures mirrored day temperatures
in the field sites (15–20 °C in winter, gradually warming to 25–30°C
in spring). Wrapping flowering plants in light, transparent fabric
(organza) ensured self-pollination and seed collection separately per
mother. Of the resulting second-generation seed material, we randomly
picked 40 mothers (hereafter ‘genotypes’) per rainfall treatment and
site (in total 320 genotypes) for the subsequent greenhouse experiment.