2.2. Pollen limitation
Supplemental hand-pollination was applied on randomly selected
individuals (20-30 per species) with at least two open flowers in
similar phenological phases. One flower was supplemented by conspecific
pollen from plants minimally two meters apart to reduce the genetic
closeness and the second was left to natural pollination as a control.
Both hand-pollinated and control flowers were marked by coloured cotton
yarn loosely knotted under the flowers. In species producing just a
single flower (Anemone nemorosa ) or compact inflorescences
(Bistorta major ) on a single shoot, we applied the treatments on
two neighbouring individuals. In Asteraceae species (Crepis
paludosa and Tephroseris crispa ) the treatments were applied on
two whole capitula, and in Apiaceae species with compound umbels
(Aegopodium podagraria and Chaerophyllum aromaticum ) the
treatments were applied on two umbellets from two different umbels. All
species with flowers in compact and sequentially opening inflorescences
(i.e. Aegopodium podagraria , Bistorta major ,Chaerophyllum aromaticum , Crepis paludosa andTephroseris crispa ) were hand-pollinated repeatedly for several
consecutive days throughout the whole flowering period. After the marked
flowers wilted, their maturing ovaries were enclosed in fine nylon mesh
bags to avoid any seed loss. We counted all viable seeds from the
collected flowers or inflorescences and measured mean weight per seed.
Because Dactylorhiza majalis produced numerous very small seeds,
we used the capsule weight as a proxy for the number of developed seeds.