2.3 Sampling for genetic analyses
In the spring of 2019, we randomly collected leaf tissue from 11–30
individuals in each of the 22 natural populations described above for a
total of 613 individuals. Leaf tissue was dried in silica gel. We
sampled the same number of each floral morph where possible; the sampled
individuals were at least 1–2 meters apart from each other. We
additionally collected leaf tissue and 2–3 capsules with seeds from
nine individuals (four pins and five thrums) of one dimorphic
heterostylous population (D07) and seven individuals from the fully
homostylous population (M01). Collected seeds of each individual were
sowed in a single pot; seedlings from a single individual formed a
family. All pots were placed in water containing gibberellic acid (70
ppm) for 24 hours and later placed in growth chambers at 18 °C with
12:12 h light-dark period. The proportion of germinated seeds per family
was recorded after eight weeks and leaf tissue from a total of ten
seedlings per family was collected whenever possible and dried in silica
gel. DNA of individuals from natural populations and progeny arrays was
extracted with a modified CTAB protocol (Doyle & Doyle, 1987) and used
for all subsequent genetic analyses.