Seed production
To document the effects of population of origin and seed maturation environment on seed dormancy, we grew plants in three different environments: the greenhouse and at the field sites from which two of the sampled populations originated, one located in central Italy (Castelnuovo di Porto 42°07’N, 12°29’E) and one in north-central Sweden (Rödåsen 62°48’N, 18°12’E). To produce plants for the three environments, seeds of five maternal lines from each population (a total of 225 lines) were sown on agar (on 18-21 August 2015 for plants to be grown in the greenhouse and at the Swedish site, and on 15-16 October 2015 for plants to be grown at the Italian site), stratified for 7 days at 4°C in darkness and then moved to a growth room for 8 days (22 °C 16 h light with photosynthetically active radiation [PAR] of 150 μmol m-2 s-1 and 16 °C 8 h dark). Only one line did not succeed in producing seedlings for the transplant. The following five days, emerging seedlings to be grown in the greenhouse were transplanted to 5 × 5 cm pots filled with commercial potting soil (two pots per line; one seedling per pot), whereas plants to be grown at the field sites were transferred to randomized positions in plug trays (299 plugs per tray, plug size 2 × 2 × 4 cm) filled with local soil in Italy and with a mixture of sand, soil and unfertilized peat in Sweden. The three outer rows in each tray (39 positions) were considered edge positions. At each field site, 24 replicates per line were planted in non-edge positions, for a total of 10,800 plants at the two sites.
In the greenhouse, plants were grown in a randomized design at 20 °C 16 h light and 16 °C 8 h dark, and were divided into four cohorts based on their timing of bolting and seed maturation. After 3 weeks, 23 lines (3 Fennoscandian and 20 Italian) had bolted. These plants (cohort 1) were kept in the greenhouse, whereas the remaining plants were moved to a growth room for vernalization. The latter plants were in the growth room exposed to 12 °C 8 h light with PAR of 150 μmol m-2s-1 and 12 °C 16 h dark for one week, 6 °C 8 h light with PAR of 50 μmol m-2 s-1 and 6 °C 16 h dark for six weeks, and then back to 12 °C 8 h light at 150 μmol m-2 s-1 and 12 °C 16 h dark for one week, after which they were returned to the greenhouse. Seeds produced by plants in the first cohort were harvested when the plants had spent a total of 8 weeks in the greenhouse. Seeds produced by the following cohorts were harvested 7 weeks (cohort 2), 8 weeks (cohort 3), and 11 weeks (cohort 4) after return to the greenhouse. At harvest, seeds from the 20 most recently matured fruits of each plant were collected. All collected seeds were stored in paper envelopes under dry conditions in the growth room used for the germination assays (20 °C 16 h and 16 °C 8 h) until germinability was tested.
Plants to be grown in the field were transported to the field sites within six days after the transfer to trays. At the field sites, trays with plants were sunk into the ground (Italy, 7 November 2015; Sweden, 11 September 2015; following procedures outlined in Ågren & Schemske, 2012; Postma & Ågren, 2016), and exposed to natural field conditions until harvest of seeds (Italy, 22-24 April 2016; Sweden, 1-3 July 2016). At the time of transfer to the field, seedlings had produced one-two pairs of true leaves, and were at the same stage of development as naturally establishing seedlings at the experimental sites. At harvest, seeds were collected from plants in non-edge positions.