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.