Study material and experimental design
The northern populations of V. alpina came from
Scandinavia (Oppland, Norway and Västerbotten, Sweden; hereafter
referred to as NOR and SWE, respectively ; Fig. 1), while the
southern populations came from two Italian mountain regions (Monte
Sobretta, Central Alps and Monte Prado, Northern Apennines; hereafter
referred to as SOB and PRA, respectively; Fig. 1). The four provenance
sites differed in terms of climatic conditions besides latitude and
corresponding location within the species’ range: NOR and SOB were
colder sites, while SWE and PRA were warmer sites (Abeli et al. 2015;
Fig. 1). In summer 2012, seeds of V. alpina plants were collected
at each site. The plants were cultivated at the Botanical Garden of the
University of Pavia, at approximately 85 m above sea level, and produced
seeds for the first time in 2015. Seeds of all populations were
harvested and kept under standard seedbank conditions (-20°C after
drying at 15%RH, 15°C, FAO 2013) until used for cultivation in
controlled conditions. Leaves of six to eight mature plants per
population were harvested and dried in silica gel for subsequent
molecular analyses.
In 2016, the seeds were sown in Petri Dishes filled with 1%
Agar in distilled water + 125 mg/L of Gibberellic Acid
(GA3) and incubated at 20°C in light- and
temperature-controlled incubators. Because the northern and the southern
populations of V. alpina differed in germination timing and
dormancy levels (Mondoni et al. 2018), we used GA3 to
obtain synchronous germination and avoid different developmental stages
at the beginning of the growth chamber experiment. The selected amount
of GA3 proved to synchronise germination without
inducing deformities in the seedlings. Soon after radicle emission, 120
seedlings per population were transferred each into a 3-cm plastic pot
with standard soil (Hochmoor substrate, Terflor®, Italy) and assigned to
two treatments (60 plants per treatment in 20 replicates of three plants
each), using a light- and temperature-controlled growth chamber
(ClimaCell®, MMM Group, Germany) as detailed below. The base treatment
simulated the average summer temperature at the warmest southernmost
site (PRA) based on temperature measured with a datalogger (MLog5W,
Geoprecision, Germany) placed in that site from 30 August 2013 to 24
July 2015. The extreme treatment simulated the temperature values
recorded by the same datalogger at PRA during a summer heat wave in July
2016. In particular, the extreme treatment differed from the base
treatment for two day-temperature peaks of 22°C at dawn and
30°C during the day, respectively. Both temperature peaks lasted one
hour (Fig. 2). In both treatments the plants were exposed to