DOI: 10.5061/dryad.np5hqbzx9
title: Effects of warming temperatures on germination responses and trade-offs between seed traits in an alpine plant

description: 1. Climate warming may affect multiple aspects of plant life history,
 including important factors such as germination responses and the key
 trade-off between offspring size and number. As a case study to address
 these concepts, we used an alpine plant (waxy bluebell, Wahlenbergia
 ceracea; Campanulaceae) that shows plasticity to warming in seed traits
 and in which seed dormancy status regulates germination. We chose an
 alpine species because alpine environments are ecosystems particularly
 under threat by climate change. 2. We conducted germination assays under
 cool and warm temperatures using seeds produced by individuals that were
 grown under historical (cooler) and future (warmer) temperature scenarios.
 We assessed the presence of a seed size vs number trade-off, and then
 examined the effects of seed number and size on germination percentage,
 the fractions of dormant and viable seeds, and germination velocity.
 Further, we examined whether warming during parental growth and during
 germination affected these relationships. 3. We found evidence for a seed
 size vs number trade-off only under historical parental temperatures.
 Indeed, under future growth temperatures, parental plants produced fewer
 and smaller seeds and there was no evidence of a trade-off. However, the
 reductions in both seed traits under warming did not affect germination,
 despite correlations of seed size and number with germination traits.
 Warming increased germination, particularly of larger seeds, but overall
 it resulted in more than fourfold reductions in parental fitness. 4.
 Synthesis. Our study shows the importance of growth conditions when
 evaluating the seed size vs number trade-off. Stressful conditions, such
 as warmer temperatures, can restrain the ability of plants to reach
 optimal investment in reproduction, masking the trade-off. By analysing
 responses across the whole life cycle, we show here an overall detrimental
 effect of warming, highlighting the potential risk of climate change for
 W. ceracea, and, potentially, for alpine plant communities more widely.
description: Experiments were conducted using the plant species
 <em>Wahlnebrgia</em> <em>ceracea</em> (waxy
 bluebells). Two datasets were used in this manuscript.
 1) Seed size vs number trade-off: Parental individuals from a
 total of 30 lines ('Line') were grown in growth chambers for 191
 days under temperature conditions of a historical/cooler (1960–1970) or a
 projected future/warmer (2090–2100) climate
 ('Parental_Temperature'). The parental individuals were randomly
 assigned to one of three blocks, which corresponded to positions inside
 the chambers, and each block was equivalent in all chambers
 ('Block'). Day and night temperatures during the experiment were
 changed every 15 days to mimic seasonality, with the maximum day
 temperatures during the peak of summer being 24°C and 29°C for the
 historical and future parental temperatures, respectively. After 100 days
 since the imposition of the temperature treatments (during the peak of the
 summer), half of the plants were moved for 5 days to new chambers, where
 the temperature was 5°C above the respective treatments, i.e., 29°C and
 34°C ('Heatwave'). After this time, the parental individuals
 were moved back to their respective historical or future temperature
 treatments. We collected the seeds throughout the 191 days of parental
 growth, and we stored them in desiccators for at least 11 weeks. After
 this time, we calculated seed size ('Seed_Size') as the average
 mass of three lots of 50 seeds divided by 50. We calculated seed number
 ('Seed_Number') as the ratio between the cumulative mass of the
 seeds produced by each parental individual and seed size. The 30 lines of
 the parental individuals were obtained by crossing plants that originated
 from seeds that were collected at the same elevation, either high or low
 elevation ('Elevation') in sites within Kosciuszko National
 Park, NSW, Australia. Therefore, 14 lines originated from high elevations
 and 14 lines from low elevations. 2) Germination
 responses - seed traits correlations: The seeds were harvested from the
 parental individuals grown under historical/cooler or projected
 future/warmer temperatures ('Parental_Temperature') (see above)
 from a subset of 14 lines ('Line'). These seeds were used in
 germination assays in the glasshouse under cool (25°C) or warm
 temperatures (30°C) ('Germination_Temperature'). We measured
 seed size ('Seed_Size') as the average mass of three lots of 50
 seeds; then these seeds were sowed in agar dishes (25 seeds per dish, 2
 dishes per temperature treatment from each parental individual). Seed
 number (‘Seed_Number’) was the same as above. Dishes were left under
 temperature treatments for 4 weeks to allow germination of the non-dormant
 fraction of the seeds ('Not_Dormant_Seeds') and germination was
 checked once per week. Then, all the dishes were moved to a cold room at
 4–5°C in the dark for 4 weeks to allow cold stratification. After this
 time, dishes were moved back to the glasshouse under the same temperature
 treatments as before to allow germination of the dormant seeds. We
 considered seeds to be dormant ('Dormant_seeds') if they
 germinated during or after cold stratification or if they did not
 germinate at all but were still determined to be viable at the end of the
 experiment. We considered seed to be viable (‘Viable_Seeds’) if they
 germinated (‘Germinated_Seeds’) as well as the seeds that contained an
 endosperm but still did not germinate (‘Not_Germinated_Seeds’), while we
 considered empty seeds as non-viable (‘Not_Viable_Seeds’). Germinated and
 not germinated seeds (as above) were used to calculate the germination
 percentage. We calculated germination velocity (‘Germination_Velocity’) as
 the reciprocal of the mean germination time (germination velocity
 (germination (%) week-1) GV = (G1 + G2 +…+ Gn) / (G1 x T1 + G2 x T2 +…+ Gn
 x Tn), where Gn is the number of new germinating seeds at each sampling
 point, and Tn is the time between each sampling point (= one
 week). The files provided present the datasets in their
 first sheet and keys with the definitions of each term in the second
 sheet.
description: Files can be opened using Excel and analysed using R.