How to analyse plant phenotypic plasticity in response to a changing climate

Plant biology is experiencing a renewed interest in the mechanistic underpinnings and evolution of phenotypic plasticity that calls for a re‐evaluation of how we analyse phenotypic responses to a rapidly changing climate. We suggest that dissecting plant plasticity in response to increasing temperature needs an approach that can represent plasticity over multiple environments, and considers both population‐level responses and the variation between genotypes in their response. Here, we outline how a random regression mixed model framework can be applied to plastic traits that show linear or nonlinear responses to temperature. Random regressions provide a powerful and efficient means of characterising plasticity and its variation. Although they have been used widely in other fields, they have only recently been implemented in plant evolutionary ecology. We outline their structure and provide an example tutorial of their implementation.This research was supported by the Australian Research Council (DP170101681)

Germination at extreme temperatures : implications for alpine shrub encroachment

Worldwide, shrub cover is increasing across alpine and tundra landscapes in response to warming ambient temperatures and declines in snowpack. With a changing climate, shrub encroachment may rely on recruitment from seed occurring outside of the optimum temperature range. We used a temperature gradient plate in order to determine the germination niche of 14 alpine shrub species. We then related the range in laboratory germination temperatures of each species to long-term average temperature conditions at: (1) the location of the seed accession site and (2) across each species geographic distribution. Seven of the species failed to germinate sufficiently to be included in the analyses. For the other species, the germination niche was broad, spanning a range in temperatures of up to 17 ◦C, despite very low germination rates in some species. Temperatures associated with the highest germination percentages were all above the range of temperatures present at each specific seed accession site. Optimum germination temperatures were consistently within or higher than the range of maximum temperatures modelled across the species’ geographic distribution. Our results indicate that while some shrub species germinate well at high temperatures, others are apparently constrained by an inherent seed dormancy. Shrub encroachment in alpine areas will likely depend on conditions that affect seed germination at the microsite-scale, despite overall conditions becoming more suitable for shrubs at high elevations

Effects of warming temperatures on germination responses and trade-offs between seed traits in an alpine plant

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.Files can be opened using Excel and analysed using R.Funding provided by: Australian Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000923Award Number: DP170101681Experiments were conducted using the plant species Wahlnebrgia ceracea (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

Tolerance of warmer temperatures does not confer resilience to heatwaves in an Alpine herb

Climate change is generating both sustained trends in average temperatures and higher frequency and intensity of extreme events. This poses a serious threat to biodiversity, especially in vulnerable environments, like alpine systems. Phenotypic plasticity is considered to be an adaptive mechanism to cope with climate change in situ, yet studies of the plastic responses of alpine plants to high temperature stress are scarce. Future weather extremes will occur against a background of warmer temperatures, but we do not know whether acclimation to warmer average temperatures confers tolerance to extreme heatwaves. Nor do we know whether populations on an elevational gradient differ in their tolerance or plasticity in response to warming and heatwave events. We investigated the responses of a suite of functional traits of an endemic Australian alpine herb, Wahlenbergia ceracea, to combinations of predicted future (warmer) temperatures and (relative) heatwaves. We also tested whether responses differed between high- vs. low-elevation populations. When grown under warmer temperatures, W. ceracea plants showed signs of acclimation by means of higher thermal tolerance (Tcrit, T50, and Tmax). They also invested more in flower production, despite showing a concurrent reduction in photosynthetic efficiency (Fv/Fm) and suppression of seed production. Heatwaves reduced both photosynthetic efficiency and longevity. However, we found no evidence that acclimation to warmer temperatures conferred tolerance of the photosynthetic machinery to heatwaves. Instead, when exposed to heatwaves following warmer growth temperatures, plants had lower photosynthetic efficiency and underwent a severe reduction in seed production. High- and low-elevation populations and families exhibited limited genetic variation in trait means and plasticity in response to temperature. We conclude that W. ceracea shows some capacity to acclimate to warming conditions but there is no evidence that tolerance of warmer temperatures confers any resilience to heatwaves.This research was supported by the Australian Research Council (DP170101681), an International Ph.D. Scholarship to RN and an ARC Future Fellowship FT110100453 to LK. Research grants funded all research related costs (such as renting growth chambers or buying equipment), while the scholarship paid a stipend to RN

The influence of leaf size and shape on leaf thermal dynamics: Does theory hold up under natural conditions?

Laboratory studies on artificial leaves suggest that leaf thermal dynamics are strongly influenced by the two-dimensional size and shape of leaves and associated boundary layer thickness. Hot environments are therefore said to favour selection for small, narrow or dissected leaves. Empirical evidence from real leaves under field conditions is scant and traditionally based on point measurements that do not capture spatial variation in heat load. We used thermal imagery under field conditions to measure the leaf thermal time constant (τ) in summer and the leaf-to-air temperature difference (∆T) and temperature range across laminae (Trange) during winter, autumn and summer for 68 Proteaceae species. We investigated the influence of leaf area and margin complexity relative to effective leaf width (we), the latter being a more direct indicator of boundary layer thickness. Normalized difference of margin complexity had no or weak effects on thermal dynamics, but we strongly predicted τ and ∆T, whereas leaf area influenced Trange. Unlike artificial leaves, however, spatial temperature distribution in large leaves appeared to be governed largely by structural variation. Therefore, we agree that small size, specifically we, has adaptive value in hot environments but not with the idea that thermal regulation is the primary evolutionary driver of leaf dissection.This work was supported by an Australian Geographic research grant and an Australian Postgraduate Award to A. Leigh; and by an Australian Research Council grant A00103546 to A.B. Nicotra

Effects of initial planting density on branch development in 4-year-old plantation grown Eucalyptus pilularis and Eucalyptus cloeziana trees

The effect of planting density on branch development was examined in 4-year-old Eucalyptus pilularis Sm. and Eucalyptus cloeziana F. Muell. plantations located near the coast of north-eastern NSW. Branch diameter, angle and status (live or dead) were measured along the entire stem of trees established at 1250, 1667 and 3333 stems per hectare (sph). Measurements of tree height and stem diameter at breast height over bark (DBH) were also recorded. Results showed that with an increase in initial planting density from 1250 to 1667 sph, branch size decreased, branch mortality on the lower stem increased, branch angle became more acute and DBH decreased in trees of both E. pilularis and E. cloeziana. A further increase in initial planting density from 1667 to 3333 sph did not significantly reduce branch size or branch angle but did result in increased branch mortality and decreased DBH in both species. These results suggest that increasing initial planting density from 1250 to 1667 sph will improve early branch control. However, there is no advantage in establishing trees at 3333 sph rather than 1667 sph to reduce branch size or increase branch mortality in either species. Clearwood production on the lower stem in all stocking treatments of both species was negligible at age 4

Predicting effects of warming requires a whole-of-life cycle perspective: A case study in the alpine herb Oreomyrrhis eriopoda

Global warming is affecting plant phenology, growth and reproduction in complex ways and is particularly apparent in vulnerable alpine environments. Warming affects reproductive and vegetative traits, as well as phenology, but seldom do studies assess these traits in concert and across the whole of a plant's life cycle, particularly in wild species. Thus, it is difficult to extrapolate from such effects to predictions about the persistence of species or their conservation and management. We assessed trait variation in response to warming in Oreomyrrhis eriopoda, an Australian native montane herb, in which populations vary in germination strategy (degree of dormancy) and growth characteristics as a function of ecological factors. Warming accelerated growth in the early stages of development, particularly for populations with non-dormant seed. The differences in growth disappeared at the transition to reproduction, when an accelerating effect on phenology emerged, to varying degrees depending on germination strategy. Overall, warming reduced flower and seed production and increased mortality, indicating a reduction in reproductive opportunities, particularly for populations with dormant seed. Developmental condition affected germination strategy of the next generation seed, leading to increased degree of dormancy and slowed germination rate. But there were no whole-scale shifts in strategy or total germination percent. Following through the life cycle reveals that warming will have some potentially positive effects (early growth rates) and some negative effects (reduced reproductive output). Ultimately, warming impacts will depend on how those effects play out in the field: early establishment and an accelerated trajectory to seed maturity may offset the tradeoff with overall seed production. Small differences among germination strategies likewise may cascade to larger effects, with important implications for persistence of species in the alpine landscape. Thus, to understand and manage the response of wild species to warming takes a whole-of-life perspective and attention to ecologically significant patterns of within-species variation.A.S. was supported by an Australian Government Research Training Program Scholarship

A high-throughput method for measuring critical thermal limits of leaves by chlorophyll imaging fluorescence

Plant thermal tolerance is a crucial research area as the climate warms and extreme weather events become more frequent. Leaves exposed to temperature extremes have inhibited photosynthesis and will accumulate damage to PSII if tolerance thresholds are exceeded. Temperature-dependent changes in basal chlorophyll fluorescence (T-F0) can be used to identify the critical temperature at which PSII is inhibited. We developed and tested a high-throughput method for measuring the critical temperatures for PSII at low (CTMIN) and high (CTMAX) temperatures using a Maxi-Imaging fluorimeter and a thermoelectric Peltier plate heating/cooling system. We examined how experimental conditions of wet vs dry surfaces for leaves and heating/cooling rate, affect CTMIN and CTMAX across four species. CTMAX estimates were not different whether measured on wet or dry surfaces, but leaves were apparently less cold tolerant when on wet surfaces. Heating/cooling rate had a strong effect on both CTMAX and CTMIN that was species-specific. We discuss potential mechanisms for these results and recommend settings for researchers to use when measuring T-F0. The approach that we demonstrated here allows the high-throughput measurement of a valuable ecophysiological parameter that estimates the critical temperature thresholds of leaf photosynthetic performance in response to thermal extremes.This research was supported by the Australian Research Council (DP170101681)