Shifts in vernalization and phenology at the rear edge hold insight into the adaptation of temperate plants to future milder winters

Abstract

Introduction

Organisms exposed to cyclical environmental changes often evolve mechanisms to sense these fluctuations and to time developmental shifts to occur under favorable conditions (Preston & Sandve, 2013). In temperate plants, vernalization, the prolonged exposure to nonlethal seasonal cold (Chouard, 1960), serves as an important cue so that key life-cycle transitions will occur after winter, for example vegetative growth and reproduction (Amasino, 2005). However, relying on such cues may be detrimental under rapidly changing environments. This is of particular concern in the context of ongoing global warming, as it is expected to affect the strength and timing of seasonal temperatures experienced by natural and agricultural species (Willis et al., 2008; Blackman, 2017). Species requiring vernalization will likely be negatively affected by climate warming as shorter and milder winters become more common (Luedeling et al., 2011; Anderson, 2023). Such conditions are expected to lead to a reduction in reproduction (Padhye & Cameron, 2009; Liu et al., 2012; Satake et al., 2013) or shifts in reproductive phenology outside of optimal time windows (Fitter & Fitter, 2002; Parmesan & Yohe, 2003; Love & Mazer, 2021; Faidiga et al., 2023; Geissler et al., 2023). It is therefore critical to understand the response to seasonal cuing, and variation in this response, to determine possible consequences of expected warmer climates for temperate plant species.

Vernalization requirements and cue responses often vary within and between species in response to differences in winter conditions (Andrés & Coupland, 2012; Blackman, 2017; Preston & Fjellheim, 2022). This suggests that while these mechanisms are crucial to complete the life cycle, they vary enough to allow persistence across heterogeneous environments. Studies have explored variation in cueing reproduction across environments by testing for clines in vernalization requirements and phenological response to temperature gradients (Blackman, 2017). This body of work provides evidence of a reduction in vernalization requirements with increases in temperature (Wesselingh et al., 1994; Dijk et al., 1997; Boudry et al., 2002; Stinchcombe et al., 2005; Jokela et al., 2015). Reproductive phenology also varies over temperature gradients though a general pattern is less clear. For example, flowering is later with the later arrival of spring-like conditions at high latitudes than at lower ones in Arabidopsis thaliana (Stinchcombe et al., 2004; Lempe et al., 2005). By contrast, earlier flowering occurs at higher latitudes in other species (e.g. Paccard et al., 2014; Vest & Sobel, 2021). These studies reveal the potential for both the mechanism of environmental cuing as well as the pattern of reproductive phenology to evolve to track warming climates.

Populations at the warmer limits of temperate species ranges are particularly important for understanding adaptation to shorter winters. This part of the range, colloquially known as the ‘rear edge’ (Hampe & Petit, 2005), often consists of relict populations that have persisted more or less in place at least since the last glacial maximum (LGM, c. 23 000–19 000 yr ago; Hughes et al., 2013). Long-term persistence at the warmer range limits over multiple glacial cycles and under continuously warming climates after the LGM has likely resulted in adaptation to warmer climates (Hampe & Petit, 2005). With this history, the rear edge may provide insight into how species requiring vernalization adapt to mild winters, and which adaptations may be best suited to future warming climates. However, these populations are typically not sampled in studies investigating clines, and therefore our knowledge of phenology and vernalization requirements in populations from the warmest habitats is limited (but see Vest & Sobel, 2021).

Here we investigate whether rear-edge populations show patterns of differentiation consistent with adaptation to milder winters compared to populations from elsewhere in the range in the North American herb Campanula americana. This species requires vernalization but occupies a wide latitudinal and temperature gradient (Fig. 1a), with ancestral rear-edge populations occurring in distinctively warmer climates in the southern parts of the range (Barnard-Kubow et al., 2015). To test for differentiation between the rear edge and the rest of the range, we first characterized differences in flowering phenology across latitude using range-wide observations in natural populations gathered from citizen science data and then tested whether this variation reflects differences in winter or growing season climate (observational study). We also assessed genetic differences in phenology by testing for variation in flowering among populations from across latitudes raised under common glasshouse conditions (Experiment 1). We then evaluated plasticity in reproductive phenology in response to a range of natural growing season cues by raising populations sampled across latitudes in common gardens (CGs) along a latitudinal gradient (Experiment 2). Finally, we tested differences in vernalization requirement and phenological plasticity in response to these winter cues by raising populations under experimentally manipulated vernalization length (Experiment 3). In total, these four approaches allow us to comprehensively test for differences in phenology and its regulation across the range of this species. The results of these experiments allow us to understand not only how rear-edge populations have adapted to their distinct habitats, but also shed light on how populations across the range vary in their response to milder winter, and ultimately the adaptation that may evolve to allow persistence under future conditions.

Fig. 1

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Variation in flowering phenology observed in nature and associated climatic factors across latitudes (observational study). (a) Location of Campanula americana observations (black dots) selected from iNaturalist 2018–2022. The range of the species is outlined with a gray line; red arrows represent the latitudinal delimitation of the rear edge. Shading indicates the latitudinal temperature gradient based on 30 yr minimum temperature in January (1991–2020) from Prism climate data (https://prism.oregonstate.edu). (b) The day of first flower inferred for observations in (a). (c) The length of vernalization and (d) the time between the start of the growing season and flowering inferred for each observation from climate data. Colors represent whether observations occurred below (red) or above (black) the identified breakpoint (34.98°N, dashed vertical lines) in the latitudinal cline on the day of first flower. Solid lines represent the significant model-predicted slope of the relationship between each variable and latitude, respective to the breakpoint, with the 95% confidence interval indicated as shading. Test statistics are reported in Table 1.

Table 1. Test of variation in the day of first flower, length of vernalization and time between the start of the growing season and flowering across the latitudinal range of Campanula americana relative to a predicted breakpoint.

Dependent variable	Latitude (L)			Breakpoint (BP)		L × BP		$$	$$
	β < BP	β > BP	χ2	β	χ2	β	χ2
Day of first flower	−5.96	1.88	23.59***	−273.10	39.52***	7.85	37.84***	0.18	0.18
Length of vernalization	2.63	10.75	13.57***	−289.84	130.99***	8.12	119.59***	0.93	0.95
Time between flowering and the start of the growing season	−15.58	−1.37	107.87***	−493.68	85.97***	14.25	82.81***	0.46	0.54

• All dependent variables were assumed to follow Gaussian distributions. Each model was optimized with the bobyqa optimizer to improve convergence. Test statistics include the slope of each effect (β), the chi-squared value and the R² of the model, considering fixed effects only (marginal, $$), or both fixed and random effects (conditional, $$)). For the effect of latitude, β was estimated for the parts of the range below or above the predicted breakpoint (BP) in latitude (34.98°N). ***, P < 0.001. Bonferroni corrections were applied for P-values of each fixed effect for the day of first flower and the time between flowering and the start of the growing season as they are nonindependent, but significance thresholds did not change. Results for random effects are not shown.