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More than half a century of research on adaptation in plants has shown that local adaptations are relatively common (Clausen et al., 1940; Linhart & Grant, 1996; Leimu & Fischer, 2008). Adaptive differentiation occurs when selection varies and requires conflicting optimization of plant form and function at different sites (Endler, 1986; Kawecki & Ebert, 2004). Especially givenaccelerating anthropogenic climate change (Bradshaw & Holzapfel, 2006; Parmesan, 2006), it is necessary to improve our understanding of the functional bases of local adaptations (Feder, 2007; Mitchell-Olds et al., 2008; Anderson et al., 2011, 2012). One classic and powerful approach to understanding historical and future adaptation is the study of trait divergence along environmental gradients (Endler, 1986) and this approach has been used successfully in studies of clinal variation in plant traits, including leaf physiology and morphology (Martin et al., 2007), above- and below-ground carbon–nutrient balance (Freschet et al., 2010), phenology and architecture (Petrů et al., 2006). In this study we examine how a suite of life-history, physiological and allocation traits provide an integrated adaptive response to increasing heat and drought during the reproductive season in an annual plant.
Range-wide studies of the model plant Arabidopsis thaliana (Brassicaceae) provide an excellent system for clinal studies of geographically varying adaptation and their genetic bases in annual plants (Mitchell-Olds, 2001; Tonsor et al., 2005; Mitchell-Olds & Schmitt, 2006; Wilczek et al., 2009; Fournier-Level et al., 2011, 2013; Ågren & Schemske, 2012; Grillo et al., 2013). A broad suite of differentiated traits is emerging in studies of adaptation across A. thaliana's latitudinal range. These include variable freezing tolerance (Hannah et al., 2006; Zhen & Ungerer, 2008), vernalization requirements (Hopkins et al., 2008), responses to light quality (Stenøien et al., 2002), heat shock protein expression (Tonsor et al., 2008), growth rate (Li et al., 1998), seed dormancy and season of germination (Kronholm et al., 2012; Montesinos-Navarro et al., 2012) and age at onset of flowering (Stinchcombe et al., 2004; Wilczek et al., 2009).
Populations in northeastern (NE) Spain occur across an altitudinal range from near sea level at the Mediterranean coast to near treeline (c. 2200 m above sea level (asl)) in the Pyrenees mountain range. Along this gradient, low-elevation sites are hotter and drier overall compared with high elevations. Low elevations experience temperatures above freezing for most of the autumn and winter, but a relatively short spring reproductive period with rapid warming and drying. By contrast, high elevations experience periodic below-freezing temperatures and snow cover during the winter, but have a relatively prolonged, cooler and wetter spring reproductive period (Montesinos et al., 2009; Montesinos-Navarro et al., 2011).
Importantly, genetic analyses indicate that the populations in this region are genetically distinct from surrounding regions and are probably descended from a common ancestor (Picó et al., 2008). There are two possible evolutionary genetic causes that could result in clinal trait variation. The first is historic colonization of high- and low-elevation sites by genetically distinct ancestors followed by spread from both ends towards mid-elevations and a subsequent isolation by distance-driven clinal pattern. As we do not detect isolation-by-distance among these populations and gene flow is very low (Montesinos et al., 2009), trait–environment covariation must therefore result from a response to a gradient in natural selection (Montesinos-Navarro et al., 2011).
Shifts in the timing of life-history transitions appear to be an important mechanism of adaptation across this climate gradient (Montesinos et al., 2009; Montesinos-Navarro et al., 2011). Temporal duration of seed primary dormancy, sensitivity of seeds to induction of secondary dormancy by high temperatures (Montesinos-Navarro et al., 2012), probability of germinating in autumn vs spring, and age at bolting (Montesinos-Navarro et al., 2011) vary with climate of origin in our study populations. Under the constant cool, moist, mid-elevation conditions used in Montesinos-Navarro et al. (2011), late-bolting high- and mid-elevation plants exhibited the highest seed production.
High temperatures and low water availability are important stresses for virtually all plants (Parmesan, 2006; Wahid et al., 2007) and water availability and temperature have been proposed as determinants of the geographic range limits of A. thaliana (Hoffmann, 2002, 2005). Therefore, in this study we test for adaptive divergence associated with variation in traits hypothesized to play a role in adaptation to hot, dry conditions. The climate gradient from the Mediterranean coast to near treeline in the Pyrenees compresses much of A. thaliana's range-wide climate gradient into a logistically manageable distance (see the 'Results' section). In particular, the coastal conditions are near the southern environmental limit for A. thaliana. The sites of the coastal populations are especially strongly differentiated from the inland, higher altitude by a rapidly warming and drying spring (Montesinos et al., 2009). We therefore focus particularly on clinal variation expressed under conditions of increasing temperature and decreasing water availability during the reproductive period. We can assess functional significance under our experimental conditions by quantifying the link between fitness and clinally varying traits. Additionally, we can use these relationships to generate hypotheses about fitness consequences of functional variation in the field.
Plants facing drought and increasing temperatures during the reproductive period potentially experience two forms of selection: for stress avoidance and/or for stress tolerance. Heat and drought stress during the spring reproductive season might accelerate A. thaliana's developmental program leading to completion of the life cycle before conditions become unsuitable, thus avoiding stress. Alternatively, A. thaliana populations under reproductive season stress might adapt their physiology, allocation strategy and morphology so as to complete the life cycle in spite of stress, thus tolerating it (Grime, 1977). In this study, we investigate a suite of plant characters that are hypothesized to represent aspects of either avoidance or tolerance of spring heat and drought.
Clinal variation in photosynthetic parameters has yet to be investigated in A. thaliana. Variation in photosynthetic parameters might be expected to be associated with adaptation across the NE Spanish climate gradient for several reasons. First, in response to heat, plants may alleviate heat stress by opening stomata and increasing transpiration (Farquhar & Sharkey, 1982). Alternatively, stomata may be closed for increased water-use efficiency (WUE) (Kalisz & Teeri, 1986; Chaves et al., 2002). Clinal variation in WUE might be expected to coincide with the previously observed cline in bolting time along the Spanish climate gradient (Montesinos-Navarro et al., 2011), as age at bolting has been shown to correlate with WUE in A. thaliana (McKay et al., 2003). Finally, clinal variation in photosynthetic rates has previously been observed both within (Arntz & Delph, 2001) and among (Wright et al., 2004) species.
Montesinos-Navarro et al. (2011) observed clinal variation in allocation in which later-bolting high-elevation plants produced larger rosettes but smaller root systems than did earlier-bolting low-elevation plants. In the face of spring heating and drying, two opposing allocation patterns could hypothetically be beneficial. Plants fitting Grime's (1977) definition of ruderals would shift resources from vegetative to reproductive structures in the face of stress. By contrast, stress-tolerant plants might show increased allocation to vegetative structures, potentially allowing continued survival in the spring (Grime, 1977). At the leaf level, changes in dry mass allocation per unit area (specific leaf area; SLA) are known to vary clinally in A. thaliana (Li et al., 1998) and other species, with leaves from higher elevation or colder climates being thicker. By contrast, hot, dry spring conditions might favor plants with low-investment leaves, as these have frequently been associated with shorter life spans and thus, possibly, with stress avoidance (Wright et al., 2004; Levey & Wingler, 2005).
We present results from a laboratory-controlled environment study of multiple lineages collected from across an elevation gradient in NE Spain. We subjected the experimental population to warming and drying during the reproductive period, placing plants under conditions similar to those in the field at the sites of origin of our low-elevation populations. With this study, we accomplish the following aims: we test for a correspondence between the elevation gradient across which the populations were collected and a major climate gradient; we quantify the extent of adaptive divergence at the genotype level by testing in a common environment for correlations between trait values and elevation- and climate-of-origin; we quantify the fitness effects under our experimental conditions of a suite of traits including leaf-level gas exchange, SLA, photochemical quantum efficiency of photosystem II (PSII), dry mass production of roots, rosettes and inflorescences, and the timing of bolting and fruit ripening under conditions of increasing spring heat and drought.
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To test the extent of adaptation to local climate, we grew plants from across an elevation gradient under dynamic growth chamber conditions that produced an accelerated winter annual life cycle and subjected plants to increasing heat and drought stress during the reproductive season, as is observed at low elevations in the field. We identified significant associations between climate and 10 of the 12 traits investigated. Populations from low-elevation coastal Mediterranean sites developed from germination to seed more quickly than those from high elevations. Low-elevation plants had relatively more mass and time invested in inflorescence structures and less in vegetative growth and rosette photosynthesis than high-elevation plants.
The first PC of climate indicated a gradient of increasing heat and drought with decreasing elevation (Fig. 2). Interestingly, most regression analyses conducted with elevation as the independent variable performed better than those using climate PC1 scores. Indeed, elevation explained 54% of the variance in trait PC1 (not shown) while climate PC1 explained only 36% of trait PC1 variation (Fig. 4). One explanation is that while elevation is accurately measured for each collection site, the climate data used are values averaged over 50 yr interpolated from nearby weather station data (Hijmans et al., 2005) and do not account for local and microclimatic site characteristics. There are other factors that may covary with elevation and contribute to clinally varying selection, including soil, atmosphere and light environments. Additionally, there is a possible influence of the biotic community, which could vary in composition and phenology across the elevation gradient.
In this study, relative adaptedness to spring heat and drought scaled with elevation of origin, favoring low-elevation phenotypes under the imposed warming and drying regime (Fig. 5). Our findings form a complement with those of Montesinos-Navarro et al. (2011), who found, using the same study populations and an overlapping but not identical set of genotypes, that higher-elevation plants outproduced low-elevation plants when spring conditions were cool and wet. The clinal trait and fitness variation observed in this study indicate adaptive divergence as a result of differential selection among sites. It is, however, important to point out that in all cases, the direct agents of selection are not known and neither the conditions nor the developmental sequences or phenotypes of the plants in our chambers perfectly match the conditions or phenotypes seen in the field. Our source populations exhibit repeated bouts of germination during favorable conditions from autumn to spring in the field (Picó, 2008). The life cycle can be as long as 9 months or as short as 50–60 d (Picó et al., 2008). Thus there is no one life cycle or set of seasonal conditions that most accurately describes the patterns of phenotype and selection in the field. Despite this variation in the early phases of the life cycle, all plants in low-elevation populations experience spring heat and drought similar to that imposed in our chambers. It is also possible that the characters measured evolved as a result of indirect selection via correlated traits (Lande & Arnold, 1983). With these caveats in mind, we now consider the relationship among traits, elevation and climate to better understand the phenotypic mechanisms underlying adaptive differentiation across the elevation gradient described earlier.
Low-elevation plants exhibited characteristics consistent with avoidance of heat and drought stress during the spring reproductive season, including faster bolting and fruit ripening. Avoiding stress through shortening the vegetative phase and rapidly shifting resources to the reproductive structures is a key to the ruderal plant strategy described by Grime (1977) and has been observed in Arabidopsis thaliana and other species (Chaves et al., 2002; McKay et al., 2003; Griffith & Watson, 2005; Heschel & Riginos, 2005). We observed that genotypes from lower elevations bolted significantly earlier than genotypes from high elevations. This pattern of earlier bolting at hotter, drier low-elevation sites is in accordance with the range-wide pattern of earlier bolting at lower latitudes (Caicedo et al., 2004; Stinchcombe et al., 2004; Lempe et al., 2005; Wilczek et al., 2009). Earlier flowering was associated with functional shifts in the distribution of biomass among plant parts and in physiological rates.
Low-elevation plants’ distribution of dry mass reflects, in part, their earlier flowering time. Low-elevation plants had smaller rosettes and larger inflorescences relative to those from high elevations (Figs 4, 5). Initiation of primary rosette leaves ceases at bolting, because the primary meristem activity transitions to inflorescence production. This ends or slows accumulation of biomass in the rosette, depending on leaf production by axillary meristems in the rosette short-shoot (Bonser & Aarssen, 2001).
The advantage of earlier flowering in the field and its influence on the ratio of inflorescence to rosette may partly be the result of the distinct thermal niches occupied by these organs. At rosette level, radiated heat from the ground and the associated still air layer lead to significantly warmer conditions when compared with air at the inflorescence level above the ground (Geiger, 1950). Thus early flowering may not just ensure earlier reproduction, but also successful carbon gain during warm, dry spring months and increasing carbon uptake capacity while avoiding further self-shading in the rosette. In support of this hypothesis, Earley et al. (2009) showed that, on average, A. thaliana inflorescences contribute a greater proportion of lifetime carbon gain than rosettes. Earley et al. (2009) also found that the inflorescence had greater WUE than the rosette, which may be advantageous during a hot, dry low-elevation spring. Future studies of lifetime carbon gain and water use by the rosette and inflorescence along climate gradients will provide important functional understanding of variation in flowering time.
Low-elevation plants’ greater inflorescence mass may be explained in part by their greater number of basal inflorescence-forming axillary meristems (Fig. 5). This trait may also allow earlier increase in the number of fruits matured, as for n basal inflorescences, a plant will produce n fruits more or less simultaneously at approximately the same age that an single inflorescence will ripen a solitary first fruit.
One final factor explaining the relationship between elevation and the distribution of above-ground dry mass is variation in senescence and reallocation of rosette resources to the inflorescence. It is possible that maximum rosette mass is greater than the final rosette mass observed for early bolting plants. Earlier bolting may lead to earlier rosette senescence. The adaptive role of nutrient and carbon reallocation during senescence may be particularly important in environments where a rapid decrease in water availability can limit the ability of the plant to acquire nutrients from the soil, maximizing the importance of repurposing of stored nutrients.
Leaves of low-elevation plants had significantly lower Fv/Fm, carbon assimilation and transpiration rates and greater WUE than high-elevation plants (Fig. 5). McKay et al. (2003) found that earlier bolting genotypes of A. thaliana were less water-use-efficient, as evidenced by decreased carbon isotope discrimination. Low-elevation plants that bolt earlier produce much larger inflorescences both overall (Fig. 5) and relative to the rosette (not shown). Inflorescences can contribute greater lifetime carbon gain while being more water-use-efficient than rosettes (Earley et al., 2009). Thus, low-elevation plants in this study may circumvent the expected tradeoff between drought avoidance and tolerance mechanisms observed in McKay et al. (2003). This result is likely to be dependent on the timing of the measurements relative to the life-history stage and the imposition of stress.
Our low values of gas exchange rates and Fv/Fm among low-elevation plants may indicate senescence of the measured leaves. This is in line with indications that low-elevation conditions produce plants that develop more rapidly than their high-elevation counterparts. The leaves we measured showed no visible sign of senescence at the time measurements were taken. However, recent studies of the molecular and physiological underpinnings of senescence indicate that the process itself begins before visible signs appear (Balazadeh et al., 2008).
We conducted gas exchange measurements in the late afternoon to assess the ability of experimental plants to photosynthesize under heat stress. Our results would not necessarily correlate with measurements taken earlier in either the daily or the developmental cycle. Nevertheless, our measures of gas exchange, WUE and quantum efficiency were all strongly related to the elevation of each population, indicating that measured or correlated unmeasured traits played a significant role in adaptation to the environmental gradient.
There was strong selection for high inflorescence masses (Table 2). This is expected, as the greater the inflorescence size, the greater the number of fruits borne. Additionally, there was selection for earlier flowering and shorter time until fruits ripen, which matches the life histories of low-elevation plants. Multiple regression analysis indicates a much stronger relationship between inflorescence mass and fitness, when controlling for correlated traits such as bolting time. Inflorescence mass is correlated with basal branch number (r = 0.52) and rosette dry mass (r = −0.62) neither of which are significant in the selection analysis (Table S6).
Inflorescence mass explained only 63% of the variance in fitness in a univariate regression (results not shown). Indeed, while low-elevation plants produced the most fruit overall, they also produced more fruit length per unit inflorescence mass (result not shown), that is, they exhibited greater mass use efficiency in the production of fruits under the conditions of this experiment. Given that inflorescences contribute significantly to lifetime carbon gain and have greater WUE than vegetative rosette tissue (Earley et al., 2009), they are likely to possess adaptive function above and beyond structurally supporting fruit production, further reflecting the advantage of conserving water via stomatal closure in the rosette while photosynthesizing in the inflorescence.
This study demonstrates variation in relative adaptedness of plants from across a climate gradient to heating and drying during the spring reproductive phase. Low-elevation plants from NE Spain were able to maximize seed production given the short reproductive season we imposed because they bolted early and allocated more to reproductive than to vegetative structures and because they ripened fruit more quickly. We propose that this is a syndrome of avoidance through early flowering accompanied by restructuring of the organism that adapts A. thaliana to low-elevation Mediterranean climates.