DISCUSSION
Our overall results demonstrate rapid evolution in three out of ten traits under in situ climate manipulations in natural plant communities after merely 10 years, i.e. at most 10 generations of our annual study species. This is a remarkably short time span, given that numerous interacting factors may hamper evolution in natural communities (Hoffmann & Sgró 2011; Shaw & Etterson 2012). The fact that this evolutionary response was consistent in two independent sites renders chance effects, e.g. genetic drift, unlikely to have affected these results and underpins that the evolutionary response was directly driven by manipulated rainfall. Intriguingly, our multiple independent lines of evidence corroborate that these changes were adaptive.
After 10 years of artificial drought, phenology had evolved in chronological time (days to flowering) and ontogenetic time (leaf number at flowering). Theory suggests accelerated life-cycles as a drought avoidance strategy that reduces the risk of mortality before reproduction (Cohen 1976; Kigel et al. 2011). Yet, early reproduction comes at the cost of smaller plant size and hence possibly lower competitive ability (Liancourt & Tielbörger 2009; Kigel et al. 2011). In line with theory, plants from dry-manipulated plots flowered earlier and with fewer leaves than plants from control and wet plots. Moreover, this rapid evolutionary response paralleled the long-term evolutionary response of B. didyma along the natural rainfall gradient where plants from more arid sites flowered earlier; a trend found in many other annuals along natural rainfall gradients (Stinchcombe et al. 2004; Kigel et al . 2011; Wolfe & Tonsor 2014; Kurze et al . 2017). Interestingly, the observed 3-4 days acceleration in phenology corresponds to an ecological distance ofc. 100 mm lower rainfall at origin for annuals along our study gradient (Kigel et al . 2011; Kurze et al. 2017). Given the magnitude of rainfall reduction in dry plots (-90 mm in SA, -160 mm in M), this suggests that a substantial part of the ‘required’ acceleration in phenology could be realized within ten years. The adaptivity of accelerated phenology under drought was furthermore corroborated by our selection analyses under controlled watering conditions in the greenhouse, which eliminated potentially confounding factors along natural environmental gradients (Mitchell-Olds & Schmitt 2006, De Frenne et al. 2013). Here, earlier flowering with fewer leaves was stronger favored under low than under high water availability. These multiple lines of evidence – theory, natural rainfall gradient, selection analyses, and consistency in both sites – provide compelling evidence that the observed rapid evolution in phenology was adaptive.
Rapid evolution of earlier flowering under drought was found also in other climate change studies; it is thus far the trait most often reported to evolve under drought in natural conditions (Franks et al. 2007; Vigouroux et al . 2011; Nevo et al. 2012; Nguyenet al. 2016). Accelerated phenology therefore emerges as a key pathway for rapid evolutionary adaptation to drier climates. While this underpins the central role of phenology for drought adaptation in annuals (Cohen 1976; Kigel et al. 2011; Kurze et al.2017), it may also signpost that phenology evolves easier than other, possibly more complex traits. However, this conclusion is still hampered by the few tests beyond our study reporting multiple traits besides phenology (Ravenscroft et al. 2014; Nguyen et al. 2016).
Here, we also observed rapid evolution in reproductive allocation. As competition is reduced in drier sites along our gradient (Schiffers & Tielbörger 2006), theory suggests reduced investment in vegetative tissue for outgrowing neighbors and increased allocation to reproduction (Aronson et al. 1990; 1993). In line with theory and in both sites, plants from dry manipulated plots produced 10-15% more seeds per vegetative biomass than control plants. Although reproductive allocation was rarely assessed in climate manipulation studies, a similar evolutionary tendency was reported for a perennial herb (Ravenscroftet al . 2014). This evolutionary response was again congruent with our selection analyses in the greenhouse, and it matched the clinal trend in reproductive allocation along our natural rainfall gradient, and parallel clines in other species (summarized in Kurze et al.2017). Thus, in all traits showing rapid evolution in the field, our independent lines of evidence demonstrate that these changes were adaptive. Intriguingly, parallel studies had reported remarkable resistance in many plant community parameters to imposed climate manipulations (Tielbörger et al . 2014; Bilton et al . 2016). The present findings highlight that rapid adaptive evolution played an important role for climate change responses in annual species, and probably contributed significantly to community resistance.
Notably, all evolutionary changes occurred solely in the dry manipulated plots, i.e. the treatment which increased, rather than decreased stress for resident plants. This appears intuitive because drought may directly lead to rapid exclusion of drought-sensitive and late-flowering genotypes, especially in dry study years. In wet plots, selection among genotypes was probably driven by competition for additional resources (Schiffers & Tielbörger 2006), resulting in weaker, more gradual fitness differences, as was shown for B. didyma in a cross-transplant with and without competition (Ariza & Tielbörger 2011), and hence slower progression of exclusion and adaptive evolution.
However, seven further candidate traits did not evolve. This is surprising because five of them exhibited clinal shifts along the natural rainfall gradient, suggesting that they contribute to B. didyma’ s long-term evolutionary response to drier climates: germination fraction, stomata density, height, vegetative biomass and seed number. In conjunction with existing theory we had expected corresponding evolution of these traits under climate manipulations (Westoby 1998; Liuet al. 2012; Tielbörger et al. 2012; ten Brink et al. 2020). Selection analyses supported this expectation for vegetative biomass, although not for stomata density and height, and they were not possible for germination fraction (no differential watering) and seed number (response variable in selection analyses). One possible explanation for the lack of evolution in other candidate traits is that sufficient adaptation was ensured by those traits that did evolve, and hence evolution of further traits was unnecessary. Alternatively, the multiple potential constraints for evolution under natural conditions hindered adaptation in other traits (Hoffmann & Sgró 2011; Shaw & Etterson 2012). In that case, the observed rapid evolution in a subset of traits may indicate incomplete adaptation to new conditions, cautioning that climate change may imperil species despite rapid evolution. Unfortunately, almost all available evidence for rapid evolution under natural conditions was restricted to very few traits (e.g. Franks et al. 2018; Nguyen et al . 2016; Grossmanet al. 2014; but see Frachon et al. 2017), i.e. there is lacking information and focus on the importance of non-evolving traits for adaptation to climate change. Our results thus highlight that studies relying on few traits might be misleading.
High trait plasticity was intensely debated as a mechanism retarding adaptive evolution to climate change, yet rarely tested in nature (Merilä & Hendry 2014; Kelly 2019; Fox et al. 2019). Here, each of the three rapidly evolving traits had a completely different degree of plasticity. This first assessment under field conditions suggests that traits may evolve rapidly irrespective of their degree of plasticity, and that rapid evolution appears rather restricted by other mechanisms such as the genetic architecture of traits, potential underlying trade-offs, or limited genetic variation within populations (Barrett & Schluter 2007; Hoffmann & Sgró 2011; Shaw & Etterson 2012). Our findings furthermore give little support for the idea that climate change leads to increased plasticity as a means to rapidly adjust the phenotype to novel conditions (Crispo 2007; Lande 2009; Merilä & Hendry 2014; Kelly 2019, Fox et al. 2019), because only a single trait (diaspore weight) showed increased plasticity under drought. Evolution of increased plasticity is therefore unlikely to be a pathway for climate change adaptation in our system, corroborating the few existing climate change studies which found plasticity either unchanged (Franks 2011) or lower (Grossman & Rice 2014).
Overall, our study demonstrates that rapid evolution plays an important role for climate change adaptation in natural annual plant communities. The novel setup of our study – combining for the first time in situ climate manipulations with a natural climatic gradient and selection analyses under controlled conditions – provided independent, compelling lines of evidence that observed evolutionary shifts were adaptive. However, with rapid evolution in merely a subset of candidate traits, our study emphasizes the importance of multi-trait studies for assessing whether rapid in situ evolution may safeguard species under climate change.