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The regulation of flowering time has substantial biological significance because it defines the vegetative-to-reproductive transition and determines the length of the post-embryonic life history of annual plants. Variation in flowering time may affect different plant fitness components, so the switch from vegetative to reproductive development is expected to be under strong selective pressure (Coupland, 1995; Ausín et al., 2005; Roux et al., 2006; Anderson et al., 2011). For this reason, unraveling the genetic basis and environmental control of flowering time has represented a major study issue for the Arabidopsis thaliana research community for many years (Coupland, 1995; Koornneef et al., 1998, 2004; Simpson & Dean, 2002; He et al., 2003; Ausín et al., 2005; Flowers et al., 2009; Brachi et al., 2010).
Given the evolutionary relevance of flowering time, research efforts have focused on the detection and assessment of natural selection on flowering time as well as its underlying genetic mechanisms in controlled conditions (Le Corre, 2005; Stenøien et al., 2005; Li et al., 2006; Scarcelli et al., 2007; Kover et al., 2009) and/or natural field settings (Weinig et al., 2002; Caicedo et al., 2004; Stinchcombe et al., 2004; Korves et al., 2007; Brock et al., 2009; Wilczek et al., 2009; Ågren & Schemske, 2012). These studies have shown that the genetic basis of flowering time variation depends strongly on the environment in which flowering time is estimated (Weinig et al., 2002; Olsen et al., 2004; Malmberg et al., 2005; Li et al., 2006; Korves et al., 2007; Brachi et al., 2010). Therefore, significant genotype × environment (G × E) interactions may reflect the wide array of mechanisms accounting for flowering time plasticity across different environments (Pigliucci & Schlichting, 1998; Stratton, 1998; Pigliucci, 2003). As the genetic architecture of a trait depends on the population and environment where it has evolved (Remington & Purugganan, 2003; Atwell et al., 2010), systematic comparisons of experiments performed in different environmental settings are essential if we aim to investigate properly the evolutionary genetics of flowering time in A. thaliana.
In the Iberian Peninsula, adaptive variation in flowering time among natural accessions of A. thaliana seems to be largely determined by variation in winter temperature: accessions from cold environments exhibit late-flowering behaviors and stronger vernalization requirements (Méndez-Vigo et al., 2011). Furthermore, different polymorphisms in flowering genes of the vernalization pathway, such as FRI and FLC, show significant associations with flowering traits and winter temperatures, revealing some of the genetic mechanisms underlying flowering time variation in Iberian A. thaliana accessions (Méndez-Vigo et al., 2011; Sánchez-Bermejo et al., 2012). As these findings were obtained from experiments conducted in glasshouse conditions, we ought to conduct experiments in realistic field settings also and compare results with those obtained in controlled conditions. These comparisons may enable us to evaluate the extent of shared genetic bases for flowering time variation among controlled and natural conditions (Weinig et al., 2002; Brachi et al., 2010).
Here, we report on the comparison between the genetic variation for flowering time in A. thaliana in controlled and natural conditions. Natural conditions were represented by a field experiment in southwest Spain where the species occurs naturally (Picó et al., 2008; Méndez-Vigo et al., 2011). Under controlled conditions, we tested the flowering inductive effect of low temperature – so-called vernalization – by comparing experiments with and without vernalization treatment. In both conditions, we conducted population-based experiments using a well-known collection of A. thaliana populations from the Iberian Peninsula (Picó et al., 2008; Méndez-Vigo et al., 2011; Kronholm et al., 2012). The evolutionary relevance of flowering time was studied by comparing quantitative genetic differentiation (QST) in the various conditions with neutral genetic differentiation (FST) estimated with two sets of neutral molecular markers. Although comparisons of QST vs FST can be constrained and biased for multiple reasons (O'Hara & Merilä, 2005; Leinonen et al., 2008; Miller et al., 2008; Holsinger & Weir, 2009; Edelaar et al., 2011), they are still valid to identify promising traits that may be under selection (Whitlock, 2008). We also analysed nucleotide variation in four well-known flowering genes (FRI, FLC, CRY2 and PHYC; Méndez-Vigo et al., 2011) from two different developmental pathways (vernalization and photoperiod pathways) to assess the association of these genes with among- and within-population patterns of variation in flowering time in different conditions.
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In this study, we evaluated the extent of among- and within-population variation in life-history traits of A. thaliana in a natural field setting. In general, the results indicated that natural A. thaliana populations contain significant genetic variation for life-history traits. The results also revealed that individuals from the GRA population exhibited the highest performance for relevant demographic traits, such as the maximum number of vegetative rosettes and the total number of fruiting plants (Table 2). As GRA represents the local population, this result suggests adaptation of GRA individuals to the local environment. Nevertheless, given the strong spatial component of local adaptation to abiotic environmental conditions, further research is needed to address this important issue in A. thaliana. For example, a macro-ecological approach may assess the effects of environmental similarities between native and experimental environments on fitness components on a large-scale spatially explicit collection of A. thaliana accessions.
We have shown that flowering time estimated in field and glasshouse conditions were all significantly positively correlated in this set of Iberian A. thaliana populations. At the population level, flowering time in field and glasshouse conditions with vernalization exhibited a very similar pattern while the among-population pattern of variation in flowering time in glasshouse conditions without vernalization was more variable (Fig. S3). This result indicates that the environmental conditions at El Castillejo Botanical Garden in southwest Spain also promoted the vernalization pathway for flowering in A. thaliana. Despite that fact that winters are mild at El Castillejo Botanical Garden (Fig. S2), the mean monthly minimum temperatures of 5°C recorded in January may have been enough to activate the response to vernalization of A. thaliana study individuals. A recent study has shown that vegetative rosettes from populations from much colder environments, such as high-altitude montane locations, hardly survive the winter and that such populations are chiefly composed by spring-germinated plants that overwinter as seeds in the soil seed bank (Picó, 2012). Hence, we hypothesize that natural environments with mild and moderately cold winters activate the vernalization pathway to promote flowering during winter. When winters are too severe, A. thaliana mostly behaves as a spring annual to complete its life cycle in such environments.
The evolutionary relevance of flowering time over the remaining life-history traits estimated in the field experiment was highlighted in this study through QST–FST comparisons, an approach that has previously been conducted in A. thaliana, but only for flowering time estimates under glasshouse conditions (Le Corre, 2005; Stenøien et al., 2005; Porcher et al., 2006). Given the inherent caveats of the QST–FST approach (Leinonen et al., 2008), these comparisons must be treated with caution. However, different results from our study support the conclusion that flowering time in A. thaliana is likely to be a trait under local divergent selection in our set of Iberian populations. First, flowering time estimated in field conditions was the only trait exhibiting significantly higher QST values than the FST value estimated with SNPs (Fig. 4). In addition, QST for flowering traits estimated without vernalization in glasshouse conditions were also significantly higher than the FST value derived from microsatellites, in agreement with previous studies (Le Corre, 2005). It must be emphasized that the comparison of different molecular markers to estimate FST values revealed the strong dependence of this approach on marker type. Our results support the suggestion that SNPs are more suitable than microsatellites for QST–FST comparisons because high mutation rates of microsatellites tend to upwardly bias the difference between QST and FST (Edelaar et al., 2011). Second, the comparison of different flowering time values estimated in different conditions indicated that flowering time or closely related traits, such as leaf number, exhibit high QST values in comparison with the other life-history traits (Fig. 4). Third, the distinct isolation-by-distance pattern observed for FST and QST values obtained for flowering time in the field support the idea that other evolutionary factors different from demography contribute to the strong population differentiation for this trait. Overall, our results reinforce the widely accepted view that flowering time plays an important role in shaping A. thaliana's life history and illustrate how inherent demographic processes (i.e. isolation-by-distance) and divergent selection mainly on flowering time may jointly account for phenotypic and genotypic population differentiation in A. thaliana.
Our experiments also revealed that there exists an important amount of within-population genetic variation for flowering time in natural A. thaliana populations whose expression varies among environments. Studies addressing the genetic and molecular basis of flowering time variation in A. thaliana have successfully detected latitudinal (Caicedo et al., 2004; Stinchcombe et al., 2004; Lempe et al., 2005; Shindo et al., 2005; Samis et al., 2008, 2012; Méndez-Vigo et al., 2011), and recently altitudinal (Méndez-Vigo et al., 2011), geographical patterns of variation. It is noteworthy that we still largely ignore the underlying mechanisms that generate and maintain within-population variation in flowering time, a key component to comprehensive understanding of evolutionary change in this important trait.
One way to evaluate the causes of within-population variation in flowering time observed in our experiments is illustrated by the analysis of the effect of polymorphisms in important flowering genes. In this study, we found two loss-of-function FRI alleles and a previously described potential FLC polymorphism (Méndez-Vigo et al., 2011) that were significantly associated with flowering time variation in this set of Iberian A. thaliana populations. Interestingly, these FRI and FLC polymorphisms, and the two major haplogroups described for PHYC and CRY2 (Olsen et al., 2004; Balasubramanian et al., 2006) segregated within one to three populations, indicating their contribution to within-population variation. In addition, we have been able to observe several important demographic and phenotypic effects of some of these polymorphisms. For example, individuals bearing FRI truncations exhibited lower recruitment rates throughout the field experiment (Fig. 5), which suggests FRI pleiotropy on major life-history traits in A. thaliana. This finding does not represent an exception because FLC has also been found to play a role in temperature-mediated germination (Chiang et al., 2009) and seed dormancy may also influence flowering time in A. thaliana (Rubio de Casas et al., 2012). Furthermore, FRI truncations altered the relationship between flowering time values estimated in different environments (Fig. 6), as individuals carrying FRI truncations flowered earlier in the glasshouse but later in field conditions. This result is in agreement with the early flowering behavior observed under natural over-wintered conditions in accessions carrying FRI functional alleles in combination with FLC alleles of haplogroup A (Caicedo et al., 2004; Samis et al., 2012), the only haplogroup found in the Iberian Peninsula (Méndez-Vigo et al., 2011). Therefore, FRI may also contribute to the observed G × E interactions, reinforcing the importance of plasticity in flowering time to the understanding of gene function in A. thaliana. Nevertheless, given the low number of individuals carrying FRI truncations included in these analyses and the close genetic relationships among them, further studies are necessary to ensure that the observed FRI effects are not caused by other genes in linkage disequilibrium with FRI.
Overall, this study stresses the need to adopt multidisciplinary and integrative approaches to study comprehensively the evolutionary genetics of flowering time variation in A. thaliana. The increase in available genomic data for world-wide natural A. thaliana accessions (Weigel, 2012) will facilitate the simultaneous study of natural variation in multiple gene polymorphisms. However, further efforts are needed to generate phenotypic data and reaction norms from different environments, including more populations and especially more individuals per population, which represent an important limitation for the advance of evolutionary genetics of ecologically important life-history traits.