Mutation bias reflects natural selection in Arabidopsis thaliana.

Since the first half of the twentieth century, evolutionary theory has been dominated by the idea that mutations occur randomly with respect to their consequences1. Here we test this assumption with large surveys of de novo mutations in the plant Arabidopsis thaliana. In contrast to expectations, we find that mutations occur less often in functionally constrained regions of the genome-mutation frequency is reduced by half inside gene bodies and by two-thirds in essential genes. With independent genomic mutation datasets, including from the largest Arabidopsis mutation accumulation experiment conducted to date, we demonstrate that epigenomic and physical features explain over 90% of variance in the genome-wide pattern of mutation bias surrounding genes. Observed mutation frequencies around genes in turn accurately predict patterns of genetic polymorphisms in natural Arabidopsis accessions (r = 0.96). That mutation bias is the primary force behind patterns of sequence evolution around genes in natural accessions is supported by analyses of allele frequencies. Finally, we find that genes subject to stronger purifying selection have a lower mutation rate. We conclude that epigenome-associated mutation bias2 reduces the occurrence of deleterious mutations in Arabidopsis, challenging the prevailing paradigm that mutation is a directionless force in evolution.

A large-effect fitness trade-off across environments is explained by a single mutation affecting cold acclimation.

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes of Arabidopsis thaliana from Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism in CBF2. This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes of CBF2 on 5-y mean fitness and fitness components at the native field sites by comparing near-isogenic lines with alternate genotypes of CBF2 to their genetic background ecotypes. The effects of CBF2 were validated at the nucleotide level using gene-edited lines in the native genetic backgrounds grown in simulated parental environments. The foreign CBF2 genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene-edited lines confirmed that CBF2 encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.

Evolvability and trait function predict phenotypic divergence of plant populations.

Understanding the causes and limits of population divergence in phenotypic traits is a fundamental aim of evolutionary biology, with the potential to yield predictions of adaptation to environmental change. Reciprocal transplant experiments and the evaluation of optimality models suggest that local adaptation is common but not universal, and some studies suggest that trait divergence is highly constrained by genetic variances and covariances of complex phenotypes. We analyze a large database of population divergence in plants and evaluate whether evolutionary divergence scales positively with standing genetic variation within populations (evolvability), as expected if genetic constraints are evolutionarily important. We further evaluate differences in divergence and evolvability-divergence relationships between reproductive and vegetative traits and between selfing, mixed-mating, and outcrossing species, as these factors are expected to influence both patterns of selection and evolutionary potentials. Evolutionary divergence scaled positively with evolvability. Furthermore, trait divergence was greater for vegetative traits than for floral (reproductive) traits, but largely independent of the mating system. Jointly, these factors explained ~40% of the variance in evolutionary divergence. The consistency of the evolvability-divergence relationships across diverse species suggests substantial predictability of trait divergence. The results are also consistent with genetic constraints playing a role in evolutionary divergence.

Adaptation of perennial flowering phenology across the European range of Arabis alpina.

Flowering phenology is important in the adaptation of many plants to their local environment, but its adaptive value has not been extensively studied in herbaceous perennials. We used Arabis alpina as a model system to determine the importance of flowering phenology to fitness of a herbaceous perennial with a wide geographical range. Individual plants representative of local genetic diversity (accessions) were collected across Europe, including in Spain, the Alps and Scandinavia. The flowering behaviour of these accessions was documented in controlled conditions, in common-garden experiments at native sites and in situ in natural populations. Accessions from the Alps and Scandinavia varied in whether they required exposure to cold (vernalization) to induce flowering, and in the timing and duration of flowering. By contrast, all Spanish accessions obligately required vernalization and had a short duration of flowering. Using experimental gardens at native sites, we show that an obligate requirement for vernalization increases survival in Spain. Based on our analyses of genetic diversity and flowering behaviour across Europe, we propose that in the model herbaceous perennial A. alpina, an obligate requirement for vernalization, which is correlated with short duration of flowering, is favoured by selection in Spain where the plants experience a long growing season.

Ecological genetics of local adaptation in Arabidopsis: An 8-year field experiment.

There is considerable evidence for local adaptation in nature, yet important questions remain regarding its genetic basis. How many loci are involved? What are their effect sizes? What is the relative importance of conditional neutrality versus genetic trade-offs? Here we address these questions in the self-pollinating, annual plant Arabidopsis thaliana. We used 400 recombinant inbred lines (RILs) derived from two locally adapted populations in Italy and Sweden, grew the RILs and parents at the parental locations, and mapped quantitative trait loci (QTL) for mean fitness (fruits/seedling planted). We previously published results from the first 3 years of the study, and here add five additional years, providing a unique opportunity to assess how temporal variation in selection might affect QTL detection and classification. We found 10 adaptive and one maladaptive QTL in Italy, and six adaptive and four maladaptive QTL in Sweden. The discovery of maladaptive QTL at both sites suggests that even locally adapted populations are not always at their genotypic optimum. Mean effect sizes for adaptive QTL, 0.97 and 0.55 fruits in Italy and Sweden, respectively, were large relative to the mean fitness of the RILs (approximately 8 fruits/seedling planted at both sites). Both genetic trade-offs (four cases) and conditional neutrality (seven cases) contribute to local adaptation in this system. The 8-year dataset provided greater power to detect QTL and to estimate their locations compared to our previous 3-year study, identifying one new genetic trade-off and resolving one genetic trade-off into two conditionally adaptive QTL.

Climate drives rhizosphere microbiome variation and divergent selection between geographically distant Arabidopsis populations.

Disentangling the contribution of climatic and edaphic factors to microbiome variation and local adaptation in plants requires an experimental approach to uncouple their effects and test for causality. We used microbial inocula, soil matrices and plant genotypes derived from two natural Arabidopsis thaliana populations in northern and southern Europe in an experiment conducted in climatic chambers mimicking seasonal changes in temperature, day length and light intensity of the home sites of the two genotypes. The southern A. thaliana genotype outperformed the northern genotype in the southern climate chamber, whereas the opposite was true in the northern climate chamber. Recipient soil matrix, but not microbial composition, affected plant fitness, and effects did not differ between genotypes. Differences between chambers significantly affected rhizosphere microbiome assembly, although these effects were small in comparison with the shifts induced by physicochemical differences between soil matrices. The results suggest that differences in seasonal changes in temperature, day length and light intensity between northern and southern Europe have strongly influenced adaptive differentiation between the two A. thaliana populations, whereas effects of differences in soil factors have been weak. By contrast, below-ground differences in soil characteristics were more important than differences in climate for rhizosphere microbiome differentiation.

Effects of primary seed dormancy on lifetime fitness of Arabidopsis thaliana in the field.

BACKGROUND AND AIMS: Seed dormancy determines the environmental niche of plants in seasonal environments, and has consequences for plant performance that potentially go far beyond the seed and seedling stages. In this study, we examined the cascading effects of seed dormancy on the expression of subsequent life-history traits and fitness in the annual herb Arabidopsis thaliana. METHODS: We planted seeds of >200 recombinant inbred lines (RILs) derived from a cross between two locally adapted populations (Italy and Sweden), and both parental genotypes at the native site of the Swedish population in three consecutive years. We quantified the relationship between primary seed dormancy and the expression of subsequent life-history traits and fitness in the RIL population with path analysis. To examine the effects of differences in dormancy on the relative fitness of the two parental genotypes, we planted dormant seeds during the seed dispersal period and non-dormant seeds during the germination period of the local population. KEY RESULTS: In the RIL population, strong primary dormancy was associated with high seedling survival, but with low adult survival and fecundity, and path analysis indicated that this could be explained by effects on germination timing, rosette size and flowering start. The relationship between primary seed dormancy and germination proportion varied among years, and this was associated with differences in seasonal changes in soil moisture. The planting of dormant and non-dormant seeds indicated that the lower primary dormancy of the local Swedish genotype contributed to its higher germination proportion in two years and to its higher fecundity in one year. CONCLUSIONS: Our results show that seed dormancy affects trait expression and fitness components across the life cycle, and suggest that among-year variation in the incidence of drought during the germination period should be considered when predicting the consequences of climatic change for population growth and evolution.

Mechanisms of Male-Male Interference during Dispersal of Orchid Pollen.

AbstractSiring success of flowering plants depends on the fates of male gametophytes, which compete for access to stigmas, stylar resources, and ovules. Although rarely considered, pollen may often compete during dispersal, affecting the processes required for export to stigmas: pollen pickup, transport, and deposition. We quantified dispersal interference by tracking bee-mediated dispersal of stained Anacamptis morio (Orchidaceae) pollen from individual donor flowers and inferred the affected dispersal mechanisms on the basis of the fit of a process-based model. During individual trials, all recipient flowers were either emasculated, precluding interference with donor pollen, or intact, adding potentially interfering pollen to the pollinator. The presence of competing pollinaria on bees reduced pickup of additional pollinaria, doubled the overall proportion of lost donor pollen, and reduced total pollen export by 27%. Interference specifically increased loss of donor pollen between successive flower visits and variation in deposition among trials, and it likely also reduced pollen contact with stigmas and pollen deposition when contact occurred. Thus, by altering pollen removal, transport, and deposition, male-male interference during pollen dispersal can significantly-and perhaps commonly-limit plant-siring success.

Life-history trade-offs and the genetic basis of fitness in Arabidopsis thaliana.

Resources allocated to survival cannot be used to increase fecundity, but the extent to which this trade-off constrains adaptation depends on overall resource status. Adaptation to local environmental conditions may therefore entail the evolution of traits that increase the amount of resources available to individuals (their resource status or 'condition'). We examined the relative contribution of trade-offs and increased condition to adaptive evolution in a recombinant inbred line population of Arabidopsis thaliana planted at the native sites of the parental ecotypes in Italy and Sweden in 2 years. We estimated genetic correlations among fitness components based on genotypic means and explored their causes with QTL mapping. The local ecotype produced more seeds per fruit than did the non-local ecotype, reflected in stronger adaptive differentiation than was previously shown based on survival and fruit number only. Genetic correlations between survival and overall fecundity, and between number of fruits and number of seeds per fruit, were positive, and there was little evidence of a trade-off between seed size and number. Quantitative trait loci for these traits tended to map to the same regions of the genome and showed positive pleiotropic effects. The results indicate that adaptive differentiation between the two focal populations largely reflects the evolution of increased ability to acquire resources in the local environment, rather than shifts in the relative allocation to different life-history traits. Differentiation both in phenology and in tolerance to cold is likely to contribute to the advantage of the local genotype at the two sites.

Evolution of floral scent in relation to self-incompatibility and capacity for autonomous self-pollination in the perennial herb Arabis alpina.

BACKGROUND AND AIMS: The transition from outcrossing to selfing is a frequent evolutionary shift in flowering plants and is predicted to result in reduced allocation to pollinator attraction if plants can self-pollinate autonomously. The evolution of selfing is associated with reduced visual floral signalling in many systems, but effects on floral scent have received less attention. We compared multiple populations of the arctic-alpine herb Arabis alpina (Brassicaceae), and asked whether the transition from self-incompatibility to self-compatibility has been associated with reduced visual and chemical floral signalling. We further examined whether floral signalling differ between self-compatible populations with low and high capacity for autonomous self-pollination, as would be expected if benefits of signalling decrease with reduced dependence on pollinators for pollen transfer. METHODS: In a common garden we documented flower size and floral scent emission rate and composition in eight self-compatible and nine self-incompatible A. alpina populations. These included self-compatible Scandinavian populations with high capacity for autonomous self-pollination, self-compatible populations with low capacity for autonomous self-pollination from France and Spain, and self-incompatible populations from Italy and Greece. KEY RESULTS: The self-compatible populations produced smaller and less scented flowers than the self-incompatible populations. However, flower size and scent emission rate did not differ between self-compatible populations with high and low capacity for autonomous self-pollination. Floral scent composition differed between self-compatible and self-incompatible populations, but also varied substantially among populations within the two categories. CONCLUSIONS: Our study demonstrates extensive variation in floral scent among populations of a geographically widespread species. Contrary to expectation, floral signalling did not differ between self-compatible populations with high and low capacity for autonomous self-pollination, indicating that dependence on pollinator attraction can only partly explain variation in floral signalling. Additional variation may reflect adaptation to other aspects of local environments, genetic drift, or a combination of these processes.

Combining population genomics and fitness QTLs to identify the genetics of local adaptation in Arabidopsis thaliana.

Evidence for adaptation to different climates in the model species Arabidopsis thaliana is seen in reciprocal transplant experiments, but the genetic basis of this adaptation remains poorly understood. Field-based quantitative trait locus (QTL) studies provide direct but low-resolution evidence for the genetic basis of local adaptation. Using high-resolution population genomic approaches, we examine local adaptation along previously identified genetic trade-off (GT) and conditionally neutral (CN) QTLs for fitness between locally adapted Italian and Swedish A. thaliana populations [Ågren J, et al. (2013) Proc Natl Acad Sci USA 110:21077-21082]. We find that genomic regions enriched in high FST SNPs colocalize with GT QTL peaks. Many of these high FST regions also colocalize with regions enriched for SNPs significantly correlated to climate in Eurasia and evidence of recent selective sweeps in Sweden. Examining unfolded site frequency spectra across genes containing high FST SNPs suggests GTs may be due to more recent adaptation in Sweden than Italy. Finally, we collapse a list of thousands of genes spanning GT QTLs to 42 genes that likely underlie the observed GTs and explore potential biological processes driving these trade-offs, from protein phosphorylation, to seed dormancy and longevity. Our analyses link population genomic analyses and field-based QTL studies of local adaptation, and emphasize that GTs play an important role in the process of local adaptation.

Demography and mating system shape the genome-wide impact of purifying selection in Arabis alpina.

Plant mating systems have profound effects on levels and structuring of genetic variation and can affect the impact of natural selection. Although theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population-genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles, whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, mating system and demography shape the impact of purifying selection on genomic variation in A. alpina These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies.

Strong stabilizing selection on timing of germination in a Mediterranean population of Arabidopsis thaliana.

PREMISE: Timing of germination can strongly influence plant fitness by affecting seedling survival and by having cascading effects on later life-history traits. In seasonal environments, the period favorable for seedling establishment and growth is limited, and timing of germination is likely to be under stabilizing selection because of conflicting selection through survival and fecundity. Moreover, optimal germination time may vary among genotypes because of inherent differences in later life-history traits. METHODS: To examine how germination time affects survival, fecundity, and the relative fitness of two genotypes differing in time to first flower, we conducted a field experiment in an Italian population of the winter annual Arabidopsis thaliana, in which seedling establishment occurs mainly in November. We transplanted seedlings of the local genotype and of a Swedish genotype monthly from August to December and monitored survival and fecundity. RESULTS: Only seedlings transplanted in November and December survived until reproduction, and fitness of the November cohort was 35 times higher than that of the December cohort, indicating strong stabilizing selection on timing of germination. There was no evidence of conflicting selection: seedling survival, adult survival, and fecundity were all highest in the November cohort. Moreover, the relative fitness of the two genotypes did not differ significantly between cohorts. CONCLUSIONS: The very narrow window of opportunity for seedling establishment was related to rapid seasonal changes in soil moisture and temperature, suggesting that rate of seasonal change is an important aspect to consider for understanding spatiotemporal variation in selection on phenological traits.

Conflicting selection on floral scent emission in the orchid Gymnadenia conopsea.

Floral scent is a crucial trait for pollinator attraction. Yet only a handful of studies have estimated selection on scent in natural populations and no study has quantified the relative importance of pollinators and other agents of selection. In the fragrant orchid Gymnadenia conopsea, we used electroantennographic data to identify floral scent compounds detected by local pollinators and quantified pollinator-mediated selection on emission rates of 10 target compounds as well as on flowering start, visual display and spur length. Nocturnal pollinators contributed more to reproductive success than diurnal pollinators, but there was significant pollinator-mediated selection on both diurnal and nocturnal scent emission. Pollinators selected for increased emission of two compounds and reduced emission of two other compounds, none of which were major constituents of the total bouquet. In three cases, pollinator-mediated selection was opposed by nonpollinator-mediated selection, leading to weaker or no detectable net selection. Our study demonstrates that minor scent compounds can be targets of selection, that pollinators do not necessarily favour stronger scent signalling, and that some scent compounds are subject to conflicting selection from pollinators and other agents of selection. Hence, including floral scent traits into selection analysis is important for understanding the mechanisms behind floral evolution.

Heterosis is common and inbreeding depression absent in natural populations of Arabidopsis thaliana.

The importance of genetic drift in shaping patterns of adaptive genetic variation in nature is poorly known. Genetic drift should drive partially recessive deleterious mutations to high frequency, and inter-population crosses may therefore exhibit heterosis (increased fitness relative to intra-population crosses). Low genetic diversity and greater genetic distance between populations should increase the magnitude of heterosis. Moreover, drift and selection should remove strongly deleterious recessive alleles from individual populations, resulting in reduced inbreeding depression. To estimate heterosis, we crossed 90 independent line pairs of Arabidopsis thaliana from 15 pairs of natural populations sampled across Fennoscandia and crossed an additional 41 line pairs from a subset of four of these populations to estimate inbreeding depression. We measured lifetime fitness of crosses relative to parents in a large outdoor common garden (8,448 plants in total) in central Sweden. To examine the effects of genetic diversity and genetic distance on heterosis, we genotyped parental lines for 869 SNPs. Overall, genetic variation within populations was low (median expected heterozygosity = 0.02), and genetic differentiation was high (median FST  = 0.82). Crosses between 10 of 15 population pairs exhibited significant heterosis, with magnitudes of heterosis as high as 117%. We found no significant inbreeding depression, suggesting that the observed heterosis is due to fixation of mildly deleterious alleles within populations. Widespread and substantial heterosis indicates an important role for drift in shaping genetic variation, but there was no significant relationship between fitness of crosses relative to parents and genetic diversity or genetic distance between populations.

Early life stages contribute strongly to local adaptation in Arabidopsis thaliana.

The magnitude and genetic basis of local adaptation is of fundamental interest in evolutionary biology. However, field experiments usually do not consider early life stages, and therefore may underestimate local adaptation and miss genetically based tradeoffs. We examined the contribution of differences in seedling establishment to adaptive differentiation and the genetic architecture of local adaptation using recombinant inbred lines (RIL) derived from a cross between two locally adapted populations (Italy and Sweden) of the annual plant Arabidopsis thaliana We planted freshly matured, dormant seeds (>180 000) representing >200 RILs at the native field sites of the parental genotypes, estimated the strength of selection during different life stages, mapped quantitative trait loci (QTL) for fitness and its components, and quantified selection on seed dormancy. We found that selection during the seedling establishment phase contributed strongly to the fitness advantage of the local genotype at both sites. With one exception, local alleles of the eight distinct establishment QTL were favored. The major QTL for establishment and total fitness showed evidence of a fitness tradeoff and was located in the same region as the major seed dormancy QTL and the dormancy gene DELAY OF GERMINATION 1 (DOG1). RIL seed dormancy could explain variation in seedling establishment and fitness across the life cycle. Our results demonstrate that genetically based differences in traits affecting performance during early life stages can contribute strongly to adaptive differentiation and genetic tradeoffs, and should be considered for a full understanding of the ecology and genetics of local adaptation.

Among-year variation in selection during early life stages and the genetic basis of fitness in Arabidopsis thaliana.

Incomplete information regarding both selection regimes and the genetic basis of fitness limits our understanding of adaptive evolution. Among-year variation in the genetic basis of fitness is rarely quantified, and estimates of selection are typically based on single components of fitness, thus potentially missing conflicting selection acting during other life-history stages. Here, we examined among-year variation in selection on a key life-history trait and the genetic basis of fitness covering the whole life cycle in the annual plant Arabidopsis thaliana. We planted freshly matured seeds of >200 recombinant inbred lines (RILs) derived from a cross between two locally adapted populations (Italy and Sweden), and both parental genotypes at the native site of the Swedish population in three consecutive years. We quantified selection against the nonlocal Italian genotype, mapped quantitative trait loci (QTL) for fitness and its components, and quantified selection on timing of germination during different life stages. In all 3 years, the local Swedish genotype outperformed the nonlocal Italian genotype. However, both the contribution of early life stages to relative fitness, and the effects of fitness QTL varied among years. Timing of germination was under conflicting selection through seedling establishment vs. adult survival and fecundity, and both the direction and magnitude of net selection varied among years. Our results demonstrate that selection during early life stages and the genetic basis of fitness can vary markedly among years, emphasizing the need for multiyear studies considering the whole life cycle for a full understanding of natural selection and mechanisms maintaining local adaptation.

Grazers affect selection on inflorescence height both directly and indirectly and effects change over time.

Selection mediated by one biotic agent will often be modified by the presence of other biotic interactions, and the importance of such indirect effects might change over time. We conducted an 11-yr field experiment to test the prediction that large grazers affect selection on floral display of the dimorphic herb Primula farinosa not only directly through differential grazing damage, but also indirectly by affecting vegetation height and thereby selection mediated by pollinators and seed predators. Exclusion of large grazers increased vegetation height and the strength of pollinator-mediated selection for tall inflorescences and seed-predator-mediated selection for short inflorescences. The direct effect of grazers on selection resulting from differential grazing damage to the two scape morphs showed no temporal trend. By contrast, the increase in vegetation height in exclosures over time was associated with an increase in selection mediated by pollinators and seed predators. In the early years of the experiment, the indirect effects of grazers on selection mediated by pollinators and seed predators were weak, whereas at the end of the experiment, the indirect effects were of similar magnitude as the direct effect due to differential grazing damage. The results demonstrate that the indirect effects of a selective agent can be as strong as its direct effects, and that the relative importance of direct vs. indirect effects on selection can change over time. A full understanding of the ecological processes governing variation in selection thus requires that both direct and indirect effects of biotic interactions are assessed.