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Balancing selection is frequently invoked as a mechanism that maintains variation within and across populations. However, there are few examples of balancing selection operating on loci underpinning complex traits, which frequently display high levels of variation. We investigated mechanisms that may maintain variation in a focal polymorphism—leaf chemical profiles of a perennial wildflower (Boechera stricta, Brassicaceae)—explicitly interrogating multiple ecological and genetic processes including spatial variation in selection, antagonistic pleiotropy, and frequency-dependent selection. A suite of common garden and greenhouse experiments showed that the alleles underlying variation in chemical profile have contrasting fitness effects across environments, implicating two ecological drivers of selection on chemical profile: herbivory and drought. Phenotype-environment associations and molecular genetic analyses revealed additional evidence of past selection by these drivers. Together, these data are consistent with balancing selection on chemical profile, likely caused by pleiotropic effects of secondary chemical biosynthesis genes on herbivore defense and drought response., Editor’s Summary: Evidence for balancing selection acting on loci that control complex traits is limited. Here, the authors show evidence for past selection on chemical profile in a perennial wildflower by two ecological drivers, herbivory and drought, consistent with balancing selection on this trait.

Premise Industrialization and human activities have elevated temperatures and caused novel precipitation patterns, altering soil moisture and nutrient availability. Predicting evolutionary responses to climate change requires information on the agents of selection that drive local adaptation and influence resource acquisition and allocation. Here, we examined the contribution of nutrient and drought stress to local adaptation, and we tested whether trade-offs across fitness components constrain or facilitate adaptation under resource stress. Methods We exposed 35 families of Boechera stricta (Brassicaceae) to three levels of water and two levels of nutrient supply in a factorial design in the greenhouse. We sourced maternal families from a broad elevational gradient (2499-3530 m a.s.l.), representing disparate soil moisture and nutrient availability. Results Concordant with local adaptation, maternal families from arid, low-elevation populations had enhanced fecundity under severe drought over those from more mesic, high-elevation sites. Furthermore, fitness trade-offs between growth and reproductive success depended on the environmental context. Under high, but not low, nutrient levels, we found a negative phenotypic relationship between the probability of reproduction and growth rate. Similarly, a negative phenotypic association only emerged between fecundity and growth under severe drought stress, not the benign water treatment levels, indicating that stressful resource environments alter the direction of trait correlations. Genetic covariances were broadly concordant with these phenotypic patterns. Conclusions Despite high heritabilities in all fitness components across treatments, trade-offs between growth and reproduction could constrain adaptation to increasing drought stress and novel nutrient levels.

Heritable genetic variation is necessary for populations to evolve in response to anthropogenic climate change. However, antagonistic genetic correlations among traits may constrain the rate of adaptation, even if substantial genetic variation exists. We examine potential genetic responses to selection by comparing multivariate genetic variance-covariances of traits and fitness (multivariate Robertson-Price identities) across different environments in a reciprocal transplant experiment of the forb Boechera stricta in the Rocky Mountains. By transplanting populations into four common gardens arrayed along an elevational gradient, and exposing populations to control and snow removal treatments, we simulated future and current climates and snowmelt regimes. Genetic variation in flowering and germination phenology declined in plants moved downslope to warmer, drier sites, suggesting that these traits may have a limited ability to evolve under future climates. Simulated climate change via snow removal altered the strength of selection on flowering traits, but we found little evidence that genetic correlations among traits are likely to affect the rate of adaptation to climate change. Overall, our results suggest that climate change may alter the evolutionary potential of B. stricta, but reduced expression of genetic variation may be a larger impediment to adaptation than constraints imposed by antagonistic genetic correlations.

Most transplant experiments across species geographic range boundaries indicate that adaptation to stressful environments outside the range is often constrained. However, the mechanisms of these constraints remain poorly understood. We used extended generation crosses from diverged high and low elevation populations. In experiments across low elevation range boundaries, there was selection on the parental lines for abiotic stress‐tolerance and resistance to herbivores. However, in support of a defense‐tolerance trade‐off, extended generation crosses showed nonindependent segregation of these traits in the laboratory across a drought‐stress gradient and in the field across the low elevation range boundary. Genotypic variation in a marker from a region of the genome containing a candidate gene (MYC2) was associated with change in the genetic trade‐off. Thus, using crosses and forward genetics, we found experimental genetic and molecular evidence for a pleiotropic trade‐off that could constrain the evolution of range expansion., Evolutionary constraints are important to understand range limits, yet the mechanisms are poorly understood. Here we used an integrative approach to show that a trade‐off between abiotic stress‐tolerance and chemical defense against herbivores may inhibit the evolution of low elevation range expansion in the ecological model Boechera stricta.

When pleiotropy is present, genetic correlations may constrain the evolution of ecologically important traits. We used a quantitative genetics approach to investigate constraints on the evolution of secondary metabolites in a wild mustard, Boechera stricta. Much of the genetic variation in chemical composition of glucosinolates in B. stricta is controlled by a single locus, BCMA1/3. In a large-scale common garden experiment under natural conditions, we quantified fitness and glucosinolate profile in two leaf types and in fruits. We estimated genetic variances and covariances (the G-matrix) and selection on chemical profile in each tissue. Chemical composition of defenses was strongly genetically correlated between tissues. We found antagonistic selection between defense composition in leaves and fruits: compounds that were favored in leaves were disadvantageous in fruits. The positive genetic correlations and antagonistic selection led to strong constraints on the evolution of defenses in leaves and fruits. In a hypothetical population with no genetic variation at BCMA1/3, we found no evidence for genetic constraints, indicating that pleiotropy affecting chemical profile in multiple tissues drives constraints on the evolution of secondary metabolites.

Differential local adaptation restricts gene flow between populations inhabiting distinct environments, resulting in isolation by adaptation. In addition to the statistical inferences of genotype–environment associations, an integrative approach is needed to investigate the effect of local adaptation on population divergence at the ecological, genetic and genomic scale. Here, we combine reciprocal transplant, genome–environment association and QTL mapping to investigate local adaptation in Boechera stricta (Drummond's rockcress). With reciprocal transplant experiment, we found local genetic groups exhibit phenotypic characteristics corresponding to the distinct selection forces from different water availability. At the genetic level, the local allele of a major fitness QTL confers higher and sturdier flowering stalks, maximizing the fecundity fitness component under sufficient water supply, and its genetic variation is associated with precipitation across the landscape. At the genomewide scale, we further showed that multiple loci associated with precipitation are highly differentiated between genetic groups, suggesting that local adaptation has a widespread effect on reducing gene flow. This study provides one of the few comprehensive examples demonstrating how local adaptation facilitates population divergence at the trait, gene and genome level.

Investment in current reproduction can reduce future fitness by depleting resources needed for maintenance, particularly under environmental stress. These trade-offs influence life-history evolution. We tested whether climate change alters the future-fitness costs of current reproduction in a large-scale field experiment ofBoechera stricta(Brassicaceae). Over 6 years, we simulated climate change along an elevational gradient in the Rocky Mountains through snow removal, which accelerates snowmelt and reduces soil water availability. Costs of reproduction were greatest in arid, lower elevations, where high initial reproductive effort depressed future fitness. At mid-elevations, initial reproduction augmented subsequent fitness in benign conditions, but pronounced costs emerged under snow removal. At high elevation, snow removal dampened costs of reproduction by prolonging the growing season. In most scenarios, failed reproduction in response to resource limitation depressed lifetime fecundity. Indeed, fruit abortion only benefited high-fitness individuals under benign conditions. We propose that climate change could shift life-history trade-offs in an environment-dependent fashion, possibly favouring early reproduction and short lifespans in stressful conditions.

The genetic basis of flowering time changes across environments, and pleiotropy may limit adaptive evolution of populations in response to local conditions. However, little information is known about how genetic architecture changes among environments. We used genome-wide association studies (GWAS) in Boechera stricta (Graham) Al-Shehbaz, a relative of Arabidopsis, to examine flowering variation among environments and associations with climate conditions in home environments. Also, we used molecular population genetics to search for evidence of historical natural selection. GWAS found 47 significant quantitative trait loci (QTLs) that influence flowering time in one or more environments, control plastic changes in phenology between experiments, or show associations with climate in sites of origin. Genetic architecture of flowering varied substantially among environments. We found that some pairs of QTLs showed similar patterns of pleiotropy across environments. A large-effect QTL showed molecular signatures of adaptive evolution and is associated with climate in home environments. The derived allele at this locus causes later flowering and predominates in sites with greater water availability. This work shows that GWAS of climate associations and ecologically important traits across diverse environments can be combined with molecular signatures of natural selection to elucidate ecological genetics of adaptive evolution.

Even after decades of research, the evolution of sex remains an enigma in evolutionary biology. Typically, research addresses the costs of sex and asexuality to characterize the circumstances in which one reproductive system is more favorable. Yet surprisingly few studies address the influence of common traits that are obligately correlated with asexuality, including hybridization and polyploidy; even though these traits have substantial impacts on selective patterns. In particular, hybridization is well-known to alter trait expression; these alterations may themselves represent a cost of sex. We examined the role of reproductive isolation in the formation of de novo hybrid lineages between two widespread species in the ecological model systemBoechera. Of 664 crosses betweenBoechera strictaandBoechera retrofracta, 17% of crosses produced F1 fruits. This suggests that postmating prezygotic barriers, i.e. pollen-pistil interactions, form the major barrier to hybrid success in this system. These interactions are asymmetrical, with 110 F1 fruits produced whenB. strictawas the maternal parent. This asymmetry was confirmed using a chloroplast phylogeny of wild-collectedB. stricta,B. retrofracta, and hybrids, which showed that most hybrids have aB. strictachloroplast haplotype. We next compared fitness of F2 hybrids and selfed parentalB. strictalines, finding that F2 fitness was reduced by substantial hybrid sterility. Our results suggest that multiple reproductively isolating barriers likely influence the formation and fitness of hybrid lineages in the wild, and that these costs of hybridization likely have profound impacts on the costs of sex in the natural environment.

The circadian clock is a molecular timekeeper that matches endogenous rhythms in diverse traits with 24-h cycles in the external environment. Although a lack of clock resonance to the environment is detrimental to performance, clock phenotypes in wild populations nevertheless deviate substantially from the predicted optimal cycle length of 24 h, and significant genetic variation exists for circadian parameters. Here, we describe covariation between 2 traits considered to reflect adaptation to different aspects of temporal environmental heterogeneity, circadian rhythms (adaptation to daily environmental cycles) and flowering time (adaptation to seasonal cycles), in a Rocky Mountain population of the mustard Boechera stricta, a North American relative of Arabidopsis thaliana. We found that 18 families that differ in circadian period in leaf movement by 3.5 h expressed genetic diversity in first-year growth, reproductive phenology, vegetative size at reproduction, and starch concentration following vernalization. The families exhibited a large (~90-day) range in mean flowering time, even though the spatial scale of population sampling covered only a few hundred meters. Circadian period covaried with other traits such that longer-period families flowered earlier and at a larger size, a trait combination predicted to yield a fitness benefit in the wild. Circadian clock research in model systems has previously shown that mutations in clock genes influence phenology. Our results widen the scope of this research by illustrating a link between naturally segregating clock variation and reproductive phenology among wild genotypes, suggesting that the causes of genetic diversity in the clock lie partly in adaptation to seasonal environmental heterogeneity.

In this research, we isolated three CBF (C-repeat-Binding Factors) genes from two genotypes of Boechera stricta with contrasting freezing tolerance and characterized their structure and expression patterns in response to cold treatment. An amino acid sequence comparison revealed that the CBF genes in B. stricta showed high conservation in the AP2 domain and PKKP/RAGR motif like other cold adaptable Brassicaceae. The pairwise sequence alignment of the CBF genes isolated from two genotypes of B. stricta showed non-synonymous mutations in CBF 2 and 3. Gene expression analysis demonstrated that CBF genes in B. stricta have expression patterns similar to CBFs in A. thaliana in response to cold treatment, while differential expression at the molecular level in CBF and COR genes was presented between two genotypes of B. stricta. Our results suggest that signal transduction of three CBF genes can be one of the central pathways in the development of freezing tolerance in B. stricta.

Intraspecific variation in flower color is often attributed to pollinator-mediated selection, yet this mechanism cannot explain flower color polymorphisms in self-pollinating species. Indirect selection mediated via biotic and abiotic stresses could maintain flower color variation in these systems. The selfing forb, Boechera stricta, typically displays white flowers, but some individuals produce purple flowers. We quantified environmental correlates of flower color in natural populations. To disentangle plasticity from genotypic variation, we performed a multiyear field experiment in five gardens. In controlled conditions, we evaluated herbivore preferences and the effects of drought stress and soil pH on flower color expression. In natural populations, purple-flowered individuals experienced lower foliar herbivory than did their white-flowered counterparts. This pattern also held in the common gardens. Additionally, low-elevation environments induced pigmented flowers (plasticity), and the likelihood of floral pigmentation decreased with source elevation of maternal families (genetic cline). Viability selection favored families with pigmented flowers. In the laboratory, herbivores exerted greater damage on tissue derived from white- vs purple-flowered individuals. Furthermore, drought induced pigmentation in white-flowered lineages, and white-flowered plants had a fecundity advantage in the well-watered control. Flower color variation in selfing species is probably maintained by herbivory, drought stress, and other abiotic factors that vary spatially.

Background Pachycladon cheesemanii is a close relative of Arabidopsis thaliana and is an allotetraploid perennial herb which is widespread in the South Island of New Zealand. It grows at altitudes of up to 1000 m where it is subject to relatively high levels of ultraviolet (UV)-B radiation. To gain first insights into how Pachycladon copes with UV-B stress, we sequenced its genome and compared the UV-B tolerance of two Pachycladon accessions with those of two A. thaliana accessions from different altitudes. Results A high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologs. Synteny analysis with genomes from other species of the Brassicaceae family found a close phylogenetic relationship of P. cheesemanii with Boechera stricta from Brassicaceae lineage I. While UV-B radiation caused a greater growth reduction in the A. thaliana accessions than in the P. cheesemanii accessions, growth was not reduced in one P. cheesemanii accession. The homologues of A. thaliana UV-B radiation response genes were duplicated in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii appears to differ from that in A. thaliana. Conclusion Although the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies allowing the plant to tolerate relatively high UV-B radiation.

Adaptation to large‐scale spatial heterogeneity in the environment accounts for a major proportion of genetic diversity within species. Theory predicts the erosion of adaptive genetic variation on a within‐population level, but considerable genetic diversity is often found locally. Genetic diversity could be expected to be maintained within populations in temporally or spatially variable conditions if genotypic rank orders vary across contrasting microenvironmental settings. Taking advantage of fine‐resolution environmental data, we tested the hypothesis that temperature heterogeneity among years could be one factor maintaining quantitative genetic diversity within a natural and genetically diverse plant population. We sampled maternal families of Boechera stricta, an Arabidopsis thaliana relative, at one location in the central Rocky Mountains and grew them in three treatments that, based on records from an adjacent weather station, simulated hourly temperature changes at the native site during three summers with differing mean temperatures. Treatment had a significant effect on all traits, with 2–3‐fold increase in above‐ and belowground biomass and the highest allocation to roots observed in the treatment simulating the warmest summer on record at the site. Treatment affected bivariate associations between traits, with the weakest correlation between above‐ and belowground biomass in the warmest treatment. The magnitude of quantitative genetic variation for all traits differed across treatments: Genetic variance of biomass was 0 in the warmest treatment, while highly significant diversity was found in average conditions, resulting in broad‐sense heritability of 0.31. Significant genotype × environment interactions across all treatments were found only in root‐to‐shoot ratio. Therefore, temperature variation among summers appears unlikely to account for the observed levels of local genetic variation in size in this perennial species, but may influence family rank order in growth allocation. Our results indicate that natural environmental fluctuations can have a large impact on the magnitude of within‐population quantitative genetic variance.

Circadian clocks have evolved independently in all three domains of life, and fitness benefits of a functional clock have been demonstrated in experimental genotypes in controlled conditions. Still, little is known about genetic variation in the clock and its fitness consequences in natural populations from heterogeneous environments. Using Wyoming populations of the Arabidopsis relative Boechera stricta as our study system, we demonstrate that genetic variation in the clock can occur at multiple levels: means of circadian period among populations sampled at different elevations differed by less than 1 h, but means among families sampled within populations varied by as much as 3.5 h. Growth traits also varied among and within populations. Within the population with the most circadian variation, we observed evidence for a positive correlation between period and growth and a negative correlation between period and root-to-shoot ratio. We then tested whether performance tradeoffs existed among families of this population across simulated seasonal settings. Growth rankings of families were similar across seasonal environments, but for root-to-shoot ratio, genotype × environment interactions contributed significantly to total variation. Therefore, further experiments are needed to identify evolutionary mechanisms that preserve substantial quantitative genetic diversity in the clock in this and other species.

In angiosperms, meiotic failure coupled with the formation of genetically unreduced gametophytes in ovules (apomeiosis) constitute major components of gametophytic apomixis. These aberrant developmental events are generally thought to be caused by mutation. However, efforts to locate the responsible mutations have failed. Herein, we tested a fundamentally different hypothesis: apomeiosis is a polyphenism of meiosis, with meiosis and apomeiosis being maintained by different states of metabolic homeostasis. Microarray analyses of ovules and pistils were used to differentiate meiotic from apomeiotic processes in Boechera (Brassicaceae). Genes associated with translation, cell division, epigenetic silencing, flowering, and meiosis characterized sexual Boechera (meiotic). In contrast, genes associated with stress responses, abscisic acid signaling, reactive oxygen species production, and stress attenuation mechanisms characterized apomictic Boechera (apomeiotic). We next tested whether these metabolic differences regulate reproductive mode. Apomeiosis switched to meiosis when premeiotic ovules of apomicts were cultured on media that increased oxidative stress. These treatments included drought, starvation, and H2O2 applications. In contrast, meiosis switched to apomeiosis when premeiotic pistils of sexual plants were cultured on media that relieved oxidative stress. These treatments included antioxidants, glucose, abscisic acid, fluridone, and 5-azacytidine. High-frequency apomeiosis was initiated in all sexual species tested: Brassicaceae, Boechera stricta, Boechera exilis, and Arabidopsis thaliana, Fabaceae, Vigna unguiculata, Asteraceae, Antennaria dioica. Unreduced gametophytes formed from ameiotic female and male sporocytes, first division restitution dyads, and nucellar cells. These results are consistent with modes of reproduction and types of apomixis, in natural apomicts, being regulated metabolically.

Freezing limits plant growth and crop productivity, and plant species in temperate zones have the capacity to develop freezing tolerance through complex modulation of gene expression affecting various aspects of metabolism and physiology. While many components of freezing tolerance have been identified in model species under controlled laboratory conditions, little is known about the mechanisms that impart freezing tolerance in natural populations of wild species. Here, we performed a quantitative trait locus (QTL) study of acclimated freezing tolerance in seedlings of Boechera stricta, a highly adapted relative of Arabidopsis (Arabidopsis thaliana) native to the Rocky Mountains. A single QTL was identified that contained the gene encoding ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (BstDGAT1), whose expression is highly cold responsive. The primary metabolic enzyme DGAT1 catalyzes the final step in assembly of triacylglycerol (TAG) by acyl transfer from acyl-CoA to diacylglycerol. Freezing tolerant plants showed higher DGAT1 expression during cold acclimation than more sensitive plants, and this resulted in increased accumulation of TAG in response to subsequent freezing. Levels of oligogalactolipids that are produced by SFR2 (SENSITIVE TO FREEZING2), an indispensable element of freezing tolerance in Arabidopsis, were also higher in freezing-tolerant plants. Furthermore, overexpression of AtDGAT1 led to increased freezing tolerance. We propose that DGAT1 confers freezing tolerance in plants by supporting SFR2-mediated remodeling of chloroplast membranes.

Phenotypic plasticity is thought to impact evolutionary trajectories by shifting trait values in a direction that is either favored by natural selection (“adaptive plasticity”) or disfavored (“nonadaptive” plasticity). However, it is unclear how commonly each of these types of plasticity occurs in natural populations. To answer this question, we measured glucosinolate defensive chemistry and reproductive fitness in over 1,500 individuals of the wild perennial mustard Boechera stricta, planted in four common gardens across central Idaho, USA. Glucosinolate profiles—including total glucosinolate quantity as well as the relative abundances and overall diversity of different compounds—were strongly plastic both among habitats and within habitats. Patterns of glucosinolate plasticity varied greatly among genotypes. More often than expected by chance, glucosinolate profiles shifted in a direction that matched the direction of natural selection, indicating that plasticity among habitats tended to increase relative fitness. In contrast, we found no evidence for within-habitat selection on glucosinolate reaction norm slopes (i.e., plasticity along a continuous environmental gradient). Together, our results indicate that glucosinolate plasticity may improve the ability of B. stricta populations to persist after migration to new habitats.

Background and aims In the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality. Methods A progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy. Key results Data from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation. Conclusion These results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

Climate change has induced pronounced shifts in the reproductive phenology of plants, yet we know little about which environmental factors contribute to interspecific variation in responses and their effects on fitness. We integrate data from a 43 yr record of first flowering for six species in subalpine Colorado meadows with a 3 yr snow manipulation experiment on the perennial forb Boechera stricta (Brassicaceae) from the same site. We analyze shifts in the onset of flowering in relation to environmental drivers known to influence phenology: the timing of snowmelt, the accumulation of growing degree days, and photoperiod. Variation in responses to climate change depended on the sequence in which species flowered, with early-flowering species reproducing faster, at a lower heat sum, and under increasingly disparate photoperiods relative to later-flowering species. Early snow-removal treatments confirm that the timing of snowmelt governs observed trends in flowering phenology of B. stricta and that climate change can reduce the probability of flowering, thereby depressing fitness. Our findings suggest that climate change is decoupling historical combinations of photoperiod and temperature and outpacing phenological changes for our focal species. Accurate predictions of biological responses to climate change require a thorough understanding of the factors driving shifts in phenology.

Fixed chromosomal inversions can reduce gene flow and promote speciation in two ways: by suppressing recombination and by carrying locally favoured alleles at multiple loci. However, it is unknown whether favoured mutations slowly accumulate on older inversions or if young inversions spread because they capture pre-existing adaptive quantitative trait loci (QTLs). By genetic mapping, chromosome painting and genome sequencing, we have identified a major inversion controlling ecologically important traits in Boechera stricta. The inversion arose since the last glaciation and subsequently reached local high frequency in a hybrid speciation zone. Furthermore, the inversion shows signs of positive directional selection. To test whether the inversion could have captured existing, linked QTLs, we crossed standard, collinear haplotypes from the hybrid zone and found multiple linked phenology QTLs within the inversion region. These findings provide the first direct evidence that linked, locally adapted QTLs may be captured by young inversions during incipient speciation.Chromosome inversions play an important role in local adaptation and speciation1,2, and selectively important inversions have been identified in many species3,4. Selection due to different environmental factors or stages in the life cycle1 may favour inversions carrying locally adapted alleles at several loci. In addition, established inversions are predicted to accumulate selectively important genetic differences, which may contribute to reproductive isolation during speciation1.Although few studies have identified the actual loci that influence selection on inversions2,4,5, rearrangements may be favoured due to gene alterations near breakpoints6, chromatin changes7 or combinations of advantageous, co-adapted alleles8. Inversions suppress recombination, so locally advantageous alleles may segregate together, causing higher fitness than recombinant haplotypes9. Most evolutionary studies have focused on widespread, older inversions, so we have little knowledge of the evolutionary processes that guide their initial increase in frequency. For example, do inversions drift to higher frequency, and then acquire new advantageous mutations after they are common? Or are multiple linked, advantageous alleles captured in a new inversion, allowing them to spread together? Analysis of younger inversions may elucidate the evolutionary forces controlling the initial spread of chromosome inversions, which therefore influence their role in adaptation and speciation4,8.Related species often differ for chromosome inversions that carry locally favoured alleles at multiple loci10,11. A key distinction among models for the evolution of inversions is whether early frequency increase is due to genetic drift or natural selection. Genetic drift might predominate initially, with subsequent accumulation of advantageous variants12. Alternatively, the Kirkpatrick–Barton model9 argues that linked, locally adapted alleles exist first, and subsequently are captured within a new, selectively favoured inversion13. In this ‘inversion-late’ evolutionary sequence1,5, linked quantitative trait loci (QTLs), similar to the ancestral haplotype that gave rise to the inversion, may still exist in non-inverted genotypes9. Here, we test these predictions of the Kirkpatrick–Barton model. First, we introduce ecologically diverged subspecies of Boechera stricta. Next, we examine a young inversion to infer the selective forces controlling its early increase in frequency. Finally, we cross collinear, standard genotypes from the hybrid zone to ask whether old, linked QTLs can be found within the inversion region.

In order to deter herbivores, plants evolve defenses suited to different tissues and varying patterns of herbivory. If the defense profiles of different tissues share common genetic controls but differ in selective pressures, evolution of effective defenses within the whole plant may be constrained. In my dissertation, I investigate how glucosinolates, biologically active secondary metabolites, evolve in multiple tissues of the crucifer Boechera stricta. I quantify variation in glucosinolate concentration and the relative ratio of each glucosinolate compound (hereafter called the glucosinolate profile) between leaves and fruits and find support for the optimal defense hypothesis, which predicts a higher concentration of glucosinolates in higher-value tissues. Using a large-scale common garden experiment within a population, I demonstrate that antagonistic selection between tissues and genetic covariances in glucosinolate profile between leaves and fruits exercise strong constraint on the evolution of defenses. Additionally, I compare the structure of genetic variances and covariances (the G-matrix) among four genetically differentiated groups of B. stricta and find that the structure of the G-matrix can be altered within approximately 5,000 generations. An Fst-Qst analysis reveals that some differences in glucosinolate profile between were driven by selection.

Plants employ highly variable chemical defenses against a broad community of herbivores, which vary in their susceptibilities to specific compounds. Variation in chemical defenses within the plant has been found in many species; the ecological and evolutionary influences on this variation, however, are less well-understood. One central theory describing the allocation of defenses in the plant is the Optimal Defense Hypothesis (ODH), which predicts that defenses will be concentrated in tissues that are of high fitness value to the plant. Although the ODH has been repeatedly supported within vegetative tissues, few studies have compared vegetative and reproductive tissues, and the results have not been conclusive. We quantified variation in glucosinolate profile and tissue value between vegetative and reproductive tissues in Boechera stricta, a close relative of Arabidopsis. B. stricta manufactures glucosinolates, a set of defensive compounds that vary genetically and are straightforward to quantify. Genetic diversity in glucosinolate profile has been previously demonstrated to be important to both herbivory and fitness in B. stricta; however, the importance of glucosinolate variation among tissues has not. Here, we investigate whether allocation of glucosinolates within the plant is consistent with the ODH. We used both clipping experiments on endogenous plants and ambient herbivory in a large-scale transplant experiment at three sites to quantify fitness effects of loss of rosette leaves, cauline leaves, and flowers and fruits. We measured glucosinolate concentration in leaves and fruits in the transplant experiment, and asked whether more valuable tissues were more defended. We also investigated within-plant variation in other aspects of the glucosinolate profile. Our results indicated that damage to fruits had a significantly larger effect on overall fitness than damage to leaves, and that fruits had much higher concentrations of glucosinolates, supporting the ODH. This is, to the best of our knowledge, the first study to explicitly compare both tissue value and chemical defense concentrations between vegetative and reproductive tissues under natural conditions.

Development of chilling and freezing tolerance is complex and can be affected by photoperiod, temperature and photosynthetic performance; however, there has been limited research on the interaction of these three factors. We evaluated 108 recombinant inbred lines of Boechera stricta, derived from a cross between lines originating from Montana and Colorado, under controlled long day (LD), short-day (SD) and in an outdoor environment (OE). We measured maximum quantum yield of photosystem II, lethal temperature for 50% survival and electrolyte leakage of leaves. Our results revealed significant variation for chilling and freezing tolerance and photosynthetic performance in different environments. Using both single- and multi-trait analyses, three main-effect quantitative trait loci (QTL) were identified. QTL on linkage group (LG)3 were SD specific, whereas QTL on LG4 were found under both LD and SD. Under all conditions, QTL on LG7 were identified, but were particularly predictive for the outdoor experiment. The co-localization of photosynthetic performance and freezing tolerance effects supports these traits being co-regulated. Finally, the major QTL on LG7 is syntenic to the Arabidopsis C-repeat binding factor locus, known regulators of chilling and freezing responses in Arabidopsis thaliana and other species.

Fitness trade-offs across episodes of selection and environments influence life-history evolution and adaptive population divergence. Documenting these trade-offs remains challenging as selection can vary in magnitude and direction through time and space. Here, we evaluate fitness trade-offs at the levels of the whole organism and the quantitative trait locus (QTL) in a multiyear field study of Boechera stricta (Brassicaceae), a genetically tractable mustard native to the Rocky Mountains. Reciprocal local adaptation was pronounced for viability, but not for reproductive components of fitness. Instead, local genomes had a fecundity advantage only in the high latitude garden. By estimating realized selection coefficients from individual-level data on viability and reproductive success and permuting the data to infer significance, we examined the genetic basis of fitness trade-offs. This analytical approach (Conditional Neutrality-Antagonistic Pleiotropy, CNAP) identified genetic trade-offs at a flowering phenology QTL (costs of adaptation) and revealed genetic trade-offs across fitness components (costs of reproduction). These patterns would not have emerged from traditional ANOVA-based QTL mapping. Our analytical framework can be applied to other systems to investigate fitness trade-offs. This task is becoming increasingly important as climate change may alter fitness landscapes, potentially disrupting fitness trade-offs that took many generations to evolve.

Ecological factors may contribute to reproductive isolation if differential local adaptation causes immigrant or hybrid fitness reduction. Since local adaptation results from the interaction between natural selection and adaptive traits, it is crucial to investigate both in order to understand ecological speciation. Previously, we used niche modeling to identify local water availability as an environmental correlate of incipient ecological speciation between two subspecies in Boechera stricta, a close relative of Arabidopsis. Here, we performed several large-scale greenhouse experiments to investigate the divergence of various physiological, phenological, and morphological traits. Although we found no significant difference in physiological traits, the Western subspecies has significantly faster growth rate, larger leaf area, less succulent leaves, delayed reproductive time, and longer flowering duration. These trait differences are concordant with previous results that habitats of the Western genotypes have more consistent water availability, while Eastern genotypes inhabit locations with more ephemeral water supplies. In addition, by comparing univariate and multivariate divergence of complex traits (QST) to the genome-wide distribution of SNP FST, we conclude that aspects of phenology and morphology (but not physiology) are under divergent selection. In addition, we also identified several highly diverged traits without obvious water-related functions.

Genetically based trait variation across environmental gradients can reflect adaptation to local environments. However, natural populations that appear well-adapted often exhibit directional, not stabilizing, selection on ecologically relevant traits. Temporal variation in the direction of selection could lead to stabilizing selection across multiple episodes of selection, which might be overlooked in short-term studies that evaluate relationships of traits and fitness under only one set of conditions. Furthermore, nonrandom mortality prior to trait expression can bias inferences about trait evolution if viability selection opposes fecundity selection. Here, we leveraged fitness and trait data to test whether phenotypic clines are genetically based and adaptive, whether temporal variation in climate imposes stabilizing selection, and whether viability selection acts on adult phenotypes. We monitored transplants of the subalpine perennial forb, Boechera stricta (Brassicaceae), in common gardens at two elevations over 2–3 years that differed in drought intensity. We quantified viability, and fecundity fitness components for four heritable traits: specific leaf area, integrated water-use efficiency, height at first flower, and flowering phenology. Our results indicate that genetic clines are maintained by selection, but their expression is context dependent, as they do not emerge in all environments. Moreover, selection varied spatially and temporally. Stabilizing selection was most pronounced when we integrated data across years. Finally, viability selection prior to trait expression targeted adult phenotypes (age and size at flowering). Indeed, viability selection for delayed flowering opposed fecundity selection for accelerated flowering; this result demonstrates that neglecting to account for viability selection could lead to inaccurate conclusions that populations are maladapted. Our results suggest that reconciling clinal trait variation with selection requires data collected across multiple spatial scales, time frames, and life-history stages.

Bacteria living on and in leaves and roots influence many aspects of plant health, so the extent of a plant's genetic control over its microbiota is of great interest to crop breeders and evolutionary biologists. Laboratory-based studies, because they poorly simulate true environmental heterogeneity, may misestimate or totally miss the influence of certain host genes on the microbiome. Here we report a large-scale field experiment to disentangle the effects of genotype, environment, age and year of harvest on bacterial communities associated with leaves and roots of Boechera stricta (Brassicaceae), a perennial wild mustard. Host genetic control of the microbiome is evident in leaves but not roots, and varies substantially among sites. Microbiome composition also shifts as plants age. Furthermore, a large proportion of leaf bacterial groups are shared with roots, suggesting inoculation from soil. Our results demonstrate how genotype-by-environment interactions contribute to the complexity of microbiome assembly in natural environments., Laboratory-based studies of the effect of plant genotype on plant microbiome composition often do not consider the influence of environmental heterogeneity. Here, Wagner et al. use a large-scale field experiment to assess the extent of host genetic and environmental factors on the microbiome of Boechera stricta.

Display Omitted The structure of the DNMTs genes in apomict and sexual Boechera species are highly similar to A. thaliana orthologs.While the expression levels of MET1 and CMT3 do not differ much, DRM2 upregulated in developing seeds of apomictic Boechera spp.Our results suggest that a deregulation in the DNA methylation machinery during endosperm development may require for apomixis. In this study, we determined the structure of DNA methyltransferase (DNMT) genes in apomict and sexual Boechera species and investigated the expression levels during seed development. Protein and DNA sequences of diploid sexual Boechera stricta DNMT genes obtained from Phytozome 10.3 were used to identify the homologues in apomicts, Boechera holboellii and Boechera divaricarpa. Geneious R8 software was used to map the short-paired reads library of B. holboellii whole genome or B. divaricarpa transcriptome reads to the reference gene sequences. We determined three DNMT genes; for Boechera spp. METHYLTRANSFERASE1 (MET1), CHROMOMETHYLASE 3 (CMT3) and DOMAINS REARRANGED METHYLTRANSFERASE 1/2 (DRM2). We examined the structure of these genes with bioinformatic tools and compared with other DNMT genes in plants. We also examined the levels of expression in silique tissues after fertilization by semi-quantitative PCR. The structure of DNMT proteins in apomict and sexual Boechera species share common features. However, the expression levels of DNMT genes were different in apomict and sexual Boechera species. We found that DRM2 was upregulated in apomictic Boechera species after fertilization. Phylogenetic trees showed that three genes are conserved among green algae, monocotyledons and dicotyledons. Our results indicated a deregulation of DNA methylation machinery during seed development in apomicts.

Low elevation “trailing edge” range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, but that antagonistic crosstalk prevents their simultaneous co-option. To test this hypothesis, we compared high and low elevation populations of Boechera stricta that have diverged with respect to constitutive levels of glucosinolate defenses and root:shoot ratios; neither population has high levels of both traits. If constraints imposed by antagonistic signaling underlie this divergence, one would predict that high constitutive levels of traits would coincide with lower plasticity. To test this prediction, we compared the genetically diverged populations in a double challenge drought-herbivory growth chamber experiment. Although a glucosinolate defense response to the generalist insect herbivore Spodoptera exigua was attenuated under drought conditions, the plastic defense response did not differ significantly between populations. Similarly, although several potential drought tolerance traits were measured, only stomatal aperture behavior, as measured by carbon isotope ratios, was less plastic as predicted in the high elevation population. However, RNAseq results on a small subset of plants indicated differential expression of relevant genes between populations as predicted. We suggest that the ambiguity in our results stems from a weaker link between the pathways and the functional traits compared to transcripts.

Anthropogenic climate change has already altered the timing of major life-history transitions, such as the initiation of reproduction. Both phenotypic plasticity and adaptive evolution can underlie rapid phenological shifts in response to climate change, but their relative contributions are poorly understood. Here, we combine a continuous 38 year field survey with quantitative genetic field experiments to assess adaptation in the context of climate change. We focused on Boechera stricta (Brassicaeae), a mustard native to the US Rocky Mountains. Flowering phenology advanced significantly from 1973 to 2011, and was strongly associated with warmer temperatures and earlier snowmelt dates. Strong directional selection favoured earlier flowering in contemporary environments (2010–2011). Climate change could drive this directional selection, and promote even earlier flowering as temperatures continue to increase. Our quantitative genetic analyses predict a response to selection of 0.2 to 0.5 days acceleration in flowering per generation, which could account for more than 20 per cent of the phenological change observed in the long-term dataset. However, the strength of directional selection and the predicted evolutionary response are likely much greater now than even 30 years ago because of rapidly changing climatic conditions. We predict that adaptation will likely be necessary for long-term in situ persistence in the context of climate change.

Divergent natural selection promotes local adaptation and can lead to reproductive isolation of populations in contrasting environments; however, the genetic basis of local adaptation remains largely unresolved in natural populations. Local adaptation might result from antagonistic pleiotropy, where alternate alleles are favoured in distinct habitats, and polymorphism is maintained by selection. Alternatively, under conditional neutrality some alleles may be favoured in one environment but neutral at other locations. Antagonistic pleiotropy maintains genetic variation across the landscape; however, there is a systematic bias against discovery of antagonistic pleiotropy because the fitness benefits of local alleles need to be significant in at least two environments. Here, we develop a generally applicable method to investigate polygenic local adaptation and identify loci that are the targets of selection. This approach evaluates allele frequency changes after selection at loci across the genome to distinguish antagonistic pleiotropy from conditional neutrality and deleterious variation. We investigate local adaptation at the qualitative trait loci (QTL) level in field experiments, in which we expose 177 F(6) recombinant inbred lines and parental lines of Boechera stricta (Brassicaceae) to their parental environments over two seasons. We demonstrate polygenic selection for native alleles in both environments, with 2.8% of the genome exhibiting antagonistic pleiotropy and 8% displaying conditional neutrality. Our study strongly supports antagonistic pleiotropy at one large-effect flowering phenology QTL (nFT): native homozygotes had significantly greater probabilities of flowering than foreign homozygotes in both parental environments. Such large-scale field studies are essential to elucidate the genetic basis of adaptation in natural populations.

What causes range limits is a central question in evolutionary ecology. Transplant studies indicate that areas just across range boundaries are often stressful. The recent Defense constraint (DC) hypothesis for plants states that the evolution of tolerance to stressful environments across a range boundary is constrained by allocation to chemical defense because of antagonistic crosstalk between abiotic and biotic stress signaling pathways that otherwise could be co-opted for range expansion. Abscisic acid (ABA) drought stress tolerance and jasmonic acid/ethylene (JA/ET) defense signaling pathways, for example, are known to be antagonistic to one another in Arabidopsis and other species. To test the DC hypothesis, we examined quantitative genetic variation and co-variation among marker-inferred inbred lines and sib-families of Boechera stricta, a close wild relative of Arabidopsis. The dynamics of the defense-stress tolerance tradeoff was examined across 1) years that differed in precipitation, 2) drought and ABA treatments, and 3) a NPK nutrient supply gradient. In support of the DC hypothesis, we observed the tradeoff a) in the dry year, and b) in response to water deficiency, which c) was affected by ABA treatments, but the interaction between ABA and glucosinolate (GS) toxin levels was not significant. In contrast to the effects of water deficiency, d) the effect of lower NPK supply to cause the tradeoff was only marginally significant. Because an ABA-mediated stress response is intrinsic to water deficient conditions and because of the known involvement of JA/ET in GS regulation, we suggest that these results provide circumstantial evidence implicating both of these pathways in the tradeoff and thus in the development of range limits.