Does urbanisation lead to parallel demographic shifts across the world in a cosmopolitan plant?

Urbanisation is occurring globally, leading to dramatic environmental changes that are altering the ecology and evolution of species. In particular, the expansion of human infrastructure and the loss and fragmentation of natural habitats in cities is predicted to increase genetic drift and reduce gene flow by reducing the size and connectivity of populations. Alternatively, the 'urban facilitation model' suggests that some species will have greater gene flow into and within cities leading to higher diversity and lower differentiation in urban populations. These alternative hypotheses have not been contrasted across multiple cities. Here, we used the genomic data from the GLobal Urban Evolution project (GLUE), to study the effects of urbanisation on non-adaptive evolutionary processes of white clover (Trifolium repens) at a global scale. We found that white clover populations presented high genetic diversity and no evidence of reduced Ne linked to urbanisation. On the contrary, we found that urban populations were less likely to experience a recent decrease in effective population size than rural ones. In addition, we found little genetic structure among populations both globally and between urban and rural populations, which showed extensive gene flow between habitats. Interestingly, white clover displayed overall higher gene flow within urban areas than within rural habitats. Our study provides the largest comprehensive test of the demographic effects of urbanisation. Our results contrast with the common perception that heavily altered and fragmented urban environments will reduce the effective population size and genetic diversity of populations and contribute to their isolation.

Haplotype-Resolved, Chromosome-Level Assembly of White Clover (Trifolium repens L., Fabaceae).

White clover (Trifolium repens L.; Fabaceae) is an important forage and cover crop in agricultural pastures around the world and is increasingly used in evolutionary ecology and genetics to understand the genetic basis of adaptation. Historically, improvements in white clover breeding practices and assessments of genetic variation in nature have been hampered by a lack of high-quality genomic resources for this species, owing in part to its high heterozygosity and allotetraploid hybrid origin. Here, we use PacBio HiFi and chromosome conformation capture (Omni-C) technologies to generate a chromosome-level, haplotype-resolved genome assembly for white clover totaling 998 Mbp (scaffold N50 = 59.3 Mbp) and 1 Gbp (scaffold N50 = 58.6 Mbp) for haplotypes 1 and 2, respectively, with each haplotype arranged into 16 chromosomes (8 per subgenome). We additionally provide a functionally annotated haploid mapping assembly (968 Mbp, scaffold N50 = 59.9 Mbp), which drastically improves on the existing reference assembly in both contiguity and assembly accuracy. We annotated 78,174 protein-coding genes, resulting in protein BUSCO completeness scores of 99.6% and 99.3% against the embryophyta_odb10 and fabales_odb10 lineage datasets, respectively.

Demographic consequences of an extreme heat wave are mitigated by spatial heterogeneity in an annual monkeyflower.

Heat waves are becoming more frequent and intense with climate change, but the demographic and evolutionary consequences of heat waves are rarely investigated in herbaceous plant species. We examine the consequences of a short but extreme heat wave in Oregon populations of the common yellow monkeyflower (Mimulus guttatus) by leveraging a common garden experiment planted with range-wide populations and observational studies of 11 local populations. In the common garden, 89% of seedlings died during the heat wave including >96% of seedlings from geographically local populations. Some populations from hotter and drier environments had higher fitness, however, others from comparable environments performed poorly. Observational studies of local natural populations drastically differed in the consequences of the heat wave-one population was completely extirpated and nearly half had a >50% decrease in fitness. However, a few populations had greater fitness during the heat wave year. Differences in mortality corresponded to the impact of the heat wave on soil moisture-retention of soil moisture throughout the heat wave led to greater survivorship. Our results suggest that not all populations experience the same intensity or degree of mortality during extreme events and such heterogeneity could be important for genetic rescue or to facilitate the distribution of adaptive variants throughout the region.

Unique drought resistance strategies occur among monkeyflower populations spanning an aridity gradient.

PREMISE: Annual plants often exhibit drought-escape and avoidance strategies to cope with limited water availability. Determining the extent of variation and factors underlying the evolution of divergent strategies is necessary for determining population responses to more frequent and severe droughts. METHODS: We leveraged five Mimulus guttatus populations collected across an aridity gradient within manipulative drought and quantitative genetics experiments to examine constitutive and terminal-drought induced responses in drought resistance traits. RESULTS: Populations varied considerably in drought-escape- and drought-avoidance-associated traits. The most mesic population demonstrated a unique resource conservative strategy. Xeric populations exhibited extreme plasticity when exposed to terminal drought that included flowering earlier at shorter heights, increasing water-use efficiency, and shifting C:N ratios. However, plasticity responses also differed between populations, with two populations slowing growth rates and flowering at earlier nodes and another population increasing growth rate. While nearly all traits were heritable, phenotypic correlations differed substantially between treatments and often, populations. CONCLUSIONS: Our results suggest drought resistance strategies of populations may be finely adapted to local patterns of water availability. Substantial plastic responses suggest that xeric populations can already acclimate to drought through plasticity, but populations not frequently exposed to drought may be more vulnerable.

Life history and chemical defense interact to drive patterns of adaptation in an annual monkeyflower.

Although chemical defenses and herbivore pressure are widely established as key targets and agents of selection, their roles in local adaptation and determining potential evolutionary responses to changing climates are often neglected. Here, we explore fitness differences between 11 rangewide M. guttatus populations in a field common garden experiment and assess the agents and targets of selection driving relative fitness patterns. We use piecewise structural equation models to disentangle associations between chemical defenses, (phenylpropanoid glycosides; PPGs), and life history traits with herbivory and fitness. While the historical environment of populations is not predictive of fitness differences between populations, >90% of variation in fitness can be predicted by the flowering time and foliar PPG defense arsenal of a population. Piecewise structural equation models indicate that life history traits, particularly earlier flowering time, are strongly and directly linked to fitness. However, herbivory, particularly fruit predation, is also an important agent of selection that creates indirect links between fitness and both chemical defenses and life history traits. Our results emphasize the multivariate nature of the agents and targets of selections in producing adaptation and suggest that future responses to selection must navigate a complex fitness landscape.

Evolution in response to climate in the native and introduced ranges of a globally distributed plant.

The extent to which species can adapt to spatiotemporal climatic variation in their native and introduced ranges remains unresolved. To address this, we examined how clines in cyanogenesis (hydrogen cyanide [HCN] production-an antiherbivore defense associated with decreased tolerance to freezing) have shifted in response to climatic variation in space and time over a 60-year period in both the native and introduced ranges of Trifolium repens. HCN production is a polymorphic trait controlled by variation at two Mendelian loci (Ac and Li). Using phenotypic assays, we estimated within-population frequencies of HCN production and dominant alleles at both loci (i.e., Ac and Li) from 10,575 plants sampled from 131 populations on five continents, and then compared these frequencies to those from historical data collected in the 1950s. There were no clear relationships between changes in the frequency of HCN production, Ac, or Li and changes in temperature between contemporary and historical samples. We did detect evidence of continued evolution to temperature gradients in the introduced range, whereby the slope of contemporary clines for HCN and Ac in relation to winter temperature became steeper than historical clines and more similar to native clines. These results suggest that cyanogenesis clines show no clear changes through time in response to global warming, but introduced populations continue to adapt to their contemporary environments.

Adaptive and nonadaptive causes of heterogeneity in genetic differentiation across the Mimulus guttatus genome.

Genetic diversity becomes structured among populations over time due to genetic drift and divergent selection. Although population structure is often treated as a uniform underlying factor, recent resequencing studies of wild populations have demonstrated that diversity in many regions of the genome may be structured quite dissimilar to the genome-wide pattern. Here, we explored the adaptive and nonadaptive causes of such genomic heterogeneity using population-level, whole genome resequencing data obtained from annual Mimulus guttatus individuals collected across a rugged environment landscape. We found substantial variation in how genetic differentiation is structured both within and between chromosomes, although, in contrast to other studies, known inversion polymorphisms appear to serve only minor roles in this heterogeneity. In addition, much of the genome can be clustered into eight among-population genetic differentiation patterns, but only two of these clusters are particularly consistent with patterns of isolation by distance. By performing genotype-environment association analysis, we also identified genomic intervals where local adaptation to specific climate factors has accentuated genetic differentiation among populations, and candidate genes in these windows indicate climate adaptation may proceed through changes affecting specialized metabolism, drought resistance, and development. Finally, by integrating our findings with previous studies, we show that multiple aspects of plant reproductive biology may be common targets of balancing selection and that variants historically involved in climate adaptation among populations have probably also fuelled rapid adaptation to microgeographic environmental variation within sites.

Human Urine Alters Methicillin-Resistant Staphylococcus aureus Virulence and Transcriptome.

Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of hospital-associated urinary tract infections (UTI), especially in catheterized individuals. Despite being rare, MRSA UTI are prone to potentially life-threatening exacerbations such as bacteremia that can be refractory to routine antibiotic therapy. To delineate the molecular mechanisms governing MRSA urinary pathogenesis, we exposed three S. aureus clinical isolates, including two MRSA strains, to human urine for 2 h and analyzed virulence characteristics and changes in gene expression. The in vitro virulence assays showed that human urine rapidly alters adherence to human bladder epithelial cells and fibronectin, hemolysis of sheep red blood cells (RBCs), and surface hydrophobicity in a staphylococcal strain-specific manner. In addition, transcriptome sequencing (RNA-Seq) analysis of uropathogenic strain MRSA-1369 revealed that 2-h-long exposure to human urine alters MRSA transcriptome by modifying expression of genes encoding enzymes catalyzing metabolic pathways, virulence factors, and transcriptional regulators. In summary, our results provide important insights into how human urine specifically and rapidly alters MRSA physiology and facilitates MRSA survival in the nutrient-limiting and hostile urinary microenvironment. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is an uncommon cause of urinary tract infections (UTI) in the general population. However, it is important to understand MRSA pathophysiology in the urinary tract because isolation of MRSA in urine samples often precedes potentially life-threatening MRSA bacteremia. In this report, we describe how exposure to human urine alters MRSA global gene expression and virulence. We hypothesize that these alterations may aid MRSA in acclimating to the nutrient-limiting, immunologically hostile conditions within the urinary tract leading to MRSA UTI.

Evolutionary history and ecology shape the diversity and abundance of phytochemical arsenals across monkeyflowers.

We examine the extent to which phylogenetic effects and ecology are associated with macroevolutionary patterns of phytochemical defence production across the Mimulus phylogeny. We grew plants from 21 species representing the five major sections of the Mimulus phylogeny in a common garden to assess how the arsenals (NMDS groupings) and abundances (concentrations) of a phytochemical defence, phenylpropanoid glycosides (PPGs), vary across the phylogeny. Very few PPGs are widespread across the genus, but many are common to multiple sections of the genus. Phytochemical arsenals cluster among sections in an NMDS and are not associated with total concentration of PPGs. There is a strong phylogenetic signal for phytochemical arsenal composition across the Mimulus genus, whereas ecological variables such as growing season length, latitude, and elevation do not significantly influence arsenal. In contrast, there is little phylogenetic signal for total PPG concentration, and this trait is significantly influenced by several ecological factors. Phytochemical arsenals and abundances are influenced by plant life history form. Both phylogenetic effects and ecology are related to phytochemical patterns across species, albeit in different ways. The independence of phytochemical defence concentrations from arsenal compositions indicates that these aspects of defence may continue to evolve independently of one another.

Population responses to a historic drought across the range of the common monkeyflower (Mimulus guttatus).

PREMISE: Due to climate change, more frequent and intense periodic droughts are predicted to increasingly pose major challenges to the persistence of plant populations. When a severe drought occurs over a broad geographical region, independent responses by individual populations provide replicated natural experiments for examining the evolution of drought resistance and the potential for evolutionary rescue. METHODS: We used a resurrection approach to examine trait evolution in populations of the common monkeyflower, Mimulus guttatus, exposed to a record drought in California from 2011 to 2017. Specifically, we compared variation in traits related to drought escape and avoidance from seeds collected from 37 populations pre- and post-drought in a common garden. In a parallel experiment, we evaluated fitness in two populations, one which thrived and one which was nearly extirpated during the drought, under well-watered and dry-down conditions. RESULTS: We observed substantial variation among populations in trait evolution. In the subset of populations where phenotypes changed significantly, divergence proceeded along trait correlations with some populations flowering rapidly with less vegetative tissue accumulation and others delaying flowering with greater vegetative tissue accumulation. The degree of trait evolution was only weakly correlated with drought intensity but strongly correlated with initial levels of standing variation. Fitness was higher in the post-drought than pre-drought accessions in both treatments for the thriving population, but lower in both treatments for the nearly extirpated population. CONCLUSIONS: Together, our results indicate that evolutionary responses to drought are context dependent and reflect the standing genetic variation and genetic correlations present within populations.

The Genetic Architecture of Plant Defense Trade-offs in a Common Monkeyflower.

Determining how adaptive combinations of traits arose requires understanding the prevalence and scope of genetic constraints. Frequently observed phenotypic correlations between plant growth, defenses, and/or reproductive timing have led researchers to suggest that pleiotropy or strong genetic linkage between variants affecting independent traits is pervasive. Alternatively, these correlations could arise via independent mutations in different genes for each trait and extensive correlational selection. Here we evaluate these alternatives by conducting a quantitative trait loci (QTL) mapping experiment involving a cross between 2 populations of common monkeyflower (Mimulus guttatus) that differ in growth rate as well as total concentration and arsenal composition of plant defense compounds, phenylpropanoid glycosides (PPGs). We find no evidence that pleiotropy underlies correlations between defense and growth rate. However, there is a strong genetic correlation between levels of total PPGs and flowering time that is largely attributable to a single shared QTL. While this result suggests a role for pleiotropy/close linkage, several other QTLs also contribute to variation in total PPGs. Additionally, divergent PPG arsenals are influenced by a number of smaller-effect QTLs that each underlie variation in 1 or 2 PPGs. This result indicates that chemical defense arsenals can be finely adapted to biotic environments despite sharing a common biochemical precursor. Together, our results show correlations between defense and life-history traits are influenced by pleiotropy or genetic linkage, but genetic constraints may have limited impact on future evolutionary responses, as a substantial proportion of variation in each trait is controlled by independent loci.

Lagging Adaptation to Climate Supersedes Local Adaptation to Herbivory in an Annual Monkeyflower.

While native populations are often adapted to historical biotic and abiotic conditions at their home site, populations from other locations in the range may be better adapted to current conditions due to changing climates or extreme conditions in a single year. We examine whether local populations of a widespread species maintain a relative advantage over distant populations that have evolved at sites better matching the current climate. Specifically, we grew lines derived from low- and high-elevation annual populations in California and Oregon of the common monkeyflower (Erythranthe guttata) and conducted phenotypic selection analyses in low- and high-elevation common gardens in Oregon to examine relative fitness and the traits mediating relative fitness. Californian low-elevation populations have the highest relative fitness at the low-elevation site, and Californian high-elevation populations have the highest relative fitness at the high-elevation site. Relative fitness differences are mediated by selection for properly timed transitions to flowering, with selection favoring more rapid growth rates at the low-elevation site and greater vegetative biomass prior to flowering at the high-elevation site. Fitness advantages for Californian plants occur despite incurring higher herbivory at both sites than the native Oregonian plants. Our findings suggest that a lag in adaptation causes maladaptation in extreme years that may be more prevalent in future climates, but local populations still have high growth rates and thus are not yet threatened.

Freeze-induced cyanide toxicity does not maintain the cyanogenesis polymorphism in white clover (Trifolium repens).

PREMISE OF THE STUDY: The maintenance of adaptive polymorphisms within species requires fitness trade-offs reflecting selection for each morph. Cyanogenesis, the ability to produce hydrogen cyanide (HCN) after tissue damage, occurs in >3000 plant species and exists as a discrete polymorphism in white clover. This polymorphism is spatially distributed in recurrent clines, with higher frequencies of cyanogenic plants in warmer climates. The HCN autotoxicity hypothesis proposes that cyanogenic plants are selected against where frosts are common, as freezing liberates HCN and could impair cellular respiration. METHODS: We tested the HCN autotoxicity hypothesis using a freezing chamber to examine survival, tissue damage, and physiological recovery as assessed via chlorophyll fluorescence following mild and severe freezing treatments. We utilized 65 genotypes from a single polymorphic population to eliminate effects of population structure. KEY RESULTS: Cyanogenic plants did not differ from acyanogenic plants in survival, tissue damage, or recovery following freezing. However, plants producing either of the two required cyanogenic precursors had lower survival and tissue damage after freezing than plants lacking both precursors. CONCLUSIONS: These results suggest that freezing-induced HCN toxicity is unlikely to be responsible for the maintenance of the cyanogenesis polymorphism in white clover. However, energetic trade-offs associated with costs of producing the cyanogenic precursors may confer a fitness benefit to acyanogenic plants under stressful climatic conditions. The lack of evidence for HCN toxicity suggests that cyanogenic clover uses physiological mechanisms mediated by ß-cyanoalanine synthase and alternative oxidase to maintain cellular function in the presence of HCN.

Competition drives trait evolution and character displacement between Mimulus species along an environmental gradient.

Closely related species may evolve to coexist stably in sympatry through niche differentiation driven by in situ competition, a process termed character displacement. Alternatively, past evolution in allopatry may have already sufficiently reduced niche overlap to permit establishment in sympatry, a process called ecological sorting. The relative importance of each process to niche differentiation is contentious even though they are not mutually exclusive and are both mediated via multivariate trait evolution. We explore how competition has impacted niche differentiation in two monkeyflowers, Mimulus alsinoides and M. guttatus, which often co-occur. Through field observations, common gardens, and competition experiments, we demonstrate that M. alsinoides is restricted to marginal habitats in sympatry and that the impacts of character displacement on niche differentiation are complex. Competition with M. guttatus alters selection gradients and has favored taller M. alsinoides with earlier seasonal flowering at low elevation and floral shape divergence at high elevation. However, no trait exhibits the pattern typically associated with character displacement, higher divergence between species in sympatry than allopatry. Thus, although character displacement was unlikely the process driving initial divergence along niche axes necessary for coexistence, we conclude that competition in sympatry has likely driven trait evolution along additional niche axes.

Optimal defense theory explains deviations from latitudinal herbivory defense hypothesis.

The latitudinal herbivory defense hypothesis (LHDH) postulates that the prevalence of species interactions, including herbivory, is greater at lower latitudes, leading to selection for increased levels of plant defense. While latitudinal defense clines may be caused by spatial variation in herbivore pressure, optimal defense theory predicts that clines could also be caused by ecogeographic variation in the cost of defense. For instance, allocation of resources to defense may not increase plant fitness when growing seasons are short and plants must reproduce quickly. Here we use a common garden experiment to survey genetic variation for constitutive and induced phenylpropanoid glycoside (PPG) concentrations across 35 Mimulus guttatus populations over a ~13° latitudinal transect. Our sampling regime is unique among studies of the LHDH in that it allows us to disentangle the effects of growing season length from those of latitude, temperature, and elevation. For five of the seven PPGs surveyed, we find associations between latitude and plant defense that are robust to population structure. However, contrary to the LHDH, only two PPGs were found at higher levels in low latitude populations, and total PPG concentrations were higher at higher latitudes. PPG levels are strongly correlated with growing season length, with higher levels of PPGs in plants from areas with longer growing seasons. Further, flowering time is positively correlated with the concentration of nearly all PPGs, suggesting that there may be a strong trade-off between development time and defense production. Our results reveal that ecogeographic patterns in plant defense may reflect variation in the cost of producing defense compounds in addition to variation in herbivore pressure. Thus, the biogeographic pattern predicted by the LHDH may not be accurate because the underlying factors driving variation in defense, in this case, growing season length, are not always associated with latitude in the same manner. Given these results, we conclude that LHDH cannot be interpreted without considering life history, and we recommend that future work on the LHDH move beyond solely testing the core LHDH prediction and place greater emphasis on isolating agents of selection that generate spatial variation in defense and herbivore pressure.

The evolution of drought escape and avoidance in natural herbaceous populations.

While the functional genetics and physiological mechanisms controlling drought resistance in crop plants have been intensely studied, less research has examined the genetic basis of adaptation to drought stress in natural populations. Drought resistance adaptations in nature reflect natural rather than human-mediated selection and may identify novel mechanisms for stress tolerance. Adaptations conferring drought resistance have historically been divided into alternative strategies including drought escape (rapid development to complete a life cycle before drought) and drought avoidance (reducing water loss to prevent dehydration). Recent studies in genetic model systems such as Arabidopsis, Mimulus, and Panicum have begun to elucidate the genes, expression profiles, and physiological changes responsible for ecologically important variation in drought resistance. Similar to most crop plants, variation in drought escape and avoidance is complex, underlain by many QTL of small effect, and pervasive gene by environment interactions. Recently identified major-effect alleles point to a significant role for genetic constraints in limiting the concurrent evolution of both drought escape and avoidance strategies, although these constraints are not universally found. This progress suggests that understanding the mechanistic basic and fitness consequences of gene by environment interactions will be critical for crop improvement and forecasting population persistence in unpredictable environments.

Replicate altitudinal clines reveal that evolutionary flexibility underlies adaptation to drought stress in annual Mimulus guttatus.

Examining how morphology, life history and physiology vary along environmental clines can reveal functional insight into adaptations to climate and thus inform predictions about evolutionary responses to global change. Widespread species occurring over latitudinal and altitudinal gradients in seasonal water availability are excellent systems for investigating multivariate adaptation to drought stress. Under common garden conditions, we characterized variation in 27 traits for 52 annual populations of Mimulus guttatus sampled from 10 altitudinal transects. We also assessed variation in the critical photoperiod for flowering and surveyed neutral genetic markers to control for demography when analyzing clinal patterns. Many drought escape (e.g. flowering time) and drought avoidance (e.g. specific leaf area, succulence) traits exhibited geographic or climatic clines, which often remained significant after accounting for population structure. Critical photoperiod and flowering time in glasshouse conditions followed distinct clinal patterns, indicating different aspects of seasonal phenology confer adaptation to unique agents of selection. Although escape and avoidance traits were negatively correlated range-wide, populations from sites with short growing seasons produced both early flowering and dehydration avoidance phenotypes. Our results highlight how abundant genetic variation in the component traits that build multivariate adaptations to drought stress provides flexibility for intraspecific adaptation to diverse climates.

Adaptive gains through repeated gene loss: parallel evolution of cyanogenesis polymorphisms in the genus Trifolium (Fabaceae).

Variation in cyanogenesis (hydrogen cyanide release following tissue damage) was first noted in populations of white clover more than a century ago, and subsequent decades of research have established this system as a classic example of an adaptive chemical defence polymorphism. Here, we document polymorphisms for cyanogenic components in several relatives of white clover, and we determine the molecular basis of this trans-specific adaptive variation. One hundred and thirty-nine plants, representing 13 of the 14 species within Trifolium section Trifoliastrum, plus additional species across the genus, were assayed for cyanogenic components (cyanogenic glucosides and their hydrolysing enzyme, linamarase) and for the presence of underlying cyanogenesis genes (CYP79D15 and Li, respectively). One or both cyanogenic components were detected in seven species, all within section Trifoliastrum; polymorphisms for the presence/absence (PA) of components were detected in six species. In a pattern that parallels our previous findings for white clover, all observed biochemical polymorphisms correspond to gene PA polymorphisms at CYP79D15 and Li. Relationships of DNA sequence haplotypes at the cyanogenesis loci and flanking genomic regions suggest independent evolution of gene deletions within species. This study thus provides evidence for the parallel evolution of adaptive biochemical polymorphisms through recurrent gene deletions in multiple species.

Aridity shapes cyanogenesis cline evolution in white clover (Trifolium repens L.).

Adaptive differentiation between populations is often proposed to be the product of multiple interacting selective pressures, although empirical support for this is scarce. In white clover, populations show adaptive differentiation in frequencies of cyanogenesis, the ability to produce hydrogen cyanide after tissue damage. This polymorphism arises through independently segregating polymorphisms for the presence/absence of two required cyanogenic components, cyanogenic glucosides and their hydrolysing enzyme. White clover populations worldwide have evolved a series of recurrent, climate-associated clines, with higher frequencies of cyanogenic plants in warmer locations. These clines have traditionally been hypothesized to reflect a fitness trade-off between chemical defence in herbivore-rich areas (warmer climates) and energetic costs of producing cyanogenic components in areas of low herbivore pressure (cooler climates). Recent observational studies suggest that cyanogenic components may also be beneficial in water-stressed environments. We investigated fitness trade-offs associated with temperature-induced water stress in the cyanogenesis system using manipulative experiments in growth chambers and population surveys across a longitudinal precipitation gradient in the central United States. We find that plants producing cyanogenic glucosides have higher relative fitness in treatments simulating a moderate, persistent drought stress. In water-neutral treatments, there are energetic costs to producing cyanogenic components, but only in treatments with nutrient stress. These fitness trade-offs are consistent with cyanogenesis frequencies in natural populations, where we find clinal variation in the proportion of plants producing cyanogenic glucosides along the precipitation gradient. These results suggest that multiple selective pressures interact to maintain this adaptive polymorphism and that modelling adaptation will require knowledge of environment-specific fitness effects.