The timing of visits by large and small bees differentially affects pollination success in Mimulus ringens.

PREMISE: Cross-fertilization in most flowering plants is facilitated by mobile animals that transport pollen while foraging for floral rewards. The contributions of different visitors can vary widely, depending on the amount of pollen transferred during a single visit and on the frequency and timing of the visits of each pollinator taxon. METHODS: We used three approaches to measure the pollination value of bees that visit Mimulus ringens: pollinator interviews, field population observations, and caging studies. RESULTS: The single-visit effectiveness of small bees (primarily Halictidae) was only half that of larger bees (primarily Bombus) for pollen delivery and removal. In five field populations, we found substantial temporal and spatial variation in visitation and pollination. In most sites big bees were active before 08:00 hours, and by 10:00-11:00 hours, stigmas were usually fully pollinated and closed, and little pollen remained in anthers. Small bees seldom visited before 10:00 hours. Excluding big bees from plants confirmed that pollination is reduced and delayed in this ecological context. CONCLUSIONS: Big bees are the primary pollinators of M. ringens, accounting for at least 75% of seed production. Not only are they more effective per visit, in most situations they also visit before small bees become active. Although small bees are not usually important pollinators of M. ringens, they have the potential to partially replace them as a "fail-safe" pollinator in contexts where big bees are not abundant. In a world where pollinator abundance is declining, such backup pollinators may be important for maintaining plant reproduction.

Weak response to selection on stigma-anther distance in a primarily selfing population of yellow monkeyflower.

Stebbins hypothesized that selfing lineages are evolutionary dead ends because they lack adaptive potential. While selfing populations often possess limited nucleotide variability compared with closely related outcrossers, reductions in the genetic variability of quantitative characters remain unclear, especially for key traits determining selfing rates. Yellow monkeyflower (Mimulus guttatus) populations generally outcross and maintain extensive quantitative genetic variation in floral traits. Here, we study the Joy Road population (Bodega Bay, CA, USA) of M. guttatus, where individuals exhibit stigma-anther distances (SAD) typical of primarily selfing monkeyflowers. We show that this population is closely related to nearby conspecifics on the Pacific Coast with a modest 33% reduction in genome-wide variation compared with a more highly outcrossing population. A five-generation artificial selection experiment challenged the hypothesis that the Joy Road population harbours comparatively low evolutionary potential in stigma-anther distance, a critical determinant of selfing rate in Mimulus. Artificial selection generated a weak phenotypic response, with low realized heritabilities (0.020-0.028) falling 84% below those measured for floral characters in more highly outcrossing M. guttatus. These results demonstrate substantial declines in evolutionary potential with a transition toward selfing. Whether these findings explain infrequent reversals to outcrossing or general limits on adaptation in selfers requires further investigation.

Phenotypic lags influence rapid evolution throughout a drought cycle.

Climate anomalies are increasing and posing strong selection, which can lead to rapid evolution. This is occurring on a backdrop of interannual variability that might weaken or even reverse selection. However, the effect of interannual climatic variability on rapid evolution is rarely considered. We study the climatic differences that contribute to rapid evolution throughout a 7-year period, encompassing a severe drought across 12 populations of Mimulus cardinalis (scarlet monkeyflower). Plants were grown in a common greenhouse environment under wet and dry treatments, where specific leaf area and date of flowering were measured. We examine the association between trait values and different climate metrics at different time periods, including the collection year, prior years, and cumulative metrics across sequential years. Of the climatic variables we assessed, we find that anomalies in mean annual precipitation best describe trait differences over our study period. Past climates, of 1-2 years prior, are often related to trait values in a conflicting direction to collection-year climate. Uncovering these complex climatic impacts on evolution is critical to better predict and interpret the impacts of climate change.

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.

Selfing rates vary with floral display, pollinator visitation and plant density in natural populations of Mimulus ringens.

Variation in selfing rates within and among populations of hermaphroditic flowering plants can strongly influence the evolution of reproductive strategies and the genetic structure of populations. This intraspecific variation in mating patterns may reflect both genetic and ecological factors, but the relative importance of these factors remains poorly understood. Here, we explore how selfing in 13 natural populations of the perennial wildflower Mimulus ringens is influenced by (a) pollinator visitation, an ecological factor, and (b) floral display, a trait with a genetic component that also responds to environmental variation. We also explore whether genetically based floral traits, including herkogamy, affect selfing. We found substantial variation among populations in selfing rate (0.13-0.55). Selfing increased strongly and significantly with floral display, among as well as within populations. Selfing also increased at sites with lower pollinator visitation and low plant density. However, selfing was not correlated with floral morphology. Overall, these results suggest that pollinator visitation and floral display, two factors that interact to affect geitonogamous pollinator movements, can influence the selfing rate. This study identifies mechanisms that may play a role in maintaining selfing rate variation among 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.

Two MYB Proteins in a Self-Organizing Activator-Inhibitor System Produce Spotted Pigmentation Patterns.

Many organisms exhibit visually striking spotted or striped pigmentation patterns. Developmental models predict that such spatial patterns can form when a local autocatalytic feedback loop and a long-range inhibitory feedback loop interact. At its simplest, this self-organizing network only requires one self-activating activator that also activates a repressor, which inhibits the activator and diffuses to neighboring cells. However, the molecular activators and inhibitors fully fitting this versatile model remain elusive in pigmentation systems. Here, we characterize an R2R3-MYB activator and an R3-MYB repressor in monkeyflowers (Mimulus). Through experimental perturbation and mathematical modeling, we demonstrate that the properties of these two proteins correspond to an activator-inhibitor pair in a two-component, reaction-diffusion system, explaining the formation of dispersed anthocyanin spots in monkeyflower petals. Notably, disrupting this pattern impacts pollinator visitation. Thus, subtle changes in simple activator-inhibitor systems are likely essential contributors to the evolution of the remarkable diversity of pigmentation patterns in flowers.

Individualized mating system estimation using genomic data.

The estimation of outcrossing rates in hermaphroditic species has been a major focus in the evolutionary study of reproductive strategies, and is also essential for plant breeding and conservation. Surprisingly, genomics has thus far minimally influenced outcrossing rate studies. In this article, we generalize a Bayesian inference method (BORICE) to accommodate genomic data from multiple subpopulations of a species. As an empirical demonstration, BORICE is applied to 115 maternal families of Mimulus guttatus. The analysis shows that low-level whole genome sequencing of parents and offspring is sufficient for individualized mating system estimation: 208 offspring (88.5%) were definitively called as outcrossed, 23 (9.8%) as selfed. After mating system parameters are established (each offspring as outcrossed or selfed and the inbreeding level of maternal plants), BORICE outputs posterior genotype probabilities for each SNP genomewide. Individual SNP calls are often burdened with considerable uncertainty and distilling information from closely linked sites (within genomic windows) can be a useful strategy. For the Mimulus data, principal components based on window statistics were sufficient to diagnose inversion polymorphisms and estimate their effects on spatial structure, phenotypic and fitness measures. More generally, mating system estimation with BORICE can set the stage for population and quantitative genomic analyses, particularly researchers collect phenotypic or fitness data from maternal individuals.

Patterns of Hybrid Seed Inviability in the Mimulus guttatus sp. Complex Reveal a Potential Role of Parental Conflict in Reproductive Isolation.

Genomic conflicts may play a central role in the evolution of reproductive barriers. Theory predicts that early-onset hybrid inviability may stem from conflict between parents for resource allocation to offspring. Here, we describe M. decorus: a group of cryptic species within the M. guttatus species complex that are largely reproductively isolated by hybrid seed inviability (HSI). HSI between M. guttatus and M. decorus is common and strong, but populations of M. decorus vary in the magnitude and directionality of HSI with M. guttatus. Patterns of HSI between M. guttatus and M. decorus, as well as within M. decorus, conform to the predictions of parental conflict: first, reciprocal F1s exhibit size differences and parent-of-origin-specific endosperm defects; second, the extent of asymmetry between reciprocal F1 seed size is correlated with asymmetry in HSI; and third, inferred differences in the extent of conflict predict the extent of HSI between populations. We also find that HSI is rapidly evolving, as populations that exhibit the most HSI are each others' closest relative. Lastly, although all populations appear largely outcrossing, we find that the differences in the inferred strength of conflict scale positively with π, suggesting that demographic or life history factors other than transitions to self-fertilization may influence the rate of parental-conflict-driven evolution. Overall, these patterns suggest the rapid evolution of parent-of-origin-specific resource allocation alleles coincident with HSI within and between M. guttatus and M. decorus. Parental conflict may therefore be an important evolutionary driver of reproductive isolation.

Environmental heterogeneity generates intrapopulation variation in life-history traits in an annual plant.

Environmental variation affects a plant's life cycle by influencing the timing of germination and flowering, and the duration of the growing season. Yet we know little information about how environmental heterogeneity generates variation in germination schedules and the consequences for growth and fecundity through genetic and plastic responses. We use an annual population of Mimulus guttatus in which, in nature, seeds germinate in both fall and spring. We investigate whether there is a genetic basis to the timing of germination, the effect of germination timing on fecundity, and if growth and flowering respond plastically to compensate for different season lengths. Using sibling families grown in simulated seasonal conditions, we find that families do not differ in their propensity to germinate between seasons. However, the germination season affects subsequent growth and flowering time, with significant genotype-by-environment interactions (G × E). Most G × E is due to unequal variance between seasons, because the spring cohort harbours little genetic variance. Despite their different season lengths, the cohorts do not differ in flower number (fecundity). Heterogeneous environments with unpredictable risks may maintain promiscuous germination, which then affects flowering time. Therefore, if selection at particular life stages changes with climate change, there may be consequences for the entire life cycle.

Variation in ecophysiological traits might contribute to ecogeographic isolation and divergence between parapatric ecotypes of Mimulus aurantiacus.

Many forms of reproductive isolation contribute to speciation, and early-acting barriers may be especially important, because they have the first opportunity to limit gene flow. Ecogeographic isolation occurs when intrinsic traits of taxa contribute to disjunct geographic distributions, reducing the frequency of intertaxon mating. Characterizing this form of isolation requires knowledge of both the geographic arrangement of suitable habitats in nature and the identification of phenotypes involved in shaping geographic distributions. In Mimulus aurantiacus, red- and yellow-flowered ecotypes are incompletely isolated by divergent selection exerted by different pollinators. However, these emerging taxa are largely isolated spatially, with a hybrid zone occurring along a narrow region of contact. In order to assess whether responses to abiotic conditions contribute to the parapatric distribution of ecotypes, we measured a series of ecophysiological traits from populations along a transect, including drought sensitivity, leaf area and the concentrations of vegetative flavonoids. In contrast to the abrupt transitions in floral phenotypes, we found that ecophysiological traits exhibited a continuous geographic transition that largely mirrors variation in climatological variables. These traits may impede gene flow across a continuous environmental gradient, but they would be unlikely to result in ecotypic divergence alone. Nevertheless, we found a genetic correlation between vegetative and floral traits, providing a potential link between the two forms of isolation. Although neither barrier appears sufficient to cause divergence on its own, the combined impacts of local adaptation to abiotic conditions and regional adaptation to pollinators may interact to drive discontinuous variation in the face of gene flow in this system.

Mechanisms of a locally adaptive shift in allocation among growth, reproduction, and herbivore resistance in Mimulus guttatus.

Environmental gradients can drive adaptive evolutionary shifts in plant resource allocation among growth, reproduction, and herbivore resistance. However, few studies have attempted to connect these adaptations to underlying physiological and genetic mechanisms. Here, we evaluate potential mechanisms responsible for a coordinated locally adaptive shift between growth, reproduction, and herbivore defense in the yellow monkeyflower, Mimulus guttatus. Through manipulative laboratory experiments, we found that gibberellin (GA) growth hormones may play a role in the developmental divergence between perennial and annual ecotypes of M. guttatus. Further, we detected an interaction between a locally adaptive chromosomal inversion, DIV1, and GA addition. This finding is consistent with the inversion contributing to the evolutionary divergence between inland annual and coastal perennial ecotypes by reducing GA biosynthesis/activity in perennials. Finally, we found evidence that the DIV1 inversion is partially responsible for a coordinated shift in the divergence of growth, reproduction, and herbivore resistance traits between coastal perennial and inland annual M. guttatus. The inversion has already been established to have a substantial impact on the life-history shift between long-term growth and rapid reproduction. Here, we demonstrate that the DIV1 inversion also has sizable impacts on both the total abundance and composition of phytochemical compounds involved in herbivore resistance.

Extreme copy number variation at a tRNA ligase gene affecting phenology and fitness in yellow monkeyflowers.

Copy number variation (CNV) is a major part of the genetic diversity segregating within populations, but remains poorly understood relative to single nucleotide variation. Here, we report on a tRNA ligase gene (Migut.N02091; RLG1a) exhibiting unprecedented, and fitness-relevant, CNV within an annual population of the yellow monkeyflower Mimulus guttatus. RLG1a variation was associated with multiple traits in pooled population sequencing (PoolSeq) scans of phenotypic and phenological cohorts. Resequencing of inbred lines revealed intermediate-frequency three-copy variants of RLG1a (trip+; 5/35 = 14%), and trip+ lines exhibited elevated RLG1a expression under multiple conditions. trip+ carriers, in addition to being over-represented in late-flowering and large-flowered PoolSeq populations, flowered later under stressful conditions in a greenhouse experiment (p < 0.05). In wild population samples, we discovered an additional rare RLG1a variant (high+) that carries 250-300 copies of RLG1a totalling ~5.7 Mb (20-40% of a chromosome). In the progeny of a high+ carrier, Mendelian segregation of diagnostic alleles and qPCR-based copy counts indicate that high+ is a single tandem array unlinked to the single-copy RLG1a locus. In the wild, high+ carriers had highest fitness in two particularly dry and/or hot years (2015 and 2017; both p < 0.01), while single-copy individuals were twice as fecund as either CNV type in a lush year (2016: p < 0.005). Our results demonstrate fluctuating selection on CNVs affecting phenological traits in a wild population, suggest that plant tRNA ligases mediate stress-responsive life-history traits, and introduce a novel system for investigating the molecular mechanisms of gene amplification.

Major QTL controls adaptation to serpentine soils in Mimulus guttatus.

Spatially varying selection is a critical driver of adaptive differentiation. Yet, there are few examples where the fitness effects of naturally segregating variants that contribute to local adaptation have been measured in the field. Plant adaptation to harsh soil habitats provides an ideal study system for investigating the genetic basis of local adaptation. The work presented here identifies a major locus underlying adaptation to serpentine soils in Mimulus guttatus and estimates the strength of selection on this locus in native field sites. Reciprocal transplant and common-garden studies show that serpentine and nonserpentine populations of M. guttatus differ in their ability to survive on serpentine soils. We directly mapped these field survival differences by performing a bulk segregant analysis with F2 survivors from a field transplant study and identify a single QTL where individuals that are homozygous for the nonserpentine allele do not survive on serpentine soils. Genotyping the survivors from an independent mapping population reveals that this same QTL controls serpentine tolerance in a second, geographically distant population. Finally, we show that this QTL controls tolerance to soil properties, as opposed to some other aspect of the field sites that may differ, by performing a laboratory-based common-garden experiment in native serpentine soils that replicates the survival differences observed in the field. These results indicate that despite the myriad chemical and physical challenges plants face in serpentine habitats, adaptation to these soils in M. guttatus has a simple genetic basis.

Parental experience modifies the Mimulus methylome.

BACKGROUND: Transgenerational plasticity occurs when the environmental experience of an organism modifies the growth and development of its progeny. Leaf damage in Mimulus guttatus exhibits transgenerational plasticity mediated through differential expression of hundreds of genes. The epigenetic mechanisms that facilitate this response have yet to be described. RESULTS: We performed whole genome bisulfite sequencing in the progeny of genetically identical damaged and control plants and developed a pipeline to compare differences in the mean and variance of methylation between treatment groups. We find that parental damage increases the variability of CG and CHG methylation among progeny, but does not alter the overall mean methylation. Instead it has positive effects in some regions and negative in others. We find 3,396 CHH, 203 CG, and 54 CHG Differentially Methylated Regions (DMRs) ranging from tens to thousands of base pairs scattered across the genome. CHG and CHH DMRs tended to overlap with transposable elements. CG DMRs tended to overlap with gene coding regions, many of which were previously found to be differentially expressed. CONCLUSIONS: Genome-wide increases in methylome variation suggest that parental conditions can increase epigenetic diversity in response to stress. Additionally, the potential association between CG DMRs and differentially expressed genes supports the hypothesis that differential methylation is a mechanistic component of transgenerational plasticity in M. guttatus.

The effect of range overlap on ecological niche divergence depends on spatial scale in monkeyflowers.

Patterns of niche divergence and geographical range overlap of closely related species provide insights into the evolutionary dynamics of ecological niches. When ranges overlap, shared selective pressures may preserve niche similarity along coarse-scale macrohabitat axes (e.g., bioclimates). Alternatively, competitive interactions may drive greater divergence along local-scale microhabitat axes (e.g., micro-topographical features). We tested these hypotheses in 16 species pairs of western North American monkeyflowers (Erythranthe and Diplacus, formerly Mimulus) with estimations of species' niches, geographic ranges, and a robust phylogeny. We found that macrohabitat niche divergence decreased with increasing range overlap, consistent with convergent selection operating at a coarse scale. No significant relationship was detected for microhabitat niches. Additionally, niche divergence was greater for recently diverged pairs along all macrohabitat niche axes, but greater for distantly diverged pairs along one microhabitat axis related to vegetation cover. For species pairs with partially overlapping ranges, greater microhabitat divergence was detected in sympatry than in allopatry for at least one niche axis for three of four pairs, consistent with character displacement in sympatry. Thus, coarse- and local-scale niche divergence show dissimilar patterns in relation to range overlap and divergence time, perhaps because the relative importance of convergent versus divergent selection depends on spatial scale.

Natural selection and outbreeding depression suggest adaptive differentiation in the invasive range of a clonal plant.

Analyses of phenotypic selection and demography in field populations are powerful ways to establishing the potential role of natural selection in shaping evolution during biological invasions. Here we use experimental F2 crosses between native and introduced populations of Mimulus guttatus to estimate the pattern of natural selection in part of its introduced range, and to seek evidence of outbreeding depression of colonists. The F2s combined the genome of an introduced population with the genome of either native or introduced populations. We found that the introduced × introduced cross had the fastest population growth rate owing to increased winter survival, clonality and seed production. Our analysis also revealed that selection through sexual fitness favoured large floral displays, large vegetative and flower size, lateral spread and early flowering. Our results indicate a source-of-origin effect, consistent with outbreeding depression exposed by mating between introduced and native populations. Our findings suggest that well-established non-native populations may pay a high fitness cost during subsequent bouts of admixture with native populations, and reveal that processes such as local adaptation in the invasive range can mediate the fitness consequences of admixture.

Effects of heterospecific pollen from a wind-pollinated and pesticide-treated plant on reproductive success of an insect-pollinated species.

PREMISE OF THE STUDY: Studies on the effects of heterospecific pollen (HP) transfer have been focused mainly on insect-pollinated species, despite evidence of insect visitation to wind-pollinated species and transfer of their pollen onto stigmas of insect-pollinated plants. Thus, the potential consequences of HP transfer from wind-pollinated species remain largely unknown. Furthermore, accumulation of pesticide residues in pollen of wind-pollinated crops has been documented, but its potential effects on wild plant species via HP transfer have not been tested. METHODS: We evaluated the effect of wind-dispersed Zea mays pollen on pollen tube growth of the insect-pollinated Mimulus nudatus via hand pollinations. We further evaluated whether pesticide-contaminated Z. mays pollen has larger effects on M. nudatus pollen success than non-contaminated Z. mays pollen. KEY RESULTS: We found a significant negative effect of Z. mays pollen on M. nudatus pollen tube growth even when deposited in small amounts. However, we did not observe any difference in the magnitude of this effect between pesticide-laden Z. mays pollen and non-contaminated Z. mays pollen. CONCLUSIONS: Our results suggest that wind-pollinated species can have negative effects as HP donors on insect-pollinated recipients. Thus, their role in shaping co-flowering interactions for wind- and insect-pollinated species deserves more attention. Although we did not find evidence that pesticide contamination increased HP effects, we cannot fully rule out the existence of such an effect, because pollen load and thus the pesticide dose applied to stigmas was low. This result should be confirmed using other HP donors and across a range of HP loads, pesticide types, and concentrations.

Differential adaptation to a harsh granite outcrop habitat between sympatric Mimulus species.

Understanding which environmental variables and traits underlie adaptation to harsh environments is difficult because many traits evolve simultaneously as populations or species diverge. Here, we investigate the ecological variables and traits that underlie Mimulus laciniatus' adaptation to granite outcrops compared to its sympatric, mesic-adapted progenitor, Mimulus guttatus. We use fine-scale measurements of soil moisture and herbivory to examine differences in selective forces between the species' habitats, and measure selection on flowering time, flower size, plant height, and leaf shape in a reciprocal transplant using M. laciniatus × M. guttatus F4 hybrids. We find that differences in drought and herbivory drive survival differences between habitats, that M. laciniatus and M. guttatus are each better adapted to their native habitat, and differential habitat selection on flowering time, plant stature, and leaf shape. Although early flowering time, small stature, and lobed leaf shape underlie plant fitness in M. laciniatus' seasonally dry environment, increased plant size is advantageous in a competitive mesic environment replete with herbivores like M. guttatus'. Given that we observed divergent selection between habitats in the direction of species differences, we conclude that adaptation to different microhabitats is an important component of reproductive isolation in this sympatric species pair.

The role of cold cues at different life stages on germination and flowering phenology.

PREMISE OF THE STUDY: The timing of major phenological transitions is critical to lifetime fitness, and life history theory predicts differences for annual and perennial plants. To correctly time these transitions, many plants rely on environmental cues such as exposure to extended periods of cold, which may occur at different stages throughout their lifetime. METHODS: We studied the role of cold at different life stages, by jointly exposing seed (stratification) and rosettes (vernalization) to cold. We used 23 populations of Mimulus guttatus, which vary from annuals to perennials, and investigated how cold at one or both stages affected germination, flowering, growth, and biomass. KEY RESULTS: We found that stratification and vernalization interact to affect life cycle transitions, and that cold at either stage could synchronize flowering phenology. For perennials, either stratification or vernalization is necessary for maximum flowering. We also found that germination timing covaried with later traits. Moreover, plants from environments with dissimilar climates displayed different phenological responses to stratification or vernalization. CONCLUSIONS: In general, cold is more important for seed germination in annuals and plants from environments with warm temperatures and variable precipitation. In contrast, cold is more important for flowering in perennials: it accelerates flowering in plants from lower precipitation environments, and it increases flowering proportion in plants from cooler, more stable precipitation environments. We discuss our findings in the context of the variable environments plants experience within a population and the variation encountered across the biogeographic native range of the species.