A standardized and efficient technique to estimate seed traits in plants with numerous small propagules.

Premise: Variation in seed traits is common within and among populations of plant species and often has ecological and evolutionary implications. However, due to the time-consuming nature of manual seed measurements and the level of variability in imaging techniques, quantifying and interpreting the extent of seed variation can be challenging. Methods: We developed a standardized high-throughput technique to measure seed number, as well as individual seed area and color, using a derived empirical scale to constrain area in Arabidopsis thaliana, Brassica rapa, and Mimulus guttatus. We develop a specific rational model using seed area measured at various spatial scales relative to the pixel count, observing the asymptotic value of the seed area as the modeled number of pixels approaches infinity. Results: We found that our model has high reliability in estimating seed traits and efficiently processes large numbers of images, facilitating the quantification of seed traits in studies with large sample sizes. Discussion: This technique facilitates consistency between imaging sessions and standardizes the measurement of seed traits. These novel advances allow researchers to directly and reliably measure seed traits, which will enable tests of the ecological and evolutionary causes of their variation.

Autopolyploid establishment depends on life-history strategy and the mating outcomes of clonal architecture.

Polyploidy is a significant component in the evolution of many taxa, particularly plant groups. However, new polyploids face substantial fitness disadvantages due to a lack of same-cytotype mates, and the factors promoting or preventing polyploid establishment in natural populations are often unclear. We develop spatially explicit agent-based simulation models to test the hypothesis that a perennial life history and clonal propagation facilitate the early stages of polyploid establishment and persistence. Our models show that polyploids are more likely to establish when they have longer life spans than diploids, especially when self-fertilization rates are high. Polyploids that combine sexual and clonal reproduction can establish across a wide range of life histories, but their success is moderated by clonal strategy. By tracking individuals and mating events, we reveal that clonal architecture has a substantial impact on the spatial structure of the mixed diploid-polyploid population during polyploid establishment: altering patterns of mating within or between cytotypes via geitonogamous self-fertilization, the mechanisms through which polyploid establishment proceeds, and the final composition of the polyploid population. Overall, our findings provide novel insight into the role of clonal structure in modulating the complex relationship between polyploidy, perenniality, and clonality and offer testable predictions for future empirical work.

Mating and fitness consequences of variation in male allocation in a wind-pollinated plant.

In hermaphrodites, the allocation of resources to each sex function can influence fitness through mating success. A prediction that arises from sex allocation theory is that in wind-pollinated plants, male fitness should increase linearly with investment of resources into male function but there have been few empirical tests of this prediction. In a field experiment, we experimentally manipulated allocation to male function in Ambrosia artemisiifolia (common ragweed) and measured mating success in contrasting phenotypes using genetic markers. We investigated the effects of morphological traits and flowering phenology on male siring success, and on the diversity of mates. Our results provide evidence for a linear relation between allocation to male function, mating, and fitness. We find earlier onset of male flowering time increases reproductive success, whereas later flowering increases the probability of mating with diverse individuals. Our study is among the first empirical tests of the prediction of linear male fitness returns in wind-pollinated plants and emphasizes the importance of a large investment into male function by wind-pollinated plants and mating consequences of temporal variation in sex allocation.

Assessing climate change tolerance and the niche breadth-range size hypothesis in rare and widespread alpine plants.

Species range limits often reflect niche limits, especially for ranges constrained along elevational gradients. In this study, we used elevational transplant experiments to test niche breadth and functional trait plasticity in early life stages of narrow-range Nabalus boottii and broad-range N. trifoliolatus plants to assess their climate change vulnerability and the applicability of the niche breadth-range size hypothesis to explain their range size differences. We discovered that the earliest life stage (seed germination) was the most vulnerable and the two alpine taxa, N. boottii and N. trifoliolatus var. nanus, were unable to establish at the warm low elevation site, however non-alpine N. trifoliolatus established at all three elevations, including at the high elevation (beyond-range) site. Niche limits in seed emergence may therefore contribute to range size in these taxa. In contrast, when seedlings were planted we found substantial functional trait plasticity in later life stages (average 44% across ten traits) that was highly similar for all Nabalus taxa, suggesting that differences in plasticity do not generate niche differences or restrict range size in the focal taxa. While this substantial plasticity may help buffer populations faced by climate change, the inability of the alpine taxa to establish at lower elevation sites suggests that their populations may still decline due to decreased seed recruitment under ongoing climate change. We therefore recommend monitoring alpine Nabalus populations, particularly globally rare N. boottii.

Population genomic and historical analysis suggests a global invasion by bridgehead processes in Mimulus guttatus.

Imperfect historical records and complex demographic histories present challenges for reconstructing the history of biological invasions. Here, we combine historical records, extensive worldwide and genome-wide sampling, and demographic analyses to investigate the global invasion of Mimulus guttatus from North America to Europe and the Southwest Pacific. By sampling 521 plants from 158 native and introduced populations genotyped at >44,000 loci, we determined that invasive M. guttatus was first likely introduced to the British Isles from the Aleutian Islands (Alaska), followed by admixture from multiple parts of the native range. We hypothesise that populations in the British Isles then served as a bridgehead for vanguard invasions worldwide. Our results emphasise the highly admixed nature of introduced M. guttatus and demonstrate the potential of introduced populations to serve as sources of secondary admixture, producing novel hybrids. Unravelling the history of biological invasions provides a starting point to understand how invasive populations adapt to novel environments.

Multi-level patterns of genetic structure and isolation by distance in the widespread plant Mimulus guttatus.

An understanding of genetic structure is essential for answering many questions in population genetics. However, complex population dynamics and scale-dependent processes can make it difficult to detect if there are distinct genetic clusters present in natural populations. Inferring discrete population structure is particularly challenging in the presence of continuous genetic variation such as isolation by distance. Here, we use the plant species Mimulus guttatus as a case study for understanding genetic structure at three spatial scales. We use reduced-representation sequencing and marker-based genotyping to understand dispersal dynamics and to characterise genetic structure. Our results provide insight into the spatial scale of genetic structure in a widespread plant species, and demonstrate how dispersal affects spatial genetic variation at the local, regional, and range-wide scale. At a fine-spatial scale, we show dispersal is rampant with little evidence of spatial genetic structure within populations. At a regional-scale, we show continuous differentiation driven by isolation by distance over hundreds of kilometres, with broad geographic genetic clusters that span major barriers to dispersal. Across Western North America, we observe geographic genetic structure and the genetic signature of multiple postglacial recolonisation events, with historical gene flow linking isolated populations. Our genetic analyses show M. guttatus is highly dispersive and maintains large metapopulations with high intrapopulation variation. This high diversity and dispersal confounds the inference of genetic structure, with multi-level sampling and spatially-explicit analyses required to understand population history.

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.

Assortative mating by flowering time and its effect on correlated traits in variable environments.

Reproductive timing is a key life-history trait that impacts the pool of available mates, the environment experienced during flowering, and the expression of other traits through genetic covariation. Selection on phenology, and its consequences on other life-history traits, has considerable implications in the context of ongoing climate change and shifting growing seasons. To test this, we grew field-collected seed from the wildflower Mimulus guttatus in a greenhouse to assess the standing genetic variation for flowering time and covariation with other traits. We then created full-sib families through phenological assortative mating and grew offspring in three photoperiod treatments representing seasonal variation in daylength. We find substantial quantitative genetic variation for the onset of flowering time, which covaried with vegetative traits. The assortatively-mated offspring varied in their critical photoperiod by over two hours, so that families differed in their probability of flowering across treatments Allocation to flowering and vegetative growth changed across the daylength treatments, with consistent direction and magnitude of covariation among flowering time and other traits. Our results suggest that future studies of flowering time evolution should consider the joint evolution of correlated traits and shifting seasonal selection to understand how environmental variation influences life histories.

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.

Loss of Color Pigmentation Is Maintained at High Frequency in a Monkey Flower Population.

Color polymorphisms have long been of evolutionary interest for their diverse roles, including mate choice, predator avoidance, and pollinator attraction. While color variation is often under strong selection, some taxa demonstrate unexpectedly high frequencies of presumed deleterious color forms. Here we show that a genetic variant underlying complete loss of anthocyanin pigmentation has risen to an unexpectedly high frequency of >0.2 in a natural population of the plant Mimulus guttatus. Decreased expression of MYB5 transcription factor is associated with unpigmented morphs. While the allele was found only in heterozygote adults in the wild, suggesting negative selection, experiments were unable to demonstrate a fitness cost for unpigmented plants, suggesting a cryptic selection pressure in the wild. However, life-history differences among morphs suggests that unpigmented individuals benefit from later flowering and clonal growth. Overall, our study highlights the complex interplay of factors maintaining variation in nature, even for genes of major effect.

Variation in gene regulation underlying annual and perennial flowering in Arabideae species.

The diversity of life history strategies within the angiosperms illustrates the evolutionary flexibility of reproductive characteristics. The number of times an individual reproduces is a key life history trait, and transitions from iteroparous perennials to semelparous annuals have occurred frequently in the flowering plants. Despite the frequency of this evolutionary transition, and the importance of annuality versus perenniality to both agriculture and ecology, understanding the molecular and genetic mechanisms involved in perennial flowering is in their infancy. In this issue of Molecular Ecology, Kiefer et al. () make significant progress towards understanding divergence in seasonal flowering between annual and perennial species in the Arabideae tribe of Brassicaceae. By combining a comparative approach with gene expression and sequence comparisons, they show that transcriptional differences in FLC orthologs, a floral inhibitor in Arabidopsis thaliana, have occurred repeatedly and underlie differences in flowering between annuals and perennials.

Losing one's touch: Evolution of the touch-sensitive stigma in the Mimulus guttatus species complex.

PREMISE OF THE STUDY: The stigmas of several species are touch sensitive and respond to pressure by closing. Previous research suggests that stigma closure could prevent self pollination within a flower during a pollinator's visit or enhance male function by increasing pollen export. Both factors could be favored in outcrossers, and neither would be beneficial in selfers. METHODS: We investigated variation in stigma-closing and the duration of closure in annual and perennial populations of the variable species Mimulus guttatus and whether four closely related selfing species (M. cupriphilus, M. laciniatus, M. nasutus, and M. pardalis) have lost their touch sensitivity. We grew plants in a controlled environment and performed experiments with and without the addition of pollen to the stigma. KEY RESULTS: In M. guttatus, the speed of stigma-closing was rapid and unaffected by the deposition of pollen. Populations varied significantly in closing speed, which may reflect their geographic location. For annual populations only, anther-stigma separation significantly affected closing speed. Also, stigmas that closed quickly stayed closed longer, and stigmas that received pollen remained closed longer. Finally, in the selfing species, stigma-closing was more variable; some populations have entirely lost the ability to respond to touch. CONCLUSIONS: We discuss our results in the context of traits that promote outcrossing and traits that are under selection during the evolution of selfing. This is the first characterization of variation in touch responses across multiple populations within a species and the first to demonstrate the loss of touch sensitivity in selfing lineages.

Comparative Transcriptomics Indicates a Role for SHORT VEGETATIVE PHASE (SVP) Genes in Mimulus guttatus Vernalization Response.

The timing of reproduction in response to variable environmental conditions is critical to plant fitness, and is a major driver of taxon differentiation. In the yellow monkey flower, Mimulus guttatus, geographically distinct North American populations vary in their photoperiod and chilling (vernalization) requirements for flowering, suggesting strong local adaptation to their surroundings. Previous analyses revealed quantitative trait loci (QTL) underlying short-day mediated vernalization responsiveness using two annual M. guttatus populations that differed in their vernalization response. To narrow down candidate genes responsible for this variation, and to reveal potential downstream genes, we conducted comparative transcriptomics and quantitative PCR (qPCR) in shoot apices of parental vernalization responsive IM62, and unresponsive LMC24 inbred lines grown under different photoperiods and temperatures. Our study identified several metabolic, hormone signaling, photosynthetic, stress response, and flowering time genes that are differentially expressed between treatments, suggesting a role for their protein products in short-day-mediated vernalization responsiveness. Only a small subset of these genes intersected with candidate genes from the previous QTL study, and, of the main candidates tested with qPCR under nonpermissive conditions, only SHORT VEGETATIVE PHASE (SVP) gene expression met predictions for a population-specific short-day-repressor of flowering that is repressed by cold.

Adaptive divergence in the monkey flower Mimulus guttatus is maintained by a chromosomal inversion.

Organisms exhibit an incredible diversity of life history strategies as adaptive responses to environmental variation. The establishment of novel life history strategies involves multilocus polymorphisms, which will be challenging to establish in the face of gene flow and recombination. Theory predicts that adaptive allelic combinations may be maintained and spread if they occur in genomic regions of reduced recombination, such as chromosomal inversion polymorphisms, yet empirical support for this prediction is lacking. Here, we use genomic data to investigate the evolution of divergent adaptive ecotypes of the yellow monkey flower Mimulus guttatus. We show that a large chromosomal inversion polymorphism is the major region of divergence between geographically widespread annual and perennial ecotypes. In contrast, ∼40,000 single nucleotide polymorphisms in collinear regions of the genome show no signal of life history, revealing genomic patterns of diversity have been shaped by localized homogenizing gene flow and large-scale Pleistocene range expansion. Our results provide evidence for an inversion capturing and protecting loci involved in local adaptation, while also explaining how adaptive divergence can occur with gene flow.

Genomic studies on the nature of species: adaptation and speciation in Mimulus.

Evolutionary biology is in an exciting era, in which powerful genomic tools make the answers accessible to long-standing questions about variation, adaptation and speciation. The availability of a suite of genomic resources, a shared knowledge base and a long history of study have made the phenotypically diverse plant genus Mimulus an important system for understanding ecological and evolutionary processes. An international Mimulus Research Meeting was held at Duke University in June 2014 to discuss developments in ecological and evolutionary genetic studies in Mimulus. Here, we report major recent discoveries presented at the meeting that use genomic approaches to advance our understanding of three major themes: the parallel genetic basis of adaptation; the ecological genomics of speciation; and the evolutionary significance of structural genetic variation. We also suggest future research directions for studies of Mimulus and highlight challenges faced when developing new ecological and evolutionary model systems.

The extent and genetic basis of phenotypic divergence in life history traits in Mimulus guttatus.

Differential natural selection acting on populations in contrasting environments often results in adaptive divergence in multivariate phenotypes. Multivariate trait divergence across populations could be caused by selection on pleiotropic alleles or through many independent loci with trait-specific effects. Here, we assess patterns of association between a suite of traits contributing to life history divergence in the common monkey flower, Mimulus guttatus, and examine the genetic architecture underlying these correlations. A common garden survey of 74 populations representing annual and perennial strategies from across the native range revealed strong correlations between vegetative and reproductive traits. To determine whether these multitrait patterns arise from pleiotropic or independent loci, we mapped QTLs using an approach combining high-throughput sequencing with bulk segregant analysis on a cross between populations with divergent life histories. We find extensive pleiotropy for QTLs related to flowering time and stolon production, a key feature of the perennial strategy. Candidate genes related to axillary meristem development colocalize with the QTLs in a manner consistent with either pleiotropic or independent QTL effects. Further, these results are analogous to previous work showing pleiotropy-mediated genetic correlations within a single population of M. guttatus experiencing heterogeneous selection. Our findings of strong multivariate trait associations and pleiotropic QTLs suggest that patterns of genetic variation may determine the trajectory of adaptive divergence.

Genetic determinants and epistasis for life history trait differences in the common monkeyflower, Mimulus guttatus.

Understanding the genetic basis of complex quantitative traits is a central problem in evolutionary biology, particularly for traits that may lead to adaptations in natural populations. The annual and perennial ecotypes of Mimulus guttatus provide an excellent experimental system for characterizing the genetic components of population divergence. The 2 life history ecotypes coexist throughout the geographic range. Focusing on population differences in life history traits, I examined the strength and direction of pairwise epistatic interactions between 2 target chromosomal regions (DIV1 and DIV2) when singly and cointrogressed into the alternate population's genetic background. I measured a suite of flowering and vegetative traits related to life history divergence in 804 plants from 18 reciprocal near-isogenic lines. I detected pleiotropic main effects for the DIV1 QTL in both genetic backgrounds and weaker main effects of the DIV2 QTL, primarily in the perennial background. Many of the traits showed epistatic interactions between alleles at the DIV1 and DIV2 QTL. Finally, for many traits, the magnitude of effect size was greater in the perennial background. I evaluate these results in the context of their potential role in population divergence in M. guttatus and adaptive evolution in natural populations.

Phenotypic profiling of ABC transporter coding genes in Myxococcus xanthus.

Information about a gene sometimes can be deduced by examining the impact of its mutation on phenotype. However, the genome-scale utility of the method is limited because, for nearly all model organisms, the majority of mutations result in little or no observable phenotypic impact. The cause of this is often attributed to robustness or redundancy within the genome, but that is only one plausible hypothesis. We examined a standard set of phenotypic traits, and applied statistical methods commonly used in the study of natural variants to an engineered mutant strain collection representing disruptions in 180 of the 192 ABC transporters within the bacterium Myxococcus xanthus. These strains display continuous variation in their phenotypic distributions, with a small number of "outlier" strains at both phenotypic extremes, and the majority within a confidence interval about the mean that always includes wild type. Correlation analysis reveals substantial pleiotropy, indicating that the traits do not represent independent variables. The traits measured in this study co-cluster with expression profiles, thereby demonstrating that these changes in phenotype correspond to changes at the molecular level, and therefore can be indirectly connected to changes in the genome. However, the continuous distributions, the pleiotropy, and the placement of wild type always within the confidence interval all indicate that this standard set of M. xanthus phenotypic assays is measuring a narrow range of partially overlapping traits that do not directly reflect fitness. This is likely a significant cause of the observed small phenotypic impact from mutation, and is unrelated to robustness and redundancy.

Major QTLs for critical photoperiod and vernalization underlie extensive variation in flowering in the Mimulus guttatus species complex.

Species with extensive ranges experience highly variable environments with respect to temperature, light and soil moisture. Synchronizing the transition from vegetative to floral growth is important to employ favorable conditions for reproduction. Optimal timing of this transition might be different for semelparous annual plants and iteroparous perennial plants. We studied variation in the critical photoperiod necessary for floral induction and the requirement for a period of cold-chilling (vernalization) in 46 populations of annuals and perennials in the Mimulus guttatus species complex. We then examined critical photoperiod and vernalization QTLs in growth chambers using F(2) progeny from annual and perennial parents that differed in their requirements for flowering. We identify extensive variation in critical photoperiod, with most annual populations requiring substantially shorter day lengths to initiate flowering than perennial populations. We discover a novel type of vernalization requirement in perennial populations that is contingent on plants experiencing short days first. QTL analyses identify two large-effect QTLs which influence critical photoperiod. In two separate vernalization experiments we discover each set of crosses contain different large-effect QTLs for vernalization. Mimulus guttatus harbors extensive variation in critical photoperiod and vernalization that may be a consequence of local adaptation.