Rapid evolution of a family-diagnostic trait: artificial selection and correlated responses in wild radish, Raphanus raphanistrum.

The mechanisms underlying trait conservation over long evolutionary time scales are poorly known. These mechanisms fall into the two broad and nonmutually exclusive categories of constraint and selection. A variety of factors have been hypothesized to constrain trait evolution. Alternatively, selection can maintain similar trait values across many species if the causes of selection are also relatively conserved, while many sources of constraint may be overcome over longer periods of evolutionary divergence. An example of deep trait conservation is tetradynamy in the large family Brassicaceae, where the four medial stamens are longer than the two lateral stamens. Previous work has found selection to maintain this difference in lengths, which we call anther separation, in wild radish, Raphanus raphanistrum. Here, we test the constraint hypothesis using five generations of artificial selection to reduce anther separation in wild radish. We found a rapid linear response to this selection, with no evidence for depletion of genetic variation and correlated responses to this selection in only four of 15 other traits, suggesting a lack of strong constraint. Taken together, available evidence suggests that tetradynamy is likely to be conserved due to selection, but the function of this trait remains unclear.

Predictive Models of Genetic Redundancy in Arabidopsis thaliana.

Genetic redundancy refers to a situation where an individual with a loss-of-function mutation in one gene (single mutant) does not show an apparent phenotype until one or more paralogs are also knocked out (double/higher-order mutant). Previous studies have identified some characteristics common among redundant gene pairs, but a predictive model of genetic redundancy incorporating a wide variety of features derived from accumulating omics and mutant phenotype data is yet to be established. In addition, the relative importance of these features for genetic redundancy remains largely unclear. Here, we establish machine learning models for predicting whether a gene pair is likely redundant or not in the model plant Arabidopsis thaliana based on six feature categories: functional annotations, evolutionary conservation including duplication patterns and mechanisms, epigenetic marks, protein properties including posttranslational modifications, gene expression, and gene network properties. The definition of redundancy, data transformations, feature subsets, and machine learning algorithms used significantly affected model performance based on holdout, testing phenotype data. Among the most important features in predicting gene pairs as redundant were having a paralog(s) from recent duplication events, annotation as a transcription factor, downregulation during stress conditions, and having similar expression patterns under stress conditions. We also explored the potential reasons underlying mispredictions and limitations of our studies. This genetic redundancy model sheds light on characteristics that may contribute to long-term maintenance of paralogs, and will ultimately allow for more targeted generation of functionally informative double mutants, advancing functional genomic studies.

High-throughput measurement of plant fitness traits with an object detection method using Faster R-CNN.

Revealing the contributions of genes to plant phenotype is frequently challenging because loss-of-function effects may be subtle or masked by varying degrees of genetic redundancy. Such effects can potentially be detected by measuring plant fitness, which reflects the cumulative effects of genetic changes over the lifetime of a plant. However, fitness is challenging to measure accurately, particularly in species with high fecundity and relatively small propagule sizes such as Arabidopsis thaliana. An image segmentation-based method using the software ImageJ and an object detection-based method using the Faster Region-based Convolutional Neural Network (R-CNN) algorithm were used for measuring two Arabidopsis fitness traits: seed and fruit counts. The segmentation-based method was error-prone (correlation between true and predicted seed counts, r2 = 0.849) because seeds touching each other were undercounted. By contrast, the object detection-based algorithm yielded near perfect seed counts (r2 = 0.9996) and highly accurate fruit counts (r2 = 0.980). Comparing seed counts for wild-type and 12 mutant lines revealed fitness effects for three genes; fruit counts revealed the same effects for two genes. Our study provides analysis pipelines and models to facilitate the investigation of Arabidopsis fitness traits and demonstrates the importance of examining fitness traits when studying gene functions.

Self-compatibility is over-represented on islands.

Because establishing a new population often depends critically on finding mates, individuals capable of uniparental reproduction may have a colonization advantage. Accordingly, there should be an over-representation of colonizing species in which individuals can reproduce without a mate, particularly in isolated locales such as oceanic islands. Despite the intuitive appeal of this colonization filter hypothesis (known as Baker's law), more than six decades of analyses have yielded mixed findings. We assembled a dataset of island and mainland plant breeding systems, focusing on the presence or absence of self-incompatibility. Because this trait enforces outcrossing and is unlikely to re-evolve on short timescales if it is lost, breeding system is especially likely to reflect the colonization filter. We found significantly more self-compatible species on islands than mainlands across a sample of > 1500 species from three widely distributed flowering plant families (Asteraceae, Brassicaceae and Solanaceae). Overall, 66% of island species were self-compatible, compared with 41% of mainland species. Our results demonstrate that the presence or absence of self-incompatibility has strong explanatory power for plant geographical patterns. Island floras around the world thus reflect the role of a key reproductive trait in filtering potential colonizing species in these three plant families.

Consequences of Whole-Genome Triplication as Revealed by Comparative Genomic Analyses of the Wild Radish Raphanus raphanistrum and Three Other Brassicaceae Species.

Polyploidization events are frequent among flowering plants, and the duplicate genes produced via such events contribute significantly to plant evolution. We sequenced the genome of wild radish (Raphanus raphanistrum), a Brassicaceae species that experienced a whole-genome triplication event prior to diverging from Brassica rapa. Despite substantial gene gains in these two species compared with Arabidopsis thaliana and Arabidopsis lyrata, ∼70% of the orthologous groups experienced gene losses in R. raphanistrum and B. rapa, with most of the losses occurring prior to their divergence. The retained duplicates show substantial divergence in sequence and expression. Based on comparison of A. thaliana and R. raphanistrum ortholog floral expression levels, retained radish duplicates diverged primarily via maintenance of ancestral expression level in one copy and reduction of expression level in others. In addition, retained duplicates differed significantly from genes that reverted to singleton state in function, sequence composition, expression patterns, network connectivity, and rates of evolution. Using these properties, we established a statistical learning model for predicting whether a duplicate would be retained postpolyploidization. Overall, our study provides new insights into the processes of plant duplicate loss, retention, and functional divergence and highlights the need for further understanding factors controlling duplicate gene fate.

Effect of expanded variation in anther position on pollinator visitation to wild radish, Raphanus raphanistrum.

Background and Aims: Plant-pollinator interactions shape the evolution of flowers. Floral attraction and reward traits have often been shown to affect pollinator behaviour, but the possible effect of efficiency traits on visitation behaviour has rarely been addressed. Anther position, usually considered a trait that influences efficiency of pollen deposition on pollinators, was tested here for its effect on pollinator visitation rates and visit duration in flowers of wild radish, Raphanus raphanistrum . Methods: Artificial selection lines from two experiments that expanded the naturally occurring phenotypic variation in anther position were used. In one experiment, plant lines were selected either to increase or to decrease anther exsertion. The other experiment decreased anther dimorphism, which resulted in increased short stamen exsertion. The hypothesis was that increased exsertion would increase visitation of pollen foragers due to increased visual attraction. Another hypothesis was that exsertion of anthers above the corolla would interfere with nectar foragers and increase the duration of visit per flower. Key Results: In the exsertion selection experiment, increased exsertion of both short and long stamens resulted in an increased number of fly visits per plant, and in the dimorphism experiment bee visits increased with increased short stamen exsertion. The duration of visits of nectar feeders declined significantly with increasing long stamen exsertion, which was opposite to the hypothesis. Conclusions: Until now, anther position was considered to be an efficiency trait to enhance pollen uptake and deposition. Anther position in wild radish is shown here also to have an ecological significance in attracting pollen foragers. This study suggests an additional adaptive role for anther position beyond efficiency, and highlights the multiple ecological functions of floral traits in plant-pollinator interactions.

Floral function: effects of traits on pollinators, male and female pollination success, and female fitness across three species of milkweeds (Asclepias).

PREMISE OF THE STUDY: Central questions in plant reproductive ecology are whether the functions of floral traits in hermaphrodites create conflict between sexes that could slow evolution, and whether individual floral traits function in pollinator attraction, efficiency, or both. We studied how floral traits affect pollinator visitation and efficiency, and how they affect male and female function and female fitness within and across three Asclepias species that differ in floral morphology. METHODS: Using separate multiple regressions, we regressed pollen removal, deposition, and fruit number onto six floral traits. We also used path analyses integrating these variables with pollinator visitation data for two of the species to further explore floral function and its effects on fruit production. KEY RESULTS: Most traits affected male pollination success only, and these effects often differed between species. The exception was increased slit length, which increased pollinia insertion in two of the species. There were no interspecific differences in the effects of the traits on female pollination success. All traits except horn reach affected pollination efficiency in at least one species, and horn reach and two hood dimensions were the only traits to affect pollinator attraction, but in just one species. CONCLUSIONS: Traits tended to function in only one sex, and more traits affected function through pollinator efficiency than through attraction. There was no significant link between female pollination success and female fitness in any of the three species; this pattern is consistent with fruit production not being limited by pollen deposition.

Factors influencing the effect size distribution of adaptive substitutions.

The distribution of effect sizes of adaptive substitutions has been central to evolutionary biology since the modern synthesis. Early theory proposed that because large-effect mutations have negative pleiotropic consequences, only small-effect mutations contribute to adaptation. More recent theory suggested instead that large-effect mutations could be favoured when populations are far from their adaptive peak. Here we suggest that the distributions of effect sizes are expected to differ among study systems, reflecting the wide variation in evolutionary forces and ecological conditions experienced in nature. These include selection, mutation, genetic drift, gene flow, and other factors such as the degree of pleiotropy, the distance to the phenotypic optimum, whether the optimum is stable or moving, and whether new mutation or standing genetic variation provides the source of adaptive alleles. Our goal is to review how these factors might affect the distribution of effect sizes and to identify new research directions. Until more theory and empirical work is available, we feel that it is premature to make broad generalizations about the effect size distribution of adaptive substitutions important in nature.

The scope of Baker's law.

Baker's law refers to the tendency for species that establish on islands by long-distance dispersal to show an increased capacity for self-fertilization because of the advantage of self-compatibility when colonizing new habitat. Despite its intuitive appeal and broad empirical support, it has received substantial criticism over the years since it was proclaimed in the 1950s, not least because it seemed to be contradicted by the high frequency of dioecy on islands. Recent theoretical work has again questioned the generality and scope of Baker's law. Here, we attempt to discern where the idea is useful to apply and where it is not. We conclude that several of the perceived problems with Baker's law fall away when a narrower perspective is adopted on how it should be circumscribed. We emphasize that Baker's law should be read in terms of an enrichment of a capacity for uniparental reproduction in colonizing situations, rather than of high selfing rates. We suggest that Baker's law might be tested in four different contexts, which set the breadth of its scope: the colonization of oceanic islands, metapopulation dynamics with recurrent colonization, range expansions with recurrent colonization, and colonization through species invasions.

Quantifying nonadditive selection caused by indirect ecological effects.

In natural biological communities, species interact with many other species. Multiple species interactions can lead to indirect ecological effects that have important fitness consequences and can cause nonadditive patterns of natural selection. Given that indirect ecological effects are common in nature, nonadditive selection may also be quite common. As a result, quantifying nonadditive selection resulting from indirect ecological effects may be critical for understanding adaptation in natural communities composed of many interacting species. We describe how to quantify the relative strength of nonadditive selection resulting from indirect ecological effects compared to the strength of pairwise selection. We develop a clear method for testing for nonadditive selection caused by indirect ecological effects and consider how it might affect adaptation in multispecies communities. We use two case studies to illustrate how our method can be applied to empirical data sets. Our results suggest that nonadditive selection caused by indirect ecological effects may be common in nature. Our hope is that trait-based approaches, combined with multifactorial experiments, will result in more estimates of nonadditive selection that reveal the relative importance of indirect ecological effects for evolution in a community context.

How weeds emerge: a taxonomic and trait-based examination using United States data.

Weeds can cause great economic and ecological harm to ecosystems. Despite their importance, comparisons of the taxonomy and traits of successful weeds often focus on a few specific comparisons - for example, introduced versus native weeds. We used publicly available inventories of US plant species to make comprehensive comparisons of the factors that underlie weediness. We quantitatively examined taxonomy to determine if certain genera are overrepresented by introduced, weedy or herbicide-resistant species, and we compared phenotypic traits of weeds to those of nonweeds, whether introduced or native. We uncovered genera that have more weeds and introduced species than expected by chance and plant families that have more herbicide-resistant species than expected by chance. Certain traits, generally related to fast reproduction, were more likely to be associated with weedy plants regardless of species' origins. We also found stress tolerance traits associated with either native or introduced weeds compared with native or introduced nonweeds. Weeds and introduced species have significantly smaller genomes than nonweeds and native species. These results support trends for weedy plants reported from other floras, suggest that native and introduced weeds have different stress adaptations, and provide a comprehensive survey of trends across weeds within the USA.

The Roles of Drift and Selection on Short Stamen Loss in Arabidopsis thaliana along an Elevational Gradient in the Spanish Pyrenees.

Traits that have lost function sometimes persist through evolutionary time. These traits may be maintained by a lack of standing genetic variation for the trait, if selection against the trait is weak relative to drift, or if they have a residual function. To determine the evolutionary processes shaping whether nonfunctional traits are retained or lost, we investigated short stamens in 16 populations of Arabidopsis thaliana along an elevational cline in the Spanish Pyrenees. We found a cline in short stamen number from retention of short stamens in high elevation populations to incomplete loss in low elevation populations. We did not find evidence that limited genetic variation constrains the loss of short stamens at high elevations nor evidence for divergent selection on short stamens between high and low elevations. Finally, we identified loci associated with short stamens in the Spanish Pyrenees that are different from loci associated with variation in short stamen number across latitudes from a previous study. Overall, we did not identify the evolutionary mechanisms maintaining an elevational cline in short stamen number but did identify different genetic loci underlying the variation in short stamen along similar phenotypic clines.

Strong evidence for positive and negative correlational selection revealed by recreating ancestral variation.

The study of adaptation helps explain biodiversity and predict future evolution. Yet the process of adaptation can be difficult to observe due to limited phenotypic variation in contemporary populations. Furthermore, the scarcity of male fitness estimates has made it difficult to both understand adaptation and evaluate sexual conflict hypotheses. We addressed both issues in our study of two anther position traits in wild radish (Raphanus raphanistrum): anther exsertion (long filament - corolla tube lengths) and anther separation (long - short filament lengths). These traits affect pollination efficiency and are particularly interesting due to the unusually high correlations among their component traits. We measured selection through male and female fitness on wild radish plants from populations artificially selected to recreate ancestral variation in each anther trait. We found little evidence for conflicts between male and female function. We found strong evidence for stabilizing selection on anther exsertion and disruptive selection on anther separation, indicating positive and negative correlational selection on the component traits. Intermediate levels of exsertion are likely an adaptation to best contact small bees. The function of anther separation is less clear, but future studies might investigate pollen placement on pollinators and compare species possessing multiple stamen types.

Rapid independent trait evolution despite a strong pleiotropic genetic correlation.

Genetic correlations are the most commonly studied of all potential constraints on adaptive evolution. We present a comprehensive test of constraints caused by genetic correlation, comparing empirical results to predictions from theory. The additive genetic correlation between the filament and the corolla tube in wild radish flowers is very high in magnitude, is estimated with good precision (0.85 ± 0.06), and is caused by pleiotropy. Thus, evolutionary changes in the relative lengths of these two traits should be constrained. Still, artificial selection produced rapid evolution of these traits in opposite directions, so that in one replicate relative to controls, the difference between them increased by six standard deviations in only nine generations. This would result in a 54% increase in relative fitness on the basis of a previous estimate of natural selection in this population, and it would produce the phenotypes found in the most extreme species in the family Brassicaceae in less than 100 generations. These responses were within theoretical expectations and were much slower than if the genetic correlation was zero; thus, there was evidence for constraint. These results, coupled with comparable results from other species, show that evolution can be rapid despite the constraints caused by genetic correlations.

Adaptive differentiation of quantitative traits in the globally distributed weed, wild radish (Raphanus raphanistrum).

Weedy species with wide geographical distributions may face strong selection to adapt to new environments, which can lead to adaptive genetic differentiation among populations. However, genetic drift, particularly due to founder effects, will also commonly result in differentiation in colonizing species. To test whether selection has contributed to trait divergence, we compared differentiation at eight microsatellite loci (measured as F(ST)) to differentiation of quantitative floral and phenological traits (measured as Q(ST)) of wild radish (Raphanus raphanistrum) across populations from three continents. We sampled eight populations: seven naturalized populations and one from its native range. By comparing estimates of Q(ST) and F(ST), we found that petal size was the only floral trait that may have diverged more than expected due to drift alone, but inflorescence height, flowering time, and rosette formation have greatly diverged between the native and nonnative populations. Our results suggest the loss of a rosette and the evolution of early flowering time may have been the key adaptations enabling wild radish to become a major agricultural weed. Floral adaptation to different pollinators does not seem to have been as necessary for the success of wild radish in new environments.

Tests of adaptation: functional studies of pollen removal and estimates of natural selection on anther position in wild radish.

BACKGROUND: There are a number of difficulties associated with the study of adaptation. One is a lack of variation in the trait, which is common in adaptations because past selection has removed unfit variants. This lack of variation makes it difficult to determine the relationship between trait variation and fitness. Another difficulty is proving causation in this trait-fitness relationship, because a correlated trait might be the actual adaptation. These difficulties can be ameliorated at least partially by combining studies of natural variation with studies of experimentally manipulated traits and traits whose variance has been augmented by artificial selection. SCOPE: We review here a number of our studies on the adaptive value of two aspects of anther position in wild radish (Raphanus raphanistrum, Brassicaceae): anther exsertion, i.e. the degree to which anthers protrude from the mouth of the corolla tube, and anther height dimorphism, i.e. the difference in lengths of the filaments between the two short and four long stamens. We have used both functional analyses, in which the response variable is pollen removal, and measurements of selection, in which the response variable is lifetime male fitness estimated by molecular genetic paternity analyses. In these studies we use both the natural variation in populations as well as manipulated variation, the latter through both stamen removal and artificial selection, to re-create the ancestral trait conditions. CONCLUSIONS: Our work provides convincing evidence that intermediate anther exsertion values are adaptive, and that this is probably an adaptation to a subset of the pollinator fauna, small bees. The picture for anther height dimorphism is much less clear, as the weight of current evidence suggests that current values of this trait might actually be maladaptive; however, if this is true it is difficult to understand how the dimorphism is maintained across the family Brassicaceae.

Parasite-mediated disruptive selection in a natural Daphnia population.

BACKGROUND: A mismatch has emerged between models and data of host-parasite evolution. Theory readily predicts that parasites can promote host diversity through mechanisms such as disruptive selection. Yet, despite these predictions, empirical evidence for parasite-mediated increases in host diversity remains surprisingly scant. RESULTS: Here, we document parasite-mediated disruptive selection on a natural Daphnia population during a parasite epidemic. The mean susceptibility of clones collected from the population before and after the epidemic did not differ, but clonal variance and broad-sense heritability of post-epidemic clones were significantly greater, indicating disruptive selection and rapid evolution. A maximum likelihood method that we developed for detecting selection on natural populations also suggests disruptive selection during the epidemic: the distribution of susceptibilities in the population shifted from unimodal prior to the epidemic to bimodal after the epidemic. Interestingly, this same bimodal distribution was retained after a generation of sexual reproduction. CONCLUSION: These results provide rare empirical support for parasite-driven increases in host genetic diversity, and suggest that this increase can occur rapidly.

Fitness effects of mutation accumulation in a natural outbred population of wild radish (Raphanus raphanistrum): comparison of field and greenhouse environments.

Spontaneous deleterious mutation has been measured in a handful of organisms, always under laboratory conditions and usually employing inbred species or genotypes. We report the results of a mutation accumulation experiment with an outbred annual plant, Raphanus raphanistrum, with lifetime fitness measured in both the field and the greenhouse. This is the first study to report the effects of spontaneous mutation measured under field conditions. Two large replicate populations (N(e) approximately 600) were maintained with random mating in the greenhouse under relaxed selection for nine generations before the field assay was performed and ten generations before the greenhouse assay. Each generation, every individual was mated twice, once as a pollen donor and once as a pollen recipient, and a single seed from each plant was chosen randomly to create the next generation. The ancestral population was maintained as seeds at 4 degrees C. Declines in lifetime fitness were observed in both the field (1.7% per generation; P= 0.27) and the greenhouse (0.6% per generation; P= 0.07). Significant increases in additive genetic variance for fitness were found for stems per day, flowers per stem, fruits per flower and seeds per fruit in the field as well as for fruits per flower in the greenhouse. Lack of significance of the fitness decline may be due to the short period of mutation accumulation, the use of outbred populations, or both. The percent declines in fitness are at the high end of the range observed in other mutation accumulation experiments and give some support to the idea that mutational effects may be magnified under harsher field conditions. Thus, measurement of mutational parameters under laboratory conditions may underestimate the effects of mutations in natural populations.

Weed evolution: Genetic differentiation among wild, weedy, and crop radish.

Approximately 200 weed species are responsible for more than 90% of crop losses and these comprise less than one percent of all named plant species, suggesting that there are only a few evolutionary routes that lead to weediness. Agricultural weeds can evolve along three main paths: they can be escaped crops, wild species, or crop-wild hybrids. We tested these three hypotheses in weedy radish, a weed of small grains and an emerging model for investigating the evolution of agricultural weeds, using 21 CAPS and SSR markers scored on 338 individuals from 34 populations representing all major species and sub-species in the radish genus Raphanus. To test for adaptation of the weeds to the agricultural environment, we estimated genetic differentiation in flowering time in a series of common garden experiments with over 2,400 individuals from 43 populations (all but one of the genotyped populations plus 10 additional populations). Our findings suggest that the agricultural weed radish R. r. raphanistrum is most genetically similar to native populations of R. r. raphanistrum and is likely not a feral crop or crop hybrid. We also show that weedy radish flowers more rapidly than any other Raphanus population or cultivar, which is consistent with rapid adaptation to the frequent and severe disturbance that characterizes agricultural fields.