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.

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.

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.

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.

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.

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.

Not all weeds are created equal: A database approach uncovers differences in the sexual system of native and introduced weeds.

Weedy species provide excellent opportunities to examine the process of successful colonization of novel environments. Despite the influence of the sexual system on a variety of processes from reproduction to genetic structure, how the sexual system of species influences weediness has received only limited consideration. We examined the hypothesis that weedy plants have an increased likelihood of being self-compatible compared with nonweedy plants; this hypothesis is derived from Baker's law, which states that species that can reproduce uniparentally are more likely to successfully establish in a new habitat where mates are lacking. We combined a database of the weed (weedy/nonweedy) and introduction status (introduced/native) of plant species found in the USA with a database of plant sexual systems and determined whether native and introduced weeds varied in their sexual systems compared with native and introduced nonweeds. We found that introduced weeds are overrepresented by species with both male and female functions present within a single flower (hermaphrodites) whereas weeds native to the USA are overrepresented by species with male and female flowers present on a single plant (monoecious species). Overall, our results show that Baker's law is supported at the level of the sexual system, thus providing further evidence that uniparental reproduction is an important component of being either a native or introduced weed.

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.

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.

Adaptive pattern of nectar volume within inflorescences: bumblebee foraging behavior and pollinator-mediated natural selection.

Larger floral displays increase pollinator visitation as well as among-flower self-pollination (geitonogamy) in self-compatible species. Dichogamy (temporal separation of gender expression) can limit geitonogamy and increase outcrossing but this depends on pollinator behavior within inflorescences. Declining nectar volume from lower to upper flowers is a hypothesized adaptation to increase outcrossing and pollen export by encouraging the upward movment of pollinators from female to male flowers and by reducing the number of flowers probed per inflorescence, but supporting evidence has been equivocal. We tested this hypothesis in Aconitum gymnandrum by studying floral display and rewards, pollinator visitation, and pollinator-mediated selection on floral traits. We found that larger inflorescences of A. gymnandrum attracted more pollinators, but did not increase the number of flowers probed per visit. Nectar production declined with increasing flower height on average, but the opposite pattern was also common. Bumblebees responded strongly to the nectar pattern, moving from higher to lower nectar concentration. Finally, there was significant pollinator-mediated direct selection for this pattern of declining nectar volume after correcting for correlations with flower size, number, and mean nectar volume. Together, the results strongly suggest that declining nectar production in higher flowers is an adaptation to enhance outcrossing in A. gymnandrum.

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.

Field measurements of genotype by environment interaction for fitness caused by spontaneous mutations in Arabidopsis thaliana.

As the ultimate source of genetic diversity, spontaneous mutation is critical to the evolutionary process. The fitness effects of spontaneous mutations are almost always studied under controlled laboratory conditions rather than under the evolutionarily relevant conditions of the field. Of particular interest is the conditionality of new mutations-that is, is a new mutation harmful regardless of the environment in which it is found? In other words, what is the extent of genotype-environment interaction for spontaneous mutations? We studied the fitness effects of 25 generations of accumulated spontaneous mutations in Arabidopsis thaliana in two geographically widely separated field environments, in Michigan and Virginia. At both sites, mean total fitness of mutation accumulation lines exceeded that of the ancestors, contrary to the expected decrease in the mean due to new mutations but in accord with prior work on these MA lines. We observed genotype-environment interactions in the fitness effects of new mutations, such that the effects of mutations in Michigan were a poor predictor of their effects in Virginia and vice versa. In particular, mutational variance for fitness was much larger in Virginia compared to Michigan. This strong genotype-environment interaction would increase the amount of genetic variation maintained by mutation-selection balance.

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.

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.

Patterns of phenotypic correlations among morphological traits across plants and animals.

Despite the long-standing interest of biologists in patterns of correlation and phenotypic integration, little attention has been paid to patterns of correlation across a broad phylogenetic spectrum. We report analyses of mean phenotypic correlations among a variety of linear measurements from a wide diversity of plants and animals, addressing questions about function, development, integration and modularity. These analyses suggest that vertebrates, hemimetabolous insects and vegetative traits in plants have similar mean correlations, around 0.5. Traits of holometabolous insects are much more highly correlated, with a mean correlation of 0.84; this may be due to developmental homeostasis caused by lower spatial and temporal environmental variance during complete metamorphosis. The lowest mean correlations were those between floral and vegetative traits, consistent with Berg's ideas about functional independence between these modules. Within trait groups, the lowest mean correlations were among vertebrate head traits and floral traits (0.38-0.39). The former may be due to independence between skull modules. While there is little evidence for floral integration overall, certain sets of functionally related floral traits are highly integrated. A case study of the latter is described from wild radish flowers.

Raissa L. Berg's contributions to the study of phenotypic integration, with a professional biographical sketch.

Raissa L. Berg had a remarkable career in many respects and an impact on the study of phenotypic integration that continues to increase over 50 years after the publication of her seminal paper in that area. She was born and lived most of her life in Russia, with most of her research focused on measuring spontaneous mutation rates in Drosophila. She was forced to abandon this work during the height of Lysenko's power in Russia, so she turned temporarily to the study of correlation patterns in plants; ironically, this work has had a more enduring impact than her main body of research. She showed that floral and vegetative traits become decoupled into separate correlation 'pleiades' in plants with specialized pollinators, but floral and vegetative traits remain correlated in plants that have less specialized pollination. Unfortunately, her plant work is often mis-cited as providing evidence for increased correlations among floral traits due to selection by pollinators for functional integration, a point she never made and one that is not supported by her data. Still, many studies of correlation pleiades have been conducted in plants, with the results mostly supporting Berg's hypothesis, although more studies on species with generalized pollination are needed.

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.

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.

Artificial selection on anther exsertion in wild radish, Raphanus raphanistrum.

To study the genetic architecture of anther exsertion, a trait under stabilizing selection in wild radish, artificial selection on anther exsertion was applied for 11 generations. Two replicate lines each of increased and decreased exsertion plus two randomly-mated controls were included. Full pedigree information is available from generation five. To estimate correlated responses to selection, 571 plants from all lines and matrilines were grown in the greenhouse and a number of floral, growth, and phenology traits were measured. To create an outbred F2 mapping population, all possible crosses among the two high and two low exsertion lines were made, using a multiple-family design to capture the genetic variance still present after 11 generations of selection. Six floral traits were measured on 40 parents, 240 F1, and 4,868 F2 offspring. Opportunities for reuse of these data include traits not previously analyzed, other analyses, especially using the pedigree and fitness data, and seeds from all generations and photos of flowers in the later generations are available.