Mutation bias reflects natural selection in Arabidopsis thaliana.

Since the first half of the twentieth century, evolutionary theory has been dominated by the idea that mutations occur randomly with respect to their consequences1. Here we test this assumption with large surveys of de novo mutations in the plant Arabidopsis thaliana. In contrast to expectations, we find that mutations occur less often in functionally constrained regions of the genome-mutation frequency is reduced by half inside gene bodies and by two-thirds in essential genes. With independent genomic mutation datasets, including from the largest Arabidopsis mutation accumulation experiment conducted to date, we demonstrate that epigenomic and physical features explain over 90% of variance in the genome-wide pattern of mutation bias surrounding genes. Observed mutation frequencies around genes in turn accurately predict patterns of genetic polymorphisms in natural Arabidopsis accessions (r = 0.96). That mutation bias is the primary force behind patterns of sequence evolution around genes in natural accessions is supported by analyses of allele frequencies. Finally, we find that genes subject to stronger purifying selection have a lower mutation rate. We conclude that epigenome-associated mutation bias2 reduces the occurrence of deleterious mutations in Arabidopsis, challenging the prevailing paradigm that mutation is a directionless force in evolution.

Multiple Sources of Introduction of North American Arabidopsis thaliana from across Eurasia.

Large-scale movement of organisms across their habitable range, or migration, is an important evolutionary process that can shape genetic diversity and influence the adaptive spread of alleles. Although human migrations have been studied in great detail with modern and ancient genomes, recent anthropogenic influence on reducing the biogeographical constraints on the migration of nonnative species has presented opportunities in several study systems to ask the questions about how repeated introductions shape genetic diversity in the introduced range. We present an extensive overview of population structure of North American Arabidopsis thaliana by studying a set of 500 whole-genome sequenced and over 2,800 RAD-seq genotyped individuals in the context of global diversity represented by Afro-Eurasian genomes. We use methods based on haplotype and rare-allele sharing as well as phylogenetic modeling to identify likely sources of introductions of extant N. American A. thaliana from the native range in Africa and Eurasia. We find evidence of admixture among the introduced lineages having increased haplotype diversity and reduced mutational load. We also detect signals of selection in immune-system-related genes that may impart qualitative disease resistance to pathogens of bacterial and oomycete origin. We conclude that multiple introductions to a nonnative range can rapidly enhance the adaptive potential of a colonizing species by increasing haplotypic diversity through admixture. Our results lay the foundation for further investigations into the functional significance of admixture.

Pollination-precision hypothesis: support from native honey bees and nectar bats.

The evolution of floral traits is often considered to reflect selection for increased pollination efficiency. Known as the pollination-precision hypothesis, increased pollination efficiency is achieved by enhancing pollen deposition on precise areas of the pollinator. Most research to date addressing this hypothesis has examined plant species that are a priori predicted to place pollen precisely, but we still lack comparisons with species predicted to have low pollination efficiency. We studied 39 plant species with diverse floral morphologies and measured the precision of pollen placement on two pollinator groups: honey bees (genus Apis) and nectar bats (family Pteropodidae). Pollen was collected from four locations of each pollinator's body (bees: dorsal thorax, ventral thorax, dorsal abdomen, ventral abdomen; bats: crown, face, chest, wing) to calculate pollen placement precision using Pielou's evenness index. We also quantified variation in floral design by scoring floral symmetry, corolla fusion, floral orientation and stamen number. We confirm the importance of four floral character states (bilateral symmetry, fused corollas, horizontal orientation and reduced stamen number) in promoting precise pollen placement on diverse pollinators. Our findings provide phylogenetically corrected, empirical support that the evolution of the four floral characters reflect selection for enhanced precision of pollen placed on pollinators.

Diversification in the Rosales is influenced by dispersal, geographic range size, and pre-existing species richness.

PREMISE: Biodiversity results from origination and extinction, justifying interest in identifying traits that influence this balance. Traits implicated in the success or failure of lineages include dispersal, colonization ability, and geographic range size. We investigated the impact of dispersal and range size on contemporary diversity in the Rosales. METHODS: We used the multiple-state speciation and extinction (MuSSE) method to explore the effects on genus-level diversification of two genus-level traits (geographic range size and within-genus proclivity to speciate) and two species traits (seed dispersal and growth habit) and the multiple hidden-state speciation and extinction (MuHiSSE) method for species-level associations. Finally, we conducted a PGLS (phylogenetic least-squares) analysis to distinguish between speciation within genera versus origination of new genera. RESULTS: At the species level, animal dispersal enhances diversification rate in both woody and herbaceous lineages, while woody lineages without animal dispersal have higher extinction rates than speciation rates. At the genus level, herbaceous taxa have positive diversification rates regardless of other character states. Diversification rate variation is also explained by two interactions: (1) a three-way interaction between large geographic range, animal-mediated dispersal, and high within-genus species richness, whereby genera possessing all three traits have high diversification rates, and (2) a four-way interaction by which the three-way interaction is stronger in woody genera than in herbaceous genera. CONCLUSIONS: Colonization ability may underlie the relationship between dispersal type and range size and may influence past diversification rates by decreasing extinction rates during late Cenozoic climate volatility. Thus, colonization ability could be used to predict future extinction risk to aid conservation.

Binary-state speciation and extinction method is conditionally robust to realistic violations of its assumptions.

BACKGROUND: Phylogenetic comparative methods allow us to test evolutionary hypotheses without the benefit of an extensive fossil record. These methods, however, make simplifying assumptions, among them that clades are always increasing or stable in diversity, an assumption we know to be false. This study simulates hypothetical clades to test whether the Binary State Speciation and Extinction (BiSSE) method can be used to correctly detect relative differences in diversification rate between ancestral and derived character states even as net diversification rates are declining overall. We simulate clades with declining but positive diversification rates, as well those in which speciation rates decline below extinction rates so that they are losing richness for part of their history. We run these analyses both with simulated symmetric and asymmetric speciation rates to test whether BiSSE can be used to detect them correctly. RESULTS: For simulations with a neutral character, the fit for a BiSSE model with a neutral character is better than alternative models so long as net diversification rates remain positive. Once net diversification rates become negative, the BiSSE model with the greatest likelihood often has a non-neutral character, even though there is no such character in the simulation. BiSSE's usefulness in detecting real asymmetry in speciation rates improves with clade age, even well after net diversification rates have become negative. CONCLUSIONS: BiSSE is most useful in analyzing clades of intermediate age, before they have reached peak diversity and gone into decline. After this point, users of BiSSE risk incorrectly inferring differential evolutionary rates when none exist. Fortunately, most studies using BiSSE and similar models focus on rapid, recent diversifications, and are less likely to encounter the biases BiSSE models are subject to for older clades. For extant groups that were once more diverse than now, however, caution should be taken in inferring past diversification patterns without fossil data.

Comparison of population genetic structures of the plant Silene stellata and its obligate pollinating seed predator moth Hadena ectypa.

Background and Aims: Population genetic structures and patterns of gene flow of interacting species provide important insights into the spatial scale of their interactions and the potential for local co-adaptation. We analysed the genetic structures of the plant Silene stellata and the nocturnal moth Hadena ectypa. Hadena ectypa acts as one of the important pollinators of S. stellata as well as being an obligate seed parasite on the plant. Although H. ectypa provides a substantial pollination service to S. stellata, this system is largely considered parasitic due to the severe seed predation by the Hadena larvae. Previous research on this system has found variable interaction outcomes across space, indicating the potential for a geographical selection mosaic. Methods: Using 11 microsatellite markers for S. stellata and nine markers for H. ectypa, we analysed the population genetic structure and the patterns and intensity of gene flow within and among three local populations in the Appalachians. Key Results: We found no spatial genetic structure in the moth populations, while significant differentiation was detected among the local plant populations. Additionally, we observed that gene flow rates among H. ectypa populations were more uniform and that the mean gene flow rate in H. ectypa was twice as large as that in S. stellata. Conclusions: Our results suggest that although the moths move frequently among populations, long-distance pollen carryover only happens occasionally. The difference in gene flow rates between S. stellata and H. ectypa could prevent strict local co-adaptation. Furthermore, higher gene flow rates in H. ectypa could also increase resistance of the local S. stellata populations to the parasitic effect of H. ectypa and therefore help to stabilize the Silene-Hadena interaction dynamics.

Characterization of the mating system of a native perennial tetraploid herb, Silene stellata.

PREMISE OF THE STUDY: Nursery pollination systems can range from obligate to facultative. In a system where generalists provide substantial pollination service, an important question is whether the cost of seed predation outweighs the benefit provided by the nursery pollinator to cause the plant to evolve toward more generalized pollination. Using a facultative system native to North America, we tested whether nursery pollinator vs. strictly mutualistic generalists affect mating-system parameters of the host plant and explored the implications for long-term coevolution. METHODS: We used paternity analyses with 11 microsatellite markers to characterize the mating system of Silene stellata when pollination service is primarily through the nursery pollinator Hadena ectypa and generalist moths. KEY RESULTS: Our experimental population of S. stellata was predominantly outcrossing (average outcrossing rate t = 0.83), and mating-system parameters were similar between pollinator groups. We detected significant correlations in both selfing and outcrossed paternity at the fruit and maternal family level, corresponding to limited pollen dispersal (mean = 3.9 m). Among individuals, variation in anther-stigma separation was positively associated with outcrossing rate, which suggests the importance of herkogamy in preventing selfing. CONCLUSIONS: Correlated paternity suggests that seeds from the same fruit and/or plants are sired by a limited number of pollen donors, resulting from low pollen dispersal and potential male-male competition. The similar mating-system parameters of the two pollinator groups suggest that selection for higher outcrossing in S. stellata is likely to be through floral design rather than through increased pollinator specialization with H. ectypa.

Conservation and Genetics.

Humans are responsible for a cataclysm of species extinction that will change the world as we see it, and will adversely affect human health and wellbeing. We need to understand at individual and societal levels why species conservation is important. Accepting the premise that species have value, we need to next consider the mechanisms underlying species extinction and what we can do to reverse the process. One of the last stages of species extinction is the reduction of a species to a few populations of relatively few individuals, a scenario that leads invariably to inbreeding and its adverse consequences, inbreeding depression. Inbreeding depression can be so severe that populations become at risk of extinction not only because of the expression of harmful recessive alleles (alleles having no phenotypic effect when in the heterozygous condition, e.g., Aa, where a is the recessive allele), but also because of their inability to respond genetically with sufficient speed to adapt to changing environmental conditions. However, new conservation approaches based on foundational quantitative and population genetic theory advocate for active genetic management of fragmented populations by facilitating gene movements between populations, i.e., admixture, or genetic rescue. Why species conservation is critical, the genetic consequences of small population size that often lead to extinction, and possible solutions to the problem of small population size are discussed and presented.

Non-equilibrium dynamics and floral trait interactions shape extant angiosperm diversity.

Why are some traits and trait combinations exceptionally common across the tree of life, whereas others are vanishingly rare? The distribution of trait diversity across a clade at any time depends on the ancestral state of the clade, the rate at which new phenotypes evolve, the differences in speciation and extinction rates across lineages, and whether an equilibrium has been reached. Here we examine the role of transition rates, differential diversification (speciation minus extinction) and non-equilibrium dynamics on the evolutionary history of angiosperms, a clade well known for the abundance of some trait combinations and the rarity of others. Our analysis reveals that three character states (corolla present, bilateral symmetry, reduced stamen number) act synergistically as a key innovation, doubling diversification rates for lineages in which this combination occurs. However, this combination is currently less common than predicted at equilibrium because the individual characters evolve infrequently. Simulations suggest that angiosperms will remain far from the equilibrium frequencies of character states well into the future. Such non-equilibrium dynamics may be common when major innovations evolve rarely, allowing lineages with ancestral forms to persist, and even outnumber those with diversification-enhancing states, for tens of millions of years.

Choices and consequences of oviposition by a pollinating seed predator, Hadena ectypa (Noctuidae), on its host plant, Silene stellata (Caryophyllaceae).

PREMISE OF THE STUDY: Pollinating seed predators are models for the study of mutualisms. These insects have dual effects on host-plant fitness, through pollination as adults and flower and fruit predation as larvae. A rarely examined question is whether pollinating seed-predator oviposition choices are influenced by plant floral and size traits and the potential consequences of oviposition for host-plant reproduction. • METHODS: We quantified oviposition by a pollinating seed predator, Hadena ectypa, on its host, Silene stellata, to determine if oviposition was associated with specific plant traits and whether oviposition was significantly correlated with fruit initiation or flower and fruit predation over three years. We also quantified whether stigmatic pollen loads of flowers visited by Hadena that both fed on nectar and oviposited were greater than when Hadena only fed on nectar. • KEY RESULTS: Hadena had significant preference for plants having flowers with long corolla tubes in all three years. Moth oviposition was correlated with other traits only in some years. Oviposition did not increase stigmatic pollen loads. We observed significant positive relationships between both oviposition and fruit initiation and oviposition and flower/fruit predation. • CONCLUSIONS: Hadena ectypa oviposition choices were based consistently on floral tube length differences among individuals, and the consequences of oviposition include both fruit initiation (due to pollination while feeding on nectar prior to oviposition) and larval flower/fruit predation. The positive association between oviposition and fruit initiation may explain the long-term maintenance of facultative pollinating seed-predator interactions.

Variable nursery pollinator importance and its effect on plant reproductive success.

Nursery pollination, in which insects use as hosts the very plants they pollinate, ranges from obligate mutualism to parasitism. In the non-obligate interaction between Greya moths and the host Lithophragma sp., the relative density of nursery pollinators and copollinators, which do not use plant tissues for larval development, is a key determinant of the interaction's outcome. Silene (Caryophyllaceae) nursery pollination by Hadena moths (Lepidoptera: Noctuidae), studied primarily in Europe, is considered antagonistic because copollinators comprise a substantial proportion of the pollinator community. However, there are few studies that ascertain the direction of the Silene-Hadena interaction by taking into account both pollinator service and seed predation. Here, we report a novel comprehensive evaluation of the direction of the interaction between North American Hadena ectypa on Silene stellata, by comparing the relative contributions of nursery and copollinators to S. stellata pollination and relate this to variation in fruit predation and reproductive success of S. stellata across multiple sites and years. Hadena ectypa pollinator importance (pollen deposited/visit/h) varied between years, resulting from variable visitation rate. Copollinator importance was higher than H. ectypa in 1 year and equivalent in another. In two of three sites, lowered H. ectypa activity was not correlated with a significant decrease in plant reproductive success, indicating a negative interaction. Although pollinator service by H. ectypa is substantial in this system, copollinators' service is at least as great, and when the cost of fruit predation is factored in, the net effect of the interaction is parasitism of host plants.

Experimental floral and inflorescence trait manipulations affect pollinator preference and function in a hummingbird-pollinated plant.

PREMISE OF THE STUDY: Controversy is ongoing regarding the importance of pollinator-mediated selection as a source of observed patterns of floral diversity. Although increasing evidence exists of pollinator-mediated selection acting on female reproductive success, there is still limited understanding of pollinator-mediated selection on floral traits via male reproductive success. Here we quantify potential selection by the ruby-throated hummingbird, Archilochus colubris, on four floral traits of hermaphroditic Silene exerted through male floral function. METHODS: In single trait manipulative experiments we quantified hummingbird visitation preference and/or fluorescent dye (a pollen analog) donation as a function of number of flowers displayed (inflorescence size), height of the floral display (inflorescence height), floral color, and corolla tube length. KEY RESULTS: Hummingbirds preferred to visit larger floral displays and floral displays at greater height, likely representing a general pollinator preference for larger, more visible signals and/or greater rewards. In addition, hummingbirds preferred to visit red flowers, and male function was greater in flowers manipulated to have longer corolla tubes. CONCLUSIONS: Selection pressures exerted by hummingbirds on S. virginica floral and inflorescence design through male reproductive success are consistent with the contemporary expression of floral traits of S. virginica relative to related Silene species with different pollinators, and they are consistent with the hummingbird syndrome of traits expressed by S. virginica.

Predicting the probability of outbreeding depression.

Fragmentation of animal and plant populations typically leads to genetic erosion and increased probability of extirpation. Although these effects can usually be reversed by re-establishing gene flow between population fragments, managers sometimes fail to do so due to fears of outbreeding depression (OD). Rapid development of OD is due primarily to adaptive differentiation from selection or fixation of chromosomal variants. Fixed chromosomal variants can be detected empirically. We used an extended form of the breeders' equation to predict the probability of OD due to adaptive differentiation between recently isolated population fragments as a function of intensity of selection, genetic diversity, effective population sizes, and generations of isolation. Empirical data indicated that populations in similar environments had not developed OD even after thousands of generations of isolation. To predict the probability of OD, we developed a decision tree that was based on the four variables from the breeders' equation, taxonomic status, and gene flow within the last 500 years. The predicted probability of OD in crosses between two populations is elevated when the populations have at least one of the following characteristics: are distinct species, have fixed chromosomal differences, exchanged no genes in the last 500 years, or inhabit different environments. Conversely, the predicted probability of OD in crosses between two populations of the same species is low for populations with the same karyotype, isolated for <500 years, and that occupy similar environments. In the former case, we recommend crossing be avoided or tried on a limited, experimental basis. In the latter case, crossing can be carried out with low probability of OD. We used crosses with known results to test the decision tree and found that it correctly identified cases where OD occurred. Current concerns about OD in recently fragmented populations are almost certainly excessive.

Fitness effects of mutation in natural populations of Arabidopsis thaliana reveal a complex influence of local adaptation.

Little is empirically known about the contribution of mutations to fitness in natural environments. However, Fisher's Geometric Model (FGM) provides a conceptual foundation to consider the influence of the environment on mutational effects. To quantify mutational properties in the field, we established eight sets of MA lines (7-10 generations) derived from eight founders collected from natural populations of Arabidopsis thaliana from French and Swedish sites, representing the range margins of the species in Europe. We reciprocally planted the MA lines and their founders at French and Swedish sites, allowing us to test predictions of FGM under naturally occurring environmental conditions. The performance of the MA lines relative to each other and to their respective founders confirmed some and contradicted other predictions of the FGM: the contribution of mutation to fitness variance increased when the genotype was in an environment where its fitness was low, that is, in the away environment, but mutations were more likely to be beneficial when the genotype was in its home environment. Consequently, environmental context plays a large role in the contribution of mutations to the evolutionary process and local adaptation does not guarantee that a genotype is at or close to its optimum.

Evolutionary breakdown of pollination specialization in a Caribbean plant radiation.

• Ecological generalization is postulated to be the rule in plant-pollinator interactions; however, the evolution of generalized flowers from specialized ancestors has rarely been demonstrated. This study examines the evolution of pollination and breeding systems in the tribe Gesnerieae (Gesneriaceae), an Antillean plant radiation that includes specialized and generalized species. • Phylogenetic reconstruction was based on two nDNA markers (internal transcribed spacer (ITS) and G-CYCLOIDEA (GCYC) and morphology. The total evidence Bayesian phylogeny was used for assessment of floral character evolution using Bayesian stochastic character mapping. • Mapping of the pollination system resulted in at least two origins of bat pollination and two origins of generalized pollination (bats, moths and hummingbirds). The evolution of bat pollination was associated with floral transitions reflecting the chiropterophilous floral syndrome. The evolution of generalization was associated with subcampanulate corollas. Autonomous breeding systems evolved only in hummingbird-pollinated lineages. • The correlated evolution of floral traits and pollination systems provides support for the pollination syndrome concept. Floral transitions may have been favored by the low frequency of hummingbird visitation in the Antilles, while the presence of autonomous pollination may have allowed the diversification of ornithophilous lineages. Results suggest that pollinator depauperate faunas on islands select for the evolution of reproductive assurance mechanisms, including generalization and autogamy.

Pollen limitation and reproductive assurance in Antillean Gesnerieae: a specialists vs. generalist comparison.

Pollen limitation of female fecundity is widespread among angiosperms, a signal that pollinators frequently fail to transfer pollen to fertilize all ovules. Recent surveys have suggested that pollen limitation is associated with floral specialization. This study uses a group of Antillean Gesneriaceae with contrasting pollination systems (bat, hummingbird, and generalist) to assess the premise that plants with specialized pollination systems and infrequent pollinator visitation experience greater pollen limitation of fruit and seed set than their generalist congeners. Alternatively, specialists may possess mechanisms that reduce pollen limitation, such as autonomous self-pollination. A survey of autonomous self-pollination conducted on 13 Gesneria and Rhytidophyllum species during 2006-2008 revealed no significant association between reproductive assurance mechanisms and pollination system specialization. However, high levels of potential autonomous self-pollination were only found among specialized hummingbird-pollinated species. A comparison of fruit and seed set between emasculated and unmanipulated flowers provided evidence for autonomous selfing acting as a reproductive assurance mechanism in three out of four ornithophilous species. Furthermore, the Puerto Rican population of G. reticulata relies almost exclusively on self-pollination for reproduction. Two-year pollen supplementation experiments conducted on nine Gesnerieae species from the Dominican Republic and Puerto Rico provided evidence for significant pollen limitation associated with pollination specialization including both bat- and hummingbird-pollinated Gesnerieae; no pollen limitation was detected in any of the four generalist species. No pollen limitation was detected either in two ornithophilous Gesneria species with low hummingbird visitation and high levels of autonomous self-pollination. This study provides support for the idea that generalized pollination systems may, in some cases, buffer against fluctuations in the pollinator environment. However, the use of reproductive assurance mechanisms allows the maintenance of highly specialized pollination systems in pollinator depauperate environments.

Variable and sexually conflicting selection on Silene stellata floral traits by a putative moth pollinator selective agent.

Conflicting selection is an important evolutionary mechanism because it impedes directional evolution and helps to maintain phenotypic variation. It can arise when mutualistic and antagonistic selective agents exert opposing selection on the same trait and when distinct phenotypic optima are favored by different fitness components. In this study, we test for conflicting selection through different sexual functions of the hermaphroditic plant, Silene stellata during its early and late flowering season. We find selection is consistently stronger during the early flowering season, which aligns with the activity peak of the pollinating seed predator Hadena ectypa. Importantly, we observe sex-specific selection on petal dimensions to have opposite signs. We propose that the observed sexually conflicting selection on petal design results from the negative selection through female function for the avoidance of oviposition and the subsequent fruit predation by H. ectypa larvae and the positive selection through male function for pollen export by H. ectypa adults. The Silene-Hadena interaction has previously been considered to be largely parasitic. Our findings suggest a trade-off mechanism that could thwart the evolution of an "escape route" from the nocturnal pollination syndrome by Silene spp. and contribute to the long-term maintenance of the Silene-Hadena system.