A multidimensional selective landscape drives adaptive divergence between and within closely related Phlox species.

Selection causes local adaptation across populations within species and simultaneously divergence between species. However, it is unclear if either the force of or the response to selection is similar across these scales. We show that natural selection drives divergence between closely related species in a pattern that is distinct from local adaptation within species. We use reciprocal transplant experiments across three species of Phlox wildflowers to characterize widespread adaptive divergence. Using provenance trials, we also find strong local adaptation between populations within a species. Comparing divergence and selection between these two scales of diversity we discover that one suite of traits predicts fitness differences between species and that an independent suite of traits predicts fitness variation within species. Selection drives divergence between species, contributing to speciation, while simultaneously favoring extensive diversity that is maintained across populations within a species. Our work demonstrates how the selection landscape is complex and multidimensional.

Mismatch between pollen and pistil size causes asymmetric mechanical reproductive isolation across Phlox species.

In flowering plants, pollen-pistil interactions can serve as an important barrier to reproduction between species. As the last barrier to reproduction before fertilization, interactions between these reproductive organs are both complex and important for determining a suitable mate. Here, we test whether differences in style length generate a post-mating prezygotic mechanical barrier between five species of perennial Phlox wildflowers with geographically overlapping distributions. We perform controlled pairwise reciprocal crosses between three species with long styles and two species with short styles to assess crossing success (seed set). We find that heterospecific seed set is broadly reduced compared to conspecific cross success and reveal a striking asymmetry in heterospecific crosses between species with different style lengths. To determine the mechanism underlying this asymmetric reproductive isolating barrier we assess pollen tube growth in vitro and in vivo. We demonstrate that pollen tubes of short-styled species do not grow long enough to reach the ovaries of long-styled species. We find that short-styled species also have smaller pollen and that both within and between species pollen diameter is highly correlated with pollen tube length. Our results support the hypothesis that the small pollen of short-styled species lacks resources to grow pollen tubes long enough to access the ovaries of the long-styled species, resulting in an asymmetrical, mechanical barrier to reproduction. Such mechanisms, combined with additional pollen-pistil incompatibilities, may be particularly important for closely related species in geographic proximity that share pollinators.

Phylogenomic analyses re-examine the evolution of reinforcement and hypothesized hybrid speciation in Phlox wildflowers.

The tree of life is riddled with reticulate evolutionary histories, and some clades, such as the eastern standing Phlox, appear to be hotspots of hybridization. In this group, there are two cases of reinforcement and nine hypothesized hybrid species. Given their historical importance in our understanding of plant speciation, the relationships between these taxa and the role of hybridization in their diversification require genomic validation. Using phylogenomic analyses, we resolve the evolutionary relationships of the eastern standing Phlox and evaluate hypotheses about whether and how hybridization and gene flow played a role in their diversification. Our results provide novel resolution of the phylogenetic relationships in this group, including paraphyly across some taxa. We identify gene flow during one case of reinforcement and find genomic support for a hybrid lineage underlying one of the five hypothesized homoploid hybrid speciation events. Additionally, we estimate the ancestries of four allotetraploid hybrid species. Our results are consistent with hybridization contributing to diverse evolutionary outcomes within this group; although, not as extensively as previously hypothesized. This study demonstrates the importance of phylogenomics in evaluating hypothesized evolutionary histories of non-model systems and adds to the growing support of interspecific genetic exchange in the generation of biodiversity.

Characterizing each step of pollination in Phlox drummondii reveals that a single butterfly species predominates in the pollinator assemblage.

PREMISE: A central goal of pollination biology is to connect plants with the identity of their pollinator(s). While predictions based on floral syndrome traits are extremely useful, direct observation can reveal further details of a species' pollination biology. The wildflower Phlox drummondii has a floral syndrome consistent with pollination by Lepidoptera. We tested this prediction using empirical data. METHODS: We observed each step of pollination in P. drummondii. First, we observed 55.5 h of floral visitation across the species range. We used temporal pollinator exclusion to determine the contribution of diurnal and nocturnal pollination to reproductive output. We then quantified P. drummondii pollen transfer by the dominant floral visitor, Battus philenor. Finally, we tested the effect of B. philenor visitation on P. drummondii reproduction by quantifying fruit set following single pollinator visits. RESULTS: Battus philenor is the primary pollinator of P. drummondii. Pollination is largely diurnal, and we observed a variety of lepidopteran visitors during the diurnal period. However, B. philenor was the most frequent visitor, representing 88.5% of all observed visits. Our results show that B. philenor is an extremely common visitor and also an effective pollinator by demonstrating that individuals transfer pollen between flowers and that a single visit can elicit fruit set. CONCLUSIONS: Our data are consistent with the prediction of lepidopteran pollination and further reveal a single butterfly species, B. philenor, as the primary pollinator. Our study demonstrates the importance of empirical pollinator observations, adds to our understanding of pollination mechanics, and offers a specific case study of butterfly pollination.

Hybridization and introgression are prevalent in Southern European Erysimum (Brassicaceae) species.

BACKGROUND AND AIMS: Hybridization is a common and important force in plant evolution. One of its outcomes is introgression - the transfer of small genomic regions from one taxon to another by hybridization and repeated backcrossing. This process is believed to be common in glacial refugia, where range expansions and contractions can lead to cycles of sympatry and isolation, creating conditions for extensive hybridization and introgression. Polyploidization is another genome-wide process with a major influence on plant evolution. Both hybridization and polyploidization can have complex effects on plant evolution. However, these effects are often difficult to understand in recently evolved species complexes. METHODS: We combined flow cytometry, analyses of transcriptomic sequences and pollen tube growth assays to investigate the consequences of polyploidization, hybridization and introgression on the recent evolution of several Erysimum (Brassicaceae) species from the South of the Iberian Peninsula, a well-known glacial refugium. This species complex differentiated in the last 2 million years, and its evolution has been hypothesized to be determined mainly by polyploidization, interspecific hybridization and introgression. KEY RESULTS: Our results support a scenario of widespread hybridization involving both extant and 'ghost' taxa. Several taxa studied here, most notably those with purple corollas, are polyploids, probably of allopolyploid origin. Moreover, hybridization in this group might be an ongoing phenomenon, as pre-zygotic barriers appeared weak in many cases. CONCLUSIONS: The evolution of Erysimum spp. has been determined by hybridization to a large extent. Species with purple (polyploids) and yellow flowers (mostly diploid) exhibit a strong signature of introgression in their genomes, indicating that hybridization occurred regardless of colour and across ploidy levels. Although the adaptive value of such genomic exchanges remains unclear, our results demonstrate the significance of hybridization for plant diversification, which should be taken into account when studying plant evolution.

Convergence without divergence in North American red-flowering Silene.

Combinations of correlated floral traits have arisen repeatedly across angiosperms through convergent evolution in response to pollinator selection to optimize reproduction. While some plant groups exhibit very distinct combinations of traits adapted to specific pollinators (so-called pollination syndromes), others do not. Determining how floral traits diverge across clades and whether floral traits show predictable correlations in diverse groups of flowering plants is key to determining the extent to which pollinator-mediated selection drives diversification. The North American Silene section Physolychnis is an ideal group to investigate patterns of floral evolution because it is characterized by the evolution of novel red floral color, extensive floral morphological variation, polyploidy, and exposure to a novel group of pollinators (hummingbirds). We test for correlated patterns of trait evolution that would be consistent with convergent responses to selection in the key floral traits of color and morphology. We also consider both the role of phylogenic distance and geographic overlap in explaining patterns of floral trait variation. Inconsistent with phenotypically divergent pollination syndromes, we find very little clustering of North American Silene into distinct floral morphospace. We also find little evidence that phylogenetic history or geographic overlap explains patterns of floral diversity in this group. White- and pink-flowering species show extensive phenotypic diversity but are entirely overlapping in morphological variation. However, red-flowering species have much less phenotypic disparity and cluster tightly in floral morphospace. We find that red-flowering species have evolved floral traits that align with a traditional hummingbird syndrome, but that these trait values overlap with several white and pink species as well. Our findings support the hypothesis that convergent evolution does not always proceed through comparative phenotypic divergence, but possibly through sorting of standing ancestral variation.

Plant carbohydrate storage: intra- and inter-specific trade-offs reveal a major life history trait.

Trade-offs among carbon sinks constrain how trees physiologically, ecologically, and evolutionarily respond to their environments. These trade-offs typically fall along a productive growth to conservative, bet-hedging continuum. How nonstructural carbohydrates (NSCs) stored in living tree cells (known as carbon stores) fit in this trade-off framework is not well understood. We examined relationships between growth and storage using both within species genetic variation from a common garden, and across species phenotypic variation from a global database. We demonstrate that storage is actively accumulated, as part of a conservative, bet-hedging life history strategy. Storage accumulates at the expense of growth both within and across species. Within the species Populus trichocarpa, genetic trade-offs show that for each additional unit of wood area growth (in cm2 yr-1 ) that genotypes invest in, they lose 1.2 to 1.7 units (mg g-1 NSC) of storage. Across species, for each additional unit of area growth (in cm2 yr-1 ), trees, on average, reduce their storage by 9.5% in stems and 10.4% in roots. Our findings impact our understanding of basic plant biology, fit storage into a widely used growth-survival trade-off spectrum describing life history strategy, and challenges the assumptions of passive storage made in ecosystem models today.

Predicting how pollinator behavior causes reproductive isolation.

Pollinator behavior is an important contributor to plants speciation, yet how variation in pollinator behavior causes variation in reproductive isolation (RI) is largely uncharacterized. Here I present a model that predicts how two aspects of pollinator behavior, constancy and preference, contribute to a barrier to reproduction in plants. This model is motivated by two observations: most co-occurring plants vary in frequency over space and time, and most plants have multiple pollinators that differ in behavior. Thus, my goal was to understand how relative frequencies of plants and pollinators in a community influence ethological RI between co-occurring plants. I find that RI for a focal plant generally increases with increasing relative plant frequency, but the shape of this relationship is highly dependent on the strength of pollinator behavior (constancy and preference). Additionally, when multiple pollinators express different behavior, I find that pollinators with stronger preference disproportionately influence RI. But, I show that RI caused by constancy is the average RI predicted from constancy of each pollinator weighted by pollinator frequency. I apply this model to examples of pollinator-mediated RI in Phlox and in Ipomopsis to predict the relationships between plant frequency and ethological RI in natural systems. This model provides new insights into how and why pollinator specialization causes RI, and how RI could change with changing biological communities.

Genomic analyses overturn two long-standing homoploid hybrid speciation hypotheses.

The importance of hybridization in generating biological diversity has been historically controversial. Previously, inference about hybridization was limited by dependence on morphological data; with the advent of the next-generation sequencing tools for nonmodel organisms, the evolutionary significance of hybridization is more evident. Here, we test classic hypotheses of hybrid origins of two species in the Phlox pilosa complex. Morphological intermediacy motivated the hypotheses that Phlox amoena lighthipei and Phlox pilosa deamii were independent homoploid hybrid lineages derived from P. amoena amoena and P. pilosa pilosa. We use double-digest restriction site-associated DNA sequencing of individuals from throughout the range of these taxa to conduct the most thorough analysis of evolutionary history in this system to date. Surprisingly, we find no support for the hybrid origin of P. pilosa deamii or P. amoena lighthipei. Our data do identify a history of admixture in individuals collected at a contemporary hybrid zone between the putative parent lineages. We show that three very different evolutionary histories, only one of which involves hybrid origin, have produced intermediate or recombinant morphological traits between P. amoena amoena and P. pilosa pilosa. Although morphological data are still an efficient means of generating hypotheses about past gene flow, genomic data are now the standard of evidence for elucidating evolutionary history.

Cycad-Weevil Pollination Symbiosis Is Characterized by Rapidly Evolving and Highly Specific Plant-Insect Chemical Communication.

Coevolution between plants and insects is thought to be responsible for generating biodiversity. Extensive research has focused largely on antagonistic herbivorous relationships, but mutualistic pollination systems also likely contribute to diversification. Here we describe an example of chemically-mediated mutualistic species interactions affecting trait evolution and lineage diversification. We show that volatile compounds produced by closely related species of Zamia cycads are more strikingly different from each other than are other phenotypic characters, and that two distantly related pollinating weevil species have specialized responses only to volatiles from their specific host Zamia species. Plant transcriptomes show that approximately a fifth of genes related to volatile production are evolving under positive selection, but we find no differences in the relative proportion of genes under positive selection in different categories. The importance of phenotypic divergence coupled with chemical communication for the maintenance of this obligate mutualism highlights chemical signaling as a key mechanism of coevolution between cycads and their weevil pollinators.

Homage to Felsenstein 1981, or why are there so few/many species?

If there are no constraints on the process of speciation, then the number of species might be expected to match the number of available niches and this number might be indefinitely large. One possible constraint is the opportunity for allopatric divergence. In 1981, Felsenstein used a simple and elegant model to ask if there might also be genetic constraints. He showed that progress towards speciation could be described by the build-up of linkage disequilibrium among divergently selected loci and between these loci and those contributing to other forms of reproductive isolation. Therefore, speciation is opposed by recombination, because it tends to break down linkage disequilibria. Felsenstein then introduced a crucial distinction between "two-allele" models, which are subject to this effect, and "one-allele" models, which are free from the recombination constraint. These fundamentally important insights have been the foundation for both empirical and theoretical studies of speciation ever since.

Adaptive variation and plasticity in non-structural carbohydrate storage in a temperate tree species.

Trees' total amount of non-structural carbohydrate (NSC) stores and the proportion of these stores residing as insoluble starch are vital traits for individuals living in variable environments. However, our understanding of how stores vary in response to environmental stress is poorly understood as the genetic component of storage is rarely accounted for in studies. Here, we quantified variation in NSC traits in branch samples taken from over 600 clonally transplanted black cottonwood (Populus trichocarpa) trees grown in two common gardens. We found heritable variation in both total NSC stores and the proportion of stores in starch (H2TNC  = 0.19, H2PropStarch  = 0.31), indicating a substantial genetic component of variation. In addition, we found high amounts of plasticity in both traits in response to cold temperatures and significant genotype-by-environment (GxE) interactions in the total amount of NSC stored (54% of P is GxE). This finding of high GxE indicates extensive variation across trees in their response to environment, which may explain why previous studies of carbohydrate stores' responses to stress have failed to converge on a consistent pattern. Overall, we found high amounts of environmental and genetic variation in NSC storage concentrations, which may bolster species against future climate change.

Protocol for Projecting Allele Frequency Change under Future Climate Change at Adaptive-Associated Loci.

We describe how to predict population-level allele frequency change at loci associated with locally adapted traits under future climate conditions. Our method can identify populations that are at higher risk of local extinction and those that might be prime targets for conservation intervention. We draw on previously developed community ecology statistical methods and apply them in novel ways to plant genomes. While a powerful diagnostic tool, our method requires a wealth of genomic data for use. For complete details on the use and execution of this protocol, please refer to Blumstein et al. (2020).

An ancient push-pull pollination mechanism in cycads.

Most cycads engage in brood-site pollination mutualisms, yet the mechanism by which the Cycadales entice pollination services from diverse insect mutualists remains unknown. Here, we characterize a push-pull pollination mechanism between a New World cycad and its weevil pollinators that mirrors the mechanism between a distantly related Old World cycad and its thrips pollinators. The behavioral convergence between weevils and thrips, combined with molecular phylogenetic dating and a meta-analysis of thermogenesis and coordinated patterns of volatile attraction and repulsion suggest that a push-pull pollination mutualism strategy is ancestral in this ancient, dioecious plant group. Hence, it may represent one of the earliest insect/plant pollination mechanisms, arising long before the evolution of visual floral signaling commonly used by flowering plants.

A New Perspective on Ecological Prediction Reveals Limits to Climate Adaptation in a Temperate Tree Species.

Forests absorb a large fraction of anthropogenic CO2 emission, but their ability to continue to act as a sink under climate change depends in part on plant species undergoing rapid adaptation. Yet models of forest response to climate change currently ignore local adaptation as a response mechanism. Thus, considering the evolution of intraspecific trait variation is necessary for reliable, long-term species and climate projections. Here, we combine ecophysiology and predictive climate modeling with analyses of genomic variation to determine whether sugar and starch storage, energy reserves for trees under extreme conditions, have the heritable variation and genetic diversity necessary to evolve in response to climate change within populations of black cottonwood (Populus trichocarpa). Despite current patterns of local adaptation and extensive range-wide heritable variation in storage, we demonstrate that adaptive evolution in response to climate change will be limited by a lack of heritable variation within northern populations and by a need for extreme genetic changes in southern populations. Our method can help design more targeted species management interventions and highlights the power of using genomic tools in ecological prediction to scale from molecular to regional processes to determine the ability of a species to respond to future climates.

Local adaptation mediates floral responses to water limitation in an annual wildflower.

PREMISE: Understanding how environmental stress affects the strength of mutualisms is critically important given observed and projected environmental changes. In particular, the frequency and duration of drought have been increasing worldwide. We investigated how water availability affects plant traits that mediate a pollination mutualism. METHODS: For butterfly-pollinated Phlox drummondii, we determined how moisture availability affects flower size, nectar volume, and nectar sugar amount. Furthermore, we explored the role that local adaptation may play in responses to moisture availability by including plants collected from regions that differ in aridity. Finally, we determined whether responses of plant populations to selection may differ under drought by calculating heritability of traits under control and dry conditions. RESULTS: Flower size was generally smaller in dry plants than in control plants. Early in the treatment period, nectar volume and sugar were higher in dry plants than in control plants for plants from both arid and wetter regions. With prolonged dry treatment, nectar volume and sugar remained higher only in plants from the arid region. Heritability of floral traits was lower for water-limited plants than for control plants. CONCLUSIONS: Plant investment into pollination mutualisms under environmental stress may depend on the extent to which populations are already locally adapted to such conditions, suggesting that mutualism may remain strong, at least in arid regions. However, decreases in heritability under water-limitation suggest that responses to selection imposed by pollinators may be low, even if drought-adapted plants maintain production of rewards to pollinators.

Sonicating bees demonstrate flexible pollen extraction without instrumental learning.

Pollen collection is necessary for bee survival and important for flowering plant reproduction, yet if and how pollen extraction motor routines are modified with experience is largely unknown. Here, we used an automated reward and monitoring system to evaluate modification in a common pollen-extraction routine, floral sonication. Through a series of laboratory experiments with the bumblebee, Bombus impatiens, we examined whether variation in sonication frequency and acceleration is due to instrumental learning based on rewards, a fixed behavioral response to rewards, and/or a mechanical constraint. We first investigated whether bees could learn to adjust their sonication frequency in response to pollen rewards given only for specified frequency ranges and found no evidence of instrumental learning. However, we found that absence versus receipt of a pollen reward did lead to a predictable behavioral response, which depended on bee size. Finally, we found some evidence of mechanical constraints, in that flower mass affected sonication acceleration (but not frequency) through an interaction with bee size. In general, larger bees showed more flexibility in sonication frequency and acceleration, potentially reflecting a size-based constraint on the range over which smaller bees can modify frequency and acceleration. Overall, our results show that although bees did not display instrumental learning of sonication frequency, their sonication motor routine is nevertheless flexible.

Autopolyploid lineage shows climatic niche expansion but not divergence in Arabidopsis arenosa.

PREMISE OF THE STUDY: Successful establishment of neopolyploids, and therefore polyploid speciation, is thought to be contingent on environmental niche shifts from their progenitors. We explore this niche shift hypothesis in the obligate outcrosser Arabidopsis arenosa complex, which includes diploid and recently formed autotetraploid populations. METHODS: To characterize the climatic niches for both cytotypes in Arabidopsis arenosa, we first gathered climatic data from localities with known ploidy types. We then estimated the climatic niches for diploids and autotetraploids and calculated niche overlap. Using this niche overlap statistic, we tested for niche equivalency and similarity. We explored differences in niches by estimating and comparing niche optimum and breadth and then calculated indices of niche expansion and unfilling. KEY RESULTS: Climatic niche overlap between diploids and autotetraploids is substantial. Although the two niche models are not significantly divergent, they are not identical as they differ in both optimum and breadth along two environmental gradients. Autotetraploids fill nearly the entire niche space of diploids and have expanded into novel environments. CONCLUSIONS: We find climatic niche expansion but not divergence, together with a moderate change in the niche optimum, in the autotetraploid lineage of Arabidopsis arenosa. These results indicate that the climatic niche shift hypothesis alone cannot explain the coexistence of tetraploid and diploid cytotypes.

Correlated evolution of self and interspecific incompatibility across the range of a Texas wildflower.

Selection to prevent interspecific mating can cause an increase or a decrease in self-pollination in sympatric populations. Characterizing the geographical variation in self and interspecific incompatibilities within a species can reveal if and how the evolution of self and interspecific mate choice are linked. We used controlled pollinations to characterize the variation in self and interspecific incompatibility across 29 populations of Phlox drummondii. We evaluated seed set from these pollinations and described the developmental timing of variation in pollen-pistil compatibility. There is extensive quantitative variation in self-incompatibility and interspecific-incompatibility with its close congener P. cuspidata. Phlox drummondii populations that co-occur and hybridize with P. cuspidata have significantly higher interspecific incompatibility and self-incompatibility than geographically isolated P. drummondii populations. The strength of self and interspecific incompatibility is significantly correlated among individuals and the strength of both incompatibilities is explained by the success of pollen adhesion to the stigma. The correlated strength of self and interspecific incompatibility across the range of P. drummondii and the concurrent developmental timing of the pollen-pistil interaction, suggests these incompatibilities have an overlapping molecular mechanism. The geographical distribution of variation in incompatibilities indicates that this mechanistic link between incompatibilities may affect the evolution of mate choice in plants.