A few essential genetic loci distinguish Penstemon species with flowers adapted to pollination by bees or hummingbirds.

In the formation of species, adaptation by natural selection generates distinct combinations of traits that function well together. The maintenance of adaptive trait combinations in the face of gene flow depends on the strength and nature of selection acting on the underlying genetic loci. Floral pollination syndromes exemplify the evolution of trait combinations adaptive for particular pollinators. The North American wildflower genus Penstemon displays remarkable floral syndrome convergence, with at least 20 separate lineages that have evolved from ancestral bee pollination syndrome (wide blue-purple flowers that present a landing platform for bees and small amounts of nectar) to hummingbird pollination syndrome (bright red narrowly tubular flowers offering copious nectar). Related taxa that differ in floral syndrome offer an attractive opportunity to examine the genomic basis of complex trait divergence. In this study, we characterized genomic divergence among 229 individuals from a Penstemon species complex that includes both bee and hummingbird floral syndromes. Field plants are easily classified into species based on phenotypic differences and hybrids displaying intermediate floral syndromes are rare. Despite unambiguous phenotypic differences, genome-wide differentiation between species is minimal. Hummingbird-adapted populations are more genetically similar to nearby bee-adapted populations than to geographically distant hummingbird-adapted populations, in terms of genome-wide dXY. However, a small number of genetic loci are strongly differentiated between species. These approximately 20 "species-diagnostic loci," which appear to have nearly fixed differences between pollination syndromes, are sprinkled throughout the genome in high recombination regions. Several map closely to previously established floral trait quantitative trait loci (QTLs). The striking difference between the diagnostic loci and the genome as whole suggests strong selection to maintain distinct combinations of traits, but with sufficient gene flow to homogenize the genomic background. A surprisingly small number of alleles confer phenotypic differences that form the basis of species identity in this species complex.

Ecological Diversification in an Adaptive Radiation of Plants: The Role of De Novo Mutation and Introgression.

Adaptive radiations are characterized by rapid ecological diversification and speciation events, leading to fuzzy species boundaries between ecologically differentiated species. Adaptive radiations are therefore key systems for understanding how species are formed and maintained, including the role of de novo mutations versus preexisting variation in ecological adaptation and the genome-wide consequences of hybridization events. For example, adaptive introgression, where beneficial alleles are transferred between lineages through hybridization, may fuel diversification in adaptive radiations and facilitate adaptation to new environments. In this study, we employed whole-genome resequencing data to investigate the evolutionary origin of hummingbird-pollinated flowers and to characterize genome-wide patterns of phylogenetic discordance and introgression in Penstemon subgenus Dasanthera, a small and diverse adaptive radiation of plants. We found that magenta hummingbird-adapted flowers have apparently evolved twice from ancestral blue-violet bee-pollinated flowers within this radiation. These shifts in flower color are accompanied by a variety of inactivating mutations to a key anthocyanin pathway enzyme, suggesting that independent de novo loss-of-function mutations underlie the parallel evolution of this trait. Although patterns of introgression and phylogenetic discordance were heterogenous across the genome, a strong effect of gene density suggests that, in general, natural selection opposes introgression and maintains genetic differentiation in gene-rich genomic regions. Our results highlight the importance of both de novo mutation and introgression as sources of evolutionary change and indicate a role for de novo mutation in driving parallel evolution in adaptive radiations.

How the switch to hummingbird pollination has greatly contributed to our understanding of evolutionary processes.

The evolutionary switch to hummingbird pollination exemplifies complex adaptation, requiring evolutionary change in multiple component traits. Despite this complexity, diverse lineages have converged on hummingbird-adapted flowers on a relatively short evolutionary timescale. Here, I review how features of the genetic basis of adaptation contribute to this remarkable evolutionary lability. Large-effect substitutions, large mutational targets for adaptation, adaptive introgression, and concentrated architecture all contribute to the origin and maintenance of hummingbird-adapted flowers. The genetic features of adaptation are likely shaped by the ecological and geographic context of the switch to hummingbird pollination, with implications for future evolutionary trajectories.

Opposing Patterns of Altitude-Driven Pollinator Turnover in the Tropical and Temperate Americas.

AbstractAbiotic factors (e.g., temperature, precipitation) vary markedly along elevational gradients and differentially affect major groups of pollinators. Ectothermic bees, for example, are impeded in visiting flowers by cold and rainy conditions common at high elevations, while endothermic hummingbirds may continue foraging under such conditions. Despite the possibly far-reaching effects of the abiotic environment on plant-pollinator interactions, we know little about how these factors play out at broad ecogeographic scales. We address this knowledge gap by investigating how pollination systems vary across elevations in 26 plant clades from the Americas. Specifically, we explore Cruden's 1972 hypothesis that the harsh montane environment drives a turnover from insect to vertebrate pollination at higher elevations. We compared the elevational distribution and bioclimatic attributes for a total of 2,232 flowering plants and found that Cruden's hypothesis holds only in the tropics. Above 30°N and below 30°S, plants pollinated by vertebrates (mostly hummingbirds) tend to occur at lower elevations than those pollinated by insects. We hypothesize that this latitudinal transition is due to the distribution of moist, forested habitats favored by vertebrate pollinators, which are common at high elevations in the tropics but not in the temperate Americas.

Small genetic steps lead to mechanical isolation in hummingbird-pollinated gingers.

Interactions with pollinators are a potent source of natural selection driving the spectacular array of flowering plant diversity on Earth (Kay & Sargent, 2009; Van der Niet et al., 2014). Floral traits play a central role in this process: reliable and effective pollination by animal pollinators depends on complex floral features, including traits that determine pollinator attraction and reward, as well as the mechanics of pollen transfer. Pollinators specify mating events between individuals, and thus differences in flowers have the potential to generate reproductive isolating barriers (floral isolation). A compelling case of floral isolation comes from spiral gingers (Costus), where hummingbird-adapted species have evolved distinct pollen placement strategies (on the bills vs. foreheads of pollinators) due to differences in flower shape and the arrangements of flower parts. This difference in pollen placement causes a mechanical barrier to cross-pollination. In this issue of Molecular Ecology, Kay and Surget-Groba (2022) dissect the genetic basis of these floral differences using a quantitative trait locus (QTL) mapping approach. They find small-effect QTLs that influence multiple correlated traits and allelic effects that suggest a history of directional selection. Their results indicate mechanical isolation reflects adaptive divergence that has built up piecemeal over time.

Adaptation to lower latitudes and lower elevations precedes the evolution of hummingbird pollination in western North American Penstemon.

PREMISE: A switch in pollinator can occur when a plant lineage enters a new habitat where the ancestral pollinator is less common, and a novel pollinator is more common. Because pollinator communities vary according to environmental tolerances and availability of resources, there may be consistent associations between pollination mode and specific regions and habitats. Such associations can be studied in lineages that have experienced multiple pollinator transitions, representing evolutionary replicates. METHODS: Our study focused on a large clade of Penstemon wildflower species in western North America, which has repeatedly evolved hummingbird-adapted flowers from ancestral bee-adapted flowers. For each species, we estimated geographic ranges from occurrence data and inferred environmental niches from climate, topographical, and soil data. Using a phylogenetic comparative approach, we investigated whether hummingbird-adapted species occupy distinct geographic regions or habitats relative to bee-adapted species. RESULTS: Hummingbird-adapted species occur at lower latitudes and lower elevations than bee-adapted species, resulting in a difference in their environmental niche. Bee-adapted species sister to hummingbird-adapted species are also found in relatively low elevations and latitudes, similar to their hummingbird-adapted sister species, suggesting ecogeographic shifts precede pollinator divergence. Sister species pairs-regardless of whether they differ in pollinator-show relatively little geographic range overlap. CONCLUSIONS: Adaptation to a novel pollinator may often occur in geographic and ecological isolation from ancestral populations. The ability of a given lineage to adapt to novel pollinators may critically depend on its ability to colonize regions and habitats associated with novel pollinator communities.

From pollen dispersal to plant diversification: genetic consequences of pollination mode.

Pollinators influence patterns of plant speciation, and one intuitive hypothesis is that pollinators affect rates of plant diversification through their effects on pollen dispersal. By specifying mating events and pollen flow across the landscape, distinct types of pollinators may cause different opportunities for allopatric speciation. This pollen dispersal-dependent speciation hypothesis predicts that pollination mode has effects on the spatial context of mating events that scale up to impact population structure and rates of species formation. Here I consider recent comparative studies, including genetic analyses of plant mating events, population structure and comparative phylogenetic analyses, to examine evidence for this model. These studies suggest that highly mobile pollinators conduct greater gene flow within and among populations, compared to less mobile pollinators. These differences influence patterns of population structure across the landscape. However, the effects of pollination mode on speciation rates is less predictable. In some contexts, the predicted effects of pollen dispersal are outweighed by other factors that govern speciation rates. A multiscale approach to examine effects of pollination mode on plant mating system, population structure and rates of diversification is key to determining the role of pollen dispersal on plant speciation for model clades.

Nectary size is a pollination syndrome trait in Penstemon.

Evolution of complex phenotypes depends on the adaptive importance of individual traits, and the developmental changes required to modify traits. Floral syndromes are complex adaptations to pollinators that include color, nectar, and shape variation. Hummingbird-adapted flowers have evolved a remarkable number of times from bee-adapted ancestors in Penstemon, and previous work demonstrates that color over shape better distinguishes bee from hummingbird syndromes. Here, we examined the relative importance of nectar volume and nectary development in defining Penstemon pollination syndromes. We tested the evolutionary association of nectar volume and nectary area with pollination syndrome across 19 Penstemon species. In selected species, we assessed cellular-level processes shaping nectary size. Within a segregating population from an intersyndrome cross, we assessed trait correlations between nectar volume, nectary area, and the size of stamens on which nectaries develop. Nectar volume and nectary area displayed an evolutionary association with pollination syndrome. These traits were correlated within a genetic cross, suggesting a mechanistic link. Nectary area evolution involves parallel processes of cell expansion and proliferation. Our results demonstrate that changes to nectary patterning are an important contributor to pollination syndrome diversity and provide further evidence that repeated origins of hummingbird adaptation involve parallel developmental processes in Penstemon.

Selfing Can Facilitate Transitions between Pollination Syndromes.

Pollinator-mediated selection on plants can favor transitions to a new pollinator depending on the relative abundances and efficiencies of pollinators present in the community. A frequently observed example is the transition from bee pollination to hummingbird pollination. We present a population genetic model that examines whether the ability to inbreed can influence evolutionary change in traits that underlie pollinator attraction. We find that a transition to a more efficient but less abundant pollinator is favored under a broadened set of ecological conditions if plants are capable of delayed selfing rather than obligately outcrossing. Delayed selfing allows plants carrying an allele that attracts the novel pollinator to reproduce even when this pollinator is rare, providing reproductive assurance. In addition, delayed selfing weakens the effects of Haldane's sieve by increasing the fixation probability for recessive alleles that confer adaptation to the new pollinator. Our model provides novel insight into the paradoxical abundance of recessive mutations in adaptation to hummingbird attraction. It further predicts that transitions to efficient but less abundant pollinators (such as hummingbirds in certain communities) should disproportionately occur in self-compatible lineages. Currently available mating system data sets are consistent with this prediction, and we suggest future areas of research that will enable a rigorous test of this theory.

Accessibility, constraint, and repetition in adaptive floral evolution.

Adaptive phenotypic evolution is shaped by natural selection on multiple organismal traits as well as by genetic correlations among traits. Genetic correlations can arise through pleiotropy and can bias the production of phenotypic variation to certain combinations of traits. This phenomenon is referred to as developmental bias or constraint. Developmental bias may accelerate or constrain phenotypic evolution, depending on whether selection acts parallel or in opposition to genetic correlations among traits. We discuss examples from floral evolution where genetic correlations among floral traits contribute to rapid, coordinated evolution in multiple floral organ phenotypes and suggest future research directions that will explore the relationship between the genetic basis of adaptation and the pre-existing structure of genetic correlations. On the other hand, natural selection may act perpendicular to a strong genetic correlation, for example when two traits are encoded by a subset of the same genes and natural selection favors change in one trait and stability in the second trait. In such cases, adaptation is constrained by the availability of genetic variation that can influence the focal trait with minimal pleiotropic effects. Examples from plant diversification suggest that the origin of certain adaptations depends on the prior evolution of a gene copy with reduced pleiotropic effects, generated through the process of gene duplication followed by subfunctionalization or neofunctionalization. A history of gene duplication in some developmental pathways appears to have allowed particular flowering plant linages to have repeatedly evolved adaptations that might otherwise have been developmentally constrained.

Ecological transition predictably associated with gene degeneration.

Gene degeneration or loss can significantly contribute to phenotypic diversification, but may generate genetic constraints on future evolutionary trajectories, potentially restricting phenotypic reversal. Such constraints may manifest as directional evolutionary trends when parallel phenotypic shifts consistently involve gene degeneration or loss. Here, we demonstrate that widespread parallel evolution in Penstemon from blue to red flowers predictably involves the functional inactivation and degeneration of the enzyme flavonoid 3',5'-hydroxylase (F3'5'H), an anthocyanin pathway enzyme required for the production of blue floral pigments. Other types of genetic mutations do not consistently accompany this phenotypic shift. This pattern may be driven by the relatively large mutational target size of degenerative mutations to this locus and the apparent lack of associated pleiotropic effects. The consistent degeneration of F3'5'H may provide a mechanistic explanation for the observed asymmetry in the direction of flower color evolution in Penstemon: Blue to red transitions are common, but reverse transitions have not been observed. Although phenotypic shifts in this system are likely driven by natural selection, internal constraints may generate predictable genetic outcomes and may restrict future evolutionary trajectories.

Multiplexed shotgun genotyping resolves species relationships within the North American genus Penstemon.

PREMISE OF THE STUDY: Evolutionary radiations provide opportunities to examine large-scale patterns in diversification and character evolution, yet are often recalcitrant to phylogenetic resolution due to rapid speciation events. The plant genus Penstemon has been difficult to resolve using Sanger sequence-based markers, leading to the hypothesis that it represents a recent North American radiation. The current study demonstrates the utility of multiplexed shotgun genotyping (MSG), a style of restriction site-associated DNA sequencing (RADseq), to infer phylogenetic relationships within a subset of species in this genus and provide insight into evolutionary patterns. METHODS: We sampled genomic DNA, primarily from herbarium material, and subjected it to MSG library preparation and Illumina sequencing. The resultant sequencing reads were clustered into homologous loci, aligned, and concatenated into data matrices that differed according to clustering similarity and amount of missing data. We performed phylogenetic analyses on these matrices using maximum likelihood (RAxML) and a species tree approach (SVDquartets). KEY RESULTS: MSG data provide a highly resolved estimate of species relationships within Penstemon. While most species relationships were highly supported, the position of certain taxa remains ambiguous, suggesting that increased taxonomic sampling or additional methodologies may be required. The data confirm that evolutionary shifts from hymenopteran- to hummingbird-adapted flowers have occurred independently many times. CONCLUSIONS: This study demonstrates that phylogenomic approaches yielding thousands of variable sites can greatly improve species-level resolution of recent and rapid radiations. Similar to other studies, we found that less conservative similarity and missing data thresholds resulted in more highly supported topologies.

Ecological diversification in an adaptive radiation of plants: the role of de novo mutation and introgression.

Adaptive radiations are characterized by rapid ecological diversification and speciation events, leading to fuzzy species boundaries between ecologically differentiated species. Adaptive radiations are therefore key systems for understanding how species are formed and maintained, including the role of de novo mutations vs. pre-existing variation in ecological adaptation and the genome-wide consequences of hybridization events. For example, adaptive introgression, where beneficial alleles are transferred between lineages through hybridization, may fuel diversification in adaptive radiations and facilitate adaptation to new environments. In this study, we employed whole-genome resequencing data to investigate the evolutionary origin of hummingbird-pollinated flowers and to characterize genome-wide patterns of phylogenetic discordance and introgression in Penstemon subgenus Dasanthera, a small and diverse adaptive radiation of plants. We found that magenta hummingbird-adapted flowers have apparently evolved twice from ancestral blue-violet bee-pollinated flowers within this radiation. These shifts in flower color are accompanied by a variety of inactivating mutations to a key anthocyanin pathway enzyme, suggesting that independent de novo loss-of-function mutations underlie parallel evolution of this trait. Although patterns of introgression and phylogenetic discordance were heterogenous across the genome, a strong effect of gene density suggests that, in general, natural selection opposes introgression and maintains genetic differentiation in gene-rich genomic regions. Our results highlight the importance of both de novo mutation and introgression as sources of evolutionary change and indicate a role for de novo mutation in driving parallel evolution in adaptive radiations.

Predictive Links between Petal Color and Pigment Quantities in Natural Penstemon Hybrids.

Flowers have evolved remarkable diversity in petal color, in large part due to pollinator-mediated selection. This diversity arises from specialized metabolic pathways that generate conspicuous pigments. Despite the clear link between flower color and floral pigment production, quantitative models inferring predictive relationships between pigmentation and reflectance spectra have not been reported. In this study, we analyze a dataset consisting of hundreds of natural Penstemon hybrids that exhibit variation in flower color, including blue, purple, pink, and red. For each individual hybrid, we measured anthocyanin pigment content and petal spectral reflectance. We found that floral pigment quantities are correlated with hue, chroma, and brightness as calculated from petal spectral reflectance data: hue is related to the relative amounts of delphinidin vs. pelargonidin pigmentation, whereas brightness and chroma are correlated with the total anthocyanin pigmentation. We used a partial least squares regression approach to identify predictive relationships between pigment production and petal reflectance. We find that pigment quantity data provide robust predictions of petal reflectance, confirming a pervasive assumption that differences in pigmentation should predictably influence flower color. Moreover, we find that reflectance data enables accurate inferences of pigment quantities, where the full reflectance spectra provide much more accurate inference of pigment quantities than spectral attributes (brightness, chroma, and hue). Our predictive framework provides readily interpretable model coefficients relating spectral attributes of petal reflectance to underlying pigment quantities. These relationships represent key links between genetic changes affecting anthocyanin production and the ecological functions of petal coloration.

Lessons from flower colour evolution on targets of selection.

The genetic basis of flower colour evolution provides a useful system to address the debate over the relative contribution of regulatory vs. functional mutations in evolution. The relative importance of these two categories depends on the type of flower colour transition and the genes involved in those transitions. These differences reflect differences in the degree of deleterious pleiotropy associated with functional inactivation of various anthocyanin pathway genes. Our findings illustrate how generalized statements regarding the contributions of regulatory and functional mutations to broad categories of traits, such as morphological vs. physiological, ignore differences among traits within categories and in doing so overlook important factors determining the relative importance of regulatory and functional mutations.

Amino acid polymorphisms in Arabidopsis phytochrome B cause differential responses to light.

Plants have a sophisticated system for sensing and responding to their light environment. The light responses of populations and species native to different habitats show adaptive variation; understanding the mechanisms underlying photomorphogenic variation is therefore of significant interest. In Arabidopsis thaliana, phytochrome B (PHYB) is the dominant photoreceptor for red light and plays a major role in white light. Because PHYB has been proposed as a candidate gene for several quantitative trait loci (QTLs) affecting light response, we have investigated sequence and functional variation in Arabidopsis PHYB. We examined PHYB sequences in 33 A. thaliana individuals and in the close relative Arabidopsis lyrata. From 14 nonsynonymous polymorphisms, we chose 5 for further study based on previous QTL studies. In a larger collection of A. thaliana accessions, one of these five polymorphisms, I143L, was associated with variation in red light response. We used transgenic analysis to test this association and confirmed experimentally that natural PHYB polymorphisms cause differential plant responses to light. Furthermore, our results show that allelic variation of PHYB activity is due to amino acid rather than regulatory changes. Together with earlier studies linking variation in light sensitivity to photoreceptor genes, our work suggests that photoreceptors may be a common target of natural selection.

Individualized mating system estimation using genomic data.

The estimation of outcrossing rates in hermaphroditic species has been a major focus in the evolutionary study of reproductive strategies, and is also essential for plant breeding and conservation. Surprisingly, genomics has thus far minimally influenced outcrossing rate studies. In this article, we generalize a Bayesian inference method (BORICE) to accommodate genomic data from multiple subpopulations of a species. As an empirical demonstration, BORICE is applied to 115 maternal families of Mimulus guttatus. The analysis shows that low-level whole genome sequencing of parents and offspring is sufficient for individualized mating system estimation: 208 offspring (88.5%) were definitively called as outcrossed, 23 (9.8%) as selfed. After mating system parameters are established (each offspring as outcrossed or selfed and the inbreeding level of maternal plants), BORICE outputs posterior genotype probabilities for each SNP genomewide. Individual SNP calls are often burdened with considerable uncertainty and distilling information from closely linked sites (within genomic windows) can be a useful strategy. For the Mimulus data, principal components based on window statistics were sufficient to diagnose inversion polymorphisms and estimate their effects on spatial structure, phenotypic and fitness measures. More generally, mating system estimation with BORICE can set the stage for population and quantitative genomic analyses, particularly researchers collect phenotypic or fitness data from maternal individuals.