Effective population size mediates the impact of pollination services on pollen limitation.

Inadequate pollen receipt limits flowering plant reproduction worldwide. Ecological causes of pollen limitation (PL), like pollinator scarcity and low plant abundance, have been a primary research focus. The genetic diversity of plant populations could impact both quantity and quality components of PL in concert with ecological factors, yet empirical examples are lacking. We evaluated joint effects of ecological factors (flower abundance, pollinator visitation) and genetic effective population size (NE) on PL across 13 populations of the common herb Argentina anserina. We used a histological approach with 5504 styles from 1137 flowers to separate quantity and quality components of PL, and link these to reproductive output. NE and pollinator visitation interacted to shape PL, but NE had stronger direct effects. Effectively smaller populations experienced stronger quantity PL, and controlled crosses in a pollinator-free environment revealed that stigmatic pollen quantity was an intrinsic population-level attribute that increased with NE. Pollinator visitation enhanced pollen quality, but only in effectively larger populations. Quantity and quality PL negatively impacted fruit and seed set, respectively. Results highlight that PL is dictated by plant population genetic diversity in addition to commonly evaluated ecological factors. Efforts to support pollinators will more strongly enhance plant reproduction in genetically diverse populations.

Linking pollen limitation and seed dispersal effectiveness.

Seed production and dispersal are crucial ecological processes impacting plant demography, species distributions and community assembly. Plant-animal interactions commonly mediate both seed production and seed dispersal, but current research often examines pollination and seed dispersal separately, which hinders our understanding of how pollination services affect downstream dispersal services. To fill this gap, we propose a conceptual framework exploring how pollen limitation can impact the effectiveness of seed dispersal for endozoochorous and myrmecochorous plant species. We summarize the quantitative and qualitative effects of pollen limitation on plant reproduction and use Optimal Foraging Theory to predict its impact on the foraging behaviour of seed dispersers. In doing so, we offer a new framework that poses numerous hypotheses and empirical tests to investigate links between pollen limitation and seed dispersal effectiveness and, consequently, post-dispersal ecological processes occurring at different levels of biological organization. Finally, considering the importance of pollination and seed dispersal outcomes to plant eco-evolutionary dynamics, we discussed the implications of our framework for future studies exploring the demographic and evolutionary impacts of pollen limitation for animal-dispersed plants.

Divergent gametic thermal performance and floral warming across an elevation gradient.

Thermal environments vary widely across species ranges, establishing the potential for local adaptation of thermal performance optima and tolerance. In the absence of local adaptation, selection should favor mechanisms to meet thermal optima. Floral temperature is a major determinant of reproductive success in angiosperms, yet whether gametic thermal performance shows signatures of local adaptation across temperature gradients, and how variation in gametic thermal performance influences floral evolution, is unknown. We characterized flowering season temperatures for the forb, Argentina anserina, at extremes of a 1000 m elevation gradient and generated thermal performance curves for pollen and ovule performance in populations at each extreme. Thermal optima fell between mean and maximum intrafloral temperatures. However, cooler high-elevation populations had ~4 °C greater pollen thermal optima than warmer low-elevation populations, while tolerance breadths did not differ. We then tested whether plants at elevational extremes differentially warmed the floral microenvironment. High-elevation flowers warmed significantly more than low, bringing intrafloral temperatures nearer the pollen optima. A manipulative experiment demonstrated that stronger warming in high elevation was conferred by floral tissues. Elevational divergence in floral warming may be driven, in part, by selection on flowers to meet different thermal demands of the gametophytes.

Drivers of strong isolation and small effective population size at a leading range edge of a widespread plant.

Climate change has influenced species distributions worldwide with upward elevational shifts observed in many systems. Leading range edge populations, like those at upper elevation limits, are crucial for climate change responses but can exhibit low genetic diversity due to founder effects, isolation, or limited outbreeding. These factors can hamper local adaptation at range limits. Using the widespread herb, Argentina anserina, we measured ecological attributes (population density on the landscape, area of population occupancy, and plant and flower density) spanning a 1000 m elevation gradient, with high elevation populations at the range limit. We measured vegetative clonal potential in the greenhouse for populations spanning the gradient. We combined these data with a ddRAD-seq dataset to test the hypotheses that high elevation populations would exhibit ecological and genomic signatures of leading range edge populations. We found that population density on the landscape declined towards the high elevation limit, as is expected towards range edges. However, plant density was elevated within edge populations. In the greenhouse, high elevation plants exhibited stronger clonal potential than low elevation plants, likely explaining increased plant density in the field. Phylogeographic analysis supported more recent colonization of high elevation populations which were also more genetically isolated, had more extreme heterozygote excess and had smaller effective population size than low. Results support that colonization of high elevations was likely accompanied by increased asexuality, contributing to a decline in effective population size. Despite high plant density in leading edge populations, their small effective size, isolation and clonality could constrain adaptive potential.

Disentangling the components of pollen limitation in a widespread herb with gametophytic self-incompatibility.

PREMISE: Seed production is frequently limited by the receipt of insufficient or low-quality pollen, collectively termed "pollen limitation" (PL). In taxa with gametophytic self-incompatibility (GSI), incompatible pollen can germinate on stigmas but pollen tubes are arrested in styles. This allows for estimates of pollen performance before, during, and after self-recognition, as well as insight into the factors underlying pollen quality limitation in GSI taxa. METHODS: We scored pollen performance following self and outcross pollinations in Argentina anserina to identify the location of self-recognition and establish the relationship between pollen tubes and seed production. We then estimated quantity and quality components of PL from >3300 field-collected styles. We combined our results with other studies to test the prediction that low pollen quality, but not quantity, drives higher PL in self-incompatible (SI) taxa than in self-compatible taxa (SC). RESULTS: Self and outcross pollen germinated readily on stigmas, but 96% of germinated self-pollen was arrested during early tube elongation. Reproduction in the field was more limited by pollen quality than by quantity, and pollen failure near the location of self-recognition was a stronger barrier to fertilization than pollen germination. Across 26 taxa, SI species experienced stronger pollen quality, but not quantity, limitation than SC species. CONCLUSIONS: Evaluating pollen performance at multiple points within pistils can elucidate potential causes of pollen quality limitation. The receipt of incompatible pollen inhibits fertilization success more than insufficient pollen receipt or poor pollen germination in A. anserina. Likewise, pollen quality limitation drives high overall PL in other SI taxa.

Pollinators exert selection on floral traits in a pollen-limited, narrowly endemic spring ephemeral.

PREMISE: Floral traits are frequently under pollinator-mediated selection, especially in taxa subject to strong pollen-limitation, such as those reliant on pollinators. However, antagonists can be agents of selection on floral traits as well. The causes of selection acting on spring ephemerals are understudied though these species can experience particularly strong pollen-limitation. I examined pollinator- and antagonist-mediated selection in a narrowly endemic spring ephemeral, Trillium discolor. METHODS: I measured pollen limitation in T. discolor across two years and evaluated its breeding system. I compared selection on floral traits (display height, petal size, petal color, flowering time) between open-pollinated, and pollen-supplemented plants to measure the strength and mode of pollinator-mediated selection. I assessed whether natural levels of antagonism impacted selection on floral traits. RESULTS: Trillium discolor was self-incompatible and experienced pollen limitation in both years of the study. Pollinators exerted negative disruptive selection on display height and petals size. In one year, pollinator-mediated selection favored lighter petals but in the second year pollinators favored darker petals. Antagonist damage did not alter selection on floral traits. CONCLUSIONS: Results demonstrate that pollinators mediate the strength and mode of selection on floral traits in T. discolor. Interannual variation in the strength, mode, and direction of pollinator-mediated selection on floral traits could be important for maintaining of floral diversity in this system. Observed levels of antagonism were weak agents of selection on floral traits.

Hybrid breakdown is elevated near the historical cores of a species' range.

New species form when they become reproductively isolated. A classic model of speciation posits that derived mutations appear in isolated populations and reduce fitness when combined in hybrids. While these Bateson-Dobzhansky-Muller incompatibilities are known to accumulate as populations diverge over time, they may also reflect the amount of standing genetic variation within populations. We analysed the fitness of F2 hybrids in crosses between 24 populations of a plant species (Campanula americana) with broad variation in standing genetic variation and genetic differentiation driven by post-glacial range expansions. Hybrid breakdown varied substantially and was strongest between populations near the historical cores of the species range where within-population genetic diversity was high. Nearly half of the variation in hybrid breakdown was predicted by the combined effects of standing genetic variation within populations, their pairwise genetic differentiation and differences in the climates they inhabit. Hybrid breakdown was enhanced between populations inhabiting distinct climates, likely reflecting local adaptation. Results support that the mutations causing hybrid breakdown, the raw material for speciation, are more common in long-inhabited areas of the species range. Genetic diversity harboured in refugial areas is thus an important source of incompatibilities critical to the speciation process.

Elevational divergence in pigmentation plasticity is associated with selection and pigment biochemistry.

Phenotypic plasticity is predicted to evolve in environmentally variable habitats, or those experiencing a high frequency of strong selection. The evolution of plasticity may however be constrained by costs or physiological limitations. In flowers, UV-absorbing pigmentation ameliorates UV damage to pollen, and is linked with elevated UV exposure. Whether plasticity contributes to this pattern remains unclear. Petals of Argentina anserina have larger UV-absorbing petal areas at high elevations where they experience higher and more variable UV exposure than low elevations. We measured UV-induced pigmentation plasticity in high- and low-elevation populations (hereafter, "high," "low"), and selection on pigmentation via male fitness. We dissected UV pigment biochemistry using metabolomics to explore biochemical mechanisms underlying plasticity. High displayed positive UV-induced pigmentation plasticity but low lacked plasticity. Selection favored elevated pigmentation under UV in high, supporting adaptive plasticity. In high, UV absorption was conferred by flavonoids produced in one flavonoid pathway branch. However, in low, UV absorption was associated with many compounds spanning multiple branches. Elevated plasticity was thus associated with reduced pigment diversity. These results are consistent with adaptive floral pigmentation plasticity in more extreme and variable environments. We discuss how biochemical underpinnings of pigmentation may permit or constrain the evolution of pigmentation plasticity.

A test of Sensory Drive in plant-pollinator interactions: heterogeneity in the signalling environment shapes pollinator preference for a floral visual signal.

Sensory Drive predicts that habitat-dependent signal transmission and perception explain the diversification of communication signals. Whether Sensory Drive shapes floral evolution remains untested in nature. Pollinators of Argentina anserina prefer small ultraviolet (UV)-absorbing floral guides at low elevation but larger guides at high. However, mechanisms underlying differential preference are unclear. High elevation populations experience elevated UV irradiance and frequently flower against bare substrates rather than foliage, potentially impacting signal transmission and perception. At high and low elevation extremes, we experimentally tested the effects of UV light (ambient vs reduced) and floral backgrounds (foliage vs bare) on pollinator choice for UV guide size. We examined how different signalling environments shaped pollinator-perceived flower colour using visual system models. At high elevation, pollinators preferred locally common large UV guides under ambient UV, but lacked preference under reduced UV. Flies preferred large guides only against bare substrate, the common high elevation background. Ambient UV amplified contrast of large UV guides with floral backgrounds, and flowers contrasted more with bare ground than foliage. Results support that local signalling conditions contribute to pollinator preference for a floral visual signal, a key tenet of Sensory Drive. Components of Sensory Drive could shape floral signal evolution in other plants spanning heterogeneous signalling environments.

Reply to Robson et al.

Robson et al.'s commentary1 on our article, 'Floral pigmentation has responded rapidly to global change in ozone and temperature'2, questions the study's conclusion that floral ultraviolet (UV) pigmentation has responded to global change, particularly to total column ozone (TCO). Robson et al.1 claim that our study spanned a time frame in which ozone was not declining and suggest no biological relationship between UV-B exposure and UV floral pigmentation. To support their claims, they selectively remove and reanalyze data. We respond with a critique of their interpretations of our results, and analyses of temporal patterns of TCO data from Koski et al.2. Despite Robson et al.'s concerns, our study continues to support a link between temporal changes in ozone and temperature, and temporal changes in UV floral pigmentation.

The effects of climate change on floral anthocyanin polymorphisms.

Pigmentation affords resistance to abiotic stressors, and thus can respond adaptively or plastically to drought and extreme temperatures associated with global change. Plants frequently display variability in flower coloration that is underlain by anthocyanin pigmentation. While anthocyanin polymorphisms impact plant-animal interactions, they also impact reproductive performance under abiotic stress. We used descriptions of flower colour from over 1900 herbarium records representing 12 North American species spanning 124 years to test whether anthocyanin-based flower colour has responded to global change. Based on demonstrated abiotic associations with performance of anthocyanin colour morphs, we predicted pigmentation would increase in species experiencing increased aridity, but decline in those experiencing larger increases in temperature. We found that the frequency of reports of pigmented morphs increased temporally in some taxa but displayed subtle declines in others. Pigmentation was negatively associated with temperature and positively associated with vapour pressure deficit (a metric of aridity) across taxa. Species experiencing larger temperature increases over time displayed reductions in pigmentation, while those experiencing increases in aridity displayed increases in pigmentation. Change in anthocyanin-based floral colour was thus linked with climatic change. Altered flower coloration has the strong potential to impact plant-animal interactions and overall plant reproductive performance.

Westward range expansion from middle latitudes explains the Mississippi River discontinuity in a forest herb of eastern North America.

It is often expected that temperate plants have expanded their geographical ranges northward from primarily southern refugia. Evidence for this hypothesis is mixed in eastern North American species, and there is increasing support for colonization from middle latitudes. We studied genome-wide patterns of variation in RADseq loci to test hypotheses concerning range expansion in a North American forest herb (Campanula americana). First, spatial patterns of genetic differentiation were determined. Then phylogenetic relationships and divergence times were estimated. Spatial signatures of genetic drift were also studied to identify the directionality of recent range expansion and its geographical origins. Finally, spatially explicit scenarios for the spread of plants across the landscape were compared, using variation in the population mutation parameter and Tajima's D. We found strong longitudinal subdivision, with populations clustering into groups west and east of the Mississippi River. While the southeastern region was probably part of a diverse Pleistocene refugium, there is little evidence that range expansion involved founders from these southern locales. Instead, declines in genetic diversity and the loss of rare alleles support a westward colonization wave from a middle latitude refugium near the southern Appalachian Mountains, with subsequent expansion from a Pleistocene staging ground in the Mississippi River Valley (0.51-1.27 million years ago). These analyses implicate stepping stone colonization from middle latitudes as an important mechanism of species range expansion in eastern North America. This study further demonstrates the utility of population genetics as a tool to infer the routes travelled by organisms during geographical range expansion.

Floral Pigmentation Has Responded Rapidly to Global Change in Ozone and Temperature.

Across kingdoms, organisms ameliorate UV stress by increasing UV-absorbing pigmentation. Rapid ozone degradation during the 20th century resulted in elevated UV incidence, but pigmentation responses to this aspect of global change have yet to be demonstrated. In flowering plants, UV exposure favors larger areas of UV-absorbing pigmentation on petals, which protects pollen from UV-damage. Pigmentation also affects floral thermoregulation, suggesting climate warming may additionally impact pigmentation. We used 1,238 herbarium specimens collected from 1941 to 2017 to test whether change in UV floral pigmentation was associated with altered ozone and temperature in 42 species spanning three continents. We tested three predictions: first, UV-absorbing pigmentation will increase temporally and be correlated with reduced ozone (higher UV) when accounting for effects of temperature; second, taxa that experienced larger ozone declines will display larger increases in pigmentation; and third, taxa with anthers exposed to ambient UV will respond more strongly than those with anthers protected by petals. Globally, the extent of petal UV pigmentation increased significantly across taxa by ∼2% per year. However, temporal change was species specific-increasing in some taxa but declining in others. Species with exposed anthers experiencing larger declines in ozone displayed more dramatic pigmentation increases. For taxa with anthers enclosed within petals, pigmentation declined with increases in temperature, supporting a thermoregulatory role of UV pigmentation. Results document a rapid phenotypic response of floral pigmentation to anthropogenic climatic change, suggesting that global change may alter pollination through its impact on floral color, with repercussions for plant reproductive fitness.

Macroevolution of Flower Color Patterning: Biased Transition Rates and Correlated Evolution with Flower Size.

Floral pigmentation patterns can both mediate plant-pollinator interactions and modify the abiotic environment of reproductive structures. To date, there have been no inquiries into the rate and directionality of macroevolutionary transitions between patterned and non-patterned petals despite their ecological importance and ubiquity across angiosperms. Petals in the Potentilleae tribe (Rosaceae) display color patterns in the ultraviolet (UV) and human-visible spectrum, or can be uniform in color (i.e., patternless). Using a phylogeny of Potentilleae, I test whether evolutionary transition rates between patterned and non-patterned petals are biased in either direction. I then examine whether UV and human-visible floral patterns are phylogenetically correlated and test the prediction that color patterns will evolve in concert with larger flowers if they function as guides to orient pollinators to floral rewards. I found that transition rates were biased toward petals that were uniform in color. Transition rates from patterned to uniformly colored petals were two and six times higher than the reverse for UV and human-visible pattern, respectively. The presence of UV and human-visible pattern evolved independently from one another. However, the evolution of human-visible pattern was associated with the evolution of larger flowers but the evolution of UV pattern was correlated with the evolution of smaller flowers. I posit that the transition bias toward non-patterned flowers may reflect developmental constraints on spatial regulation of pigments required to produce floral color patterning. The correlated evolution of larger flowers and human-visible pigmentation patterns support the hypothesis that nectar or pollen guides are more likely to evolve in larger-flowered species. This work provides insight into how transition rate bias and trait correlations can shape phylogenetic patterns of floral color pattern diversity.

Geographic Variation in Floral Color and Reflectance Correlates With Temperature and Colonization History.

Petal color variation within species is common and may be molded by abiotic or biotic selection pressures, or neutral population structure. For example, darker flowers may be favored in cooler environments because they absorb more solar radiation, elevating the temperature of reproductive structures. Additionally, flower color may evolve to attract the dominant or most efficient pollinator type in a given population. Here, we evaluate geographic variation in petal coloration across the range of Campanula americana in Eastern North America and test whether color covaries with abiotic factors, the pollination community, and genetic structure established through post-glacial expansion. Consistent with other studies, flowers from cooler, higher latitude populations were less reflective across the UV-NIR spectrum than those from warmer populations. Local temperature explained variation in petal reflectance better than the pollinator community or colonization history. Petal color perceived by trichromatic bee pollinators displayed a strong longitudinal pattern but was unassociated with climatic factors and the pollinator community. Instead, pollinator-perceived color was tightly correlated with the geographic distance from C. americana's glacial refugium. In total, abiotic conditions appear to shape large-scale geographic variation in the intensity of petal reflectance while genetic structure is the strongest driver of pollinator-perceived petal coloration. This study highlights the importance of abiotic factors and historical processes associated with range expansion as major evolutionary forces shaping diversity of flower coloration on large geographic scales.

The role of sensory drive in floral evolution.

Sensory drive theory posits that the evolution of communication signals is shaped by the sensory systems of receivers and the habitat conditions under which signals are received. It has inspired an enormous body of research, advancing our understanding of signal evolution and speciation in animals. In plants, the extreme diversification of floral signals has fascinated biologists for over a century. While processes involved in sensory drive probably play out in plant-pollinator communication, the theory has not been formally synthesized in this context. However, it has untapped potential to explain mechanisms underlying variation in pollinator preferences across populations, and how environmental conditions impact floral signal transmission and perception. Here I develop a framework of sensory drive for plant-pollinator interactions, identifying similarities and differences from its original conception. I then summarize studies that shed light on how the primary processes of sensory drive - habitat transmission, perceptual tuning, and signal matching - apply to the evolution of floral color and scent. Throughout, I propose research avenues and approaches to assess how sensory drive shapes floral diversity. This framework will be important for explaining patterns of extant floral diversity and examining how altered signaling conditions under global change will impact the evolutionary trajectory of floral traits.

Pollen colour morphs take different paths to fitness.

Colour phenotypes are often involved in communication and are thus under selection by species interactions. However, selection may also act on colour through correlated traits or alternative functions of biochemical pigments. Such forms of selection are instrumental in maintaining petal colour diversity in plants. Pollen colour also varies markedly, but the maintenance of this variation is little understood. In Campanula americana, pollen ranges from white to dark purple, with darker morphs garnering more pollinator visits and exhibiting elevated pollen performance under heat stress. Here, we generate an F2 population segregating for pollen colour and measure correlations with floral traits, pollen attributes and plant-level traits related to fitness. We determine the pigment biochemistry of colour variants and evaluate maternal and paternal fitness of light and dark morphs by crossing within and between morphs. Pollen colour was largely uncorrelated with floral traits (petal colour, size, nectar traits) suggesting it can evolve independently. Darker pollen grains were larger and had higher anthocyanin content (cyanidin and peonidin) which may explain why they outperform light pollen under heat stress. Overall, pollen-related fitness metrics were greater for dark pollen, and dark pollen sires generated seeds with higher germination potential. Conversely, light pollen plants produce 61% more flowers than dark, and 18% more seeds per fruit, suggesting a seed production advantage. Results indicate that light and dark morphs may achieve fitness through different means-dark morphs appear to have a pollen advantage whereas light morphs have an ovule advantage-helping to explain the maintenance of pollen colour variation.

Floral Color Properties of Serpentine Seep Assemblages Depend on Community Size and Species Richness.

Functional traits, particularly those that impact fitness, can shape the ecological and evolutionary relationships among coexisting species of the same trophic level. Thus, examining these traits and properties of their distributions (underdispersion, overdispersion) within communities can provide insights into key ecological interactions (e.g., competition, facilitation) involved in community assembly. For instance, the distribution of floral colors in a community may reflect pollinator-mediated interactions between sympatric plant species, and the phylogenetic distribution of color can inform how evolutionary contingencies can continue to shape extant community assemblages. Additionally, the abundance and species richness of the local habitat may influence the type or strength of ecological interactions among co-occurring species. To evaluate the impact of community size and species richness on mechanisms shaping the distribution of ecologically relevant traits, we examined how floral color (defined by pollinator color vision models) is distributed within co-flowering assemblages. We modeled floral reflectance spectra of 55 co-flowering species using honeybee (Apis mellifera) and syrphid fly (Eristalis tenax) visual systems to assess the distributions of flower color across 14 serpentine seep communities in California. We found that phylogenetic relatedness had little impact on the observed color assemblages. However, smaller seep communities with lower species richness were more overdispersed for flower color than larger, more species-rich communities. Results support that competitive exclusion could be a dominant process shaping the species richness of flower color in smaller-sized communities with lower species richness, but this is less detectable or overwhelmed by other processes at larger, more speciose communities.

Selfing ability and drift load evolve with range expansion.

Colonization at expanding range edges often involves few founders, reducing effective population size. This process can promote the evolution of self-fertilization, but implicating historical processes as drivers of trait evolution is often difficult and requires an explicit model of biogeographic history. In plants, contemporary limits to outcrossing are often invoked as evolutionary drivers of self-fertilization, but historical expansions may shape mating system diversity, with leading-edge populations evolving elevated selfing ability. In a widespread plant, Campanula americana, we identified a glacial refugium in the southern Appalachian Mountains from spatial patterns of genetic drift among 24 populations. Populations farther from this refugium have smaller effective sizes and fewer rare alleles. They also displayed elevated heterosis in among-population crosses, reflecting the accumulation of deleterious mutations during range expansion. Although populations with elevated heterosis had reduced segregating mutation load, the magnitude of inbreeding depression lacked geographic pattern. The ability to self-fertilize was strongly positively correlated with the distance from the refugium and mutation accumulation-a pattern that contrasts sharply with contemporary mate and pollinator limitation. In this and other species, diversity in sexual systems may reflect the legacy of evolution in small, colonizing populations, with little or no relation to the ecology of modern populations.