Phenology and foraging bias contribute to sex-specific foraging patterns in the rare declining butterfly Argynnis idalia idalia.

Variation in pollinator foraging behavior can influence pollination effectiveness, community diversity, and plant-pollinator network structure. Although effects of interspecific variation have been widely documented, studies of intraspecific variation in pollinator foraging are relatively rare. Sex-specific differences in resource use are a strong potential source of intraspecific variation, especially in species where the phenology of males and females differ. Differences may arise from encountering different flowering communities, sex-specific traits, nutritional requirements, or a combination of these factors. We evaluated sex-specific foraging patterns in the eastern regal fritillary butterfly (Argynnis idalia idalia), leveraging a 21-year floral visitation dataset. Because A. i. idalia is protandrous, we determined whether foraging differences were due to divergent phenology by comparing visitation patterns between the entire season with restricted periods of male-female overlap. We quantified nectar carbohydrate and amino acid contents of the most visited plant species and compared those visited more frequently by males versus females. We demonstrate significant differences in visitation patterns between male and female A. i. idalia over two decades. Females visit a greater diversity of species, while dissimilarity in foraging patterns between sexes is persistent and comparable to differences between species. While differences are diminished or absent in some years during periods of male-female overlap, remaining signatures of foraging dissimilarity during implicate mechanisms other than phenology. Nectar of plants visited more by females had greater concentrations of total carbohydrates, glucose, and fructose and individual amino acids than male-associated plants. Further work can test whether nutritional differences are a cause of visitation patterns or consequence, reflecting seasonal shifts in the nutritional landscape encountered by male and female A. i. idalia. We highlight the importance of considering sex-specific foraging patterns when studying interaction networks, and in making conservation management decisions for this at-risk butterfly and other species exhibiting strong intraspecific variation.

Changes in female function and autonomous selfing across floral lifespan interact to drive variation in the cost of selfing.

PREMISE: Morphological and developmental changes as flowers age can impact patterns of mating. At the same time, direct or indirect costs of floral longevity can alter their fitness outcomes. This influence has been less appreciated, particularly with respect to the timing of selfing. We investigated changes in stigma events, autonomous selfing, outcross seed set capacity, and autofertility-a measure representing the potential for reproductive assurance-across floral lifespan in the mixed-mating biennial Sabatia angularis. METHODS: We examined stigma morphology and receptivity, autonomous self-pollen deposition, and seed number and size under autonomous self-pollination and hand outcross-pollination for flowers of different ages, from 1 d of female phase until 14 d. We compared autonomous seed production to maximal outcross seed production at each flower age to calculate an index of autofertility. RESULTS: The stigmatic lobes begin to untwist 1 d post anthesis. They progressively open, sextend, coil, and increase in receptivity, peaking or saturating at 8-11 d, depending on the measure. Autonomous seed production can occur early, but on average remains low until 6 d, when it doubles. In contrast, outcross seed number and size start out high, then decline precipitously. Consequently, autofertility increases steeply across floral lifespan. CONCLUSIONS: Changes in stigma morphology and receptivity, timing of autonomous self-pollen deposition, and floral senescence can interact to influence the relative benefit of autonomous selfing across floral lifespan. Our work highlights the interplay between evolution of floral longevity and the mating system, with implications for the maintenance of mixed mating in S. angularis.

How early does the selfing syndrome arise? Associations between selfing ability and flower size within populations of the mixed-mater Collinsia verna.

PREMISE: Widespread associations between selfing rate and floral size within and among taxa suggest that these traits may evolve in concert. Does this association develop immediately because of shared genetic and/or developmental control, or stepwise with selection shaping the evolution of one trait following the other? If the former, then association ought to appear within and across selfing populations. We explore this fundamental question in three populations of the mixed-mater Collinsia verna where autonomous selfing (AS) ability has been shown to be under selection by the pollination environment. METHODS: We grew clonal replicates of C. verna in a controlled environment to characterize broad-sense genetic correlations among traits within populations and to assess whether divergence in mating system and floral traits among these populations is consistent with their previously observed selection pressures. RESULTS: As predicted by their respective pollination environments, we demonstrate significant genetic divergence among populations in AS ability. However, patterns of divergence in floral traits (petal, stamen, and style size, stigmatic receptivity, and stigma-anther distance) were not as expected. Within populations, genetic variation in AS appeared largely independent from floral traits, except for a single weak negative association in one population between flower size and AS rate. CONCLUSIONS: Together, these results suggest that associations between selfing rate and floral traits across Collinsia species are not reflected at microevolutionary scales. If C. verna were to continue evolving toward the selfing syndrome, floral trait evolution would likely follow stepwise from mating system evolution.

Causes and consequences of variation in heterospecific pollen receipt in Oenothera fruticosa.

PREMISE: Heterospecific pollen transfer, the transfer of pollen between species, is common among co-flowering plants, yet the amount of pollen received is extremely variable among species. Intraspecific variation in heterospecific pollen receipt can be even greater, but we lack an understanding of its causes and fitness consequences in wild populations. METHODS: We examined potential drivers of variation in heterospecific pollen receipt in Oenothera fruticosa. We evaluated the relationship between heterospecific and conspecific pollen receipt and considered how visitation by different pollinator groups, local floral neighborhood composition, and flowering phenology affect the total amount and proportion of heterospecific pollen received. Finally, we tested whether variation in heterospecific pollen receipt translated into lower seed production. RESULTS: Heterospecific pollen was ubiquitous on O. fruticosa stigmas, but the amount received was highly variable and unrelated to conspecific pollen receipt. Heterospecific pollen receipt depended on pollinator type, the proportion of nearby conspecific flowers, and flowering date. Significant interactions revealed that the effects of pollinator type and neighborhood were not independent, further contributing to variation in heterospecific pollen. Naturally occurring levels of heterospecific pollen were sufficient to negatively impact seed set, but large amounts of conspecific pollen counteracted this detrimental effect. CONCLUSIONS: Although selection could act on floral traits that attract quality pollinators and promote synchronous flowering in O. fruticosa, the risk of heterospecific pollen is equally dependent on local floral context. This work highlights how extrinsic and intrinsic factors contribute to intraspecific variation in heterospecific pollen receipt in wild plants, with significant fitness consequences.

Type and intensity of surrounding human land use, not local environment, shape genetic structure of a native grassland plant.

Landscape heterogeneity can shape genetic structure and functional connectivity of populations. When this heterogeneity imposes variable costs of moving across the landscape, populations can be structured according to a pattern of "isolation by resistance" (IBR). At the same time, divergent local environmental filters can limit gene flow, creating an alternative pattern of "isolation by environment" (IBE). Here, we evaluate IBR and IBE in the insect-pollinated, biennial plant Sabatia angularis (L.) Pursh (Gentianaceae) across serpentine grasslands in the fragmented landscape of SE Pennsylvania, USA using ~4500 neutral SNP loci. Specifically, we test the extent to which radical alteration of the landscape matrix by humans has fundamentally altered the cost of movement, imprinting a pattern of IBR dictated by land use type and intensity, and the potential for IBE in relation to a gradient of heavy metal concentrations found in serpentine soil. We reveal a strong signal of IBR and a weak signal of IBE across sites, indicating the greater importance of the landscape matrix in shaping genetic structure of S. angularis populations in the study region. Based on Circuitscape and least cost path approaches, we find that both low- and high-intensity urbanization resist gene flow by orders of magnitude greater than "natural" habitats, although resistance to low-intensity urbanization weakens at larger spatial scales. While cropland presents a substantially lower barrier than urban development, cumulative human land use surrounding populations predicts within-population genetic diversity and inbreeding in S. angularis. Our results emphasize the role of forest buffers and corridors in facilitating gene flow between serpentine grassland patches and averting local extinction of plant populations.

Context-dependency of resource allocation trade-offs highlights constraints to the evolution of floral longevity in a monocarpic herb.

Floral longevity is a critical component of floral display, yet there is a conspicuous paucity of empirical research on its evolution within species. Evolutionary models of floral longevity are grounded in resource allocation theory and propose that selection acts on heritable variation to optimize longevity in light of competing floral construction and maintenance costs. Key assumptions remain untested within wild species. We measured maximum floral longevity alongside protandry, flower size, flower number and flowering rate across families of the monocarpic herb Sabatia angularis grown under high and low resources. We evaluated genetic variation, plasticity and correlations between display traits, including fundamental resource-allocation trade-offs and their interactions with resource availability. All display traits showed significant genetic variation. Resource availability influenced mean floral longevity and flower number, with genetic variation in these responses. Importantly, both floral longevity-flower number and flower number-size trade-offs were significant and stronger under low resources. This study reinforces the application of resource allocation theory to floral display trait evolution. Our work highlights the context-dependency of trade-offs and the potential importance of plasticity in resource allocation, with plants investing in the construction of new flowers at faster rates when resources are high rather than in the maintenance of longer-lived flowers.

Shifts to earlier selfing in sympatry may reduce costs of pollinator sharing.

Coexisting plant congeners often experience strong competition for resources. Competition for pollinators can result in direct fitness costs via reduced seed set or indirect costs via heterospecific pollen transfer (HPT), causing subsequent gamete loss and unfit hybrid offspring production. Autonomous selfing may alleviate these costs, but to preempt HPT, selfing should occur early, before opportunities for HPT occur (i.e., "preemptive selfing hypothesis"). We evaluated conditions for this hypothesis in Collinsia sister species, C. linearis and C. rattanii. In field studies, we found virtually identical flowering times and pollinator sharing between congeners in sympatric populations. Compared to allopatric populations, sympatric C. linearis populations enjoyed higher pollinator visitation rates, whereas visitation to C. rattanii did not differ in sympatry. Importantly, the risk of HPT to each species in sympatry was strongly asymmetrical; interspecies visits comprised 40% of all flower-to-flower visits involving C. rattanii compared to just 4% involving C. linearis. Additionally, our greenhouse experiment demonstrated a strong cost of hybridization when C. rattanii was the pollen donor. Together, these results suggest that C. rattanii pays the greatest cost of pollinator sharing. Matching predictions of the preemptive selfing hypothesis, C. rattanii exhibit significantly earlier selfing in sympatric relative to allopatric populations.

Small and surrounded: population size and land use intensity interact to determine reliance on autonomous selfing in a monocarpic plant.

Background and Aims: Habitat fragmentation has transformed landscapes globally, leaving remnants embedded within a complex matrix that is rapidly becoming more developed. For many plant populations, the associated factors of decreased size and intensification of land use surrounding them are expected to increase pollen limitation ('PL'), unless autonomous self-pollination provides reproductive assurance ('RA'). Decreased pollinator visitation is often assumed to drive these patterns, but other, less studied mechanisms might include increased heterospecific pollen transfer or decreased conspecific pollen availability via florivory. I investigate how PL and RA and their potential underlying mechanisms vary with population size and land use intensity surrounding populations in the biennial Sabatia angularis (Gentianaceae). Methods: I estimated the capacity for seed production via autonomous self-pollination (i.e. autofertility). Over 2 years in 22 S. angularis populations across a fragmented landscape, I performed emasculation and pollen supplementation experiments measuring RA and PL, and quantified visitation rates of potential pollinators and a pollen consumer, conspecific pollen loads and rates of heterospecific pollen deposition. Key results: Autofertility based on fruit mass was 93 % under PL but only 51.6 % relative to maximal conditions. PL and RA were significant on average across populations in the first year of study. Variation in RA was significantly influenced by the interaction between population size and land use intensity, which in turn rendered PL independent of these factors. Visitation and heterospecific pollen deposition rates were greatest in small populations and declined with population size, while conspecific pollen loads were greatest in intermediate sized populations. Conclusions: Increased reliance on RA is predicted in small S. angularis populations surrounded by intense development, which can explain elevated selfing rates in fragmented populations of plant species more generally. Results from this study point toward forces such as heterospecific pollen transfer, self-pollen limitation or resource availability influencing the need and ability to rely on RA.

Phenotypic selection varies with pollination intensity across populations of Sabatia angularis.

Pollinators are considered primary selective agents acting on plant traits, and thus variation in the strength of the plant-pollinator interaction might drive variation in the opportunity for selection and selection intensity across plant populations. Here, we examine whether these critical evolutionary parameters covary with pollination intensity across wild populations of the biennial Sabatia angularis. We quantified pollination intensity in each of nine S. angularis populations as mean stigmatic pollen load per population. For female fitness and three components, fruit number, fruit set (proportion of flowers setting fruit) and number of seeds per fruit, we evaluated whether the opportunity for selection varied with pollination intensity. We used phenotypic selection analyses to test for interactions between pollination intensity and selection gradients for five floral traits, including flowering phenology. The opportunity for selection via fruit set and seeds per fruit declined significantly with increasing pollen receipt, as expected. We demonstrated significant directional selection on multiple traits across populations. We also found that selection intensity for all traits depended on pollination intensity. Consistent with general theory about the relationship between biotic interaction strength and the intensity of selection, our study suggests that variation in pollination intensity drives variation in selection across S. angularis populations.

Plasticity of floral longevity and floral display in the self-compatible biennial Sabatia angularis (Gentianaceae): untangling the role of multiple components of pollination.

BACKGROUND AND AIMS: Plasticity of floral traits in response to pollination can enable plants to maximize opportunities for pollen import and export under poor pollination conditions, while minimizing costs under favourable ones. Both floral longevity and display are key traits influencing pollination. While pollination-induced flower wilting is widely documented, we lack an understanding of the multifactorial complexity of this response, including the influence of other pollination components, costs of extended longevity and subsequent impacts on floral display. METHODS: Plasticity of floral longevity was experimentally evaluated in Sabatia angularis in response to multiple pollination factors: pollen addition, removal, and source (self, single-donor outcross, multiple-donor outcross) and timing of pollination. Effects of pollen quantity were further evaluated by exploiting variation in autonomous self-pollen deposition. Delayed pollination costs were tested comparing seed set from early versus late pollinations. Finally, I compared floral display metrics (peak floral display, time to peak flower, flowering duration, mean flowering rate) between experimentally pollinated and control plants. KEY RESULTS: Floral longevity was highly plastic in response to pollen addition and its timing, and the response was dose-dependent but insensitive to pollen source. Pollen removal tended to extend floral longevity, but only insofar as it precluded pollination-induced wilting via autonomous self-pollination. Under delayed pollination, the wilting response was faster and no cost was detected. Pollination further led to reduced peak floral displays and condensed flowering periods. CONCLUSIONS: Floral longevity and display plasticity could optimize fitness in S. angularis, a species prone to pollen limitation and high inbreeding depression. Under pollinator scarcity, extended floral longevities offer greater opportunities for pollen receipt and export at no cost to seed set, reproductive assurance via autonomous self-pollination and larger, more attractive floral displays. Under high pollinator availability, shortened longevities lead to smaller displays that should lower the risk of geitonogamy.

Inbreeding depression and drift load in small populations at demographic disequilibrium.

Inbreeding depression is a major driver of mating system evolution and has critical implications for population viability. Theoretical and empirical attention has been paid to predicting how inbreeding depression varies with population size. Lower inbreeding depression is predicted in small populations at equilibrium, primarily due to higher inbreeding rates facilitating purging and/or fixation of deleterious alleles (drift load), but predictions at demographic and genetic disequilibrium are less clear. In this study, we experimentally evaluate how lifetime inbreeding depression and drift load, estimated by heterosis, vary with census (Nc ) and effective (estimated as genetic diversity, He ) population size across six populations of the biennial Sabatia angularis as well as present novel models of inbreeding depression and heterosis under varying demographic scenarios at disequilibrium (fragmentation, bottlenecks, disturbances). Our experimental study reveals high average inbreeding depression and heterosis across populations. Across our small sample, heterosis declined with He , as predicted, whereas inbreeding depression did not vary with He and actually decreased with Nc . Our theoretical results demonstrate that inbreeding depression and heterosis levels can vary widely across populations at disequilibrium despite similar He and highlight that joint demographic and genetic dynamics are key to predicting patterns of genetic load in nonequilibrium systems.

In a long-term experimental demography study, excluding ungulates reversed invader's explosive population growth rate and restored natives.

A major goal in ecology is to understand mechanisms that increase invasion success of exotic species. A recent hypothesis implicates altered species interactions resulting from ungulate herbivore overabundance as a key cause of exotic plant domination. To test this hypothesis, we maintained an experimental demography deer exclusion study for 6 y in a forest where the native ungulate Odocoileus virginianus (white-tailed deer) is overabundant and Alliaria petiolata (garlic mustard) is aggressively invading. Because population growth is multiplicative across time, we introduce metrics that correctly integrate experimental effects across treatment years, the cumulative population growth rate, λc, and its geometric mean, λper-year, the time-averaged annual population growth rate. We determined λc and λper-year of the invader and of a common native, Trillium erectum. Our results conclusively demonstrate that deer are required for the success of Alliaria; its projected population trajectory shifted from explosive growth in the presence of deer (λper-year = 1.33) to decline toward extinction where deer are excluded (λper-year = 0.88). In contrast, Trillium's λper-year was suppressed in the presence of deer relative to deer exclusion (λper-year = 1.04 vs. 1.20, respectively). Retrospective sensitivity analyses revealed that the largest negative effect of deer exclusion on Alliaria came from rosette transitions, whereas the largest positive effect on Trillium came from reproductive transitions. Deer exclusion lowered Alliaria density while increasing Trillium density. Our results provide definitive experimental support that interactions with overabundant ungulates enhance demographic success of invaders and depress natives' success, with broad implications for biodiversity and ecosystem function worldwide.

Gynodioecy to dioecy: are we there yet?

BACKGROUND: The 'gynodioecy-dioecy pathway' is considered to be one of the most important evolutionary routes from hermaphroditism to separate sexes (dioecy). Despite a large accumulation of evidence for female seed fertility advantages in gynodioecious species (females and hermaphrodites coexist) in support of the first step in the gynodioecy-dioecy pathway, we still have very little evidence for the second step, i.e. the transition from gynodioecy to dioecy. SCOPE: We review the literature to evaluate whether basic predictions by theory are supported. To establish whether females' seed fertility advantage and frequencies are sufficient to favour the invasion of males, we review these for species along the gynodioecy-dioecy pathway published in the last 5 years. We then review the empirical evidence for predictions deriving from the second step, i.e. hermaphrodites' male fertility increases with female frequency, selection favours greater male fertility in hermaphrodites in gynodioecious species, and, where males and hermaphrodites coexist with females (subdioecy), males have greater male fertility than hermaphrodites. We review how genetic control and certain ecological features (pollen limitation, selfing, plasticity in sex expression and antagonists) influence the trajectory of a population along the gynodioecy-dioecy pathway. CONCLUSIONS: Females tend to have greater seed fertility advantages over hermaphrodites where the two coexist, and this advantage is positively correlated with female frequency across species, as predicted by theory. A limited number of studies in subdioecious species have demonstrated that males have an advantage over hermaphrodites, as also predicted by theory. However, less evidence exists for phenotypic selection to increase male traits of hermaphrodites or for increasing male function of hermaphrodites in populations with high female frequency. A few key case studies underline the importance of examining multiple components of male fertility and the roles of pollen limitation, selfing and plasticity, when evaluating advantages. We conclude that we do not yet have a full understanding of the transition from gynodioecy to dioecy.

Sex ratio and subdioecy in Fragaria virginiana: the roles of plasticity and gene flow examined.

Here we examined the roles of sex-differential plasticity (SDP) and gene flow in sex ratio evolution of subdioecious Fragaria virginiana. We assessed whether female frequency varied with resource availability in 17 natural populations and then characterized plasticity and mean investment in allocation to female function at flower and plant levels in the sex morphs in the glasshouse. We estimated patterns of population divergence using five microsatellite markers. We reveal SDP in fruit production substantial enough to translate into a higher equilibrium female frequency at low resources. Thus SDP can account, in part, for the strong negative relationship between female frequency and resources found in the field. Pollen-bearing morphs varied in plasticity across populations, and the degree of plasticity in fruit number was positively correlated with in situ variation in nitrogen (N) availability, suggesting an adaptive component to sex-allocation plasticity. Low neutral genetic differentiation, indicating high gene flow or recent divergence, may contribute to the absence of population differentiation in fruit-setting ability of pollen-bearing morphs despite considerable sex ratio variation. We consider how these processes, in addition to other features of this system, may work in concert to influence sex ratios and to hinder the evolution of dioecy in F. virginiana.

Genetic architecture of sexual dimorphism in a subdioecious plant with a proto-sex chromosome.

The rise of sexual dimorphism is thought to coincide with the evolution of sex chromosomes. Yet because sex chromosomes in many species are ancient, we lack empirical evidence of the earliest stages of this transition. We use QTL analysis to examine the genetic architecture of sexual dimorphism in subdioecious octoploid Fragaria virginiana. We demonstrate that the region housing the male-function locus controls the majority of quantitative variation in proportion fruit set, confirming the existence of a proto-sex chromosome, and houses major QTL for eight additional sexually dimorphic traits, consistent with theory and data from animals and plants with more advanced sex chromosomes. We also detected autosomal QTL, demonstrating contributions to phenotypic variation in sexually dimorphic traits outside the sex-determining region. Moreover, for proportion seed set we found significant epistatic interactions between autosomal QTL and the male-function locus, indicating sex-limited QTL. We identified linked QTL reflecting trade-offs between male and female traits expected from theory and positive integration of male traits. These findings indicate the potential for the evolution of greater sexual dimorphism. Involvement of linkage groups homeologous to the proto-sex chromosome in these correlations reflects the polyploid origin of F. virginiana and raises the possibility that chromosomes in this homeologous group were predisposed to become the sex chromosome.

Comparative genetic mapping points to different sex chromosomes in sibling species of wild strawberry (Fragaria).

Separate sexes have evolved repeatedly from hermaphroditic ancestors in flowering plants, and thus select taxa can provide unparalleled insight into the evolutionary dynamics of sex chromosomes that are thought to be shared by plants and animals alike. Here we ask whether two octoploid sibling species of wild strawberry--one almost exclusively dioecious (males and females), Fragaria chiloensis, and one subdioecious (males, females, and hermaphrodites), F. virginiana--share the same sex-determining chromosome. We created a genetic map of the sex chromosome and its homeologs in F. chiloensis and assessed macrosynteny between it and published maps of the proto-sex chromosome of F. virginiana and the homeologous autosome of hermaphroditic diploid species. Segregation of male and female function in our F. chiloensis mapping population confirmed that linkage and dominance relations are similar to those in F. virginiana. However, identification of the molecular markers most tightly linked to the sex-determining locus in the two octoploid species shows that, in both, this region maps to homeologues of chromosome 6 in diploid congeners, but is located at opposite ends of their respective chromosomes.

Comparative mapping reveals autosomal origin of sex chromosome in octoploid Fragaria virginiana.

Recent evolution of separate sexes in flowering plants provides unparalleled opportunities for understanding the early stages of sex chromosome evolution, including their origin from autosomes. Moreover, the transition from combined to separate sexes can be associated with speciation via polyploidization in angiosperms, suggesting that genome doubling/merger may facilitate sterility mutations required for sex chromosome formation. To gain insight into the origin of sex chromosomes in a polyploid plant, we doubled the simple sequence repeat (SSR) density and increased genome coverage in a genetic map of octoploid Fragaria virginiana, a species purported to have a "proto-sex" chromosome, where limited recombination occurs between 2 linked "loci" carrying the male- and female-sterility mutations. Incorporation of almost 3 times the number of SSR markers into the current map facilitated complete characterization of the F. virginiana proto-sex chromosome, revealing its largely autosomal nature and the location of the sex-determining region toward the distal end. Furthermore, extensive synteny between our genetic map and a map involving diploid hermaphroditic congeners allowed assignment of linkage groups to homeologous groups, identification of the proto-sex chromosome's autosomal homoeolog, and detection of a putative rearrangement near the sex-determining region. Fine mapping and additional comparative work will shed light on the intriguing possibility that rearrangements during polyploidization were involved in the evolution of sex chromosomes in Fragaria.