Pollen Diet Diversity does not Affect Gut Bacterial Communities or Melanization in a Social and Solitary Bee Species.

Pollinators face many stressors, including reduced floral diversity. A low-diversity diet can impair organisms' ability to cope with additional stressors, such as pathogens, by altering the gut microbiome and/or immune function, but these effects are understudied for most pollinators. We investigated the impact of pollen diet diversity on two ecologically and economically important generalist pollinators, the social bumble bee (Bombus impatiens) and the solitary alfalfa leafcutter bee (Megachile rotundata). We experimentally tested the effect of one-, two-, or three-species pollen diets on gut bacterial communities in both species, and the melanization immune response in B. impatiens. Pollen diets included dandelion (Taraxacum officinale), staghorn sumac (Rhus typhina), and hawthorn (Crataegus sp.) alone, each pair-wise combination, or a mix of all three species. We fed bees their diet for 7 days and then dissected out guts and sequenced 16S rRNA gene amplicons to characterize gut bacterial communities. To assess melanization in B. impatiens, we inserted microfilament implants into the bee abdomen and measured melanin deposition on the implant. We found that pollen diet did not influence gut bacterial communities in M. rotundata. In B. impatiens, pollen diet composition, but not diversity, affected gut bacterial richness in older, but not newly-emerged bees. Pollen diet did not affect the melanization response in B. impatiens. Our results suggest that even a monofloral, low-quality pollen diet such as dandelion can support diverse gut bacterial communities in captive-reared adults of these bee species. These findings shed light on the effects of reduced diet diversity on bee health.

Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization.

Crithidia bombi is a trypanosomatid parasite that infects several species of bumble bees (Bombus spp.), by adhering to their intestinal tract. Crithidia bombi infection impairs learning and reduces survival of workers and the fitness of overwintering queens. Although there is extensive research on the ecology of this host-pathogen system, we understand far less about the mechanisms that mediate internal infection dynamics. Crithidia bombi infects hosts by attaching to the hindgut via the flagellum, and one previous study found that a nectar secondary compound removed the flagellum, preventing attachment. However, approaches that allow more detailed observation of parasite attachment and growth would allow us to better understand factors mediating this host-pathogen relationship. We established techniques for genetic manipulation and visualization of cultured C. bombi. Using constructs established for Crithidia fasciculata, we successfully generated C. bombi cells expressing ectopic fluorescent transgenes using two different selectable markers. To our knowledge, this is the first genetic modification of this species. We also introduced constructs that label the mitochondrion and nucleus of the parasite, showing that subcellular targeting signals can function across parasite species to highlight specific organelles. Finally, we visualized fluorescently tagged parasites in vitro in both their swimming and attached forms, and in vivo in bumble bee (Bombus impatiens) hosts. Expanding our cell and molecular toolkit for C. bombi will help us better understand how factors such as host diet, immune system, and physiology mediate outcomes of infection by these common parasites.

Differential bumble bee gene expression associated with pathogen infection and pollen diet.

BACKGROUND: Diet and parasitism can have powerful effects on host gene expression. However, how specific dietary components affect host gene expression that could feed back to affect parasitism is relatively unexplored in many wild species. Recently, it was discovered that consumption of sunflower (Helianthus annuus) pollen reduced severity of gut protozoan pathogen Crithidia bombi infection in Bombus impatiens bumble bees. Despite the dramatic and consistent medicinal effect of sunflower pollen, very little is known about the mechanism(s) underlying this effect. However, sunflower pollen extract increases rather than suppresses C. bombi growth in vitro, suggesting that sunflower pollen reduces C. bombi infection indirectly via changes in the host. Here, we analyzed whole transcriptomes of B. impatiens workers to characterize the physiological response to sunflower pollen consumption and C. bombi infection to isolate the mechanisms underlying the medicinal effect. B. impatiens workers were inoculated with either C. bombi cells (infected) or a sham control (un-infected) and fed either sunflower or wildflower pollen ad libitum. Whole abdominal gene expression profiles were then sequenced with Illumina NextSeq 500 technology. RESULTS: Among infected bees, sunflower pollen upregulated immune transcripts, including the anti-microbial peptide hymenoptaecin, Toll receptors and serine proteases. In both infected and un-infected bees, sunflower pollen upregulated putative detoxification transcripts and transcripts associated with the repair and maintenance of gut epithelial cells. Among wildflower-fed bees, infected bees downregulated immune transcripts associated with phagocytosis and the phenoloxidase cascade. CONCLUSIONS: Taken together, these results indicate dissimilar immune responses between sunflower- and wildflower-fed bumble bees infected with C. bombi, a response to physical damage to gut epithelial cells caused by sunflower pollen, and a strong detoxification response to sunflower pollen consumption. Identifying host responses that drive the medicinal effect of sunflower pollen in infected bumble bees may broaden our understanding of plant-pollinator interactions and provide opportunities for effective management of bee pathogens.

Sunflower plantings reduce a common gut pathogen and increase queen production in common eastern bumblebee colonies.

Community diversity can reduce the prevalence and spread of disease, but certain species may play a disproportionate role in diluting or amplifying pathogens. Flowers act as both sources of nutrition and sites of pathogen transmission, but the effects of specific plant species in shaping bee disease dynamics are not well understood. We evaluated whether plantings of sunflower (Helianthus annuus), whose pollen reduces infection by some pathogens when fed to bees in captivity, lowered pathogen levels and increased reproduction in free-foraging bumblebee colonies (Bombus impatiens). Sunflower abundance reduced the prevalence of a common gut pathogen, Crithidia bombi, and reduced infection intensity, with an order of magnitude lower infection intensity at high sunflower sites compared with sites with little to no sunflower. Sunflower abundance was also positively associated with greater queen production in colonies. Sunflower did not affect prevalence of other detected pathogens. This work demonstrates that a single plant species can drive disease dynamics in foraging B. impatiens, and that sunflower plantings can be used as a tool for mitigating a prevalent pathogen while also increasing reproduction of an agriculturally important bee species.

Gut Transplants from Bees Fed an Antipathogenic Pollen Diet Do Not Confer Pathogen Resistance to Recipients.

Pollinators are threatened by diverse stressors, including microbial pathogens such as Crithidia bombi. Consuming sunflower pollen dramatically reduces C. bombi infection in the bumble bee Bombus impatiens, but the mechanism behind this medicinal effect is unclear. We asked whether diet mediates resistance to C. bombi through changes in the gut microbiome. We hypothesized that sunflower pollen changes the gut microbiome, which in turn reduces Crithidia infection. To test this, we performed a gut transplant experiment. We fed donor bees either a sunflower pollen treatment or buckwheat pollen as a control treatment and then inoculated recipient bees with homogenized guts from either sunflower-fed or buckwheat-fed donor bees. All recipient bees were then fed a wildflower pollen diet. Two days after the transplant, we infected recipients with C. bombi, and 2 days later, we provided another donor gut transplant. To quantify infection, we performed both fecal screens and dissections of the recipient bees. We found no significant differences in C. bombi infection intensity or presence between bees that received sunflower-fed microbiomes versus buckwheat-fed microbiomes. This suggests that sunflower pollen's effects on pathogen resistance are not mediated by gut microbiota.

Flowering plant composition shapes pathogen infection intensity and reproduction in bumble bee colonies.

Pathogens pose significant threats to pollinator health and food security. Pollinators can transmit diseases during foraging, but the consequences of plant species composition for infection is unknown. In agroecosystems, flowering strips or hedgerows are often used to augment pollinator habitat. We used canola as a focal crop in tents and manipulated flowering strip composition using plant species we had previously shown to result in higher or lower bee infection in short-term trials. We also manipulated initial colony infection to assess impacts on foraging behavior. Flowering strips using high-infection plant species nearly doubled bumble bee colony infection intensity compared to low-infection plant species, with intermediate infection in canola-only tents. Both infection treatment and flowering strips reduced visits to canola, but we saw no evidence that infection treatment shifted foraging preferences. Although high-infection flowering strips increased colony infection intensity, colony reproduction was improved with any flowering strips compared to canola alone. Effects of flowering strips on colony reproduction were explained by nectar availability, but effects of flowering strips on infection intensity were not. Thus, flowering strips benefited colony reproduction by adding floral resources, but certain plant species also come with a risk of increased pathogen infection intensity.

Sunflower pollen reduces a gut pathogen in the model bee species, Bombus impatiens, but has weaker effects in three wild congeners.

Commercial bumblebees have become popular models to understand stressors and solutions for pollinator health, but few studies test whether results translate to other pollinators. Consuming sunflower pollen dramatically reduces infection by the gut parasite Crithidia bombi in commercially reared Bombus impatiens. We assessed the effect of sunflower pollen on infection in wild B. impatiens, Bombus griseocollis, Bombus bimaculatus and Bombus vagans. We also asked how pollen diet (50% sunflower pollen versus wildflower pollen) and infection (yes/no) affected performance in wild B. impatiens microcolonies. Compared to controls, sunflower pollen dramatically reduced Crithidia infection in commercial and wild B. impatiens, had similar but less dramatic effects in B. bimaculatus and B. vagans, and no effect in B. griseocollis. Bombus impatiens, B. bimaculatus and B. vagans are in the same subgenus, suggesting that responses to sunflower pollen may be phylogenetically conserved. In microcolonies, 50% sunflower pollen reduced infection compared to wildflower pollen, but also reduced reproduction. Sunflower pollen could control Crithidia infections in B. impatiens and potentially close relatives, but may hinder reproduction if other resources are scarce. We caution that research using managed bee species, such as B. impatiens, be interpreted carefully as findings may not relate to all bee species.

Pollen limitation of native plant reproduction in an urban landscape.

PREMISE: Evidence suggests that bees may benefit from moderate levels of human development. However, the effects of human development on pollination and reproduction of bee-pollinated plants are less-well understood. Studies have measured natural variation in pollination and plant reproduction as a function of urbanization, but few have experimentally measured the magnitude of pollen limitation in urban vs. non-urban sites. Doing so is important to unambiguously link changes in pollination to plant reproduction. Previous work in the Southeastern United States found that urban sites supported twice the abundance of bees compared to non-urban sites. We tested the hypothesis that greater bee abundance in some of the same urban sites translates into reduced pollen limitation compared to non-urban sites. METHODS: We manipulated pollination to three native, wild-growing, bee-pollinated plants: Gelsemium sempervirens, Oenothera fruticosa, and Campsis radicans. Using supplemental pollinations, we tested for pollen limitation of three components of female reproduction in paired urban and non-urban sites. We also measured pollen receipt as a proxy for pollinator visitation. RESULTS: We found that all three plant species were pollen-limited for some measures of female reproduction. However, opposite to our original hypothesis, two of the three species were more pollen-limited in urban relative to non-urban sites. We found that open-pollinated flowers in urban sites received less conspecific and more heterospecific pollen on average than those in non-urban sites. CONCLUSIONS: These results suggest that even when urban sites have more abundant pollinators, this may not alleviate pollen limitation of native plant reproduction in urban landscapes.

Facilitative pollinator sharing decreases with floral similarity in multiple systems.

Investigating the factors that determine whether interactions are competitive or facilitative is essential to understanding community structure and trait evolution. Co-flowering plants interact indirectly through shared pollinators, and meta-analyses suggest that phylogenetic relatedness and floral trait similarity may predict the outcome of these interactions. In a comparative approach, we manipulated the floral community across five focal species to assess how floral similarity and phylogenetic relatedness affect the outcome of interactions. To assess the extent of pollinator-mediated competition versus facilitation, we compared pollen limitation in five focal species growing with floral neighbors (either congeners or neighbors from a different family) relative to a control (growing alone). We measured floral morphology, color, and nectar traits to calculate multivariate floral similarity between species pairs and inferred a phylogeny to calculate phylogenetic distance. Pollinator-mediated interaction values were regressed against floral similarity and phylogenetic distance. We found evidence of pollinator-mediated facilitation in nine of 13 species pairs. Furthermore, floral similarity and phylogenetic distance reduced facilitative interactions, but the latter relationship was not significant when controlling for the identity of the focal species. Our results suggest that facilitative pollinator sharing is more common than reported in the literature, but co-flowering plant species with similar floral traits are less likely to facilitate pollination. A better understanding of the factors that promote facilitation versus competition has important potential applications for managing rare and invasive species.

Crop pests and predators exhibit inconsistent responses to surrounding landscape composition.

The idea that noncrop habitat enhances pest control and represents a win-win opportunity to conserve biodiversity and bolster yields has emerged as an agroecological paradigm. However, while noncrop habitat in landscapes surrounding farms sometimes benefits pest predators, natural enemy responses remain heterogeneous across studies and effects on pests are inconclusive. The observed heterogeneity in species responses to noncrop habitat may be biological in origin or could result from variation in how habitat and biocontrol are measured. Here, we use a pest-control database encompassing 132 studies and 6,759 sites worldwide to model natural enemy and pest abundances, predation rates, and crop damage as a function of landscape composition. Our results showed that although landscape composition explained significant variation within studies, pest and enemy abundances, predation rates, crop damage, and yields each exhibited different responses across studies, sometimes increasing and sometimes decreasing in landscapes with more noncrop habitat but overall showing no consistent trend. Thus, models that used landscape-composition variables to predict pest-control dynamics demonstrated little potential to explain variation across studies, though prediction did improve when comparing studies with similar crop and landscape features. Overall, our work shows that surrounding noncrop habitat does not consistently improve pest management, meaning habitat conservation may bolster production in some systems and depress yields in others. Future efforts to develop tools that inform farmers when habitat conservation truly represents a win-win would benefit from increased understanding of how landscape effects are modulated by local farm management and the biology of pests and their enemies.

Herbivory and Time Since Flowering Shape Floral Rewards and Pollinator-Pathogen Interactions.

Herbivory can induce chemical changes throughout plant tissues including flowers, which could affect pollinator-pathogen interactions. Pollen is highly defended compared to nectar, but no study has examined whether herbivory affects pollen chemistry. We assessed the effects of leaf herbivory on nectar and pollen alkaloids in Nicotiana tabacum, and how herbivory-induced changes in nectar and pollen affect pollinator-pathogen interactions. We damaged leaves of Nicotiana tabacum using the specialist herbivore Manduca sexta and compared nicotine and anabasine concentrations in nectar and pollen. We then pooled nectar and pollen by collection periods (within and after one month of flowering), fed them in separate experiments to bumble bees (Bombus impatiens) infected with the gut pathogen Crithidia bombi, and assessed infections after seven days. We did not detect alkaloids in nectar, and leaf damage did not alter the effect of nectar on Crithidia counts. In pollen, herbivory induced higher concentrations of anabasine but not nicotine, and alkaloid concentrations rose and then fell as a function of days since flowering. Bees fed pollen from damaged plants had Crithidia counts 15 times higher than bees fed pollen from undamaged plants, but only when pollen was collected after one month of flowering, indicating that both damage and time since flowering affected interaction outcomes. Within undamaged treatments, bees fed late-collected pollen had Crithidia counts 10 times lower than bees fed early-collected pollen, also indicating the importance of time since flowering. Our results emphasize the role of herbivores in shaping pollen chemistry, with consequences for interactions between pollinators and their pathogens.

Assessing Chemical Mechanisms Underlying the Effects of Sunflower Pollen on a Gut Pathogen in Bumble Bees.

Many pollinator species are declining due to a variety of interacting stressors including pathogens, sparking interest in understanding factors that could mitigate these outcomes. Diet can affect host-pathogen interactions by changing nutritional reserves or providing bioactive secondary chemicals. Recent work found that sunflower pollen (Helianthus annuus) dramatically reduced cell counts of the gut pathogen Crithidia bombi in bumble bee workers (Bombus impatiens), but the mechanism underlying this effect is unknown. Here we analyzed methanolic extracts of sunflower pollen by LC-MS and identified triscoumaroyl spermidines as the major secondary metabolite components, along with a flavonoid quercetin-3-O-hexoside and a quercetin-3-O-(6-O-malonyl)-hexoside. We then tested the effect of triscoumaroyl spermidine and rutin (as a proxy for quercetin glycosides) on Crithidia infection in B. impatiens, compared to buckwheat pollen (Fagopyrum esculentum) as a negative control and sunflower pollen as a positive control. In addition, we tested the effect of nine fatty acids from sunflower pollen individually and in combination using similar methods. Although sunflower pollen consistently reduced Crithidia relative to control pollen, none of the compounds we tested had significant effects. In addition, diet treatments did not affect mortality, or sucrose or pollen consumption. Thus, the mechanisms underlying the medicinal effect of sunflower are still unknown; future work could use bioactivity-guided fractionation to more efficiently target compounds of interest, and explore non-chemical mechanisms. Ultimately, identifying the mechanism underlying the effect of sunflower pollen on pathogens will open up new avenues for managing bee health.

Bee pathogen transmission dynamics: deposition, persistence and acquisition on flowers.

Infectious diseases are a primary driver of bee decline worldwide, but limited understanding of how pathogens are transmitted hampers effective management. Flowers have been implicated as hubs of bee disease transmission, but we know little about how interspecific floral variation affects transmission dynamics. Using bumblebees ( Bombus impatiens), a trypanosomatid pathogen ( Crithidia bombi) and three plant species varying in floral morphology, we assessed how host infection and plant species affect pathogen deposition on flowers, and plant species and flower parts impact pathogen survival and acquisition at flowers. We found that host infection with Crithidia increased defaecation rates on flowers, and that bees deposited faeces onto bracts of Lobelia siphilitica and Lythrum salicaria more frequently than onto Monarda didyma bracts . Among flower parts, bracts were associated with the lowest pathogen survival but highest resulting infection intensity in bee hosts. Additionally, we found that Crithidia survival across flower parts was reduced with sun exposure. These results suggest that efficiency of pathogen transmission depends on where deposition occurs and the timing and place of acquisition, which varies among plant species and environmental conditions. This information could be used for development of wildflower mixes that maximize forage while minimizing disease spread.

Phenotypic selection on floral traits in an urban landscape.

Native species are increasingly living in urban landscapes associated with abiotic and biotic changes that may influence patterns of phenotypic selection. However, measures of selection in urban and non-urban environments, and exploration of the mechanisms associated with such changes, are uncommon. Plant-animal interactions have played a central role in the evolution of flowering plants and are sensitive to changes in the urban landscape, and thus provide opportunities to explore how urban environments modify selection. We evaluated patterns of phenotypic selection on the floral and resistance traits of Gelsemium sempervirens in urban and non-urban sites. The urban landscape had increased florivory and decreased pollen receipt, but showed only modest differences in patterns of selection. Directional selection for one trait, larger floral display size, was stronger in urban compared to non-urban sites. Neither quadratic nor correlational selection significantly differed between urban and non-urban sites. Pollination was associated with selection for larger floral display size in urban compared to non-urban sites, due to the differences in the translation of pollination into seeds rather than pollinator selectivity. Thus, our data suggest that urban landscapes may not result in sweeping differences in phenotypic selection but rather modest differences for some traits, potentially mediated by species interactions.

Disease where you dine: plant species and floral traits associated with pathogen transmission in bumble bees.

Hotspots of disease transmission can strongly influence pathogen spread. Bee pathogens may be transmitted via shared floral use, but the role of plant species and floral trait variation in shaping transmission dynamics is almost entirely unexplored. Given the importance of pathogens for the decline of several bee species, understanding whether and how plant species and floral traits affect transmission could give us important tools for predicting which plant species may be hotspots for disease spread. We assessed variation in transmission via susceptibility (probability of infection) and mean intensity (cell count of infected bees) of the trypanosomatid gut pathogen Crithidia bombi to uninfected Bombus impatiens workers foraging on 14 plant species, and assessed the role of floral traits, bee size and foraging behavior on transmission. We also conducted a manipulative experiment to determine how the number of open flowers affected transmission on three plant species, Penstemon digitalis, Monarda didyma, and Lythrum salicaria. Plant species differed fourfold in the overall mean abundance of Crithidia in foraging bumble bees (mean including infected and uninfected bees). Across plant species, bee susceptibility and mean intensity increased with the number of reproductive structures per inflorescence (buds, flowers and fruits); smaller bees and those that foraged longer were also more susceptible. Trait-based models were as good or better than species-based models at predicting susceptibility and mean intensity based on AIC values. Surprisingly, floral size and morphology did not significantly predict transmission across species. In the manipulative experiment, more open flowers increased mean pathogen abundance fourfold in Monarda, but had no effect in the other two plant species. Our results suggest that variation among plant species, through their influence on pathogen transmission, may shape bee disease dynamics. Given widespread investment in pollinator-friendly plantings to support pollinators, understanding how plant species affect disease transmission is important for recommending plant species that optimize pollinator health.

Consequences of multiple flower-insect interactions for subsequent plant-insect interactions and plant reproduction.

PREMISE OF THE STUDY: Plants often interact simultaneously with multiple antagonists and mutualists that can alter plant traits at the phenotypic or genetic level, subsequent plant-insect interactions, and reproduction. Although many studies have examined the effects of single floral antagonisms on subsequent pollination and plant reproduction, we know very little about the combined, potentially non-additive effects of multiple flower-insect interactions. METHODS: We simulated increased florivory, nectar robbing, and pollination on field-grown Impatiens capensis, which allowed us to determine interactive effects on five subsequent plant-insect interactions and 16 plant traits, including traits related to plant growth, floral attractiveness, floral defenses, and plant reproduction. KEY RESULTS: All three manipulative treatments had significant non-additive effects on the behavior of subsequent floral visitors, indicating that the effect of floral visitors generally depended on the presence or behavior of others. Pollination increased visitation by both pollinators and nectar larcenists (robbers and thieves), while florivory reduced pollinator and larcenist visits. Surprisingly, supplemental pollination also increased leaf herbivory. Florivores often responded to manipulations in opposite ways than did nectar larcenists and pollinators, suggesting different mechanisms influencing visitors that consume nectar compared to floral tissue. While our treatments did not affect any floral trait measured, they non-additively impacted plant reproduction, with florivory having a larger overall impact than either nectar robbing or pollination. CONCLUSIONS: These results emphasize the importance of understanding the context in which flower-insect interactions occur because the composition of the interacting community can have large and non-additive impacts on subsequent insect behavior and plant reproduction.

Landscape predictors of pathogen prevalence and range contractions in US bumblebees.

Several species of bumblebees have recently experienced range contractions and possible extinctions. While threats to bees are numerous, few analyses have attempted to understand the relative importance of multiple stressors. Such analyses are critical for prioritizing conservation strategies. Here, we describe a landscape analysis of factors predicted to cause bumblebee declines in the USA. We quantified 24 habitat, land-use and pesticide usage variables across 284 sampling locations, assessing which variables predicted pathogen prevalence and range contractions via machine learning model selection techniques. We found that greater usage of the fungicide chlorothalonil was the best predictor of pathogen (Nosema bombi) prevalence in four declining species of bumblebees. Nosema bombi has previously been found in greater prevalence in some declining US bumblebee species compared to stable species. Greater usage of total fungicides was the strongest predictor of range contractions in declining species, with bumblebees in the northern USA experiencing greater likelihood of loss from previously occupied areas. These results extend several recent laboratory and semi-field studies that have found surprising links between fungicide exposure and bee health. Specifically, our data suggest landscape-scale connections between fungicide usage, pathogen prevalence and declines of threatened and endangered bumblebees.

Gypsy moth herbivory induced volatiles and reduced parasite attachment to cranberry hosts.

Interactions between species can have cascading effects that shape subsequent interactions. For example, herbivory can induce plant defenses that affect subsequent interactions with herbivores, pathogens, mycorrhizae, and pollinators. Parasitic plants are present in most ecosystems, and play important roles in structuring communities. However, the effects of host herbivory on parasitic plants, and the potential mechanisms underlying such effects, are not well known. We conducted a greenhouse study to ask whether gypsy moth (Lymantria dispar) damage, host cultivar, and their interaction affected preference of the stem parasite dodder (Cuscuta spp.) on cranberry hosts (Vaccinium macrocarpum). We then assessed the mechanisms that could underlie such effects by measuring induced changes in phytohormones and secondary compounds. We found that damage by gypsy moths delayed dodder attachment by approximately 0.3 days when dodder stems were added 2 days after damage, and reduced attachment by more than 50% when dodder stems were added 1 week after host plant damage. Gypsy moth damage significantly increased jasmonic acid (JA) levels, total volatile emissions, and the flavonol, quercetin aglycone, suggesting possible mechanisms underlying variation in dodder ability to locate or attach to hosts. Dodder preference also differed between cranberry cultivars, with the highest attachment on the cultivar that had significantly lower levels of total volatile emissions and total phenolic acids, suggesting that volatile composition and phenolics may mediate dodder preference. Our results indicate that herbivory can reduce subsequent attachment by a highly damaging parasitic plant, demonstrating the potential importance of early damage for shaping subsequent species interactions.

Effects of florivory on plant-pollinator interactions: Implications for male and female components of plant reproduction.

PREMISE OF THE STUDY: Florivory could have direct negative effects on plant fitness due to consumption of floral organs, and indirect effects mediated through changes in traits important to pollination. These effects likely vary with plant sexual system, depending on sex- or morph-specific patterns of damage. We investigated the direct and indirect effects of simulated florivory on male and female components of reproduction in the native, distylous vine Gelsemium sempervirens. METHODS: We crossed floral damage and supplemental pollination treatments in a common garden array and tracked pollinator behavioral responses. We also estimated male function using fluorescent dye as an analog for pollen transfer, and measured both fruit and seed production. KEY RESULTS: The effects of floral damage varied by floral morph, the genus of floral visitor, and the component of reproduction measured. Damage reduced the number of pollinator visits to pin but not thrum plants, and increased the time some pollinators spent per flower in thrum but not pin plants. Flowers of damaged plants transferred more dye particles to recipient plants compared to undamaged plants, but only later in the season when the majority of dye transfer occurred. Damage had no effect on female reproduction. CONCLUSION: These results suggest that florivory can have positive indirect effects on estimated male plant reproduction through changes in different pollinators' behavior at flowers, but the effects of floral damage vary with male vs. female function. These results underscore the importance of other species' interactions at flowers in driving pollinator behavior and pollen transfer dynamics.

Geographic variation in resistance to nectar robbing and consequences for pollination.

PREMISE OF THE STUDY: Floral evolution is frequently ascribed to selection by pollinators, but may also be shaped by antagonists. However, remarkably few studies have examined geographic mosaics in resistance to floral antagonists or the consequences for other floral interactions. METHODS: Gelsemium sempervirens experiences frequent nectar robbing in northern Georgia, but rarely in southern Georgia. We conducted common-garden experiments in both locations using genotypes from each region and measured robbing, pollinator attraction, floral attractive and defensive traits, and plant reproduction. KEY RESULTS: Nectar robbing was more than four times higher in the north vs. south, and pollinator visits did not differ between gardens. Across both gardens, northern genotypes were half as likely to be nectar-robbed but received half as many pollinator visits as southern genotypes, suggesting evolution of resistance to robbing at a cost of reduced pollinator attraction. Plant-level traits, such as height and number of flowers, were more closely associated with resistance to robbing than floral size, shape, or chemistry. Northern genotypes had lower female and estimated male reproduction compared to southern genotypes at both locations, which could be due to costs of resistance to nectar robbing, or costs of adaptations to other biotic or abiotic differences between regions. CONCLUSIONS: Our study indicates that geographic variation can play a strong role structuring interactions with floral antagonists and mutualists and provides evidence consistent with the hypothesis that local resistance to nectar robbing imposes costs in terms of decreased pollinator attraction and reproduction.