Experimental studies of pollen carryover: Hummingbirds and Ipomopsis aggregata

Summary We present results of experiments designed to identify floral characteristics that influence patterns of pollen carryover by hummingbirds visiting Ipomopsis aggregata flowers. We used fluorescent dye powders as pollen analogues. For all four experimental treatments considered, amounts of dye deposited on recipient stigmas declined linearly as a function of flower position in a visitation sequence. The decline was significantly steeper when recipient flowers had pollen-carrying anthers than when they did not; whereas degree of stigma clogging and presence or absence of empty anthers did not influence carryover. From this we conclude that presence of pollen on recipient flowers significantly reduces the average number of subsequent flowers reached by donor pollen. We discuss mechanisms for this effect and its significance for the evolution of floral structure.     

Plant size,geitonogamy and seed set in Ipomopsis aggregata

Summary We used powdered fluorescent dyes to estimate receipt of self vs. outcross pollen in the self-incompatible species Ipomopsis aggregata (Polemoniaceae). Flowers on small and large plants received equal amounts of outcross pollen, whereas flowers on large plants received more self pollen, so the proportion of self pollen delivered through geitonogamy increased with plant size. In natural populations emasculation of all flowers on a plant raised average seed set per flower from 5.19 to 6.99 and also raised fruit set, though not significantly. From these results one expects a negative correlation between plant size and seeds per flower. The opposite trend was observed in a sample of plants in the field, suggesting that deleterious effects of geitonogamy on female fecundity in large plants can be overruled by other factors such as size-related fruit or seed abortion. Results are discussed in relation to the evolution of gynodioecy.     

Realized tolerance to nectar robbing: compensation to floral enemies in Ipomopsis aggregata

Background and Aims

Although the ecological and evolutionary consequences of foliar herbivory are well understood, how plants cope with floral damage is less well explored. Here the concept of tolerance, typically studied within the context of plant defence to foliar herbivores and pathogens, is extended to floral damage. Variation in tolerance to floral damage is examined, together with some of the mechanisms involved.

Methods

The study was conducted on Ipomopsis aggregata, which experiences floral damage and nectar removal by nectar-robbing bees. High levels of robbing can reduce seeds sired and produced by up to 50 %, an indirect effect mediated through pollinator avoidance of robbed plants. Using an experimental common garden with groups of I. aggregata, realized tolerance to robbing was measured. Realized tolerance included both genetic and environmental components of tolerance. It was hypothesized that both resource acquisition and storage traits, and traits involved in pollination would mitigate the negative effects of robbers.

Key Results

Groups of I. aggregata varied in their ability to tolerate nectar robbing. Realized tolerance was observed only through a component of male plant reproduction (pollen donation) and not through components of female plant reproduction. Some groups fully compensated for robbing while others under- or overcompensated. Evidence was found only for a pollination-related trait, flower production, associated with realized tolerance. Plants that produced more flowers and that had a higher inducibility of flower production following robbing were more able to compensate through male function.

Conclusions

Variation in realized tolerance to nectar robbing was found in I. aggregata, but only through an estimate of male reproduction, and traits associated with pollination may confer realized tolerance to robbing. By linking concepts and techniques from studies of plant–pollinator and plant–herbivore interactions, this work provides insight into the role of floral traits in pollinator attraction as well as plant defence.Key words: Compensation, herbivory, indirect effects, Ipomopsis aggregata, male reproductive success, nectar robbing, pollen donation, pollination, resistance, tolerance     

Experimental studies of pollen carryover: effects of floral variability in Ipomopsis aggregata

Summary In the montane herb Ipomopsis aggregata, size and placement of stamens and pistils vary substantially among flowers within plants, among nearby plants, and among groups of plants separated by 50–100 m. We trained captive hummingbirds to feed from flowers of this species in a flight cage, and explored the effects of different degrees of floral variability on carryover of fluorescent dyes that act as pollen mimics. We found that the slopes of linear dye carryover functions generally became more shallow as floral variability increased; this led to substantially longer carryover in the treatment with greatest variability. On the other hand, total amounts of dye transferred did not appear to be sensitive to the degree of variability. Floral variability may have a subtle but important effect on plant fitness by influencing the distance of pollen transfer.     

Factors limiting rosette recruitment in scarlet gilia, Ipomopsis aggregata: seed and disturbance limitation

We performed a factorial combination of seed additions, surface soil disturbance, and protection from potential seed predation in experimental plots to elucidate the relative importance of each factor in the recruitment of scarlet gilia seedlings. We detected a significant interaction between seed addition and surface soil disturbance on seedling recruitment over 2 years of establishment. Plots that received both supplemental seed addition and disturbance established 10.5 times as many rosettes as control plots. We did not detect main or interactive effects of caging on seedling establishment. We explored the importance of density-dependent recruitment by investigating the number of rosettes per experimental plot through time. In addition, we used planned contrasts to compare the absolute and percent changes in control and treatment plot rosette densities between the study years. We found significant downward concave curvature to the seedling recruitment curve, suggesting negative density-dependent recruitment across the experimental range of rosette densities. We found a significant difference between the control plots and the seed addition-disturbance plots in terms of absolute changes in plot density from 1997 to 1998. Greater net mortality occurred in plots that received both seed addition and disturbance. However, we found no differences among treatments in the percent change in rosette density across the study years. Our study demonstrates that seedling recruitment in natural plant populations may be limited by the interaction of seed and microsite availability, and that seedling recruitment in scarlet gilia may be negatively density dependent. Our results suggest that the fecundity effects of particular plant-animal interactions (e.g., pollination, herbivory) may affect scarlet gilia population dynamics, particularly under conditions of high soil disturbance. Received: 11 June 1999 / Accepted: 24 November 1999     

Pollination and pre-dispersal seed predation: net effects on reproduction and inflorescence characteristics in Ipomopsis aggregata

Summary We examined net seed production for the self-incompatible, monocarp, Ipomopsis aggregata, by monitoring pre-pollination seed parasite (Hylemya sp.) oviposition and hummingbird mediated fruit set on 21 plants of variable height. Both pollination and seed predation increased with inflorescence height. Net seed production (incorporating seed predator mortality) also was positively related to height, and this would have been the case if pollination or seed predation were doubled. Although results suggest pomopsis aggregata should be under selective pressure to maximize inflorescence height, generation time and resource limits could result in advantages for inflorescences of intermediate height.     

Small and Large Bumblebees Invest Differently when Learning about Flowers

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When resources don't rescue: flowering phenology and species interactions affect compensation to herbivory in Ipomopsis aggregata

The ability of plants to tolerate, or compensate for, herbivore damage is highly variable and has been the subject of much research. Although many plants can compensate for herbivore damage, and some even overcompensate, we cannot yet generalize about the conditions that promote a positive response to damage. Here, we asked how abiotic resources (i.e. plant nutrient status) coupled with biotic interactions – i.e. subsequent interactions with pollinators, seed predators and nectar robbing bumble bees – affect the compensatory ability of Ipomopsis aggregata, a monocarpic herb that has been the subject of much previous debate. We hypothesized that compensation to herbivore damage in I. aggregata (Polemoniaceae) would depend first on plants having an ample supply of resources and, second, on the outcome of subsequent interactions with mutualist pollinators and enemy pre‐dispersal seed predators and nectar robbing bumble bees. We used a fully‐factorial experiment in which plants were watered, fertilized or left as unmanipulated controls, crossed with clipping to simulate herbivore damage to the apical meristem. Resource addition enhanced both male and female components of fitness, but resource enhancement did not provide the means for plants to fully compensate for simulated herbivory. Clipped plants produced significantly more inflorescences, but at the expense of a delay in flowering and fewer total flowers. Clipping significantly reduced losses to dipteran pre‐dispersal seed predators by delaying flowering time, but early flowering plants produced higher numbers of seeds despite incurring higher rates of predation. Clipped plants incurred a higher risk to nectar robbers in one of two years. Overall, clipped plants suffered severe reductions (a nearly 50% reduction in total seed set) in female success, but clipping combined with nutrient addition enhanced male function through increases in per‐flower pollen production. However, because clipped plants produced significantly fewer flowers than unclipped plants, whole‐plant pollen production was significantly reduced by clipping. Pollinator visitation and nectar robbing were variable between clipping treatments and between years and (nectar robbing) among sites. Our results demonstrate that the variability in plant response to herbivory can, at least in part, be driven by plant interactions with mutualists and enemies. Thus, accounting for such interactions and their variability is important to fully understanding plant compensation for herbivore damage and will likely go far to explain variation in plant response that appears to be independent of resources.     

Pollen precedence and stigma closure: a mechanism of competition for pollination between Delphinium nelsonii and Ipomopsis aggregata

Summary Previous experiments showed that the sympatric herbs Delphinium nelsonii and Ipomopsis aggregata compete for hummingbird pollination and that deleterious effects of the former species on seed set of the latter involve interspecific pollen transfer. However, seed set was not reduced when pollen of both species was applied simultaneously to I. aggregata stigmas. Hence a competitive effect may require arrival of foreign pollen before conspecific pollen. To explore this possibility we subjected I. aggregata flowers to a competition treatment in which they received D. nelsonii pollen 6 h before I. aggregata pollen, or to a control in which they received only the conspecific pollen. Foreign pollen precedence decreased mean seed set by almost 50%, which is consistent with effects observed in previous experiments. Reduced seed set can be explained by the fact that foreign pollen often caused stigma lobes to close together within 1.5–6 h, reducing subsequent receptivity. Stigma closure was also elicited by conspecific pollen, but not by mechanical stimulation, and was influenced by size of the pollen load and identity of the plant being pollinated.     

The effects of grazers on the performance of individuals and populations of scarlet gilia,Ipomopsis aggregata

Summary I. aggregata exhibits considerable powers of regrowth following removal of its primary shoot by herbivores, but we found no evidence of overcompensation (i.e. of significantly higher plant performance where plants were exposed to ungulate herbivory) in a comparison between individuals on grazed and ungrazed sides of exclosure fences, in a comparison between artificially clipped and control plants in one population in the Okanagan National Forest, or in comparisons between grazed and ungrazed plants in 14 natural populations. We tested whether ungulate grazing affects the population size of Ipomopsis aggregata by comparing populations inside and outside deer exclosures at 7 sites in the Western United States. We found consistent, highly significant differences in plant population density on the grazed and ungrazed sides of these exlosure fences. Plant density was a modal 25-fold higher on the protected side of the fence, suggesting that exposure to ungulate grazing increases plant death rates at some stage in the life cycle. Our results show that the presence of ungulate grazers leads to a substantial decrease in plant density despite the fact that grazing on young bolting shoots has very little influence on fruit production. Since this decrease in population density is not correlated with a decrease in the fecundity of individuals, it must instead be due to other direct and indirect effects of ungulate grazers.     

Effects of experimental manipulation of inflorescence size on pollination and pre-dispersal seed predation in the hummingbird-pollinated plant Ipomopsis aggregata

Large floral displays should theoretically provide advantages to plants through increased pollinator visitation and resulting fruit and seed set. However empirical tests of the response of pollinators to floral display size have been limited by a lack of direct experimentation, and the results of such studies have been equivocal. In addition, other selective agents such as pre-dispersal seed predators might modulate effects of floral display on pollination. By artificially altering flower number, we examined the direct effects of floral display in the monocarpic herb, Ipomopsis aggregata (Polemoniaceae), on visitation rates by broad-tailed and rufous hummingbird pollinators, as well destruction of fruits by a pre-dispersal seed predator (Hylemya: Anthomyiidae). In addition, we quantified the ultimate effects of flower number on female reproductive success. Plants with larger floral displays were most likely to be visited first in any given foraging bout (P < 0.01). As expected, plants with more flowers received more total flower visits. However, we found no gain in the proportion of flowers visited for many- versus few-flowered plants, or the total number of approaches/hour. In fact, a significantly greater percentage of flowers were visited on few-flowered plants. Plants did not compensate for our reduction in flowers by increasing investment in the number or proportion of flowers that set fruit, the number of seeds/fruit, or seed weight. Pre-dispersal seed predation was greater for many- than for few-flowered plants (P < 0.001), but this did not offset the potential fitness gains of producing large displays. Our data support the hypothesis that large floral displays function primarily in long-distance attraction of pollinators, and enhance maternal success. Received: 21 March 1996 / Accepted: 24 October 1996     

The consequences of direct versus indirect species interactions to selection on traits: pollination and nectar robbing in Ipomopsis aggregata

Organisms experience a complex suite of species interactions. Although the ecological consequences of direct versus indirect species interactions have received attention, their evolutionary implications are not well understood. I examined selection on floral traits through direct versus indirect pathways of species interactions using the plant Ipomopsis aggregata and its pollinators and nectar robber. Using path analysis and structural equation modeling, I tested competing hypotheses comparing the relative importance of direct (pollinator-mediated) versus indirect (robber-mediated) interactions to trait selection through female plant function in 2 years. The hypothesis that provided the best fit to the observed data included robbing and pollination, suggesting that both interactors are important in driving selection on some traits; however, the direction and intensity of selection through robbing versus pollination varied between years. I then increased my scope of inference by assessing traits and species interactions across more years. I found that the potential for temporal variation in the direction and intensity of selection was pronounced. Taken together, results suggest that assessing the broader context in which organisms evolve, including both direct and indirect interactions and across multiple years, can provide increased mechanistic understanding of the diversity of ways that animals shape floral and plant evolution.     

The evolution of compensation to herbivory in scarlet gilia, Ipomopsis aggregata: herbivore-imposed natural selection and the quantitative genetics of tolerance

Tolerance is the ability of plants to maintain fitness after experiencing herbivore damage. We investigated scarlet gilia tolerance to browsing in the framework of phenotypic plasticity using both an operational and candidate trait approach. Individuals from full-sib families were split into an artificial clipping treatment, a natural-damage treatment, or left as controls. We tested for genetic variation in tolerance by evaluating family x herbivory treatment interactions on fitness in a mixed model analysis of variance. In addition, we used selection analyses to assess the function of flowering phenology and compensatory regrowth (via branch production) as candidate tolerance traits. We found a strong detrimental fitness effect of browsing and considerable variation among sire half-sib families in levels of tolerance (25% to 63% of the fitness of controls). There was no evidence of overcompensation at either the population or family level and no additive genetic variation in operationally defined tolerance. Phenotypic selection analyses provide evidence that early flowering and compensatory regrowth function as tolerance characters. We found strong linear and correlational selection for early flowering and increased branch production for damaged plants and linear selection for apical dominance (reduced branchiness) and early flowering in control plants. Moreover, reduced phenological delay and increased plasticity in branch production were correlated with tolerance. We detected significant additive genetic variation in flowering phenology in both treatments and a positive genetic correlation between the phenology of control and damaged plants. We found significant additive genetic variation in branch production in undamaged and naturally damaged plants, but not in clipped plants. Damaged plants exhibited marginally significant additive genetic variance in fitness, although its heritability was very low (approximately 3.6%). We failed to find additive genetic variation in the fitness of control plants. Our results suggest that tolerance traits are under herbivore-imposed natural selection in this population, but that responses to selection are limited by available genetic variation and selective constraints.     

PERMANENT GENETIC RESOURCES: Identification of nuclear microsatellite loci for Ipomopsis aggregata and the distribution of pairwise relatedness in a natural population

Nine microsatellite loci were developed from enriched libraries of scarlet gilia (Ipomopsis aggregata). A screen of 160 individuals from a population identified reduced levels of heterozygosity, low levels of relatedness, and weak spatial genetic patterns. The population inbreeding coefficient was calculated to be 0.19 (SE = 0.04). These patterns are consistent with those expected from low levels of biparental inbreeding in an obligate outcrosser and extensive seed and pollen dispersal. These preliminary data confirm the usefulness of microsatellite markers for breeding system studies of I. aggregata.     

Adaptive differentiation in floral traits in the presence of high gene flow in scarlet gilia (Ipomopsis aggregata)

Plant–pollinator interactions are thought to be major drivers of floral trait diversity. However, the relative importance of divergent pollinator‐mediated selection vs. neutral processes in floral character evolution has rarely been explored. We tested for adaptive floral trait evolution by comparing differentiation at neutral genetic loci to differentiation at quantitative floral traits in a putative Ipomopsis aggregata hybrid zone. Typical I. aggregata subsp. candida displays slender white tubular flowers that are typical of flowers pollinated by hawkmoths, and subsp. collina displays robust red tubular flowers typical of flowers pollinated by hummingbirds; yet, hybrid flower morphs are abundant across the East Slope of the Colorado Rockies. We estimated genetic differentiation (F_ST) for nuclear and chloroplast microsatellite loci and used a half‐sib design to calculate quantitative trait divergence (Q_ST) from collection sites across the morphological hybrid zone. We found little evidence for population structure and estimated mean F_ST to be 0.032. Q_ST values for several floral traits including corolla tube length and width, colour, and nectar volume were large and significantly greater than mean F_ST. We performed multivariate comparisons of neutral loci to genetic correlations within and between populations and found a strong signal for divergent selection, suggesting that specific combinations of floral display and reward traits may be the targets of selection. Our results show little support for historical subspecies categories, yet floral traits are more diverged than expected due to drift alone. Non‐neutral divergence for multivariate quantitative traits suggests that selection by pollinators is maintaining a correlation between display and reward traits.     

Floral Sonication is an Innate Behaviour in Bumblebees that can be Fine-Tuned with Experience in Manipulating Flowers

Bumblebees demonstrate an extensive capacity for learning complex motor skills to maximise exploitation of floral rewards. This ability is well studied in nectar collection but its role in pollen foraging is less well understood. Floral sonication is used by bees to extract pollen from some plant species with anthers which must be vibrated (buzzed) to release pollen. Pollen removal is determined by sonication characteristics including frequency and amplitude, and thus the ability to optimise sonication should allow bees to maximise the pollen collection. We investigated the ability of the buff-tailed bumblebee (Bombus terrestris) to modify the frequency and amplitude of their buzzes with increasing experience manipulating flowers of the buzz-pollinated plant Solanum rostratum. We analysed flight and feeding vibrations generated by naïve workers across feeding bouts. Feeding buzzes were of a higher frequency and a lower amplitude than flight buzzes. Both flight and feeding buzzes had reduced amplitudes with increasing number of foraging trips. However, the frequency of their feeding buzzes was reduced significantly more than their flight buzzes as bumblebee workers gained experience manipulating flowers. These results suggest that bumblebees are able to modify the characteristics of their buzzes with experience manipulating buzz-pollinated flowers. We discuss our findings in the context of bumblebee learning, and the current understanding of the optimal sonication characteristics for releasing pollen in buzz-pollinated species. Our results present a tantalising insight into the potential role of learning in floral sonication, paving the way for future research in this area.     

The Influence of Prior Learning Experience on Pollinator Choice: An Experiment Using Bumblebees on Two Wild Floral Types of Antirrhinum majus

Understanding how pollinator behavior may influence pollen transmission across floral types is a major challenge, as pollinator decision depends on a complex range of environmental cues and prior experience. Here we report an experiment using the plant Antirrhinum majus and the bumblebee Bombus terrestris to investigate how prior learning experience may affect pollinator preferences between floral types when these are presented together. We trained naive bumblebees to forage freely on flowering individuals of either A. majus pseudomajus (magenta flowers) or A. majus striatum (yellow flowers) in a flight cage. We then used a Y-maze device to expose trained bumblebees to a dual choice between the floral types. We tested the influence of training on their choice, depending on the type of plant signals available (visual signals, olfactory signals, or both). Bumblebees had no innate preference for either subspecies. Bumblebees trained on the yellow-flowered subspecies later preferred the yellow type, even when only visual or only olfactory signals were available, and their preference was not reinforced when both signal types were available. In contrast, bumblebees trained on the magenta-flowered subspecies showed no further preference between floral types and took slightly more time to make their choice. Since pollinator constancy has been observed in wild populations of A. majus with mixed floral types, we suggest that such constancy likely relies on short-term memory rather than acquired preference through long-term memory induced by prior learning.     

Radar Tracking and Motion-Sensitive Cameras on Flowers Reveal the Development of Pollinator Multi-Destination Routes over Large Spatial Scales

Central place foragers, such as pollinating bees, typically develop circuits (traplines) to visit multiple foraging sites in a manner that minimizes overall travel distance. Despite being taxonomically widespread, these routing behaviours remain poorly understood due to the difficulty of tracking the foraging history of animals in the wild. Here we examine how bumblebees (Bombus terrestris) develop and optimise traplines over large spatial scales by setting up an array of five artificial flowers arranged in a regular pentagon (50 m side length) and fitted with motion-sensitive video cameras to determine the sequence of visitation. Stable traplines that linked together all the flowers in an optimal sequence were typically established after a bee made 26 foraging bouts, during which time only about 20 of the 120 possible routes were tried. Radar tracking of selected flights revealed a dramatic decrease by 80% (ca. 1500 m) of the total travel distance between the first and the last foraging bout. When a flower was removed and replaced by a more distant one, bees engaged in localised search flights, a strategy that can facilitate the discovery of a new flower and its integration into a novel optimal trapline. Based on these observations, we developed and tested an iterative improvement heuristic to capture how bees could learn and refine their routes each time a shorter route is found. Our findings suggest that complex dynamic routing problems can be solved by small-brained animals using simple learning heuristics, without the need for a cognitive map.     

Estimates of Gene Flow among Populations, Geographic Races, and Species in the Ipomopsis Aggregata Complex

Interpopulational gene flow within a species can reduce population differentiation due to genetic drift, whereas genetic exchange among taxa can impede speciation. We used allozyme data to estimate gene flow within and among geographic races and species of perennial herbs in the Ipomopsis aggregata complex (Polemoniaceae). Estimates of interpopulational gene flow within taxa from two methods (F statistics and private alleles) were correlated with one another. Gene flow among populations within each geographic race (subspecies) of I. aggregata was relatively high (Nm greater than approximately 1.0). Gene flow was also high among populations of I. arizonica and among four northern populations of I. tenuituba. However, gene flow was low (Nm less than 1.0) for I. tenuituba when a population representing subsp. macrosiphon was included. This is consistent with previous findings that subsp. macrosiphon has had an independent origin and is reproductively, as well as geographically, isolated. A recently developed model, based on hierarchical F statistics, was employed to estimate genetic exchange among taxa. Gene flow estimates were generally high among races of I. aggregata (dNmrace greater than 1.0) but were low among subspecies of I. tenuituba (dNmrace less than 1.0). Consistent with morphological evidence, estimates of interspecific gene flow were moderate between I. aggregata and I. tenuituba, which hybridize in several areas. However, contrary to morphological evidence, we estimated relatively high levels of interspecific gene flow involving I. arizonica. Our results suggest that I. arizonica has hybridized with other species without the transfer of morphological traits.(ABSTRACT TRUNCATED AT 250 WORDS)