Non-aborted carpels, and seed production as the counts of developed seeds vs. undeveloped seeds, both of which are structurally binomial variables. Relative to a binomial distribution, however, our data for both analyses were overdispersed which we corrected by including an individual-level random effect . We ran GLMMs with binomial-errors using the default logit link in the ‘lme4’ package for R . We assessed the relative contribution of the quantities of heterospecific and conspecific pollen on reproductive output and carpel abortion using a modelcomparison framework. In these models conspecific pollen and heterospecific pollen, as well as their interaction, are the fixed effects that we assess in different combinations. We specifically compared five mixed-effects models: full model including an interaction between the two fixed effects ; additive model ; amount of conspecific pollen only; amount of heterospecific pollen only; and a model including no fixed effects . For all analyses, we used AICc, Akaike’s Information Criterioncorrected for small sample size , to determine the best model. We reported all models within two ΔAICc points of the best model. We used the ‘AICcmodavg’ package in R for model selection. We excluded observations in which there was seed set with either zero stigmatic pollen recorded , blueberries in pots or zero proportion of conspecific pollen . We assume that these observations were due to loss of stigmatic pollen in the field between fertilization and stigma collection.
To understand the association between naturally deposited heterospecific pollen and seed production, we used a field approach linking stigmatic pollen deposition to seed set in the same individual carpels in wild plants. Heterospecific pollen deposition was highly variable on D. barbeyi stigmas in the field and while some degree of heterospecific pollen was found on most sampled flowers, it was typically present in low amounts . Neither heterospecific pollen nor conspecific pollen was a good predictor of carpel abortion in our sites. Conspecific pollen deposition was positively related to viable seed production, but we also found a significant negative interaction between heterospecific pollen and conspecific pollen. That is, with increasing heterospecific pollen, the positive relationship between conspecific pollen deposition and viable seed production in D. barberyi became weaker . We found that heterospecific pollen on D. barbeyi stigmas is widespread, highly variable, and typically occurs at relatively low levels. In terms of variability, heterospecific pollen represented anywhere from 0 to 97% of the grains present in a pollen load. The widespread nature of heterospecific pollen deposition is reflected by the fact that 85% of stigmas had some heterospecific pollen present. Still, most stigmas received only low levels of heterospecific pollen deposition . These results are broadly consistent with patterns reported in a comprehensive review of studies assessing the impact of heterospecific pollen transfer, including 77 species from 17 different sources . This review found that, although receipt of heterospecific pollen was variable, all species received at least some heterospecific pollen on their stigmas. Similarly, in a community-wide analysis of pollen transfer, Fang and Huang found that of the 57 plant species they surveyed, heterospecific pollen deposition was common but highly variable, representing 0-66% of the total pollen on stigmas.
The co-flowering plant community and pollinator behavior are just two of the factors that might influence variation in HP receipt between plants of the same species within and between sites. Variation in heterospecific pollen receipt within species or even within a population obviously has important implications for evolution of coping mechanisms and remains an area ripe for more research . A large proportion of carpels aborted in our study , yet contrary to our expectations we found that neither heterospecific pollen nor conspecific pollen was a good predictor of carpel abortion in D. barbeyi. Thus, it seems likely that resource limitation, rather than pollen limitation, drove carpel abortion in our system. Extrinsic factors such as weather conditions, flower phenology, herbivory, competition , and disease can lead to within plant variation in carpel abortion . Furthermore, intrinsic factors including architecture , plant size, developmental constraints or allocation strategies are also known to affect patterns of carpel abortion . In contrast to carpel abortion, both conspecific pollen and heterospecific pollen played a role in seed production in D. barberyi. As expected, conspecific pollen deposition on its own was positively related to viable seed production. Heterospecific pollen on its own did not affect seed set, but there was a significant negative heterospecific pollen × conspecific pollen interaction. In other words, with greater heterospecific pollen deposition, a fixed amount of conspecific pollen would result in lower seed set. Because this was a correlational study, we were not able to tease apart other factors that may have lead to a decrease in seed production such as diversity of heterospecific pollen.
To our knowledge, this is the first demonstration of an interaction between heterospecific pollen and conspecific pollen in their effects on plant reproduction. We hypothesized that we would find such an interaction as we expected that the negative effects of heterospecific pollen might occur only in the context of conspecific pollen. In a case comparing deposition of a small vs. a large heterospecific pollen load in stigmas with no conspecific pollen should yield the same result: zero seeds produced. Following the same logic, we would expect the impact of depositing a medium-sized load of heterospecific pollen on a stigma would differ in stigmas with just a few conspecific pollen grains versus a large conspecific pollen load . We are aware of only one previous assessment of the statistical interaction between stigmatic pollen load quantity and the proportion ofheterospecific pollen grains . This study found no evidence for a pollen quantity × proportion heterospecific pollen interaction, but the study design was oriented at a different objective and thus included just two heterospecific pollen load sizes, which corresponded to 28% and 16% of total pollen load proportions. Finally, we were not able to distinguish between self and outcrossed conspecific pollen in this study. Self pollen has been shown to intensify the negative impact of HP receipt in Mimulus guttatus and self pollen has been shown to produce fewer seeds in D. barbeyi . More studies examining how mixed mating plant species cope with HP receipt would be valuable, particularly in a field setting where plants may receive HP from a variety of plant species. Putting this result in the context of previous findings highlights that there has been a gulf between field studies and hand-pollination studies in greenhouse settings, a gulf that, if bridged, would improve our understanding of the effects of heterospecific pollen transfer in nature. Future work in hand-pollination studies should contribute to bridging the gap by integrating what we have learned from field studies so far. First, hand-pollination studies should be designed using parameters explicitly drawn from field studies, in terms of heterospecific and conspecific pollen amounts, and heterospecific pollen diversity, considering not only means but also variability. To date, most hand-pollination studies have used only one proportion of heterospecific pollen , and we are aware of only one hand-pollination study that specifically applied a full range of heterospecific pollen deposition. Second, the results from our field study, particularly our finding of a heterospecific pollen × conspecific pollen interaction in seed production, highlights the advantage of understanding this result mechanistically, via hand-pollination studies that vary the quantity of both conspecific pollen and heterospecific pollen factorially. Third, our finding that neither conspecific pollen nor heterospecific pollen was strongly linked to carpel abortion rates underscores that hand-pollination studies that control both pollen and resource limitation in a greenhouse setting could greatly improve our understanding of how these factors interact. Similarly, field studies on the effects of heterospecific pollen can look to hand-pollination experiments—with their typically more mechanistic focus—for inspiration. A critical first step is to increase the number of field studies that directly link pollen deposition and seed production within the same carpel . To our knowledge the work we present here was the first time that this approach has been applied to understanding the effects of heterospecific pollen, which should be repeated in a wide range of plant species with different mating systems .
Another particular need is for field studies that assess multiple sites. We continue to have a poor understanding of how plant genotype and environmental factors interact to shape the effects of heterospecific pollen, square plant pots and work along environmental gradients could be informative, especially in disentangling the relative effects of pollen vs. resource limitation in shaping seed set. Similarly, experimental approaches to assessing the relative effects of resource vs. pollen limitation in the context of heterospecific pollen deposition in field settings would also be a valuable research direction. Finally, hand-pollination and field approaches should be explicitly integrated by conducting more hand-pollination experiments in field settings. Our understanding of the impact of heterospecific pollen deposition is growing, but there is still much to learn about the way that co-flowering plants interact through pollinator sharing and how heterospecific pollen deposition impacts plant reproductive fitness. In a changing world where we can expect to see both increasing disruptions in pollination as well as the emergence of new interactions via introduced species and climate change, studies that unify both field studies and controlled hand pollination studies will allow us to better understand the implications of heterospecific pollen deposition for reproductive output in natural plant communities.Pollinator foraging behavior directly impacts plant reproductive output and ecosystem function through the transfer of pollen between plant individuals within a single foraging bout. If pollinators move between different plant species they can transfer heterospecific pollen, potentially reducing reproductive output . Given the functional significance of pollinator resource use, it is important to understand the factors driving pollinator foraging behavior. The plants that pollinators forage on is a function of a number of factors including innate and learned preference, morphological traits, as well as direct and indirect competition with other pollinators in the community . This study focuses on how competitive interactions between pollinators shape pollinator foraging behavior. Decades of research has demonstrated that interspecific competition can influence pollinator foraging behavior. In one example, Pimm et al. found that in the presence of a dominant competitor, two other hummingbird species spent more time at a less rewarding feeder. In contrast, without interspecific competition, all three hummingbird species visited a feeder with high sucrose concentrations. Brosi and Briggs found that after a release from interspecific competition, bumble bees decreased their floral fidelity . These changes in foraging behavior led to a significant decrease in reproductive output in a common alpine plant species. Fründ et al. provide another example in which pollinators’ flower preferences can be flexible, and depend on community context . In their system, as competition between pollinator species increased, the species reduced their niche overlap by shifting to new plant species, which resulted in increased plant reproduction. Thus, we know bees respond to competition and often do so strongly, but we do not know if bee species vary in their response to competition in complex assemblages of bee species or what traits are important in determining how they will respond. Trait based differences between species may reduce interspecific competition and maintain diversity within a community . Generally, we still have a poor understanding of which traits influence the outcome of competition and community structure but some animal traits such as body size or bill size/shape and plant traits related to resource acquisition, such as root depth , can limit competition thus encouraging species coexistence. Bumble bee communities provide an excellent system in which to empirically explore how trait differences drive foraging plasticity in response to interspecific competition, all within a community context. Bombus assemblages are often species-rich, and sympatric species typically have substantial overlap in their life history requirements . Furthermore, traits that affect resource acquisition and foraging efficiency can influence how species partition resources within a community . Tongue length is a trait that directly determines which resources a bumble bee can access and how resource selection varies among species . In general, long-tongued bumble bee foragers visit flowers with deep corollas and short-tongued bumble bees forage on shallow flowers . Still, bumble bees are known to be labile in their foraging patterns if more rewarding floral resources become available or the competitive landscape shifts .