Heatwaves exacerbate pollen limitation through reductions in pollen production and pollen vigour.

Increasingly frequent heat waves threaten the reproduction of flowering plants; compromising the future persistence, adaptive capacity, and dispersal of wild plant populations, and also the yield of fruit-bearing crop plants. Heat damages the development of sensitive floral organs and gametes, which inhibits pollen germination, pollen tube growth, and fertilization. However, the role of heat has not been integrated into the framework of pollen quantity and quality limitation and how heat influences the success of cross and self-pollination. We exposed developing flowers to either controlled temperature (25 °C:20 °C) or extreme heat (35 °C:20 °C) over 72 h. We then hand-pollinated them with either crossed or self-derived pollen from the same temperature treatment to determine the direct and interactive effects of simulated heatwaves on pollen tube growth and resulting seed set. We also collected anthers from virgin flowers to measure heat impacts on pollen production. Under cooler control temperatures pollen tube survival of self-derived pollen was approximately 27% lower than that of crossed pollen. Pollen tube survival in heat-treated cross-pollinated and heat-treated self-pollinated flowers were 71% and 77% lower compared to flowers cross-pollinated at control temperatures. These differences in pollen tube survival rate between heat-treated cross-pollinated and heat-treated self-pollinated flowers were insignificant. Furthermore, extreme heat reduced seed set by 87%, regardless of pollen origin, and also reduced pollen production during flower development by approximately 20%. Our results suggest flowers that develop during heatwaves are likely to experience exacerbated pollen quantity and quality limitation driven by changes in pollen production and pollen vigour. Heatwave-induced pollen limitation will likely reduce crop yields in agricultural systems, and depress mating and reproduction in wild plant species, the latter of which may hinder the adaptive capacity of plants to a rapidly changing world.

Temperature and soil moisture manipulation yields evidence of drought-induced pollen limitation in bee-pollinated squash.

Climate change alters environmental conditions in ways that directly and indirectly affect plants. Flowering plants, for example, modify reproductive allocation in response to heat and drought stress, and such changes can in turn affect pollinator visitation and, ultimately, plant reproduction. Although the individual effects of warming and drought on plant reproductive allocation are well known, these factors may interact to influence reproduction. Here, we conducted a fully crossed temperature by irrigation manipulation in squash (Cucurbita pepo) to test how temperature and soil moisture variation affect pollinator-mediated reproduction. To tease apart the direct and indirect effects of temperature and soil moisture, we compared hand-pollinated plants to bee-pollinated plants and restricted bee foraging (i.e., pollen transfer) to one experimental group per day. Temperature and soil-moisture limitation acted independently of one another: warming decreased flower size and increased pollen production, whereas the effects of soil-moisture limitation were uniformly inhibitory. While treatments did not change squash bee (Xenoglossa spp.) behavior, floral visitation by the honey bee (Apis mellifera) increased with temperature in male flowers and decreased with soil moisture in female flowers. Pollen deposition by bees was independent of plant soil moisture, yet reducing soil moisture increased pollen limitation. This result stemmed at least in part from the effects of soil-moisture limitation on pollen viability; seed set declined with increasing deposition of fluorescent pigment (a proxy for pollen) from plants experiencing decreased soil moisture. These findings suggest that the transfer of lower-quality pollen from plants experiencing soil-moisture limitation led to drought-induced pollen limitation. Similar effects may occur in a wide variety of flowering plant species as climate warming and drought increasingly impact animal-pollinated systems.

Successive stamen movement in Saxifraga candelabrum is responsive to weather and pollinator visits.

BACKGROUND: Successive stamen movement is a complex plant behavior involving successive uplift of stamens and pollen release, which plays a role in reducing sexual interference, increasing pollen deposition and promoting pollen export. Although reported from several taxa, studies on whether the movement can be influenced by abiotic and biotic factors are scarce. METHODS: In this study, we here for the first time described a pattern of successive stamen movement in Saxifraga candelabrum (Saxifragaceae). We then compared the rates of stamen movement in S. candelabrum under different weather and varying pollinator visits. Pollen packaging and presentation schedule of S. candelabrum were also investigated. RESULTS: The results showed that the number of stamens bent per day in sunny days was significantly higher than overcast and rain. Flowers that receive more pollinator visits (control treatment) had significantly higher number of stamen movement than those that received fewer (removal treatment) and none (bagging treatment). Throughout the staminate phase of a flower, there was a progressive increase in both pollen quantity of individual stamens and pollen presentation during each day. CONCLUSION: Our research demonstrates that successive stamen movement in S. candelabrum was accelerated by favorable weather and increased pollinator visits, which may promote pollen export. Moreover, incremental pollen packaging is likely an adaptation to seasonal regularity in variations of sex ratio resulting from protandry.

Does pollination interact with the abiotic environment to affect plant reproduction?

BACKGROUND AND AIMS: Abiotic and biotic components of the environment both limit plant reproduction, but how they interact with one another in combination is less understood. Understanding these interactions is especially relevant because abiotic and biotic environmental components respond differently to various global change drivers. Here we aim to understand whether the effects of pollination (biotic component) on plant reproduction depend on soil moisture (abiotic component), two factors known to affect plant reproduction and that are changing with global change. METHODS: We conducted pollen supplementation experiments for two plant species, Delphinium nuttallianum and Hydrophyllum fendleri, in subalpine meadows in the Western USA across four years that varied in soil moisture. In a separate one-year field experiment, we factorially crossed water addition with pollen supplementation. We measured proportion fruit set, seeds per fruit, and seeds per plant, in addition to stomatal conductance, to determine whether plant physiology responded to watering. KEY RESULTS: In the four-year study, only H. fendleri reproduction was pollen limited, and this occurred independently of soil moisture. Experimental water addition significantly increased soil moisture and stomatal conductance for both species. The effect of pollen addition on reproduction depended on the watering treatment only for H. fendleri fruit production. Reproduction in D. nuttallianum was not significantly affected by pollen addition or water addition, but it did respond to interannual variation in soil moisture. CONCLUSIONS: Although we find some evidence for the effect of a biotic interaction depending on abiotic conditions, it was only for one aspect of reproduction in one species, and it was in an unexpected direction. Our work highlights interactions between the abiotic and biotic components of the environment as an area of further research for improving our understanding of how plant reproduction responds to global change.

Pollination efficiency and the pollen-ovule ratio.

Pollination presents a risky journey for pollen grains. Pollen loss is sometimes thought to favour greater pollen investment to compensate for the inefficiency of transport. Sex allocation theory, to the contrary, has consistently concluded that postdispersal loss should have no selective effect on investment in either sex function. But the intuitively appealing compensation idea continues to be raised despite the lack of theoretical endorsement. We address the theoretical issue with a model that directly represents pollen loss (and ovule loss through floral demise or loss of receptivity) as rate-dependent dynamical processes. These loss rates can be varied to examine the effect of pollination efficiency on optimal sex allocation. Pollen-ovule ratios follow from the sex allocation based on the resource costs of pollen and ovule production. This model confirms conventional findings that pollen loss should have essentially no effect on sexual resource allocation in large, panmictic populations. Pollen limitation of seed set does not alter this conclusion. These results force us to rethink the empirical association of pollination efficiency with low pollen-ovule ratios. This pattern could arise if efficient pollen transport commonly results in stigmatic deposition of cohorts of related pollen. Empirical evidence of correlated paternity supports this explanation.

Support for Baker's law: Facultative self-fertilization ability decreases pollen limitation in experimental colonization.

PREMISE: The ability to self-fertilize is predicted to provide an advantage in colonization because a single individual can reproduce and establish a next generation in a new location regardless of the density of mates. While there is theoretical and correlative support for this idea, the strength of mate limitation as a selective agent has not yet been delineated from other factors that can also select for self-fertilization in colonization of new habitats. We used known mating-system variation in the American bellflower (Campanula americana) to explore how plants' ability to self-fertilize can mitigate density-dependent reproduction and impact colonization success. METHODS: We created experimental populations of single individuals or a small number of plants to emulate isolated colonization events. These populations were composed of plants that differed in their ability to self-fertilize. We compared pollen limitation of the single individuals to that of small populations. RESULTS: Experimental populations of plants that readily self-fertilize produced consistent seed numbers regardless of population size, whereas plants with lower ability to self-fertilize had density-dependent reproduction with greater seed production in small populations than in populations composed of a single individual. CONCLUSIONS: We experimentally isolated the effect of mate limitation in colonization and found that it can select for increased self-fertilization. We show the benefit of self-fertilization in colonization, which helps to explain geographic patterns of self-fertilization and shows support for Baker's law, a long-held hypothesis in the field of mating-system evolution.

A test of balanced fitness limitations theory: Pollen limitation in plants.

When reproductive success is determined by the relative availabilities of a series of essential, non-substitutable resources, the theory of balanced fitness limitations predicts that the cost of harvesting a particular resource shapes the likelihood that a shortfall of that resource will constrain realized fitness. Plant reproduction through female function offers a special opportunity to test this theory; essential resources in this context include, first, the pollen received from pollinators or abiotic vectors that is used to fertilize ovules, and, second, the resources needed to provision the developing seeds and fruit. For many plants realized reproductive success through female function can be readily quantified in the field, and one key potential constraint on fitness, pollen limitation, can be assessed experimentally by manually supplementing pollen receipt. We assembled a comparative dataset of pollen limitation using only studies that supplement pollen to all flowers produced over the plant's reproductive lifespan. Pre- and post-pollination costs were estimated using the weight of flowers and fruits and estimates of fruit set. Consistent with expectations, we find self-incompatible plants make greater pre-pollination investments and experience greater pollen limitation. However, contrary to theoretical expectations, when variation due to self-compatibility is accounted for by including self-compatibility in the statistical model as a covariate, we find no support for the prediction that plants that invest more heavily in pre-pollination costs are subject to greater pollen limitation. Strong within-species, between-population variation in the expression of pollen limitation makes the quantification of mean pollen limitation difficult. We urge plant ecologists to conduct more studies of pollen limitation using whole-plant pollen supplementation to produce a richer comparative dataset that would support a more robust test of the balanced limitations hypothesis.

Lack of pollinators selects for increased selfing, restricted gene flow and resource allocation in the rare Mediterranean sage Salvia brachyodon.

Range contraction and habitat fragmentation can cause biodiversity loss by creating conditions that directly or indirectly affect the survival of plant populations. Fragmented habitats can alter pollinator guilds and impact their behavior, which may result in pollen/pollinator limitation and selection for increased selfing as a mechanism for reproductive assurance. We used Salvia brachyodon, a narrowly distributed and endangered sage from eastern Adriatic, to test the consequences of range contraction and habitat fragmentation. Molecular data indicate a severe and relatively recent species range reduction. While one population is reproductively almost completely isolated, moderate gene flow has been detected between the remaining two populations. The high pollen-to-ovule ratio and the results of controlled hand pollination indicate that S. brachyodon has a mixed mating system. Quantitative and qualitative differences in the community and behaviour of flower visitors resulted in limited pollination services in one population where no effective pollinator other than pollen and nectar robbers were observed. In this population, self-pollination predominated over cross-pollination. Various environmental factors, in which plant-pollinator interactions play a pivotal role, have likely created selection pressures that have led to genetic and phenotypic differentiation and different resource allocation strategies among populations.

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

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

Pollinator-mediated effects of landscape-scale land use on grassland plant community composition and ecosystem functioning - seven hypotheses.

Environmental change is disrupting mutualisms between organisms worldwide. Reported declines in insect populations and changes in pollinator community compositions in response to land use and other environmental drivers have put the spotlight on the need to conserve pollinators. While this is often motivated by their role in supporting crop yields, the role of pollinators for reproduction and resulting taxonomic and functional assembly in wild plant communities has received less attention. Recent findings suggest that observed and experimental gradients in pollinator availability can affect plant community composition, but we know little about when such shifts are to be expected, or the impact they have on ecosystem functioning. Correlations between plant traits related to pollination and plant traits related to other important ecosystem functions, such as productivity, nitrogen uptake or palatability to herbivores, lead us to expect non-random shifts in ecosystem functioning in response to changes in pollinator communities. At the same time, ecological and evolutionary processes may counteract these effects of pollinator declines, limiting changes in plant community composition, and in ecosystem functioning. Despite calls to investigate community- and ecosystem-level impacts of reduced pollination, the study of pollinator effects on plants has largely been confined to impacts on plant individuals or single-species populations. With this review we aim to break new ground by bringing together aspects of landscape ecology, ecological and evolutionary plant-insect interactions, and biodiversity-ecosystem functioning research, to generate new ideas and hypotheses about the ecosystem-level consequences of pollinator declines in response to land-use change, using grasslands as a focal system. Based on an integrated set of seven hypotheses, we call for more research investigating the putative pollinator-mediated links between landscape-scale land use and ecosystem functioning. In particular, future research should use combinations of experimental and observational approaches to assess the effects of changes in pollinator communities over multiple years and across species on plant communities and on trait distributions both within and among species.

The effect of experimental pollinator decline on pollinator-mediated selection on floral traits.

Human-mediated environmental change, by reducing mean fitness, is hypothesized to strengthen selection on traits that mediate interactions among species. For example, human-mediated declines in pollinator populations are hypothesized to reduce mean seed production by increasing the magnitude of pollen limitation and thus strengthen pollinator-mediated selection on floral traits that increase pollinator attraction or pollen transfer efficiency. To test this hypothesis, we measured two female fitness components and six floral traits of Lobelia siphilitica plants exposed to supplemental hand-pollination, ambient open-pollination, or reduced open-pollination treatments. The reduced treatment simulated pollinator decline, while the supplemental treatment was used to estimate pollen limitation and pollinator-mediated selection. We found that plants in the reduced pollination treatment were significantly pollen limited, resulting in pollinator-mediated selection for taller inflorescences and more vibrant petals, both traits that could increase pollinator attraction. This contrasts with plants in the ambient pollination treatment, where reproduction was not pollen limited and there was not significant pollinator-mediated selection on any floral trait. Our results support the hypothesis that human-mediated environmental change can strengthen selection on traits of interacting species and suggest that these traits have the potential to evolve in response to changing environments.

Water deficit changes patterns of selection on floral signals and nectar rewards in the common morning glory.

Understanding whether and how resource limitation alters phenotypic selection on floral traits is key to predict the evolution of plant-pollinator interactions under climate change. Two important resources predicted to decline with our changing climate are pollinators and water in the form of increased droughts. Most work, however, has studied these selective agents separately and in the case of water deficit, studies are rare. Here, we use the common morning glory (Ipomoea purpurea) to investigate the effects of experimental reduction in pollinator access and water availability on floral signals and nectar rewards and their effects on phenotypic selection on these traits. We conducted a manipulative experiment in a common garden, where we grew plants in three treatments: (1) pollinator restriction, (2) water reduction and (3) unmanipulated control. Plants in pollinator restriction and control treatments were well-watered compared to water deficit. We found that in contrast to pollinator restriction, water deficit had strong effects altering floral signals and nectar rewards but also differed in the direction and strength of selection on these traits compared to control plants. Water deficit increased the opportunity for selection, and selection in this treatment favoured lower nectar volumes and larger floral sizes, which might further alter pollinator visitation. In addition, well-watered plants, both in control and pollinator deficit, showed similar patterns of selection to increase nectar volume suggesting non-pollinator-mediated selection on nectar. Our study shows that floral traits may evolve in response to reduction in water access faster than to declines in pollinators and reinforces that abiotic factors can be important agents of selection for floral traits. Although only few experimental selection studies have manipulated access to biotic and abiotic resources, our results suggest that this approach is key for understanding how pollination systems may evolve under climate change.

Pollinator probing preference and switching mode-mediated self-interference within a monoecious plant significantly reduced reproductive fitness.

Introduction: Monoecy is usually interpreted as an important evolutionary route of the plant sexual system from hermaphroditism to dioecy. This floral mechanism can effectively reduce self-interference during the reproductive process, and the services provided by pollinators may play an essential role in monoecious species; however, relevant research is still lacking. Thus, we aimed to determine whether monoecious plants could effectively avoid self-interference and promote the evolution of monoecy under the service of pollinators. Methods: Here, we successfully performed manipulation experiments to test self-compatibility, pollinator behavior, and self-interference between male and female functions in Akebia trifoliata, a typical monoecious species. Results: We demonstrated that experimental self-pollination did not yield any fruit, and supplemental pollination significantly increased fruit set and fruit weight compared to natural pollination, suggesting that this species is completely self-incompatible and experiences strong pollen limitation. Simultaneous self- and cross-pollination and self-pollination prior to cross-pollination significantly reduced reproductive fitness, but self-pollination after cross-pollination did not, indicating self-interference in this plant. Moreover, both male flower probing preference and switching modes within inflorescences by pollinators successfully reinforced self-interference and were also responsible for decreasing reproductive fitness in A. trifoliata. Discussion: In summary, pollinator-mediated self-interference significantly reduced selfing, providing potential dynamics for the maintenance and evolution of monoecy.

Population Rescue through an Increase in the Selfing Rate under Pollen Limitation: Plasticity versus Evolution.

AbstractIncreased rates of self-fertilization offer reproductive assurance when plant populations experience pollen limitation, but self-fertilization may reduce fitness by exposing deleterious mutations. If an environmental change responsible for pollen limitation also induces plastic mating system shifts toward self-pollination, the reproductive assurance benefit and inbreeding depression cost of increased self-fertilization occur immediately, while the benefit and cost happen more gradually when increased self-fertilization occur through evolution. I built eco-evolutionary models to explore the demographic and genetic conditions in which higher self-fertilization by plasticity and/or evolution rescues populations, following deficits due to a sudden onset of pollen limitation. Rescue is most likely under an intermediate level of selfing rate increase, either through plasticity or evolution, and this critical level of selfing rate increase is higher under stronger pollen limitation. Generally, rescue is more likely through plasticity than through evolution. Under weak pollen limitation, rescue by enhanced self-fertilization may mainly occur through purging of deleterious mutations rather than reproductive assurance. The selfing rate increase conferring the highest rescue probability is lower when the initial population size is smaller. This article shows the importance of plasticity during plant population rescue and offers insights for future studies of the evolution of mating system plasticity.

Pollinator-mediated plant coexistence requires high levels of pollinator specialization.

In pollen-limited plant communities, the foraging behavior of pollinators might mediate coexistence and competitive exclusion of plant species by determining which plants receive conspecific pollen. A key question is whether realistic pollinator foraging behavior promotes coexistence or exclusion of plant species. We use a simulation model to understand how pollinator foraging behavior impacts the coexistence dynamics of pollen-limited plants. To determine whether pollinators are likely to provide a biologically important coexistence mechanism, we compare our results to bee foraging data from the literature and from a novel experimental analysis. Model results indicate that strong specialization at the level of individual foraging paths is required to promote coexistence. However, few empirical studies have robustly quantified within-bout specialization. Species-level data suggest that foraging behavior is sufficient to permit pollinator-mediated coexistence in species-poor plant communities and possibly in diverse communities where congeneric plants co-occur. Our experiments using bumblebees show that individual-level specialization does exist, but not at levels sufficient to substantially impact coexistence dynamics. The literature on specialization within natural foraging paths suffers from key limitations, but overall suggests that pollinator-mediated coexistence should be rare in diverse plant communities.

Does pollinator dependence decrease along elevational gradients?

Plants have long been thought to be less dependent on pollinators for seed production at higher elevations due to adverse pollination environments. However, recent research has yet to consistently support the generality of this expectation. In this study, we asked whether pollinator dependence decreases along an elevational gradient and how it varies with various reproductive traits. To answer these questions, we quantified pollinator-plant associations and various reproductive traits for 112 flowering plants spanning a large elevational gradient (990-4260 m a.s.l.) in the Qinghai-Tibet Plateau. We found that flowering plants in the Qinghai-Tibet Plateau region are highly dependent on pollinators for seed production (76.2% of seed production was contributed by animal pollinators and 44.6% of plants would produce no seed without pollinator visitation). Contrary to our expectation, there was no significant elevational gradient in pollinator dependence index. Although the pollinator dependence index was not significantly correlated with pollen limitation, flower size, floral longevity, or reward type, it was correlated with compatibility status and flowering time. These findings indicate that pollinator dependence does not decrease along an elevational gradient in the Qinghai-Tibet Plateau. Our study also highlights the severe vulnerability of flowering plant seed production to pollinator declines under global change in the Qinghai-Tibet Plateau region, particularly for early-flowering or self-incompatible plants growing at higher elevations (e.g., subnival belt).

Sustained mangrove reproduction despite major turnover in pollinator community composition at expanding range edge.

BACKGROUND AND AIMS: How well plants reproduce near their geographic range edge can determine whether distributions will shift in response to changing climate. Reproduction at the range edge can be limiting if pollinator scarcity leads to pollen limitation, or if abiotic stressors affect allocation to reproduction. For many animal-pollinated plants with expanding ranges, the mechanisms by which they have overcome these barriers are poorly understood. METHODS: In this study, we examined plant-pollinator interactions hypothesized to impact reproduction of the black mangrove, Avicennia germinans, which is expanding northward in coastal Florida, USA. We monitored insects visiting A. germinans populations varying in proximity to the geographic range edge, measured the pollen loads of the most common insect taxa and pollen receipt by A. germinans stigmas, and quantified flower and propagule production. KEY RESULTS: We found that despite an 84 % decline in median floral visits by insects at northernmost versus southernmost sites, range-edge pollen receipt remained high. Notably, local floral visitor assemblages exhibited substantial turnover along the study's latitudinal gradient, with large-bodied bees and hover flies increasingly common at northern sites. We also observed elevated flower production in northern populations and higher per capita reproductive output at the range edge. Furthermore, mean propagule mass in northern populations was 18 % larger than that from the southernmost populations. CONCLUSIONS: These findings reveal no erosion of fecundity in A. germinans populations at range limits, allowing rapid expansion of mangrove cover in the region. These results also illustrate that substantial turnover in the assemblage of flower-visiting insects can occur at an expanding range edge without altering pollen receipt.

Reproductive collapse in European beech results from declining pollination efficiency in large trees.

Climate warming increases tree mortality which will require sufficient reproduction to ensure population viability. However, the response of tree reproduction to climate change remains poorly understood. Warming can reduce synchrony and interannual variability of seed production ("masting breakdown") which can increase seed predation and decrease pollination efficiency in trees. Here, using 40 years of observations of individual seed production in European beech (Fagus sylvatica), we showed that masting breakdown results in declining viable seed production over time, in contrast to the positive trend apparent in raw seed count data. Furthermore, tree size modulates the consequences of masting breakdown on viable seed production. While seed predation increased over time mainly in small trees, pollination efficiency disproportionately decreased in larger individuals. Consequently, fecundity declined over time across all size classes, but the overall effect was greatest in large trees. Our study showed that a fundamental biological relationship-correlation between tree size and viable seed production-has been reversed as the climate has warmed. That reversal has diverse consequences for forest dynamics; including for stand- and biogeographical-level dynamics of forest regeneration. The tree size effects suggest management options to increase forest resilience under changing climates.

Do Silene species with exposed stigmas tolerate interference by heterospecific pollen?

PREMISE: Co-flowering species that have not evolved an avoidance mechanism may have tolerance to heterospecific pollen (HP) deposition as an adaptive strategy to minimize any deleterious effects of HP transfer, but empirical evidence for the tolerance hypothesis remains scarce. METHODS: To estimate the potential effects of heterospecific pollen deposition (HPD) on female reproductive success, we counted conspecific (CP) and HP pollen grains deposited on stigmas and assessed subsequent seed set of both open- and hand-pollinated flowers in three co-flowering Silene species with exposed stigmas that usually received numerous HP grains on the elongated receptive area. RESULTS: The percentage of HP grains per flower (HP%) varied from 16.6% to 43.0% among three species. Silene chungtienensis had lower HP%, and the CP-HP relationship was neutral; S. gracilicaulis and S. yunnanensis had a relatively higher HP% with a positive CP-HP relationship. The effects of CP and HP number on natural seed set were positive for all three species, but HP% had stronger negative effects in S. chungtienensis and S. gracilicaulis. In hand-pollinated flowers of the three Silene species, seed set did not decrease with HP whether CP was in excess or insufficient, indicating no negative effects of HPD on seed production. CONCLUSIONS: Consistent with the tolerance hypothesis, our results indicated that species with higher HP interference are likely to be tolerant to an increase in HP%. These species with generalist-pollinated flowers and exposed large stigmas may benefit from an increase of conspecific pollen deposition, despite the associated increase in heterospecific pollen deposition.