Relative impact of mate versus pollinator availability on pollen limitation and outcrossing rates in a mass‐flowering species

Plant mating systems are driven by several pre‐pollination factors, including pollinator availability, mate availability and reproductive traits. We investigated the relative contributions of these factors to pollination and to realized outcrossing rates in the patchily distributed mass‐flowering shrub Rhododendron ferrugineum. We jointly monitored pollen limitation (comparing seed set from intact and pollen‐supplemented flowers), reproductive traits (herkogamy, flower size and autofertility) and mating patterns (progeny array analysis) in 28 natural patches varying in the level of pollinator availability (flower visitation rates) and of mate availability (patch floral display estimated as the total number of inflorescences per patch). Our results showed that patch floral display was the strongest determinant of pollination and of the realized outcrossing rates in this mass‐flowering species. We found an increase in pollen limitation and in outcrossing rates with increasing patch floral display. Reproductive traits were not significantly related to patch floral display, while autofertility was negatively correlated to outcrossing rates. These findings suggest that mate limitation, arising from high flower visitation rates in small plant patches, resulted in low pollen limitation and high selfing rates, while pollinator limitation, arising from low flower visitation rates in large plant patches, resulted in higher pollen limitation and outcrossing rates. Pollinator‐mediated selfing and geitonogamy likely alleviates pollen limitation in the case of reduced mate availability, while reduced pollinator availability (intraspecific competition for pollinator services) may result in the maintenance of high outcrossing rates despite reduced seed production.     

Phenological change in a spring ephemeral: implications for pollination and plant reproduction

Climate change has had numerous ecological effects, including species range shifts and altered phenology. Altering flowering phenology often affects plant reproduction, but the mechanisms behind these changes are not well‐understood. To investigate why altering flowering phenology affects plant reproduction, we manipulated flowering phenology of the spring herb Claytonia lanceolata (Portulacaceae) using two methods: in 2011–2013 by altering snow pack (snow‐removal vs. control treatments), and in 2013 by inducing flowering in a greenhouse before placing plants in experimental outdoor arrays (early, control, and late treatments). We measured flowering phenology, pollinator visitation, plant reproduction (fruit and seed set), and pollen limitation. Flowering occurred approx. 10 days earlier in snow‐removal than control plots during all years of snow manipulation. Pollinator visitation patterns and strength of pollen limitation varied with snow treatments, and among years. Plants in the snow removal treatment were more likely to experience frost damage, and frost‐damaged plants suffered low reproduction despite lack of pollen limitation. Plants in the snow removal treatment that escaped frost damage had higher pollinator visitation rates and reproduction than controls. The results of the array experiment supported the results of the snow manipulations. Plants in the early and late treatments suffered very low reproduction due either to severe frost damage (early treatment) or low pollinator visitation (late treatment) relative to control plants. Thus, plants face tradeoffs with advanced flowering time. While early‐flowering plants can reap the benefits of enhanced pollination services, they do so at the cost of increased susceptibility to frost damage that can overwhelm any benefit of flowering early. In contrast, delayed flowering results in dramatic reductions in plant reproduction through reduced pollination. Our results suggest that climate change may constrain the success of early‐flowering plants not through plant‐pollinator mismatch but through the direct impacts of extreme environmental conditions.     

Distinct male reproductive strategies in two closely related oak species

Reproductive strategies of closely related species distributed along successional gradients should differ as a consequence of the trade‐off between competition and colonization abilities. We compared male reproductive strategies of Quercus robur and Q. petraea, two partly interfertile European oak species with different successional status. In the studied even‐aged stand, trees of the late‐successional species (Q. petraea) grew faster and suffered less from intertree competition than trees of the early‐successional species (Q. robur). A large‐scale paternity study and a spatially explicit individual‐based mating model were used to estimate parameters of pollen production and dispersal as well as sexual barriers between species. Male fecundity was found to be dependent both on a tree's circumference and on its environment, particularly so for Q. petraea. Pollen dispersal was greater and more isotropic in Q. robur than in Q. petraea. Premating barriers to hybridization were strong in both species, but more so in Q. petraea than in Q. robur. Hence, predictions based on the competition–colonization trade‐off are well supported, whereas the sexual barriers themselves seem to be shaped by colonization dynamics.     

The evolution of self-fertilization and inbreeding depression under pollen discounting and pollen limitation

We model the evolution of plant mating systems under the joint effects of pollen discounting and pollen limitation, using a dynamic model of inbreeding depression, allowing for partial purging of recessive lethal mutations by selfing. Stable mixed mating systems occur for a wide range of parameter values with pollen discounting alone. However, when typical levels of pollen limitation are combined with pollen discounting, stable selfing rates are always high but less than 1 (0.9相似文献   

Seed set variation in wild Clarkia populations: teasing apart the effects of seasonal resource depletion,pollen quality,and pollen quantity

In habitats where resource availability declines during the growing season, selection may favor early‐flowering individuals. Under such ephemerally favorable conditions, late‐blooming species (and individuals) may be particularly vulnerable to resource limitation of seed production. In California, a region prone to seasonal drought, members of the annual genus Clarkia are among the last to flower in the spring. We compared pollen limitation (PL) of seed set and outcrossing rates between early‐ and late‐flowering individuals in two mixed‐mating Clarkia taxa to detect whether flowering time is associated with changes in seed set due to resource depletion, PL, or increased selfing. In 2008–2010, we hand‐pollinated one flower on a total of 1855 individual plants either Early (near the onset of flowering) or Late (near the end of flowering) in the flowering season and compared seed set to adjacent, open‐pollinated flowers on the same stem. To assess the contribution of pollen quality to reproduction, we first (2008) used allozymes to estimate outcrossing rates of seeds produced by Early and Late open‐pollinated flowers. Second (2009), we conducted an anther‐removal experiment to estimate self‐pollen deposition. Seed set in Clarkia unguiculata was not pollen‐limited. Clarkia xantiana ssp. xantiana was pollen‐limited in 2008 and 2010, but not 2009. PL did not differ between Early and Late treatments. In both taxa, seed set of Early flowers was greater than Late flowers, but not due to PL in the latter. Reproduction was generally pollinator‐dependent. Most pollen deposition was xenogamous, and outcrossing rates were >0.7 – and similar between Early and Late periods. These results suggest that pollen receipt and pollen quality remain seasonally consistent. By contrast, the resources necessary to provision seeds decline, reducing the fitness benefits associated with resource allocation to ovules.     

Variable hybridization outcomes in trout are predicted by historical fish stocking and environmental context

Hybridization can profoundly affect the genomic composition and phenotypes of closely related species, and provides an opportunity to identify mechanisms that maintain reproductive isolation between species. Recent evidence suggests that hybridization outcomes within a species pair can vary across locations. However, we still do not know how variable outcomes of hybridization are across geographic replicates, and what mechanisms drive that variation. In this study, we described hybridization outcomes across 27 locations in the North Fork Shoshone River basin (Wyoming, USA) where native Yellowstone cutthroat trout and introduced rainbow trout co‐occur. We used genomic data and hierarchical Bayesian models to precisely identify ancestry of hybrid individuals. Hybridization outcomes varied across locations. In some locations, only rainbow trout and advanced backcrossed hybrids towards rainbow trout were present, while trout in other locations had a broader range of ancestry, including both parental species and first‐generation hybrids. Later‐generation intermediate hybrids were rare relative to backcrossed hybrids and rainbow trout individuals. Using an individual‐based simulation, we found that outcomes of hybridization in the North Fork Shoshone River basin deviate substantially from what we would expect under null expectations of random mating and no selection against hybrids. Since this deviation implies that some mechanisms of reproductive isolation function to maintain parental taxa and a diversity of hybrid types, we then modelled hybridization outcomes as a function of environmental variables and stocking history that are likely to affect prezygotic barriers to hybridization. Variables associated with history of fish stocking were the strongest predictors of hybridization outcomes, followed by environmental variables that might affect overlap in spawning time and location.     

Pollen limitation and Allee effect related to population size and sex ratio in the endangered Ottelia acuminata (Hydrocharitaceae): implications for conservation and reintroduction

Small populations may suffer more severe pollen limitation and result in Allee effects. Sex ratio may also affect pollination and reproduction success in dioecious species, which is always overlooked when performing conservation and reintroduction tasks. In this study, we investigated whether and how population size and sex ratio affected pollen limitation and reproduction in the endangered Ottelia acuminata, a dioecious submerged species. We established experimental plots with increasing population size and male sex ratio. We observed insect visitation, estimated pollen limitation by hand‐pollinations and counted fruit set and seed production per fruit. Fruit set and seed production decreased significantly in small populations due to pollinator scarcity and thus suffered more severe pollen limitation. Although frequently visited, female‐biased larger populations also suffered severe pollen limitation due to few effective visits and insufficient pollen availability. Rising male ratio enhanced pollination service and hence reproduction. Unexpectedly, pollinator preferences did not cause reduced reproduction in male‐biased populations because of high pollen availability. However, reproductive outputs showed more variability in severe male‐biased populations. Our results revealed two component Allee effects in fruit set and seed production, mediated by pollen limitation in O. acuminata. Moreover, reproduction decreased significantly in larger female‐biased populations, increasing the risk of an Allee effect.     

Between a rock and a hard place: adaptive sensing and site‐specific dispersal

Environmental variability can lead to dispersal: why stay put if it is better elsewhere? Without clues about local conditions, the optimal strategy is often to disperse a set fraction of offspring. Many habitats contain environmentally differing sub‐habitats. Is it adaptive for individuals to sense in which sub‐habitat they find themselves, using environmental clues, and respond plastically by altering the dispersal rates? This appears to be done by some plants which produce dimorphic seeds with differential dispersal properties in response to ambient temperature. Here we develop a mathematical model to show, that in highly variable environments, not only does sensing promote plasticity of dispersal morph ratio, individuals who can sense their sub‐habitat and respond in this way have an adaptive advantage over those who cannot. With a rise in environmental variability due to climate change, our understanding of how natural populations persist and respond to changes has become crucially important.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.     

Evolutionary consequences of ecological factors: pollinator reliability predicts mating‐system traits of a perennial plant

The reproductive‐assurance hypothesis predicts that mating‐system traits will evolve towards increased autonomous self‐pollination in plant populations experiencing unreliable pollinator service. We tested this long‐standing hypothesis by assessing geographic covariation among pollinator reliability, outcrossing rates, heterozygosity and relevant floral traits across populations of Dalechampia scandens in Costa Rica. Mean outcrossing rates ranged from 0.16 to 0.49 across four populations, and covaried with the average rates of pollen arrival on stigmas, a measure of pollinator reliability. Across populations, genetically based differences in herkogamy (anther–stigma distance) were associated with variation in stigmatic pollen loads, outcrossing rates and heterozygosity. These observations are consistent with the hypothesis that, when pollinators are unreliable, floral traits promoting autonomous selfing evolve as a mechanism of reproductive assurance. Extensive covariation between floral traits and mating system among closely related populations further suggests that floral traits influencing mating systems track variation in adaptive optima generated by variation in pollinator reliability.     

动物传粉植物花粉呈现时序的进化意义

由动物传粉的植物为了使供体花粉能够被高效地传递到受体柱头上, 进化出了多种多样的花部特征, 花粉呈现时序便是其中之一。植物主要通过包装机制和分摊机制控制花粉呈现的速度以限制昆虫一次拜访转移的花粉量。花粉呈现理论(PPT)认为: 花粉逐步呈现策略倾向于出现在传粉者数量丰富但传粉效率低的植物中, 而花粉同时呈现策略则多出现在传粉者少但传粉效率高的植物中。本文对花粉呈现时序的相关研究进行了总结, 重点介绍: (1) 限制花粉转移的花部机制; (2)花粉呈现理论; (3)花粉呈现时序的适应意义。目前的研究主要集中在花粉呈现时序对传粉动物的适应性上, 但环境因子对花粉呈现时序也有一定的影响。PPT数学模型还不能完全预测特定环境中植物的花粉呈现时序。因此, 有必要选择合适的植物类群, 从花部综合特征、传粉系统、交配系统和环境等方面进行综合研究, 进一步揭示动物传粉植物花粉呈现时序的适应意义。     

Quantitative and qualitative consequences of reduced pollen loads in a mixed‐mating plant

Greater pollination intensity can enhance maternal plant fitness by increasing seed set and seed quality as a result of more intense pollen competition or enhanced genetic sampling. We tested experimentally these effects by varying the pollen load from a single pollen donor on stigmas of female flowers of Dalechampia scandens (Euphorbiaceae) and measuring the effects on seed number and seed mass. Seed set increased rapidly with pollen number at low to moderate pollen loads, and a maximum set of three seeds occurred with a mean pollen load of 19 pollen grains. We did not detect a trade‐off between the number of seeds and seed mass within a fruit. Seed mass increased with increasing pollen load, supporting the hypothesis of enhanced seed quality via increased pollen‐competition intensity or genetic sampling. These results suggest that maternal fitness increases with larger pollen loads, even when the fertilization success is already high. Our results further highlight the importance of high rates of pollen arrival onto stigmas, as mediated by reliable pollinators. Comparing the pollen‐to‐seed response curve obtained in this experiment with those observed in natural populations suggests that pollen limitation may be more severe in natural populations than predicted from greenhouse studies. These results also indicate that declines in pollinator abundance may decrease plant fitness through lowered seed quality before an effect on seed set is detected.     

Introgressive hybridization as a mechanism for species rescue

Rapid evolution on ecological time scales can play a key role in species responses to environmental change. One dynamic that has the potential to generate the diversity necessary for evolution rapid enough to allow response to sudden environmental shifts is introgressive hybridization. However, if distinct sub-species exist before an environmental shift, mechanisms that impede hybridization, such as assortative mating and hybrid inferiority, are likely to be present. Here we explore the theoretical potential for introgressive hybridization to play a role in response to environmental change. In particular, we incorporate assortative mating, hybrid inferiority, and demographic stochasticity into a two-locus, two-allele population genetic model of two interacting species where one locus identifies the species and the other determines how fitness depends on the changing environment. Simulation results indicate that moderately high values for the strength of assortative mating will allow enough hybridization events to outweigh demographic stochasticity but not so many that continued hybridization outweighs backcrossing and introgression. Successful introgressive hybridization also requires intermediate relative fitness at the allele negatively affected by environmental change such that hybrid survivorship outweighs demographic stochasticity but selection remains strong enough to affect the genetic dynamics. The potential for successful introgression instead of extinction with greater environmental change is larger with monogamous rather than promiscuous mating due to lower stochasticity in mating events. These results suggest species characteristics (e.g., intermediate assortative mating and mating systems with low variation in mating likelihood) which indicate a potential for rapid evolution in response to environmental change via introgressive hybridization.     

A high‐throughput FTIR spectroscopy approach to assess adaptive variation in the chemical composition of pollen

The two factors defining male reproductive success in plants are pollen quantity and quality, but our knowledge about the importance of pollen quality is limited due to methodological constraints. Pollen quality in terms of chemical composition may be either genetically fixed for high performance independent of environmental conditions, or it may be plastic to maximize reproductive output under different environmental conditions. In this study, we validated a new approach for studying the role of chemical composition of pollen in adaptation to local climate. The approach is based on high‐throughput Fourier infrared (FTIR) characterization and biochemical interpretation of pollen chemical composition in response to environmental conditions. The study covered three grass species, Poa alpina, Anthoxanthum odoratum, and Festuca ovina. For each species, plants were grown from seeds of three populations with wide geographic and climate variation. Each individual plant was divided into four genetically identical clones which were grown in different controlled environments (high and low levels of temperature and nutrients). In total, 389 samples were measured using a high‐throughput FTIR spectrometer. The biochemical fingerprints of pollen were species and population specific, and plastic in response to different environmental conditions. The response was most pronounced for temperature, influencing the levels of proteins, lipids, and carbohydrates in pollen of all species. Furthermore, there is considerable variation in plasticity of the chemical composition of pollen among species and populations. The use of high‐throughput FTIR spectroscopy provides fast, cheap, and simple assessment of the chemical composition of pollen. In combination with controlled‐condition growth experiments and multivariate analyses, FTIR spectroscopy opens up for studies of the adaptive role of pollen that until now has been difficult with available methodology. The approach can easily be extended to other species and environmental conditions and has the potential to significantly increase our understanding of plant male function.     

Pollen limitation and its influence on natural selection through seed set

Stronger pollen limitation should increase competition among plants, leading to stronger selection on traits important for pollen receipt. The few explicit tests of this hypothesis, however, have provided conflicting support. Using the arithmetic relationship between these two quantities, we show that increased pollen limitation will automatically result in stronger selection (all else equal) although other factors can alter selection independently of pollen limitation. We then tested the hypothesis using two approaches. First, we analysed the published studies containing information on both pollen limitation and selection. Second, we explored how natural selection measured in one Ontario population of Lobelia cardinalis over 3 years and two Michigan populations in 1 year relates to pollen limitation. For the Ontario population, we also explored whether pollinator‐mediated selection is related to pollen limitation. Consistent with the hypothesis, we found an overall positive relationship between selection strength and pollen limitation both among species and within L. cardinalis. Unexpectedly, this relationship was found even for vegetative traits among species, and was not found in L. cardinalis for pollinator‐mediated selection on nearly all trait types.     

Pollen limitation,species’ floral traits and pollinator visitation: different relationships in contrasting communities

Failures in the process of pollen transfer among conspecific plants can severely impact female reproductive success. Thus, pollen limitation can cause selection on plant mating systems and floral traits. The relationships between pollen limitation and floral traits might be partly mediated by the quantity and identity of pollinator visits. However, very little is known about the relationship between pollinator visits and pollen limitation. We examined the relationships between pollen limitation and floral traits at the community level to connect them to community ecology processes. We used 48 plant species from two contrasting communities: one species‐rich lowland community and one species‐poor alpine community. In addition, we calculated visitation rates and ecological pollination generalization for 38 of the species to examine the relationship between pollinator visitation and pollen limitation at the community level. We found low overall levels of pollen limitation that did not differ significantly between the alpine and the lowland community. In both communities, species with evolutionary specialized flowers were more pollen limited than species with unspecialized flowers. Species’ visitation rates and selfing capability were negatively related to pollen limitation in the alpine community, where pollinators are scarcer. However, flower size/number, ecological generalization of plants and flowering onset had greater effects on pollen limitation levels at the lowland community, indicating that the identity of the visitors and plant‐plant competitive interactions are more decisive for plant reproduction in this species‐rich community. There, pollen limitation increased with flower size and flowering onset, and decreased with ecological generalization, but only in species with evolutionary specialized flowers. Our study suggests that selection on plant mating system and floral traits may be idiosyncratic to each particular community and highlights the benefits of conducting community‐level studies for a better understanding of the processes underlying evolutionary responses to pollen limitation.     

Effects of tree architecture on pollen dispersal and mating patterns in Abies pinsapo Boiss. (Pinaceae)

Plant architecture is crucial to pollination and mating in wind‐pollinated species. We investigated the effect of crown architecture on pollen dispersal, mating system and offspring quality, combining phenotypic and genotypic analyses in a low‐density population of the endangered species Abies pinsapo. A total of 598 embryos from three relative crown height levels (bottom, middle and top) in five mother plants were genotyped using eleven nuclear microsatellite markers (nSSRs). Paternity analysis and mating system models were used to infer mating and pollen dispersal parameters. In addition, seeds were weighed (N = 16 110) and germinated (N = 736), and seedling vigour was measured to assess inbreeding depression. Overall, A. pinsapo shows a fat‐tailed dispersal kernel, with an average pollen dispersal distance of 113–227 m, an immigration rate of 0.84–26.92%, and a number of effective pollen donors (N_ep) ranging between 3.5 and 11.9. We found an effect of tree height and relative crown height levels on mating parameters. A higher proportion of seeds with embryo (about 50%) and a higher rate of self‐fertilization (about 60%) were found at the bottom level in comparison with the top level. Seed weight and seedling vigour are positively related. Nevertheless, no differences were found in seed weight or in seedling‐related variables such as weight and length of aerial and subterranean parts among the different relative crown height levels, suggesting that seeds from the more strongly inbred bottom level are not affected by inbreeding depression. Our results point to vertical isotropy for outcross‐pollen and they suggest that self‐pollen may ensure fertilization when outcross‐pollen is not available in low‐density population.     

Pollination of a bee‐dependent forb in restored prairie: no evidence of pollen limitation in landscapes dominated by row crop agriculture

Restoration is used to conserve biodiversity; however, it is unclear to what extent restoration impacts ecosystem functions. Pollination is an ecosystem function that is critical to plant reproduction and thus restoration success. Few studies have assessed whether pollination is restored within restoration areas themselves. Plant–animal interactions may be affected by factors beyond the scale of the restoration. For example, surrounding landscape context may influence pollinator abundance and consequently the amount of pollen deposited. Decreased pollen receipt might then limit seed set. We hypothesized that in restorations surrounded by more agriculture, pollinator‐dependent forbs would experience greater pollen limitation. This would likely be due to declines in pollinator abundance within the restorations with an increase in surrounding agriculture. We deployed potted Chamaecrista fasciculata (Fabaceae), an obligatorily bee‐pollinated forb, and sampled bee communities in restored prairies in Minnesota, U.S.A. We measured pollen limitation by comparing seed set among open and supplementally pollinated plants. We also sampled native bees in seven of the eight sites. We tested for a relationship between proportion row crop agriculture (corn and soy) surrounding a restoration and pollen limitation, as well as an effect of agriculture on bee abundance. We did not find evidence that increasing proportion of surrounding agriculture negatively affected pollen limitation or bee abundance. Our results indicate that greater surrounding agriculture may not influence pollination of C. fasciculata through declines in pollinator availability, and suggest for some plants that landscape context might not limit pollination in restorations.     

Self‐ and intra‐morph incompatibility and selection analysis of an inconspicuous distylous herb growing on the Tibetan plateau (Primula tibetica)

There is discussion over whether pollen limitation exerts selection on floral traits to increase floral display or selects for traits that promote autonomous self‐fertilization. Some studies have indicated that pollen limitation does not mediate selection on traits associated with either pollinator attraction or self‐fertilization. Primula tibetica is an inconspicuous cross‐fertilized plant that may suffer from pollen limitation. We conducted a selection analysis on P. tibetica to investigate whether pollen limitation results in selection for an increased floral display in case the evolution of autonomous self‐fertilization has been difficult for this plant. The self‐ and intra‐morph incompatibility features, the capacity for autonomous self‐fertilization, and the magnitude of pollen limitation were examined through hand‐pollination experiments. In 2016, we applied selection analysis on the flowering time, corolla width, stalk height, flower tube length, and flower number in P. tibetica by tagging 76 open‐pollinated plants and 37 hand‐pollinated plants in the field. Our results demonstrated that P. tibetica was strictly self‐ and intra‐morph incompatible. Moreover, the study population underwent severe pollen limitation during the 2016 flowering season. The selection gradients were found to be significantly positive for flowering time, flower number, and corolla width, and marginally significant for the stalk height. Pollinator‐mediated selection was found to be significant on the flower number and corolla width, and marginally significant on stalk height. Our results indicate that the increased floral display may be a vital strategy for small distylous species that have faced difficulty in evolving autonomous self‐fertilization.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.