The evolution of postpollination reproductive isolation in Costus

Reproductive isolation is critical to the diversification of species. Postpollination barriers may be important in limiting gene flow between closely related species, but they are relatively cryptic and their evolution is poorly understood. Here, we review the role of postpollination reproductive isolation in plants, including the various stages at which it operates and the hypotheses for how it may evolve. We then review empirical studies in the plant genus Costus, evaluating documented postpollination barriers in light of these hypotheses. We summarize isolation due to parental style length differences and present evidence supporting the hypothesis that the differences are in part a by-product of selection on floral morphology. Additionally, we show that reduced pollen adhesion, germination, and tube growth contribute to reproductive isolation between two closely related sympatric species of Costus. Geographic variation in the strength of these crossing barriers supports the hypothesis that they evolved under reinforcement, or direct natural selection to strengthen isolation.     

Genic rather than genome‐wide differences between sexually deceptive Ophrys orchids with different pollinators

High pollinator specificity and the potential for simple genetic changes to affect pollinator attraction make sexually deceptive orchids an ideal system for the study of ecological speciation, in which change of flower odour is likely important. This study surveys reproductive barriers and differences in floral phenotypes in a group of four closely related, coflowering sympatric Ophrys species and uses a genotyping‐by‐sequencing (GBS) approach to obtain information on the proportion of the genome that is differentiated between species. Ophrys species were found to effectively lack postpollination barriers, but are strongly isolated by their different pollinators (floral isolation) and, to a smaller extent, by shifts in flowering time (temporal isolation). Although flower morphology and perhaps labellum coloration may contribute to floral isolation, reproductive barriers may largely be due to differences in flower odour chemistry. GBS revealed shared polymorphism throughout the Ophrys genome, with very little population structure between species. Genome scans for F_ST outliers identified few markers that are highly differentiated between species and repeatable in several populations. These genome scans also revealed highly differentiated polymorphisms in genes with putative involvement in floral odour production, including a previously identified candidate gene thought to be involved in the biosynthesis of pseudo‐pheromones by the orchid flowers. Taken together, these data suggest that ecological speciation associated with different pollinators in sexually deceptive orchids has a genic rather than a genomic basis, placing these species at an early phase of genomic divergence within the ‘speciation continuum’.     

Divergent selection on flowering phenology but not on floral morphology between two closely related orchids

Closely related species often differ in traits that influence reproductive success, suggesting that divergent selection on such traits contribute to the maintenance of species boundaries. Gymnadenia conopsea ss. and Gymnadenia densiflora are two closely related, perennial orchid species that differ in (a) floral traits important for pollination, including flowering phenology, floral display, and spur length, and (b) dominant pollinators. If plant–pollinator interactions contribute to the maintenance of trait differences between these two taxa, we expect current divergent selection on flowering phenology and floral morphology between the two species. We quantified phenotypic selection via female fitness in one year on flowering start, three floral display traits (plant height, number of flowers, and corolla size) and spur length, in six populations of G. conopsea s.s. and in four populations of G. densiflora. There was indication of divergent selection on flowering start in the expected direction, with selection for earlier flowering in two populations of the early‐flowering G. conopsea s.s. and for later flowering in one population of the late‐flowering G. densiflora. No divergent selection on floral morphology was detected, and there was no significant stabilizing selection on any trait in the two species. The results suggest ongoing adaptive differentiation of flowering phenology, strengthening this premating reproductive barrier between the two species. Synthesis: This study is among the first to test whether divergent selection on floral traits contribute to the maintenance of species differences between closely related plants. Phenological isolation confers a substantial potential for reproductive isolation, and divergent selection on flowering time can thus greatly influence reproductive isolation and adaptive differentiation.     

Floral isolation is the main reproductive barrier among closely related sexually deceptive orchids

Floral isolation is an important component of pollinator-driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators' mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F(1) hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination.     

Floral scent and species divergence in a pair of sexually deceptive orchids

Speciation is typically accompanied by the formation of isolation barriers between lineages. Commonly, reproductive barriers are separated into pre‐ and post‐zygotic mechanisms that can evolve with different speed. In this study, we measured the strength of different reproductive barriers in two closely related, sympatric orchids of the Ophrys insectifera group, namely Ophrys insectifera and Ophrys aymoninii to infer possible mechanisms of speciation. We quantified pre‐ and post‐pollination barriers through observation of pollen flow, by performing artificial inter‐ and intraspecific crosses and analyzing scent bouquets. Additionally, we investigated differences in mycorrhizal fungi as a potential extrinsic factor of post‐zygotic isolation. Our results show that floral isolation mediated by the attraction of different pollinators acts apparently as the sole reproductive barrier between the two orchid species, with later‐acting intrinsic barriers seemingly absent. Also, the two orchids share most of their fungal mycorrhizal partners in sympatry, suggesting little or no importance of mycorrhizal symbiosis in reproductive isolation. Key traits underlying floral isolation were two alkenes and wax ester, present predominantly in the floral scent of O. aymoninii. These compounds, when applied to flowers of O. insectifera, triggered attraction and a copulation attempt of the bee pollinator of O. aymoninii and thus led to the (partial) breakdown of floral isolation. Based on our results, we suggest that adaptation to different pollinators, mediated by floral scent, underlies species isolation in this plant group. Pollinator switches may be promoted by low pollination success of individuals in dense patches of plants, an assumption that we also confirmed in our study.     

Modeling the two‐locus architecture of divergent pollinator adaptation: how variation in SAD paralogs affects fitness and evolutionary divergence in sexually deceptive orchids

Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n‐alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species‐specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl–acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator‐mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two‐locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator‐mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator‐driven ecological speciation.     

Genetics of floral traits influencing reproductive isolation between Aquilegia formosa and Aquilegia pubescens

Abstract: Reproductive isolation between Aquilegia formosa and Aquilegia pubescens is influenced by differences in their flowers through their effects on pollinator visitation and pollen transfer. Here, we investigate the genetic basis of floral characters differentiating these species. We found that in addition to the effects of flower orientation and the length of nectar spurs previously described, other characters such as flower color or odor affect hawkmoth visitation. Repeatability of measurements in an F2 population ranged from 0.53 to 0.83 among five floral traits, indicating that using the means of multiple measures per plant will substantially increase the power of a quantitative trait locus (QTL) analysis. Integration of floral traits was indicated by significant correlations among traits in an F2 population. In a separate F2 population we found that QTL for different floral traits were often closely associated, indicating that linkage or pleiotropy cause at least some of this integration. In addition, we found QTL for all floral traits examined. Because Aquilegia species are largely interfertile and vary extensively in both floral morphology and ecology, they offer the opportunity for QTL studies of a wide range of characters affecting reproductive isolation.     

Floral isolation is the major reproductive barrier between a pair of rewarding orchid sister species

The crucial role of reproductive isolation in speciation has long been recognized; however, a limited number of studies quantify different isolation barriers and embed reproductive isolation in a phylogenetic context. In this study, we investigate reproductive isolation between the often sympatrically occurring orchid species, Gymnadenia conopsea and G. odoratissima. We examine the phylogenetic relationship between the two species and analyse floral isolation, fruit set and seed viability from interspecies crosses, as well as the ploidy level. Additionally, we quantify interspecies differences in floral signals and morphology. The results suggest that the two species have a sister–species relationship. In terms of reproductive isolation, we found complete floral isolation between the two species, but little to no post‐pollination isolation; the species also mostly had the same ploidy level in the studied populations. We also show clear distinctions in floral signals, as well as in floral size and spur length. We propose that respective adaptation to short‐ vs. long‐tongued pollinators was the driver of speciation in the here studied Gymnadenia species. Our study supports the key role of floral isolation in orchid speciation and shows that floral isolation is not restricted to highly specialized pollination systems, but can also occur between species with less specialized pollination.     

Selective pollination by fungus gnats potentially functions as an alternative reproductive isolation among five Arisaema species

Background and AimsInterspecific difference in pollinators (pollinator isolation) is important for reproductive isolation in flowering plants. Species-specific pollination by fungus gnats has been discovered in several plant taxa, suggesting that they can contribute to reproductive isolation. Nevertheless, their contribution has not been studied in detail, partly because they are too small for field observations during flower visitation. To quantify their flower visitation, we used the genus Arisaema (Araceae) because the pitcher-like spathe of Arisaema can trap all floral visitors.MethodsWe evaluated floral visitor assemblage in an altitudinal gradient including five Arisaema species. We also examined interspecific differences in altitudinal distribution (geographic isolation) and flowering phenology (phenological isolation). To exclude the effect of interspecific differences in altitudinal distribution on floral visitor assemblage, we established ten experimental plots including the five Arisaema species in high- and low-altitude areas and collected floral visitors. We also collected floral visitors in three additional sites. Finally, we estimated the strength and contribution of these three reproductive barriers using a unified formula for reproductive isolation.Key ResultsEach Arisaema species selectively attracted different fungus gnats in the altitudinal gradient, experimental plots and additional sites. Altitudinal distribution and flowering phenology differed among the five Arisaema species, whereas the strength of geographic and phenological isolations were distinctly weaker than those in pollinator isolation. Nevertheless, the absolute contribution of pollinator isolation to total reproductive isolation was weaker than geographic and phenological isolations, because pollinator isolation functions after the two early-acting barriers in plant life history.ConclusionsOur results suggest that selective pollination by fungus gnats potentially contributes to reproductive isolation. Since geographic and phenological isolations can be disrupted by habitat disturbance and interannual climate change, the strong and stable pollinator isolation might compensate for the weakened early-acting barriers as an alternative reproductive isolation among the five Arisaema species.     

POLLINATOR SPECIFICITY DRIVES STRONG PREPOLLINATION REPRODUCTIVE ISOLATION IN SYMPATRIC SEXUALLY DECEPTIVE ORCHIDS

Few studies have quantified the full range of pre‐ and postzygotic barriers that limit introgression between closely related plant species. Here, we assess the strength of four isolating mechanisms operating between two morphologically similar and very closely related sympatric orchid taxa, Chiloglottis valida and C. aff. jeanesii. Each taxon sexually attracts its specific wasp pollinator via distinct floral volatile chemistry. Behavioral experiments with flowers and synthetic versions of their floral volatiles confirmed that very strong pollinator isolation is mediated by floral odor chemistry. However, artificially placing flowers of the two taxa in contact proximity revealed the potential for rare interspecific pollination. Although we found hybrid vigor in F1 hybrids produced by hand‐crossing, genetic analysis at both nuclear and chloroplast loci showed significant and moderate‐to‐strong genetic differentiation between taxa. A Bayesian clustering method for the detection of introgression at nuclear loci failed to find any evidence for hybridization across 571 unique genotypes at one site of sympatry. Rather than inhibiting gene flow, postpollination barriers surveyed here show no contribution to overall reproductive isolation. This demonstrates the primacy of pollinators in maintaining species boundaries in these orchids, which display one of the strongest known examples of prepollination floral isolation.     

Experimental sympatry suggests geographic isolation as an essential reproductive barrier between two sister species of Pedicularis

Although mechanical isolation mediated by shared pollinators has been considered as a classic model of pollinator-mediated floral isolation in Pedicularis, a superdiverse genus in Hengduan Mountains, southwest China, there has been no empirical study of interspecific pollen flow between closely related species. We examined reproductive barriers at six different stages between Pedicularis cranolopha and Pedicularis tricolor, two sister species. The two sister species were geographically isolated from each other based on our field survey and the records of herbarium specimens. Translocation experiments showed that flowering phenology partly overlapped and bumblebee pollinators did not discriminate between flowers of the two species. Bumblebee interspecific moves could mediate interspecific pollination as traced using fluorescent powder, in which pollen analogs placed on one species were transferred to the stigmas of the other species in experimental plots containing both species. Heterospecific pollen tubes grew in the style as well as conspecific pollen in hand-pollination experiments. Reciprocal hybridization between the two species could produce (partially) viable seeds, suggesting weak post-pollination barriers. Our results showed that geographic isolation was an important barrier of two species, and the total reproductive isolation between two species was incomplete when without geographical isolation. The formation of Big Snow Mountains could introduce an important pre-zygotic reproductive barrier between the two sister species of Pedicularis; such geographical isolation could be responsible for allopatric speciation, giving a clue to understanding the rapid radiation on mountain areas.     

Reproductive isolation between two closely related hummingbird-pollinated neotropical gingers

Empirical estimates of the relative importance of different barriers to gene flow between recently diverged species are important for understanding processes of speciation. I investigated the factors contributing to reproductive isolation between Costus pulverulentus and C. scaber (Costaceae), two closely related hummingbird-pollinated understory Neotropical herbs. I studied broad-scale geographic isolation, microhabitat isolation, flowering phenology, overlap in pollinator assemblages, floral constancy by pollinators, mechanical floral isolation, pollen-pistil interactions, seed set in interspecific crosses, and postzygotic isolation (hybrid seed germination, greenhouse survival to flowering, and pollen fertility). Aside from substantial geographic isolation, I found evidence for several factors contributing to reproductive isolation in the sympatric portion of their geographic ranges, but the identity and relative strength of these factors varied depending on the direction of potential gene flow. For C. pulverulentus as the maternal parent, mechanical floral isolation was the most important factor, acting as a complete block to interspecific pollen deposition. For C. scaber as the maternal parent, microhabitat isolation, pollinator assemblage, mechanical floral isolation, and postpollination pollen-pistil incompatibility were important. Overall, prezygotic barriers were found to be strong, resulting in 100% reproductive isolation for C. pulverulentus as the maternal parent and 99.0% reproductive isolation for C. scaber as the maternal parent. Some postzygotic isolation also was identified in the F1 generation, increasing total isolation for C. scaber to 99.4%. The results suggest that ecological factors, including habitat use and plant-pollinator interactions, contributed to speciation in this system and evolved before extensive intrinsic postzygotic isolation.     

Associations between floral specialization and species diversity: cause, effect, or correlation?

It has been proposed frequently, from Darwin’s time onwards, that specialized pollination increases speciation rates and thus the diversity of plant species (i.e. clade species richness). We suggest here that the correlation between clade species richness and floral specialization is real, but that clade species richness is frequently the cause, not the result of floral specialization. We urge a broader, variance-partitioning perspective for assessing the causes of this correlation by suggesting four models of how the diversity-specialization correlation might come about: (1) floral specialization promotes initial reproductive isolation (“Initial-RI” model), (2) floral specialization promotes reinforcement of reproductive isolation upon secondary contact (“Reinforcement” model), (3) floral specialization reduces the extinction rate by promoting tighter species packing (“Extinction” model), (4) floral specialization is the result of high clade species richness, which increases the number of related species in communities, and thus selects for floral character displacement (“Character-Displacement” model). These hypotheses are evaluated by comparing the relationships between species richness, speciation mechanisms, and pollination precision, accuracy, and specialization in the broader literature and, more specifically, in four study systems: Dalechampia (Euphorbiaceae), Collinsia (Plantaginaceae), Burmeistera (Campanulaceae), and Stylidium (Stylidiaceae). These systems provide stronger support for the character-displacement hypothesis, wherein local species diversity drives the evolution of specialized pollination. Although the two reproductive-isolation hypotheses may hold for plants like orchids, with extremely precise pollination systems, the reproductive character-displacement hypothesis seems likely to be more important for plant groups with less precise pollination systems.     

Digest: Adaptation and isolation: Testing genetic and environmental barriers to hybridization in Silene*

Species differences are maintained by the cumulative effect of factors that reduce gene flow between divergent lineages. In this issue, Karrenberg et al. quantify multiple genetic and environmental barriers to gene exchange between two closely related plant species and find that adaptation to divergent environments has the greatest effect on reproductive isolation.     

Character displacement of male nuptial colour in threespine sticklebacks (Gasterosteus aculeatus)

To study the isolation mechanism of two commonly intermingled louseworts, Pedicularis rhinanthoides and Pedicularis longiflora , pollination biology in three mixed populations with the two species was investigated during a 3-year project. The results indicated that higher flowering density could help to enhance pollinator activity, and thus increase reproductive output. Bumblebees are the exclusive pollinator for the two louseworts and are essential for their reproductive success. Reproductive isolation between the two species is achieved by a combination of pre- and postzygotic isolation mechanisms. Although both species are pollinated by bumblebees, the present study indicates they successfully avoid interspecific pollen transfer due to floral isolation. Mechanical isolation is achieved by the stigma in the two species picking up pollen from different parts of the pollinator's body, whereas ethological isolation occurs due to flower constancy. Additionally, strong postzygotic isolation was demonstrated by non seed set after artificial cross-pollination even with successful pollen tube growth. We describe the hitherto unreported role of variation in the tightness and direction of the twist of the corolla beak in maintaining mechanical isolation between Pedicularis species. Although floral isolation in Pedicularis is incomplete, it is considered to be an important mechanism in maintaining species boundaries in the genus.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 37–48.     

Sibling competition arena: selfing and a competition arena can combine to constitute a barrier to gene flow in sympatry

Closely related species coexisting in sympatry provide critical insight into the mechanisms underlying speciation and the maintenance of genetic divergence. Selfing may promote reproductive isolation by facilitating local adaptation, causing reduced hybrid fitness in parental environments. Here, we propose a novel mechanism by which selfing can further impair interspecific gene flow: selfing may act to ensure that nonhybrid progeny systematically co-occur whenever hybrid genotypes are produced. Under a competition arena, the fitness differentials between nonhybrid and hybrid progeny are then magnified, preventing development of interspecific hybrids. We investigate whether this "sibling competition arena" can explain the coexistence in sympatry of closely related species of the plant fungal pathogens (Microbotryum) causing anther-smut disease. The probabilities of intrapromycelial mating (automixis), outcrossing, and sibling competition were manipulated in artificial inoculations to evaluate their contribution to reproductive isolation. We report that both intrapromycelial selfing and sibling competition significantly reduced rates of hybrid infection beyond that expected based solely upon selfing rates and noncompetitive fitness differentials between hybrid and nonhybrid progeny. Our results thus suggest that selfing and a sibling competition arena can combine to constitute a barrier to gene flow and diminish selection for additional barriers to gene flow in sympatry.     

The bees don't know and the flowers don't care: the effect of heterospecific pollen on reproduction in co‐occurring Thysanotus species (Asparagaceae) with similar flowers

Species integrity relies on the maintenance of reproductive isolation, particularly between closely related species. Further, it has been hypothesized that the presence of heterospecific pollen on flower stigmas adversely affects plant reproduction with increasing effect in closely related species. Using two pairs of co‐occurring buzz‐pollinated Thysanotus spp. in the Mediterranean climate region of Perth, Western Australia, we quantified the effect of heterospecific pollen on fruit and seed set. We first determined the mating systems of the two focal species using self‐ and outcross pollen, followed by separate treatments with heterospecific pollen within each species pair. Of the two species receiving pollen, Thysanotus triandrus had a mixed mating system, but with significantly lower fruit and seed set from self‐pollen relative to outcross pollen. Thysanotus tenellus was autogamous with no difference in fruit or seed set between autogamous, self‐ or outcross pollinations. Heterospecific pollen had no effect on fruit or seed set of either focal species. These outcomes point to post‐pollination reproductive isolation, consistent with a floral morphology that causes low specificity of pollen placement and thus a poor capacity for pre‐pollination discrimination. Negative effects of heterospecific pollen, therefore, do not appear to play a role in the reproduction in this group of buzz‐pollinated flowers.     

Both morph‐ and species‐dependent asymmetries affect reproductive barriers between heterostylous species

The interaction between floral traits and reproductive isolation is crucial to explaining the extraordinary diversity of angiosperms. Heterostyly, a complex floral polymorphism that optimizes outcrossing, evolved repeatedly and has been shown to accelerate diversification in primroses, yet its potential influence on isolating mechanisms remains unexplored. Furthermore, the relative contribution of pre‐ versus postmating barriers to reproductive isolation is still debated. No experimental study has yet evaluated the possible effects of heterostyly on pre‐ and postmating reproductive mechanisms. We quantify multiple reproductive barriers between the heterostylous Primula elatior (oxlip) and P. vulgaris (primrose), which readily hybridize when co‐occurring, and test whether traits of heterostyly contribute to reproductive barriers in unique ways. We find that premating isolation is key for both species, while postmating isolation is considerable only for P. vulgaris; ecogeographic isolation is crucial for both species, while phenological, seed developmental, and hybrid sterility barriers are also important in P. vulgaris, implicating sympatrically higher gene flow into P. elatior. We document for the first time that, in addition to the aforementioned species‐dependent asymmetries, morph‐dependent asymmetries affect reproductive barriers between heterostylous species. Indeed, the interspecific decrease of reciprocity between high sexual organs of complementary floral morphs limits interspecific pollen transfer from anthers of short‐styled flowers to stigmas of long‐styled flowers, while higher reciprocity between low sexual organs favors introgression over isolation from anthers of long‐styled flowers to stigmas of short‐styled flowers. Finally, intramorph incompatibility persists across species boundaries, but is weakened in long‐styled flowers of P. elatior, opening a possible backdoor to gene flow through intramorph pollen transfer between species. Therefore, patterns of gene flow across species boundaries are likely affected by floral morph composition of adjacent populations. To summarize, our study highlights the general importance of premating isolation and newly illustrates that both morph‐ and species‐dependent asymmetries shape boundaries between heterostylous species.     

Floral traits and isolation of three sympatric Aquilegia species in the Qinling Mountains, China

Floral isolation has been considered to be an important reproductive mechanism governing the species diversification in many genera. In a classic example Aquilegia, sympatric species from North America with diverse floral traits are generally associated with specialized pollinators that prohibit interspecific hybridization. It remains unclear whether species diversification in the genus from Eurasia is also maintained by floral isolation. We investigated floral phenology, floral characteristics and pollinators in three sympatric Aquilegia species (A. ecalcarata, A. incurvata and A. yabeana) in the Qinling Mountains, Shanxi Province, China from 2001 to 2005. The spurless A. ecalcarata flowers earlier than the other two species with nectar spurs but their floral phenology overlaps. Major pollinators of A. ecalcarata are syrphid flies while bumblebees are major for A. incurvata and A. yabeana. Therefore our observations confirm that mechanical isolation through differential pollinators could contribute reproductive isolation between spurless and spurred species, as demonstrated by studies from North America. Whether floral isolation plays a major role in the reproductive isolation between two spurred species (A. incurvata and A. yabeana), however, remains to be seen. Further studies are required to quantify the potential role of geographical isolation because they occupy different habitats.     

Pollen competition as an asymmetric reproductive barrier between two closely related Silene species

Reproductive barriers are important determinants of gene flow between divergent populations or species. We studied pollen competition as a post‐mating reproductive barrier between Silene dioica and S. latifolia. Gene flow between these species is extensive, but early‐generation hybrids are rare. In an experiment with conspecific, heterospecific and 50 : 50 mixed pollinations in the two species, pollination treatments did not significantly affect seed set and seed weight. However, molecular determination of siring success after mixed pollinations showed that fewer than expected hybrids were produced in S. latifolia (18% hybrids) but not in S. dioica (51% hybrids). This constitutes an asymmetric post‐mating reproductive barrier and likely contributes to the rarity of early‐generation hybrids. Our study shows that pollen competition can be an effective barrier to hybridization between closely related species that likely acts in concert with other reproductive barriers.