NATURAL HYBRIDIZATION AMONG FOUR SPECIES OF BAPTISIA (LEGUMINOSAE)

Alston , R. E., and B. L. Turner . (U. Texas, Austin.) Natural hybridization among four species of Baptisia (Leguminosae). Amer. Jour. Bot. 50(2): 159–173. Illus. 1963.— Interspecific hybridization involving 4 species of Baptisia (B. leucophaea, B. sphaerocarpa, B. nuttalliana, and B. leucantha) has been studied by means of extensive field work and subsequent morphological and chromatographic analyses. As a result of these studies, numerous hybridizing populations involving any 2, 3 and, in 1 instance, 4 species have been located. Near Dayton, Texas, all 4 species and all 6 of the possible 2-way hybrid combinations have been found in a single field. Approximately 125 different chemical compounds have now been detected in the 4 species. Many of these compounds serve as species specific markers useful in the validation of specific hybrid types. Hybrids between B. leucophaea and B. sphaerocarpa and between B. leucophaea and B. nuttalliana are numerous, and in these large hybrid swarms a chromatographic and morphological analysis of population structure is possible. The former combination provides an excellent opportunity for the utilization of chemical markers as criteria for introgressive hybridization. The hybrid B. leucantha × B. sphaerocarpa is frequently encountered and contains a large number of compounds species-specific for one or the other parental species. The other 3 hybrid types have been found infrequently. Certain hybrid types are generally similar morphologically (e.g., B. leucantha × B. sphaerocarpa as opposed to B. leucantha × B. nuttalliana), and chromatographic techniques are of great value in the absolute identification of such plants, especially in complex populations where backcrossing further complicates the interpretation of the background of a plant from exomorphic features alone.     

Variation in pollinator preference between two Ipomopsis contact sites that differ in hybridization rate

Pollinator-mediated reproductive isolation is often a principal factor in determining the rate of hybridization between plant species. Pollinator preference and constancy can reduce interspecific pollen transfer between otherwise interfertile, coflowering species. The importance of this ethological isolation can be assessed by comparing the strength of preference and constancy of pollinators in contact sites that differ in the frequency of hybrid individuals. We observed visitation by hummingbirds and hawkmoths in natural single-species patches and artificial mixed-species arrays in two Ipomopsis aggregata/I. tenuituba contact sites-one with few hybrids, and one in which hybrids are abundant. Pollinator preference and constancy were stronger at the low-frequency hybrid site, especially for hawkmoths (Hyles lineata). Hawkmoths at the low-frequency hybrid site showed significant preference and constancy for I. tenuituba, while at the high-frequency site hawkmoths visited both species equally. One hypothesis that might explain these differences in hawkmoth foraging is that warmer nights at the low-frequency hybrid site allow for nocturnal foraging where the light-colored corollas of I. tenuituba have a visibility advantage. These differences in hawkmoth behavior might in turn affect hummingbirds differently at the two sites, through changes in nectar resources, leading to greater pollinator-mediated isolation at the low-frequency hybrid site. Our results suggest that differences in pollinator behaviors between sites can have both direct and indirect effects on hybridization rates between plant species.     

The genetic architecture of reproductive isolation in Louisiana irises: pollination syndromes and pollinator preferences

In animal-pollinated plants, pollinator preferences for divergent floral forms can lead to partial reproductive isolation. We describe regions of plant genomes that affect pollinator preferences for two species of Louisiana Irises, Iris brevicaulis and Iris fulva, and their artificial hybrids. Iris brevicaulis and I. fulva possess bee and bird-pollination syndromes, respectively. Hummingbirds preferred I. fulva and under-visited both I. brevicaulis and backcrosses toward this species. Lepidopterans preferred I. fulva and backcrosses toward I. fulva, but also under-visited I. brevicaulis and I. brevicaulis backcrosses. Bumblebees preferred I. brevicaulis and F1 hybrids and rarely visited I. fulva. Although all three pollen vectors preferred one or the other species, these preferences did not prevent visitation to other hybrid/parental classes. Quantitative trait locus (QTL) mapping, in reciprocal BC1 mapping populations, defined the genetic architecture of loci that affected pollinator behavior. We detected six and nine QTLs that affected pollinator visitation rates in the BCIb and BCIf mapping populations, respectively, with as many as three QTLs detected for each trait. Overall, this study reflects the possible role of quantitative genetic factors in determining (1) reproductive isolation, (2) the pattern of pollinator-mediated genetic exchange, and thus (3) hybrid zone evolution.     

Spatial–temporal patterns of flowering asynchrony and pollinator fidelity in hybridizing species of Narcissus

Speciation requires the evolution of reproductive barriers to achieve isolation between species. In this paper, we examine the role of two major pre-zygotic barriers in reducing the chance of F₁ hybrid formation between two pairs of Narcissus species. Field experiments were performed over 5?years in eight natural populations to determine whether flowering phenology and pollinator fidelity could act as reproductive isolation barriers in Narcissus. Our results show that reproductive isolation due to flowering phenology is highly variable and asymmetric. In some populations, pollinator fidelity was so strong that the quantification of reproductive isolation was complete and a strong negative correlation was found between the strength of this barrier and the abundance of hybrids. Nevertheless, the degree of pollinator fidelity was quite variable among populations indicating that reproductive isolation varies geographically but very consistent across years indicating that plant-pollinator interactions are well established. In fact, the finding that hybrid formation between these species occurs only in sites where pollinator fidelity is incomplete suggests that hybrid formation also varies geographically and that divergent evolutionary outcomes may occur in different sympatric populations of Narcissus.     

Genetic Evidence for Natural Hybridization between Species of Dioecious Ficus on Island Populations1

Natural hybrids between Ficus septica and two closely related dioecious species, F. fistulosa and F. hispida, were confirmed using amplified fragment length polymorphisms (AFLP) and chloroplast DNA markers. Ficus species have a highly species‐specific pollination mutualism with agaonid wasps. Therefore, the identification of cases in which breakdown in this sophisticated system occurs and the circumstances under which it happens is of interest. Various studies have confirmed that Ficus species are able to hybridize and that pollinator‐specificity breakdown can occur under certain conditions. This study is the first example in which hybrid identity and the presence of hybrids in the natural distribution of parental species for Ficus have been confirmed with molecular markers. Hybrid individuals were identified on three island locations in the Sunda Strait region of Indonesia. These findings support Janzen's (1979) hypothesis that breakdown in pollinator specificity is more likely to occur on islands. We hypothesized that hybrid events could occur when the population size of pollinator wasps was small or had been small in one of the parental species. Later generation hybrids were identified, indicating that backcrossing and introgression did occur to some extent and that therefore, hybrids could be fertile. The small number of hybrids found indicated that there was little effect of hybridization on parental species integrity over the study area. Although hybrid individuals were not common, their presence at multiple sites indicated that the hybridization events reported here were not isolated incidences. Chloroplast DNA haplotypes of hybrids were not derived solely from one species, suggesting that the seed donor was not of the same parental species in all hybridization events.     

The strength of reproductive isolation in two hybridizing food-deceptive orchid species

Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. In orchids, the wide variety of pollination systems and highly diverse floral traits have traditionally suggested a prominent role for pollinator isolation, and thus for prezygotic isolation, as an effective barrier to gene flow among species. Here, we examined the nature of reproductive isolation between Anacamptis morio and Anacamptis papilionacea, two sister species of Mediterranean food-deceptive orchids, in two natural hybrid zones. Comparative analyses of the two hybrid zones that are located on soils with volcanic origin and have different and well-dated ages consistently revealed that all hybrid individuals were morphologically and genetically intermediate between the parental species, but had strongly reduced fitness. Molecular analyses based on nuclear ITS1 and (amplified fragment length polymorphism) AFLP markers clearly showed that all examined hybrids were F1 hybrids, and that no introgression occurred between parental species. The maternally inherited plastid DNA markers indicated that hybridization between A. morio and A. papilionacea was bidirectional, as confirmed by the molecular analysis of seed families. The genetic architecture of the two hybrid zones suggests that the two parental species easily and frequently hybridize in sympatry as a consequence of partial pollinator overlap but that strong postzygotic barriers reduce hybrid fitness and prevent gene introgression. These results corroborate that chromosomal divergence is instrumental for reproductive isolation between these food-deceptive orchids and suggest that hybridization is of limited importance for their diversification.     

Reproductive Isolation of Hybrid Populations Driven by Genetic Incompatibilities

Despite its role in homogenizing populations, hybridization has also been proposed as a means to generate new species. The conceptual basis for this idea is that hybridization can result in novel phenotypes through recombination between the parental genomes, allowing a hybrid population to occupy ecological niches unavailable to parental species. Here we present an alternative model of the evolution of reproductive isolation in hybrid populations that occurs as a simple consequence of selection against genetic incompatibilities. Unlike previous models of hybrid speciation, our model does not incorporate inbreeding, or assume that hybrids have an ecological or reproductive fitness advantage relative to parental populations. We show that reproductive isolation between hybrids and parental species can evolve frequently and rapidly under this model, even in the presence of substantial ongoing immigration from parental species and strong selection against hybrids. An interesting prediction of our model is that replicate hybrid populations formed from the same pair of parental species can evolve reproductive isolation from each other. This non-adaptive process can therefore generate patterns of species diversity and relatedness that resemble an adaptive radiation. Intriguingly, several known hybrid species exhibit patterns of reproductive isolation consistent with the predictions of our model.     

Hybridization between the endemic brown algae Carpophyllum maschalocarpum and Carpophyllum angustifolium (Fucales): Genetic and morphological evidence

Hybridization is an important evolutionary process, which can have significant effects on biodiversity. While hybridization is well known in plants, less is known about the prevalence of hybridization in other kingdoms. Hybridization in the field has been confirmed in brown algae in a few cases, mainly in the northern hemisphere genus Fucus. Putative hybrids have been found in the New Zealand endemic species Carpophyllum angustifolium and Carpophyllum maschalocarpum. We used nuclear‐encoded molecular data (ITS2) and morphometrics to confirm hybridization between C. angustifolium and C. maschalocarpum. Putative hybrid thalli were collected that had heterozygous ITS2, each copy corresponding to one of the parental species from that population. Morphological analysis also showed that the three classes (two parental species and hybrids) were easily distinguishable in these populations. It was found that the hybrids had an intermediate morphology to the parent species. Some individuals with C. angustifolium morphology had hybrid ITS2 ribotypes suggestive of backcrossing between the hybrids and C. angustifolium. Our data reveal another case of hybridization within the Fucales and suggests that further research on how these species remain separate is needed.     

Hybridization in closely related Rhododendron species: half of all species‐differentiating markers experience serious transmission ratio distortion

An increasing number of studies of hybridization in recent years have revealed that complete reproductive isolation between species is frequently not finalized in more or less closely related organisms. Most of these species do, however, seem to retain their phenotypical characteristics despite the implication of gene flow, highlighting the remaining gap in our knowledge of how much of an organism's genome is permeable to gene flow, and which factors promote or prevent hybridization. We used AFLP markers to investigate the genetic composition of three populations involving two interfertile Rhododendron species: two sympatric populations, of which only one contained hybrids, and a further hybrid‐dominated population. No fixed differences between the species were found, and only 5.8% of the markers showed some degree of species differentiation. Additionally, 45.5% of highly species‐differentiating markers experienced significant transmission distortion in the hybrids, which was most pronounced in F1 hybrids, suggesting that factors conveying incompatibilities are still segregating within the species. Furthermore, the two hybrid populations showed stark contrasting composition of hybrids; one was an asymmetrically backcrossing hybrid swarm, while in the other, backcrosses were absent, thus preventing gene flow.     

Pollination patterns limit hybridization between two sympatric species of Narcissus (Amaryllidaceae)

Natural hybrids between rare and common sympatric species are commonly eradicated to avoid the potential extinction of the rare species, although there is currently no clear predictive framework to quantify this risk. As hybrids can have intrinsic value as new evolutionary pathways, further knowledge on the factors controlling hybridization is needed. In this study we evaluated the role of pollination patterns in hybridization events in two sympatric populations of Narcissus cavanillesii and N. serotinus in Portugal. Narcissus cavanillesii is a rare species, while N. serotinus is widely distributed across the Mediterranean. The hybrid, N. ×perezlarae, is quite frequent in southeastern Spain but is scarce in Portugal. Reciprocal manual crossings confirmed compatibility between the two species, although hybridization was more successful when N. cavanillesii participated as female. Narcissus cavanillesii and N. serotinus only shared one pollinator, Megachile sp. (Hymenoptera), which had low visitation rates and high flower constancy. No single isolation mechanism was fully effective in preventing hybridization. Temporal displacement of flowering peaks, strong pollinator specificity, and high flower constancy in the shared pollinator all contributed to limiting hybridization in this site. In other sympatric occurrences, different phenological windows and pollination assemblages may allow greater frequency of the hybrid.     

Pollinator convergence and the nature of species' boundaries in sympatric Sardinian Ophrys (Orchidaceae)

Background and Aims

In the sexually deceptive Ophrys genus, species isolation is generally considered ethological and occurs via different, specific pollinators, but there are cases in which Ophrys species can share a common pollinator and differ in pollen placement on the body of the insect. In that condition, species are expected to be reproductively isolated through a pre-mating mechanical barrier. Here, the relative contribution of pre- vs. post-mating barriers to gene flow among two Ophrys species that share a common pollinator and can occur in sympatry is studied.

Methods

A natural hybrid zone on Sardinia between O. iricolor and O. incubacea, sharing Andrena morio as pollinator, was investigated by analysing floral traits involved in pollinator attraction as odour extracts both for non-active and active compounds and for labellum morphology. The genetic architecture of the hybrid zone was also estimated with amplified fragment length polymorphism (AFLP) markers, and pollination fitness and seed set of both parental species and their hybrids in the sympatric zone were estimated by controlled crosses.

Key Results

Although hybrids were intermediate between parental species in labellum morphology and non-active odour compounds, both parental species and hybrids produced a similar odour bouquet for active compounds. However, hybrids produced significantly lower fruit and seed set than parental species, and the genetic architecture of the hybrid zone suggests that they were mostly first-generation hybrids.

Conclusions

The two parental species hybridize in sympatry as a consequence of pollinator overlap and weak mechanical isolation, but post-zygotic barriers reduce hybrid frequency and fitness, and prevent extensive introgression. These results highlight a significant contribution of late post-mating barriers, such as chromosomal divergence, for maintaining reproductive isolation, in an orchid group for which pre-mating barriers are often considered predominant.Key words: AFLP markers, floral scent variation, hybrid zone, hybrid fitness, Ophrys iricolor, Ophrys incubacea, reproductive isolation, sexual deception     

Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post‐mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid hybrid speciation to occur. Here, we investigate the potential of behavioural mate choice to generate assortative mating among hybrids and parental species. We made three‐first‐generation hybrid crosses between different species of African cichlid fish. In three‐way mate‐choice experiments, we allowed hybrid and nonhybrid females to mate with either hybrid or nonhybrid males. We found that hybrids generally mated nonrandomly and that hybridization can lead to the expression of new combinations of traits and preferences that behaviourally isolate hybrids from both parental species. Specifically, we find that the phenotypic distinctiveness of hybrids predicts the symmetry and extent of their reproductive isolation. Our data suggest that behavioural mate choice among hybrids may facilitate the establishment of isolated hybrid populations, even in proximity to one or both parental species.     

ECOLOGICAL SEGREGATION IN A SMALL MAMMAL HYBRID ZONE: HABITAT‐SPECIFIC MATING OPPORTUNITIES AND SELECTION AGAINST HYBRIDS RESTRICT GENE FLOW ON A FINE SPATIAL SCALE

The degree to which closely related species interbreed is determined by a complex interaction of ecological, behavioral, and genetic factors. We examine the degree of interbreeding between two woodrat species, Neotoma bryanti and N. lepida, at a sharp ecological transition. We identify the ecological association of each genotypic class, assess the opportunity for mating between these groups, and test whether they have similar patterns of year‐to‐year persistence on our study site. We find that 13% of individuals have a hybrid signature but that the two parental populations and backcrosses are highly segregated by habitat type and use. Also, we find that adult hybrids are comparable to parental types in terms of year‐to‐year persistence on our site but that, among juveniles, significantly fewer hybrids reach adulthood on site compared to their purebred counterparts. Our analyses show that this hybrid zone is maintained by occasional nonassortative mating coupled with hybrid fertility, but that these factors are balanced by lower apparent survival of juvenile hybrids and habitat‐based preference or selection that limits heterospecific mating while promoting backcrossing to habitat‐specific genotypes. This system presents a novel example of the role that sharp resource gradients play in reproductive isolation and the potential for genetic introgression.     

POLLEN TRANSFER BY NATURAL HYBRIDS AND PARENTAL SPECIES IN AN IPOMOPSIS HYBRID ZONE

Models of hybrid zones differ in their assumptions about the relative fitnesses of hybrids and the parental species. These fitness relationships determine the form of selection across the hybrid zone and, along with gene flow, the evolutionary dynamics and eventual outcome of natural hybridization. We measured a component of fitness, export and receipt of pollen in single pollinator visits, for hybrids between the herbaceous plants Ipomopsis aggregata and I. tenuituba and for both parental species. In aviary experiments with captive hummingbirds, hybrid flowers outperformed flowers of both parental species by receiving more pollen on the stigma. Although hummingbirds were more effective at removing pollen from anthers of I. aggregata, hybrid flowers matched both parental species in the amount of pollen exported to stigmas of other flowers. These patterns of pollen transfer led to phenotypic stabilizing selection, during that stage of the life cycle, for a stigma position intermediate between that of the two species and to directional selection for exserted anthers. Pollen transfer between the species was high, with flowers of I. aggregata exporting pollen equally successfully to conspecific and I. tenuituba flowers. Although this study showed that natural hybrids enjoy the highest quality of pollinator visits, a previous study found that I. aggregata receives the highest quantity of pollinator visits. Thus, the relative fitness of hybrids changes over the life cycle. By combining the results of both studies, pollinator-mediated selection in this hybrid zone is predicted to be strong and directional, with hybrid fitness intermediate between that of the parental species.     

Predicting patterns of mating and potential hybridization from pollinator behavior

Hybridization in flowering plants is determined in part by the rate at which animal pollinators move between species and by the effectiveness of such movements in transferring pollen. Pollinator behavior can also influence hybrid fitness by determining receipt and export of pollen. We incorporated information on pollinator effectiveness and visitation behavior into a simulation model that predicts pollen transfer between Ipomopsis aggregata, Ipomopsis tenuituba, and hybrids. These predictions were compared with estimates of pollen transfer derived from movement of fluorescent dyes in experimental plant arrays. Interspecific pollen transfer was relatively uncommon in these arrays, whereas transfer between hybrids and the parental species was at least as common as conspecific transfer. Backcrossing was asymmetrical; I. aggregata flowers frequently received mixed loads of hybrid and conspecific pollen. The simulation suggests that these patterns of pollen transfer are largely explained by the visitation sequences of hummingbird and insect pollinators, with little contribution from mechanical isolation. Pollen receipt by hybrids exceeded that of both parental species in a year when pollinators preferred to visit F(1) and F(2) hybrids and was intermediate in another year when they preferred to visit I. aggregata. This suggests that natural variation in pollination may produce spatiotemporal variation in hybridization and hybrid fitness.     

Pollinator preferences for Nicotiana alata, N. forgetiana, and their F1 hybrids

The role of pollinators in plant speciation and maintenance of species boundaries is dubious because most plant species are visited by several types of pollinators, and most pollinator species visit several species of plants. We investigated pollinator preferences and their efficacy as ethological isolation mechanisms between two interfertile species, Nicotiana alata and N. forgetiana and their F1 hybrids. Hawkmoths pollinate N. alata, while primarily hummingbirds and occasionally small hawkmoths visit N. forgetiana. F1 hybrids are easily produced in the greenhouse and although the species grow in similar habitats, hybrids have not been found in nature. In Rio Grande do Sul, Brazil, near where both species are found, experimental plots were studied containing both species, and both species plus F1 hybrids. In the mixed-species plots, hawkmoths showed a strong preference for N. alata. Hummingbirds were less common and only visited N. forgetiana. Hybrid seed was produced but plants made significantly fewer hybrid offspring than predicted by the frequency of interspecific pollinator movements. Nicotiana forgetiana was the seed parent of 97% of the F1 offspring, suggesting an asymmetry in pollen delivery or postpollination processes. In plots containing F1 hybrids plus both parental species, hawkmoths preferred N. alata and undervisited the other two phenotypes, except that in the third plot they visited hybrids in proportion to the hybrid frequency. Hummingbirds strongly preferred N. forgetiana in all plots but also visited F1 hybrids in proportion to their frequency in the third plot. Overall, F1 hybrids were well pollinated and were frequently visited immediately before or after one of the parental species. Thus hybrids could facilitate gene flow between the parental species. We conclude that pollinator discrimination among species is strong but is an imperfect isolation mechanism, especially if hybrids are present.     

Hybridization between tetraploid species of Alopecurus L. (Poaceae): morphological studies of natural and artificial hybrids

The morphology of individuals of mixed natural populations of three species of Alopecurus, A. pratensis, A. geniculars and A. arundinaceus , is described. It is suggested that plants morphologically intermediate between the parental species are hybrids or of hybrid origin. Hybridization occurs more readily between A. pratensis and A. arundinaceus than between A. pratensis and A. geniculate , since intermediate forms were recovered from all mixed populations of A. pratensis and A. arundinaceus. The intermediate forms occur only in a few populations of A. pratensis and A. geniculars where an appropriate habitat is available. Evidence of backcrossing and therefore introgression comes from the continuum of intermediate forms that exists between the species. Scatter diagrams for individual populations show distinct differences in the degree of introgression. Observations on artificially produced hybrids are similar to those on naturally occurring hybrids.     

Evidence for natural hybridization between Primula beesiana and P. bulleyana,two heterostylous primroses in NW Yunnan,China

Natural hybridization was assumed to play an essential role for the diversification of Primula; however, only one study of hybridization in the region of the Himalayas has been undertaken. In the present study, we examined another natural hybrid zone where morphologically putative hybrids as well as P. beesiana Forrest, P. bulleyana Forrest, and P. poissonii Franch. co-occurred. We used molecular data to confirm the parental species of putative hybrids and the unidirectional hybridization pattern between P. beesiana and P. bulleyana. Moreover, with reference to synthetic F₁s, most hybrids examined are possibly advanced generations, although the possibility of F₁ hybrids currently examined could not be completely excluded. In addition, pollinator observations on experimental arrays of transplanted parental species showed interspecific pollen flows during visitations of shared pollinators, indicating an incomplete pre-zygotic barrier between P. beesiana and P. bulleyana. Seed productions from both flower morphs of putative hybrids were significantly lower than parental species, suggesting lower reproductive success in these hybrids. Combined with the evidence of recent habitat disturbance in the study area, we might witness the early process of hybridization between P. beesianaand P. bulleyana.     

Genetic Analysis of an Interspecific Hybrid Swarm of Populus: Occurrence of Unidirectional Introgression

Restriction fragment length polymorphisms were used to distinguish genotypes of two species of Populus, P. fremontii ('Fremont') and P. angustifolia ('narrowleaf'). Both inter- and intraspecific polymorphisms were detected in these cottonwood trees. The interspecific variation was much greater than the intraspecific variation. This permitted identification of parental genotypes within individual trees of a hybrid swarm which exists in an overlap zone between the two species. Within this hybrid swarm, individual trees are either F1 hybrids or backcrosses with a pure 'narrowleaf' parent; no progeny were found that could be attributed to crossing between F1 hybrid trees, or to backcrossing between F1 hybrid trees and 'Fremont'.     

Differential pollinator effectiveness and importance in a milkweed (Asclepias, Apocynaceae) hybrid zone

? Premise of the study: Exceptions to the ideal of complete reproductive isolation between species are commonly encountered in diverse plant, animal, and fungal groups, but often the causative ecological processes are poorly understood. In flowering plants, the outcome of hybridization depends in part on the effectiveness of pollinators in interspecific pollen transport. In the Asclepias exaltata and A. syriaca (Apocynaceae) hybrid zone in Shenandoah National Park, Virginia, extensive introgression has been documented. The objectives of this study were to (1) determine the extent of pollinator overlap among A. exaltata, A. syriaca, and their hybrids and (2) identify the insect taxa responsible for hybridization and introgression. ? Methods: We observed focal plants of parental species and hybrids to measure visitation rate, visit duration, and per-visit pollinia removal and deposition, and we calculated pollinator effectiveness and importance. ? Key results: Visitation rates varied significantly between the 2 yr of the study. Overall, Apis mellifera, Bombus sp., and Epargyreus clarus were the most important pollinators. However, Bombus sp. was the only visitor that was observed to both remove and insert pollinia for both parent species as well as hybrids. ? Conclusions: We conclude that Bombus may be a key agent of hybridization and introgression in these sympatric milkweed populations, and hybrids are neither preferred nor selected against by pollinators. Thus, we have identified a potential mechanism for how hybrids act as bridges to gene flow between A. exaltata and A. syriaca. These results provide insights into the breakdown of prezygotic isolating mechanisms.