Absence of conspecific pollen advantage in the dynamics of an Ipomopsis (Polemoniaceae) hybrid zone

The frequency of hybrid formation in angiosperms depends on how often heterospecific pollen is transferred to the stigma and on the success of that heterospecific pollen at fertilizing ovules. Even if heterospecific pollen is capable of effecting fertilization it may perform poorly when conspecific pollen is also available on the stigma. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Ipomopsis (Polemoniaceae) in Colorado. Plants of both parental species and natural hybrids were pollinated with I. aggregata and I. tenuituba pollen in ratios of 100:0, 80:20, 50:50, 20:80, and 0:100 by mass. Plants were homozygous for different alleles at an isozyme marker, allowing us to distinguish the type of pollen parent for 2166 viable seeds from 273 fruits. In contrast to studies of many other hybridizing taxa, there was no evidence of an advantage to conspecific pollen, nor did composition of the stigmatic pollen load affect seed set. Instead, the frequency of seeds sired by a given species was proportional to its representation in the pollen load. In this hybrid zone, both the frequency of first-generation hybrid formation and the relative male fitness of the two parental species should be predictable from the rates of pollen transfer to stigmas.     

Variation in frequency of hybrids and spatial structure among Ipomopsis (Polemoniaceae) contact sites

Rates of hybridization vary among angiosperm taxa. Among-taxon variation in hybridization rate has been used to compare the importance of pre- and post-zygotic reproductive isolating mechanisms. Variation in rates of hybridization within a single-species pair would suggest that local conditions also affect reproductive isolation within a single taxonomic context. In this study, contact sites of Ipomopsis aggregata-Ipomopsis tenuituba were surveyed for variation in frequency of hybrids, and spatial structure. Floral morphology was used to identify parent species and hybrids in seven contact sites in the western Rocky Mountains, USA. Contact sites varied widely in elevational range, the degree to which morphological variation was clinal rather than mosaic and the frequency of hybrids. Two sites provided a strong contrast between a clinal, unimodal site and a mosaic, bimodal site. This natural variation among contact sites of the same species pair provides an opportunity to assess the effect of local ecological conditions and spatial structure of parent populations on reproductive isolation, while controlling for between-taxon variation.     

Characterization of microsatellite loci in Ipomopsis (Polemoniaceae) wildflowers

Members of the Ipomopsis aggregata species complex in the phlox family (Polemoniaceae) often hybridize when they occur in sympatry, and thus have been extensively studied to examine processes involved in plant speciation. I developed 12 microsatellite loci in I. aggregata that are also polymorphic in closely related Ipomopsis tenuituba, producing an average of eight alleles per locus in test populations of 12 individuals per species. Several of these markers also successfully amplified in other Ipomopsis species and more distant members of the Polemoniaceae, suggesting they should prove useful for a broad range of evolutionary studies in this widely distributed system.     

Patterns of color and nectar variation across an Ipomopsis (Polemoniaceae) hybrid zone

Hybridization may uncouple adaptive trait combinations that are present in parental species. I studied variation in flower color and reward quality across a hybrid zone of Ipomopsis aggregata and I. tenuituba. Individuals from hybrid populations showed considerable variation in flower color using corolla reflectance measurements. Flower spectra of such individuals were either intermediate or else resembled those flowers from the parental species. Ipomopsis aggregata populations had consistently higher nectar production rates and higher nectar standing crops than either I. tenuituba or hybrids. Ipomopsis aggregata flowers also produced more dilute nectar than those of hybrids and I. tenuituba, but the actual concentration values were quite variable among populations of the same type. Overall, the nectar quality of hybrid flowers most resembled that of I. tenuituba flowers. Based on the observed interpopulation patterns of color-reward associations in this hybrid zone, pollinators should be able to discriminate against I. tenuituba and hybrid populations and against most individuals within hybrid populations. However, they may visit those hybrids that resemble the most rewarding flower type (I. aggregata). The results emphasize the need for studies that address how hybridization affects subsequent plant fitness and the evolutionary dynamics of the species involved.     

Cytoplasmic and nuclear markers reveal contrasting patterns of spatial genetic structure in a natural Ipomopsis hybrid zone

Spatial variation in natural selection may play an important role in determining the genetic structure of hybridizing populations. Previous studies have found that F1 hybrids between naturally hybridizing Ipomopsis aggregata and Ipomopsis tenuituba in central Colorado differ in fitness depending on both genotype and environment: hybrids had higher survival when I. aggregata was the maternal parent, except in the centre of the hybrid zone where both hybrid types had high survival. Here, we developed both maternally (cpDNA PCR-RFLP) and biparentally inherited (nuclear AFLP) species-diagnostic markers to characterize the spatial genetic structure of the natural Ipomopsis hybrid zone, and tested the prediction that the majority of natural hybrids have I. aggregata cytoplasm, except in areas near the centre of the hybrid zone. Analyses of 352 individuals from across the hybrid zone indicate that cytoplasmic gene flow is bidirectional, but contrary to expectation, most plants in the hybrid zone have I. tenuituba cytoplasm. This cytotype distribution is consistent with a hybrid zone in historical transition, with I. aggregata nuclear genes advancing into the contact zone. Further, nuclear data show a much more gradual cline than cpDNA markers that is consistent with morphological patterns across the hybrid populations. A mixture of environment- and pollinator-mediated selection may contribute to the current genetic structure of this hybrid system.     

Airborne‐pollen pool and mating pattern in a hybrid zone between Pinus pumila and P. parviflora var. pentaphylla

The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne‐pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne‐pollen was determined by single‐pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne‐pollen showed that the two pine species, particularly P. pumila, still have chances to form F₁ hybrid seeds. Both parental species showed a strong assortative mating pattern; F₁ seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross‐incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross‐incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.     

Species-independent, geographical structuring of chloroplast DNA haplotypes in a montane herb Ipomopsis (Polemoniaceae)

Two hypotheses have been proposed to explain the occurrence of hybrid zones between red-flowered Ipomopsis aggregata and white-flowered I. tenuituba . Either local adaptation to hummingbird and hawkmoth pollinators has given rise to sympatric (or parapatric) divergence of flower colour and morphology (primary intergradation at hybrid zones), or alternatively two previously allopatric species are coming into contact at several geographical areas of secondary intergradation. We examined restriction site patterns in nuclear DNA (nrDNA), chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) from populations of I. aggregata and I. tenuituba representing seven zones of sympatry. No variation was detected in a 350-bp fragment of mtDNA and uninformative levels of variation were observed for nrDNA. We detected 22 potentially informative restriction site polymorphisms in cpDNA, all of which united geographical areas containing populations of both species. We detected no informative species-specific markers. Studies of other species (e.g. oaks) have detected similar species-independent geographical structure of cpDNA. However, in these cases secondary interegradation could be inferred from species-specific nuclear alleles. The pattern in Ipomopsis is consistent with both primary intergradation (independent speciation in each area of sympatry) or secondary intergradation involving complete cytoplasmic replacement. Thus, additional data are needed to explain the origin of hybrid zones in Ipomopsis .     

Pollen tube competition as a mechanism of prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor M. guttatus

This paper considers the extent to which differences in pollen tube growth rates can provide prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor, Mimulus guttatus . Mimulus nasutus is partially cleistogamous, but its larger chasmogamous flowers offer appreciable opportunity for outcrossing. Mimulus nasutus was found to have smaller pollen grains and shorter styles than M. guttatus . No differences were observed in pollen grain germination on conspecific and heterospecific stigmas. However, pollen tube growth rates of M. nasutus were found to be much slower than those of M. guttatus in the styles of that species. Consequently, any M. nasutus pollen transferred to an M. guttatus stigma was found to be competitively disadvantaged in an M. gutattus style. By contrast, no difference in pollen tube growth rate was detected between the species when growing in M. nasutus styles, possibly because M. nasutus styles are unable to support fast pollen tube growth. We tested the prediction from the pollen tube studies that a 50:50 mix of M. guttatus and M. nasutus pollen would produce 50% hybrid seeds when M. nasutus was the maternal parent, and near to 0% hybrid seed when M. guttatus was the maternal parent. The results were found to support this prediction. We conclude that pollen–pistil interactions can effect strong reproductive isolation between these species, as M. guttatus pollen tubes have a competitive advantage over those of M. nasutus in an M. guttatus style, but not in an M. nasutus style.     

Components of reproductive isolation between the monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae)

Evolutionists have long recognized the role of reproductive isolation in speciation, but the relative contributions of different reproductive barriers are poorly understood. We examined the nature of isolation between Mimulus lewisii and M. cardinalis, sister species of monkeyflowers. Studied reproductive barriers include: ecogeographic isolation; pollinator isolation (pollinator fidelity in a natural mixed population); pollen competition (seed set and hybrid production from experimental interspecific, intraspecific, and mixed pollinations in the greenhouse); and relative hybrid fitness (germination, survivorship, percent flowering, biomass, pollen viability, and seed mass in the greenhouse). Additionally, the rate of hybridization in nature was estimated from seed collections in a sympatric population. We found substantial reproductive barriers at multiple stages in the life history of M. lewisii and M. cardinalis. Using range maps constructed from herbarium collections, we estimated that the different ecogeographic distributions of the species result in 58.7% reproductive isolation. Mimulus lewisii and M. cardinalis are visited by different pollinators, and in a region of sympatry 97.6% of pollinator foraging bouts were specific to one species or the other. In the greenhouse, interspecific pollinations generated nearly 50% fewer seeds than intraspecific controls. Mixed pollinations of M. cardinalis flowers yielded >75% parentals even when only one-quarter of the pollen treatment consisted of M. cardinalis pollen. In contrast, both species had similar siring success on M. lewisii flowers. The observed 99.915% occurrence of parental M. lewisii and M. cardinalis in seeds collected from a sympatric population is nearly identical to that expected, based upon our field observations of pollinator behavior and our laboratory experiments of pollen competition. F1 hybrids exhibited reduced germination rates, high survivorship and reproduction, and low pollen and ovule fertility. In aggregate, the studied reproductive barriers prevent, on average, 99.87% of gene flow, with most reproductive isolation occurring prior to hybrid formation. Our results suggest that ecological factors resulting from adaptive divergence are the primary isolating barriers in this system. Additional studies of taxa at varying degrees of evolutionary divergence are needed to identify the relative importance of pre- and postzygotic isolating mechanisms in speciation.     

Interspecific mating in the Piriqueta caroliniana (Turneraceae) complex: effects of pollen load size and composition

Two taxa of Piriqueta (P. caroliniana and P. viridis) form a broad hybrid zone that extends over much of the central Florida peninsula. We used genetic markers to examine the strength of the isolation barriers between these taxa and the patterns of mating at the initial stages of hybridization. Regression models were employed to analyze the effects of pollen load size and the proportions of intra- and interspecific pollen on the frequency of first-generation (F1) hybrid formation. Overall, the postpollination mating barriers between these two taxa were relatively weak. However, there were significant effects of pollen load composition and size on the patterns of hybridization in both taxa with frequency-dependent responses to composition in both taxa. The frequency of F1 hybrid formation was generally lower than expected based on the frequency of each pollen type on the stigma for P. caroliniana recipients. The lower frequencies of F1 seeds in this taxon were apparently due to a greater competitive ability for intraspecific pollen, since hybrid seed formation decreased with increasing pollen load size. Pollen from P. caroliniana donors was also competitively superior on P. viridis recipients, leading to higher than expected frequencies of hybrid seed formation. Pollen from P. caroliniana did suffer higher rates of pollen-tube attrition than intraspecific pollen on P. viridis recipients, so the frequency of hybrid seed formation would be lower when pellen load sizes were small. In general, reproductive isolation mechanisms were stronger in P. caroliniana, suggesting that introgression should occur into P. viridis when these taxa come into close contact. Comparison of these expected patterns of mating to the distribution of hybrid genotypes in Florida provides insights into the relative roles of mating and selection in the evolution of hybrid populations of Piriqueta.     

Multiple paternity in fruits of Ipomopsis aggregata (Polemoniaceae)

Two different mechanisms can result in multiple paternity within fruits: deposition of a mixed pollen load due to carryover of pollen from flower to flower and multiple pollinator visits in close succession. I investigated the extent of multiple paternity within fruits of Ipompsis aggregata containing from 2 to 14 seeds. A paternity analysis based on ten polymorphic isozyme markers revealed multiple paternity in a minimum of 68% (based on simple paternity exclusion) and up to 100% (based on identification of the most likely father) of the multiseeded fruits. The estimated number of fathers increased with the number of seeds in a fruit, with an average of four sires, and up to nine sires, represented in a single fruit. To explore whether this level of multiple paternity could be explained solely by simultaneous deposition of a mixed pollen load, I constructed a computer simulation model based on previous measurements of movement patterns and pollen carryover by the hummingbird pollinators. Model predictions provided a good match to observed values for number of sires per fruit. Thus, the extensive pollen carryover in this species and consequent mixed pollen loads can explain the high levels of multiple paternity in natural populations.     

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.     

Reproductive isolation and the period gene of Drosophila

The identification of genes of large effect on ecologically important traits is an important aim of molecular ecology. The period gene of Drosophila is a candidate for a gene with a large influence on premating isolation between Drosophila species, as it determines species specific aspects of courtship behaviour. Strains of D. melanogaster are available which have been genetically transformed with the period gene of either D. melanogaster or D. simulans. Here we show that D. melanogaster females do not discriminate between two such strains. This suggests that period may only make a small contribution to total premating isolation between these species. We discuss the use of genetically transformed strains in assessing the influence of single genes on complex traits.     

Sexual isolation evolves faster than hybrid inviability in a diverse and sexually dimorphic genus of fish (Percidae: Etheostoma)

Abstract .Theory predicts that sexual (or behavioral) isolation will be the first form of reproductive isolation to evolve in lineages characterized by sexual selection. Here I directly compare the rate of evolution of sexual isolation with that of hybrid inviability in a diverse and sexually dimorphic genus of freshwater fish. The magnitude of both sexual isolation and hybrid inviability were quantified for multiple pairs of allopatric species. Rates of evolution were inferred by comparing genetic distances of these species pairs with the magnitude of each form of reproductive isolation: the slope of the regression of genetic distance on the magnitude of reproductive isolation represents the rate of evolution. Of the two forms of isolation, the magnitude of sexual isolation exhibited the steeper slope of regression, indicating that sexual isolation will tend to evolve to completion earlier than hybrid inviability, strictly as a by-product of evolution in geographically isolated populations. Additional evidence from the literature is used to qualitatively compare rates of evolution of sexual isolation with that of other forms of reproductive isolation. Preliminary comparisons support the prediction that sexual isolation will evolve more rapidly than other forms. Because Etheostoma is characterized by striking sexual dimorphism, these results are consistent with the hypothesis that sexual selection for exaggerated mate-recognition characters causes the relatively rapid evolution of sexual isolation.     

The genetics of reproductive isolation and the potential for gene exchange between Drosophila pseudoobscura and D. persimilis via backcross hybrid males

Hybrid male sterility, hybrid inviability, sexual isolation, and a hybrid male courtship dysfunction reproductively isolate Drosophila pseudoobscura and D. persimilis. Previous studies of the genetic bases of these isolating mechanisms have yielded only limited information about how much and what areas of the genome are susceptible to interspecies introgression. We have examined the genetic basis of these barriers to gene exchange in several thousand backcross hybrid male progeny of these species using 14 codominant molecular genetic markers spanning the five chromosomes of these species, focusing particularly on the autosomes. Hybrid male sterility, hybrid inviability, and the hybrid male courtship dysfunction were all associated with X-autosome interactions involving primarily the inverted regions on the left arm of the X-chromosome and the center of the second chromosome. Sexual isolation from D. pseudoobscura females was primarily associated with the left arm of the X-chromosome, although both the right arm and the center of the second chromosome also contributed to it. Sexual isolation from D. persimilis females was primarily associated with the second chromosome. The absence of isolating mechanisms being associated with many autosomal regions, including some large inverted regions that separate the strains, suggests that these phenotypes may not be caused by genes spread throughout the genome. We suggest that gene flow between these species via hybrid males may be possible at loci spread across much of the autosomes.     

Multiple herbivores and coevolutionary interactions in an Ipomopsis hybrid swarm

Studies focusing on pairwise interactions between plants and herbivores may not give an accurate picture of the overall selective effect of herbivory, given that plants are often eaten by a diverse array of herbivore species. The outcome of such interactions may be further complicated by the effects of plant hybridization. Hybridization can lead to changes in morphological, phenological and chemical traits that could in turn alter plant–herbivore interactions. Here we present results from manipulative field experiments investigating the interactive effects of multiple herbivores and plant hybridization on the reproductive success of Ipomopsis aggregata formosissima X I. tenuituba. Results showed that ungulate herbivores alone had a net positive effect on plant relative fitness, increasing seed production approximately 2-fold. Caterpillars had no effect on plant relative fitness when acting alone, with caterpillar-attacked plants producing the same number of flowers, fruits and seeds as the uneaten controls. Caterpillars, however, significantly reduced flower production of ungulate browsed plants. Flower production in these plants, however, was still significantly greater (approximately 1.7-fold greater) than uneaten controls, likely leading to an increase in reproductive success through the paternal component of fitness given that fruit and seed production was not significantly different from that of herbivore-free controls. Although results suggest that herbivore imposed selection is pairwise, ungulates likely have a large influence on the abundance of, and hence the amount of damage caused by, caterpillar herbivores. Thus, because of the ecological interactions between ungulates and caterpillars, selection on Ipomopsis may be diffuse rather than pairwise, assuming such interactions translate into differential effects on plant fitness as herbivore densities vary. Plant hybridization had no significant effect on patterns of ungulate or caterpillar herbivory; i.e., no significant interactions were detected between herbivory and plant hybridization for any of the fitness traits measured in this study nor did plant hybridization have any significant effect on host preference. These results may be due to patterns of introgression or the lack of species-specific differences between I. aggregate formosissima and I. tenuituba. Plant hybridization per se resulted in lowered reproductive success of white colored morphs due in part to the effects of pollination. Although it appears that there would be strong directional selection favoring darker flower colors due to the lower reproductive success of the white colored morphs in the short run, the natural distribution of hybrids suggest that over the long run selection either tends to average out or there are no fitness differences among morphs in most years due to the additive fitness effects of hawkmoth and hummingbird pollinators.     

Testing for mating isolation between ecotypes: laboratory experiments with lake, stream and hybrid stickleback

Mating isolation is a frequent contributor to ecological speciation – but how consistently does it evolve as a result of divergent selection? We tested for genetically based mating isolation between lake and stream threespine stickleback (Gasterosteus aculeatus L.) from the Misty watershed, Vancouver Island, British Columbia. We combined several design elements that are uncommon in the studies of stickleback mate choice: (i) we used second‐generation laboratory‐reared fish (to reduce environmental and maternal effects), (ii) we allowed for male–male competitive interactions (instead of the typical no‐choice trials) and (iii) we included hybrids along with pure types. Males of different types (Lake, Inlet, hybrid) were paired in aquaria, allowed to build nests and then exposed sequentially to females of all three types. We found that Lake and Inlet males differed in behaviours thought to influence stickleback mate choice (inter‐ and intra‐sexual aggression, display and nest activities), whereas hybrids were either intermediate or apparently ‘inferior’ in these behaviours. Despite these differences, Lake and Inlet fish did not mate assortatively and hybrid males did not have a mating disadvantage. Our study reinforces the noninevitability of mating isolation evolving in response to ecological differences and highlights the need to further investigate the factors promoting and constraining progress towards ecological speciation.