Pollen competition between two sympatric Orchis species (Orchidaceae): the overtaking of conspecific of heterospecific pollen as a reproductive barrier

The frequency of hybrid formation in angiosperms depends on how and when heterospecific pollen is transferred to the stigma, and on the success of that heterospecific pollen at fertilising ovules. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Mediterranean deceptive orchid. Plants of Orchis italica and O. anthropophora were pollinated with conspecific and heterospecific pollen (first conspecific pollen then heterospecific pollen and vice versa) and molecular analysis was used to check the paternity of the seeds produced. In this pair of Mediterranean orchids, competition between conspecific and heterospecific pollen functions as a post‐pollination pre‐zygotic barrier limiting the frequency of the formation of hybrids in nature. Flowers pollinated with heterospecific pollen can remain receptive for the arrival of conspecific pollen for a long time. There is always an advantage of conspecific pollen for fruit formation, whether it comes before or after heterospecific pollen, because it overtakes the heterospecific pollen. The conspecific pollen advantage exhibited in O. italica and O. anthropophora is likely to result from the reduced germination of heterospecific pollen or retarded growth of heterospecific pollen tubes in the stigma and ovary. Overall, the results indicate that our hybrid zone represents a phenomenon of little evolutionary consequence, and the conspecific pollen advantage maintains the genetic integrity of the parental species.     

Pollen competition as a reproductive isolating mechanism between two sympatric Hibiscus species

Differences in pollen tube growth rates (certation) between heterospecific (foreign) and conspecific pollen may strongly influence whether hybrid offspring are produced after mixed pollen loads are delivered to a stigma. For both members of a sympatric species pair, Hibiscus moscheutos and H. laevis, pollination by pure loads of foreign pollen resulted in fruit set that was not significantly different from conspecific pollination, indicating that pure loads of foreign pollen could readily result in hybrid offspring. However, the number of seeds per fruit from pure foreign pollinations was significantly less than that of pure conspecific pollination. Simultaneous mixed pollination resulted in a proportion of hybrid seeds (detected by an electrophoretic marker enzyme) that was significantly lower than expected based upon the capacity of foreign pollen to effect fertilization when applied in pure pollinations. After these 50/50% pollen mixtures were applied to stigmas, 8.0 and 7.4% hybrids were produced when H. moscheutos and H. laevis were the ovule parents, respectively. For these Hibiscus species, pollen competition appears to function as a barrier to hybridization that is of moderate intensity compared with similar barriers occurring between other recently studied sympatric species pairs.     

Pollen competition as a unilateral reproductive barrier between sympatric diploid and tetraploid Chamerion angustifolium

Speciation requires the evolution of barriers to gene exchange between descendant and progenitor populations. Cryptic reproductive barriers in plants arise after pollination but before fertilization as a result of pollen competition and interactions between male gametophytes and female reproductive tissues. We tested for such gametic isolation between the polyploid Chamerion angustifolium and its diploid progenitor by conducting single (diploid or tetraploid) and mixed ploidy (1 : 1 diploid and tetraploid) pollinations on both cytotypes and inferring siring success from paternity analysis and pollen-tube counts. In mixed pollinations, polyploids sired most (79%) of their own seeds as well as those of diploids (61%) (correcting for triploid block, siring success was 70% and 83%, respectively). In single donor pollinations, pollen tubes from tetraploids were more numerous than those from diploids at four different positions in each style and for both diploid and tetraploid pollen recipients. The lack of a pollen donor x recipient interaction indicates that the tetraploid siring advantage is a result of pollen competition rather than pollen-pistil interactions. Such unilateral pollen precedence results in an asymmetrical pattern of isolation, with tetraploids experiencing less gene flow than diploids. It also enhances tetraploid establishment in sympatric populations, by maximizing tetraploid success and simultaneously diminishing that of diploids through the production of inviable triploid offspring.     

Interspecific pollen transfer and competition between co-occurring plant species

Summary Computer simulations of a pollinator foraging in a mixture of two species were used to explore how plant reproduction can be influenced by interspecific pollination movements. Interspecific pollen transfer led to strong competitive effects when availabilities of pollen, receptive stigma surfaces, or pollinator movements were limited relative to the total number of fertilizations possible in the mixed population. Results from simulations suggest that competition for pollination through interspecific pollen transfer can result in rapid exclusion of one of two species, and that such competition represents a selective force promoting stable divergence of potential competitors in habitat affinity, flowering time, or other characteristics related to pollinator sharing.     

In vitro allelopathic effects of pollen from three Hieracium species (Asteraceae) and pollen transfer to sympatric Fabaceae

Pollen allelopathy occurs when pollen toxins inhibit sexual reproduction in heterospecifics. To quantify pollen allelopathic effects in vitro, pollen of Hieracium aurantiacum, H. floribundum, and H. pratense was extracted with double distilled water into concentrations of 0 (control), 0.1, 1, 3, 5, 10, and 25 pollen grains/μl. A subset of these extracts was partitioned by ion-exchange chromatography into acidic, basic, and neutral fractions. Pollen from six sympatric species of Fabaceae (Lotus corniculatus, Medicago sativa, Trifolium hybridum, T. pratense, T. repens, Vicia cracca) and the species of Hieracium was germinated on agar media containing different extract concentrations. As extract concentrations from intact pollen or from acidic fractions increased, mean percent germination of pollen of all Fabaceae decreased nonlinearly. Extract concentrations of 0.1 grains/μl and above inhibited pollen germination in the Fabaceae. In no case was any Hieracium species affected, i.e., there was no autotoxicity. Similar results were obtained using plants from nine populations of H. floribundum and H. pratense across eastern Canada. Observation of floral visitors indicated that there were relatively few movements between Hieracium and the Fabaceae (about 15% of total visits). Hence there is limited opportunity for heterospecific pollen transfer. This was consistent with censuses of stigma, i.e., few pollen grains of Hieracium were found on stigma of the Fabaceae. Thus, pollen allelopathy in Hieracium is unlikely to affect reproductive success in these six species of Fabaceae.     

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.     

Mentor effects in wild species of Helianthus (Asteraceae)

Self-incompatibility (SI) is an effective method for limiting self-fertilization in flowering plant species, but there are circumstances in which an otherwise functional SI system may fail. One of the most intriguing of these is the induction of selfing by mixed loads of self and heterospecific pollen (the mentor effect) because it is likely to occur under natural conditions, such as in hybrid zones. Here we conducted a series of controlled crosses to determine whether mentor effects operate in two SI annual species, Helianthus annuus and H. petiolaris, and whether the failure of SI results in a decrease in the frequency of hybridization between these two species. Of the 1396 achenes examined from pollen mixtures that included varying ratios of self, intraspecific compatible, and interspecific pollen, 71 (5.1%) were selfed. Selfing frequencies were significantly less than expected based on pollen ratios, except when the proportion of intraspecific compatible pollen was low. Hybridization frequencies from these same pollen ratios averaged 41.8% with H. annuus as the maternal species and 13.3% with H. petiolaris as the mother. Analysis of 1404 achenes from pollen mixtures that excluded self pollen resulted in hybridization frequencies for H. annuus (42.2%) and H. petiolaris (18.2%) that do not differ significantly from those including self pollen. Thus, mentor effects do not appear to play an important role in reproductive isolation between these species. On the other hand, even a modest increase in self-fertilization in hybrid populations, such as that due to mentor effects, could enhance the probability of hybrid species establishment.     

Interspecific interactions between three sympatric species of kangaroo rats (Dipodomys)

Interspecific interactions between Dipodomys merriami, D. ordii, and D. panamintinus were observed in the laboratory. The largest species, D. panamintinus, was the most dominant while the smallest species, D. merriami, was the least dominant. All interactions were characterized by extreme fighting. Laboratory and field evidence indicate that interspecific aggression in the field may be one mechanism keeping these sympatric species of kangaroo rats ecologically separate.     

Genomic relationships between hexaploid Helianthus resinosus and diploid Helianthus annuus (Asteraceae)

Genus Helianthus comprises diploid and polyploid species. An autoallopolyploid origin has been proposed for hexaploid species but the genomic relationships remain unclear. Mitotic and meiotic studies in annual Helianthus annuus (2n = 2x = 34) and perennial Helianthus resinosus (2n = 6x = 102) as well as the F₁ hybrids between both species were carried out. Chromosome counting confirmed the hybrid origin of the latter plants and their tetraploid condition. Bivalents in hybrids ranged from 12 to 28 ( $ \bar{x} $  = 20.8). Univalents, trivalents and quadrivalents were also observed. Meiotic products comprised dyads, triads and normal tetrads and pollen grains were heterogeneous in size. These observations suggest the occurrence of 2n pollen in addition to the expected n. Genomic in situ hybridization (GISH) of total H. annuus DNA on H. resinosus chromosomes rendered weak but uniform signals; similar hybridization pattern was observed using three other annual species. Hybridization with H. annuus probe performed on root tip cells of F₁ H. annuus × H. resinosus hybrids revealed 17 chromosomes with a strong hybridization signal. GISH in hybrid meiocytes distinguished chromosomes from parental species and revealed autosyndetic pairing of H. resinosus chromosomes, allosyndetic pairing in bivalents, trivalents and quadrivalents, and the presence of univalents derived from parents, H. annuus and H. resinosus. Results obtained from classical and molecular cytogenetics do not support H. annuus as a direct ancestor of H. resinosus. The occurrence of allosyndetic pairing and the relatively high fertility of the F₁ hybrids point to the possibility that useful genes could be transferred from H. resinosus to cultivate sunflower, although the effective rate of recombination has not been evaluated. GISH method proved effective to recognize parental chromosomes in H. annuus × H. resinosus progeny.     

Origin(s) of the diploid hybrid species Helianthus deserticola (Asteraceae)

Homoploid hybrid speciation has traditionally been considered a rare event, dependent on the establishment of both a novel, balanced genotype and reproductive isolating barriers between the new species and its progenitors. However, more recent studies have shown that synthetic hybrids converge toward the chromosomal structure of natural hybrids after only a few generations, suggesting that this phenomenon may be more frequent than previously assumed. Here, the possibility that the diploid hybrid species Helianthus deserticola arose from more than one hybrid speciation event was investigated using patterns of variation from cpDNA, 18 nuclear microsatellite loci, and population interfertility. Helianthus deserticola contains cpDNA haplotypes characteristic of both parental species, is polyphyletic with one parental species based on nine microsatellite loci, and has a high degree of interfertility among populations. The data are consistent with either a single origin followed by introgression with the parental species or multiple origins. Analysis of microsatellite variation places the origin of H. deserticola between 170?000 and 63?000 years before present, making it unlikely that anthropogenic disturbances influenced its origin. Finally, the hybrid species generally has lower levels of genetic diversity but higher levels of differentiation among populations than either parental species.     

Maintenance of species boundaries in three sympatric Ligularia (Senecioneae,Asteraceae) species

The key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages. Although species boundaries are frequently investigated between two species across many taxa, reproductive isolating barriers among multiple species (>2) that would represent the most common phenomenon in nature, remain to be clarified. Here, we use double digest restriction‐site associated DNA (ddRAD) sequencing to examine patterns of hybridization at a sympatric site where three Ligularia species grow together and verify whether those patterns contribute to the maintenance of boundaries among species. The results based on the RAD SNP datasets indicated hybridization Ligularia cyathiceps × L. duciformis and L. duciformis × L. yunnanensis were both restricted to F₁s plus a few first‐generation backcrosses and no gene introgression were identified, giving rise to strong reproductive isolation among hybridizing species. Moreover, hybrid swarm simulation, using HYBRIDLAB, indicated the RAD SNP datasets had sufficient discriminatory power for accurate hybrid detection. We conclude that parental species show strong reproductive isolation and they still maintain species boundaries, which may be the key mechanism to maintain species diversity of Ligularia in the eastern Qinghai‐Tibetan Plateau and adjacent areas. Moreover, this study highlights the effectiveness of RAD sequencing in hybridization studies.     

Interspecific interference competition between two field cricket species

Species interactions, such as interspecific competition, determine population dynamics and community structure. In a park of Amami Oshima Island, Japan, two field cricket species Teleogryllus occipitalis (Audinet‐Serville) (Gryllidae) and Loxoblemmus equestris Saussure (Gryllidae) are dominant species. We examined the interference competition at the nymph and adult stages in the two crickets under controlled laboratory conditions. Survival and emergence rates of T. occipitalis were not affected by the density of L. equestris, whereas survival and emergence rates of L. equestris decreased as the density of T. occipitalis increased. These results showed that T. occipitalis was competitively superior over L. equestris in species interactions. We discuss inconsistency between the results and coexistence of these two species under field conditions.     

Heterospecific pollen transfer between sympatric species in a midsuccessional old-field community

The cumulative (season-long) incidence of heterospecific pollen transfer (HPT) was examined using nine sympatric species in a midsuccessional old field. Inflorescences were collected weekly during the flowering season, and the proportion of foreign pollen/stigma was recorded. Flowering phenologies of sympatric species and ovule and seed counts of study species were also recorded. Heterospecific pollen was detected on some stigmas of each species. Medicago sativa (Fabaceae) received the most foreign pollen; in some cases, all of the grains on a stigma were heterospecific. Lotus corniculatus (Fabaceae) received the least amount of foreign pollen; the incidence of heterospecific pollen was near zero in most cases. The mean and range of foreign pollen received varied by as much as an order of magnitude between species. The six species with zygomorphic flowers, all Fabaceae, received more heterospecific pollen than the three species with actinomorphic flowers, Potentilla recta and P. simplex (Rosaceae) and Ranunculus acris (Ranunculaceae). This probably reflects a bias because our data were analyzed on a cumulative basis and the Fabaceae had longer flowering phenologies. HPT was not correlated with the species' relative abundance within the community. Proportion of foreign pollen received varied temporally within species, and this variation generally was not related to phenology of any sympatric taxa or the species' own phenology. Pollen grain diameter was positively related to levels of foreign pollen received by species. This might be caused by poor adhesion of large pollen grains to small stigmatic papillae or if generalist pollinators carrying large amounts of heterospecific pollen visit the large-grained species and specialists with little foreign pollen visit the small-grained species. The large proportions of heterospecific pollen on stigmas of many species indicate that HPT occurs frequently in the community we studied and the implications may include reduced seed set because of occlusion by foreign grains. As yet, however, it is unclear how important a factor HPT is in mediating pollen limitation of reproductive success.     

Interspecific competition during transmission of two sympatric malaria parasite species to the mosquito vector

The role of species interactions in structuring parasite communities remains controversial. Here, we show that interspecific competition between two avian malaria parasite species, Plasmodium gallinaceum and P. juxtanucleare, occurs as a result of interference during parasite fertilization within the bloodmeal of the mosquito. The significant reduction in the transmission success of P. gallinaceum to mosquitoes, due to the co-infecting P. juxtanucleare, is predicted to have compromised its colonization of regions occupied by P. juxtanucleare and, thus, may have contributed to the restricted global distribution of P. gallinaceum. Such interspecies interactions may occur between human malaria parasites and, thus, impact upon parasite species epidemiology, especially in regions of seasonal transmission.     

Interspecific competition between alien and native congeneric species

A good way to check hypotheses explaining the invasion of ecosystems by exotic plants is to compare alien and native congeneric species. To test the hypothesis that invasive alien plants are more competitive than natives, we designed a replacement series experiment to evaluate interspecific competition between three Senecio species representing the same bushy life form: two alien species (S. inaequidens and S. pterophorus, both from South Africa) and a native species from the south-east of the Iberian Peninsula and Maghreb (S. malacitanus). While S. inaequidens is widespread throughout western Europe and is expanding towards the south of Spanish–French border, the geographical distribution of the recently introduced S. pterophorus is still limited to north-eastern Spain. Plants from each species were grown in pure and in mixed cultures with one of their congeners, and water availability was manipulated to evaluate the effects of water stress on competitive abilities. Our results show that the alien S. inaequidens is the most competitive species for all water conditions. The native S. malacitanus is more competitive that the alien S. pterophorus in water stress conditions, but this situation is reversed when water availability is not limiting.     

Interspecific competition between two species of the bean weevil

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Contributions from the Entomological Laboratory, Kyoto University, No. 110.     

Ecogeographic isolation: a reproductive barrier between species and between cytotypes in Houstonia (Rubiaceae)

‘Ecogeographic isolation’ describes the combined role of ecology and geography as a reproductive barrier, and an important component in speciation. Evidence increasingly shows that this form of isolation is important for maintaining the genetic integrity of populations and species. Further, ecogeographic isolation can be a reproductive barrier between polyploid individuals and their diploid progenitors. New ecoinformatic methods, which includes niche modeling and associated statistical assessments of these models with spatially explicit environmental data, allow us to test if ecogeographic isolation is a contributing isolating barrier between species and between cytotypes within a species. We tested the hypothesis that ecogeographic isolation contributes to isolation of species and cytotypes within species of the plant genus Houstonia. We found that species in this group occupied significantly different niches, which suggests ecogeographic isolation is a contributing reproductive barrier. We also found that diploid and tetraploid forms of H. longifolia show some level of ecogeographic isolation, but H. purpurea diploids and tetraploids did not. Our results suggest that ecogeographic isolation plays a role in reproductive isolation between Houstonia species and between cytotypes of H. longifolia.     

Natural hybridization and introgression in sympatric Ligularia species (Asteraceae, Senecioneae)

The difficulty in clarifying species of genus Ligularia Cass.has been attributed to rapid and continuous allopatric speciation in small and isolated populations,combined with interspecific diploid hybridization in the Qinghai-Tibetan Plateau and adjacent areas.However,no concrete example has been reported to prove this hypothesis.We studied a natural mixed population of six species of Ligularia in which some individuals were morphologically intermediate between L.subspicata and L.nelumbifolia.Based on DNA sequences (trnC-E trnL-rpL32,trnQ5'rpsl6,trnK-rpsl6,and internal transcribed spacer) and inter-simple sequence repeat data,we concluded that putative hybrids are primarily products of hybridization between L.nelumbifolia and L.subspicata.The other four species or additional,unknown species may also be involved in hybridization.This hybridization is bidirectional but asymmetrical.Hybrid individuals were mostly the first generation,but F2 and later-generation hybrids were also present.Moreover,the backcrossed individuals detected indicate that natural gene flow occurs among at least three Ligularia species.Hybrids may become stabilized to form new species or may function as intermediates in evolutionary diversification.