Asymmetrical introgression between two Morus species (M. alba, M. rubra) that differ in abundance

Asymmetrical introgression is an expected genetic consequence of hybridization when parental taxa differ in abundance; however, evidence for such effects in small populations is scarce. To test this prediction, we estimated the magnitude and direction of hybridization between red mulberry (Morus rubra L.), an endangered species in Canada, and the introduced and more abundant white mulberry (Morus alba L.) using nuclear (randomly amplified polymorphic DNA) and cytoplasmic (chloroplast DNA sequence) markers. Parentage of 184 trees (n = 42 using cpDNA) from four sympatric populations was estimated using a hybrid index and related to six morphological characters and population frequencies of the parental classes. Overall, the frequency of nuclear hybrids was 53.7% (n = 99) and ranged from 43% to 67% among populations. The parental and hybrid taxa differed with respect to all of the morphological traits. Sixty-seven percent of all hybrids contained more nuclear markers from M. alba than M. rubra (hybrid index x = 0.46); among populations, the degree of M. alba bias was correlated with the frequency of M. alba. In addition, the majority of hybrids (68%) contained the chloroplast genome of white mulberry. These results suggest that introgression is bidirectional but asymmetrical and is related, in part, to the relative frequency of parental taxa.     

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.     

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.     

Apomixis versus sexuality in blackberries (Rubus subgen.Rubus,Rosaceae)

InRubus L. a connection seems to exist between the degree of meiotic disturbances on the one hand, and the production of unreduced embryo sacs, pollen fertility and relative seed set on the other hand. Severe meiotic disturbances commonly encountered in apomictic taxa decrease pollen fertility and thereby seed set since pollen is necessary for endosperm development. By contrast interspecific hybrids between apomictic taxa appear to be sexual and exhibit high pollen fertilities, probably due to an improved meiosis. Thus, apomixis leads to a decreased fertility inRubus, not the opposite, as often discussed.     

Experimental hybridization of north-west European species in Carex section Acutae (Cyperaceae)

Experiments were performed on the synthesis and fertility of interspecific hybrids in Carex section Acutae.
Crosses between members of a few pairs of species gave as high a seed set as crosses within species. Other pairs of species showed some degree of intersterility in their primary crosses, but the level of seed set varied widely according to which individuals of the species were crossed. The investigated species formed two groups, within which interfertility was high, and between which interfertility was low. These groups are compared with the taxonomic subsections of the Acutae and some amendments in the composition of the subsections are suggested.
In interspecific backcrosses, it was found that seed set was impaired only if the hybrid was the female parent. The pollen grains of interspecific hybrids were found to be more variable in size than those of non-hybrids.
Results obtained in these investigations confirm that C. juncella is conspecific with C. nigra and that north-west European plants of C. recta are actually interspecific hybrids of two types.     

Maternal and paternal contributions to the fitness of hybrids between red and white mulberry (Morus, Moraceae)

The fitness of hybrids depends on the genetic disparity between parental taxa and the magnitude of their nuclear and non-nuclear contributions. To estimate the role of non-nuclear effects, we crossed red (R), white (W) and hybrid (H) mulberry in all combinations and compared the magnitude of maternal and paternal effects on offspring fitness (seed set, germination, survival and aboveground biomass) in a greenhouse environment. Variation in offspring fitness was determined largely by the identity of the maternal parent; specifically, progeny with white mothers had the highest cumulative fitness. As fathers, red, white, and hybrid mulberry had no effect on fitness, and maternal × paternal interactions were significant only for survival. Individual cross-types differed significantly for all fitness components except seed set. Offspring from hybrid crosses (W × R, H × R, H × W) often differed from at least one of the within-parent crosses (W × W, R × R) as well as from other hybrid crosses, although their fitness values never exceeded the most fit parent. Reciprocal crosses differed in only two of 15 possible parental combinations: W × H (cumulative fitness) and W × R (aboveground biomass). Overall, the strong asymmetry in magnitude of maternal and paternal effects suggests that fitness of hybrid mulberry is governed largely by non-nuclear, parental effects.     

Pollen competition among two species of Senecio (Asteraceae) that form a hybrid zone on Mt. Etna, Sicily

Hybridization between interfertile, sympatric or parapatric, plant species can be reduced significantly by conspecific pollen advantage (CPA), whereby conspecific pollen has an advantage over heterospecific pollen in terms of ovule fertilization. We examined CPA in two interfertile species of Senecio, S. aethnensis, and S. chrysanthemifolius (Asteraceae), which form a hybrid zone on Mt. Etna, Sicily. Individuals of both species were pollinated with pollen mixtures containing 0, 25, 50, 75, or 100% heterospecific pollen, and offspring were genotyped to determine if they were products of conspecific or heterospecific pollen fertilizing the ovules. The mean proportion of hybrid offspring produced on S. aethnensis plants was not significantly different to that expected based on the proportion of heterospecific pollen applied to the flower head. However, S. chrysanthemifolius mother plants showed moderate CPA, with the proportion of hybrid offspring significantly less than expected. Seed set or seed germination was not reduced, hence the CPA found for S. chrysanthemifolius acts before ovule fertilization. The consequences of asymmetry in CPA on the reproductive isolation of S. aethnensis are briefly discussed, along with other mechanisms that may play a role in the maintenance of the hybrid zone on Mt. Etna.     

Variability in Pollen Load: Implications for Reproduction and Seedling Vigor in a Rare Plant, Silene douglasii var. Oraria

Habitat fragmentation can markedly influence the levels of pollen deposition and seed production in natural populations, and rare plants may be especially susceptible to any associated reductions in pollen quantity and quality. In order to ascertain the potential for pollen limitation of maternal fitness in a rare plant, Silene douglasii var. oraria, which is endemic to western coastal prairies, we counted ovules and measured conspecific and heterospecific pollen deposition on stigmas collected from open-pollinated plants. We further investigated the effect of increasing pollen intensity on fruit production, seed number and weight, as well as several measures of progeny vigor. Three levels of outcross pollen were added to plant stigmas for comparison with autogamous and open pollination in the largest naturally occurring population. Both seed and fruit production were significantly greater (P<0.05) for supplemented versus nonsupplemented stigmas, but flowers receiving different levels of pollen addition were statistically indistinguishable. Seed germination and seedling survival were also lowest for the offspring of nonsupplemented flowers; however, in natural populations, opportunities for pollen competition are very limited since open-pollinated flowers averaged fewer viable pollen grains than ovules. Seed production was equivalent for open- and autogamously pollinated flowers in 1996, indicating that natural pollen transfer may have involved mostly self pollen. Overall, the low reproductive success of var. oraria likely reflects both low pollen quantity and quality. Multiyear empirical studies of pollen intensity in field populations are needed so that we can better understand the fitness consequences of pollen limitation in rare perennials.     

Quantification of the zygotic barrier between interbreeding taxa using gene flow data

Hybridization and introgression via interspecific gene flow are common processes in the plant kingdom. The effectiveness of these processes is governed by the strengths of multiple zygotic barriers. These barriers have often been quantified in artificial settings using laborious and time‐consuming hand‐pollination experiments, but their quantification is nonexistent in the landscape. In this study, we utilized gene flow data within a spatially explicit simulation to assess the strengths of zygotic barriers. Our model system consisted of Populus nigra and its hybrid, P. × canadensis, which interbreed under natural conditions. The study population was located in the floodplain of the Eder River in central Germany. Pollen‐mediated introgression rates from hybrid males into the seeds of individual female trees were used as the target pattern using an inverse modeling approach. Simulations that treated pollen from both taxa equally revealed a large discrepancy between the observed and modeled rates of introgression for both taxa. The discrepancy was reduced by introducing a zygotic barrier against the pollen from the hybrid males. The best model outcome values indicated comparably strong zygotic barriers acting against pollen‐mediated introgressive gene flow into the two parental taxa, P. nigra and P. × canadensis. The sensitivity of our model was tested by applying different dispersal functions. Four common probability density functions were used along with a pollen dispersal function that had previously been fitted to gene flow data from the same dataset. The best barrier value was almost independent of the dispersal functions used here. Moreover, it was within the range previously determined in hand‐pollination‐based investigations, validating our model. These data indicate that the inverse modeling approach is a powerful method for quantifying hidden processes, and we discuss its use as a valuable tool for generating new insights into plant mating systems that are relevant to evolutionary biology and risk analyses in conservation efforts.     

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield. Received: 31 August 1999 / Revision accepted: 28 February 2000     

PATTERNS OF MATING IN WILD SUNFLOWER HYBRID ZONES

Theory predicts that homoploid hybrid speciation will be facilitated by selfing, yet most well-documented hybrid species are outcrossers. One possible explanation for this puzzle is that conditions in hybrid populations may favor selfing, even in otherwise outcrossing species. For example, in self-incompatible plants, mixtures of self and interspecific pollen often induce selfing. Here, we examine patterns of mating in three hybrid zones and four “pure” populations of Helianthus annuus and H. petiolaris, wild, self-incompatible sunflower species that are thought to have parented three homoploid hybrid species. Fourteen to 16 maternal families from each pure population and 44–46 maternal families from each hybrid zone were analyzed for seven polymorphic isozyme loci. Maximum-likelihood (ML) methods were used to estimate multilocus outcrossing rates (T_m) and hybridization frequencies for each maternal family, each phenotypic group within each hybrid zone (annuus-like, hybrid, and petiolaris-like), and each population. As predicted for self-incompatible species, all four parental populations have outcrossing rate ML estimates of 1.0. Within the hybrid zones, outcrossing rates were lowest in the H. annuus–like fraction of the population (0.73, 0.72, and 0.74 in the three hybrid zones, respectively), largely intermediate in the H. petiolaris–like group (0.94, 0.90, and 0.94), and highest in the hybrid group (0.97, 0.93, and 0.97). Although outcrossing rates are lower in hybrid zones than in pure populations, it is unlikely that the observed decrease facilitates hybrid speciation because outcrossing rates in the critical hybrid fraction of the population do not differ significantly from 1.0. Dividing the outcrossed pollen pool into intraspecific and interspecific components revealed that maternal plants are largely fertilized by conspecific pollen, confirming an important role for pollen competition as a reproductive barrier. Highly sterile hybrid plants do not appear to discriminate between parental species pollen, but hybrids with higher fertility tend to be fertilized by pollen from the parental group they resemble genetically. Thus, gametic selection leads to substantial assortative mating in these hybrid zones.     

Geographical variation in postzygotic isolation and its genetic basis within and between two Mimulus species

The aim of this study is to investigate the evolution of intrinsic postzygotic isolation within and between populations of Mimulus guttatus and Mimulus nasutus. We made 17 intraspecific and interspecific crosses, across a wide geographical scale. We examined the seed germination success and pollen fertility of reciprocal F₁ and F₂ hybrids and their pure-species parents, and used biometrical genetic tests to distinguish among alternative models of inheritance. Hybrid seed inviability was sporadic in both interspecific and intraspecific crosses. For several crosses, Dobzhansky–Muller incompatibilities involving nuclear genes were implicated, while two interspecific crosses revealed evidence of cytonuclear interactions. Reduced hybrid pollen fertility was found to be greatly influenced by Dobzhansky–Muller incompatibilities in five out of six intraspecific crosses and nine out of 11 interspecific crosses. Cytonuclear incompatibilities reduced hybrid fitness in only one intraspecific and one interspecific cross. This study suggests that intrinsic postzygotic isolation is common in hybrids between these Mimulus species, yet the particular hybrid incompatibilities responsible for effecting this isolation differ among the populations tested. Hence, we conclude that they evolve and spread only at the local scale.     

Barriers to interspecific hybridization between Juglans nigra L. and J. regia L species

Juglans nigra and Juglans regia are phylogenetically divergent species. Despite the economic interest in Juglans?×?intermedia (J. nigra?×? J. regia), walnut hybridization is rare under natural conditions and still difficult using controlled pollination. Here, we evaluated some reproductive mechanisms that may prevent successful natural hybridization. The study of flowering phenology of 11 J. nigra and 50 J. regia trees growing in a plantation provided information regarding the opportunity for interspecific crosses. Variation in flower size, pollen quality of putative donors, and variation in seed yield and rate of hybrid production among putative maternal trees were examined. DNA fingerprinting and parentage analyses based on nine microsatellites permitted the identification of hybrids and hybridogenic parent. Our data indicated that overlap occurred between the staminate flowering of protogynous J. regia and the beginning of pistillate flowering of protogynous J. nigra. Differences in floral size were computed between walnut species. Only three hybrids among 422 offspring of eleven J. nigra progenies were identified. Interspecific hybridization involving pollination of one early-flowering-protogynous J. nigra by three protogynous J. regia trees was detected. The correct development of J. regia male gametophytes, high pollen viability (86.5 %), and germination (57.6 %) ruled out the possibility that low pollen quality contributed to depressed hybrid production. Our findings indicated that these two species tended to remain reproductively isolated. The substantial disjunction in flowering time and additional prezygotic barriers such as differences in floral size and conspecific pollen advance may affect interspecific gene flow between J. regia and J. nigra.     

Preferential exclusion of hybrids in mixed pollinations between oilseed rape (Brassica napus) and weedy B. campestris (Brassicaceae)

In most experimental hybridizations between oilseed rape (Brassica napus) and weedy B. campestris, either intra- or interspecific pollen has been applied to individual flowers. Under field conditions, however, stigmas will often receive a mixture of the two types of pollen, thereby allowing for competition between male gametophytes and/or seeds within pods. To test whether competition influences the success of hybridization, pollen from the two species was mixed in different proportions and applied to stigmas of both species. The resulting seeds were scored for paternity by isozyme and randomly amplified polymorphic DNA analysis. Using data on the proportion of fully developed seeds and the proportion of these seeds that were hybrids, a statistical model was constructed to estimate the fitness of conspecific and heterospecific pollen and the survival of conspecific and heterospecific zygotes to seeds. B. campestris pollen in B. napus styles had a significantly lower fitness than the conspecific pollen, whereas no difference between pollen types was found in B. campestris styles. Hybrid zygotes survived to significantly lower proportions than conspecific zygotes in both species, with the lowest survival of hybrid zygotes in B. napus pods. This is in contrast to the higher survival of hybrid seeds in B. napus than in B. campestris pods when pollinations are made with pure pollen. Altogether, the likelihood of a foreign pollen grain producing a seed was much lower on B. napus than on B. campestris. In addition, pods on B. napus developed to a lower extent the more heterospecific pollen was in the mix, whereas this had no effect on B. campestris.     

Microgametophyte population sizes and plant reproductive output in the insect-pollinated Prunella grandiflora (Lamiaceae)

Intraspecific variation in pollen deposition and number of pollen tubes per style is rarely quantified, but is essential for assessing the occurrence of pollen limitation and pollen competition and their evolutionary implications. Moreover, pollen deposition, pollen tube growth, and the fate of fertilized ovules are rarely distinguished in field studies. Here we present such a study in eight natural populations of Prunella grandiflora. We quantified microgametophyte population sizes and inferred pollen limitation when the number of fertilizable ovules exceeded pollen tubes, and assessed seed set and fate after open pollination. Two and three populations had on average significantly fewer pollen grains and pollen tubes per flower, respectively, than the fixed number of fertilizable ovules per fruit, while one population experienced significant pollen competition. Style length was positively correlated with the number of pollen tubes. While pollen availability was very variable, seed abortion was significantly less frequent in denser populations, and in one population the proportion of well-developed seeds was significantly, positively correlated with the number of pollen tubes in the style. Less pollen deposition, lower numbers of pollen tubes reaching the base of the style, lower pollen quality and therefore increased abortion of fertilized ovules can all reduce seed set in natural P. grandiflora stands. Substantial intraspecific variability implies that microgametophyte competition also occurs in this species. Finally, style morphology may affect pollen receipt.     

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.     

Polymorphism for interspecific hybridisation within a population of wild radish (Raphanus raphanistrum) pollinated by oilseed rape (Brassica napus)

The within-population polymorphism of wild radish (Raphanus raphanistrum) for interspecific hybridisation with two cultivars of oilseed rape (Brassica napus) was investigated by hand crossing experiments and fluorescence microscopy. Wide variability among plants was observed in the ability of oilseed rape pollen to germinate on the wild radish stigma; the frequency of pistils showing pollen tubes ranged from 0 to 1, depending on the female plant. The ratio of fertilised ovules to the total number of ovules in ovaries where pollen tubes arrived ranged from 0.02 to 0.51. Overall, the results provide evidence for the presence of different phenotypes. In 40% of the plants, pistils had no or very few pollen tubes and few fertilised ovules. In 23%, the foreign pollen tubes grew through the style towards the ovary, but had low ovule fertilisation efficiency. The remaining 37% showed a large number of pollen tubes in the style and frequent ovule fertilisation, and two plants showed no difference between foreign and conspecific pollen. With regard to post-zygotic barriers, pollen germination and ovule fertilisation represent minor barriers to interspecific hybridisation between oilseed rape and wild radish. It is suggested that the effectiveness of these barriers could be improved through plant breeding; this could reduce the risk of gene flow from transgenic oilseed rape to populations of wild relatives. Received: 15 April 2001 / Accepted: 24 May 2001     

Pollen tube growth and early ovule development following self- and cross-pollination in<Emphasis Type="Italic"> Eucalyptus nitens</Emphasis>

Controlled self- and cross-pollinations were conducted on flowers of five mature Eucalyptus nitens trees. Levels of self-sterility of the trees ranged from 25.8 to 93.6%. Pollen tube numbers in styles and ovule penetration by pollen tubes was investigated 2 weeks after pollination by fluorescence microscopy. There were no significant differences between treatments in the number of pollen tubes present in styles or in the percentage of ovules penetrated by pollen tubes. Embryology of material harvested 2 and 4 weeks after pollination was investigated by bright-field microscopy. Fertilisation had taken place by 2 weeks after pollination with nearly every ovule showing evidence of fertilisation. Cross-pollination resulted in a greater proportion of healthy, developing ovules, at both 2 and 4 weeks after pollination, compared with self-pollination. The proportion of degenerating ovules increased from 2 to 4 weeks after pollination. The reduced ability of E. nitens to set self-pollinated seed compared with cross-pollinated seed appears to be controlled by a post-zygotic mechanism. Differences in ovule size may potentially assist in the identification of trees incapable of setting self-pollinated seed.     

Unique growth paths of heterospecific pollen tubes result in late entry into ovules in the gynoecium of Sagittaria (Alismataceae)

• Pollen‐pistil interactions are a fundamental process in the reproductive biology of angiosperms and play a particularly important role in maintaining incipient species that exist in sympatry. However, the majority of previous studies have focused on species with syncarpous gynoecia (fused carpels) and not those with apocarpous gynoecia (unfused carpels).
• In the present study, we investigated the growth of conspecific pollen tubes compared to heterospecific pollen tubes in Sagittaria species, which have apocarpous gynoecia. We conducted controlled pollinations between S. pygmaea and S. trifolia and observed the growth of conspecific and heterospecific pollen tubes under a fluorescence microscope.
• Heterospecific and conspecific pollen tubes arrived at locules within the ovaries near simultaneously. However, conspecific pollen tubes entered into the ovules directly, whereas heterospecific tubes passed through the carpel base and adjacent receptacle tissue, to ultimately fertilize other unfertilized ovules. This longer route taken by heterospecific pollen tubes therefore caused a delay in the time required to enter into the ovules. Furthermore, heterospecific pollen tubes displayed similar growth patterns at early and peak pollination. The growth pattern of heterospecific pollen tubes at late pollination was similar to that of conspecific pollen tubes at peak pollination.
• Heterospecific and conspecific pollen tubes took different routes to fertilize ovules. A delayed entry of heterospecific pollen into ovules may be a novel mechanism of conspecific pollen advantage (CPA) for apocarpous species.