Effects of pollen load size and composition on pollen donor performance in wild radish, Raphanus sativus (Brassicaceae)

A critical concern in the debate over the importance of sexual selection in plants is whether the nonrandom mating demonstrable in greenhouse crosses can occur in the field. Field populations likely experience smaller and more variable pollen load sizes than those that have been used in many greenhouse experiments. Therefore, we performed a greenhouse experiment in which we varied both pollen load size and composition in wild radish, Raphanus sativus, and examined the paternity of seeds. We used five maternal plants and four pairs of pollen donors. We were able to produce pollen loads of 40, 118, and 258 grains per stigma. The smallest of the pollen loads was scant enough to result in a slight, but significant reduction in seed number per fruit. While variation in pollen load composition significantly affected the proportions of seeds fathered by different donors, variation in pollen load size did not. The relative performance of different donors was constant across pollen load sizes, suggesting that, for this species, differential performance of pollen donors can occur at pollen load sizes that are likely to occur in field populations.     

Do differences in plant and flower age change mating patterns and alter offspring fitness in Raphanus sativus (Brassicaceae)?

When more pollen is present on stigmas than needed to fertilize all ovules, selection among pollen grains may occur due to effects of both pollen donors and maternal plants. We asked whether increasing plant age and flower age, two changes in maternal condition, altered the pattern of seed paternity after mixed pollination. We also asked whether changes in seed paternity affected offspring success in an experimental garden. While flower age did not affect seed paternity, there was a dramatic shift in pollen donor performance as plants aged. These differences were seen in the offspring as well, where the offspring of one pollen donor, which sired more seeds on young plants, flowered earlier in the season, and the offspring of another pollen donor, which sired more seeds on old plants, flowered later in the season. Thus, change in maternal condition resulted in altered seed paternity, perhaps because the environment for pollen tube growth was different. The pattern of seed paternity and offspring performance suggests that pollen donors may show temporal specialization.     

How robust is nonrandom mating in wild radish: do small pollen loads coupled with more competing pollen donors lead to random mating?

In previous studies of the weedy annual Raphanus sativus we have demonstrated that mating is nonrandom in greenhouse plants, suggesting that sexual selection is possible. To investigate how these greenhouse results might translate to conditions more similar to the field, we manipulated both pollen load size and the number of competing pollen donors on stigmas. While the smallest pollen loads (22 grains per stigma) were small enough to reduce fruit and seed set, seed siring success was unaffected by pollen load size. When the number of competing donors in a mixed pollination was increased to four, the proportion of seeds sired by the pollen donors was the furthest from expectation, suggesting that nonrandom mating increases as the number of donors per pollination increases. There was no significant interaction between pollen load size and number of competitors per pollination. Overall, mating remained nonrandom across all treatments. Thus differential seed paternity is likely to occur in the field as well as in the greenhouse.     

The effects of pollen load size and donor diversity on pollen performance, selective abortion, and progeny vigor in Mirabilis jalapa

The influence of pollen competitive environment on pollen performance (pollen germination, stigmatic penetration, and pollen tube growth rate), the maturation or abortion of initiated fruit, seed size, and seedling vigor was explored by manipulating the size and diversity of stigmatic pollen loads on Mirabilis jalapa. All aspects of pollen performance significantly increased with the number of pollen grains on a stigma or pollen tubes in a style, but was not influenced by the diversity of pollen donors. Plants tended to mature single-ovulate fruits that came from flowers where pollen load size and diversity were greatest and aborted those where these were lowest. No plants from seeds resulting from pollinations with a single pollen grain survived, but other fitness measures were mostly determined by maternal plant. The data suggest that pollen performance is influenced by pollen competitive environment, and both the genetic diversity of the pollen load and number of competing pollen tubes are important determinants of seed/fruit abortion.     

Mechanisms of differential pollen donor performance in wild radish, Raphanus sativus (Brassicaceae)

In order to understand the characters on which sexual selection might operate in plants, it is critical to assess the mechanisms by which pollen competition and mate choice occur. To address this issue we measured a number of postpollination characters, ranging from pollen germination and pollen tube growth to final seed paternity, in wild radish. Crosses were performed using four pollen donors on a total of 16 maternal plants (four each from four families). Maternal plants were grown under two watering treatments to evaluate the effects of maternal tissue on the process of mating. The four pollen donors differed significantly in number of seeds sired and differed overall in the mating characters measured. However, it was difficult to associate particular mechanistic characters with ability to sire seeds, perhaps because of interactions among pollen donors within styles or among pollen donors and maternal plants. The process of pollen tube growth and fertilization differed substantially among maternal watering treatments, with many early events occurring more quickly in stressed plants. Seed paternity, however, was somewhat more even among pollen donors used on stressed maternal plants, suggesting that when maternal tissue is more competent, mating is slowed and is more selective.     

Differential siring success of Pgi genotypes in Clarkia Unguiculata (Onagraceae)

Competition among pollen grains for the fertilization of ovules can play an important role in determining the male and female reproductive success of flowering plants. To examine the influence of pollen-donor genotype on male reproductive success, hand-pollinations were conducted on Clarkia unguiculata and the siring success of pollen-donor plants was compared between donors homozygous for different allelomorphs of the allozyme PGI (phosphoglucoisomerase). Donors homozygous for the B allele sired more seeds than C-allele donors. Single-donor crosses indicated that C-donor-sired seeds are aborted more often than are B-donor-sired seeds, suggesting that the B-allele donor's advantage in mixed pollinations was a result of differential abortion. A negative relationship between pollen load and the siring success of B-allele donors implies that pollen from B-allele donors has reduced performance relative to C-allele donors when pollen loads are high. These data demonstrate consistent differences in siring success between individuals homozygous for different alleles at a single locus and suggest that variation at the Pgi locus may be maintained by a post-pollination trade-off.     

POSTPOLLINATION EFFECTS ON SEED PATERNITY: MECHANISMS IN ADDITION TO MICROGAMETOPHYTE COMPETITION OPERATE IN WILD RADISH

After pollen arrives on a stigma, the paternity of seeds may be influenced by microgametophyte competition, maternal choice, genetic complementation between parents, and embryo competition. While microgametophyte competition has been well accepted, the other mechanisms are more difficult to demonstrate, and their effects are often confounded. Here, wild radish plants were pollinated with single and mixed pollen loads, and some plants were stressed such that reproduction was reduced. Effects of pollen donors, maternal families, maternal × paternal interaction, pollen donor number, and stress on fruit abortion, seed number per fruit, seed weight, and total seed weight per fruit were measured. Maternal-plant × pollen-donor interaction effects were found for all variables, indicating that genetic complementation or maternal choice occurred. Values of the components of reproduction were generally higher for multiply sired fruits than for singly sired fruits, indicating that either competition among embryos changed under multiple paternity or maternal choice for multiply sired fruits occurred. Finally, when maternal plants were stressed, the components of reproduction were more strongly affected by seed and fruit paternity. This result indicates that either competitive regimes among embryos were affected by stress or maternal plants become more selective under stress. In both cases where embryo competition might have been an explanation of the results, variation in seed weight within fruits was unaffected, suggesting that competitive regimes were unchanged. Clearly, mechanisms in addition to microgametophyte competition are important in sorting the pollen that arrives on stigmas of wild radish. These data suggest that maternal choice is likely to be important. In addition, these processes are likely to occur in the field, since the effects are stronger in stressed than in control plants.     

DIFFERENTIAL SUCCESS OF POLLEN DONORS IN A SELF-COMPATIBLE LILY

If pollen donors are equally effective at siring seeds, the presence of equal proportions of pollen from two pollen donors on a stigma will lead to equal proportions of seeds sired by each pollen donor. Variation in germination rates, pollen-tube growth, and embryo viability may cause one donor to sire more seed than another. We looked for differential donor success in the field by simultaneously applying equal amounts of pollen from two pollen donors. We simultaneously applied equal amounts of self and outcross pollen to receptive stigmas and simultaneously applied pollen from two donors at different physical distances from the recipient. Following simultaneous application of self and outcross pollen, significantly more of the seeds were sired by outcross pollen donors. Seed set following simultaneous application of two outcross donors was also nonrandom. Pollen donors from 100 m were more likely to sire seeds when competing with pollen from plants nearby (1 m). To determine whether pollen-tube growth rates were responsible for these patterns of paternity, we varied the timing of deposition of outcross pollen allowing self pollen tubes a head start on the stigma. Outcross pollen was applied 3 or 24 h after self pollen. In spite of this time delay, the majority of the seeds were again sired by outcross pollen. There was no significant difference in the amount of seeds sired by self pollen between the two delay treatments. This result suggests that mechanisms operating after ovule fertilization may contribute to the discordance between the proportions of the pollen present and the proportions of seeds sired.     

Effects of sequential pollination on the success of "fast" and "slow" pollen donors in Hibiscus moscheutos (Malvaceae)

Competition among pollen grains for the chance to fertilize ovules typically involves two stages: arrival times on stigmas and/or the growth of pollen tubes through styles. In a previous study of Hibiscus moscheutos, we found that individual pollen donors often differed in pollen tube competitive ability. Here we determined whether short delays in pollen arrival time altered the average success of "fast" and "slow" pollen donors when both types of pollen experienced the same delays. Hand-pollination experiments were carried out using four pairs of pollen donors that differed in competitive ability. We allowed delays of 15 or 30 min between the first and second pollen donor and then determined seed paternity using allozyme markers. The second donor typically sired fewer seeds than pollen that arrived earlier, but, contrary to expectation, "faster" pollen did not always sire significantly more seeds than "slower" pollen when each was applied after delays of the same duration. In two of the four pairs of donors, differences that were seen following simultaneous pollinations disappeared when each type of pollen was applied following identical delays of 15 or 30 min. This unexpected response suggests that the dynamics of pollen tube competition are more complex than anticipated.     

DIFFERENTIAL POLLEN-TUBE GROWTH RATES AND NONRANDOM FERTILIZATION IN HIBISCUS MOSCHEUTOS (MALVACEAE)

The prevalence of nonrandom fertilization due to postpollination events has rarely been studied in natural populations, despite important implications for outcrossing rates, mate choice, and plant fitness. Nonrandom paternity within fruits can be caused by both unequal fertilization and unequal embryo abortion. Using self-compatible Hibiscus moscheutos, we studied the potential for nonrandom fertilization by comparing growth rates of pollen-tubes from different donors. The branched style of Hibiscus allowed within-flower comparisons between pollen donors. Relative pollen-tube growth rates were determined by applying pollen from pairs of donors to different stigmas on adjacent stylar branches. We then measured the number of callose plugs per tube in cross-sectional transects across the style after 3 hr. We demonstrate that rates of callose plug formation can be used as a sensitive indicator of relative pollen-tube growth rate. Differences between pollen donors were common and repeatable. Self-pollen-tubes grew slower than outcross pollen-tubes in some crosses and faster in others. Allozyme variation in glucose phosphate isomerase was used to show that individuals with fast-growing pollen-tubes sired a disproportionate number of seeds following mixed pollinations (up to 72%). Since seed abortion was negligible, we conclude that variation in pollen-tube growth rates leads to nonrandom paternity within fruits.     

EFFECTS OF POLLEN DONOR IDENTITY ON OFFSPRING QUALITY IN WILD RADISH,RAPHANUS SATIVUS

If pollen donor performance during mating correlates with differences in offspring growth and fitness, processes that sort among potential mates may directly improve offspring fitness. Here seeds sired by three pollen donors on ten maternal plants were grown for eight weeks in the greenhouse. The performance of the pollen donors during pollination and fertilization was known from a previous experiment. There were significant effects of paternity on two measures of early growth: leaf number and plant height. Paternal effects on three measures more closely related to fitness; final plant weight, day of first flower production, and total flower number were also significant. Under the conditions of this experiment, final plant weight was probably the best predictor of fitness. The pollen donor that sired the largest seeds in the previous experiment sired offspring that were largest after 8 weeks of growth. Half of the plants were grown under low-water conditions. Paternal effects on growth were not masked by the environmental effects. In fact, some paternal effects became stronger under stress. This suggests that paternal effects could also be important in the field. Plants sired by donor A bolted very early when water was limited and would probably have an advantage in a season that was very short due to an early and severe drought. During fertilization and seed filling, seeds sired by this donor were more frequent on water-stressed maternal plants than on control maternal plants (Marshall, 1988). The data from this experiment indicate a connection between pollen donor performance during mating and offspring growth. These results suggest that the processes that sort among potential fathers during pollination, fertilization, and seed filling, may improve offspring quality.     

Under how wide a set of conditions will nonrandom mating occur in Raphanus sativus (Brassicaceae)?

Studies of the weedy annual Raphanus sativus have demonstrated that nonrandom mating, a prerequisite for sexual selection, can occur in greenhouse plants. To determine whether this nonrandom mating pattern can occur under a wide range of conditions, including conditions that might occur in the field, we considered variation in both maternal condition and pollen load size. Maternal condition was varied by altering the watering regime. Pollen load size was varied from approximately 26 to 343 pollen grains per stigma. At the smallest pollen load size, patterns of seed paternity were altered in two of the three pollen donor pairs; seed paternity became more equal among donors. For one of three pollen donor pairs, seed paternity was more divergent among donors on stressed maternal plants. Finally, for one pollen donor pair, rank order of pollen donor performance changed from the medium to the small pollen loads on stressed vs. control maternal plants. Thus, some field conditions may alter patterns of nonrandom mating in wild radish.     

Pollen load size, reproductive success, and progeny kinship of naturally pollinated flowers of the tropical dry forest tree Pachira quinata (Bombacaceae)

Several studies have demonstrated, using controlled pollinations, that the number and identity of pollen grains deposited onto a flower's stigma affect the reproductive success of plants. However, few studies have shown this relationship under conditions of natural pollination. Using the tropical dry forest tree Pachira quinata, we evaluated the relationship between the number of microgametophytes per pistil and the number of sires with respect to the production of fruits and seeds in a natural population of Pachira quinata. Our study demonstrates that fruit and seed production are directly related to the number of microgametophytes per pistil in natural populations of P. quinata. Only 6% of the marked flowers developed into mature fruits and 10% of the marked flowers initiated fruits but later aborted them. A mean of 23 pollen grains were required to produce a seed. Flowers with >400 pollen grains on the stigma always developed into mature fruits, whereas flowers that received <200 grains never matured fruits. Half of the pollen grains transferred to a flower stigma germinated and developed pollen tubes to the base of the style. The number of pollen grains on a stigma explained 34% of the variation in seed number per fruit, and the number of seeds produced per fruit is positively correlated with the size of the seeds. The population of P. quinata studied is predominantly outcrossing, and seeds within fruits are sired by one or a few donors. The total seed crop within trees was sired by three to five donors. Our study examined the implications of the variation in size of microgametophyte loads per pistil with respect to the breeding system and the paternity of progeny under natural conditions. The competitive ability of pollen and pollen tube attrition are important factors regulating fruit production in P. quinata.     

Seed paternity in flowering plants: an evolutionary perspective

The ultimate importance of paternal contributions to fitness and of post-pollination selection in flowering plants have remained elusive, largely because of the technical difficulty of assigning paternity. I review empirical studies that use heritable markers to investigate per-fruit seed paternity in natural populations and after experimental multiple-donor pollination. Thirty-one studies covering 23 species from 16 plant families document that in natural populations seeds from a single fruit are often fathered by multiple pollen donors (5 species from 5 families), that donors can differ significantly in seed-siring success (8 species from 6 families), that variation in pollen tube growth rates can be heritable (n = 1 out of 4 studies), that donor and recipient genotypes can simultaneously affect paternity (n = 2), and that temporal order of pollen deposition (n = 1) and environmental effects(n = 2) affect the outcome of pollen competition. These studies also investigate the role of post-pollination selection in the avoidance of inbreeding and for species boundaries. Most studies of male reproductive success in plants to date base on isozyme electrophoresis. The availability in the last decade of highly polymorphic molecular markers such as microsatellite DNA has been expected to open new possibilities to investigate competition and selection during the gametophytic phase. Yet, to date, there is still need for greater data wealth on seed paternity to test theories of sex allocation and to gain deeper understanding of floral trait evolution and of the evolutionary consequences of post-pollination selection in flowering plants.     

POLLEN DISPERSAL DYNAMICS IN AN ALPINE WILDFLOWER,POLEMONIUM VISCOSUM

The alpine wildflower, Polemonium viscosum, depends on insect visitors for effective pollination. Here, I examine experimentally the effects of pollinator visitation on pollen removal, pollen dispersal success, paternity, and gene flow. Bumble bee pollinators visited donor individuals homozygous for marker alleles at an isozyme (GOT-2) encoding locus and then were presented with arrays of recipient plants lacking the marker alleles. Four aspects of male fitness were estimated for each donor: the number of pollen grains dispersed to flowers of the first recipient visited, the number of offspring sired on that recipient, the proportion of offspring sired in the full array, and the proportion of mates in the array bearing seeds of the donor. Pollen removal was strongly influenced by the number of bee visits to donor flowers. The amount of pollen removed in turn significantly affected the number of pollen grains reaching flowers of the first recipient. However, because seed production decelerates with stigma pollen load, the relationship between pollen export and paternal success at this proximate scale showed diminishing returns. The probability of reaching mates within the array also increased with pollen export. These findings show that floral characters enhancing pollinator visitation rate in P. viscosum have positive effects on paternity and gene flow.     

Effects of soil phosphorus on pollen production,pollen size,pollen phosphorus content,and the ability to sire seeds in Cucurbita pepo (Cucurbitaceae)

To determine the effects of soil phosphorus on pollen production, pollen grain size, phosphate concentration per pollen grain, and the siring ability of pollen, two cultivars of the common zucchini (Cucurbita pepo) were grown under two soil phosphorus conditions in an experimental garden. Overall, soil phosphorus availability had a significant effect on reproductive output through the female function and on traits affecting the male function of plants (staminate flower production, pollen production per flower, and pollen grain size). In addition, pollen produced by plants in the high phosphorus soils had a higher phosphate concentration than pollen produced by plants in the low phosphorus soils. A pollen mixture experiment revealed that pollen produced by plants in the high phosphorus treatment sired significantly more seeds than pollen produced by plants in the low phosphorus treatment. This study showed that growing conditions such as soil phosphorus can influence the size of a pollen grain and its chemical composition, which, in turn, can affect its ability to sire mature seeds.     

Seed germination patterns of Salvia mellifera in fire-prone environments

Summary I examined the effects of pollen loads containing pollen from one, three and five donors on fruit production and fruit quality in Campsis radicans. Number of pollen donors had no significant effect on % fruit production, seed number, seed weight or seed germination. In singledonor pollinations the identity of the donor did have a strong effect on the above parameters. Furthermore, the best single donor sired fruits with more seeds and heavier seeds than any mixture containing this donor. This pattern indicates interference of pollens or preemption of some ovules by the inferior pollen. In Campsis, therefore, the number of pollen donors contributing to a pollen load is less important than the identity of these donors in determining fruit production and fruit quality. Seeds from fruits resulting from mixed pollination were slightly more variable than seeds from fruits resulting from single-donor pollinations.     

Effect of pollen load size and source (self, outcross) on seed and fruit production in highbush blueberry cv. 'Bluecrop' (VACCINIUM CORYMBOSUM; Ericaceae)

Reproductive fitness of a plant is ultimately determined by both number and quality of seed offspring. This is determined by sexual selection of pollen microspores and ovules during pollination and fertilization. These processes may include pollen competition and seed abortion, which reduce the number of microspores and ovules available for final seed production. Thus, even an excess of pollen microspores to ovules does not result in fertile seeds equal to ovule number. We investigated pollen requirements of highbush blueberry (Vaccinium corymbosum cultivar 'Bluecrop') for maximal seed production and how fertile seed number translates into fruit quality, since fruit quality would ultimately determine the dispersal of its offspring. We demonstrate that individual blueberry flowers with a mean of 106 ovules reach their maximum fruit set and mass and minimum time to ripen when 125 outcross pollen tetrads pollinate a flower, compared to 10 or 25. Three hundred tetrads resulted in the increase of fertile seeds, but did not result in a further increase of fruit mass or fruit set, or decrease in time to ripen. We also examined the effect of pure and mixed loads of self and outcross pollen (25 and 125 tetrads), and found no differences in fertile seed number, fruit mass, or percentage fruit set when pollen loads were either 25 self or outcross pollen tetrads, although number of days to ripen was significantly shorter by 8 d with 25 outcross tetrads. When the pollen load of 125 tetrads consisted of self or a 50:50 mixture of self and outcross pollen, fruit mass, days to ripen, and percentage fruit set were not different from loads of 125 outcross pollen. In addition, a pollen load of 25 outcross tetrads resulted in fertile seed number and fruit quality in between that of 25 self, and 125 self, 125 mixed, or 125 outcross tetrads. Large, small, and flat seed types were identified, and only large seeds (length = 1.7 mm) were fertile. These results improve our understanding of pollen load size and source requirements of a crop plant and the limits to pollen transfer when translated to fruit growth.     

Does multiple paternity affect seed mass in angiosperms? An experimental test in Dalechampia scandens

Flowers fertilized by multiple fathers may be expected to produce heavier seeds than those fertilized by a single father. However, the adaptive mechanisms leading to such differences remain unclear, and the evidence inconsistent. Here, we first review the different hypotheses predicting an increase in seed mass when multiple paternity occurs. We show that distinguishing between these hypotheses requires information about average seed mass, but also about within‐fruit variance in seed mass, bias in siring success among pollen donors, and whether siring success and seed mass are correlated. We then report the results of an experiment on Dalechampia scandens (Euphorbiaceae), assessing these critical variables in conjunction with a comparison of seed mass resulting from crosses with single vs. multiple pollen donors. Siring success differed among males when competing for fertilization, but average seed mass was not affected by the number of fathers. Furthermore, paternal identity explained only 3.8% of the variance in seed mass, and siring success was not correlated with the mass of the seeds produced. Finally, within‐infructescence variance in seed mass was not affected by the number of fathers. These results suggest that neither differential allocation nor sibling rivalry has any effect on the average mass of seeds in multiply sired fruits in D. scandens. Overall, the limited paternal effects observed in most studies and the possibility of diversification bet hedging among flowers (but not within flowers), suggest that multiple paternity within fruits or infructescence is unlikely to affect seed mass in a large number of angiosperm species.     

DOES INTERFERENCE COMPETITION AMONG POLLEN GRAINS OCCUR IN WILD RADISH?

Interest in the possibility of sexual selection in plants has focused primarily on competition among pollen donors based on the speed of pollen-tube growth. However, when pollen arrives on stigmas, there is the opportunity for both races for access to ovules (exploitation competition) and interference with the germination and growth of pollen from other donors (interference competition). We considered whether this second form of competition might occur among pollen grains of wild radish in two experiments. In the first, interference likely occurred because the amount of pollen germination was less in mixed-donor than in single-donor pollinations. This result was duplicated in a second experiment, which also showed that interference occurred only when pollen grains from different donors were in direct contact with each other. In addition, in the second experiment, the opportunity for interference affected the frequency of seeds sired by different pollen donors. Because pollen loads are often mixed in nature, interference competition among pollen grains may be important in the ecology and evolution of plant reproduction.