Effects of flower size and number on pollinator visitation to wild radish,Raphanus raphanistrum

Plant traits that increase pollinator visitation should be under strong selection. However, few studies have demonstrated a causal link between natural variation in attractive traits and natural variation in visitation to whole plants. Here we examine the effects of flower number and size on visitation to wild radish by two taxa of pollinators over 3 years, using a combination of multiple regression and experimental reductions in both traits. We found strong, consistent evidence that increases in both flower number and size cause increased visitation by syrphid flies. The results for small bees were harder to interpret, because the multiple regression and experimental manipulation results did not agree. It is likely that increased flower size causes a weak increase in small-bee visitation, but strong relationships between flower number and small-bee visitation seen in 2 years of observational studies were not corroborated by experimental manipulation of this trait. Small bees may actually have responded to an unmeasured trait correlated with flower number, or lower small-bee abundances when the flower number manipulation was conducted may have reduced our ability to detect a causal relationship. We conclude that studies using only 1 year, one method, or measuring only one trait may not provide an adequate understanding of the effects of plant traits on pollinator attraction.     

Sources of phenotypic variation in floral traits in wild radish, Raphanus raphanistrum (Brassicaceae)

Pollinator-mediated natural selection has been shown to act on phenotypic variation in floral morphology, and this variation has often been demonstrated to be heritable, but few details are available concerning the sources of floral variation. We examined phenotypic variation in seven floral traits in wild radish (Raphanus raphanistrum) at six levels: between two populations grown in a common garden, among plants within populations, among flowers measured on different weeks, between flowers on two flowering stalks measured on the same day, between adjacent flowers on a flowering stalk, and within individual flowers. There were no significant differences between plants derived from the two source populations, which were ～800 km apart. Most of the variance was within individual plants; repeatabilities were all <0.35. There were highly significant differences between flowers measured in different weeks and also highly significant plant by week interactions, indicating that the among-plant variation was not consistent over time. There was substantial variance among adjacent flowers on the same stalk, particularly in the gynoecium. This high within-plant variance is partly responsible for the low heritability of floral traits in the field and the weak selection on floral traits found in previous studies of wild radish.     

Heritable variation in stomatal responses to elevated CO2 in wild radish, Raphanus raphanistrum (Brassicaceae)

Rising atmospheric carbon dioxide may affect plant populations in the short term through effects on photosynthesis and carbon allocation, and over the long term as an agent of natural selection. To test for heritable effects of elevated CO2 on stomatal responses and plant fecundity in Raphanus raphanistrum, we grew plants from 12 paternal families in outdoor open-top chambers at ambient (35 Pa) or elevated (67 Pa) CO2. Contrary to results from a previous study of this species, total flower and fruit production were marginally lower under elevated CO2. Across families, stomatal index and guard cell length showed little response to CO2 enrichment, but these characters varied significantly among paternal families in both the direction and magnitude of their response to changing CO2. Although these family-by-CO2 interactions suggest that natural selection might affect stomatal characters when ambient CO2 levels increase, we found no significant correlation between either character and flower or fruit production. Therefore, our data suggest that while heritable variation for stomatal index and guard cell length exists in this population, selection due to increasing CO2 is not likely to act on these traits because they had no detectable effect on lifetime fecundity.     

Pollination thoroughness and maternal yield regulation in wild radish,Raphanus raphanistrum (Brassicaceae)

Summary Under conditions where resources are limited, there are often negative correlations between components of maternal yield, or between fruit and flower production. Pollination, in turn, may vary among individuals and influence total maternal expenditure. We examined the impact of variation in pollination thoroughness upon yield components and overall plant growth in wild radish (R. raphanistrum) plants grown in the greenhouse. Plants received different pollination treatments in which 0% to 100% of all flowers produced were hand-pollinated. Fruit set was increased by hand-pollination, but rarely exceeded 30%, even when more than 50% of the flowers were pollinated. Plants receiving more thorough pollination or having greater proportion fruit set produced significantly smaller seeds. Seed number per fruit was not influenced by pollination treatment. Mean values of yield components and interactions between components often varied among plants from different maternal families. Increasing pollination thoroughness also resulted in dramatic decreases in flower production. If male fitness is related to flower number, there may be a tradeoff between maternal fecundity and successful pollen export operating at the whole-plant level in this species.     

Patterns of genetic variability within and among populations of wild radish,Raphanus raphanistrum (Brassicaceae)

The genetic structure of populations is an important determinant of the evolutionary potential of a species. Colonizing plants tend to be characterized by low within- and high among-population variability. Genetic differentiation of both floral traits and isozymes was studied in six populations of wild radish (Raphanus raphanistrum). Evidence for differentiation in both sets of traits was found, but patterns of differentiation of floral traits did not coincide with isozyme differentiation. Contrary to most colonizing species, wild radish showed high within- and only moderate among-population variability at isozyme loci. In addition, levels of differentiation did not correspond to geographic distance between the populations. These results are likely due at least in part to the self-incompatibility system of this species, long-distance movement of large numbers of wild radish seeds by humans, and introgression from cultivated radish (R. sativus).     

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.     

Pollinator preferences and the persistence of crop genes in wild radish populations (Raphanus raphanistrum, Brassicaceae)

Crop-weed hybridization can potentially influence the evolutionary ecology of wild populations. Many crops are known to hybridize with wild relatives, but few studies have looked at the long-term persistence of crop genes in the wild. This study investigated one factor in the hybridization process in radish: differential pollinator visitation to wild radish (Raphanus raphanistrum) vs. crop-wild F1 hybrids (R. sativus x R. raphanistrum). Wild genotypes had yellow flowers, a recessive single-locus trait, whereas hybrids always had white or pale pink flowers. In experimental arrays in northern Michigan, total pollinator visitation was significantly biased toward wild plants when the frequencies of wild and hybrid plants were equal. Syrphid flies, the most frequent visitors, preferred wild plants while bumble bees showed no preference. This pattern was also observed when hybrid plants were overrepresented in the array (12 hybrid:2 wild). In contrast, when hybrid plants were rare (2 hybrid:12 wild), neither morph was preferred by any pollinator group. Later in the summer, pollinators were also observed in a large experimental garden with nearly equal frequencies of wild and hybrid plants. Cabbage butterflies (Pieris rapae) strongly overvisited wild plants, while bumble bees showed a slight preference for hybrids. Taken together, these studies suggest that F1 hybrids may not be at a disadvantage with regard to pollinator visits when they occur at low frequencies or when bumble bees are frequent flower visitors. Thus, variation in the proportion of white-flowered morphs among wild radish populations could be influenced by different histories of crop-to-wild hybridization, as well as by variation in the composition of local pollinator taxa.     

Genotype-specific effects of elevated CO2 on fecundity in wild radish (Raphanus raphanistrum)

Rising atmospheric CO₂ may lead to natural selection for genotypes that exhibit greater fitness under these conditions. The potential for such evolutionary change will depend on the extent of within-population genetic variation in CO₂ responses of wild species. We tested for heritable variation in CO₂-dependent life history responses in a weedy, cosmopolitan annual, Raphanus raphanistrum. Progeny from five paternal families were grown at ambient and twice ambient CO₂ using outdoor open-top chambers (160 plants per CO₂ treatment). Elevated CO₂ stimulated net assimilation rates, especially in plants that had begun flowering. Across paternal families, elevated CO₂ led to significant increases in flower and seed production (by 22% and 13% respectively), but no effect was seen on time to bolting, leaf area at bolting, fruit set, or number of seeds per fruit. Paternal families differed in their response to the CO₂ treatment: in three families there were no significant CO₂ effects, while in one family lifetime fecundity increased by >50%. These genotype-specific effects altered fitness rankings among the five paternal families. Although we did not detect a significant genotype x CO₂ interaction, our results provide evidence for heritable responses to elevated CO₂. In a subset of plants, we found that the magnitude of CO₂ effects on fecundity was also influenced by soil fertility.     

Visitation, effectiveness, and efficiency of 15 genera of visitors to wild radish, Raphanus raphanistrum (Brassicaceae)

Plant-pollinator interactions are one of the most important and variable mutualisms in nature. Multiple pollinators often visit plants and can vary in visitation rates, pollen removal and deposition, and spatial and temporal distribution, altering plant reproduction and patterns of pollinator-mediated selection. Although some visitors may not be effective pollinators, pollinator effectiveness is rarely estimated directly as seed set resulting from a single visit by each taxon visiting generalist plants. For two years, effectiveness of visitors to wild radish, Raphanus raphanistrum, was quantified by counting seeds set and pollen grains removed as a result of a single visit. We calculated a pollinator's importance to plant reproduction as the product of visitation rate and single-visit seed set, and regressed pollinator body size on pollen-removal and on seed set effectiveness. Although pollinators differed in effectiveness and visitation rates, pollinator importance was primarily determined by visitation rates. In contrast to similar 2-yr studies, pollinator assemblage composition varied little, suggesting pollinator-mediated selection can be consistent across years for this generalist. Larger pollinators were more effective than smaller at effecting seed set, but body size was a poor predictor of pollen removal ability. Instead, pollen-removal effectiveness may be more influenced by foraging behavior than size.     

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.     

Self-pollen interference is absent in wild radish (Raphanus raphanistrum, Brassicaceae), a species with sporophytic self-incompatibility

Explaining the diversity of mating systems and floral forms in flowering plants is a long-standing concern of evolutionary biologists. One topic of interest is the conditions under which self-pollination can interfere with seed set for flowering plants with a self-incompatibility system. We investigated the effect of self-pollen interference for wild radish, Raphanus raphanistrum, which has sporophytic self-incompatibility. We performed pollinations and determined seed set for plants grown in the greenhouse, using pollen mixtures representing either self- with outcross-pollen or outcross-pollen alone. Stigmas were collected for a subset of pollinated flowers to determine the number of pollen grains applied. Average seed set for the self/cross (5.13 seeds/pollination) and cross treatments (5.09 seeds/pollination) did not differ significantly. Stigmatic pollen loads averaged around 700 grains, an amount close to observed natural pollen loads on R. raphanistrum. We concluded that for R. raphanistrum in natural populations, self-pollen is unlikely to interfere with outcross-pollen success. This study is the first to investigate effects of self-pollen interference on seed set for a homomorphic species with sporophytic self-incompatibility where rejection occurs at the stigmatic surface.     

Adaptive differentiation of quantitative traits in the globally distributed weed, wild radish (Raphanus raphanistrum)

Weedy species with wide geographical distributions may face strong selection to adapt to new environments, which can lead to adaptive genetic differentiation among populations. However, genetic drift, particularly due to founder effects, will also commonly result in differentiation in colonizing species. To test whether selection has contributed to trait divergence, we compared differentiation at eight microsatellite loci (measured as F(ST)) to differentiation of quantitative floral and phenological traits (measured as Q(ST)) of wild radish (Raphanus raphanistrum) across populations from three continents. We sampled eight populations: seven naturalized populations and one from its native range. By comparing estimates of Q(ST) and F(ST), we found that petal size was the only floral trait that may have diverged more than expected due to drift alone, but inflorescence height, flowering time, and rosette formation have greatly diverged between the native and nonnative populations. Our results suggest the loss of a rosette and the evolution of early flowering time may have been the key adaptations enabling wild radish to become a major agricultural weed. Floral adaptation to different pollinators does not seem to have been as necessary for the success of wild radish in new environments.     

Tests for major genes affecting quantitative traits in wild radish,Raphanus raphanistrum

The number of gene loci coding for quantitative traits is an important issue in genetics. However, there are still very few empirical data on this point, especially in natural populations. I tested for major gene effects on ten quantitative traits in wild radish, using an indirect method based on the patterns of family means and within and between family variances for traits. This method should reveal whether a single locus is responsible for most of the variation in a trait. Eight of the traits measured were morphological dimensions of leaves and flowers; no strong evidence for major gene effects on these traits was found. In contrast, evidence for major gene effects was found in the other two traits, emergence time and flowering time.     

Leaf damage by herbivores affects attractiveness to pollinators in wild radish, Raphanus raphanistrum

 We carried out two experiments to determine the effect of leaf damage on plant attractiveness to pollinators using wild radish, Raphanus raphanistrum (Brassicaceae), a self-incompatible annual herb. Pairs of plants from 36 full-sib families were grown in pots in the greenhouse. One member of each pair was damaged by Pieris rapae larvae that were allowed to remove half of the leaf area of each of the first four rosette leaves. The plants were subsequently taken out for pollinator observations once a week from the beginning of flowering in late June until the end of August. We conducted two experiments to examine how foliar damage affected visitation by pollinators. In the first experiment, numbers of pollinators visiting plants were compared between damaged and control sibling plants. In the second experiment, the number of open flowers during observations was controlled to be the same for both damaged and undamaged sibs. Damage significantly decreased the number and size of flowers during the first observations in late June. Damaged plants received fewer visits by native bees during the first week of observations. Since damage did not affect native bee visits when the number of open flowers was equalized between treatments, flower number was probably the main cue attracting native bees to plants. In the experiment without flower number control, syrphid flies, the other abundant pollinator taxon, spent more time per flower on the undamaged than on the damaged plants. When flower number was controlled, flies probed significantly more flowers during each visit on the undamaged than on the damaged plants and had higher visitation rates to undamaged plants early in the season. Since syrphid flies preferred undamaged plants both with and without flower number control, they apparently used cues apart from flower number for visitation. The difference between undamaged and damaged plants in floral characteristics and pollinator visitation vanished within a few weeks after the start of flowering. This result suggests that early damage may not have a strong fitness effect through reduction in mating success. However, poor weather conditions can cause early mortality of plants in the field, and nutrient depletion and competition decrease fruit set of later flowers. Therefore, conditions exist under which visitation to early flowers may affect plant fitness. Received: 30 July 1996 / Accepted: 10 February 1997     

Fitness effects of mutation accumulation in a natural outbred population of wild radish (Raphanus raphanistrum): comparison of field and greenhouse environments

Spontaneous deleterious mutation has been measured in a handful of organisms, always under laboratory conditions and usually employing inbred species or genotypes. We report the results of a mutation accumulation experiment with an outbred annual plant, Raphanus raphanistrum, with lifetime fitness measured in both the field and the greenhouse. This is the first study to report the effects of spontaneous mutation measured under field conditions. Two large replicate populations (N(e) approximately 600) were maintained with random mating in the greenhouse under relaxed selection for nine generations before the field assay was performed and ten generations before the greenhouse assay. Each generation, every individual was mated twice, once as a pollen donor and once as a pollen recipient, and a single seed from each plant was chosen randomly to create the next generation. The ancestral population was maintained as seeds at 4 degrees C. Declines in lifetime fitness were observed in both the field (1.7% per generation; P= 0.27) and the greenhouse (0.6% per generation; P= 0.07). Significant increases in additive genetic variance for fitness were found for stems per day, flowers per stem, fruits per flower and seeds per fruit in the field as well as for fruits per flower in the greenhouse. Lack of significance of the fitness decline may be due to the short period of mutation accumulation, the use of outbred populations, or both. The percent declines in fitness are at the high end of the range observed in other mutation accumulation experiments and give some support to the idea that mutational effects may be magnified under harsher field conditions. Thus, measurement of mutational parameters under laboratory conditions may underestimate the effects of mutations in natural populations.     

Fitness of backcross six of hybrids between transgenic oilseed rape (Brassica napus) and wild radish (Raphanus raphanistrum)

The process of introgression between a transgenic crop modified for better agronomic characters and a wild relative could lead potentially to increased weediness and adaptation to the environment of the wild species. However, the formation of hybrid and hybrid progeny could be associated with functional imbalance and low fitness, which reduces the risk of gene escape and establishment of the wild species in the field. Our work compares the fitness components of parents and different types of backcross in the sixth generation of hybrids between transgenic oilseed rape (Brassica napus, AACC, 2n = 38) resistant to the herbicide glufosinate and wild radish (Raphanus raphanistrum, RrRr, 2n = 18). The backcross with oilseed rape cytoplasm (OBC) has a fitness value 100 times lower than that of the backcross with wild radish cytoplasm (RBC). The herbicide-resistant RBC has similar growth to the susceptible RBC, but final male and female fitness values are two times lower. In turn, susceptible RBC exhibit similar fitness to the control wild radishes. The relative fitnesses of the different types are the same whether or not they grow under competitive conditions. The consequence on fitness of the chromosome location of the transgene conferring resistance and the relevance of these results to the impact of gene flow on the environment are discussed.     

Within-population spatial variation in pollinator visitation rates,pollen limitation on seed set,and flower longevity in an alpine species

Pollen limitation through insufficient pollen deposition on stigmas caused by too infrequent pollinator visitation may influence the reproductive outcome of plants. In this study we investigated how pollinator visitation rate, the degree of pollen limitation, and flower longevity varied spatially among three sites at different altitudes within a population of the dwarf shrub Dryas octopetala L. in alpine southern Norway. Significant pollen limitation on seed set only occurred at the mid-elevation site, while seed set at the other sites appeared to be mainly resource limited, thus indicating a spatial variation in pollen limitation. There was no association between the spatial variation in the extent of pollen limitation and pollinator visitation rate to flowers. However, pollinator visitation rates were related to flower longevity of Dryas; sites with low visitation rates had long-lived flowers and vice versa. Thus, our results suggest within-population spatial co-variation between pollinator visitation rates, pollen limitation, and a developmental response to these factors, flower longevity.     

Within-population spatial variation in pollinator visitation rates, pollen limitation on seed set, and flower longevity in an alpine species

Pollen limitation through insufficient pollen deposition on stigmas caused by too infrequent pollinator visitation may influence the reproductive outcome of plants. In this study we investigated how pollinator visitation rate, the degree of pollen limitation, and flower longevity varied spatially among three sites at different altitudes within a population of the dwarf shrub Dryas octopetala L. in alpine southern Norway. Significant pollen limitation on seed set only occurred at the mid-elevation site, while seed set at the other sites appeared to be mainly resource limited, thus indicating a spatial variation in pollen limitation. There was no association between the spatial variation in the extent of pollen limitation and pollinator visitation rate to flowers. However, pollinator visitation rates were related to flower longevity of Dryas; sites with low visitation rates had long-lived flowers and vice versa. Thus, our results suggest within-population spatial co-variation between pollinator visitation rates, pollen limitation, and a developmental response to these factors, flower longevity.     

The role of the persistent fruit wall in seed water regulation in Raphanus raphanistrum (Brassicaceae)

Background and Aims

Dry fruits remain around the seeds at dispersal in a number of species, especially the Brassicaceae. Explanations for this vary, but usually involve mechanisms of innate dormancy. We speculate that, instead, a persistent fruit may give additional protection through control of dehydration, to species growing in arid or Mediterranean environments where water is sporadic.

Methods

X-rays and weight measurements were used to determine the extent to which Raphanus raphanistrum seeds within mature fruits imbibe water, and germination tests determined the roles of the fruit and seed coat in seed dormancy. Rates of water uptake and desiccation, and seedling emergence were compared with and without the fruit. Finally, germinability of seeds extracted from fruits was determined after various periods of moist conditions followed by a range of dry conditions.

Key Results

Most seeds rapidly take up water within the fruit, but they do not fully imbibe when compared with naked seeds. The seed coat is more important than the dry fruit wall in maintaining seed dormancy. The presence of a dry fruit slows emergence from the soil by up to 6–8 weeks. The fruit slows the rate of desiccation of the seed to a limited extent. The presence of the fruit for a few days during imbibition somehow primes more seeds to germinate than if the fruit is absent; longer moist periods within the pod appear to induce dormancy.

Conclusions

The fruit certainly modifies the seed environment as external conditions change between wet and dry, but not to a great extent. The major role seems to be: (a) the physical restriction of imbibition and germination; and (b) the release and then re-imposition of dormancy within the seed. The ecological significance of the results requires more research under field conditions.     

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