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.     

Pollen donor performance can be consistent acrossmaternal plants in wild radish (Raphanus sativus,Brassicaceae): a necessary condition for the action of sexualselection

The possibility that sexual selection has played a role in theevolution of plant reproductive characters remains interesting, butcontroversial. One reason is that clear demonstration of the necessaryconditions for sexual selection is lacking. For sexual selection tooccur, differences in pollen donor performance must be consistent acrossmaternal plants and not be due to mechanisms such asself-incompatibility that produce interactions between maternal plantsand pollen donors. Here, I performed two experiments with wild radish totest whether differences in pollen donor performance are consistentacross maternal plants and not due to subtle effects of theincompatibility system. In the first, all maternal and paternal lineageshad different S-alleles. There were 16 maternal plants, four in each offour lineages and four pollen donors, one in each of four lineages. Rankorder of pollen donor performance, in terms of number of seeds siredafter mixed pollination, was highly consistent across maternal plantsand maternal lineages. In addition, maternal stress treatment had aneffect on mating success of pollen donors, but the effect was subtle anddid not affect the rank order of seeds sired by the four pollen donors.In the second experiment, pollinations were performed on both mature andimmature stigmas. Immature stigmas allowed some self seed set, so theincompatibility system was compromised. There was some nonrandom seedpaternity on both mature and immature stigmas. However, the amount ofnonrandom mating was less on immature stigmas. Taken together, theseexperiments show that the kind of consistent nonrandom mating necessaryfor sexual selection occurs in wild radish, but that the incompatibilitysystem and the mechanisms for sorting among compatible mates may overlapin time of development or in somepathways.     

Bidirectional history of hybridization in California wild radish, Raphanus sativus (Brassicaceae), as revealed by chloroplast DNA

The evolutionary processes that take place in invasive plant populations are not well documented or understood. Interspecific hybridization between cultivated radish (Raphanus sativus) and R. raphanistrum is known to be responsible for the origin of the invasive California wild radish, but little is known about the nature of the hybridization events that produced the hybrid-derived lineage. We analyzed the trnL-rpl32 intergenic region of chloroplast DNA (cpDNA) obtained from 37 cultivated radish individuals from four different cultivars, 53 R. raphanistrum individuals from six European populations and 104 California wild radish individuals from 11 populations covering its entire range throughout the state. We found that cultivated radish and R. raphanistrum shared no cpDNA haplotypes but that they both shared haplotypes with California wild radish, evidence for bidirectional hybridization between the progenitor species in the creation of the California lineage. We also found evidence that multiple cultivars and multiple European source populations contributed to the diversity of cpDNA haplotypes within California. Studies like this will continue to be important for our understanding of the origin of invasive populations and the mechanisms by which they succeed.     

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.     

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.     

The effects of natural variation in pollinator visitation on rates of pollen removal in wild radish,Raphanus raphanistrum (Brassicaceae)

Pollen removal from flowers is an important component of male fitness, but the effects of natural variation in visitation rates on pollen removal are poorly understood. We measured pollen removal over 2 yr in experimental field populations of wild radish, Raphanus raphanistrum. Pollen removal and pollinator visitation over 1-hr periods were measured on previously unvisited flowers. The effects of pollen production and visitation by different insect taxa on pollen removal were determined using multiple regression. Pollen removal rates were extremely high; a median of 84% of pollen produced was removed in 1 hr. Pollen production was far more important than visitation in determining the number of pollen grains removed. Pollen removal increased with increasing numbers of visits by honey bees and small native bees, but increased numbers of syrphid fly visits had no effect. Average visit duration had no effect on pollen removal in 1991, and a marginally negative effect in 1992.     

Evolution of enhanced reproduction in the hybrid-derived invasive, California wild radish (Raphanus sativus)

Evolution is receiving increased attention as a potentially important factor in invasions. For example, hybridization may have stimulated the evolution of invasiveness in several well-known plant pests. However, the mechanism for success of such hybrid-derived lineages remains unknown in the majority of the cases studied. Here we ask whether increased reproductive success (in terms of maternal fitness) has evolved in an invasive lineage with confirmed hybrid ancestry. We compare the relative fitness of the non-native, hybrid-derived California wild radish (Raphanus sativus) to that of its two progenitor species in field experiments at different sites and in different years. We found that California wild radish has high survivorship and produces more fruits per plant and more seeds per plant than either of its progenitors in several environments. Furthermore, populations of California wild radish display a strong genotype-by-environment interaction, indicating that maintenance of genetic and phenotypic diversity between populations may be responsible for the weed’s ability to invade a wide breadth of California habitats. Our results suggest that hybridization may contribute the evolution of enhanced invasiveness and, also, that by limiting the introduction and subsequent hybridization of congeners, we may be able to prevent the evolution of new invasive lineages.     

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).     

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.     

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.     

Allozyme diversity and population structure of Japanese and Korean populations of wild radish, Raphanus sativus var. hortensis f. raphanistroides (Brassicaceae)

Raphanus satiuus var. hortensis f. raphanistroides (wild radish: Brassicaceae) is an insect-pollinated wild plant that grows mainly on beaches in East Asia. Starch gel electrophoresis was used to investigate the allozyme diversity and genetic structure of 25 Japanese and 9 Korean populations of this plant. Although the Korean populations were small, isolated, and patchily distributed, they maintained a high level of genetic diversity; the average percentage of polymorphic loci was 63.1%, the mean number of alleles per locus was 2.27, and the average heterozygosity was 0.278. The corresponding estimates for these parameters in the Japanese populations were 53.3%, 2.26, and 0.278. These estimates are considerably higher than those from species with similar life history and ecological characteristics, but they are lower than those from R. raphanistrum, the wild radish that grows in Europe and the U.S.A. The combination of an insect-pollinated, outcrossing breeding system, large population sizes, gene flow from cultivated radish population, and a propensity for high fecundity may explain the high level of genetic diversity within wild populations.     

Selecting for floral character associations in wild radish,Raphanus sativus L.

ESS models of reproductive allocation have been used extensively to explain patterns of floral diversity in angiosperms. These theoretical explorations assume that proportional allocation to pollen, ovules, and seeds, as well as to secondary features such as showy petals and nectar rewards, can evolve independently within the limits set by total resource availability. In populations of California wild radish, we have shown previously that petal size, a strong determinant of visitation by honey bee pollinators, is positively correlated with both pollen and nectar production, but not with ovule or seed number per flower. These phenotypic associations may reflect selection, environmental correlation, and/or genetic constraint. By exerting selection on the petal size : pollen number ratio over two generations, we eliminated the positive correlation between petal size and pollen production, with both characters showing significant change after a single selection episode. Once these two floral traits became uncoupled, nectar sugar production was significantly correlated only with petal size. Our results suggest that natural selection could readily alter reproductive allocation in these flowers, and that the phenotypic correlations observed in nature may be maintained by selection for effective reproductive phenotypes.     

Responses of radish (Raphanus sativus) to drought stress

With this study the effects of water deficit on radish growth, dry mass production and partitioning, source–sink relations, physiological responses and accumulation of secondary metabolites in storage roots and leaves have been investigated. Three water stress treatments were applied: (Control) 100% of water holding capacity (WHC), (WS50) 50% of WHC, (WS30) 30% of WHC. Water stress was effectively acquired starting from 287 growing degree days (GDD) as demonstrated by the lower values of relative water content (RWC), higher leaf temperature and NIR‐based water indices values in water stressed conditions. Water stress reduced storage root dry weight by 27% at WS50 and 53% at WS30 as well as leaf dry matter accumulation (by 23% and 31% in WS50 and WS30, respectively), expansion (by 28% and 45% in WS50 and WS30, respectively) and specific leaf area (by 7% and 20% in WS50 and WS30, respectively) at 403 GDD. The increasing of leaf‐to‐root mass (L/R) and leaf area‐to‐root mass (LA/R) ratios indicated less dry matter allocation into storage organs under water stress conditions. Besides, water shortage increased leaf greenness as estimated by the higher soil–plant analysis development (SPAD) values (+14% and +20% on average for WS50 and WS30, respectively); other reflectance indices only partially confirmed SPAD readings. Substantially, water limitation did not significantly influence total anthocyanin content, ABTS‐radical scavenging activity and total free phenolic compounds in storage roots, as well as the total free phenolic compounds in leaves. Radish showed a strong plasticity in its adaptation to drought thanks to avoidance mechanisms such as constrained leaf development, increased leaf thickness and adjusted source–sink relationships.     

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.     

Growth of radish (Raphanus sativus) seedlings in magnetic fields

Radish seedlings were grown in asymmetric magnetic fields. The number of seeds germinated and the dry weight of the plants were the two criteria by which possible effects were examined. Two experiments were done; in the first the plants were grown for an average of 7.7 days and in the second for 14.1 days. A statistical analysis of the results failed to reveal any significant difference between control plants grown in dummy magnets and those subjected to the magnetic influence.     

Effects of ethylene on tuberization in radish (Raphanus sativus)

The effects of ethylene on the elongation of radish hypocotyls and on dry matter partitioning between tubers and shoots were analysed in order to gain insight into the possible role of ethylene in the regulation of tuberization. Treatment of very young seedlings with ethylene results in heavier tubers (Vreugdenhil et al. 1984). Here we report that addition of ethylene or ethephon two days after germination inhibited the elongation of the hypocotyl; trapping of endogenously produced ethylene had a stimulative effect on elongation. Ethephon, sprayed at a later stage, changed the partitioning of assimilates between tubers and shoots, resulting in lower tuber weights. It is concluded that ethylene had a dual effect on tuberization in radish: at a very early stage of development it inhibited elongation of the hypocotyl, resulting in earlier tuber formation and heavier tubers. At a later stage, it had a negative effect on tuber weight by changing dry matter partitioning.     

Florivores prefer white versus pink petal color morphs in wild radish, Raphanus sativus

Many hypotheses suggest that pollinators act to maintain or change floral color morph frequencies in nature, although pollinator preferences do not always match color morph frequencies in the field. Therefore, non-pollinating agents may also be responsible for color morph frequencies. To test this hypothesis, we examined whether Raphanus sativus plants with white flowers received different amounts of florivory than plants with pink flowers, and whether florivores preferred one floral color over the other. We found that white-flowered plants received significantly more floral damage than pink-flowered plants in eight populations over 4 years in northern California. Both generalists and specialists on Brassicaceae preferred white petals in choice and short-term no choice tests. In performance tests, generalists gained more weight on white versus pink petals whereas specialists gained similar amounts of weight on pink and white morphs. Because our results suggest that florivores prefer and perform better on white versus pink flowers, these insects may have the opportunity to affect the frequency of color morphs in the field.     

Relative fitness of females and hermaphrodites in a natural gynodioecious population of wild radish,Raphanus sativus L. (Brassicaceae): comparison based on molecular genotyping

In many gynodioecious species, sex determination involves both cytoplasmic male‐sterility (CMS) genes and nuclear genes that restore male function. Differences in fitness among genotypes affect the dynamics of those genes, and thus that of gynodioecy. We used a molecular marker to discriminate between hermaphrodites with and without a CMS gene in gynodioecious Raphanus sativus. We compared fitness through female function among the three genotypes: females, hermaphrodites with the CMS gene and those without it. Although there was no significant difference among the genotypes in seed size, hermaphrodites without the CMS gene produced significantly more seeds, and seeds with a higher germination rate than the other genotypes, suggesting no fitness advantage for females and no benefit to bearing the CMS gene. Despite the lack of fitness advantage for females in the parameter values we estimated, a theoretical model of gynodioecy shows it can be maintained if restorer genes impose a cost paid in pollen production. In addition, we found that females invest more resources into female reproduction than hermaphrodites when they become larger. If environmental conditions enable females to grow larger this would facilitate the dynamics of CMS genes.     

Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus)

Background and Aims

Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development.

Methods

Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages.

Key Results

In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress.

Conclusions

The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these controls may change over time. Thus, there may be times in development when selection on the maternal, paternal or embryo contributions to development are more and less likely.     

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.