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).     

Expression of additive genetic variances and covariances for wild radish floral traits: comparison between field and greenhouse environments

Measurements of the genetic variation and covariation underlying quantitative traits are crucial to our understanding of current evolutionary change and the mechanisms causing this evolution. This fact has spurred a large number of studies estimating heritabilities and genetic correlations in a variety of organisms. Most of these studies have been done in laboratory or greenhouse settings, but it is not well known how accurately these measurements estimate genetic variance and covariance expressed in the field. We conducted a quantitative genetic half-sibling analysis on six floral traits in wild radish. Plants were grown from seed in the field and were exposed to natural environmental variation throughout their lives, including herbivory and intra- and interspecific competition. The estimates of heritabilities and the additive genetic variance-covariance matrix (G) obtained from this analysis were then compared to previous greenhouse estimates of the same floral traits from the same natural population. Heritabilities were much lower in the field for all traits, and this was due to both large increases in environmental variance and decreases in additive genetic variance. Additive genetic covariance expressed was also much lower in the field. These differences resulted in highly significant differences in the G matrix between the greenhouse and field environments using two complementary testing methods. Although the G matrices shared some principal components in common, they were not simply proportional to each other. Therefore, the greenhouse results did not accurately depict how the floral traits would respond to natural selection in the field.     

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 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.     

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     

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.     

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.     

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.     

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.     

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.     

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.     

Fitness change in relation to mutation number in spontaneous mutation accumulation lines of Chlamydomonas reinhardtii

Although all genetic variation ultimately stems from mutations, their properties are difficult to study directly. Here, we used multiple mutation accumulation (MA) lines derived from five genetic backgrounds of the green algae Chlamydomonas reinhardtii that have been previously subjected to whole genome sequencing to investigate the relationship between the number of spontaneous mutations and change in fitness from a nonevolved ancestor. MA lines were on average less fit than their ancestors and we detected a significantly negative correlation between the change in fitness and the total number of accumulated mutations in the genome. Likewise, the number of mutations located within coding regions significantly and negatively impacted MA line fitness. We used the fitness data to parameterize a maximum likelihood model to estimate discrete categories of mutational effects, and found that models containing one to two mutational effect categories (one neutral and one deleterious category) fitted the data best. However, the best‐fitting mutational effects models were highly dependent on the genetic background of the ancestral strain.     

Variation of female frequency and cytoplasmic male-sterility gene frequency among natural gynodioecious populations of wild radish (Raphanus sativus L.)

In gynodioecious plant populations, sex determination often involves both cytoplasmic male-sterility (CMS) genes and specific nuclear genes that restore male function. How gynodioecy is maintained under the joint dynamics of CMS and restorer genes remains controversial. Although many theoretical models deal with interactions between CMS genes and restorer genes with sexual phenotypes and predict changes in their frequencies, it is difficult to observe the frequencies because no molecular markers have been established for either CMS or restorer genes in well-studied gynodioecious plants. This is the first report of the frequency of a CMS gene determined using a molecular marker in natural populations of a gynodioecious plant. Using a set of CMS gene-specific polymerase chain reaction primers, we compared female and CMS gene frequencies in 18 natural populations of Raphanus sativus. Female frequency was relatively low, ranging from 0 to 0.21. In contrast, the CMS gene frequency was highly variable among populations, ranging from 0 to 1. Estimated restorer gene frequency seemed less variable than observed CMS gene frequency, probably due to higher gene flow than in the CMS gene. Genetic drift may play a role in maintaining high variability of the CMS gene, although other possibilities are not excluded.     

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.     

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.     

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.     

Increase in calmodulin level in the early phases of radish seed (Raphanus sativus) germination

Abstract Calmodulin (Cam), the heat-stable, ubiquitous, Ca²⁺-dependent regulator protein, has been purified to apparent homogeneity from germinating radish seeds (Raphanus sativus). The characteristics of radish Cam-molecular weight, absorption spectrum, Ca²⁺-dependent activation of brain phosphodiesterase (PDE)-are very similar to those described for Cam from other plant materials. Radish Cam, like other plant Cam, shows some differences to Cam of calf brain. The total amount of Cam in radish embryos at 24 h of germination is ca. 37 μg g⁻¹ fresh weight. Approximately 95% of the total amount of Cam is present in the soluble fraction (supernatant at 100,000 g). The level in the embryo axis strongly increases in the first 24 h of germination (+540%); this increase is strongly reduced when the germination is inhibited by abscisic acid (ABA). In the presence of Ca²⁺, no ‘free’ Cam (i.e. not bound to other structures) is present in the soluble fraction, suggesting that, during early germination, Cam level is a limiting factor for the activities of Ca²⁺ -Cam-dependent systems. These studies suggest that Cam plays an important role in the early phases of seed germination. An inhibitor of the Ca²⁺-Cam-dependent phosphodiesterase is present in the soluble fraction from radish embryos; this substance decreases during germination. A possible role of this inhibitor during the early germination phases is hypothesized.     

Competition alters life history and increases the relative fecundity of crop-wild radish hybrids (Raphanus spp.)

The evolutionary impact of crop-to-wild gene flow depends on the fitness of hybrids under natural, competitive conditions. Here, we measured the performance of third-generation (F3) radish hybrids (Raphanus raphanistrum x Raphanus sativus) and weedy R. raphanistrum to understand how competitive interactions affect life history and relative fecundity. Three wild and three F1 crop-wild hybrid radish populations were established in semi-natural, agricultural conditions in Michigan, USA. The effects of competition on life-history traits and fecundity of F3 progeny were measured 2 yr later in a common garden experiment. Third-generation hybrid plants generally produced fewer seeds per fruit and set fewer fruits per flower than wild plants, resulting in lower lifetime fecundity. With increasing competition, age at reproduction was delayed, the relative number of seeds per fruit was reduced in wild plants and differences between hybrid and wild fecundity diminished. Competition may enhance the fecundity of advanced-generation hybrids relative to wild plants by reducing differences in life history, potentially promoting the introgression of crop alleles into weed populations.     

Fitness decline in spontaneous mutation accumulation lines of Caenorhabditis elegans with varying effective population sizes

The rate and fitness effects of new mutations have been investigated by mutation accumulation (MA) experiments in which organisms are maintained at a constant minimal population size to facilitate the accumulation of mutations with minimal efficacy of selection. We evolved 35 MA lines of Caenorhabditis elegans in parallel for 409 generations at three population sizes (N = 1, 10, and 100), representing the first spontaneous long-term MA experiment at varying population sizes with corresponding differences in the efficacy of selection. Productivity and survivorship in the N = 1 lines declined by 44% and 12%, respectively. The average effects of deleterious mutations in N = 1 lines are estimated to be 16.4% for productivity and 11.8% for survivorship. Larger populations (N = 10 and 100) did not suffer a significant decline in fitness traits despite a lengthy and sustained regime of consecutive bottlenecks exceeding 400 generations. Together, these results suggest that fitness decline in very small populations is dominated by mutations with large deleterious effects. It is possible that the MA lines at larger population sizes contain a load of cryptic deleterious mutations of small to moderate effects that would be revealed in more challenging environments.