Allison A. Snow

WHAT MOLECULES CAN TELL US ABOUT POPULATIONS: CHOOSING ANDUSING A MOLECULAR MARKER

The rapid development of molecular techniques offers a palette of technical approaches for population biologists interested in a wide range of questions. For example, these tools can be used to determine individual reproductive success or to measure rates of genetic divergence among populations. Which technique is most appropriate for a par- ticular question depends upon (1) the extent of genetic polymorphism required to best answer the question, (2) the analytical or statistical approaches available for the techniques application, and (3) the pragmatics of time and costs of materials. Here we evaluate the application of several major techniques (protein electrophoresis, nuclear and mitochondrial RFLPs (restriction fragment length polymorphisms), minisatellite and microsatellite VNTRs (variable number tandem repeats), RAPDs (random amplified polymorphic DNA), and DNA sequencing) to an array of questions regarding individual identification, exclusion and assignment of parentage, and various levels of population structure. In our evaluation, we briefly explain the technical components of each molecular approach and assess whether the typical outcomes expected from each approach will provide useful information as applied to each level of inquiry. For studies of population genetic structure, protein electrophoresis remains a powerful tool for most taxa, although techniques based on nucleic acids (par- ticularly DNA sequencing and mitochondrial DNA RFLPs) are useful here as well. Recently developed nucleic acid techniques (e.g., VNTRs) can often identify enough genetic vari- ability to address questions of self-identification or parentage. Some of the newest tech- niques (RAPDs and microsatellites) are potentially useful across a number of levels of inquiry, although procedures for adopting them are still developing.

GENETICALLY ENGINEERED ORGANISMS AND THE ENVIRONMENT: CURRENT STATUS AND RECOMMENDATIONS1

The Ecological Society of America has evaluated the ecological effects of current and potential uses of field-released genetically engineered organisms (GEOs), as described in this Position Paper. Some GEOs could play a positive role in sustainable agriculture, forestry, aquaculture, bioremediation, and environmental management, both in developed and developing countries. However, deliberate or inadvertent releases of GEOs into the environment could have negative ecological effects under certain circumstances. Possible risks of GEOs could include: (1) creating new or more vigorous pests and pathogens; (2) exacerbating the effects of existing pests through hybridization with related transgenic organisms; (3) harm to nontarget species, such as soil organisms, non-pest insects, birds, and other animals; (4) disruption of biotic communities, including agroecosystems; and (5) irreparable loss or changes in species diversity or genetic diversity within species. Many potential applications of genetic engineering extend beyond traditional breeding, encompassing viruses, bacteria, algae, fungi, grasses, trees, insects, fish, and shellfish. GEOs that present novel traits will need special scrutiny with regard to their environmental effects. The Ecological Society of America supports the following recommendations. (1) GEOs should be designed to reduce environmental risks. (2) More extensive studies of the environmental benefits and risks associated with GEOs are needed. (3) These effects should be evaluated relative to appropriate baseline scenarios. (4) Environmental release of GEOs should be prevented if scientific knowledge about possible risks is clearly inadequate. (5) In some cases, post-release monitoring will be needed to identify, manage, and mitigate environmental risks. (6) Science-based regulation should subject all transgenic organisms to a similar risk assessment framework and should incorporate a cautious approach, recognizing that many environmental effects are GEO- and site-specific. (7) Ecologists, agricultural scientists, molecular biologists, and others need broader training and wider collaboration to address these recommendations. In summary, GEOs should be evaluated and used within the context of a scientifically based regulatory policy that encourages innovation without compromising sound environmental management. The Ecological Society of America is committed to providing scientific expertise for evaluating and predicting the ecological effects of field-released transgenic organisms.

Commercialization of Transgenic Plants: Potential Ecological Risks

transgenic crop varieties is increasing exponentially (Figure 1). As of 1996, several transgenic crop plants have already been approved for commercial release in the United States, including disease-resistant squash, herbicide-resistant soybean, and insect-resistant potato and cotton. Many more crop varieties are nearly ready for commercialization. At this rate of development, the majority of all widely cultivated plants in the United States may possess genetically engineered traits within the next few decades.

A Bt TRANSGENE REDUCES HERBIVORY AND ENHANCES FECUNDITY IN WILD SUNFLOWERS

Gene flow from transgenic crops can introduce novel traits into related spe- cies, but the ecological importance of this process is unknown. Here, we report the first empirical evidence that wild plants can benefit from a bacterial transgene under uncaged, natural conditions. Cultivated sunflower ( Helianthus annuus) is known to hybridize fre- quently with wild sunflower ( H. annuus) in the western and midwestern United States. We studied a crop-developed Bacillus thuringiensis(Bt) transgene, cry1Ac, in backcrossed wild sunflower populations. Lepidopteran damage on transgenic plants was strongly reduced relative to control plants at our two study sites, while damage by several weevil and fly species was unaffected. Our results suggest that reduced herbivory caused transgenic plants to produce an average of 55% more seeds per plant relative to nontransgenic controls at the field site in Nebraska. A similar but nonsignificant trend was seen at the site in Colorado (14% more seeds per plant). In a greenhouse experiment the transgene had no effect on fecundity, suggesting that it was not associated with a fitness cost. If Bt sunflowers are released commercially, we expect that Bt genes will spread to wild and weedy populations, limit damage from susceptible herbivores on these plants, and increase seed production when these herbivores are common.

Gene Flow from Genetically Modified Rice and Its Environmental Consequences

Abstract Within the next few years, many types of transgenic rice (Oryza sativa) will be ready for commercialization, including varieties with higher yields, greater tolerance of biotic and abiotic stresses, resistance to herbicides, improved nutritional quality, and novel pharmaceutical proteins. Although rice is primarily self-pollinating, its transgenes are expected to disperse to nearby weedy and wild relatives through pollen-mediated gene flow. Sexually compatible Oryza species often co-occur with the crop, especially in tropical countries, but little is known about how quickly fitness-enhancing transgenes will accumulate in these populations and whether this process will have any unwanted environmental consequences. For example, weedy rice could become much more difficult to manage if it acquires herbicide resistance, produces more seeds, or occurs in a wider range of habitats because of the spread of certain transgenes. Rice-growing countries urgently need publicly available ecological assessments of the risks and benefits of transgenic rice before new varieties are released.

Pollination dynamics in Epilobium canum (Onagraceae): consequences for gametophytic selection

Several hand-pollination experiments have shown that pollen tube competition leads to gametophytic selection, and thus affects genotypic and phenotypic characteristics of the next generation. This study is one of the first to quantify natural levels of pollen tube competition. In a population of Epilobium canum, I measured both the amount of pollen deposited on stigmas and the timing of deposition. Approximately 20 tetrads were required for full seed set within fruits. Hummingbirds deposited >20 tetrads at 50-70% of the flowers examined, often in a single load. When pollen arrived in 2 loads, a portion of the ovules within each ovary was probably sired by competing pollen from the second load. Competition may be relatively weak unless at least 30 tetrads compete for ovules. About 20% of the flowers received >30 tetrads in the first load, and 13% acquired > 30 tetrads in 2 loads. The frequency and intensity of pollen tube competition varied among plants. In some styles, 80% of the pollen tubes were excluded from access to ovules, but in others no competition occurred. Further studies of pollination rates and progeny fitness are needed before we can assess the role of pollen tube competition in natural populations. Potential effects of gametophytic selection are discussed.

Costs of transgenic herbicide resistance introgressed from Brassica napus into weedy B. rapa

Wild relatives of genetically engineered crops can acquire transgenic traits such as herbicide resistance via spontaneous crop–wild hybridization. In agricultural weeds, resistance to herbicides is often a beneficial trait, but little is known about possible costs that could affect the persistence of this trait when herbicides are not used. We tested for costs associated with transgenic resistance to glufosinate when introgressed into weedy Brassica rapa. Crosses were made between transgenic B. napus and wild B. rapa from Denmark. F1 progeny were backcrossed to B. rapa and BC1 plants were selected for chromosome numbers similar to B. rapa. Further backcrossing resulted in a BC2 generation that was hemizygous for herbicide resistance. We quantified the reproductive success of 457 BC3 progeny representing six full‐sib families raised in growth rooms (plants were pollinated by captive bumblebees). Pollen fertility and seed production of BC3 plants were as great as those of B. rapa raised in the same growth rooms. Segregation for herbicide resistance in BC3 plants was 1:1 overall, but the frequency of resistant progeny was lower than expected in one family and higher than expected in another. There were no significant differences between transgenic and nontransgenic plants in survival or the number of seeds per plant, indicating that costs associated with the transgene are probably negligible. Results from this growth‐chamber study suggest that transgenic resistance to glufosinate is capable of introgressing into populations of B. rapa and persisting, even in the absence of selection due to herbicide application.

The Ecology of Geitonogamous Pollination

Working as a natural historian in the 1700s, C.K. Sprengel wrote a pioneering book demonstrating that many hermaphroditic species require pollinator visits in order to produce seed (see Chapters 1 and 2). He did not provide a scientific explanation as to why cross-pollination is important, but in the next century Darwin, H. and F. Muller, and others proposed that various outcrossing mechanisms have evolved to avoid selfing and the consequences of inbreeding (Darwin, 1876; see Baker, 1983). Darwin also recognized that the potential for selfing is greatest in species with massive floral displays because having many flowers promotes the transfer of self-pollen to other flowers on the same genetic individual (geitonogamy). Following Darwin’s lead, many authors have suggested that the avoidance of selfing has been a major factor in the evolution of traits such as dioecy, self-incompatibility, monoecy, temporal separation of male and female organs (dichogamy), spatial separation of anthers and stigmas within flowers (herkogamy), and having few open flowers per day (see reviews by Arroyo, 1976; Lloyd, 1979; Bawa and Beach, 1981; Willson, 1983; Wyatt, 1983; Richards, 1986; Charlesworth and Charlesworth, 1987; Thomson and Brunet, 1990; de Jong, et al., 1992a; Harder and Barrett 1995; Hodges, 1995; also see Chapters 6, 8, and 14).

Long-term introgression of crop genes into wild sunflower populations

Abstract A strategy of DNA pooling aimed at identifying markers linked to quantitative trait loci (QTLs), ‘Sequential Bulked Typing’ (SBT), is presented. The method proposed consists in pooling DNA from consecutive pairs of individuals ranked phenotypically, i.e., pools are formed with individuals ranked (1st, 2nd), (3rd, 4th),…, (N-1st, Nth). The N/2 pools are subsequently amplified using the polymerase chain reaction (PCR). If the whole population is typed the number of PCRs per marker is halved with respect to individual typing (IT). But if this strategy is combined with selective genotyping of extreme individuals savings can be further increased. Two extreme cases are considered: in the first one (SBT0), it is assumed that only presence or absence of a given allele can be ascertained in a pool; in the second one (SBT1), it is further assumed that differences between allele band intensities can be distinguished. The theory to estimate by maximum likelihood the QTL effect and its position with respect to flanking markers is presented. The behaviour of IT and SBT was studied using stochastic computer simulation in backcross and F2 populations. Three percentages of subpattern distinction (0, 50 and 100%) two population sizes (n=1200 and 600) and two QTL effects (a=0.1 and 0.25 standard deviations) were considered. SBT1 had the same power as individual genotyping at half the genotyping costs in all situations studied. Accuracy of QTL location is not increased with a dense number of markers, as opposed to individual typing. As a result DNA pooling is not useful for accurate location of the QTL but rather to pick up genome regions containing QTLs of at least moderate effect. The theory developed provides the general theoretical framework to deal with any DNA pooling strategy aimed at detecting QTLs.

The impact of a flower-color polymorphism on mating patterns in experimental populations of wild radish (Raphanus raphanistrum L.)

We conducted field experiments to determine how a naturally occurring petal‐color polymorphism influences mating patterns in wild radish (Raphanus raphanistrum). The polymorphism is controlled at a single genetic locus, with white petal color being completely dominant to yellow. In experimental populations with equal numbers of yellow‐ and white‐flowered homozygous individuals, insect visitors strongly discriminated against white flowers. Pieris rapae, the most frequent pollinator, was almost 50% more likely to visit yellow than white flowers. Maternal fecundity did not differ between the morphs and was not significantly influenced by a plants compatibility with potential donors, suggesting that seed production was not limited by receipt of compatible pollen. In contrast, the yellow‐flowered morph sired approximately 75% of all seeds produced during the study. This paternity proportion was consistently greater than that expected on the basis of postpollination compatibility measures and was indistinguishable from that expected on the basis of pollinator‐visitation frequency. We conclude that the male‐fitness advantage of the yellow morph resulted from enhanced pollen export due to the greater attractiveness of its flowers to insect pollinators. With color morphs evenly distributed in experimental arrays, insects did not move assortatively on the basis of petal color, and we found no evidence for assortative pollen flow due to the floral polymorphism. Once postpollination compatibility relationships within populations were taken into account, paternal success of yellow donors did not differ between yellow‐ and white‐flowered maternal plants.