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List of publicationsheadline bar For full text see or request articles at https://www.researchgate.net/profile/Johanne_Brunet  **In review** **62. Fragoso, F. and J. Brunet. Honey bees exhibit greater patch fidelity than bumble bees when foraging in a common environment.** **61. Brunet, J. and D. F. Minahan. Honey bee pollination ecology. In “The foraging behavior of the honey bee”, John Purdy (ed.), Elsevier, New York, New York. 2023.**  **Preprints** **60. Hernandez, F. Palmeira, L. Brunet, J. 2022. Persistence of cultivar alleles in wild carrot (*Daucus carota* L.) populations in the United States. BioRxiv https://doi.org/10.1101/2022.12.05.519198** **59. Minahan, D. and J. Brunet. 2020. Foraging strategy predicts species-specific patterns of pollen foraging by honey bees and bumble bees. Authorea May 06, 2020**. https://doi.org/10.22541/au.158879116.62887966 **** **Published Articles** **58. Fragoso FP, Brunet J. 2023. Differential ability of three bee species to move genes via pollen. PLoS ONE 18(4): e0271780.**http://doi.org/10.1371/journal.pone.0271780 Since the release of genetically engineered (GE) crops, there has been increased concernabout the introduction of GE genes into non-GE fields of a crop and their spread to feral orwild cross-compatible relatives. More recently, attention has been given to the differentialimpact of distinct pollinators on gene flow, with the goal of developing isolation distancesassociated with specific managed pollinators. To examine the differential impact of bee specieson gene movement, we quantified the relationship between the probability of getting aGE seed in a pod, and the order in which a flower was visited, or the cumulative distancetraveled by a bee in a foraging bout. We refer to these relationships as ‘seed curves’ andcompare these seeds curves among three bee species. The experiments used *Medicago**sativa* L. plants carrying three copies of the glyphosate resistance (GR) allele as pollendonors (*M. sativa* is a tetraploid), such that each pollen grain carried the GR allele, and conventionalplants as pollen recipients. Different foraging metrics, including the number of GRseeds produced over a foraging bout, were also quantified and contrasted among bee species.The lowest number of GR seeds set per foraging bout, and the GR seeds set at theshortest distances, were produced following leafcutting bee visits. In contrast, GR seedswere found at the longest distances following bumble bee visits. Values for honey beeswere intermediate. The ranking of bee species based on seed curves correlated well withfield-based gene flow estimates. Thus, differential seed curves of bee species, whichdescribe patterns of seed production within foraging bouts, translated into distinct abilities ofbee species to move genes at a landscape level. Bee behavior at a local scale (foragingbout) helps predict gene flow and the spread of GE genes at the landscape scale. **57. Fragoso, FP. and J. Brunet. 2023. The decision-making process of leafcutting bees when selecting patches. Biology Letters 19:20220411.**https://doi.org/10.1098/rsbl.2022.0411 Change in land configuration is an important driver of pollinator decline. Understanding patch selection by bees in fragmented landscapes has therefore become imperative to guide the design of habitats that support pollinators and ensure their conservation. This is especially true for solitary bees that make up most bee species in the world. To elucidate the decision-making process of a solitary bee when selecting patches, we tested four models of patch attractiveness that differed in the role of patch size and isolation distance in the selection process. In these models, bees used both patch size and patch distance, only patch distance, or chose randomly among patches. When patch size was included, bees could estimate patch resources fully or partially. An experiment with a centre patch, surrounded by four peripheral patches of different sizes and distances from the centre, provided observed transition data to test against predictions derived from each of the models. The alfalfa leafcutting bee, *Megachile rotundata*, does not move randomly among patches. This bee uses both patch size and isolation distance when selecting a patch but can only evaluate patch resources partially. This knowledge can guide the design of habitats in fragmented landscapes to facilitate solitary bee conservation. **56. Brunet, J., Jiang, Q., Zhao, Y., Thairu, M.W., Clayton, M.K. 2023. Bee Species Perform Distinct Foraging Behaviors That Are Best Described by Different Movement Models. Scientific Reports. (2023) 13:71. 2023**. https://doi.org/10.1038/s41598-022-26858-9 Linking animal behavior to models of animal movement has been an important, yet challenging goal for animal ecologists. In insect-pollinated plants, the foraging behavior of pollinators can influence their movement and ultimately affect how genes, including genetically engineered (GE) genes, are moved via pollen. We recorded the fine scale movement of three bee species foraging in patches of alfalfa (Medicago sativa), the European honey bee, Apis mellifera, the common eastern bumble bee, Bombus impatiens, and the alfalfa leafcutting bee, Megachile rotundata. Using the resulting distance and direction data, four models of bee movement were compared for each bee species. Each model simulated a bee moving a given distance and direction between consecutive racemes during a foraging bout. The models differed in whether the distance or direction traveled between consecutive racemes were randomly selected from the empirical distribution of distances (Random Distance) or directions (Random Direction) or were derived from the best model for distance (Modeled Distance) and transition probabilities for direction (Modeled Direction). The Modeled Distance-Modeled Direction model best described bumble bee movement. For leafcutting bees, a Random Distance-Random Direction model was favored. If we assume the lower sample sizes provided lower power to discriminate among models, the Modeled Distance-Modeled Direction likely describes honey bee movement. Using these models of pollinator movement, we obtained shortest net distances traveled by leafcutting bees, followed by honey bees and last, bumble bees. Pollen dispersal curves and seed curves provided similar rankings of bee species with respect to pollen dispersal and gene flow. Rankings for leafcutting bees and honey bees were supported by gene flow data. Leafcutting bee is the recommended pollinator to limit adventitious presence in alfalfa seed production fields. This study provides a mechanistic explanation for the lower gene flow by leafcutting bees and the approach developed here can be extended to other crops and their pollinators. Linking pollinator foraging behavior to pollinator movement can guide the design of pollinator strategies to minimize gene flow and facilitate coexistence in agricultural crops. **55. Keefover-Ring, K., Hetherington, M., Brunet, J. 2022. Population-specific responses of floral volatiles to abiotic factors in changing environments. American Journal of Botany. 109(5):676-688.** https://doi.org/10.1002/ajb2.1846 Changes in water availability associated with climate change can affect many plant traits, including floral volatiles involved in pollinator attraction. To examine whether floral volatiles responded to water availability, and whether this phenotypic plasticity differed among populations, we grew Rocky Mountain columbine plants (Aquilegia coerulea), started from seeds collected in three wild populations in Colorado (CO), Utah, and Arizona, under two moisture regimes. We observed a phenotypic response to water availability, with an increase in three volatile organic compounds (VOCs) in the wetter environment. Moreover, the phenotypic response of six VOCs to water differed among populations. Emissions of alpha-thujene, (-)-alpha-pinene, and (+)-ß-pinene, for example, increased under drier conditions for the CO and UT populations but decreased for the AZ population. When plants from the CO population were grown under the two water regimes (wet and dry) at 20° C (cool) or 25.5o C (warm) daytime temperature, there was no phenotypic response of VOC to water at either temperature. Finally, when comparing day to evening emission rates of VOCs for plants from the wet treatment in the cool room, emission of most individual compounds was greater during the day. The population-specific phenotypic responses of floral volatiles to water complicate predictions of the effects of climate change on a plant and its pollinators. **54. Dieterich Mabin, M.E., J. Brunet, H. Riday and L. Lehmann. 2021 Self-fertilization, inbreeding and yield in alfalfa seed production.** **Frontiers in Plant Science 12:700708.** https://doi.org/10.3389/fpls.2021.700708 Selfing (or self-pollination) is the ultimate form of inbreeding, or mating among close relatives. Selfing can create yield loss when inbreeding depression, defined as a lower survival and reproduction of inbred relative to outbred progeny, is present. To determine the impact of selfing in alfalfa (Medicago sativa L.), we quantified the selfing rate of 32 alfalfa seed-production fields located in three regions, the Pacific Northwest, the Central Valley and the Imperial Valley of California. Selfing rates (the proportion of selfed seeds) varied between 5.3 and 30% with an average of 12.2% over the 32 seed production fields. In both the parents and their progeny, we observed an excess of heterozygotes relative to Hardy-Weinberg expectations. We detected notable levels of inbreeding in parents (0.231 ± 0.007 parental inbreeding coefficient) and progeny (0.229 ± 0.005). There was a 15% decrease in the number of seeds per stem (seed set) and a 13% decline in the number of seeds per pod in selfed relative to outcrossed stems, but negligible inbreeding depression for pods per raceme and seed weight. The number of racemes on selfed stems increased significantly in fields with greater selfing rates, supporting the presence of geitonogamous or among flower selfing. Despite the significant level of inbreeding depression, seed set did not decrease in fields with higher selfing rates, where the greater number of racemes on the selfed stems increased seed set. The effects of field selfing rate on seed yield metrics were mostly indirect with direct effects of the number of racemes per stem. Available data indicate that the majority of selfing in alfalfa is pollinator-mediated and thus eliminating selfing in alfalfa seed production would require the selection of self-incompatible varieties while providing a 15% potential increase in seed yield and an increase in future hay yield. **53. Santa-Martinez, E., Cardoso Castro, C., Flick, A., Sullivan, M., Riday, H., Clayton, M.K. and Brunet, J. 2021. Bee species visiting *Medicago sativa* differ in pollen deposition rates with consequences for gene flow. American Journal of Botany 108(6): 1016-1028.**https://doi.org/10.1002/ajb2.1683 The pattern of pollen deposition or how pollinators deposit pollen from a particular donor onto successive flowers visited can affect pollen dispersal. In this study, we compared the pattern of pollen deposition over successive flowers and over distances for two grooming pollinators, one social bee, Bombus impatiens, and one solitary bee, Megachile rotundata foraging on Medicago sativa flowers. Plants carrying three copies of the ß-glucuronidase (GUS) allele were used as pollen donors for this tetraploid species. One individual bee was allowed to visit the GUS plants before being released on a linear array of untransformed M. sativa plants. Plant, raceme and flower visited in succession were marked and distance traveled between successive flowers measured. Stigmas were collected, stained and the blue pollen grains (GUS pollen) on each stigma counted. Different mixed effect Poisson regression models, which allowed for different rates of decay in pollen deposition, were fitted to the pollen data for each bee species. For both bee species, the regular Poisson model best fitted the pattern of pollen decay over successive flowers. Pollen decay over distance exhibited a slower rate of decay. For both successive flowers or distance, leafcutting bees exhibited steeper curves with shorter tails relative to bumble bees. The Poisson model is equivalent to an exponential model of pollen decay. This finding challenges whether the long dispersal tails detected in earlier studies are true biological phenomena or mere artifacts of the experimental or statistical methods used in these studies. This is the first report of a difference in pollen dispersal curves between two bee species that are both grooming pollinators. Differences in pollen dispersal curves could be used in agriculture to help predict differences in risk of gene escape between distinct pollinators. **52. Fragoso, F.P., Jiang, Q., Clayton, M.K. and Brunet, J. 2021. Patch selection by bumble bees navigating discontinuous landscapes. Scientific Reports****11, 8986.**https://doi.org/10.1038/s41598-021-88394-2 Resources are unevenly distributed over space and bees must determine how to exploit such patchily distributed resources. Identifying the rules used by bees to select patches in a spatially variable environment has represented a daunting task. Here, we propose a simple and novel approach to identify the rules used by bees when navigating patchy resources and show how bumble bees use both distance between patches and patch size to select a patch. Bumble bees follow rules of optimal foraging when selecting patches and prefer nearby patches with more resources. We developed mathematical models of patch attractiveness and tested these models against empirical data. **51. Kesoju, S.R, Kramer, M., Brunet, J., Greene, SL, Jordan A, Martin R.C. 2021. Gene flow in commercial alfalfa (*Medicago sativa* *sativa* L.) seed production fields: Distance is the primary but not the sole influence on adventitious presence. PLoS ONE 16(3): e0248746.**https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248746 In insect-pollinated crops, gene flow is affected not only by a crop’s characteristics, its mating system, life history and its pollinators, but also by pollinator and planting management practices. Previous studies have concentrated on the impact of distance to genetically engineered (GE) fields on adventitious presence (AP), the unwanted presence of a GE trait in non-GE material. However, other variables, including pollinators and different aspects of their management, field size, proximity to riparian and rangeland areas and various environmental and topographical factors may affect AP. In addition, AP may be present in the parent seed lots used to establish conventional fields. To identify the explanatory variables that influence the proportion of AP in conventional alfalfa fields, we performed variable selection regression analyses at two levels, a sample-level and field-level analysis. The two approaches gave broadly similar, though not identical results. For the sample-level model, distance from the GE field explained 66% of the variance in AP, confirming its importance in affecting AP. The relative area of GE fields within pollinator foraging range explained an additional 30% of the variation in AP in the model. The density of alfalfa leafcutting bee domiciles was also significant in both models. This study suggested that management procedures to minimize AP in conventional seed should focus on managing isolation distances, while taking into account the size of the GE pollen pool within pollinator foraging range, and the unique foraging behavior of pollinators. **50. Brunet, J., Flick A.F. and Bauer, A.A. 2021. Phenotypic selection on flower color and floral display size by three bee species. Frontiers in Plant Science****11:2244.**https://www.frontiersin.org/article/10.3389/fpls.2020.587528 Plants exhibit a wide array of floral forms and pollinators can act as agents of selection on floral traits. Two trends have emerged from recent reviews of pollinator-mediated selection in plants. First, pollinator-mediated selection on plant-level attractants such as floral display size is stronger than on flower-level attractant such as flower color. Second, when comparing plant species, distinct pollinators can exert different selection patterns on floral traits. In addition, many plant species are visited by a diverse array of pollinators but very few studies have examined selection by multiple, distinct pollinators. In the current study, we examined phenotypic selection on flower color and floral display size by three distinct bee species, the European honey bee, Apis mellifera, the common eastern bumble bee, Bombus impatiens, and the alfalfa leafcutting bee, Megachile rotundata, foraging on Medicago sativa. To estimate phenotypic selection by each bee species and for all bees combined simultaneously and on the same group of plants, we combine pollinator visitation data to seed set and floral trait measurements data typical of phenotypic selection study. When comparing floral traits, all bee species selected on the number of racemes per stem and the number of stems per raceme, two components of floral display size. However, only leafcutting bees selected on hue or flower color and only bumble bees selected on chroma or darkness of flowers. Selection on chroma occurred via correlational selection between chroma and number of flowers per raceme and we examine how correlational selection may facilitate the evolution of flower color in plant populations. Distinct bee species exerted similar selection on some floral traits but different patterns on other floral traits. The trends detected were consistent with previous studies and we advocate the approach introduced here for future studies examining the impact of distinct pollinators on floral trait evolution. **49. Boyer, K.J., Fragoso F.P., Dieterich Mabin, M.E. and Brunet, J. 2020. Netting and pan traps fail to identify the pollinator guild of an agricultural crop. Scientific Reports 10:13819.**https://doi.org/10.1038/s41598-020-70518-9 Pollinator decline is expected to cause significant reductions in food production and plant reproduction. To properly quantify the impact of pollinator decline on food production, we need survey methods that can identify insect and bee species responsible for pollination of specific crops. To address this issue, we compared the effectiveness of two survey methods, pan trapping and netting, at capturing the pollinators of alfalfa, *Medicago sativa.* Alfalfa is a major component of forage for cows and an important ingredient in chicken feed. We also examined the impact of the two survey methods on species richness and diversity and compared these measures among three different colors of pan traps. Netting was more effective at capturing the pollinators of alfalfa, especially those belonging to the *Bombus* and *Apis*genera. Pan traps captured a higher bee diversity relative to netting and each method was more efficient at capturing certain bee genera. Similarly, while the three colors of pan traps captured similar bee richness and diversity, each color was more effective at catching specific bee genera. Whether netting or pan traps best capture the pollinators of a plant species will depend on the pollinator guild of the plant species. We therefore recommend using both methods, with multiple colors of traps. A crucial step in these studies will be to identify the insects/bees that carry the pollen on their body or pollen sacs. A stronger link between survey methods and pollination will provide a more realistic quantification of the impact of pollinator decline on food production. **48. Sapir, Y., Brunet, J., Byers, D., Lambert, E., Staedler, Y. and Shonemberger, J. Floral evolution, breeding systems, pollinators, and beyond. International Journal of Plant****Sciences 180: 929-933. Special Issue on Floral Evolution.** https://www.journals.uchicago.edu/doi/10.1086/706240 Flowering plants exhibit an amazing diversity of flower types. Flowers are often viewed as organs that attract pollinators and pollinator-mediated selection has been considered the major mode of selection on floral traits. However, a review of phenotypic selection on floral traits highlighted many low estimates of phenotypic selection with more than half of the studies having estimates that did not differ statistically from zero. In addition, alternative hypotheses emphasizing the role of abiotic factors and non-pollinator biotic agents of selection on floral traits have been proposed. Finally, a recent meta-analysis supported the presence of pollinator-mediated selection on floral traits but detected similar strength of selection by abiotic factors. The paradigm of pollinator-driven floral trait evolution must therefore be reexamined. The collection of articles in this special issue broadens the scope of floral evolution research beyond plant-pollinator interactions and expands the perception of flowers beyond mere organs of pollinator attraction. The studies in this special issue convincingly show that non-pollinator biotic and abiotic selection agents can shape the evolution of flowers. Some of the studies indicate how more controlled factorial experiments that estimate the relative contribution of pollinator-mediated selection relative to other factors on floral traits are warranted. Other studies demonstrate how phenotypic plasticity and sexual selection should be considered in studies of floral evolution. By moving a floral trait closer or further from its new optimum, phenotypic plasticity can, respectively, slow down or accelerate selection on standing genetic variation. Sexual selection could accelerate or oppose the effect of natural selection on floral traits. One study examines the impact of plant physiology on selection on floral traits and determines that reducing the constraints on the water and carbon costs of flowers can increase the lability of flowers and enable stronger pollinator-mediated selection. Another study presents some evidence for the role of abiotic factors in the maintenance of a flower color polymorphism. Finally, one study reexamines a well-accepted although weakly supported hypothesis and determines that competition for pollinators does not select for staggered flowering. By presenting and combining different perspectives, this special issue on floral evolution broadens our understanding of the mechanisms responsible for the floral diversity prevalent in the world today. **47. Brunet, J. and M. Van Etten. 2019. The response of floral traits associated with pollinator attraction to environmental changes expected under anthropogenic climate change in high-altitude habitats. International Journal of Plant Sciences, 180: 954-964. Special Issue on Floral Evolution.**https://www.journals.uchicago.edu/doi/pdfplus/10.1086/705591 *Premise of the research.*While climate change will affect all plant species, those in high-altitude habitats cannot move to a more suitable habitat and must therefore adjust to their new environment. To determine how floral traits associated with pollinator attraction may respond to environmental changes, we examine whether these traits are phenotypically plastic, whether genetic variation exists for these traits to enable adaptation and whether phenotypic plasticity itself has a genetic basis.*Methodology.*In high-altitude habitats, climate models predict increases in temperature and in spring and early summer rainfall. We grew *Aquilegia coerulea* plants from three natural populations in two greenhouse rooms, each set at a different temperature, with two water regimes per room. There were 2 water X 3 populations X 17 families X 3-4 plants in each room. We measured floral display size, flower size, flower color, and reward traits. We used linear mixed models to examine the effects of family, population, water and their interactions on the different traits within each room.*Pivotal Results.*All floral traits showed genetic variation either within or among populations. Many floral traits exhibited phenotypic plasticity in response to water and genetic variation in phenotypic plasticity was identified for two traits. The number of flowers increased while spur lenth and reflectivity decreased between the cool-dry and the warm-wet treatments.*Conclusions.* All floral traits examined had the potential to adjust to the changes in water expected under anthropogenic climate change in high-altitude habitats. As the plant adjusts to the new environmental conditions, the potential roles of phenotypic plasticity versus selection on a trait mean varies among floral traits. We discuss the implications of these expected changes in floral traits on pollinators and plant fitness. **46. Brunet, J. 2019. Pollinator decline: Implications for Food Security and Environment. *Scientia Global.***https://doi.org/10.33548/SCIENTIA371 Bees are particularly important pollinators, with over 16,000 bee species known worldwide. The great majority of bee species are solitary; they do not have colonies and castes with a queen, workers (females) and drones (males). Both social and solitary bees contribute to crop pollination and to the pollination of wild plant species. Unfortunately, bees are in decline and the decrease in bee populations has been attributed to various causes – the three major culprits are pests and pathogens, exposure to agrochemicals and habitat loss and degradation. These factors can interact with one another, where habitat loss or pesticides can lead to poor nutrition and poor nutrition makes bees more prone to disease. Human actions impact many of the factors responsible for bee decline. Habitat degradation and fragmentation result mainly from the expansion of human dwellings and from a more extensive use of the land for agriculture. Furthermore, the use of pesticides in agriculture and home gardens is directly influenced by the need for food and for enhanced urban and suburban landscapes. Humans may have facilitated the movement of pathogens across continents. There is also concern that climate change will disrupt the synchrony between pollinator and plant emergence, limiting interactions between plants and their pollinators with potential negative effects on pollinator health and plant reproduction. A decline of pollinators can seriously impact the food supply. Fruit production would be strongly affected as most fruits require insects for pollination. Propagation of vegetables would become very problematic. A loss of pollinators would not lead to the complete disappearance of plants on earth, as various plants are wind-pollinated and other plants rely entirely on self-pollination for seed production. However, it would shift the composition of plant communities with serious implications for the animals that feed on them or use them for shelter. These changes would have many ramifications, many of which are difficult to predict. Humans depend on plants and plants depend on pollinators. A balance must be maintained in order to sustain life on earth and protect human survival and health. **45. Guzman, A., Gaines-Day H.R., Lois, A.N., Steffan, S.A., Brunet, J., Zalapa J. and C. Guedot. 2019. Surrounding landscape and spatial arrangement of honey bee hives affect pollen foraging and yield in cranberry. *Agriculture, Ecosystems and Environment* 286, 1 December 2019, 106624.**https://doi.org/10.1016/j.agee.2019.106624 Honey bees are the most important managed pollinator in the world. Recent trends suggest, however, that the demand for their pollination services is growing faster than the available supply. Therefore, it is critical to determine the most efficient management practices to maximize their use for crop production. One factor known to influence the efficiency of crop pollination is the availability of alternative, non-crop floral resources. These resources can vary as a function of the landscape surrounding a farm as well as local management practices within a farm. However, little is known about how the foraging behavior of honey bees on the target crop responds to the spatial arrangement of hives or the composition of the surrounding landscape. In this study, we collected pollen from pollen traps on honey bee hives placed on commercial cranberry marshes in central Wisconsin (USA). Individual marshes were selected to fall across a gradient of surrounding landscape from high- to low-woodland. Within each marsh, hives were placed either adjacent to wooded habitat, adjacent to a water reservoir, or in the center of the marsh. Honey bees from hives near water reservoirs collected a lower proportion of cranberry pollen than honey bees near the wooded area or at the center of the marsh. However, honey bees collected the same number of cranberry pollen grains and total pollen biomass irrespective of hive location or the surrounding landscape. Honey bees from hives located near water reservoirs, relative to the other two hive locations, tended to collect more pollen from fewer plants (low evenness). Cranberry yield did not vary as a function of the proportion of cranberry pollen collected or total number of cranberry pollen grains collected, but yield was higher at marshes located in low woodland landscapes relative to those in high-woodland landscapes. We conclude that the location of hives on a cranberry marsh in relation to non-crop habitat does not affect yield allowing growers to place hives where it is convenient, although placing hives near water reservoirs should provide bees with a more diverse pollen diet. **44. Brunet, J., Y. Zhao and M.K. Clayton. 2019. Linking the foraging behavior of three bee species to pollen dispersal and gene flow. *PLoS ONE* 14(2): 0212561.** https://doi.org/10.1371/journal.pone.0212561 Foraging behaviors that impact gene flow can guide the design of pollinator strategies to mitigate gene flow. Reduced gene flow is expected to minimize the impact of genetically engineered (GE) crops on feral and natural populations and to facilitate the coexistence of different agricultural markets. The goal of this study is to link foraging behavior to gene flow and identify behaviors that can help predict gene flow for different bee species. To reach this goal, we first examined and compared the foraging behaviors of three distinct bee species, the European honey bee, *Apis mellifera L*.*,*the common eastern bumble bee, *Bombus impatiens Cr.,*and the alfalfa leafcutting bee, *Megachile rotundata F.*, foraging on *Medicago sativa* flowers. Each foraging behavior investigated differed among bee species. Both social bees exhibited directionality of movement and had similar residence, in contrast to the random movement and shorter residence of the solitary bee. Tripping rate and net distance traveled differed among the three bee species. We ranked each behavior among bee species and used the relative ranking as gene flow predictor before testing the predictions against empirical gene flow data. Tripping rate and net distance traveled, but not residence, predicted relative gene dispersal among bee species. Linking specific behaviors to gene flow provides mechanisms to explain differences in gene flow among bee species and guides the development of management practices to reduce gene flow. Although developed in one system, the approach developed here can be generalized to different plant/pollinator systems. **43. Dieterich Mabin M., F.P. Fragoso and J. Brunet. 2019. A modified seedling phenotypic assay to identify glyphosate resistance in different alalfa varieties. *Proceedings for the 2019 Winter Seed School Conference, WASGA*, January 27-29, New Orleans, Louisiana pps. 34-45.**https://www.wasga.org/uploads/5/5/0/6/55065965/2019_wasga_proceed_book.pdf To determine the general applicability of a seedling phenotypic assay, previously developed by Boyle and colleagues to distinguish between conventional and glyphosate-resistant seedlings, we extended the assay to four conventional and four glyphosate- resistant alfalfa varieties. We also tested the assay under more generic laboratory conditions, using petri dishes placed in an incubator. Finally, we tested and confirmed that a 16h light: 8h dark regime, 80 ppm glyphosate concentration and a 14-day germination period, represented the best conditions to separate conventional and glyphosate-resistant seedlings using the phenotypic assay. Contrary to Boyle and colleagues, we did not observe the presence of root hair in all glyphosate-resistant seedlings exposed to 80 ppm glyphosate, although none of the conventional seedlings had root hair. Variation in the percentage of glyphosate-resistant seedlings with root hair was detected among varieties in both the control and the 80 ppm glyphosate treatment. We identified the presence of secondary roots as an additional phenotypic trait useful for this phenotypic assay. Variation among varieties was also detected for percent seedlings with secondary roots. Such variation will affect the sensitivity of the phenotypic seedling assay among varieties. We therefore do not expect the presence/absence of root hair and secondary roots between glyphosate-resistant and conventional seedlings to be 100% accurate when extended to different varieties. We propose a methodology that combines the phenotypic seedling assay with the use of the RUR test strips to detect glyphosate-resistant seedlings. This methodology can be applied to different alfalfa varieties under more generic laboratory conditions and will be useful to detect gene flow events and AP presence in alfalfa seed-production fields. **42. Palmieri, L, S. Ellison, D. Senalik, P. Simon and J. Brunet. 2019. Genetic markers to detect introgression of cultivar genes in wild carrot populations. *Acta Horticulturae*****1264, 165-174.**https://doi.org/10.17660/ActaHortic.2019.1264.20 Wild and cultivated carrots easily hybridize and cultivar genes can infiltrate wild carrot populations when pollinators move pollen from cultivated to wild carrots or when cultivar seeds migrate into wild carrot populations. Cultivar genes may then spread within and among wild carrot populations, a process called introgression. Wild carrots are widespread in the USA, can be weedy and have been declared invasive in some states. However, the extent of cultivar gene introgression into wild US carrot populations has yet to be been quantified. The goal of this study is to identify genetic markers to detect the presence of cultivar genes in wild carrot populations. To reach this goal, we sampled leaf tissue from individuals in each of four wild carrot populations near a site that has been used to breed carrot cultivars for about 40 years. We also collected leaf tissue from individuals in wild carrot populations further away from the breeding site (3.3-20.8 km). We extracted DNA and performed genotyping by sequencing (GBS) on these samples. We identified single nucleotide polymorphisms (SNPs) from the combined samples of cultivated and wild carrots. We used these SNPs to examine, using fastSTRUCTURE, the population structure of wild and cultivated carrots and then only the wild carrot. We detected strong genetic differences between cultivated and wild carrots and between the near wild and far wild carrot populations. We identified markers capable of detecting introgression by comparing the gene frequencies of these SNPs between the far wild, near wild, and cultivar groups. Depending on the cultivars used in the analyses, we detected between 52 and 863 such markers. These markers did not aggregate over some chromosomes but were dispersed over all nine carrot chromosomes, suggesting that no chromosome was free of introgression. **41. Mandel, J. and J. Brunet (2019).  Gene flow in carrots. Pps. 59-76. In “The carrot genome, Compendium of plant Genomes”, P.W. Simon, M. Iorizzo, D. Grzebelus and R. Baranski (eds). Springer, Switzerland. (Book Chapter)**DOI: 10.1007/978-3-030-03389-7_4 Carrot is an important crop and maintaining the purity of cultivars is a priority for the carrot industry. Gene flow among cultivars is reduced to maintain cultivar purity by setting minimum isolation distances among fields. But gene flow from wild to cultivated carrot can also affect cultivar purity and carrot quality. Such gene flow is often noted by the presence of early bolters in carrot fields. Cultivated and wild carrot belong to the same species, are interfertile, and often grow in close proximity such that gene flow from wild to cultivated carrot and from cultivated to wild is a great concern. Given wild carrots are weedy throughout much of their range and have been declared invasives in a number of states in the United States, the escape of cultivar genes and their spread into wild populations is a question of interest as there is a chance that escaped genes could render wild carrot even more invasive. The process of introgression of cultivar genes in wild carrot populations includes the spread of the genes within and among wild carrot populations. Therefore, to understand the process of introgression in wild carrots it is important to obtain estimates of gene flow among wild carrot populations. In this chapter, we first present characteristics of carrots that will affect gene flow and discuss dispersal via pollen by insect pollinators and via seeds by wind and animals. Although it is often referred to as a biennial, we introduce the various life history strategies observed in wild carrot populations as these can impact population growth and the range expansion of wild carrots over the landscape. We then review the studies of gene flow between crops, between crop and wild carrot and among wild carrot populations, concentrating on studies that used molecular markers. The consequences of these different types of gene flow (among cultivars, between crop and wild, and among wild) are then discussed. A major goal of biotechnology risk assessment is to improve predictions of the fate of escaped genes. We suggest as a priority for future studies to incorporate population dynamics with population genetics when modeling the fate of introduced genes. Improving our understanding of the factors that affect the spread of escaped genes will lead to the design of better management strategies to contain and limit their spread. **40. Brunet, J., R. Ziobro, J. Osvatic and M.K. Clayton. 2019. The effects of time, temperature and plant variety on pollen viability and its implications for gene flow risk. *Plant Biology*21(4) 715-722.**DOI: 10.1111/plb.12959 Pollen viability affects the probability that a pollen grain deposited on a plant’s stigma will produce a viable seed. Because a mature seed is needed before a gene flow event can occur, pollen viability will influence the risk of escape for genetically-engineered (GE) crops.
Pollen viability was measured at intervals for up to two hours following removal of the pollen from the anthers. It was quantified at three temperatures and for different alfalfa varieties, including both conventional and Roundup Ready (RR) varieties. Pollen viability was assessed using *in vitro*germination.
Time since removal from the anthers was the most prevalent factor affecting pollen viability in alfalfa. Pollen viability declined with increasing time at all three temperatures and for all varieties tested. Pollen viability was not affected by temperatures ranging between 25° and 37°C and did not vary among plant varieties, including conventional and RR varieties.
Bee foraging behavior suggested pollen viability within the first ten minutes following pollen removal from a flower to most affect seed production. Pollen longevity was predicted to have little impact on seed set and gene flow. Linking pollinator behavior to pollen viability improved our understanding of its impact on gene flow risk. **39. Minahan, D.F.  and J. Brunet. 2018. Strong interspecific differences in foraging effort observed between bumble bees and honeybees using miniaturized radio frequency identification (RFID). *Frontiers in Ecology and Evolution*. 05 October 2018.**https://doi.org/10.3389/fevo.2018.00156 Central place foragers depart from and return to a central location with enough resources for themselves, and in many cases, for the group. Honey bees and bumble bees are eusocial central place foragers. Honey bees have large perennial colonies while bumble bee colonies are annual and considerably smaller. Foraging range, body size and division of labor also vary between these two bee species. Honey bees use their unique dance language to recruit foragers to the most profitable patches. Bumble bees exploit patches individually and develop trapline foraging patterns. We expect such differences among bee species to engender differences in foraging activity. Moreover, variation in resource availability and in colony needs over the flowering season, can affect bee foraging activity. Finally, spatial variation in resource availability may impact bumble bees to a greater extent than honey bees due to their smaller foraging range. Using miniaturized radio frequency identification (RFID), we tracked the foraging activity of individual bees to and from hives at three sites and over five time periods. Pollen pellets were also collected from bees returning to the hive. We compared the European honey bee, *Apis mellifera*, and the common eastern bumble bee, *Bombus impatiens*. Linear mixed effect models determined the impact of bee species, time of season (period) and site, and their interactions, on multiple foraging metrics calculated from the RFID data and on pollen dry weight. Relative to honey bees, individual bumble bees made more foraging trips each day, resulting in a greater time spent foraging. A greater proportion of RFID tagged bumble bees foraged each day and bumble bees brought heavier pollen sacs to the hive compared to honey bees. Foraging bout duration did not vary between bee species and none of the foraging metrics varied among time periods or among sites. Both bee species brought heavier pollen sacs back to the hive at the beginning and the end of the flowering season. These results are discussed in terms of species differences in foraging strategies, size of individuals and colonies, and temporal variation in colony needs and resource availability. **38. Brunet, J. 2018. A conceptual framework that links pollinator foraging behavior to gene flow. *Proceedings for the 2018 Winter Seed School Conference, WASGA*, January 28-30, San Antonio, Texas pps. 63-67.** **https://www.wasga.org/uploads/5/5/0/6/55065965/wsga_proceedings_book_2018_final.pdf** The foraging behavior of pollinators can influence pollen dispersal and gene flow. In order to facilitate linking pollinator behavior to gene flow, we divide the process into three separate components. The first component comprises pollinator movement within patches. Pollinators move between flowers, racemes and plants within patches and one must understand the behaviors that influence within patch movements and the factors that influence such behaviors. Within patch movement is equivalent to a pollinator moving within a continuous landscape. Moreover, as they move from flower to flower, pollinators deposit pollen on stigmas. Pollen deposition is the second component of linking pollinator behavior to gene flow. Pollen deposition curves describe the number of pollen grains from a source that are deposited on stigmas of flowers visited in succession during a foraging trip and the steepness of the pollen deposition curve will affect gene flow. The third component includes patch choice or pollinator movement between patches. Pollinators will tend to move between patches over the agricultural landscape and, when doing so, they must decide which patch to move to next. Although this has been a difficult question to tackle, an understanding of the patch choice decision process would greatly improve our ability to predict pollinator movements and subsequent gene flow in discontinuous landscapes. Here, we examine the foraging behaviors likely to influence each of these three components, the factors likely to affect such behaviors and the subsequent potential impact on gene flow. **37. Hirsch H., J. Brunet, J.E. Zalapa, H. von Wehrden, M. Hartmann, C. Kleindienst B. Schlautman, E. Kosman, K. Wesche, D. Renison, I. Hensen. 2017. Intra- and interspecific hybridization in invasive Siberian elm. *Biological Invasions*****19:****1889–1904.** https://doi.org/10.1007/s10530-017-1404-6 Hybridization creates unique allele combinations which can facilitate the evolution of invasiveness. Frequent interspecific hybridization between the Siberian elm, *Ulmus pumila*, and native elm species has been detected in the Midwestern United States, Italy and Spain. However, *Ulmus pumila* also occurs in the western United States and Argentina, regions where no native elm species capable of hybridizing with it occurs. We examined whether inter- or intraspecific hybridization could be detected in these regions. Nuclear markers and the program STRUCTURE helped detect interspecific hybridization and determine the population genetic structure in both the native and the two non-native ranges. Chloroplast markers identified sources of introduction into these two non-native ranges. No significant interspecific hybridization was detected between *U. pumila* and *U. rubra* in the western United States or between *U. pumila* and *U. minor* in Argentina and vice versa. However, the genetic findings supported the presence of intraspecific hybridization and high levels of genetic diversity in both non-native ranges. The evidence presented for intraspecific hybridization in the current study, combined with reports of interspecific hybridization from previous studies, identifies elm as a genus where both inter- and intraspecific hybridization may occur and help maintain high levels of genetic diversity potentially associated with invasiveness. **36. Bauer A.A, M.K Clayton and J. Brunet. 2017. Floral traits influencing plant attractiveness to three bee species: Consequences for plant reproductive success. *American Journal of Botany*. 104: 1-10.**https://doi.org/10.3732/ajb.1600405 The ability to attract pollinators is crucial to plants that rely on insects for pollination. We contrasted the roles of floral display size and flower color in attracting three bee species and determined the relationships between plant attractiveness (number of pollinator visits) and seed set for each bee species. We recorded pollinator visits to plants, measured plant traits, and quantified plant reproductive success. A zero-inflated Poisson regression model indicated plant traits associated with pollinator attraction. It identified traits that increased the number of bee visits and traits that increased the probability of a plant not receiving any visits. Different components of floral display size were examined and two models of flower color contrasted. Relationships between plant attractiveness and seed set were determined using regression analyses. Plants with more racemes received more bee visits from all three bee species. Plants with few racemes were more likely not to receive any bee visits. The role of flower color varied with bee species and was influenced by the choice of the flower color model. Increasing bee visits increased seed set for all three bee species, with the steepest slope for leafcutting bees, followed by bumble bees, and finally honey bees. Floral display size influenced pollinator attraction more consistently than flower color. The same plant traits affected the probability of not being visited and the number of pollinator visits received. The impact of plant attractiveness on female reproductive success varied, together with pollinator effectiveness, by pollinator species. **35. Brunet J. and R.P. Guries. 2017. Elm genetic diversity and hybridization in the presence of Dutch elm disease. Pps 99-107 in: Pinchot, Cornelia C.****; Knight, Kathleen S.; Haugen, Linda M.; Flower, Charles E.; Slavicek, James M., eds. Proceedings of the American elm restoration workshop 2016; 2016 October 25-27; Lewis Center, OH. Gen. Tech. Rep. NRS-P-174. Newtown Square, PA: U.S. Department of Agriculture, Forest Service, Northern Research Station: 99-1** https://www.fs.usda.gov/research/treesearch/54921 The impact of DED on the genetic diversity of slippery elm is summarized and its potential impact on the genetic diversity of other North American native elms, *U. americana, U. thomasii, U. alata and U. serotina*, is discussed. The potential for hybridization between introduced *U. pumila* and the native North American elms is considered given previous findings with *U. rubra*. We do not expect DED to reduce the genetic diversity of these native elms. The only exception may be *U. serotina* if its restricted range leads to genetic discontinuities among populations. We do not expect hybridization between *U. americana* and *U. pumila* due to incompatibility barriers, but hybridization between *U. pumila* and other native elms appears more likely and could have negative effects on the long term conservation of these species. This information is timely given the current efforts to restore American elm across the US landscape. **34. Brunet J. and Syed Z. 2017. Enhancing pollination by attracting and retaining leafcutting bees *(Megachile rotundata)*in alfalfa seed production fields. *Proceedings for the 2017 Winter Seed School Conference*, January 29-31, Las Vegas, Nevada p. 67-73. https://www.wasga.org/uploads/5/5/0/6/55065965/wasga_proceedings_-_final.pdf** The alfalfa leafcutting bee, *Megachile rotundata (F.)*, has become an important managed pollinator of alfalfa, *Medicago sativa L.* One problem when using alfalfa leafcutting bees as managed pollinator, is the dispersal of many females upon release, even when adequate nesting sites are present. While dispersal of female bees from the site of emergence may represent a successful evolutionary adaptation to avoid inbreeding, this behavior becomes problematic when these pollinators are used as managed pollinators of alfalfa. Reducing the dispersal of females upon release into the fields, would facilitate the maintenance of viable commercial populations of alfalfa leafcutting bees. While female leafcutting bees prefer old, relative to new nesting boards, the use of old nesting boards is not a viable option because it facilitates disease propagation and increases bee mortality. Odors emitted from old nest contents could influence the preference of bees for older nesting boards and the use of chemical attractants may offer a viable alternative to retain female bees at a site. The objectives of this research were, therefore, to extract and isolate the biologically active constituents from empty alfalfa leafcutting bee cells (where eggs were laid and bees developed) and to quantify the attraction of these chemicals to the bees. If positive behavioral responses are detected, we will identify the biologically active constituents using bee’s antenna as a biological detector. Long range attractants (LRA) and short range arrestants (SRA) were isolated from empty bee cells and behavioral tests were performed to quantify attraction of the different extracts to alfalfa leafcutting bees. When individually tested against a control, we observed no preference of leafcutting bees for either long range attractants or short range arrestants. Bees visited the bee boards with solvent (control) as frequently as the bee boards with the chemical being tested. Due to the lack of positive behavioral responses, we did not conduct identification of the biologically active constituents using bee’s antenna as a biological detector. Although more behavioral tests would help confirm the generality of these findings, the behavioral responses obtained to date do not support the utility of field deployable attractive baits, using the attracting chemicals from empty bee cells, to facilitate the maintenance of alfalfa leafcutting bees in seed-production fields and improve alfalfa pollination. **33. Van Etten, M.L. and J. Brunet. 2017. Using population matrix models to reduce the spread of wild carrot. *Acta Horticulturae.*** DOI: 10.17660/ActaHortic.2017.1153.40 Wild carrot was most likely introduced to North America from Europe as a weed. It has since spread to every state in the USA and has been declared invasive. Wild carrot can easily hybridize with cultivated carrots leading to the potential transfer of genes from the crop to wild carrot. Hybridization could become an issue if the genes transferred to wild carrot conferred a selective advantage and increased its competitiveness or invasiveness. A better understanding of the demography of wild carrot would permit the identification of the life history stages that most affect its population growth. Such knowledge would facilitate the design of management practices to best control its spread. In this study, we used data  collected from wild carrot populations on reproduction, germination rate, overwinter survival and flowering rate to parameterize a stage structure model for a biennial lifecycle with a non-reproductive and a reproductive stage. Carrot populations were predicted to increase in size (λ>1), with growth rate (λ) of 1.9 when germination was low and 6.1 with high germination. Overwinter survival and flowering rate were the life history parameters that most affected population growth. Therefore, milder winter temperatures resulting from global warming could increase overwinter survival and the potential for spread of wild carrots. Effective management methods to control the spread of wild carrot and of the genes introduced into wild carrot populations should focus on lowering flowering rate and overwinter survival. For example, mowing should occur before flowering because our models predict that a single plant setting seeds could increase the population to 382 individuals within 3 years. **32. Brunet J., J.E. Zalapa and R.P. Guries. 2016. Conservation of genetic diversity in Slippery elm (*Ulmus rubra*) in Wisconsin despite the devastating impact of Dutch elm disease. *Conservation Genetics*. 17: 1001-1010.**https://doi.org/10.1007/s10592-016-0838-1 Forest pest epidemics are responsible for many population declines reported in forest trees. While forest tree populations tend to be genetically diverse, in principle mortality resulting from disease could diminish that genetic diversity and alter the genetic structure of the remnant populations with consequences for the ability of a species to adapt to changing environments. Slippery elm (*Ulmus rubra*Muhl.) is a long-lived, wind-pollinated forest tree with a native range covering essentially all of eastern North America. Dutch elm disease (DED) caused by an introduced fungal pathogen (*Ophiostoma ulmi*) devastated North American elm populations, including slippery elm, beginning in the 1930s.  Estimates of the numbers of elms lost to DED are unknown but range into the hundreds of millions of trees given their former abundance. In this study, the genotypes of 77 herbarium specimens collected between 1890 and 2004 in Wisconsin, and of 100 slippery elm trees from five wild Wisconsin populations, were characterized using 13 microsatellite loci. Levels of genetic diversity were compared between the herbarium specimens collected pre- and post-DED spread in Wisconsin. In addition, the levels of genetic diversity and degree of genetic differentiation were quantified in the five wild populations. The allelic diversity and expected levels of heterozygosity were similar between the pre- and post-DED herbarium specimens. The five wild populations were only slightly differentiated and no genetic bottleneck was detected for any population. At least in Wisconsin, slippery elm apparently has maintained levels of genetic diversity that could facilitate adaptation to future climatic and environmental changes. **31. Brunet, J., M. Thairu, J. Henss, R. Link and J. Kluever 2015. The effects of flower, floral display and reward sizes on bumble bee foraging behavior when pollen is the reward and plants are dichogamous. *International Journal of Plant Sciences*176: 811-819.**https://doi.org/10.1093/aob/mcv028 *Premise of the Research:*The size of floral display, flower and reward can influence pollinator foraging behavior. However, the importance of correlations between floral traits and rewards in affecting pollinator preferences is unclear. Moreover, in monoecious plants and plants with strong within flower dichogamy, some flowers do not offer a pollen reward and pollinator attraction may depend on the number of rewarding flowers rather than floral display size. We examined these questions in the Rocky Mountain columbine, where bumble bees can only forage for pollen produced in male-phase flowers.*Methodology:*We used dual choice assays and experimental arrays to determine whether a correlation between pollen reward and flower size was necessary before bees could distinguish between flowers with different pollen rewards or sizes. The impact of floral display size on bee foraging was first examined using inflorescences where all flowers provided a reward, either small or large. We then varied either floral display size or number of rewarding flowers while keeping the other variable constant.*Pivotal Results:*Bumble bees preferred flowers with a larger pollen reward or larger flowers only when the size of the pollen reward was correlated with flower size. Bees selected larger floral displays when all flowers presented a similar amount of pollen but preferred inflorescences with more pollen-rewarding flowers when only a proportion of the flowers offered a pollen reward.*Conclusions*: Bees could identify whether a flower offered pollen or not and could quantify the number of pollen-rewarding flowers. Correlations between floral traits and pollen reward affected pollinator preferences and may facilitate selection of floral traits by pollinators. Pollinators preferred inflorescences with more pollen-producing flowers rather than larger floral displays which suggests that selection of floral display size by pollinators may differ between plants with different mating systems. **30. Heathcliffe Riday, Peter Raisen, John Raasch, Emmanuel Santa-Martinez and Johanne Brunet. 2015. Selfing rate in an alfalfa seed production field pollinated with alfalfa leaf cutter bees. *Crop Science* 55: 1087- 1095.**https://doi.org/10.2135/cropsci2014.04.0295 Self-pollination or “selfing” in autotetraploid alfalfa *(Medicago sativa* L.) (2n = 4x = 32) leads to severe inbreeding depression. Investigating factors that influence the rate of selfing in commercial alfalfa seed production fields may allow mitigation strategy development against potential negative impacts of selfing on varietal performance. Using molecular markers permits in situ selfing rate estimation in commercial alfalfa seed production fields. The objective of this study was to measure in situ selfing rates from seed sampled from random plants in a commercial 0.84 ha alfalfa seed production field pollinated by leafcutter bee (*Megachile rotundata* F.). Selfing rates of alfalfa plants were estimated by genotyping ~24 progeny from each of 38 maternal plants at 18 SSR loci. The distance of the maternal plant from pollinator domicile, the positions of pods on racemes, the position of racemes on stems, and the number of seeds per pod were noted during seed and tissue collection. Field wide selfing rates averaged 11.8% while individual plant selfing rates ranged between 0% and 52.2%. Seed from pods collected from the upper part of racemes had, on average, lower selfing rates (9.1%) compared to pods from the lower part of the raceme (15.1%). When “low” self-compatible (<15% selfing rate in 3+ seeded pods) and “high” self-compatible (≥ 15% selfing rate in 3+ seeded pods) plants were examined separately, however, this pattern remained significant only for the low self-compatible plants (selfing rate of upper raceme 3.1% vs. lower raceme 8.3%). Low self-compatible plants also had higher selfing rates in 1-2 seeded pods (12.9%) compared to 3+ seeded pods (3.8%) while high self-compatible plants showed no differences in selfing rates based on number of seeds per pod. Genetic differences in the ability of self-pollen to outcompete outcross pollen when growing down the style best explained the observed differences between low and high self-compatible plants. We discuss how best management practices and selection could help reduce but would not eliminate selfing in alfalfa seed production fields. **29. Margaret Thairu and Johanne Brunet. 2015.The role of pollinators in maintaining variation in flower colour in the Rocky Mountain columbine, *Aquilegia coerulea*. *Annals of Botany* 115: 971-979.**https://doi.org/10.1093/aob/mcv028 *Background and aims:* Flower colour varies within and among populations of the Rocky Mountain columbine in conjunction with the abundance of its two major pollinators, hawkmoths and bumble bees. This study seeks to understand whether the choice of flower colour by these major pollinators can help explain the variation in flower colour observed in *Aquilegia coerulea* populations.*Methods:* Dual choice assays and experimental arrays of blue and white flowers were used to determine the preference of hawkmoths and bumble bees for flower colour. We tested whether a differential preference for flower colour, with bumble bees preferring blue and hawkmoths white flowers, could explain the variation in flower colour. We examined whether a single pollinator could maintain a flower colour polymorphism by testing whether preference for a flower colour varied between day and dusk for hawkmoths and whether bumble bees preferred novel or rare flower colour morphs.*Key Results:* Hawkmoths preferred blue flowers under both day and dusk light conditions. Naïve bumble bees preferred blue flowers but quickly learned to forage randomly on the two colour morphs when similar rewards were presented in the flowers. Bees quickly learned to associate a flower colour with a pollen reward. Prior experience affected the choice of flower colour by bees but they did not preferentially visit novel flower colours or rare or common colour morphs.*Conclusions:* Differences in flower colour preference between the two major pollinators could not explain the variation in flower colour observed in *A. coerulea*. The preference of hawkmoths for flower colour did not change between day and dusk and bumble bees did not prefer a novel or a rare flower colour morph. Other factors, besides pollinators, may affect the flower colour variation observed in *A. coerulea*. **28. Brandon Schlautman, Vera Pfeiffer, Juan Zalapa, and Johanne Brunet. 2014. The use of sequence-based SSR mining for the development of a vast collection of microsatellites in *Aquilegia formosa*. *American Journal of Plant Sciences* 5: 2402-2412.**https://doi.org/10.4236/ajps.2014.515253 Numerous microsatellite markers were developed for *Aquilegia formosa* from sequences deposited within the Expressed Sequence Tag (EST), Genomic Survey Sequence (GSS), and Nucleotide databases in NCBI. Microsatellites (SSRs) were identified and primers were designed for 9 SSR containing sequences in the Nucleotide database, 3803 sequences in the EST database, and 2226 sequences in the GSS database. For validation purposes, 45 primer pairs were used to amplify DNA from 16 *A. formosa* individuals from the H. J. Andrews experimental forest in Oregon, a Long Term Ecological Research (LTER) site. Genetic polymorphisms were identified at 30 of the 45 microsatellite loci with an average of 13.2 alleles per locus, and the observed level of heterozygosity was greater than 0.8 for 21 of the 30 loci. The use of these polymorphic loci was sufficient to individually separate the 16 individuals using a principal coordinate analysis. This comprehensive collection of primers significantly increases the availability of microsatellite primers for *Aquilegia spp.* and provides ample material for future studies that require highly variable SSRs such as mapping and association studies and investigation of plant mating system and gene flow. **27. Johanne Brunet, Juan Zalapa, Francesco Pecori and Alberto Santini. 2013. Hybridization and introgression between the exotic Siberian elm, *Ulmus pumila*, and the native Field elm, *minor*, in Italy. *Biological Invasions* 15: 2717-2730.**https://doi.org/10.1007/s10530-013-0486-z In response to the first Dutch elm disease (DED) pandemic, Siberian elm, *Ulmus pumila*, was planted to replace the native elm, *U. minor*, in Italy. The potential for hybridization between these two species is high and repeated hybridization could result in the genetic swamping of the native species and facilitate the evolution of invasiveness in the introduced species. We used genetic markers to examine the extent of hybridization between these two species and to determine the pattern of introgression. We quantified and compared the level of genetic diversity between the hybrids and the two parental species. Hybrids between *U. pumila* and *U. minor* were common. The pattern of introgression was not as strongly biased towards *U. pumila* as was previously observed for hybrids between *U. rubra* and *U. pumila* in the United States. The levels of heterozygosity were similar between *U. minor* and the hybrids and both groups had higher levels of heterozygosity relative to *U. pumila*. The programs Structure and NewHybrids indicated the presence of first- (F1) and second- generation (F2) hybrids and of backcrosses (BC) in the hybrid population. The presence of healthy DED resistant U. minor individuals combined with the self-compatibility of U. minor could help explain the presence of F2 individuals in Italy. The presence of F2 individuals, where most of the variability present in the hybrids will be released could facilitate rapid evolution and the potential evolution of invasiveness of *U. pumila* in Italy. **26. Van Etten and J. Brunet, 2013. The impact of global warming on floral traits that affect the selfing rate in a high-altitude plant. *International Journal of Plant Sciences* 174: 1099-1108.** https://doi.org/10.1086/671805 *Premise of research.* Changes in the abiotic environment can influence plant mating systems through phenotypic changes in floral traits that affect selfing. Herkogamy (spatial separation of male and female functions within a flower), dichogamy (temporal separation) and total flower number affect within (autogamy) and/or among flower selfing (geitonogamy). We examined the potential effects of environmental changes associated with global warming on herkogamy, dichogamy and number of flowers per plant and discussed the impacts on the mating system in a high-altitude species, *Aquilegia coerulea*.*Methodology.* In the high-altitude habitats in which this species grows, global warming is expected to increase both temperature and spring precipitation in the form of rain. We grew plants from 17 families from each of three natural populations of *A. coerulea* at two temperature and two water treatments, leading to 692 experimental plants (2 temperature * 2 water * 3 populations * 17 families * 3-4 individuals per family). We measured herkogamy and dichogamy, recorded total flower number for each plant and examined variation in floral traits due to genes and the environment.*Pivotal results.* Herkogamy, dichogamy and total flower number, traits known to influence selfing rate, were all phenotypically plastic in response to changes in water or temperature. Genetic variation for all three traits was detected within and/or among populations. *Conclusions.* Based on our results, we predict that the increase in total flower number as a plastic response to global warming will have the strongest influence on the selfing rate and increase the level of geitonogamous selfing. Because inbreeding depression is high in A. coerulea, selection will act to reduce floral display in order to reduce inbreeding depression but there will be a balance between the negative impact of inbreeding depression and the advantage provided by large floral displays in attracting pollinators. **25. Massimo Iorizzo, Douglas A. Senalik, Shelby L. Ellison, Dariusz Grzebelus, Pablo F. Cavagnaro, Charlotte Allender, Johanne Brunet, David M. Spooner, Allen Van Deynze and Philipp W. simon. 2013. Genetic structure and domestication of carrots. *American Journal of Botany* 100: 930-938.**https://doi.org/10.3732/ajb.1300055 *Premise of the study*: Analyses of genetic structure and phylogenetic relationships illuminate the origin and domestication of modern crops. Despite being an important world-wide vegetable, the genetic structure and domestication of carrot (*Daucus carota L.*) is poorly understood. We provide the first such study using a large data set regarding molecular markers and accessions, widely dispersed around the world.*Methods*: Sequencing data from the carrot transcriptome were used to develop 4000 Single Nucleotide Polymorphisms (SNP). Eight-four genotypes including a geographically well-dispersed subset of wild and cultivated carrots were genotyped using a KASPar assay.*Key results*: Analysis of allelic diversity of SNP data revealed no reduction of genetic diversity in cultivated vs. wild accessions. Structure and phylogenetic analysis revealed a clear separation between wild and cultivated accessions as well as between eastern and western cultivated carrot. Among the wild carrots, those from Central Asia were genetically most similar to cultivated accessions. Furthermore, we found that wild carrots from North America were most closely related to European wild accessions.*Conclusions*: Comparative genetic diversity of wild and cultivated accessions suggested that there was not a genetic bottleneck during carrot domestication. In conjunction with historical documents, our results suggest an origin of domesticated carrot from Central Asia. Wild carrots from North America were likely introduced as weeds to the USA with European colonization. This study provides a framework for future genomic studies. **24. Brunet, J., Z. Larson-Rabin and C. M. Stewart. 2012. The distribution of genetic diversity within and among populations of the Rocky Mountain columbine: the impact of gene flow, pollinators, and mating system. *International Journal of Plant Sciences* 173(5): 484-494.**https://doi.org/10.1086/665263 The distribution of genetic diversity of a plant species over the landscape provides insight into the evolutionary process and affects conservation strategies. We used both conventional methods (F-statistic and AMOVA) and more recently developed methods (Structure and Population Graphs) to describe the genetic structure of 13 *Aquilegia coerulea*populations across four geographic areas. Gene flow played an important role in explaining the distribution of genetic diversity over the landscape in *A. coerulea*populations. We detected significant isolation by distance and identified long-distance dispersal events and instances of restricted gene flow. The long-distance dispersal events explained some of the genetic similarities observed among populations from different regions. Restricted gene flow contributed to the genetic differentiation detected among populations within some regions. Populations in Utah represented the most likely center of the range for this group of populations. The mating system had little influence on the distribution of genetic diversity and the abundance of the two main pollinators did not explain differences among populations from different regions. Fire and differences in flowering phenology with altitude, combined with aggregated populations, limited gene flow and increased the genetic differentiation of some geographically close populations. **23. Brunet, J. and Z. Larson-Rabin. 2012. The response of flowering time to global warming in a high-altitude plant: the impact of genetics and the environment. *Botany* 90(4): 319-326.**https://doi.org/10.1139/b2012-001 In high-altitude habitats, an increase in temperature and greater precipitation in the form of rain represent climate changes typically associated with global warming. We determined whether phenotypic plasticity and genetic changes in the mean phenotype could affect the adaptation of flowering time to changes in the environment resulting from global warming in a montane plant species, *Aquilegia coerulea* James. We collected seeds from 17 plants from each of three natural populations. For each of these 51 families, we assigned 3–4 individuals to each of four water and temperature treatments. We observed phenotypic plasticity in flowering time in response to both temperature and water availability but no genetic variance or genetic differentiation in phenotypic plasticity. These results indicate that phenotypic plasticity could provide a quick response to environmental changes but provides little evolutionary potential. In contrast to phenotypic plasticity in flowering time, the mean flowering time did vary among families and among populations, suggesting a genetic basis to flowering time and adaptation in the different populations. The most likely scenario for the adaptation of this plant species to climate change is a rapid response via phenotypic plasticity followed by selection and micro-evolutionary changes in the mean phenotype. **22. Soza, V., J. Brunet, A. Liston, P. Smith and V. Di Stilio. 2012. Phylogenetic insights into the correlates of dioecy in meadow-rues (*Thalictrum*, *Ranunculaceae*). *Molecular Phylogenetics and Evolution* 63(1): 180-192.**https://doi.org/10.1016/j.ympev.2012.01.009 Numerous studies have examined the evolution of sexual systems in angiosperms, but few explore the interaction between these and the evolution of pollination mode. Wind pollination is often associated with unisexual flowers, but which evolved first and played a causative role in the evolution of the other is unclear. *Thalictrum*, meadow-rues (*Ranunculaceae*), provides a unique opportunity to study the evolution of these traits because it contains insect and wind pollination and four sexual systems. We used a phylogenetic approach to reconstruct ancestral states for sexual system, pollination mode, and geographic distribution in *Thalictrum*, and tested for correlations to uncover the factors involved in the evolution of unisexuality and wind pollination. Our results show that dioecy, andro- and gynomonoecy evolved at least twice from hermaphroditism. Wind pollination, unisexual flowers, and New World distribution were all significantly correlated. Wind pollination may have evolved early in the genus, followed by multiple losses and gains, and likely preceded the origin of unisexual flowers in several cases; we found no evidence for unisexual flowers evolving prior to wind pollination. Given a broad scale study showing the evolution of dioecy before wind pollination, our results from a finer scale analysis highlight that different evolutionary pathways are likely to occur throughout angiosperms. **21. Zalapa, J. E., J. Brunet and R. P. Guries. 2010. The extent of hybridization and its impact on the genetic diversity and population structure of an invasive tree, *Ulmus pumila* (*Ulmaceae*). *Evolutionary Applications* 3: 157-168.**https://doi.org/10.1111/j.1752-4571.2009.00106.x *Ulmus pumila* is considered an invasive tree in 41 of the United States. In this study, we examined the extent of hybridization in naturalized populations of *U. pumila,* its impact on genetic diversity and genetic structure and its potential role in explaining the invasion process of *U. pumila.* Genetic analyses indicated widespread hybridization with native *U. rubra* in naturalized *U. pumila* populations. Hybridization increased the genetic diversity of *U. pumila* populations and affected their genetic structure. The level of genetic diversity in ‘mature’ accessions, many of which may represent original plantings throughout the USA, was high and similar to the diversity of East Asian accessions. Hybridization with the native red elm may play an important role in the success of Siberian elm as an invader in temperate regions of the USA. **20. Brunet, J. and C. M. Stewart. 2010. Impact of bee species and plant density on alfalfa pollination and potential for gene flow. *Psyche: A Journal of Entomology Volume 2010,* Article ID 201858. Special Issue on Foraging Biology of Neglected Social Pollinators.**https://doi.org/10.1155/2010/201858 In outcrossing crops like alfalfa, various bee species can contribute to pollination and gene flow in seed production fields. With the increasing use of transgenic crops, it becomes important to determine the role of these distinct pollinators on alfalfa pollination and gene flow. The current study examines the relative contribution of honeybees, three bumble bee species, and three solitary bee species to pollination and gene flow in alfalfa. Two wild solitary bee species and one wild bumble bee species were best at tripping flowers, while the two managed pollinators commonly used in alfalfa seed production, honeybees and leaf cutting bees, had the lowest tripping rate. Honeybees had the greatest potential for gene flow and risk of transgene escape relative to the other pollinators. For honeybees, gene flow and risk of transgene escape were not affected by plant density although for the three bumble bee species gene flow and risk of transgene escape were the greatest in high-density fields. **19. Brunet, J., and K. G. A. Holmquist. 2009. The influence of distinct pollinators on female and male reproductive success in the Rocky Mountain columbine. *Molecular Ecology* 18(17): 3745-3758.**https://doi.org/10.1111/j.1365-294X.2009.04304.x Although there are many reasons to expect distinct pollinator types to differentially affect a plant’s reproductive success, few studies have directly examined this question. Here, we contrast the impact of two kinds of pollinators on reproductive success via male and female functions in the Rocky Mountain columbine, *Aquilegia coerulea.* We set up pollinator exclusion treatments in each of three patches where *Aquilegia* plants were visited by either day pollinators (majority bumble bees), by evening pollinators (hawkmoths), or by both (control). Day pollinators collected pollen and groomed, whereas evening pollinators collected nectar but did not groom. Maternal parents, potential fathers and progeny arrays were genotyped at five microsatellite loci. We estimated female outcrossing rate and counted seeds to measure female reproductive success and used paternity analysis to determine male reproductive success. Our results document that bumble bees frequently moved pollen among patches of plants and that, unlike hawkmoths, pollen moved by bumble bees sired more outcrossed seeds when it remained within a patch as opposed to moving between patches. Pollinator type differentially affected the outcrossing rate but not seed set, the number of outcrossed seeds or overall male reproductive success. Multiple visits to a plant and more frequent visits by bumble bees could help to explain the lack of impact of pollinator type on overall reproductive success. The increase in selfing rate with hawkmoths likely resulted from the abundant pollen available in experimental flowers. Our findings highlighted a new type of pollinator interactions that can benefit a plant species. **18. Brunet, J. 2009. Pollinators of the Rocky Mountain columbine: temporal variation, functional groups and associations with floral traits. *Annals of Botany* 103(9): 1567-1578.**https://doi.org/10.1093/aob/mcp096 *Background and Aims:* Pollinators together with other biotic and some abiotic factors can select for floral traits. However, variation in pollinator abundance over time and space can weaken such selection. In the present study, the variation in pollinator abundance over time and space was examined in populations of the Rocky Mountain columbine. The variation in three floral traits is described and correlations between pollinator type, functional pollinator groups or altitude and floral traits are examined.*Methods:* Pollinator observations took place in six *Aquilegia coerulea* populations over 1–4 years and spur length, flower colour and sepal length were measured in 12 populations. Pollinator abundance, measured as visits per flower per hour, was compared among populations and years. Pollinators were grouped into two functional groups: pollen or nectar collectors. The following associations were examined: annual presence of hawkmoths and whiter flowers with longer spurs; the presence of Sphinx vashti and longer spurs; and higher altitudes and whiter flowers. The study looked at whether an increase in the proportion of hawkmoths in a population was associated with whiter and larger flowers with longer spurs.*Key Results:* The abundance of different pollinator groups varied over time and space. Floral traits varied among populations. Higher altitude was correlated with bluer flowers. Whiter flowers were associated with the annual presence of hawkmoths. Populations visited by *Sphinx vashti* had longer spurs than populations visited only by *Hyles lineata.* Populations with greater percentage of nectar-collecting pollinators did not have whiter, larger flowers with longer spurs.*Conclusions:* Despite the large variation in pollinator abundance over time and space, one species of bumble-bee or hawkmoth tended to predominate in each population each year. Future studies of *Aquilegia coerulea* should examine the specific influences of pollinators and the environment on flower colour and of hawkmoth species on spur length. **17. Zalapa, J. E., J. Brunet and R. P. Guries. 2009. Patterns of hybridization and introgression between invasive *Ulmus pumila (Ulmaceae)* and native *rubra.* *American Journal of Botany* 96(6): 1116-1128.**https://doi.org/10.3732/ajb.0800334 Natural hybridization between introduced species and their native congeners occurs frequently and can create serious conservation concerns. *Ulmus pumila* (Siberian elm) is an introduced Asian elm species that has naturalized in the United States and is now considered invasive in 41 states. Red elm (*U. rubra*), a native to the eastern United States, often occurs in sympatry with Siberian elm, and the two species are thought to hybridize. Here, we genetically characterized reference populations of the two elm species to identify species-specific microsatellite alleles. These markers were used to classify individuals in putative hybrid zones as parental species or hybrids, assess the extent of hybridization, and track patterns of introgression. We identified nine *U. rubra,* 32 *U. pumila,* and 51 hybrid individuals in our hybrid zones. Of the 51 hybrids, 35 were classified as first-generation hybrids and 16 as backcrosses. The majority of the backcrosses (88%) were introgressed towards *U. pumila.* Our classification of genotypes was consistent whether we used manual classification, principal coordinate analyses or Bayesian clustering. We observed greater genetic diversity and new combination of alleles in the hybrids. Our study indicates widespread hybridization between *U. pumila* and *U. rubra* and an asymmetric pattern of introgression towards *U. pumila.* **16. Zalapa, J. E., J. Brunet and R. P. Guries. 2008. Genetic diversity and relationships among Dutch elm disease tolerant *Ulmus pumila L.* accessions from China. *Genome* 51(7): 492-500.**https://doi.org/10.1139/G08-034 Elm breeding programs worldwide have relied heavily on Asian elm germplasm, particularly *Ulmus pumila,* for the breeding of Dutch elm disease tolerant cultivars. However, the extent and patterning of genetic variation in Asian elm species is unknown. Therefore, the objective of this research was to determine the extent of genetic diversity among 53 *U. pumila* accessions collected throughout the People’s Republic of China. Using 23 microsatellite loci recently developed in the genus Ulmus, a total of 94 alleles were identified in 15 polymorphic and 4 monomorphic loci. The average number of alleles per locus was 4.9, with a range of 1–11 alleles. Gene diversity estimates per locus ranged from 0.08 to 0.87, and the non-exclusion probability for the 15 polymorphic loci combined was 0.7 x 10–9. Nineteen region-specific alleles were identified, and regional gene diversity estimates were moderately high (0.48–0.57). The genetic relationships among accessions and regions were estimated by UPGMA and principal coordinate analysis. Both techniques discriminated all accessions and regions. Two microsatellite markers (UR175 + UR123 or Ulm-3) were sufficient to discriminate up to 99.7% of the accessions studied. This research provides useful information for DNA-based fingerprinting, breeding, ecological studies, and diversity assessment of elm germplasm. **15. Zalapa, J. E., J. Brunet and R. P. Guries. 2008. Isolation and characterization of microsatellite markers for red elm (*Ulmus rubra Muhl.*) and cross-species amplification with Siberian elm (*Ulmus pumila L.*). *Molecular Ecology Resources* 8(1): 109-112.**https://doi.org/10.1111/j.1471-8286.2007.01805.x *Ulmus pumila* is an elm species, non-native to the USA that hybridizes with *Ulmus rubra.* In order to study the genetic structure and hybridization patterns between these two elm species, we developed 15 primer pairs for microsatellite loci in *U. rubra* and tested their cross amplification in *U. pumila.* All 15 primers amplified in both species, 11 of which possessed species-specific alleles. Eight loci were polymorphic in *U. pumila* and eight in *U. rubra,* each with two to eight alleles per locus. In addition, five primer pairs previously developed in *U. laevis* and *U. carpinifolia* (syn. *U. minor*) cross-amplified and showed polymorphic loci in *U. pumila* and/or *U. rubra.* These markers will facilitate the study of genetic structure and gene flow between *U. rubra* and exotic, invasive *U. pumila.* **14. Mundt, C. C., J. Brunet and K. E. Sackett. 2008. Impact of density and disease on frequency-dependent selection and genetic polymorphism: experiments with stripe rust and wheat. *Evolutionary Ecology* 22(5): 637-657.**https://doi.org/10.1007/s10682-007-9187-3 Frequency-dependent disease impacts may contribute to the maintenance of genetic diversity and sexual reproduction in plant populations. In earlier work with experimental wheat (*Triticum aestivum*) populations at a single density, we found that stripe rust (caused by *Puccinia striiformis*) created frequency-dependent selection on its host but competitive interactions between host genotypes reduced the potential for disease to maintain genetic polymorphisms in this highly self-pollinated species; the weaker competitor actually exhibited positive disease mediated frequency-dependent selection. Based on these results we predicted that at low density, where the overall level of competition is lower, disease would have a stronger impact relative to competition and thus be more likely to maintain genetic polymorphisms; at low densities the greatest effect of disease for negative frequency-dependent selection should be seen in the weak competitor. Here we report on results with wheat stripe rust in which we altered both the frequency and density of host genotypes in factorial combinations of two-way mixtures where each host genotype was attacked by its own specialized race of rust. Within each density disease levels increased with genotype frequencies, creating frequency-dependent disease attack at all densities. Similarly, disease created negative frequency-dependent selection on its host at all densities, as a genotype’s fitness was often greater at low than high frequency when disease was present. Disease levels increased with plant density in 1997 but decreased in 1998. While increasing plant density reduced absolute fitness, presumably as a result of increased competition, a genetic polymorphism was not more likely to be maintained at low than high density as we had predicted. Within each density, the impact of disease was insufficient to reverse the slope of the relationship between absolute fitness and planted frequency from positive to negative for the less competitive host genotype, thus preventing the maintenance of a genetic polymorphism. **13. Brunet, J. and H. R. Sweet. 2006. Impact of insect pollinator group and floral display size on outcrossing rate. *Evolution* 60(2): 234-246.**https://doi.org/10.1111/j.0014-3820.2006.tb01102.x Despite the strong influence of pollination ecology on the evolution of selfing, we have little information on how distinct groups of insect pollinators influence outcrossing rate. However, differences in behavior between pollinator groups could easily influence how each group affects outcrossing rate. We examined the influence of distinct insect pollinator groups on outcrossing rate in the rocky mountain columbine, *Aquilegia coerulea.* The impact of population size, plant density, size of floral display, and herkogamy (spatial separation between anthers and stigmas) on outcrossing rate was also considered as these variables were previously found to affect outcrossing rate in some plant species. We quantified correlations between all independent variables and used simple and two-factor regressions to determine direct and indirect impact of each independent variable on outcrossing rate. Outcrossing rate increased significantly with hawkmoth abundance but not with the abundance of any of the other groups of floral visitors, which included bumblebees, solitary bees, syrphid flies, and muscidae. Outcrossing rate was also significantly affected by floral display size and together, hawkmoth abundance and floral display size explained 87% of the variation in outcrossing rate. None of the other independent variables directly affected the outcrossing rate. This is the first report of a significant impact of pollinator type on outcrossing rate. Hawkmoths did not visit fewer flowers per plant relative to other pollinator groups but preferred visiting female-phase flowers first on a plant. Both the behavior of pollinators and floral display size affected outcrossing rate via their impact on the level of geitonogamous (among flower) selfing. Given that geitonogamous selfing is never advantageous, the variation in outcrossing rate and maintenance of mixed mating systems in populations of *A. coerulea* may not require an adaptive explanation. **12. Brunet, J. and H. R. Sweet. 2006. The maintenance of selfing in a population of the Rocky Mountain columbine. *International Journal of Plant Sciences* 167(2): 213-219.**https://doi.org/10.1086/498322 Pollination biology plays a major role in the evolution of selfing and the maintenance of mixed mating systems. We examine whether selfing in a population of *Aquilegia coerulea* is selected for or whether it is merely a nonadaptive consequence of adaptations for cross-pollination in a self-compatible plant with more than one flower open simultaneously. The comparison of selfing rate between groups of emasculated and control flowers on experimental plants helped quantify the contributions of geitonogamous and autogamous selfing to the overall selfing rate. While an increased seed set in control flowers indicated reproductive assurance, the genotypic data indicated that geitonogamous selfing made up the majority of the selfing and that autogamous selfing was negligible in the population. We examined whether bumblebees, the major pollinator in the population that year, visited control and emasculated flowers with equal frequency. We quantified the impact of emasculated flowers on the level of geitonogamous selfing in control flowers. After examining the reasons behind the discrepancy between seed set and genotypic data, we concluded that selfing in A. coerulea is a nonadaptive consequence of adaptations for cross-pollination in a plant with large floral displays. **11. Brunet, J. 2005. Plant-Pollinator Interactions and Pollen Dispersal. “Practical Pollination Biology”, A. Dafni, P. Kevan, and B. Husband (eds.), pps 56-82, *Enviroquest,* Cambridge, Canada.** This book chapter introduces different aspects of plant-pollinator interactions and pollen dispersal: methods for estimating pollen dispersal; pollen discounting; phenotypic selection by pollinators; and the impact of dichogamy on pollen transfer among flowers. **10. Brunet, J. and A. Liston. 2001. Polyploidy and gender dimorphism. *Science* 291: 1441.**https://doi.org/10.1126/science.291.5508.1441a This is a technical comment on a paper published in Science on polyploidy and the evolution of gender dimorphism in plants by Miller and Venable. **9. Brunet, J. and C. C. Mundt. 2000. Effects of competition on resistance gene polymorphism in a plant/pathogen system. *Heredity* 85(4): 393-400.**https://doi.org/10.1046/j.1365-2540.2000.00767.x *Puccinia striiformis,* a fungal pathogen, has been shown to cause direct, negative frequency-dependent selection on its host, wheat (*Triticum aestivum*). This disease-induced frequency-dependent selection was not sufficient to maintain polymorphism for resistance genes in the host populations. The present study examines whether interactions between disease and competition could impact upon the maintenance of genetic polymorphism in a highly self-pollinated species such as wheat, where strong associations between traits are likely. Four different two-way mixtures of wheat genotypes, susceptible to different races of *P. striiformis,* were planted at different frequencies in both the presence and absence of disease. In order to examine the influence of competition and disease on the maintenance of genetic polymorphism, relationships between host absolute fitness and host frequency were studied for each genotype in the mixtures of plants both in the presence and in the absence of disease. In the absence of disease, the absolute fitness of the stronger competitor was often negatively frequency-dependent, or else it did not vary with host frequency; the absolute fitness of the weaker competitor was often positively frequency-dependent. Disease typically rendered the slopes between absolute fitness and genotype frequency more negative for the stronger competitor. However, the influence of disease was not strong enough to reverse the sign of the slope between absolute fitness and genotype frequency for the genotype that was the weaker competitor in the absence of disease. Thus, disease was unable to reverse the relative ranking of the two genotypes caused by competition and create the negative frequency dependence on both genotypes in a mixture that is required for the maintenance of genetic polymorphism. **8. Brunet, J. and C. Mundt. 2000. Combined effects of disease and competition on plant fitness. *Canadian Journal of Botany* 78(5): 646-654.**https://doi.org/10.1139/b00-037 Wheat genotypes susceptible to different races of a pathogen, *Puccinia striiformis,* were planted in pure stands and in three different 1:1 mixtures, in both the presence and absence of disease, in two sites, and over 3 years. Using analyses of variance, we tested whether disease and intergenotypic competition influenced a genotype’s fitness and whether significant interactions existed between the effects of disease and competition on genotype fitness. Seed weight, number of inflorescences per seed planted, seeds per inflorescence, and absolute fitness were estimated for each genotype in each treatment. Absolute fitness was determined as the number of seeds collected per seed planted. Disease reduced seed weight. The other fitness measures were influenced by either disease or competition, and the impact of each factor often varied among site–year combinations. In general, interactions between the effects of disease and competition on genotype fitness were not significant. The few significant interactions indicated a less than additive effect of competition and disease on genotype fitness. The overall lack of interaction may be, in part, due to lesser disease levels in mixed as compared with pure stands, or reduced level of competition under diseased conditions. **7. Brunet, J. and C. C. Mundt. 2000. Disease, frequency-dependent selection, and genetic polymorphisms: experiments with stripe rust and wheat. *Evolution* 54(2): 406-415.**https://doi.org/10.1111/j.0014-3820.2000.tb00043.x Pathogens have the potential to maintain genetic polymorphisms by creating frequency-dependent selection on their host. This can occur when a rare host genotype is less likely to be attacked by a pathogen (frequency-dependent disease attack) and has higher fitness at low frequency (negative frequency-dependent selection). In this study, we used wheat genotypes that were susceptible to different races of the pathogen *Puccinia striiformis* to test whether disease created frequency-selection on its host and whether such selection could maintain polymorphisms for resistance genes in the wheat populations. Four different two-way mixtures of wheat genotypes were planted at different frequencies in both the presence and absence of disease. Disease created frequency-dependent selection on its host in some populations. Unknown factors other than disease also created frequency-dependent selection in this system because, in some instances, rare genotype advantage was observed in the absence of disease. Although the pathogen created frequency-dependent selection on its host, this selection was not sufficient to maintain genetic polymorphism in the host populations. In all cases where frequency-dependent selection occurred only in the diseased plots, one of the two genotypes was predicted to dominate in the population and the same genotype was predicted to dominate in both the presence and absence of disease. Only in cases where frequency-dependent selection was not caused by disease was there evidence that genetic polymorphisms would be maintained in the population. The frequency-dependent selection described in this study is a consequence of epidemiological effects of disease and differs from the time-lagged frequency-dependent selection resulting from coevolution between hosts and parasites. The impact of this direct frequency-dependent selection on the maintenance of genetic polymorphisms in the host population is discussed. **6. Brunet, J. and C. G. Eckert. 1998. Effects of floral morphology and display on outcrossing in blue columbine, *Aquilegia caerulea* (Ranunculaceae). *Functional Ecology* 12(4): 596-606.**https://doi.org/10.1046/j.1365-2435.1998.00231.x 1. The effect of variation in floral morphology and display on the level of outcrossing was investigated in a Rocky Mountain population of the self-compatible, protandrous, perennial herb, *Aquilegia caerulea.* ANOVA revealed substantial variation among 36 plants in floral traits including herkogamy (CV = 35%), protandry (23%), pollen production (12%), flower size (8%), the daily number of flowers in male phase (58%) and autofertility, seed production in the absence of pollinators (192%).2. Whether this floral variation affected the outcrossing rates of individual plants was assessed using both a categorical analysis, in which outcrossing rates were estimated separately for groups of plants that differed with respect to floral features, and a correlation analysis involving outcrossing rates estimated for individual plants from large progeny arrays (n≥ 40). Both analyses indicated significant positive correlations between the outcrossing rate and herkogamy (r = + 0·71) and protandry (+ 0·54), negative correlations with the number of flowers in male phase during each day of female phase (– 0·42) and autofertility (– 0·36), but no correlation with flower size or pollen production.3. Partial correlation indicated that both herkogamy and dichogamy, although weakly intercorrelated, were positively correlated with the outcrossing rate and that these effects were only partly mediated by their negative correlation with autofertility.4. The extent to which floral features and the outcrossing rate covaried across the flowering sequence within plants was also examined in this study. As expected for a plant with strong protandry and sequential blooming, the number of flowers in male phase decreased strongly with flowering sequence position. However, categorical analysis did not support the prediction that the first flowers to open on an inflorescence outcrossed at a lower rate than later-opening flowers.5. These results suggest that self-fertilization in *A. caerulea* is the result of all three major modes: autonomous and facilitated autogamy as well as geitonogamy. Although the geitonogamous component of selfing cannot be selected for directly, autogamy may be selected if it provides reproductive assurance. **5.****Brunet, J. 1996. Male reproductive success and variation in fruit and seed set in *Aquilegia caerulea* (Ranunculaceae). *Ecology* 77(8): 2458-2471.**https://doi.org/10.2307/2265746 The timing of fruit initiation and the proximity of fruits to resources influence the probability of fruit and seed initiation in many hermaphroditic plants. In two populations of *Aquilegia caerulea,* both fruit and seed set decreased significantly between early and late flowers. Low fruit and seed set of late flowers was not due to pollen limitation. Fruit and seed set of late flowers remained low when extra pollen was added to these flowers (pollen quantity), or when all flowers on an inflorescence received self or outcross pollen (pollen quality). While competition for resources occurred among flowers, resource limitation was not responsible for the low fruit and seed set of late flowers. Indeed, preventing pollination of late flowers significantly increased both fruit and seed set of early flowers, but fruit and seed set of late flowers remained low when early flowers were prevented from setting fruits. Late flowers were not just smaller or larger replicates of early flowers, as they allocated more reproductive resources to male function relative to early flowers. Neither herbivory nor architectural constraints could explain the low fruit and seed set of late flowers. While previously published adaptive explanations proposed to explain the low fruit and seed set of late flowers have emphasized a positive aspect to female reproductive success, it is suggested here that both male and female functions should be considered. Morphological data and estimates of male and female reproductive success demonstrate how, both morphologically and functionally, late flowers on inflorescences of *A. caerulea* specialize as males, early flowers as females. It will be argued that protandry in this sequential bloomer is expected to select for an increase in relative male allocation between early and late flowers, and that the observed pattern of resource allocation and the decrease in fruit and seed set between early and late flowers is consistent with this prediction. **4. Brunet, J. and D. Charlesworth. 1995. Floral sex allocation in sequentially blooming plants. *Evolution* 49(1): 70-79.**https://doi.org/10.1111/j.1558-5646.1995.tb05959.x In plants whose flowers develop in a sequence, different flowers may exhibit temporal variation in pollen donation and receipt such that the fitness contributions through male and female functions can vary among flowers. Dichogamy, or directional pollinator movements within inflorescences, can create situations where flowers in different stages in the sequence may differ in the numbers of flowers in the female stage available as potential mates. We present an evolutionarily stable strategy (ESS) analysis of the resource allocations expected in different flowers in hermaphroditic plants when the mating environments vary among flowers. This introduces a modular element into sex-allocation models. Our analysis shows that such variation in the mating environments of flowers can select for differences in sex allocation between flowers. When male and female fertilities are nonlinear functions of the allocations, variation in resource availability can also select for variation in sex allocation among flowers. The influence of dichogamy and pollinator directionality on floral sex allocation is discussed, and the empirical evidence supporting the predictions derived from the model is briefly reviewed. The implications of our results for the evolution of andromonoecy and monoecy are discussed. **3. Rigney, L. P., J. D. Thomson, M. B. Cruzan, and J. Brunet. 1993. Differential success of pollen donors in a self-compatible lily. *Evolution* 47(3): 915-924.**https://doi.org/10.1111/j.1558-5646.1993.tb01244.x If pollen donors are equally effective at siring seeds, the presence of equal proportions of pollen from two pollen donors on a stigma will lead to equal proportions of seeds sired by each pollen donor. Variation in germination rates, pollen-tube growth, and embryo viability may cause one donor to sire more seed than another. We looked for differential donor success in the field by simultaneously applying equal amounts of pollen from two pollen donors. We simultaneously applied equal amounts of self and outcross pollen to receptive stigmas and simultaneously applied pollen from two donors at different physical distances from the recipient. Following simultaneous application of self and outcross pollen, significantly more of the seeds were sired by outcross pollen donors. Seed set following simultaneous application of two outcross donors was also nonrandom. Pollen donors from 100 m were more likely to sire seeds when competing with pollen from plants nearby (1 m). To determine whether pollen-tube growth rates were responsible for these patterns of paternity, we varied the timing of deposition of outcross pollen allowing self pollen tubes a head start on the stigma. Outcross pollen was applied 3 or 24 h after self pollen. In spite of this time delay, the majority of the seeds were again sired by outcross pollen. There was no significant difference in the amount of seeds sired by self pollen between the two delay treatments. This result suggests that mechanisms operating after ovule fertilization may contribute to the discordance between the proportions of the pollen present and the proportions of seeds sired. **2. Brunet, J. 1992. Sex allocation in hermaphroditic plants. *Trends in Ecology and Evolution* 7(3): 79-84.**https://doi.org/10.1016/0169-5347(92)90245-7 Hermaphroditic plants allocate their reproductive resources to different functions: male, female, and pollinator attraction. While earlier sex-allocation models considered only male and female functions, more recent ones can divide reproductive resources into multiple functions. The basic predictions derived from these models are similar. While most models predict sex allocation at the fruit stage (pollen and seeds), some have examined allocation at the flower stage (pollen and ovules). Selfing rate, mode of pollination and competition among offspring of the same parent are some of the factors that can influence sex allocation among populations. Although the empirical evidence lags behind the theoretical development, sex-allocation theory has been quite successful at predicting trends among populations. **1. Thomson, J. D. and J. Brunet. 1990. Hypotheses for the evolution of dioecy in seed plants. *Trends in Ecology and Evolution* 5(1): 11-16.**https://doi.org/10.1016/0169-5347(90)90006-Y Over the last decade new hypotheses have been proposed for the evolution of dioecy in plants. Most of the selective mechanisms invoked have been suggested and supported by phylogenetic correlations. Here we review (1) the validity of the correlations (especially in light of recent critiques of the comparative method), and (2) the conformity of the proposed mechanisms to empirical data. None of the hypotheses can be flatly rejected on existing evidence, but the strength of their support varies. Future correlational studies must explicitly consider phylogeny; more importantly, such broad studies should also be supplemented by detailed studies of particular transitions to dioecy (e.g. within genera) – studies of the sort that have clarified analogous issues such as heterostyly.


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