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  DOI 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.

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. Int J Plant Sciences 176: 811-819.

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.

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.                                                                                             doi: 10.2135/cropsci2014.04.0295; Posted 7 Jan. 2015.

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.

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.

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.

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.

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.

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, U. minor, in Italy. Biological Invasions 15: 2717-2730.

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.

M. 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.

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.

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.

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.

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.

Abstract: 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.

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.

Abstract: 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.

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.

Abstract: 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.

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.

Abstract: 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.

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 (2010), Article ID 201858, doi: 10.1155/2010/201858. Special Issue on Foraging Biology of Neglected Social Pollinators.

Abstract: 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.

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.

Abstract: 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.

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.

Abstract: 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.

Zalapa, J. E., J. Brunet and R. P. Guries. 2009. Patterns of hybridization and introgression between invasive Ulmus pumila (Ulmaceae) and native U. rubra. American Journal of Botany 96(6): 1116-1128.

Abstract: 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.

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.

Abstract: 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.

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.

Abstract: 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.

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.

Abstract: 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.

Brunet, J. and H. R. Sweet. 2006. Impact of insect pollinator group and floral display size on outcrossing rate. Evolution 60(2): 234-246.

Abstract: 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.

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.

Abstract: 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.

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.

Brunet, J. and A. Liston. 2001. Polyploidy and gender dimorphism. Science 291: 1441.

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.

Brunet, J. and C. C. Mundt. 2000. Effects of competition on resistance gene polymorphism in a plant/pathogen system. Heredity 85(4): 393-400.

Abstract: 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.

Brunet, J. and C. Mundt. 2000. Combined effects of disease and competition on plant fitness. Canadian Journal of Botany 78(5): 646-654.

Abstract: 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.

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.

Abstract: 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.

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.


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.

Brunet, J. 1996. Male reproductive success and variation in fruit and seed set in Aquilegia caerulea (Ranunculaceae). Ecology 77(8): 2458-2471.

Abstract: 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.

Brunet, J. and D. Charlesworth. 1995. Floral sex allocation in sequentially blooming plants. Evolution 49(1): 70-79.

Abstract: 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.

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.

Abstract: 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.

Brunet, J. 1992. Sex allocation in hermaphroditic plants. Trends in Ecology and Evolution 7(3): 79-84.

Abstract: 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.

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.

Abstract: 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.

Department of Entomology, University of Wisconsin-Madison