Orientador: Felipe Wanderley Amorim / Resumo: As mudanças globais mediadas pela ação antrópica estão alterando a biodiversidade e os ecossistemas em um ritmo acelerado. Um dos principais impulsionadores dessas mudanças é a introdução de espécies exóticas em ecossistemas nativos. Entre os grupos de organismos afetados por este processo, o das plantas é reconhecido um dos mais preocupantes, uma vez que a produção primária limita o tamanho e a composição das comunidades e participa através de efeitos em cascata em interações multi-tróficas. Uma das principais relações ecológicas influenciada por esse efeito é a relação entre plantas e polinizadores, cujo papel é importante para estrutura e funcionamento das comunidades biológicas, não apenas porque as plantas fornecem recursos alimentares essenciais para muitos grupos de animais que visitam flores, mas também porque o sucesso reprodutivo da maioria das plantas com flores depende dos serviços bióticos fornecidos por estes animais. Neste contexto, a introdução de espécies de plantas exóticas invasoras pode ter impactos críticos nas interações planta-polinizador ao nível da comunidade, principalmente através da competição com espécies nativas. Como as interações planta-polinizador são cruciais para determinar a estrutura da comunidade, nesse estudo nós testamos como o crescimento rápido de uma gramínea invasora altera a composição das espécies de plantas nativas em um campo cerrado, juntamente com os impactos deste processo sobre a estrutura das interações planta-polinizador. ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The global change mediated by anthropic action is altering biodiversity and ecosystems in a fast pace. One major driver of these changes is the introduction of alien species in native ecosystems. Among the groups of organisms that are affected by this process, plants are recognized to be one of the most concern, since primary production limit global communities’ sizes and composition, and participate through cascade effects on multitrophic interactions. One crucial type of interaction that is influenced by this effect is the plant-pollinator relationship, which have an important role in the structure and functioning of biological communities, not only because plants provide essential food resources for many groups of animals that visit flowers, but also because the reproductive success of most flowering plants depends on the biotic services provided by animals. In this context, the introduction of invasive alien plant species may have critical impacts on plant-pollinator interactions at community level, mainly through competition with native species. Since plantpollinator interactions are determinants of community structure, here we evaluated how the rising of a fast-growing invasive alien grass species changes plant species composition of a Neotropical grassland community along with its impacts on the structure of plant-pollinator interactions. For this, we analyzed the changes in community composition and plantpollinator interactions over time, through the temporal turnover... (Complete abstract click electronic access below) / Mestre

1. Insect pollinators have been shown to alter their foraging patterns in response to habitat and landscape composition, particularly in relation to changes in the availability of floral resources which provide essential pollen and nectar provisions. Changes to pollinator behaviour and community composition, may alter the distance, directness and frequency of pollen movement and thus, the compatibility and genetic relatedness of pollen transferred between plants. We still lack good understanding of how variation in the spatial and temporal availability of floral resources drives pollinator responses and in turn, affects the fitness of outcrossing plants. Knowledge in this area could contribute to improved management interventions to enhance pollination services for plant conservation. 2. Through a combination of habitat and landscape scale field experiments, I explored how the availability of floral resources at different spatial scales affected plant-pollinator interactions, pollen transfer and mating success in plant populations, particularly those isolated from conspecifics. This involved introducing different species of plants in experimental arrays across a range of study systems that varied in structure and floral availability. Over the course of the thesis, I measured the community composition and behaviour of pollinators visiting experimental arrays; focusing on traits considered important for pollen transfer (e.g. Inter-tegular ('IT') span). Pollen movement was quantified within and between populations (5-150m) and the resulting plant outcrossing rates were measured using different methods including paternity analysis and the use of a dominance inheritance system. In addition, the implications of variations in pollinator foraging and pollination services can be attributed to pollen and gene flow and subsequently the reproduction and fitness of plants were assessed as a means of predicting the impacts on longer-term plant survival. 3. Findings from this thesis demonstrate reductions in the activity density (the abundance of actively foraging pollinators) and richness of pollinators and thus, the potential for plant visitation in response to a high abundance of floral resources within a habitat. This led to disruptions in pollen transfer, illustrated through a lower incidence of intra and inter-population pollen movement, and ultimately, reduced plant outcrossing rates. In parallel, plant seed set and germination rates were also reduced in habitats with high resource availability. Changes to pollinator communities and pollination services varied with the spatial scale at which floral resources were measured. Pollinator communities (activity density, richness and IT span) were most affected by floral resource abundance at a local scale (1-50m), particularly within a 20m radius of a plant population. Intra-population pollen movement was similarly affected by floral resources at a local spatial scale (within a 1m radius of a plant population). In contrast, no effect was observed on pollinator communities, intra-population pollen movement or plant reproduction when floral resources were measured at a landscape scale (within a 100-1500m radius of a plant population). However, findings were variable across different experiments at the same scale of measurement. For instance, the availability of floral resources at a local scale did not always elicit an effect on plant reproduction. This reflects differences in plant species identity and the effects of breeding system and floral traits, illustrated through variations in visitation rates between plant species. Inconsistencies were further observed with pollinator activity density and richness, which were not related to floral resources at a habitat scale in one chapter. 4. This thesis highlights the importance of the availability of floral resources at a local scale on plant-pollinator interactions and pollination services to plants. Co-flowering plants within florally rich habitats compete for pollinators and subsequently, visitation and pollen transfer between individuals of low density plant populations is diluted rather than facilitated. This suggests that although pollinator abundance and diversity may be enhanced through florally rich habitats (e.g. habitats implemented under the agri-environment scheme), pollination services are not automatically improved for plants which are present at low frequency in the landscape. This needs to be considered when designing and implementing management for threatened or isolated plants where plants may instead benefit from focused interventions. For instance, pollination services may be increased by efforts to maximise the facilitative effect of surrounding habitats, while increasing the ability of threatened or isolated plants to withstand competition from co-flowering plants.

The inadvertent spread of exotic pests and pathogens has resulted in devastating losses for bees. The vast majority of bee disease research has focused on a single species of managed bee, the European honey bee (Apis mellifera). More recently, pathogen spillover from managed bees is implicated in the decline of several bumble bee species (Bombus spp.) demonstrating a need to better understand the mechanisms driving disease prevalence in bees, transmission routes, and spillover events. RNA viruses, once considered specific to honey bees, are suspected of spilling over from managed honey bees into wild bumble bee populations. To test this, I collected bees and flowers in the field from areas with and without honey bee apiaries nearby. Prevalence of deformed wing virus (DWV) and black queen cell virus (BQCV) as well as replicating DWV infections in Bombus vagans and B. bimaculatus were highest in bumble bees collected near honey bee apiaries (χ 12 < 6.531, P < 0.05). My results suggest that honey bees are significant contributors of viruses to bumble bees. Flowers have been suspected as bridges in virus transmission among bees. I detected bee viruses on 18% of the flowers collected within honey bee apiaries and detected no virus on flowers in areas without apiaries, thus providing evidence that viruses are transmitted at flowers from infected honey bees. In controlled experiments using captive colonies in flight cages, I found that honey bees leave viruses on flowers but not equally across plant species. My results suggest that there are differences in virus ecology mediated by floral morphology and/or pollinator behavior. No bumble bees became infected in controlled experiments, indicating that virus transmission through plants is a rare event that is likely to require repeated exposure. The few studies examining viruses in bumble bees are generally limited to virus detection, resulting in little understanding of the conditions affecting virus titers. In honeybees, infections may remain latent, capable of replicating under certain conditions, such as immunosuppression induced by pesticide exposure. I tested whether exposure to imidacloprid, a neonicotinoid pesticide, affects virus titers in bumble bees. In previous honey bee studies, imidacloprid exposure increased virus titers. In contrast, I found that bumble bee exposure to imidacloprid decreased BQCV and DWV titers (χ42 < 20.873, p < 0.02). My findings suggest that virus-pesticide interactions are species-specific and results from honey bee studies should not be generalized across other bee species. Having found that honey bees are significant contributors of viruses to wild bees and flowers, I investigated how honey bee management practices affect disease spread and developed recommendations and tools to lesson the risk of spillover events. Honey bee disease may be exacerbated by migratory beekeeping which increases stress and opportunities for disease transmission. I experimentally tested whether migratory conditions contribute to disease spread in honey bees and found negative yet varying effects on bees suggesting that the effects of migratory practices may be ameliorated with rest time between pollination events. State apiary inspection programs are critical to controlling disease spread and reducing the risk of spillover. However, these programs are often resource constrained. I developed and deployed a toolkit that enables state programs to prioritize inspections and provide a platform for beekeeper education. Using novel data collected in Vermont, I discovered several promising avenues for future research and provided realistic recommendations to improve bee health.

Herbivory can have many effects on plant fitness, including altering plant-pollinator interactions and sexual reproduction in angiosperms. Pollinator-mediated interactions may be impacted when herbivores alter plant traits, such as floral display size, that can influence pollinator visitation rates, and, ultimately, the reproductive component of plant fitness. Here I describe an investigation into the indirect effects of feeding by beetles released as a biological control agent, Galerucella calmariensis and G. pusilla, on plant-pollinator interactions and reproductive output in the invasive plant Lythrum salicaria L. (purple loosestrife). During the summer of 2011, three treatments (low, ambient and mechanical herbivory) were applied to 105 plants during the pre-flowering period of growth. At the onset of flowering, a series of pollinator observations were conducted over the course of approximately 1.5 weeks. Several aspects of floral display were affected by the herbivory treatments, including increased inflorescence and flower production in the ambient and mechanical herbivory treatments, relative to the low herbivory treatment. Treatment type did not have a significant effect on the number of pollinator foraging bouts, but had marginally significant effects on the number of flowers probed per pollinator foraging bout and per 30-minutes. Moreover, treatment had a significant effect on the number of switches among the inflorescences on a single plant. I discuss the possibility that the differences in pollinator visitation were mediated by differences in the architecture and the size of floral display. There were no detectable differences in fruit or seed production (i.e., female fitness) among treatments. However, as I discuss, differences in pollinator visitation may affect other unmeasured aspects of fitness, such as the level of inbreeding or the number of seeds sired through male function.

Pollinator visitation patterns should affect pollination success and mating patterns in flowering species. In the orchid family, about one third of the species do not provide any reward for their pollinators. Pollination by deceit is typically associated with low fruit set but may increase the chance of cross-pollination since the pollinator should soon leave the individual plant when there is no reward in the flowers. This may be beneficial if self-fertilisation results in inbreeding depression. I studied the mating patterns of one rewarding and one deceptive orchid in two closely related genera by tracking the fate of stained pollinia. I also conducted controlled crosses to estimate inbreeding depression. The results show that the deceptive orchid Dactylorhiza lapponica has lower pollination success, but higher cross-pollination rate (ca. 90%) than the nectariferous orchid Gymnadenia conopsea (ca. 18% cross-pollination). The results further suggest that in G. conopsea, nocturnal visitors mediate higher geitonogamous pollination rate (ca. 100%) than diurnal visitors (ca. 60%). In both study species, fruits produced from cross-pollination were heavier than fruits produced from selfing. Inbreeding depression for fruit mass did not differ significantly between the two species (δ = 0.21 in D. lapponica and δ = 0.29 in G. conopsea). These data support the hypothesis that pollination by deceit can enhance cross-pollination. A literature study including several rewarding and non-rewarding orchid species indicated lower geitonogamy in the deceptive orchids, but the difference was not statistically significant.

<p>Evolution can be studied at many levels, from phenotypic to molecular, and from a variety of disciplines. An integrative approach can help provide a more complete understanding of the complexities of evolutionary change. This dissertation examines the ecology, genetics, and molecular mechanisms of the evolution of floral anthocyanin pigmentation in four species of <em>Mimulus</em> native to central Chile. Anthocyanins, which create red and purple colors in many plants, are a valuable model for studying evolutionary processes. They are ecologically important and highly variable both within and between species, and the underlying biosynthetic pathway is well characterized. The focus of this dissertation is dramatic diversification in anthocyanin coloration, in four taxa that are closely related to the genomic model system <em>M. guttatus</em>. I posed three primary questions: (1) Is floral diversification associated with pollinator divergence? (2) What is the genetic basis of the floral diversification? (3) What is the molecular mechanism of the increased production of anthocyanin pigment? The first question was addressed by evaluating patterns of pollinator visitation in natural populations of all four study taxa. The second question was explored using segregation analysis for a series of inter- and intraspecific crosses. One trait, increased petal anthocyanins in <em>M. cupreus</em>, was further dissected at the molecular level, using candidate gene testing and quantitative gene expression analysis. Pollinator studies showed little effect of flower color on pollinator behavior, implying that pollinator preference probably did not drive pigment evolution in this group. However, segregation analyses revealed that petal anthocyanin pigmentation has evolved three times independently in the study taxa, suggesting an adaptive origin. In addition to pollinator attraction, anthocyanins and their biochemical precursors protect against a variety of environmental stressors, and selection may have acted on these additional functions. Molecular analysis of petal anthocyanins in <em>M. cupreus</em> revealed that this single-locus trait maps to a transcription factor, <em>McAn1</em>, which is differentially expressed in high- versus low-pigmented flowers. Expression of the anthocyanin structural genes is tightly correlated with <em>McAn1</em> expression. The results suggest that <em>M. cupreus</em> pigmentation evolved by a mutation cis to <em>McAn1</em> that alters the intensity of anthocyanin biosynthesis.</p> / Dissertation

Der tropische Regenwald im südwestlichen Amazonasgebiet ist durch den zunehmenden Bevölkerungsdruck, die wirtschaftliche Ausbeutung, die Abholzung und das Abbrennen großer Flächen für die Viehwirtschaft bedroht. Allgemein zeichnen sich tropische Regenwälder durch eine hohe Diversität von Baumarten und daraus folgenden niedrigen Populationsdichten und entsprechend hohen Distanzen zwischen den Artgenossen aus. Die räumliche Verteilung der Individuen hat grundlegende Konsequenzen auf die Bewegung der Bestäuber und den intraspezifischen Genfluss, was somit die sexuelle Fortpflanzung beeinflusst. Daher ist das Wissen über den Genfluss grundlegend für das Verständnis von Reproduktionserfolgen und dem Management tropischer Baumarten. In dieser Studie untersuchte ich unterschiedliche Aspekte der sexuellen Fortpflanzung von wildem und kultiviertem Kakao (Theobroma cacao) und zwei weiteren Theobroma-Arten in Bolivien. Folgende Hypothesen wurden angenommen: 1) Wilde und kultivierte Pflanzen von T. cacao in Bolivien sind genetisch unterschiedlich; 2) Die Verteilungsdistanz der Pollen wilder Kakaopflanzen ist größer als die der kultivierten Pflanzen; 3) Es existiert ein gewisser Genfluss zwischen den wilden und den kultivierten Pflanzen von T. cacao; 4) Die Ansammlung der Bestäuber unterscheidet sich zwischen den Populationen wilder und kultivierter Pflanzen von T. cacao, sowohl in Bezug auf die taxonomische Zusammensetzung als auch in der Abundanz der Blütenbesucher; 5) Die kleinräumige genetische Struktur der Theobroma-Arten ist aufgrund der Abhängigkeit von Tieren bezüglich Bestäubung und Samenverbreitung gemäßigt. Zu diesem Zweck wurden im Tiefland von Bolivien drei verbreitete Arten der Gattung Theobroma ausgewählt: T. cacao L. (wild und kultiviert), T. speciosum Willd. ex Spreng und T. subincanum Mart. Diese Arten sind stammblütige Bäume des Unterwuchses. Sie haben zahlreiche kleine Blüten und schwere Früchte mit essbaren Samen, welche von Fruchtfleisch umgeben sind. Um den Pollenfluss des Kakaos zu untersuchen, schätzte ich den Grad der Selbstbestäubung und die Distanz der Pollenverbreitung sowie den Genfluss zwischen wildem und kultiviertem Kakao. Ich untersuchte zudem, ob sich die wilden von den kultivierten Kakaobäumen genetisch unterscheiden. Um die Kakaobestäuber zu identifizieren, dokumentierte ich die Zusammensetzung der Blütenbesucher auf wildem und kultiviertem Kakao. Weiterhin ermittelte ich die genetische Diversität und die kleinräumige genetische Struktur (SGS) der drei Theobroma-Arten, um die Distanzen der genetischen Verbreitung aus den Mustern der SGS zu schätzen. Die Ergebnisse zeigen, dass wilder und kultivierter Kakao sich genetisch unterscheiden, woraus geschlussfolgert wird,  dass die „wilden“ Populationen des Kakaos im Tiefland Boliviens tatsächlich wilde Formen oder zumindest sehr alte Sorten darstellen. Wilde Populationen zeigten eine geringere genetische Diversität als kultivierter Kakao, was möglicherweise durch die unterschiedliche Herkunft der kultivierten Sorten (z.B. Costa Rica, Trinidad y Tobago) begründet ist. Im Unterschied zu früheren Studien konnte ich Hinweise für die Selbstbestäubung in wildem und kultiviertem Kakao finden, auch wenn die Fremdbestäubung klar dominiert. Selbstbestäubung war häufiger in kultiviertem als im wilden Kakao festzustellen. Die gesamtdurchschnittliche Distanz der Pollenverbreitung betrug 867 m. Die Distanz der Pollenverbreitung des wilden Kakaos lag in einem Bereich von 10 und 3007 m, während die der Pollen des kultivierten Kakaos über Distanzen von 13 bis 2360 m verbreitet wurden. Die Bestäubungsdistanzen des Kakaos waren höher als die der typischen Unterwuchsbaumarten, wenn man die geringen Größen der bestäubenden Mücken berücksichtigt. Der relativ hohe Pollenaustausch von kultiviertem zu wildem Kakao (20%) gefährdet die genetische Identität der wilden Populationen. Weiterhin wurde eine große Auswahl an Blütenbesuchern dokumentiert und erhebliche Unterschiede zwischen den Besuchern des wilden und des kultivierten Kakaos festgestellt. Die Ergebnisse lassen vermuten, dass die Mücken als Haupt- oder gar alleinige Bestäuber in unserem Untersuchungsgebiet nicht in Betracht kommen, da sie zu selten vorkommen. Potenzielle zusätzliche Bestäuber sind die kleine Diptera (z.B. Chloropidae und Phoridae) und Hymenoptera (z.B. Eulophidae und Platygastridae). Die wilden Verwandten des T. cacao, T. speciosum und T. subincanum zeigten eine relativ hohe genetische Diversität. Die Arten von Theobroma hatten eine niedrige, aber signifikant kleinräumige genetische Struktur. T. speciosum zeigte die höchste Distanz der Pollenverbreitung, was auf eine effiziente Samen- und Pollenverbreitung hindeutet, wohingegen T. subincanum die strukturiertere Art mit der geringsten Distanz der Genverbreitung war.  Abschließend sollten bei verbessertem Management der Kakao-Populationen folgende Punkte beachtet werden: Die hohe Distanz der Pollenverbreitung bei Kakao und der relativ hohe Pollenaustausch von kultiviertem zu wildem Kakao zeigt die Notwendigkeit des Schutzes weitläufiger Naturwälder. Um die genetische Identität des wilden Kakaos in Bolivien zu schützen und zusätzlich den genetischen Austausch zwischen den wilden Populationen zu unterstützen, sollte die genetische Variabilität lebensfähiger Populationen zu erhalten werden. Die kleinräumige genetische Struktur in Theobroma macht die Bedeutung des Schutzes der samenverbreitenden Tiere deutlich, um die genetischen Ressourcen dieser tropischen Baumgattung zu erhalten.

Understanding the functional relationship between characters and components of fitness is a central goal of evolutionary biology. The studies in this thesis examined the ecological causes and evolutionary consequences underlying differences in fitness among individuals of Lobelia cardinalis. Flowering plants experience selection from many sources, which may enhance or oppose selection by pollinators. In the second chapter of this thesis, the role of pollinators and herbivores in shaping selection on floral characters was investigated. Floral traits experienced pollinator-mediated selection and weak selection by weevil larvae and slugs. Because pollinators also forage according to local density of flowers, in the fourth chapter I explored how local density of individual plants and flowers influences fitness of individual plants. Plants at dense sites produced more seeds, consistent with pollinator preference for denser patches. Individual female-phase flowers produced more seeds as the density of surrounding male-phase flowers increased and female-phase flowers decreased. This study highlights how plant phenotype and local density influence pollination and subsequent plant fitness. In L. cardinalis rosette formation (a life-history character) partly shapes the distribution of plants, and may influence plant survival and fitness. In the fifth chapter, I explored how variation in allocation to clonal reproduction among plants (ramets) and genets influenced survival and fitness. Plants that produced more and larger rosettes realized higher survival independent of the phenotype of the parental. Plants that produced one rosette in 2009 produced more seeds in 2010 than plants that produced more than one rosette. This pattern was reversed in the following time period; plants that produced more rosettes in 2010 produced more seeds in 2011. The relative importance of pollinators versus other selective agents in shaping floral traits, as well as the intensity of competition among individual plants and flowers likely depend on the extent to which reproduction is pollen limited. In the third chapter, I explored how pollen limitation affected selection on floral traits via female fitness and found a weak relationship. Although this seems to contradict intuition, several reasons may limit the influence of pollen limitation on selection.

The goal of this study was to evaluate several sustainable turf maintenance techniques for their potential to increase beneficial insect populations, which could then provide ecosystem services including pest suppression and pollination. The three techniques in question were 1) raising mowing height in commercial and residential lawns, 2) establishing naturalized roughs on golf courses, and 3) creating pollinator refuges on golf courses through the program Operation Pollinator. We found that raising mowing heights did increase populations of some predators such as spiders and staphylinids, but did not increase predation, which was ubiquitously high because ant populations were unaffected by mowing height. In addition, we found that pests reared in high-mowed grass were less likely to survive and gained weight more slowly than when raised in low-mowed grass. On golf courses, we found that naturalized roughs and traditional roughs supported different populations of predators, but contrary to our original hypothesis, naturalized roughs had little impact on biological control on the rest of the golf course. Operation Pollinator was successful in supporting 49 species of pollinators, including rare and declining bumble bees, demonstrating that turf systems can provide valuable pollinator conservation services, especially in urban systems where pollinator habitats are already rare.

Herbivory can have many effects on plant fitness, including altering plant-pollinator interactions and sexual reproduction in angiosperms. Pollinator-mediated interactions may be impacted when herbivores alter plant traits, such as floral display size, that can influence pollinator visitation rates, and, ultimately, the reproductive component of plant fitness. Here I describe an investigation into the indirect effects of feeding by beetles released as a biological control agent, Galerucella calmariensis and G. pusilla, on plant-pollinator interactions and reproductive output in the invasive plant Lythrum salicaria L. (purple loosestrife). During the summer of 2011, three treatments (low, ambient and mechanical herbivory) were applied to 105 plants during the pre-flowering period of growth. At the onset of flowering, a series of pollinator observations were conducted over the course of approximately 1.5 weeks. Several aspects of floral display were affected by the herbivory treatments, including increased inflorescence and flower production in the ambient and mechanical herbivory treatments, relative to the low herbivory treatment. Treatment type did not have a significant effect on the number of pollinator foraging bouts, but had marginally significant effects on the number of flowers probed per pollinator foraging bout and per 30-minutes. Moreover, treatment had a significant effect on the number of switches among the inflorescences on a single plant. I discuss the possibility that the differences in pollinator visitation were mediated by differences in the architecture and the size of floral display. There were no detectable differences in fruit or seed production (i.e., female fitness) among treatments. However, as I discuss, differences in pollinator visitation may affect other unmeasured aspects of fitness, such as the level of inbreeding or the number of seeds sired through male function.