<div>To compensate for their sessile existence, plants synthesize and emit a wide diversity of volatile organic compounds (VOCs) that serve important biological functions pertaining to defense, reproduction, and plant-plant signaling. In addition to their importance in plant secondary metabolism, VOCs are used as fragrances, flavoring agents, and therapeutics. Plant metabolic engineering has successfully been implemented towards the design of value-added plants with enhanced defense, improved aroma and flavor, and increased production of specialty chemicals. However, rational design requires rigorous characterization of the mechanisms controlling metabolic fluxes in a network. Thus, the major aims of this dissertation are to study biological and physical mechanisms controlling the synthesis and emission of plant VOCs. This dissertation focuses on (i) modeling 2-phenylethanol biosynthesis in Arabidopsis and (ii) characterization of the biophysical properties of flower cuticles with respect to the emission of VOCs.</div><div><br></div><div>2-Phenylethanol (2-PE) is a naturally-occurring aromatic volatile with properties that make it a candidate oxygenate for petroleum-derived gasoline. In plants, 2-PE biosynthesis competes with the phenylpropanoid pathway for the common precursor L-phenylalanine (Phe). The phenylpropanoid pathway directs up to 30% of fixed carbon towards the production of lignin, a major constituent of plant cell walls that renders biomass recalcitrant to pretreatment techniques impeding the economical production of biofuels. An initial genetic engineering approach was proposed, whereby a portion of the carbon flux towards lignin production is diverted towards the biosynthesis 2-PE. Transgenic Arabidopsis thaliana expressing enzymes catalyzing the biosynthetic steps from Phe to 2-PE were generated. Excised stems from transgenic Arabidopsis were supplied 13C6-ring labeled Phe, and isotopic enrichment of downstream metabolites were quantified to calculate fluxes. By combining flux measurements with predictions from a kinetic model of the Phe metabolic network, we hypothesized that 2-PE biosynthesis in transgenic Arabidopsis was limited by endogenous pools of cytosolic Phe. Multiple independent genetic strategies were proposed based on model-guided predictions, such as inducing Phe hyper-accumulation, reduction of the activity of the competing phenylpropanoid pathway, and sequestering the 2-PE biosynthesis pathway in plastids. Combining kinetic modeling with time-course in vivo metabolomics led to successful rational engineering of 2-PE accumulating plants.</div><div><br></div><div>The plant cuticle is the physical interface between the flower and its surrounding environment. Passage of VOCs through the cuticle is driven solely by diffusion and is thus dependent on the cuticle physicochemical properties. Wax compounds in the cuticular matrix self-assemble into a multiphase system of crystalline and amorphous regions, where their relative amounts and arrangements govern VOC diffusion. To investigate the effect of wax composition on the crystallinity and permeability of the cuticle, we characterized the cuticular waxes of Petunia hybrida petals using GC-MS, FTIR, DSC, and XRD. Petal waxes were found to be enriched with long-chain hydrocarbons forming semi-crystalline waxes localized on petal surfaces. A ternary system of wax compounds was proposed as a model for petal cuticles to investigate the effect of wax composition on cuticle crystallinity and permeability. Atomistic simulations of VOC displacement in waxes of varying chemical composition were performed at 298 K and 1 bar under NPT conditions to estimate diffusivities. Wax anisotropy was found to be highly dependent on the elongation of methylene chains, restricting the molecular diffusion path. Changes in crystalline symmetry were found to have measurable effects on VOC diffusion. Simulations of compositional variants of the model cuticle shows that changes in relative crystallinity exert differential control on the dynamics of VOC emissions.</div><div><br></div><div>To directly determine the effect of the cuticle on VOC emissions in petunia flowers, the wax exporter PhABCG12 was silenced using RNA interference, resulting in flowers with thinner cuticles. However, VOC emissions were found to have significantly decreased in transgenic flowers relative to the wild-type control. Dewaxing wild-type and transgenic petunia revealed that the cuticle serves as a site of VOC build-up during emission, and deficient coverage limits the extent to which compounds can accumulate. In addition, the cuticle was found to impart differing levels of mass transfer resistance for certain VOCs, suggesting that the cuticle controls the dynamics of VOC emissions. Taken together, petal cuticles provide an additional layer of regulation in emission of VOCs from plants.</div><div><br></div>

Callitriche (water-starwort) belongs among difficult and insufficiently known genera of the Czech flora. The presented work provides the first critical taxonomic revision of the genus in the Czech Republic, with information relevant also to the broader region of Central Europe. Morphological and cytometric investigation resulted in identification of six Callitriche species in the Czech Republic. The genome size of all six Czech representatives of the genus was estimated using flow cytometry. The hybrid C. × vigens Martinsson (C. cophocarpa × C. platycarpa) was found for the first time in the Czech Republic. An individual related to C. hamulata was found in the Tichá Orlice river, which has aberrant genome size and aborted flowers; further study of this taxon is necessary. Multivariate morphometric analyses of fruits and cultivation were used to check and define reliable distinctive features and an impact of phenotypic plasticity. Key to the identification of species (including the first key for sterile plants), its detailed descriptions and ecological demands were provided. Each species differ significantly from the others in genome size, morphological features as well as in ecology. The reproductive strategy has the main importance for ecology and morphology of the studied species. The distribution of...

L’environnement a un effet considérable sur les végétaux par différents facteurs abiotiques (climat, sol, urbanisation) ou biotiques (pollinisateurs, herbivores). Les fortes pressions de sélection exercées par ces facteurs sur certains traits phénotypiques aboutissent souvent à des adaptations chez les plantes. Les pollinisateurs exercent une pression de sélection sur les traits floraux qui résultent en des adaptations convergentes que l’on appelle syndromes de pollinisation. L’étude de ses syndromes, mais aussi de la performance de pollinisation de chaque visiteur, permet de mieux comprendre l’intensité de cette pression de sélection ainsi que l’évolution florale. Dans cette thèse, nous nous sommes appuyés sur l’utilisation de deux modèles d’études végétaux : la famille des Gesneriaceae des Antilles et l’Impatiente du Cap. Ces deux modèles, de par leurs stratégies de pollinisation et leurs variations florales, sont appropriés pour étudier l’adaptation florale aux pollinisateurs. Nous avons ainsi pu tester 1) si la forme florale est expliquée par la forme du bec des colibris pollinisateurs chez les Gesneriaceae antillaises ; 2) si l’espèce Rhytidophyllum bicolor Urb. est généraliste en pollinisation et si ses différents types fonctionnels de pollinisateurs ont une performance de pollinisation similaire ; 3) si l’urbanisation affecte la forme florale de l’espèce Impatiens capensis Meerb., à travers des changements dans les communautés de pollinisateurs. Pour tester ces hypothèses, des approches de morphométries géométriques ont été utilisées (hypothèses 1 et 3), ainsi que des observations de pollinisation in situ et la mesure du taux de visite (hypothèses 2 et 3). La mesure de performance de pollinisation a été réalisé via le comptage des grains de pollen déposés sur le stigmate après chaque visite, et à la mesure du taux de visite. Enfin, des tests statistiques (ANOVA, régressions linéaires) et analyses multivariées (analyses de redondance, analyse en composantes principales) ont été réalisées sur les données obtenues pour chaque étude. Les résultats de la première étude montrent une corrélation positive entre la longueur de la corolle de fleurs spécialistes aux colibris et la longueur du bec des pollinisateurs. Bien que moins significatifs, les résultats des comparaisons de formes globales, obtenues par application de morphométrie géométrique, indiquent que cette approche est prometteuse pour ce genre d’analyse. Nous avons démontré que la forme florale des fleurs généralistes est impactée par les colibris pollinisateurs, bien que d’une manière différente des spécialistes. 4 Pour l’hypothèse 2, la prédiction de stratégie de pollinisation de l’espèce Rhytidophyllum bicolor a été validée, puisque cette plante est pollinisée par des colibris, chauves-souris et abeilles. Cependant, face au déclin des populations de colibris après le passage de l’ouragan Matthew sur Haïti, seules les performances de pollinisation des abeilles et des chauves-souris ont pu être mesurées. Les résultats montrent que les chauves-souris sont des pollinisateurs efficaces et conséquents, bien que la performance des abeilles ne soit pas négligeable. Il a ainsi pu être mis de l’avant que la stratégie de pollinisation généraliste semble être un avantage pour les plantes présentes dans les zones sujettes aux fluctuations de populations de pollinisateurs, comme cela peut souvent être le cas sur les îles à la suite du passage d’un ouragan. La troisième étude montre que l’urbanisation n’a pas d’effet direct sur la forme florale de l’Impatiente du Cap, mais qu’elle a des effets indirects significatifs via les changements causés sur les communautés de pollinisateurs. Sur les six sites échantillonnées les pollinisateurs principaux, Bombus sp. Latreille et Apis mellifera Linnaeus, sont les mêmes. Cependant les taux de diversité obtenus montrent une variation entre les sites, due à la présence de certaines espèces de pollinisateurs occasionnels dans certains sites et pas dans d’autres. Ces taux ne sont pas plus faibles dans les sites les plus urbains. Les résultats indiquent que certaines formes florales sont associées à des espèces de pollinisateurs particulières. Les différentes espèces de pollinisateurs ayant une pression de sélection différente sur les traits floraux, l’urbanisation a ainsi un impact indirect sur la forme florale chez l’espèce étudiée. À travers trois études différentes, cette thèse a mis en avant l’impact que l’environnement peut avoir sur les traits floraux, de manière indirecte, via les pollinisateurs. Alors que la deuxième et troisième étude ont montré la pression exercée par les pollinisateurs sur les traits floraux dans différents cas de perturbations de l’habitat, la première étude a permis de mieux comprendre l’adaptation remarquable des fleurs à leurs pollinisateurs, même pour des espèces généralistes en pollinisation. / The environment has a considerable effect on plants through various abiotic (climate, soil, urbanization) or biotic (pollinators, herbivores) factors. The strong selection pressures exerted by these factors on phenotypic traits often results in adaptations. Pollinators exert selection pressure on floral traits that result in converging adaptations called pollination syndromes. The study of syndromes, as well as the pollination performance of each floral visitor, allows us to better understand the intensity of a/biotic selection pressure and floral evolution. In this thesis, we relied on two plant models: the Gesneriaceae family in the Antilles, and the common Jewelweed, Impatiens capensis Meerb. Due to their pollination strategies and their floral variation, both of these models are suitable for studying floral adaptation to biotic factors, specifically, their pollinators. We were thus able to test the following hypotheses: 1) whether the floral form is explained by the beak shape of pollinating hummingbirds in West Indies Gesnericeae; 2) whether the Gesnericeae Rhytidophyllum bicolor Urb. has a generalist strategy for pollination and whether their different functional types of pollinators have similar pollination performances; 3) whether urbanization affects the floral form of the common Jewelweed, through changes in pollinator communities. To test these hypotheses, geometric morphometric approaches were used (hypotheses 1 and 3), as well as in situ pollination observations, and estimation of the visitation rate (hypotheses 2 and 3). Pollination performance was measured by counting pollen grains deposited on the stigma after each visit, and by measuring the visitation rate. Finally, statistical tests (ANOVA, linear regressions) and multivariate analyses (redundancy analysis, principal component analysis) were carried out on the data obtained to test each hypothesis. The first results show a positive correlation between the length of the corolla of flowers specialized for hummingbirds and the beak length of pollinators. Although less significant, our results of the comparisons of global shapes, obtained by applying geometric morphometry, indicate that this approach is promising for this kind of analysis. We show that the floral form of generalist flowers is impacted by pollinating hummingbirds, albeit in a different way from specialists. For the second hypothesis, our prediction that R. bicolor has a generalist pollination strategy was validated, since this plant is pollinated by hummingbirds, bats and bees. However, faced with the decline in hummingbird populations after Hurricane Matthew hit Haiti in 2016, only the pollination performance of bees and bats could be measured. Our results show that bats are efficient and consistent pollinators of R. bicolor, although the performance of bees is not negligible. Thus, it has been possible to put forward that the generalist pollination strategy seems to be an advantage for plants present in areas subject to fluctuations in their pollinator populations, as can often be the case on hurricane-prone islands. The third study shows that urbanization does not have a direct effect on the flower form of common Jewelweed, but that urbanization does have significant indirect effects through changes caused on pollinator communities. At the six sites sampled, the main pollinators, Bombus sp. Latreille and Apis mellifera Linnaeus, are the same. However, diversity rates show variation between sites, due to the presence of certain species of occasional pollinators in some sites and not in others. These rates are not lower in the most urban sites. Our results indicate that certain floral forms are associated with particular pollinator species. Since different pollinator species have different selection pressures on floral traits, urbanization has an indirect impact on the floral form in the species studied. Through three different studies, this thesis highlighted the impact that the environment can have on floral traits, indirectly, via pollinators. While the second and third studies showed the pressure exerted by pollinators on floral traits in different cases of habitat disturbance, the first study helped to better highlight the remarkable adaptation of flowers to their pollinators, even for generalist species in pollination.

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[ES] En los cítricos, la baja temperatura promueve la inducción floral en otoño-invierno aumentando la expresión del gen promotor CiFT3 (homólogo en los cítricos del gen FLOWERING LOCUS T). La presencia de un gran número de frutos en el árbol durante ese momento inhibe la expresión de CiFT3 y la floración, pero se desconoce la señal inhibitoria que genera el fruto. Las hipótesis mayormente aceptadas proponen que la señal puede ser hormonal o nutricional. En el primer caso, el efecto inhibidor se atribuye a las hormonas que el fruto produce y exporta durante su desarrollo. En el segundo caso, el efecto inhibidor se atribuye a la alta demanda y consumo de carbohidratos por los frutos en desarrollo. Ambas hipótesis son complementarias y no excluyentes entre sí. Además, se ha demostrado que el fruto promueve la activación epigenética del represor de la floración CcMADS19 (homólogo en los cítricos del gen FLOWERING LOCUS C), que inhibe la expresión del gen CiFT3. Con el objetivo de determinar qué señal produce el fruto para inhibir la floración, en esta Tesis se propone la siguiente hipótesis: El fruto inhibe la floración a través de la síntesis y exportación de auxinas que activa la síntesis de giberelinas y, a su vez, la expresión de CcMADS19. Mediante experimentos con tratamientos exógenos de auxinas, giberelinas, y sus antagonistas, aclareo de frutos, y la interrupción del transporte por el floema entre el fruto y las yemas, los resultados indican que ni las giberelinas ni las auxinas se relacionan de forma consistente con la activación de la expresión de CcMADS19 en las hojas. En las yemas, las giberelinas se relacionan con la activación del gen inhibidor CENTRORRADIALIS (CEN), cuando hay fruto por aumento de la síntesis de GA4, y cuando no hay fruto por su aplicación exógena. La presencia del fruto aumenta la concentración de auxinas en el tallo y la yema en el momento de la inducción, y reprime su síntesis y trasporte. Pero esto no impide que, en la yema, el gen CcMADS19 esté epigenéticamente silenciado y que el silenciamiento se transmita a los nuevos brotes vegetativos. Estos brotes florecen en el siguiente ciclo, y, en sus yemas, la diferenciación floral se relaciona con un aumento de la síntesis y trasporte de auxinas y una reducción de la síntesis de giberelinas. / [CA] Als cítrics, les baixes temperatures promouen la inducció floral a la tardor i l'hivern augmentant l'expressió del gen promotor CiFT3 (homòleg en els cítrics del gen FLOWERING LOCUS T). La presència d'un gran nombre de fruita a l'arbre en aquest moment inhibeix l'expressió de CiFT3 i la floració, però es desconeix la senyal inhibidora que genera la fruita. Les hipòtesis majoritàriament acceptades proposen que la senyal pot ser hormonal o nutricional. En el primer cas, l'efecte inhibidor s'atribueix a les hormones que la fruita produeix i exporta durant el seu desenvolupament. En el segon cas, l'efecte inhibidor s'atribueix a la alta demanda i consum de carbohidrats per part de la fruita en desenvolupament. Ambdues hipòtesis són complementàries i no es descarten mútuament. A més, s'ha demostrat que la fruita promou l'activació epigenètica del repressor de la floració CcMADS19 (homòleg en els cítrics del gen FLOWERING LOCUS C), que inhibeix l'expressió del gen CiFT3. Amb l'objectiu de determinar quina senyal produeix la fruita per inhibir la floració, en aquesta Tesi es proposa la següent hipòtesi: La fruita inhibeix la floració mitjançant la síntesi i exportació d'auxines que activa la síntesi de giberelines i, al seu torn, l'expressió de CcMADS19. Mitjançant experiments amb tractaments exògens d'auxines, giberelines i els seus antagonistes, aclarida de fruita i la interrupció del transport pel floema entre la fruita i les brots, els resultats indiquen que ni les giberelines ni les auxines es relacionen de manera consistent amb l'activació de l'expressió de CcMADS19 a les fulles. A les gemmes, les giberelines es relacionen amb l'activació del gen inhibidor CENTRORRADIALIS (CEN) quan hi ha fruita per l'augment de la síntesi de GA4 i quan no hi ha fruita per la seua aplicació exògena. La presència de la fruita augmenta la concentració d'auxines a la tija i la gemma en el moment de la inducció i reprimeix la seua síntesi i transport. Però això no impedeix que, a la gemma, el gen CcMADS19 estigui epigenèticament silenciat i que el silenciament es transmeti als nous brots vegetatius. Aquests brots floreixen al següent cicle i, a les seues gemmes, la diferenciació floral es relaciona amb un augment de la síntesi i transport d'auxines i una reducció de la síntesi de giberelines. / [EN] In citrus, low temperature promotes flower induction in autumn-winter by increasing the expression of the CiFT3 promoter gene (citrus homologue of the FLOWERING LOCUS T gene). The presence of large numbers of fruits on the tree at this time inhibits CiFT3 expression and flowering, but the inhibitory signal produced by the fruits is unknown. The most widely accepted hypotheses are that the signal is hormonal or nutritional. In the first case, the inhibitory effect is attributed to hormones produced and exported by the fruit during development. In the second case, the inhibitory effect is attributed to the high demand and consumption of carbohydrates by the developing fruit. The two hypotheses are complementary and not mutually exclusive. In addition, it has been shown that the fruit promotes the epigenetic activation of the flowering repressor CcMADS19 (citrus homolog of the FLOWERING LOCUS C gene), which inhibits the expression of the CiFT3 gene. To determine which signal is produced by the fruit to inhibit flowering, the following hypothesis is proposed in this thesis: The fruit inhibits flowering through the synthesis and export of auxins, which activates the synthesis of gibberellins and, in turn, the expression of CcMADS19. Experiments with exogenous treatments of auxins, gibberellins and their antagonists, fruit thinning, and disruption of phloem transport between fruit and buds indicate that neither gibberellins nor auxins are consistently associated with the activation of CcMADS19 expression in leaves. In buds, gibberellins are associated with the activation of the flowering inhibitor CENTRORADIALIS (CEN), in the presence of fruit by increasing GA4 synthesis, and in the absence of fruit by its exogenous application. The presence of fruit increases the concentration of auxin in the stem and bud at the time of induction and suppresses its synthesis and transport. However, this does not prevent the epigenetic silencing of the CcMADS19 gene in the bud, which is transmitted to the leaves of the new vegetative shoots. These shoots flower in the following cycle, where floral differentiation is associated with an increase in auxin synthesis and transport and a decrease in gibberellin synthesis in the bud. / Marzal Blay, A. (2024). Study of the Fruit Inhibitory Mechanism on Citrus flowering. Nutritional, Hormonal and Genetic Factors [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203155

In this thesis, I combined comparative and experimental approaches to examine selection on reproductive traits and population differentiation in the insect-pollinated, self-incompatible, perennial herb Primula farinosa. More specifically, I (1) determined whether the effects of floral display and interactions with pollinators and seed predators, and plant reproductive success were frequency-dependent and affected by surrounding vegetation context, (2) examined the consequences of intermittent drought years on population dynamics using numerical simulations based on demographic data collected over seven years, (3) analyzed among-population differentiation in flowering phenology and reproductive allocation, and its relationship to soil-depth at the site of origin. A field experiment suggested that conspicuous plants facilitate inconspicuous plants in terms of pollinator attraction, and that the facilitation effect is contingent on the height of the surrounding vegetation. Further experiments revealed that both mutualistic and antagonistic interactions can result in frequency-dependent selection on floral display. Among inconspicuous plants, both fruit initiation, and damage from seed predators increased with the proportion of the conspicuous morph. The relative strength of these effects, and therefore their net outcome on the relationship between morph ratio and seed production varied among years. I combined information on vital rates and their relation to environmental conditions in simulations to predict future population viability in changing environments. Simulated stochastic population growth rate decreased with increasing frequency of drought years. Reproductive allocation varied significantly among populations both in the field and in a common-garden experiment, but was correlated with soil depth at the site of origin only in the field. The results suggest that among-population variation in reproductive effort in the field mainly reflects plastic responses to environmental conditions, and that this plasticity may be adaptive. The common-garden experiment suggested that the study populations have diverged genetically in flowering time.

Cis-regulatory changes in e.g. promoters or enhancers that affect the expression of a linked focal gene have long been thought to be important for adaptation. In this thesis, I investigate the selective importance and genomic correlates of cis-regulatory variation and divergence in the genus Capsella, using massively parallel sequencing data. This genus provides an opportunity to investigate cis-regulatory changes in response to polyploidization and mating system shifts, as it harbors three diploid species, the outcrosser Capsella grandiflora and the selfers Capsella orientalis and Capsella rubella, as well as the tetraploid Capsella bursa-pastoris. We first identify cis-regulatory changes associated with adaptive floral evolution in connection with the recent switch to self-fertilization in C. rubella and show that cis-regulatory changes between C. rubella and its outcrossing close relative C. grandiflora are associated with differences in transposable element content. Second, we show that variation in positive and purifying selection is important for the distribution of cis-regulatory variation across the genome of C. grandiflora. Interestingly, the presence of polymorphic transposable elements is strongly associated with cis-regulatory variation in C. grandiflora. Third, we show that the tetraploid C. bursa-pastoris is of hybrid origin and investigate the contribution of both parental species to gene expression. We show that gene expression in the tetraploid is partly explained by cis-regulatory divergence between the parental species. Nonetheless, within C. bursa-pastoris there is a great deal of variation in homeolog expression. In summary, this thesis explores the role of cis-regulatory changes for adaptive morphological changes in connection to a shift in mating system, the role of cis-regulatory divergence between progenitor species for an allopolyploid as well as the impact of positive and purifying selection on cis-regulatory variation within a species.

<div>Grand Sable Dunes, as a perched dune field on the shore of Lake Superior, is a sensitive</div><div>ecosystem subject to continual disturbance. Repeated natural disturbances necessitate specialized</div><div>plant communities to develop. There were two objectives of my research in this system that are</div><div>treated in separate chapters. They include: 1) the quantification of successional changes in the</div><div>plant community over time, and the identification of population demography changes for rare</div><div>species within the dunes and 2) the evaluation of evaluate in pollinator species for two plants</div><div>Hieracium caespitosum (Yellow Hawkweed) and Lithospermum caroliniense (Carolina</div><div>Puccoon).</div><div>For the first objective, target plant community composition and structures (i.e. richness,</div><div>diversity) were quantified in 2011 and 2018 across Grand Sable Dunes in 1 m² quadrats.</div><div>Additionally, two relatively rare plant species (Cirsium pitcheri and Tanacetum bipinnatum)</div><div>were selected to quantify demographic (i.e. flowering, non-flowering) patterns and changes over</div><div>time. Samples for C. pitcheri and T. bipinnatum were acquired via circle-plots 2.5 m in diameter.</div><div>Population comparisons between 2011 and 2018 illustrate minimal change in community</div><div>structure (richness and diversity). Composition increased slightly with eight species occurring in</div><div>2018, but not 2011. Additionally, community similarity was high (~78%) between the two years.</div><div>C. pitcheri occurrence was inversely related to presence of other species. Plant community</div><div>composition in eastern and western survey zones within the dunes appear to be diverging. Minor</div><div>changes in the plant community composition and structures indicate successional changes have</div><div>occurred, but without major disturbance. This divergence in community composition may be</div><div>related to weather related incidents associated with Lake Superior disturbance potential.</div><div>The secondary objective concerns pollinator species on two similar plant species found in</div><div>the Grand Sable Dunes. Individuals of H. caespitosum and L. caroliniense were observed and all</div><div>floral visitors were identified to family. The majority of arthropod families were observed</div><div>10</div><div>visiting both H. caespitosum and L. caroliniense, with an absence of typically important families</div><div>(e.g. Apidae, Bombiliidae). Halictidae, Muscidae and Syrphidae were the most common visitors,</div><div>with L. caroliniense attracting far more Muscidae than their H. caespitosum competitors.</div><div>Overlap in visitors for both species was observed, which may lead to decreased reproduction in</div><div>L. caroliniense.</div>

Doctor of Philosophy (PhD) / A renewed focus on generalised pollinator systems has inspired a conceptual framework which highlights that spatial and temporal interactions among plants and their assemblage of pollinators can vary across the individual, population, regional and species levels. Pollination is clearly a dynamic interaction, varying in the number and interdependence of participants and the strength of the outcome of the interaction. Therefore, the role of variation in pollination is fundamental for understanding ecological dynamics of plant populations and is a major factor in the evolution and maintenance of generalised and specialised pollination systems. My study centred on these basic concepts by addressing the following questions: (1) How variable are pollinators in a generalised pollination system? To what degree do insect visitation rates and assemblage composition vary spatially among populations and temporally among flowering seasons? (2) How does variation in pollinators affect plant reproductive success? I chose to do this using a model system, Trachymene incisa subsp. incisa (Apiaceae), which is a widespread Australian herbaceous species with simple white flowers grouped into umbels that attract a high diversity of insect visitors. The Apiaceae are considered to be highly generalist in terms of pollination, due to their simple and uniform floral display and easily accessible floral rewards. Three populations of T. incisa located between 70 km and 210 km apart were studied over 2-3 years. The few studies investigating spatial and temporal variation simultaneously over geographic and yearly/seasonal scales indicate that there is a trend for more spatial than temporal variation in pollinators of generalist-pollinated plants. My study showed both spatial and temporal variation in assemblage composition among all populations and variation in insect visitation rates, in the form of a significant population by year interaction. However, removing ants from the analyses to restrict the assemblage to flying insects and the most likely pollinators, resulted in a significant difference in overall visitation rate between years but no difference in assemblage composition between the Myall Lakes and Tomago populations. These results indicate more temporal than spatial variation in the flying insect visitor assemblage of T. incisa. Foraging behaviour provides another source of variation in plant-pollinator interactions. Trachymene incisa exhibits umbels that function as either male or female at any one time and offer different floral rewards in each phase. For successful pollination, pollinators must visit both male and female umbels during a foraging trip. Insects showed both preferences and non-preferences for umbel phases in natural patches where the gender ratio was male biased. In contrast, insects showed no bias in visitation during a foraging trip or in time spent foraging on male and female umbels in experimental arrays where the gender ratio was equal. Pollinator assemblages consisting of a mixture of different pollinator types coupled with temporal variation in the assemblages of populations among years maintains generalisation at the population/local level. In addition, spatial variation in assemblages among populations maintains generalisation at the species level. Fire alters pollination in T. incisa by shifting the flowering season and reducing the abundance of flying insects. Therefore, fire plays an important role in maintaining spatial and temporal variation in this fire-prone system. Although insect pollinators are important in determining the mating opportunities of 90% of flowering plant species worldwide, few studies have looked at the effects of variation in pollinator assemblages on plant reproductive success and mating. In T. incisa, high insect visitation rates do not guarantee high plant reproductive success, indicating that the quality of visit is more important than the rate of visitation. This is shown by comparing the Agnes Banks and Myall Lakes populations in 2003: Agnes Banks received the highest visitation rate from an assemblage dominated by ants but produced the lowest reproductive output, and Myall Lakes received the lowest visitation rate by an assemblage dominated by a native bee and produced the highest seedling emergence. Interestingly, populations with different assemblage composition can produce similar percentage seed set per umbel. However, similar percentage seed set did not result in similar percentage seedling emergence. Differences among years in reproductive output (total seed production) were due to differences in umbel production (reproductive effort) and proportion of umbels with seeds, and not seed set per umbel. Trachymene incisa is self-compatible and suffers weak to intermediate levels of inbreeding depression through early stages of the life cycle when seeds are self-pollinated and biparentally inbred. Floral phenology, in the form of synchronous protandry, plays an important role in avoiding self-pollination within umbels and reducing the chance of geitonogamous pollination between umbels on the same plant. Although pollinators can increase the rate of inbreeding in T. incisa by foraging on both male and female phase umbels on the same plant or closely related plants, most consecutive insect movements were between plants not located adjacent to each other. This indicates that inbreeding is mostly avoided and that T. incisa is a predominantly outcrossing species, although further genetic analyses are required to confirm this hypothesis. A new conceptual understanding has emerged from the key empirical results in the study of this model generalised pollination system. The large differences among populations and between years indicate that populations are not equally serviced by pollinators and are not equally generalist. Insect visitation rates varied significantly throughout the day, highlighting that sampling of pollinators at one time will result in an inaccurate estimate and usually underestimate the degree of generalisation. The visitor assemblage is not equivalent to the pollinator assemblage, although non-pollinating floral visitors are likely to influence the overall effectiveness of the pollinator assemblage. Given the high degree of variation in both the number of pollinator species and number of pollinator types, I have constructed a model which includes the degree of ecological and functional specialisation of a plant species on pollinators and the variation encountered across different levels of plant organisation. This model describes the ecological or current state of plant species and their pollinators, as well as presenting the patterns of generalisation across a range of populations, which is critical for understanding the evolution and maintenance of the system. In-depth examination of pollination systems is required in order to understand the range of strategies utilised by plants and their pollinators, and I advocate a complete floral visitor assemblage approach to future studies in pollination ecology. In particular, future studies should focus on the role of introduced pollinators in altering generalised plant-pollinator systems and the contribution of non-pollinating floral visitors to pollinator assemblage effectiveness. Comparative studies involving plants with highly conserved floral displays, such as those in the genus Trachymene and in the Apiaceae, will be useful for investigating the dynamics of generalised pollination systems across a range of widespread and restricted species.