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CYCLOIDEA (CYC) and DICHOTOMA (DICH) of the CYC2 clade of the TCP gene family have been shown to play a significant role in regulating the identity of the dorsal petals and abortion of the single dorsal stamen in Antirrhinum majus. It is believed that CYC2-like genes are responsible for the convergent evolution of floral zygomorphy, but their role in the development of actinomorphic flowers is still unknown. In Solanaceae, previous analysis has identified two paralogs of CYC2-like genes, CYC2A and CYC2B, resulting from a gene duplication that predates the origin the family. Virus-induced gene silencing (VIGS) is a technique to study the gene function by silencing specific target genes of interest, which is shown to be useful in diverse plant species. Here, we report on the role of CYC2-like genes during floral development in Solanaceae based on the results of VIGS using tobacco rattle virus (TRV)-based vector in Solanum lycopersicum having completely actinomorphic flowers and Nicotiana obtusifolia having slightly zygomorphic flowers. Our VIGS experiments in So. lycopersicum show that downregulation of both CYC2A and CYC2B leads to misshaped petals, the unequal growth of the petals, and most frequently increased number of petals, stamens and sepals, while the carpel and ovule morphology remain the same as the wild type. On the contrary, downregulation of CYC2A and CYC2B in N. obtusifolia results in reduced number of flower organs in sepals, stamens, and petals, however carpels remained the same. For both solanaceous species, silencing CYC2A and CYC2B changes the property of cytoplasm and retards the rate of pollen germination. Our findings suggest that the CYC2-like genes are likely involved in the floral development, mainly regulating the number of floral organs and pollen development in Solanaceae.

Observations of floral development indicate that floral organ initiation in pentapetalous flowers more commonly results in a medially positioned abaxial petal (MAB) than in a medially positioned adaxial petal (MAD), where the medial plane is defined by the stem and the bract during early floral development. It was proposed that the dominant MAB petal initiation might impose a developmental constraint that leads to the evolution of limited patterns of floral zygomorphy in Asteridae, a family in which the floral zygomorphy develops along the medial plane and results in a central ventral (CV) petal in mature flowers. Here, I investigate whether the pattern of floral organ initiation may limit patterns of floral zygomorphy to evolve in pentapetalous angiosperms. I analyzed floral diagrams representing 405 species in 330 genera of pentapetalous angiosperms to reconstruct the evolution of floral organ initiation and the evolution of developmental processes that give rise to floral zygomorphy on a phylogenetic framework. Results indicate that MAB petal initiation is the most common; it occupies 86.2% of diversity and represents the ancestral state of floral organ initiation in pentapetalous angiosperms. The MAD petal initiation evolved 28 times independently from the ancestral MAB petal initiation. Among the 34 independent originations of floral zygomorphy, 76.5% of these clades represent MAB petal initiation, among which only 47% of the clades result a CV petal in mature flowers. The discrepancy is explained by the existence of developmental processes that result in floral zygomorphy along oblique planes of floral symmetry in addition to along the medial plane. Findings suggest that although the early floral organ initiation plays a constraining role to the evolution of patterns of floral zygomorphy, the constraint diverges along phylogenetically distantly related groups that allow the independent originations of floral zygomorphy through distinct development processes in pentapetalous angiosperms. In additional study, the butterfly-like flowers of Schizanthus are adapted to pollination by bees, hummingbirds, and moths. I investigated the genetic basis of the zygomorphic corolla, for which development is key to the explosive pollen release mechanism found in the species of Schizanthus adapted to bee pollinators. I examined differential gene expression profiles across the zygomorphic corolla of Schizanthus pinnatus, a bee-pollinated species, by analyzing RNA transcriptome sequencing (RNA- seq). Data indicated that CYC2 is not expressed in the zygomorphic corolla of Sc. pinnatus, suggesting CYC2 is not involved in the development of floral zygomorphy in Schizanthus (Solanaceae). The data also indicated that a number of genes are differentially expressed across the corolla.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em plantas dependentes da polinização biótica, traços florais podem incluir pistas táteis, olfativas e visuais e fatores como cor, formato, odor ou combinações dessas pistas podem ser utilizados para identificar as flores como fonte de recompensas.Um dos componentes que atraem os polinizadores é a simetria, utilizada para discriminação entre flores com diferentes quantidades de recursos e pode ser alterada pela atividade de herbívoros florais, capazes de interferir na quantidade de visitas do polinizador ou alterar a eficiência da polinização. As espécies de Malpighiaceae são conhecidas por ofertar pólen e óleo aos seus visitantes, além de ser uma família importante no Cerrado, um dos ecossistemas que mais sofre degradação no mundo. Diante disso, os objetivos desse estudo foram: a) observar a relação entre simetria floral natural e recursos ofertados aos polinizadores e se b) há discriminação pelos polinizadores entre flores simétricas e flores experimentalmente assimétricas, ou seja, artificialmente danificadas simulando herbivoria e seus efeitos sobre a polinização e consequente produção de frutos. A coleta de dados foi realizada em uma área de cerrado do Clube de Caça e Pesca Itororó de Uberlândia, de setembro de 2013 a julho de 2014. A taxa de simetria floral natural e os recursos florais foram observados nas flores de 24 indivíduos de Byrsonima intermedia (murici-pequeno), das quais 3 inflorescências foram ensacadas na pré-antese e três flores de cada inflorescência tiveram suas medidas tomadas. A partir do eixo longitudinal central da pétala estandarte foram medidas as larguras dos lados direito e esquerdo. Foram medidos os comprimentos das duas pétalas imediatamente ao lado da pétala estandarte (pétalas proximais) pelos seus eixos longitudinais centrais. Foram medidas, ainda, as distâncias do ápice da pétala estandarte até o ápice das duas pétalas mais distantes, ou pétalas distais. Cada flor medida foi também utilizada para a quantificação do volume de óleo de seus elaióforos com tubo capilar e quantificação dos grãos de pólen, retirados e corados com Carmin Acético. Nas mesmas plantas, foram escolhidas quatro flores que tiveram 10% das áreas das pétalas estandarte, proximais ou distais retiradas e quatro controles, que não receberam manipulação. Essas flores foram acompanhadas até a frutificação. Não foram encontradas diferenças significativas entre a quantidade de óleo ofertada aos polinizadores e a simetria floral (r= -0.1407, df= 22, p= 0.5118), bem como para quantidade de pólen (r= -0.0079, df= 22, p= 0.9707), o que indica que, para esse sistema de estudo, a simetria não pode ser utilizada como parâmetro dos recursos disponíveis. Foi encontrada diferença significativa na proporção de frutos formados entre o grupo controle e o grupo tratamento (X²=91,147, gl=3, p<0,01), mas não entre os tratamentos (X²=2,026, gl=2, p=0,363), o que sugere que as manipulações realizadas nas pétalas influenciaram a visitação e consequente polinização, mas o local onde essa manipulação foi realizada não é tão importante. / For plants which rely on biological pollination, floral traits related to pollination can include tactile, olfactory and visual cues and color, shape and odor or combinations of these cues can be used to identify flowers as source of rewards. One of the components that attract pollinators is symmetry, used to discriminate flowers with different levels of rewards. Symmetry can be altered by the activity of floral herbivores, capable of interfering on pollinator visitation rate or alter pollination efficacy. Malpighiaceae species are known to offer pollen and oil to their floral visitors and compose an important family on Brazilian savannah (Cerrado), one of the most degraded ecosystems in the world. The aim of this study was to: a) observe the relation between natural floral symmetry and floral rewards and b) assess pollinator discrimination of experimentally asymmetrical flowers, simulating floral herbivory, and its effects over pollination and fruit set. Data collection was performed in a Cerrado area inside Clube Caca e Pesca Itororo de Uberlandia, from September 2013 to July 2014. Natural floral symmetry and floral rewards were observed in flowers of 24 bushes of Byrsonima intermedia (Malpighiaceae), in which 3 inflorescences were bagged in pre-anthesis phase and three flowers from each inflorescence had their measures taken. The width of right and left sides of the standard petal was measured as well as the length of proximal petals from the central longitudinal axis . The distance of the apex of standard petal to the distal petals was also measured. Each measured flower was then used to the quantification of elaiophores oil volume with a capillary tube and pollen grain quantification, dyed with Acetic Carmin. On the same plants, we chose four plants that had 10% of petal area removed, on standard, proximal or distal petal and four control plants, which were not manipulated. These flowers were followed to fructification and the seeds were placed in gerbox boxes over germination paper. There were no statistical differences between oil quantity and floral symmetry (r= -0.1407, df= 22, p= 0.5118) as well as pollen quantity (r= -0.0079, df= 22, p= 0.9707), which indicates that, for this particular plant, symmetry is not a reliable sign of floral rewards. There was statistical differences in the proportion of fruits formed from control and treatment groups (X²=91,147, gl=3, p<0,01), but not among treatments (X²=2,026, gl=2, p=0,363), what suggests that petal manipulation have an effect on pollinator visitation, but the point of manipulation is not that significant. / Dissertação (Mestrado)

Les Angiospermes forment un clade connu pour sa grande diversité d’espèces et une répartition inégale de cette diversité en son sein. Ce travail se concentre sur la reconstruction d’états ancestraux de caractères floraux sur les phylogénies dans le but d’étudier leur impact sur la diversification. En premier lieu, nous nous sommes focalisés sur deux innovations-clés potentielles chez les Proteaceae, la symétrie bilatérale du périanthe et la présence dans les hotspots à climat méditerranéen. L’utilisation sur une phylogénie de modèles de diversification dépendants des états de caractère a permis de montrer que le premier état caractère n’a pas d’impact significatif sur la diversification des espèces dans ce groupe, contrairement au second. Ensuite, nous avons reconstruit les états ancestraux de la symétrie du périanthe à l’échelle de toutes les Angiospermes, avec une stratégie d’échantillonnage permettant de refléter toutes les transitions de ce caractère. Nous avons mis en évidence un minimum de 130 origines de la symétrie bilatérale, suivies de 69 réversions vers la symétrie radiaire. Enfin, la même méthode a été étendue à l’étude de quatre autres caractères du périanthe. Différents modèles de reconstruction d’états ancestraux ont été utilisés sur une phylogénie de 1232 espèces pour examiner l’influence des changements de modèle sur les états ancestraux reconstruits. Nos résultats montrent que les changements de modèles de reconstruction ont une influence sur l’état de certains nœuds seulement, jamais sur l’ensemble. Les différents résultats trouvés pour la symétrie, caractère commun aux trois chapitres, révèlent que notre méthode d’échantillonnage présente l’inconvénient d’estimer des taux de transition trop élevés pour donner des résultats concluants avec la méthode du maximum de vraisemblance. Les résultats sont en revanche beaucoup moins biaisés lorsque l’on examine plusieurs caractères simultanément. / Angiosperms are a clade known for its great species diversity and the uneven distribution of this diversity among its lineages. This work focuses on the ancestral state reconstruction of floral characters on phylogenies in the purpose of studying their impact on diversification. We first focused on two potential key innovations in Proteaceae, bilateral perianth symmetry and presence in Mediterranean-climate hotspots. Using character state dependent diversification models, we found that the first character state did not have any significant impact on species diversification in this group, contrary to the second one. We then reconstructed ancestral states for perianth symmetry in angiosperms as a whole, using a sampling strategy aimed at capturing all of the transitions in this character. We found a minimum of 130 origins of bilateral symmetry, followed by 69 subsequent reversals to radial symmetry. Lastly, the same approach was extended to the study of four other perianth characters. Different ancestral state reconstruction models were used on a phylogenetic tree of 1232 species to test the influence of model changes on reconstructed ancestral states. Our results show that changes in reconstruction models have an impact on the inferred ancestral state of some nodes, but not all of them. The various results obtained for symmetry, a character shared among the three chapters, indicate that our sampling method has the drawback of estimating transition rates too high to give conclusive results with maximum likelihood. On the other hand, results are much less biased when several characters are examined simultaneously.

Specialization on insect and animal pollinators is thought to be the driving force for the evolution of floral traits. Specifically in the New World (NW), the oil-bee pollination syndrome has led to the convergence of floral characters in two distantly related families of core eudicots, Malpighiaceae and Krameriaceae. Both families display a flag-like structure that establishes a zygomorphic flower and floral oil rewards in epithelial elaiophores. These traits work concomitantly to attract and reward female oil-bees that help fertilize these flowers and in return receive oils. The underlying genetics of floral zygomorphy were studied in several clades of core eudicots, which implicated CYCLOIDEA2-(CYC2-)like genes of the TCP gene family to play a role in the establishment and maintenance of this trait. In Malpighiaceae, previous work demonstrated that two CYC2-like genes, CYC2A and CYC2B, are expressed during development correlated with establishing zygomorphy in flowers of NW Malpighiaceae. In this thesis work, I investigated the underlying developmental and genetic basis for the establishment of non-homologous and yet functionally similar traits in the oil-bee pollinated flowers of Malpighiaceae and Krameriaceae. In Chapter 1, I investigated the modification of a conserved CYC2 genetic program in the Old World (OW) acridocarpoids of Malpighiaceae following a major biogeographic disjunction. And in Chapter 2, I studied the floral ontogeny and genetic basis of floral zygomorphy development in Krameria lanceolata Torrey of Krameriaceae. This thesis work sheds light on the significance of the interdisciplinary study of floral symmetry evolution through comparative pollination ecology, development, and genetics.