Background: Relatively little is known about the genomic basis and evolution of wood- feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. Results: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. Conclusions: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

Philosophiae Doctor - PhD / Plants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.

Brachyspira species are bacteria that colonize the intestines of some mammalian and avian species with different degrees of pathogenicity. Brachyspira intermedia is a mild pig and bird pathogen with an unknown genomic sequence. In this project, we completed the genome of Brachyspira intermedia PWS/AT and did a comparative genomic analysis between B. intermedia PWS/AT and the already completed genomes of B. hyodysenteriae WA1, B. murdochii 56-150T and B. pilosicoli 95/1000. A table containing 15 classes of unique and shared genes was developed and analyzed in order to gain a better understanding of species-specific traits and clues behind the different degree of pathogenicity. Our result shows that genes are overall poorly annotated and further studies are of great importance for understanding different and shared properties. The largest number of unique features was found in B. intermedia and B. murdochii. B. hyodysenteriae and B. pilosicoli has most likely developed independently towards different biological niches and B. pilosicoli has undergone a major reductive evolution. One plasmid and six prophages were found in B. intermedia, where two of the phages appear to be capable of horizontal gene transfer. Further genome sequencing of more strains will probably increase the understanding of species-specific traits even more.

The angiosperm order Malpighiales includes \(\sim 16,000\) species and constitutes up to 40% of the understory tree diversity in tropical rain forests. Despite remarkable progress in angiosperm phylogenetics during the last 20 years, relationships within Malpighiales have remained poorly resolved, possibly due to its rapid rise during the mid-Cretaceous. Using phylogenomic approaches, including analyses of 82 plastid genes from 58 species, we identified 12 new clades in Malpighiales and substantially increased resolution along the backbone (Chapter 1). This greatly improved phylogeny revealed a dynamic history of shifts in net species’ diversification rates across Malpighiales, with bursts of diversification noted in the Barbados cherries (Malpighiaceae), cocas (Erythroxylaceae), and passion flowers (Passifloraceae). We also found that commonly used a priori approaches for partitioning data in similar large-scale analyses, by gene or by codon position, performed poorly relative to the use of partitions identified a posteriori using a Bayesian mixture model. Another aspect of my thesis focused on investigating horizontal gene transfer (HGT) in Malpighiales. Recent studies have suggested that plant genomes have undergone potentially rampant HGT. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. Using phylogenomic approaches, we analyzed the nuclear transcriptome (Chapter 2) and mitochondrial genome (Chapter 3) of the holoparasite Rafflesiaceae, which represents an enigmatic subclade of Malpighiales. Our analyses show that several dozen actively transcribed nuclear genes, and as many as 34–47% of its mitochondrial gene sequences, show evidence of HGT depending on the species. Some of these HGTs appear to have maintained synteny with their donor and recipient lineages suggesting that vertically inherited genes have likely been displaced via homologous recombination, as is common in bacteria. Finally, our results establish for the first time that although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. Moreover, the elevated rate of unidirectional host-to-parasite gene transfer raises the possibility that HGTs may provide a fitness benefit to Rafflesiaceae for maintaining these genes.

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Many diverse microbes have been detected in or isolated from glaciers, including novel taxa exhibiting previously unrecognized physiological properties with significant biotechnological potential. Of 29 unique phylotypes isolated from Victoria Upper Glacier, Antarctica (VUG), 12 were related to the poorly studied bacterial genus Hymenobacter including several only distantly related to previously described taxa. Further study of these microorganisms revealed genotypic, phenotypic, morphological and chemotaxonomic divergence from named species and suggested that they likely represent novel Hymenobacter species. These studies also indicated, however, that the systematics of Hymenobacter and related microorganisms is more complex than previously realized, and may exhibit poorly defined species boundaries due to cosmopolitan dispersal, significant rates of horizontal gene transfer and reintroduction of archived genotypes, e.g., from glacial ice. These processes are reflected in the carotenoid composition of Hymenobacter and related organisms, which includes several novel methyl- and xylosyl-derivatives of 2'-hydroxyflexixanthin with distributions indicative of horizontal gene transfer or differential gain and/or loss of terminal biosynthetic pathway steps. These processes have been previously underappreciated in assessments of microbial carotenoid diversity and suggest the need for fine-scale phylogenetic study of carotenoid distribution in other microbial taxa. Further comparative genomics-based evaluation of microbial carotenoid biosynthesis indicated its wide phylogenetic distribution and diversification, controlled by several lineage-specific modes of evolution including horizontal transfer, de novo enzyme evolution followed by differential gene loss, co-evolution with biochemically associated structures and elevated mutation rates. The latter especially interacts with horizontal transfer depending on metabolic pathway topology, exemplified by the evolution of purple bacterial carotenoid biosynthesis. Exploration of VUG microbial diversity, therefore, not only revealed novel taxa and biotechnologically interesting compounds but also spurred broader evaluation of the mechanisms of metabolic pathway evolution applicable to many other taxa and biochemical pathways. / Microbiology and Cell Biotechnology

Orientador: Maria do Carmo Bittencourt de Oliveira / Banca: Luiz Henrique Zanini Branco / Banca: Mariana Cabral de Oliveira / Resumo: As cianobactérias apresentam uma ampla variabilidade fenotípica e ecológica. Porém, esta variabilidade, muitas vezes, não corresponde à sua diversidade genética. Assim, o uso de marcadores moleculares é fundamental para os estudos de filogenia neste grupo. Entretanto, a filogenia molecular enfrenta um desafio na seleção dos marcadores devido à ocorrência relativamente frequente da transferência de genes de forma lateral entre os procariotos. Em cianobactérias os marcadores dos espaçadores dos genes ribossomais (16S-23S-ITS rDNA) e do operon da ficocianina (PC-IGS) estão entre os mais utilizados nestes estudos. Contudo, alguns trabalhos sugerem que o PC-IGS possa ter sito transferido lateralmente em sua história evolutiva. A identificação de morfoespécies dos gêneros Microcystis e Geitlerinema é baseada em caracteres morfológicos que em geral não correspondem à sua variabilidade genética. Com o objetivo de investigar a transferência lateral do operon da ficocianina em Geitlerinema e Microcystis, foram obtidas e comparadas árvores filogenéticas de ambas espécies baseadas nos marcadores PC-IGS e 16S-23S-ITS rDNA. As topologias das árvores obtidas para ambos os marcadores foram muito semelhantes e indicaram que o PC-IGS é estável e indicado para os estudos de taxonomia e filogenia de linhagens de Geitlerinema e Microcystis. Assim, hipótese inicial de transferência lateral foi refutada. Algumas linhagens tiveram seu posicionamento divergente entre um marcador e outro, o que ressalta a importância do uso de mais de um marcador em estudos de filogenia. O marcador PC-IGS apresentou melhor desempenho que 16S-23S-ITS rDNA. As árvores filogenéticas de Geitlerinema baseadas em ambos os marcadores indicaram a ocorrência de espécies crípticas dentre as linhagens estudadas e corroboraram que G. amphibium e G. unigranulatum devem ser consideradas sinonímias... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cyanobacteria show a wide phenotypic and ecological variability, but frequently this variability does not correspond to their genetic variation. Therefore, the use of molecular markes is critical for phylogenetic studies in this group. At the same time, the selection of molecular markers represents a challenge for the molecular phylogeny due to the horizontal gene transfer, witch is a relatively common process among the prokaryotes. In cyanobacteria, makers for the ribosomal genes spacer (16S-23S-ITS rDNA) and for the phycocyanin operon spacer (PC-IGS) are among of the most used for phylogeny. However, some studies suggest that the PC-IGS marker may have been horizontally transferred during its evolutionary history. The identification of the morphospecies from the genus Microcystis and Geitlerinema is based in their morphological characters, but they generally do not correspond to genetic variability. In order to investigate the possibility of horizontal transfer of the phycocyanin operon in Microcystis and Geitlerinema, phylogenetic trees based on the PC-IGS and 16S- 23S-ITS rDNA were generated and compared. The topologies obtained for both markers were very similar, indicating that the PC-IGS marker is stable and suitable for taxonomical and phylogenetic studies in Microcystis and Geitlerinema. Therefore, the initial hypothesis of horizontal transfer was rejected. Some strains were found to have divergent positions between the trees based on the two molecular markes, witch highlights the importance of using more than one marker in phylogenetic studies. The PC-IGS marker performed better than 16S-23SITS rDNA. The Geitlerinema phylogenetic trees based on both markers indicated the occurrence of cryptic species among the strains and corroborated that G. amphibium and G. unigranulatum should be treated as synonyms. The phylogenetic tree based on PC-IGS formed a monophyletic clade... (Complete abstract click electronic access below) / Mestre

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Organellar genomes are remnants of more complex bacterial genomes reduced until reach the simplest and most efficient content. Regularly depicted as circular, these genomes can form other structures, like linear, ramified, or entangled chromosomes, or a combination of those. Nonetheless, their gene content is nearly constant throughout flowering plants based on the multiple plastid genomes (plastomes) and the comparatively few mitochondrial genomes (mitogenomes) sequenced to date. Here, I explored the evolution of the organellar genomes in orchids from a phylogenetic perspective. For this research, plastomes and mitogenomes were assembled from short pair-ended reads obtained using Illumina sequencing technology. I developed a workflow to confidently recover plastid and mitochondrial sequences, even for regions without references in databases (chapter 1). The comparison among taxa from all orchid subfamilies identified patterns of gain, loss and rearrangement of coding and non-coding DNA. Plastid and mitochondrial protein-coding genes present in all samples were used to reconstruct the phylogenetic history of orchids that was coincident in terms of topology (chapter 1). Plastomes can suffer degradation in heterotrophic species, however that is not true for mixotrophic species, as I discovered by comparing albino and green individuals of the orchid Epipactis helleborine. I found that albino plants did not suffer loss of any genes and that the sequence was almost identical to the photosynthetic plants (chapter 2). In contrast to what it is observed in angiosperm plastomes, for which the structure, content and size is conserved, plant mitogenomes are highly variable in size, which can increase by the acquisition of external DNA via horizontal gene transfer. In some orchids, the mitogenome hosts a sixteen-gene sequence transferred from a fungal mitogenome to a clade of epidendroid orchids 12-60 My ago, and has been fragmented, conserved, or fully lost since (chapter 3). Transfer RNA genes are variable in number and origin throughout orchid evolution. I identified that they had four different sources, three novel possible replacement events of the native genes with plastid-origin genes, seven tRNA remodeling events in orchids and three more in other angiosperms (chapter 4). Our comparative studies conclude that there are three main dynamics that shape the organellar genomes: gain, loss and rearrangement of genomic content. I presented examples of them in orchids (chapter 5). Additionally, I created two sets of genomic resources: one comprises eighteen new orchid mitogenomes and plastomes, and the second consists of a well-curated set of references of tRNA genes in mitogenomes discriminated by origin. These results contribute to increasing the knowledge of angiosperm organellar genomes and highlight the importance of comprehensive studies that allow the interpretation of the genomic changes in the light of the phylogenetic evolution.

Communities are defined as the ensemble of populations that interact with each other and with the environment in a specific time and location. Community ecology studies how communities assemble, what are the patterns of diversity, abundance, and composition of species, and the processes driving these patterns. It includes four basic mechanisms for the assembly of communities: dispersal, drift, selection, and speciation, with each mechanism influencing how the communities change in a different way. Dispersal, the movement of species from one geographical location to another, plays a major role in the recolonization of barren environments and the introduction of new species to established environments. Drift (i.e., random birth and death events within a community) could, theoretically, be negligible in bacterial communities where the high population densities are expected to buffer its effect. Conversely, horizontal gene transfer can be a strong selective force, as horizontally transferred genetic material is a source of functional traits that may provide selective advantages to the recipient cells, especially in environments where strong selection pressure occurs. In my Ph.D. thesis, I aim to examine these three contrasting mechanisms in controlled, simplified bacterial communities that are designed and studied through a synthetic ecology approach. I found that even at low dispersal rates, the species abundance of planktonic bacterial communities can be homogenized by migration. This homogenization can occur even when there are strong variable selection forces interacting in each environment. I also found strong evidence on the importance of stochasticity in communities. Drift can decrease the community similarity by up to 6.3%, and increases the probabilities that species become extinct, especially in the case of rare taxa. In contrast, I found that naturally competent bacteria are favored to uptake more DNA in communities that are highly productive and phylogenetically diverse. This pattern is explained by a potential higher availability of naked DNA for naturally competent bacteria, presumably because there are more cells and the predation systems are more effective. Altogether, our findings support the theory on the importance of stochastic forces and their interaction with deterministic forces on the shaping of microbial community assembly.