The evolution of novel protein functions and protein families is a fundamental question within both evolutionary biology and biochemistry. While many gene families follow predictable patterns of molecular tinkering, many protein families exist with completely novel functions now essential. The guanylate kinase protein interaction domain (GKPID) of the membrane associated guanylate kinases (MAGUK) represents a model system for the study of protein evolution in which a protein scaffolding domain has evolved from a nucleotide kinase ancestor. Here we elucidate the ancient mechanisms by which these new functions evolved by combining ancestral protein reconstruction with in vitro and cell-biological molecular experiments. We found that the GKPID's capacity to serve as a mitotic spindle-orienting scaffold evolved by duplication and divergence of an ancient guanylate kinase enzyme before the divergence of animals and choanoflagellates. Re-introducing a single historical substitution into the ancestral guanylate kinase is sufficient to abolish the ancestral enzyme activity, confer the derived scaffolding function, and establish the capacity to mediate spindle orientation in cultured cells. This substitution appears to have revealed a latent protein-binding site, rather than constructing a novel interaction interface, apparently by altering the dynamics or conformational occupancy of a hinge region that determines whether the binding site is exposed or hidden. Three further substitutions also conveyed a measure of ligand specificity to phosphorylated Pins, which is necessary in metazoan spindle orientation pathways. These findings show how a small number of simple, ancient genetic changes caused the evolution of novel molecular functions crucial for the evolution of complex animals and laid the groundwork for an entirely new family of metazoan scaffolding proteins. This dissertation contains previously unpublished, co-authored material.

Deciphering the rapid emergence of bilaterian animals around the time of the Cambrian Explosion and reconstructing the interrelationships of animal groups have long been two of the most elusive problems in Zoology. This thesis concerns the phylogenetic interrelationships within and among Acoela and Nemertodermatida, two groups of small worms that are believed to be basal bilaterians and which may provide important clues for understanding the early evolution of animals. In addition to trying to resolve the phylogenetic positions of these groups, major focus is put on inferring how ancestral animals might have looked, given the phylogenetic hypotheses put forward. The data used to infer phylogenies include nuclear ribosomal DNA, the mitochondrial COI gene and microRNAs. Based on phylogenetic analyses of a large number of 18S SSU ribosomal DNA sequences, it is proposed that Cnidaria is the sister taxon to Bilateria. Poor taxon sampling is suggested to be one of the reasons for why earlier assessments of the interrelationships among the most basal animal groups have yielded many conflicting results using the same gene. Analyses of new 18S SSU rDNA and 28S LSU rDNA sequences from six of the nine known species of nemertodermatids corroborate earlier indications that Acoela and Nemertodermatida are not sister taxa, as once thought. Being separate basal bilaterian animal groups, it is suggested that the last common ancestor of all bilaterians shared much of their comparatively simple morphology. Many methods are deployed to assess whether the phylogenetic results are mainly due to long-branch attraction, but no indication of this artifact is detected. The first comprehensive phylogenetic framework of Acoela is reconstructed from the 18S SSU, 28S LSU and COI genes, in combination with morphological data. The ancestral acoel worm is reconstructed using Bayesian methods and morphological observations in extant species. Two indeces, posterior similarity and reconstruction signal, are implemented to assess how similar different species are to the last common ancestor of all acoels and illustrate how clearly different characters or nodes are reconstructed. It is suggested that the ancestral acoel looked much like extant species of Diopisthoporus. The phylogenetic positions of Acoela and Nemertodermatida are assessed using new data on microRNAs in the acoel Hofstenia miamia and the nemertodermatid Meara stichopi. Acoela and Nemertodermatida are again found to be basal bilaterians, in congruence with earlier results. Using the work-flow and indeces developed earlier, it is concluded that the bilaterian ancestral microRNA repertoire can not yet be reconstructed with high confidence. All papers stress the importance of inclusive taxon sampling for making generalized inferences about ancestral features in animals.

xvii, 167 p. : ill. (some col.) / The vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit enzyme complex responsible for acidification of cellular organelles. The V-ATPase hydrolyzes ATP to pump protons across membranes to create an electrochemical gradient. Acidification of vesicular compartments is critical in numerous biological processes including protein trafficking, endocytosis, and ion homeostasis; defects in V-ATPase function can also lead to human diseases. While the function of the V-ATPase enzyme is highly conserved across eukaryotes, the molecular architecture of this protein complex has undergone unique structural changes through evolutionary time. The goal of this work is to investigate the assembly, transport, and evolution of this critical molecular machine in the model organism <italic>Saccharomyces cerevisiae</italic>. A series of genetic screens was performed in budding yeast to identify factors and pathways that are involved in promoting full V-ATPase function. I utilized several "assembly factor" alleles to serve as sensitized genetic backgrounds to partially reduce enzyme function; this work implicated sphingolipid composition in promoting full vacuolar ATPase enzyme function. I also used ancestral gene reconstruction to analyze the two isoforms of subunit a of the V₀ subdomain (Vph1p and Stv1p) by recreating the most recent common ancestral subunit (Anc.a). Characterization of Anc.a demonstrated that this ancient subunit was able to properly assemble and function within a hybrid V-ATPase complex. While the Vph1p-containing complex localized to the vacuole membrane and the Stv1p-containing complex was present on the Golgi/endosome, incorporation of Anc.a caused the V-ATPase to localize to both types of cellular compartments. Finally, I used ancestral reconstruction to investigate the lineage-specific gene duplication of one of the proteolipid subunits of the V₀ subcomplex that occurred within the fungal clade. I demonstrate that inclusion of a third proteolipid subunit within fungi (as compared to two subunits within metazoans) could have occurred via neutral processes by asymmetric degeneration of subunit-subunit interfaces that "ratcheted" the duplicated subunit with the V₀ ring. These results present a model that describes how macromolecular machines can increase in complexity through evolutionary time. This dissertation includes previously published co-authored material and unpublished co-authored material. / Committee in charge: George Sprague, Chairperson; Tom H. Stevens, Advisor; Victoria Herman, Member; Bruce Bowerman, Member; Ken Prehoda, Outside Member

Nickel enzymes have been suggested, through numerous phylogenetic studies, to have been among the very first catalytic compounds on the early Earth, possibly present in the last universal common ancestor (LUCA) or prior to the onset of life. This is because of the type of reactions catalyzed by some of these enzymes, the nature of organisms that utilize them, their distribution in the tree of life, and their key roles in what is now thought of as possibly one of the oldest carbon fixation pathways, the Wood-Ljungdahl (WL) pathway. Additionally, nickel is generally thought to have been an abundant element on the early Earth, highly soluble in what were, theoretically, euxinic (anoxic and sulfidic) ocean waters. This combined with the fact that the enzymes involved in the WL pathway have an active center configuration that resembles that of minerals found in hydrothermal vent walls, makes nickel enzymes a likely candidate to have evolved from what were proto-enzymes, responsible for the prebiotic catalysis of the first simple organic molecules prior to the origins of life, according to the so-called submarine alkaline hydrothermal vent theory, first presented by Michael J. Russell in 1993 (Russell et al. 1994). In this study, I expand the known coverage on the distribution of these enzymes by mapping them in 10,575 OTUs of microbial taxa. Using their pattern of distribution, I reconstruct their histories along the branches of a reference phylogenetic tree of the same taxa through methods of ancestral reconstruction of discrete traits. Additionally, I construct an individual gene tree for each of the enzymes in order to consolidate gene history with species history. My results showed that the redox nickel enzymes (except methyl-coenzyme M reductase) are ancestral to all prokaryotes, while non-redox enzymes are derived and with multiple origins, possibly due to lateral gene transfer events or convergent evolution. I propose that the patterns observed are a product of the drastic changes during early Earth history, namely a hypothesized “nickel famine” or the Great Oxidation Event, which acted as selective pressures.

Orientador: Silvia Rodrigues Machado / Resumo: Leguminosae é a terceira maior família dentre as Angiospermas, é cosmopolita, podendo ocorrer em florestas tropicais úmidas, florestas secas, savanas, regiões mediterrâneas e desérticas. Papilionoideae, subfamília com maior riqueza de espécies de Leguminosae, apresenta Phaseoleae como uma de suas tribos de maior importância econômica e alto número de espécies. Cajaninae, por sua vez, é a maior subtribo de Phaseoleae, destacando-se por apresentar aproximadamente 490 espécies distribuídas em 10 gêneros. Os representantes desta subtribo possuem distribuição pantropical, com apenas Rhynchosia e Eriosema ocorrendo nos neotrópicos. De modo geral, as espécies desta subtribo ocorrem em campos graminosos, florestas tropicais secas, regiões semiáridas, áreas degradadas e ambientes propensos à passagem do fogo. A ocorrência das espécies de Cajaninae nestes diferentes ambientes pode estar relacionada à presença de estruturas secretoras, como glândulas vesiculares e tricomas de base bulbosa. Apesar do conhecimento sobre a existência destas estruturas peculiares neste grupo desde o século 19, nenhum estudo detalhado de anatomia, ultraestrutura ou estudos com enfoque evolutivo haviam sido realizados. A subtribo tem sido considerada monofilética, porém os poucos trabalhos filogenéticos existentes abordam Phaseoleae como um todo, possuindo uma amostragem muito baixa dos representantes de Cajaninae, o que não esclarece sua monofilia. Considerando o pouco conhecimento sobre as relações filogené... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leguminosae is the third largest family among Angiosperms, is cosmopolitan, occurring in humid tropical forests, dry forests, savannas, Mediterranean and desert regions. Papilionoideae, a subfamily with the highest species richness of Leguminosae, presents Phaseoleae as one of its tribes of major economic importance and high number of species. Cajaninae, in turn, is the largest subtribe of Phaseoleae, standing out to present approximately 490 species distributed in 10 genera. The representatives of this subtribe have pantropical distribution, with only Rhynchosia and Eriosema occurring in the neotropics. The species of this subtribe occur in greenfields, dry tropical forests, semi-arid regions, degraded areas and environments prone to fire. The occurrence of Cajaninae species in these different environments may be related to the presence of secretory structures, such as vesicular glands and bulbous based-trichomes. Despite the knowledge about the existence of these peculiar structures in this group since the 19th century, no detailed study of anatomy, ultrastructure or evolutionary approach studies had been performed. The subtribe has been considered monophyletic, but the few existing phylogenetic works address Phaseoleae as a whole, having a very low sampling of Cajaninae representatives, which does not clarify its monophyly. Considering the little knowledge about the phylogenetic relationships in Cajaninae, on the secretory structures that are peculiar to this group and the... (Complete abstract click electronic access below) / Doutor

La théorie de l’évolution repose sur la biologie moderne. Toutes les nouvelles espèces émergent d’une espèce existante. Il en résulte que différentes espèces partagent une ascendance commune, telle que représentée dans la classification phylogénétique. L’ascendance commune peut expliquer les similitudes entre tous les organismes vivants, tels que la chimie générale, la structure cellulaire, l’ADN comme matériau génétique et le code génétique. Les individus d’une espèce partagent les mêmes gènes mais (d’ordinaire) différentes séquences d’allèles de ces gènes. Un individu hérite des allèles de leur ascendance ou de leurs parents. Le but des études phylogénétiques est d’analyser les changements qui se produisent dans différents organismes pendant l’évolution en identifiant les relations entre les séquences génomiques et en déterminant les séquences ancestrales et leurs descendants. Une étude de phylogénie peut également estimer le temps de divergence entre les groupes d’organismes qui partagent un ancêtre commun. Les arbres phylogénétiques sont utiles dans les domaines de la biologie, comme la bio informatique, pour une phylogénétique systématique et comparative. L’arbre évolutif ou l’arbre phylogénétique est une exposition ramifiée les relations évolutives entre divers organismes biologiques ou autre existence en fonction des différences et des similitudes dans leurs caractéristiques génétiques. Les arbres phylogénétiques sont construits à partir de données moléculaires comme les séquences d’ADN et les séquences de protéines. Dans un arbre phylogénétique, les nœuds représentent des séquences génomiques et s’appellent des unités taxonomiques. Chaque branche relie deux nœuds adjacents. Chaque séquence similaire sera un voisin sur les branches extérieures, et une branche interne commune les reliera à un ancêtre commun. Les branches internes sont appelées unités taxonomiques hypothétiques. Ainsi, les unités taxonomiques réunies dans l’arbre impliquent d’être descendues d’un ancêtre commun. Notre recherche réalisée dans cette dissertation met l’accent sur l’amélioration des prototypes évolutifs appropriés et des algorithmes robustes pour résoudre les problèmes d’inférence phylogénétiques et ancestrales sur l’ordre des gènes et les données ADN dans l’évolution du génome complet, ainsi que leurs applications.[...] / The theory of evolution is based on modern biology. All new species emerge of an existing species. As a result, different species share common ancestry,as represented in the phylogenetic classification. Common ancestry may explainthe similarities between all living organisms, such as general chemistry, cell structure,DNA as genetic material and genetic code. Individuals of one species share the same genes but (usually) different allele sequences of these genes. An individual inheritsalleles of their ancestry or their parents. The goal of phylogenetic studies is to analyzethe changes that occur in different organisms during evolution by identifying therelationships between genomic sequences and determining the ancestral sequences and theirdescendants. A phylogeny study can also estimate the time of divergence betweengroups of organisms that share a common ancestor. Phylogenetic trees are usefulin the fields of biology, such as bioinformatics, for systematic phylogeneticsand comparative. The evolutionary tree or the phylogenetic tree is a branched exposure the relationsevolutionary between various biological organisms or other existence depending on the differences andsimilarities in their genetic characteristics. Phylogenetic trees are built infrom molecular data such as DNA sequences and protein sequences. Ina phylogenetic tree, the nodes represent genomic sequences and are calledtaxonomic units. Each branch connects two adjacent nodes. Each similar sequencewill be a neighbor on the outer branches, and a common internal branch will link them to acommon ancestor. Internal branches are called hypothetical taxonomic units. Thus,Taxonomic units gathered in the tree involve being descended from a common ancestor. Ourresearch conducted in this dissertation focuses on improving evolutionary prototypesappropriate and robust algorithms to solve phylogenetic inference problems andancestral information about the order of genes and DNA data in the evolution of the complete genome, as well astheir applications.

The placement of genetic information within bacterial genomes is intentionally organized, creates predictable gradients of molecular properties along the origin-terminus of replication axis. Previous studies have reported that genes located near the origin of replication generally have a higher expression level, increased dosage, and are more conserved than genes located near the terminus of replication. Additionally, substitution rates usually increases with increasing distance from the origin of replication. However, the constant reorganization of genetic information is often overlooked when considering spatial molecular trends. Here, we explore the interplay of genomic reorganization along the origin and terminus of replication axis of gene expression and substitutions in Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti. Using ancestral reconstruction to account for genome reorganization, we demonstrated that the correlation between the number of substitutions and distance from the origin of replication is significant but small and inconsistent in direction. In another study, we looked at the overall expression levels of all genes from the same bacteria, and confirmed that gene expression tends to decrease when moving away from the origin of replication. We looked specifically at how inversions - one type of genomic reorganization - impact gene expression between closely related strains of E. coli. Some inversions cause significant differences in gene expression compared to non-inverted regions, however, the variation in expression does not significantly differ between inverted and non-inverted regions. This change in gene expression may be due to the expression regulation properties of two nucleoid proteins, Histone-like Nucleoid-Structuring (H-NS) and Factor for inversion stimulation (Fis), who’s binding sites had a significant positive correlation with inverted regions. In conclusion, we highlight the impact that genomic rearrangements and location have on molecular trends in bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis, and to ensure accurate generalization of previously determined trends. Assuming that molecular trends are exclusively in one direction can be problematic. / Dissertation / Doctor of Philosophy (PhD)

En bio-informatique, comprendre comment les molécules d’ADN ont évolué au cours du temps reste un problème ouvert etcomplexe. Des algorithmes ont été proposés pour résoudre ce problème, mais ils se limitent soit à l’évolution d’un caractèredonné (par exemple, un nucléotide précis), ou se focalisent a contrario sur de gros génomes nucléaires (plusieurs milliardsde paires de base), ces derniers ayant connus de multiples événements de recombinaison – le problème étant NP completquand on considère l’ensemble de toutes les opérations possibles sur ces séquences, aucune solution n’existe à l’heureactuelle. Dans cette thèse, nous nous attaquons au problème de reconstruction des séquences ADN ancestrales en nousfocalisant sur des chaînes nucléotidiques de taille intermédiaire, et ayant connu assez peu de recombinaison au coursdu temps : les génomes de chloroplastes. Nous montrons qu’à cette échelle le problème de la reconstruction d’ancêtrespeut être résolu, même quand on considère l’ensemble de tous les génomes chloroplastiques complets actuellementdisponibles. Nous nous concentrons plus précisément sur l’ordre et le contenu ancestral en gènes, ainsi que sur lesproblèmes techniques que cette reconstruction soulève dans le cas des chloroplastes. Nous montrons comment obtenirune prédiction des séquences codantes d’une qualité telle qu’elle permette ladite reconstruction, puis comment obtenir unarbre phylogénétique en accord avec le plus grand nombre possible de gènes, sur lesquels nous pouvons ensuite appuyernotre remontée dans le temps – cette dernière étant en cours de finalisation. Ces méthodes, combinant l’utilisation d’outilsdéjà disponibles (dont la qualité a été évaluée) à du calcul haute performance, de l’intelligence artificielle et de la biostatistique,ont été appliquées à une collection de plus de 450 génomes chloroplastiques. / In Bioinformatics, understanding how DNA molecules have evolved over time remains an open and complex problem.Algorithms have been proposed to solve this problem, but they are limited either to the evolution of a given character (forexample, a specific nucleotide), or conversely focus on large nuclear genomes (several billion base pairs ), the latter havingknown multiple recombination events - the problem is NP complete when you consider the set of all possible operationson these sequences, no solution exists at present. In this thesis, we tackle the problem of reconstruction of ancestral DNAsequences by focusing on the nucleotide chains of intermediate size, and have experienced relatively little recombinationover time: chloroplast genomes. We show that at this level the problem of the reconstruction of ancestors can be resolved,even when you consider the set of all complete chloroplast genomes currently available. We focus specifically on the orderand ancestral gene content, as well as the technical problems this raises reconstruction in the case of chloroplasts. Weshow how to obtain a prediction of the coding sequences of a quality such as to allow said reconstruction and how toobtain a phylogenetic tree in agreement with the largest number of genes, on which we can then support our back in time- the latter being finalized. These methods, combining the use of tools already available (the quality of which has beenassessed) in high performance computing, artificial intelligence and bio-statistics were applied to a collection of more than450 chloroplast genomes.