The present dissertation aims to shed light on the molecular machinery involved in the process of shell formation (thecagenesis) in Arcella (Arcellinida : Amoebozoa). Arcellinida are single-celled testate amoebae organisms, characterized by the presence of an outer shell (test or carapace); it is a monophyletic lineage of Amoebozoa, sister group to a naked amoeboid lineage. No homologous structure to shell is present in the sister group of Arcellinida, thus it is considered an evolutionary novelty. The origin and evolution of the shell in Arcellinida are currently open questions; deciphering its formation process is a key step to address these questions. During each reproductive process by budding division, these organisms build a new shell. In the span of more than a century, several authors have described the thecagenesis process on Arcellinida, primarily focusing on the genus \\textit, based on cyto-morphological evidence. Conversely, the absence of molecular data has impaired advances on describing the molecular aspects of shell formation. In this study, we designed and applied a molecular framework to identify candidate genes and develop a molecular model for the shell formation process in Arcella; we based this framework on single-cell RNA-sequencing, gene expression profiling, Gene Ontology analysis, and comparative analysis of cyto-morphological with newly generated molecular data. We identify and propose a set of 539 genes as the candidate genes for shell formation, based on expression profiling and biological process assignment. We propose a model for the the shell formation process, which describes the mechanistic aspect of this process, hypothetically based on a molecular machinery conserved in Eukaryotes. Additionally, we identified a massive expansion of the Rab GTPase family, a protein likely to be involved on the process of shell formation. In the lights of the present study, we briefly discuss possible evolutionary scenarios involved on the origin and evolution of the shell and present future perspectives; we propose the shell of Arcellinida as a prosperous model to study the origin and evolution of evolutionary novelties, as well as other evolutionary questions / A presente dissertação tem como objetivo lançar luz sobre a maquinaria molecular envolvida no processo de formação de teca (tecagênese) em \\textit (Arcellinida: Amoebozoa). Arcellinida são amebas tecadas unicelulares, caracterizadas pela presença de uma teca (carapaça ou concha) externa; é uma linhagem monofilética de Amoebozoa, grupo irmão de alguns organismos amebóides nus. Nenhuma estrutura homóloga à carapaça está presente no grupo irmão de Arcellinida, sendo considerada como uma novidade evolutiva. A origem e evolução da carapaça em Arcellinida são questões em aberto; Decifrar seu processo de formação é um passo fundamental para abordar essas questões. Durante todo processo reprodutivo, por divisão por brotamento, estes organismo constroem uma nova concha. No decorrer de mais de um século, vários autores descreveram o processo de tecagênese nestes organismos, focando principalmente no gênero \\textit, baseados em evidências cito-morfológicas. Enquanto isso, a ausência de dados moleculares impede avanços na descrição dos aspectos moleculares da formação de conchas. Neste estudo, projetamos e aplicamos uma \\textit molecular para identificar genes candidatos e desenvolver um modelo molecular para o processo de formação de teca em \\textit; Baseamos este \\textit em sequenciamento de RNA \\textit, perfil de expressão gênica, análise de \\textit{Gene Ontology} e análise comparativa de dados cito-morfológicos e moleculares. Nós identificamos e propomos um conjunto de 539 genes como genes candidatos para a formação de carapaça, com base no perfil de expressão e na atribuição de processos biológica. Propomos um modelo para o processo de formação de carapaça, que descreve o aspecto mecanicista deste processo, hipoteticamente baseado em um mecanismo molecular conservado em Eucariotos. Além disso, identificamos uma expansão maciça da família gênica das Rab GTPase, gene provavelmente envolvida no processo de formação de carapaça. À luz do presente estudo, discutimos brevemente possíveis cenários evolutivos envolvidos na origem e evolução da teca e apresentamos perspectivas futuras; propomos a teca dos Arcellinida como próspero modelo para estudar a origem e evolução das novidades evolutivas, bem como outras questões evolutivas

This thesis consists of seven published papers on free-living amoebae (FLA), members of Amoebozoa, Excavata: Heterolobosea, and Cercozoa, and covers three main topics: (i) FLA as potential fish pathogens, (ii) diversity and phylogeography of FLA, and (iii) FLA as hosts of prokaryotic organisms. Diverse methodological approaches were used including culture-dependent techniques for isolation and identification of free-living amoebae, molecular phylogenetics, fluorescent in situ hybridization, and transmission electron microscopy.

My dissertation explores several aspects of the relationship between morphological and molecular evolution in amoeboid lineages: Chapter 1 - General Introduction: This chapter provides an overview of the most pressing issues in Amoebozoa phylogeny that are dealt with in the remainder of the thesis Chapter 2 - Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new generation high fidelity DNA polymerase: This chapter addresses the methodological difficulty in the study of large gene families, the generation of artifactual sequences by recombination during PCR. Chapter 3 - Evolution of the actin gene family in testate lobose amoebae (Arcellinida) is characterized by two distinct clades of paralogs and recent independent expansions: This chapter explores intriging patterns of evolution in the actin gene families of testate amoebae. Chapter 4 - Comprehensive phylogenetic reconstruction of Amoebozoa based on concatenated analysis of SSU-rDNA and actin genes: A deep phylogenetic analyses of the Amoebozoa, enables exploration of well supported taxonomic units within the group. Chapter 5 - Interpreting the evolutionary history of the Tubulinea (Amoebozoa), in light of a multigene phylogeny: This chapter explores a more restrict taxonomic unit within the Amoebozoa - the Tubulinea - based on an expanded sample of genes and taxa. Chapter 6 - The chastity of amoebae: re-evaluating evidence for sex in amoeboid organisms: This chapter asks whether the null-hypothesis that amoebae are asexual is consistent with current phylogenetic evidence

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals (including us humans) and Fungi. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. Here we have isolated a naked eukaryotic amoeba with filose subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a wholistic approach including light, electron, fluorescence microscopy and SSU rDNA, we find that this amoeboid organism fails to match any previously described eukaryote genus. Our isolate amoebae are most similar to some variosean amoebae which also possess acutely pointed filose subpseudopodia. Maximum likelihood and Bayesian tree of the SSU-rDNA gene places our isolate in Variosea of Amoebozoa as a novel lineage with high statistical support closely related to the highly diverse protosteloid amoebae Protostelium. This novel variosean is herein named “Hodorica filosa” n. g. n. sp. We present a robust phylogeny of Amoebozoa based on a broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea and Tevosa. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features Integrins are transmembrane receptors that activate signal transduction pathways upon extracellular matrix binding. The Integrin Mediated Adhesion Complex (IMAC), mediates various cell physiological processes and are key elements that are associated animal multicellularity. The IMAC was thought to be specific to animals. Over the last decade however, the IMAC complexes were discovered throughout Obazoa. We show the presence of an ancestral complex of integrin adhesion proteins that predate the evolution of the Amoebozoa. Co-option of an ancient protein complex was key to the emergence of animal multicellularity. The role of the IMAC in a unicellular context is unknown but must also play a critical role for at least some unicellular organisms.