The aim of this thesis was to study the evolution of reproductive systems and reproductive traits in the fungal genus Neurospora. More specifically, I have investigated the evolutionary forces shaping the genes involved in sexual reproduction, focusing on mating-type (mat) and pheromone receptor (pre) genes. Neurospora contains species exhibiting three different mating systems, i.e., heterothallism (self-incompatibility), homothallism (self-compatibility) and pseudohomothallism (partial self-incompatibility). First, a robust phylogeny of Neurospora was established. The phylogenetic analyses revealed multiple independent transitions in reproductive life style during the evolutionary history of the genus. We argued for a heterothallic ancestor of the genus, although our subsequent ancestral reconstruction analyses favored a homothallic ancestor. To be able to settle the ancestral mating system, we zoomed in on the structural architecture of the mat-locus in four homothallic species of Neurospora, thought to have arisen from independent transitions. Our results led us to suggest two different genetic mechanisms (translocation and unequal crossover) to explain the transitions in mating system from heterothallism to homothallism. We pointed out that the mating-system transitions in Neurospora are unidirectional, and suggested that transposable elements might be driving the transitions. In conclusion, we suggest a heterothallic ancestor for Neurospora, and that at least six transitions to homothallism and two transitions to pseudohomothallism have occurred in its evolutionary history. Further, we used the phylogeny of Neurospora as a framework to test if the evolution of pre-genes (pre-1 and pre-2) in hetero- and homothallic Neurospora is dependent on mating systems and/or even the homothallic clades themselves (i.e., mating-system and/or switch-dependent). The molecular evolution results suggest that pre-1 and pre-2 are overall functional in both homothallic and heterothallic Neurospora. The molecular evolution of pre-1 seems to be independent of mating-system or homothallic clade, and we detected signs for positive selection in the C-terminal tail. For pre-2 we found no support for mating-system dependent evolution, but indications for switch-dependent evolution. In this study we also included expression analyses of both pre- as well as mat-genes, with the prospect to assess functionality and regulation. During this thesis work, we also performed a phylogenetic study were we found that reproductive genes might be more permeable to introgression than other genes, which is in contrast to theoretical expectations. In the last study, we confirmed the co-existence of two alternative splice variants of the pheromone receptor gene pre-1 in Neurospora crassa, and performed expression profiles studies using quantitative RT-PCR. I hope this thesis work will further strengthen Neurospora as a model for research in evolutionary genetics.

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T17:23:36Z (GMT). No. of bitstreams: 1 Almeida_LudimilaDias_M.pdf: 4371531 bytes, checksum: 15deaceac3a09118222822415b70daaf (MD5) Previous issue date: 2015 / Resumo: O ciclo sexual de basidiomicetos é controlado pelo sistema mating type. Este é formado por dois loci multigênicos não ligados A e B, o locus A codifica duas proteínas homeodomínio HD1 e HD2, capazes de heterodimerização, enquanto o locus B apresenta genes para receptores de feromônio e feromônios. Em fungos heterotálicos, o desenvolvimento sexual depende da especificidade entre os quatro alelos, sistema este chamado tetrapolar, e é ativado apenas por interações específicas entre alelos parentais necessariamente diferentes, assegurando que hifas geneticamente iguais sejam incompatíveis. Em contrapartida, a condição na qual hifas geneticamente iguais são compatíveis é denominada homotalismo. Fungos basidiomicetos são tipicamente heterotálicos, no entanto, apesar de pertencer a este filo, o fitopatógeno Moniliophthora perniciosa, causador da doença Vassoura de Bruxa no cacaueiro, é classificado como homotálico primário. Curiosamente, apesar desta classificação, M. perniciosa contém um sistema genético tetrapolar, sendo o primeiro fungo descrito com essa característica. Neste trabalho, foi realizada a caracterização dos loci mating type em M. perniciosa e verificamos o perfil transcricional destes genes com o objetivo de entender os mecanismos moleculares que atuam no seu comportamento homotálico. Primeiramente, foram identificados no genoma um locus A e um locus B, além de genes atuantes no processamento e sinalização em resposta aos feromônios. O estudo do perfil transcricional destes genes revelou que um receptor tem um perfil de expressão condizente com a fase do ciclo de vida do fungo na qual ocorre o processo de dicariotização. A análise funcional dos receptores foi realizada em um sistema expressão heteróloga, promissor para o estudo de GPCRs (G coupled proteins receptors), porém não permitiu confirmar a presença de alelos compatíveis de receptores e precursores de feromônios no genoma de M. perniciosa como uma possível explicação ao comportamento homotálico. Tendo em vista o locus A, este é formado por um par MpHD1 e MpHD2, o que difere de outros basidiomicetos devido a inserção de uma sequência (11,958kb) interrompendo seus promotores. A hipótese neste cenário é que o transposon encontrado no locus A poderia ter permitido um crossover desigual que trariam genes compatíveis para o mesmo alelo, sendo responsável pelo homotalismo na espécie. Contrariando essa hipótese, os dados obtidos neste projeto indicam que uma possível transição prévia ao homotalismo resultou em uma pressão seletiva relaxada sobre os loci mating type, cuja consequência foi a degeneração nos genes destes loci. Neste contexto, os genes do mating type poderiam não estar mais envolvidos na dicariotização. Este trabalho, portanto, fornece importantes dados para o entendimento da biologia sexual deste fungo, o que futuramente poderá ser correlacionado a sua fitopatogenicidade / Abstract: The basidiomycetes¿ sexual cycle is controlled by the mating type system. The structure of this system comprises two unlinked multigenic loci, A and B. The A locus codes for homeodomain proteins, HD1 e HD2 which form a heterodimer, and B locus presents pheromone receptors and pheromones. In outcrossing (heterothallic) fungi, sexual development depends on the compatibility of four genes in two different allelic versions in a so-called tetrapolar system, and is strictly activated by specific interactions between different parental alleles, ensuring that genetically identical hyphae are incompatible. The phytopathogen Moniliophthora perniciosa causes Witches¿ broom disease in cacao plants, and it is a typical basidiomycete fungi. However, it completes its sexual development through the crossing of genetically identical hyphae, and is the first described homothallic fungi with a complete tetrapolar genetic system. Here we show the characterization of the mating type loci of M. perniciosa and the transcriptional profile of these genes, to uncover the mechanisms underpinning its homothallic behavior. First, we identified an A locus, a B locus and a set of genes that participates in pheromone processing and signalization. Considering the transcriptional profile of these genes, one receptor shows an expression profile consistent with an involvement in dikaryotization. The functional evaluation of the receptors was performed in a heterologous expression system, a promising tool for GPCR (G coupled proteins receptors) proteins study. This system did not allow the confirmation if M. perniciosa contains compatible alleles for receptors and pheromones, one possible explanation for homothallism. Considering A locus, it codes for a pair MpHD1 and MpHD2, which has a sequence insertion (11,958kb) interrupting their promoters, differing from others basidiomycetes. The hypothesis in this scenario is that the insertion of a transposon could have allowed an unequal crossover that brought together compatible genes in the same allele, causing the homothallism in this species. Interestingly, in an opposite direction, our data indicates that a previous transition for homothallism could have resulted in a relaxed selective pressure on mating type loci, with consequences such as the presence of degenerated genes on these loci. In this context, the mating type genes could not necessarily play a role in dikaryotization process. This work provides valuable data for understanding the sexual biology of M. perniciosa, which hereafter could be correlated with its phytopathogenicity / Mestrado / Genetica de Microorganismos / Mestra em Genética e Biologia Molecular