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The creation of biodiversity involves the evolution of new species. Recent trends in the study of speciation have increased the emphasis on the role of ecology in adaptation and the evolution of reproductive isolation. This includes examining the relative contributions of different types of selection, the role of gene flow and the genomic changes that occur during ecological speciation. The search for speciation genes continues, however our growing knowledge of how the genome translates into phenotypes means we should now consider a broader molecular basis of speciation, which includes genetic, transcriptomic and potentially epigenetic variation that contribute to phenotypic variation. This thesis addresses the molecular basis of speciation by using three different complementary methods to examine the early stages of ecological speciation and the evolution of premating reproductive isolation between two incipient species of the cactophilic fly, Drosophila mojavensis. First, the genetic basis was examined through the sequencing of two candidate genes underlying reproductive isolation (Chapter 2). Second, the historical biogeography of population divergence was uncovered using multiple sequenced loci (Chapter 3). Lastly, gene expression across the whole transcriptome associated with phenotypic plasticity and mating success was assessed (Chapter 4). Further, the role of epigenetic imprinting in the population divergence of a freshwater fish, Girardinichthys multiradiatus, was examined through sequencing of a well known gene involved in sexual conflict (Chapter 5). These studies find that uncovering the genetic variation underlying speciation is difficult, especially when there is extensive phenotypic plasticity. Further, gene expression plasticity may play an important role in the evolution of premating isolation, and this includes a role for epigenetic mechanisms of gene expression. Additionally, it is important to assess the demographic scenario of population divergence to put into context the ecological and functional data on divergent groups. Through these studies this thesis examines the genetic, expression and epigenetic variation associated with on-going population divergence, and emphasises the need to consider the potential role of the full range of gene expression changes during ecological speciation.

Orientador: Claudia Marcia Aparecida Carareto / Banca: Luis Gustavo da Conceição Galego / Banca: Francisco Langeani Neto / Resumo: A despeito de ter sido intensivamente estudado, muitas das questões sobre o processo de especiação ainda permanecem sem respostas, principalmente em relação às alterações genéticas que conduzem ao isolamento reprodutivo. Neste trabalho foram analisados dois genes candidatos a genes de especiação, fruitless (fru) e Odysseus (OdsH), em duas espécies cactofílicas estreitamente relacionadas de Drosophila, D. mojavensis e D. arizonae, que divergiram de um ancestral comum a menos que 1 milhão de anos atrás. O gene fru exerce um papel predominante na especificação do potencial para o comportamento sexual de machos de Drosophila, podendo influenciar no isolamento reprodutivo pré-zigótico. O gene OdsH, que contém um homeodomínio que sofreu rápida evolução no subgrupo melanogaster, associa-se a esterilidade híbrida em Drosophila. Foram amplificados e sequenciados fragmentos destes genes em quatro linhagens de D. mojavensis e duas de D. arizonae e realizadas análises de diversidade, testes de seleção e análises filogenéticas. Ambos os genes apresentaram baixa diversidade entre as sequências das duas espécies, o que está de acordo com a divergência recente entre elas. Os testes de seleção realizados para as sequências de ambos os genes revelaram que os fragmentos analisados estão sob seleção purificadora, o que está de acordo com dados em outras espécies de Drosophila considerando o gene fru, mas é diferente do observado no subgrupo melanogaster para o gene OdsH, onde o gene mostra sinal de seleção positiva. As relações filogenéticas reconstruídas para as sequências dos dois genes, bem como redes de haplótipos permitiram fazer inferências sobre a história evolutiva das espécies e suas populações. Análises de Neighbor Joining e de Máxima Verossimilhança agruparam todas a sequências de D. mojavensis em um clado monofilético, como também as sequências de cada uma de suas linhagens corroborando... / Abstract: Despite having been intensively studied, many questions about the process of speciation remain unanswered, especially regarding genetic changes that lead to reproductive isolation. In this study we analyzed two genes candidates for speciation genes, fruitless (fru) and Odysseus (OdsH) in two closely related cactophilic Drosophila species, D. mojavensis and D. arizonae, which diverged from a common ancestor less than 1 million years ago. The fru gene plays a predominant role in the specification of the potential for sexual behavior of Drosophila male, thus influencing the pre-zygotic isolation. OdsH, a gene with a homeodomain that has undergone rapid evolution in the melanogaster subgroup of species, has been associated to hybrid sterility in Drosophila. Sequences of these two genes, known to be variable in Drosophila and other insects, were amplified and sequenced in four strains of D. mojavensis and two of D. arizonae. Analyses of diversity, selection tests and phylogenetic analyses were performed. Both genes show low diversity between the sequences from the two species, which is consistent with the recent divergence between them. The selection tests performed for the sequences showed that the fragments analyzed of both genes are under purifying selection, which is consistent with data in other Drosophila species considering the fru gene, but is different from data for OdsH in species of the subgroup melanogaster, in which is under positive selection. Phylogenetic relationships reconstructed for the sequences of the two genes, as well as networks of haplotypes allowed to make inferences about the evolutionary history of species and populations. Analyzes of Neighbor Joining and Maximum Likelihood clustered all the sequences of D. mojavensis in a monophyletic clade, as well as the sequences of each of its strains, reinforcing the classification of subspecies for the populations from where they were colected. However, the monophyly... / Mestre

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The cactophilic Drosophila mojavensis species group living in the deserts and dry tropical forests of the Southwestern United States and Mexico provides a valuable system for studies in diversification and speciation. My dissertation addresses a variety of evolutionary genetic questions using this system.Rigorous studies of the relationships between host races of D. mojavensis and the relationships among the members of the species group (D. mojavensis, D. arizona, and D. navojoa) are lacking. I used mitochondrial CO1 sequence data to address the phylogenetics and population genetics of this species group (Appendix A). In this study I have found that the sister species D. mojavensis and D. arizonae share no mitochondrial haplotypes and thus show no evidence for recent introgression. I estimate the divergence time between D. mojavensis and D. arizonae to be between 0.66 and 0.99 million years ago. I performed additional population genetic analyses of these species to provide a basis for future hypothesis testing.In Appendix B, I report the first example of substantial intraspecific polymorphism for genetic factors contributing to hybrid male sterility. I show that the occurrence of hybrid male sterility in crosses between Drosophila mojavensis and its sister species, D. arizonae is controlled by factors present at different frequencies in different populations of D. mojavensis. In addition, I show that hybrid male sterility is a complex phenotype; some hybrid males with motile sperm still cannot sire offspring.The large degree of variation between isofemale lines in producing sterile hybrid sons suggests a complex genetic basis to hybrid male sterility warranting quantitative genetic analysis. Since the genes underlying hybrid male sterility in these species are not yet fixed, I am able to perform explicit genetic analysis of this reproductive isolating mechanism. In Appendix C, I present the results of mapping QTL for hybrid male sterility within species. The genetic architecture underlying hybrid male sterility when analyzed directly in the F1 is highly complex. Thus, hybrid male sterility arises as a complex trait in this system and we propose a drift-based model for the evolution of this phenotype.

Made available in DSpace on 2015-09-17T15:24:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-05-16. Added 1 bitstream(s) on 2015-09-17T15:47:53Z : No. of bitstreams: 1 000846718.pdf: 1350026 bytes, checksum: ccde94ea62cfa0bca8ae7ebc162a76d7 (MD5) / A despeito de ter sido intensivamente estudado, muitas das questões sobre o processo de especiação ainda permanecem sem respostas, principalmente em relação às alterações genéticas que conduzem ao isolamento reprodutivo. Neste trabalho foram analisados dois genes candidatos a genes de especiação, fruitless (fru) e Odysseus (OdsH), em duas espécies cactofílicas estreitamente relacionadas de Drosophila, D. mojavensis e D. arizonae, que divergiram de um ancestral comum a menos que 1 milhão de anos atrás. O gene fru exerce um papel predominante na especificação do potencial para o comportamento sexual de machos de Drosophila, podendo influenciar no isolamento reprodutivo pré-zigótico. O gene OdsH, que contém um homeodomínio que sofreu rápida evolução no subgrupo melanogaster, associa-se a esterilidade híbrida em Drosophila. Foram amplificados e sequenciados fragmentos destes genes em quatro linhagens de D. mojavensis e duas de D. arizonae e realizadas análises de diversidade, testes de seleção e análises filogenéticas. Ambos os genes apresentaram baixa diversidade entre as sequências das duas espécies, o que está de acordo com a divergência recente entre elas. Os testes de seleção realizados para as sequências de ambos os genes revelaram que os fragmentos analisados estão sob seleção purificadora, o que está de acordo com dados em outras espécies de Drosophila considerando o gene fru, mas é diferente do observado no subgrupo melanogaster para o gene OdsH, onde o gene mostra sinal de seleção positiva. As relações filogenéticas reconstruídas para as sequências dos dois genes, bem como redes de haplótipos permitiram fazer inferências sobre a história evolutiva das espécies e suas populações. Análises de Neighbor Joining e de Máxima Verossimilhança agruparam todas a sequências de D. mojavensis em um clado monofilético, como também as sequências de cada uma de suas linhagens corroborando... / Despite having been intensively studied, many questions about the process of speciation remain unanswered, especially regarding genetic changes that lead to reproductive isolation. In this study we analyzed two genes candidates for speciation genes, fruitless (fru) and Odysseus (OdsH) in two closely related cactophilic Drosophila species, D. mojavensis and D. arizonae, which diverged from a common ancestor less than 1 million years ago. The fru gene plays a predominant role in the specification of the potential for sexual behavior of Drosophila male, thus influencing the pre-zygotic isolation. OdsH, a gene with a homeodomain that has undergone rapid evolution in the melanogaster subgroup of species, has been associated to hybrid sterility in Drosophila. Sequences of these two genes, known to be variable in Drosophila and other insects, were amplified and sequenced in four strains of D. mojavensis and two of D. arizonae. Analyses of diversity, selection tests and phylogenetic analyses were performed. Both genes show low diversity between the sequences from the two species, which is consistent with the recent divergence between them. The selection tests performed for the sequences showed that the fragments analyzed of both genes are under purifying selection, which is consistent with data in other Drosophila species considering the fru gene, but is different from data for OdsH in species of the subgroup melanogaster, in which is under positive selection. Phylogenetic relationships reconstructed for the sequences of the two genes, as well as networks of haplotypes allowed to make inferences about the evolutionary history of species and populations. Analyzes of Neighbor Joining and Maximum Likelihood clustered all the sequences of D. mojavensis in a monophyletic clade, as well as the sequences of each of its strains, reinforcing the classification of subspecies for the populations from where they were colected. However, the monophyly...

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Made available in DSpace on 2015-04-09T12:28:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-08Bitstream added on 2015-04-09T12:47:33Z : No. of bitstreams: 1 000809550.pdf: 1357515 bytes, checksum: 11c72eee17175a1530dc9a966eb6c180 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Essa tese teve como principal objetivo investigar como os elementos de transposição (TEs), sequências capazes de se movimentarem no genoma hospedeiro, e entre diferentes espécies, se comportam em híbridos interespecíficos. Buscou-se compreender a atividade do TEs em cruzamentos entre Drosophila mojavensis e D. arizonae devido a algumas características particulares desse par de espécies. São espécies irmãs, com divergência muito recente (< 1 milhão de anos), apresentam isolamento reprodutivo pré- e pós-zigótico incompleto em laboratório e uma delas, D. mojavensis, teve seu genoma sequenciado, o que permitiu a realização de análises in silico de TEs dessa espécie. A tese foi dividida em quatro capítulos. O primeiro apresenta uma análise detalhada do retrotransposon NLTR denominado I, conhecido por causar disgenesia híbrida em D. melanogaster. Foram identificadas e caracterizadas sequências putativamente ativas de elementos semelhantes ao I no genoma de D. mojavensis e realizadas análises filogenéticas que mostraram que as sequências I de D. mojavensis e aquelas encontradas em outras espécies de Drosophila pertencem a diferentes famílias I. Análises de expressão por RTq-PCR mostraram que esse elemento é transcricionalmente ativo nos ovários e testículos de ambas as espécies, e em seus híbridos, e que têm elevada expressão em testículos, mas não em ovários dos híbridos, o que poderia ser associado ao fenótipo de esterilidade do macho hibrido. No segundo capítulo são apresentadas análises por RNA-Seq de TEs expressos em ovários de duas linhagens parentais e seus híbridos F1. Os resultados mostram expressão espécie-específica de diferentes elementos nas espécies parentais e híbridos; e, de forma inédita, que apesar de alguns TEs estarem superexpressos nos híbridos, encontram-se de forma geral regulados em relação aos parentais. No terceiro capítulo são apresentados resultados de expressão ... / This thesis aimed to investigate how transposable elements (TEs), sequences capable of moving into the host genome and between different species, behave in interspecific hybrids. We seek to understand the activity of TEs in crosses between Drosophila mojavensis and D. arizonae due to some particular characteristics of this species pair. They are sister species, with very recent divergence ( < 1 million years ), present incomplete pre- and post- zygotic reproductive isolation in laboratory and one of them, D. mojavensis, had its genome sequenced, which allowed for in silico analyses of TEs. The thesis is divided into four chapters. The first presents a detailed analysis of an non- LTR retrotransposon called I, known to cause hybrid dysgenesis in D. melanogaster. Putatively active sequences similar to the I element were identified and characterized in the genome of D. mojavensis. The performed phylogenetic analyzes showed that the I sequences in D. mojavensis and those harbored by other Drosophila species belong to different I families. Expression analyses by RTq-PCR showed that this element is transcriptionally active in ovaries and testes of both species and their hybrids, and have high expression in the testes, but not in the hybrids ovaries, which could be associated with the male hybrid sterility phenotype. In the second chapter are presented analyses of expressed TEs in the ovaries of two parental strains and their hybrids by RNA-Seq. The results show species-specific expression of TEs in the parents and hybrids; and, in an unprecedented manner, that TEs are generally regulated in hybrids regarding with their parents, although some of them are overexpressed. In the third chapter are presented results of expression of four retrotransposons (Helena , I, Copia and Osvaldo) quantified by RTq-PCR; and finally, in the last chapter, we presented estimates of the genome size ( C - value), in both parental species and reciprocal hybrids ...

Orientador: Claudia Marcia Aparecida Carareto / Coorientador: Cristina Vieira / Banca: Élgion Lúcio da Silva Loreto / Banca: Marie France Sagot / Banca: Lilian Madi Ravazzi / Banca: Cristina Vieira / Resumo: Essa tese teve como principal objetivo investigar como os elementos de transposição (TEs), sequências capazes de se movimentarem no genoma hospedeiro, e entre diferentes espécies, se comportam em híbridos interespecíficos. Buscou-se compreender a atividade do TEs em cruzamentos entre Drosophila mojavensis e D. arizonae devido a algumas características particulares desse par de espécies. São espécies irmãs, com divergência muito recente (< 1 milhão de anos), apresentam isolamento reprodutivo pré- e pós-zigótico incompleto em laboratório e uma delas, D. mojavensis, teve seu genoma sequenciado, o que permitiu a realização de análises in silico de TEs dessa espécie. A tese foi dividida em quatro capítulos. O primeiro apresenta uma análise detalhada do retrotransposon NLTR denominado I, conhecido por causar disgenesia híbrida em D. melanogaster. Foram identificadas e caracterizadas sequências putativamente ativas de elementos semelhantes ao I no genoma de D. mojavensis e realizadas análises filogenéticas que mostraram que as sequências I de D. mojavensis e aquelas encontradas em outras espécies de Drosophila pertencem a diferentes famílias I. Análises de expressão por RTq-PCR mostraram que esse elemento é transcricionalmente ativo nos ovários e testículos de ambas as espécies, e em seus híbridos, e que têm elevada expressão em testículos, mas não em ovários dos híbridos, o que poderia ser associado ao fenótipo de esterilidade do macho hibrido. No segundo capítulo são apresentadas análises por RNA-Seq de TEs expressos em ovários de duas linhagens parentais e seus híbridos F1. Os resultados mostram expressão espécie-específica de diferentes elementos nas espécies parentais e híbridos; e, de forma inédita, que apesar de alguns TEs estarem superexpressos nos híbridos, encontram-se de forma geral regulados em relação aos parentais. No terceiro capítulo são apresentados resultados de expressão ... / Abstract: This thesis aimed to investigate how transposable elements (TEs), sequences capable of moving into the host genome and between different species, behave in interspecific hybrids. We seek to understand the activity of TEs in crosses between Drosophila mojavensis and D. arizonae due to some particular characteristics of this species pair. They are sister species, with very recent divergence ( < 1 million years ), present incomplete pre- and post- zygotic reproductive isolation in laboratory and one of them, D. mojavensis, had its genome sequenced, which allowed for in silico analyses of TEs. The thesis is divided into four chapters. The first presents a detailed analysis of an non- LTR retrotransposon called I, known to cause hybrid dysgenesis in D. melanogaster. Putatively active sequences similar to the I element were identified and characterized in the genome of D. mojavensis. The performed phylogenetic analyzes showed that the I sequences in D. mojavensis and those harbored by other Drosophila species belong to different I families. Expression analyses by RTq-PCR showed that this element is transcriptionally active in ovaries and testes of both species and their hybrids, and have high expression in the testes, but not in the hybrids ovaries, which could be associated with the male hybrid sterility phenotype. In the second chapter are presented analyses of expressed TEs in the ovaries of two parental strains and their hybrids by RNA-Seq. The results show species-specific expression of TEs in the parents and hybrids; and, in an unprecedented manner, that TEs are generally regulated in hybrids regarding with their parents, although some of them are overexpressed. In the third chapter are presented results of expression of four retrotransposons (Helena , I, Copia and Osvaldo) quantified by RTq-PCR; and finally, in the last chapter, we presented estimates of the genome size ( C - value), in both parental species and reciprocal hybrids ... / Doutor