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Universidade De Pernambuco - UPE) Universidade Federal de Pernambuco Centro de Biociências Programa de Pós-Graduação em Genética Zilpa das Graças Silva de Melo Estudo da variabilidade genética de Drosophila willistoni (Insecta: Diptera) e sua infecção pelo endossimbionte Wolbachia pipientis Recife 2018 Zilpa das Graças Silva de Melo Estudo da variabilidade genética de Drosophila willistoni (Insecta: Diptera) e sua infecção pelo endossimbionte Wolbachia pipientis Tese de Doutorado apresentada ao Programa de Pós-Graduação em Genética da Universidade Federal de Pernambuco como parte dos requisitos exigidos para obtenção do título de Doutor em Genética. Orientadora: Profa. Dra. Rita de Cássia de Moura - UPE Coorientadora: Profa. Dra. Claudia Rohde - UFPE Recife 2018 FICHA CATALOGRÁFICA Melo, Zilpa das Graças Silva de Estudo da variabilidade genética de Drosophila willistoni (Insecta: Diptera) e sua infecção pelo endossimbionte Wolbachia pipientis / Zilpa das Graças Silva de Melo - 2018. 211 folhas: il., fig., tab. Orientadora: Rita de Cássia de Moura Coorientadora: Claudia Rohde Tese (doutorado) – Universidade Federal de Pernambuco. Centro de Biociências. Genética, Recife, 2018. Inclui referências e anexos. 1. Díptero 2. Marcadores genéticos 3. Florestas I. Moura, Rita de Cássia (orient.) II. Rohde, Claudia (coorient.) III. Título 595.77 CDD (22.ed.) UFPE/CB-2018- 058 ZILPA DAS GRAÇAS SILVA DE MELO Estudo da variabilidade genética de Drosophila willistoni (Insecta: Diptera) e sua infecção pelo endossimbionte Wolbachia pipientis Tese apresentada ao Programa de Pós-Graduação em Genética, Área de concentração Biologia Molecular, da Universidade Federal de Pernambuco, como requisito parcial para obtenção do título de Doutor em Genética. Aprovado em: 29/08/2017 BANCA AVALIADORA ____________________________________________ Dra. Rita de Cássia de Moura (Universidade de Pernambuco - UPE) ____________________________________________ Dr. José Eduardo Garcia (Universidade Federal de Pernambuco - UFPE) ____________________________________________ Dr. Artur Maia Wanderley (Universidade Federal de Pernambuco - UFPE) ____________________________________________ Dra. Vilma Loreto da Silva (Universidade Federal de Pernambuco - UFPE) ____________________________________________ Dra. Anna Carolina S. Almeida (Universidade Federal Rural de Pernambuco - UFRPE) À minha mãe Graça, e meu pai Pedro (in memorian), meus exemplos. Agradecimentos À Deus, fonte de vida, razão de tudo, minha maior inspiração. À minha família, em especial à minha mãe, uma mulher guerreira, ‘de fibra’, que sempre cuidou de mim, me apoiou em todas as minhas escolhas, me aconselhando e ajudando em tudo. Ao meu pai (in memorian), homem de grande superação, que perdi no primeiro ano do doutorado. Foram dias muito difíceis para mim e minha família. Ao meu esposo Cristian pelo amor, companheirismo, apoio e barras de chocolates. À minha filha Giovanna Melissa, o presente mais lindo que a vida me deu! Amor sublime e inexplicável. À minha orientadora Dra. Rita Moura, por ter aceitado me orientar, pela paciência, pelos ensinamentos, por ter persistido em me orientar mesmo quando eu falei da minha gravidez no início do doutorado e ainda ter permitido eu assumir o concurso da Compesa, no segundo ano do doutorado. Sua postura ética e profissional me motivou a continuar na luta. Minha eterna gratidão por tudo! À minha co-orientadora Dra. Claudia Rohde, por ter aceitado me orientar desde o mestrado. Por ter me apresentado o universo das drosófilas e por tantos ensinamentos, tanto científicos quanto humanísticos! Foi uma experiência incrível! Não posso deixar de agradecer por ter sido minha motorista durante todos esses anos (risos). Foram muitas caronas e reuniões científicas a bordo do seu ‘drosomóvel’. Suas habilidades e coragem me inspiram. Minha eterna gratidão por tudo! Aos meus colaboradores e co-orientadores por consideração, Dra. Ana Garcia e Dr. Martín Montes, pelos ensinamentos, e pelos ‘choques de realidade’, que foram importantes para me impulsionar a continuar fazendo um trabalho bem feito. Aos companheiros de trabalho do Laboratório de Genética do CAV- UFPE, vocês foram de extrema importância para a realização deste trabalho de doutorado. O apoio de vocês e os momentos de descontração foram fundamentais para superar meus momentos de pessimismo e dificuldade. Foi um privilégio poder conviver, aprender e fazer amizade com excelentes pessoas: Jorge, Rita, Gleyse, André, Anadeje, Érima, Danubia, Ícaro, Samuel, Jéssica, Phelipe, Rafaela, e outros que passaram pelo laboratório. Às minhas queridas ICs: Jaqueline, Robervânia, Ana Patrícia e Kelly, que embarcaram comigo nesta grande aventura científica. A todos que me ajudaram a preparar meios e repicar aquela estante repleta de linhagens de Drosophila willistoni. Eu não teria conseguido sem a ajuda vocês. Às minhas amigas e colaboradoras: Dra. Geórgia Oliveira, a ‘doutora mor’ do laboratório (risos) e Msc. Paloma da Silva, ‘Palometch a poderosa’ (risos). Meninas, foi um prazer enorme trabalhar com vocês. Sou grata pela parceria e amizade. Geo, você para mim foi uma co-orientadora nos temas ecológicos. Aos colegas do laboratório de Biodiversidade e Genética de Insetos (LBGI) da UPE Ígor, Crislaine e Adriana, foi um prazer ter trabalhado com vocês também. Em especial, Celso que muito me ajudou com os programas de análises de dados moleculares. Sou grata também a Geyner pelos ensinamentos e críticas construtivas ao trabalho, sua ajuda e parceria foram fundamentais. À minha sogra Cilene por ter cuidado muitas vezes da minha princesa, enquanto eu estava realizando meus experimentos e aulas. À Universidade Federal de Pernambuco, ao qual tenho muito orgulho em ser aluna. Ao Programa de Pós-Graduação em Genética e aos professores, pelos ensinamentos. Aos colegas do Programa de Pós-Graduação em Genética pelos grupos de estudo e leitura de artigos. Aos meus amigos que fiz ao longo da minha vida, em especial: Késia, Midiã, Fabíola, Jeneffer, Michele, Stella e Sheila, que me deram incentivo, e compreenderam minha ausência em alguns ‘encontrinhos’. E por fim, à FACEPE pela bolsa de pesquisa recebida em parte deste doutorado, e aos demais Órgãos de Fomento CNPq, CAPES e PROPESQ-UFPE. “A ciência é uma ferramenta absolutamente essencial para qualquer sociedade que tenha a esperança de sobreviver bem, neste e nos próximos séculos” Carl Sagan Resumo Biodiversidade tem sido um tema recorrente e envolve o estudo da diversidade genética intraespecífica e os processos associados à origem, manutenção e distribuição da diversidade. Drosophila willistoni é uma das espécies mais abundantes da família Drosophilidae na região neotropical. Neste trabalho foi caracterizada a diversidade genética desta espécie em diferentes biomas do Brasil através de marcadores moleculares, bem como sua infecção pelo endossimbionte Wolbachia pipientis. Após coleta e identificação taxonômica da espécie, foram estabelecidos cultivos de linhagens isofêmeas durante todo o trabalho. Aqui estão apresentados: i) resultados com o marcador ISSR em 156 linhagens, que indicaram significativa diversidade genética, baixa diferenciação populacional e elevado fluxo gênico; ii) caracterização da diversidade genética dos genes mitocondriais COI (114 indivíduos) e COII (83) e do gene nuclear HCF (28 indivíduos), que indicaram eventos recentes de expansão populacional nas amostras do Brasil; iii) resultados da presença do endossimbionte W. pipientis em 85 indivíduos de nove populações de D. willistoni e a ubiquidade da variante wWil em 50 amostras sequenciadas; iv) diversidade ecológica de espécies de drosofilídeos nos fragmentos de floresta úmida estudados. O conjunto dos resultados a partir das linhagens investigadas possibilitou compreender melhor a diversidade genética de D. willistoni, cujas populações naturais não apresentam sinais de estruturação genética, apesar da distância geográfica entre as amostras e das diferenças entre os ambientes estudados. Nossos dados reforçam que D. willistoni apresenta elevada diversidade genética, possui uma ampla associação com W. pipientis e ainda é uma das espécies de Drosophilidae mais abundante em florestas tropicais do Brasil. Palavras-chave: Marcador ISSR. Gene mitocondrial. Gene Host Cell Factor. Wolbachia pipientis. Abstract Biodiversity has been a recurring theme and involves the study of intraspecific genetic diversity and processes associated with the origin, maintenance and distribution of diversity. Drosophila willistoni is one of the most abundant species of the family Drosophilidae in the neotropical region. In this work we characterized the genetic diversity of the species in different biomes of Brazil through molecular markers, and its infection by the endosymbionte Wolbachia pipientis. After collection and taxonomic identification of the species, cultures of isofines lineages were established throughout the work. In this thesis are presented: i) results with the ISSR marker in 156 lines, which indicated significant genetic diversity, low population differentiation and high gene flow; ii) characterization of the genetic diversity of the mitochondrial COI (114 individuals) and COII (83) and nuclear HCF genes (28 individuals), which indicated recent population expansion events in the Brazilian samples; iii) results of presence of W. pipientis endosymbionts in 85 individuals from nine D. willistoni populations and the ubiquity of the wWil variant in 50 sequenced samples; iv) ecological diversity of drosofilídeos species in the fragments of rainforest studied. The set of results
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