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Filogenia, Filogeografia E Avaliação Do Código De Barras De DNA Em Roedores Do Gênero Euryoryzomys (Sigmodontinae: Oryzomyini)

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Filogenia, Filogeografia E Avaliação Do Código De Barras De DNA Em Roedores Do Gênero Euryoryzomys (Sigmodontinae: Oryzomyini) Dissertação de Mestrado Keila Aparecida de Almeida Filogenia, filogeografia e avaliação do código de barras de DNA em roedores do gênero Euryoryzomys (Sigmodontinae: Oryzomyini) Phylogeny, phylogeography and DNA barcoding in the identification of the genus Euryoryzomys (Sigmodontinae: Oryzomyini) São Paulo 2014 1 Dissertação de Mestrado Keila Aparecida de Almeida Filogenia, filogeografia e avaliação do código de barras de DNA em roedores do gênero Euryoryzomys (Sigmodontinae: Oryzomyini) Phylogeny, phylogeography and DNA barcoding in the identification of the genus Euryoryzomys (Sigmodontinae: Oryzomyini) Dissertação apresentada ao Instituto de Biociências da Universidade de São Paulo, para a obtenção de Título de Mestre em Biologia (Genética), na Área de Biologia (Genética). Orientador(a): Dra Maria José de Jesus Silva São Paulo 2014 2 Ficha Catalográfica Almeida, Keila Aparecida de Filogenia, Filogeografia e avaliação do código de barras de DNA em roedores do gênero Euryoryzomys (Sigmodontinae: Oryzomyini) Número de páginas 152 Dissertação (Mestrado) - Instituto de Biociências da Universidade de São Paulo. Departamento de Genética e Biologia Evolutiva. 1. Euryoryzomys 2. Filogenia molecular 3. Filogeografia I. Universidade de São Paulo. Instituto de Biociências. Departamento de Genética e Biologia Evolutiva. Comissão Julgadora: ________________________ _______________________ Prof(a). Dr(a). Prof(a). Dr(a). ______________________ Dra. Maria José de Jesus Silva Orientadora 3 Dedico esta dissertação à memória da minha amada tia Maria Aparecida (Ieda), que viveu uma vida de doações, caridade, perseverança e respeito ao próximo nas mais diversas situações. Sempre presente. 4 Agradecimentos Agradeço ao Instituto de Biociências da Universidade de São Paulo por fornecer a infraestrutura da pós-graduação e a todos os funcionários que se empenham todos os dias, nos dando suporte e fornecendo informações. Aos professores do departamento, por compartilharem seus conhecimentos. À CAPES pela bolsa concedida e à FAPESP pelo fomento à pesquisa. À Dra. Maria José de Jesus Silva pela orientação. Ao Dr. Miguel Trefaut Rodrigues, da Universidade de São Paulo e ao Dr. Pablo Jayat, da Universidade Nacional de Tucumán, pela cessão de amostras de tecidos e ao Dr. James L. Patton, Universidade da Califórnia, Berkeley, pela cessão de algumas sequências utilizadas neste trabalho. Ao Laboratório de Biotecnologia do Instituto Butantan, dirigido na época pelo professor Dr. Paulo Lee Ho, e ao técnico Msc. Leonardo Kobashi, cujas colaborações possibilitaram o sequenciamento das amostras. Aos pesquisadores, alunos e funcionários do Laboratório de Ecologia e Evolução do Instituto Butantan, onde esse trabalho foi desenvolvido. À MSc. Taís Machado pela ajuda com os programas e sugestões e a MSc. Lorena Corina Bezerra de Lima pela ajuda e amizade ao longo do projeto. Ao Dr. Elkin Suarez-Villota por compartilhar seu conhecimento com simplicidade e companheirismo, e pela leitura de uma versão preliminar. À MSc. Camilla Bruno Di Nizo, também pela colaboração e leitura da versão preliminar do primeiro capítulo texto. À MSc. Natália Araújo, por toda ajuda e disposição sempre que eu enviava um e-mail desesperado. Obrigada pela ajuda desde a graduação. Ao Dr. João Morgante por ter aberto as portas do LABEC/USP na época em que eu estava na graduação e à Dra Caroline Cotrim Aires e MSc. Ana Carolina d’Oliveira Pavan pelas técnicas moleculares pacientemente ensinadas. Obrigada por terem me acolhido no início dessa empreitada. Ao prof. Dr. Fábio Mesquita, pelo incentivo desde a graduação e agora como colega de trabalho na Universidade Paulista. Obrigada por compartilhar seu entusiasmo científico. 5 Às minhas novas companheiras de trabalho Ana Paula e Natália, por tornarem o Laboratório de Ciências da Saúde da Universidade Paulista um local de trabalho agradável, sempre companheiras e dispostas a ajudar. Aos melhores amigos do mundo que tornaram essa fase da vida muito mais divertida: Denis, Fernanda, Gordo, Roberta, Sá, Junior, Roni, Vera, Rafael, Renata, Fabrício e Carlos Fernando. Obrigada pela amizade, viagens, risadas e os finais de semana divertidos que passamos juntos, vocês são especiais. À amiga Mariana, pela amizade que desenvolvemos desde a graduação e que vai muito além da vida acadêmica. Obrigada também pela enorme ajuda que me deu na finalização da edição desse documento. Igualmente, agradeço ao Richard Capestrini, que juntamente com ela me ajudou na edição e finalização. À amiga Adriana, por estar presente sempre. Não há parágrafos suficientes para demonstrar a imensa gratidão que tenho por você. Muito obrigada. À minha família que participou direta e indiretamente dessa dissertação. Cada encontro de família sempre é uma renovação dos nossos valores, obrigada por me proporcionarem isso sempre que nos reunimos. Ao meu pai Mário e minha mãe Edna pelos valores que me ensinaram, pela boa educação que me deram e por todo o sacrifício que passaram para que eu pudesse chegar aonde desejasse. Obrigada por compreenderem minha ausência. Ao meu sobrinho Murillo pelo sorriso e esperteza que só uma criança cinco anos pode ter. Finalmente, agradeço ao meu namorado Fernando pela paciência nas horas em que me ausentei para o estudo e por toda ajuda no tratamento das imagens, agradeço também pela amizade, carinho, amor e por estar comigo sempre. Meu amigo, companheiro e confidente, te amo. 6 Sumário CAPÍTULO I - INTRODUÇÃO ............................................................................................... 9 1. INTRODUÇÃO .............................................................................................. 10 1.1 ORDEM RODENTIA ................................................................................. 10 1.2 TRIBO ORYZOMYINI ................................................................................ 12 1.3 O GÊNERO EURYORYZOMYS .................................................................... 14 1.4 CONSIDERAÇÕES SOBRE INFERÊNCIAS FILOGENÉTICAS BASEADAS EM SEQUÊNCIAS DE DNA .................................................................................. 20 1.5 IDENTIFICAÇÃO DE ESPÉCIES BASEADA EM SEQUÊNCIAS DO GENE CITOCROMO OXIDASE I - DNA BARCODING ........................................................................ 22 1.6 CONSIDERAÇÕES SOBRE FILOGEOGRAFIA ................................................. 24 1.8 REFERÊNCIAS BIBLIOGRÁFICAS ............................................................ 36 CAPÍTULO II – SISTEMÁTICA MOLECULAR EM ROEDORES DO GÊNERO EURYORYZOMYS ...... 45 2. INTRODUÇÃO .............................................................................................. 48 2.1 MATERIAL E MÉTODOS ............................................................................ 51 2.1.1 AMOSTRAGEM, EXTRAÇÃO DE DNA, PCR E OBTENÇÃO DAS SEQUÊNCIAS 51 2.1.2 ANÁLISES FILOGENÉTICAS E DISTÂNCIAS GENÉTICAS ............................ 52 2.2 RESULTADOS......................................................................................... 64 2.2.1 FILOGENIA ....................................................................................... 64 2.3 DISCUSSÃO ........................................................................................... 72 2.4 CONCLUSÕES ........................................................................................ 78 2.5 REFERÊNCIAS BIBLIOGRÁFICAS ............................................................... 79 CAPÍTULO III – O POTENCIAL DO DNA BARCODING NA IDENTIFICAÇÃO DE ROEDORES DO GÊNERO EURYORYZOMYS (CRICETIDAE: SIGMODONTINAE: ORYZOMYINI)........................... 84 3. INTRODUÇÃO .............................................................................................. 87 3.1 MATERIAL E MÉTODOS ..................................................................... 88 3.1.2 ANÁLISE DAS SEQUÊNCIAS ................................................................ 89 3.1.3 IDENTIFICAÇÃO DOS ESPÉCIMES NO BOLD E O TESTE DE DELIMITAÇÃO DE ESPÉCIES ................................................................................................... 89 3.2 RESULTADOS......................................................................................... 90 3.2.1 TOPOLOGIA NEIGHBOR-JOINING ........................................................ 91 3.2.2 DISTÂNCIAS INTRA E INTERESPECÍFICAS ......................................... 92 7 3.2.3 DELIMITAÇÃO DE ESPÉCIES APLICADA AO DNA BARCODING ............ 93 3.3 DISCUSSÃO ........................................................................................... 94 3.4 CONCLUSÕES ........................................................................................ 99 CAPÍTULO IV – FILOGEOGRAFIA DE EURYORYZOMYS RUSSATUS (SIGMODONTINAE: ORYZOMYINI): ESTRUTURA GENÉTICA E HISTÓRIA DEMOGRÁFICA ..................................... 115 4. INTRODUÇÃO ............................................................................................ 118 4.2 MATERIAL E MÉTODOS ......................................................................... 121 4.2.1 AMOSTRAGEM, EXTRAÇÃO DE DNA, PCR, OBTENÇÃO DAS SEQUÊNCIAS E ALINHAMENTO ........................................................................................... 121 4.2.2 ANÁLISES FILOGENÉTICAS ............................................................... 121 4.2.3 GENÉTICA DE POPULAÇÕES E ANÁLISES DEMOGRÁFICAS .................... 122 4.3 RESULTADOS....................................................................................... 122 4.3.1 ANÁLISES FILOGENÉTICAS .............................................................
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