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Oecomys (RODENTIA: SIGMODONTINAE) DA AMAZÔNIA BRASILEIRA

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Oecomys (RODENTIA: SIGMODONTINAE) DA AMAZÔNIA BRASILEIRA INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA Programa de Pós-graduação em Genética, Conservação e Biologia Evolutiva CARACTERIZAÇÃO CITOGENÔMICA E DNA BARCODING DE TRÊS ESPÉCIES DE Oecomys (RODENTIA: SIGMODONTINAE) DA AMAZÔNIA BRASILEIRA Renan Gabriel Gomes Júnior Manaus - AM Fevereiro, 2014 Renan Gabriel Gomes Júnior CARACTERIZAÇÃO CITOGENÔMICA E DNA BARCODING DE TRÊS ESPÉCIES DE Oecomys (RODENTIA: SIGMODONTINAE) DA AMAZÔNIA BRASILEIRA Dissertação apresentada ao Programa de Pós-graduação do Instituto Nacional de Pesquisas da Amazônia, como parte dos requisitos para obtenção do título de mestre em Genética, Conservação e Biologia Evolutiva. Orientadora: Dra. Maria Claudia Gross - UFAM Coorientadora: Dra. Maria Nazareth Ferreira da Silva – INPA Fonte Financiadora: Rede de pesquisa para ampliação do conhecimento sobre a biodiversidade de vertebrados da Amazônia brasileira com aplicações sobre seu uso e conservação – Rede BioPHAM (CNPq/FAPEAM) Manaus - AM Fevereiro, 2014 ii G633 Gomes Júnior, Renan Gabriel Caracterização citogenômica e DNA barcoding de três espécies de Oecomys (Rodentia: Sigmodontinae) da Amazônia brasileira / Renan Gabriel Gomes Júnior. --- Manaus: [s. n.], 2014. xiv, 84 f. : il. color. Dissertação (Mestrado) --- INPA, Manaus, 2013. Orientador: Maria Claudia Gross. Coorientador: Maria Nazareth Ferreira Da Silva. Área de concentração: Genética, Conservação e Biologia Evolutiva. 1. Citogenética. 2. FISH. 3. COI. I. Título. CDD 599.3233 iii Dedico esta dissertação àqueles que me servem de exemplo todos os dias e me dão forças para buscar meus sonhos: Rosangela e Renan iv “Quem tem esperança sabe que nenhuma tristeza é eterna. Sabe que, após a chuva, virá o Sol. Que amanhã será um outro dia, cheio de surpresas e de boas novidades." Hugo Schlesinger “A fé é a força da vida. Se o homem vive é porque acredita em alguma coisa.” Léon Tolstoi v AGRADECIMENTOS Gostaria de agradecer a todos que colaboraram e tornaram o desenvolvimento dessa tese possível, e os dias na cidade de Manaus mais prazerosos. Primeiramente à Professora Doutora Maria Claudia Gross, pela presteza, conhecimento transmitido, confiança, e incentivo no desenvolvimento deste trabalho, às inúmeras oportunidades oferecidas, e às doses diárias de histórias e bom humor. À Doutora Maria Nazareth Ferreira da Silva, pela coorientação, pelas leituras e sugestões na redação desta dissertação, pela paciência, calma e paixão ao me apresentar um pouco sobre os pequenos mamíferos. À Doutora Eliana Feldberg, por me receber de portas abertas em seu laboratório, e por ser tão acessível como coordenadora do curso. Ao professor Doutor Carlos Schneider, “vice-orientador”, pelos ensinamentos nas práticas laboratoriais, sugestões na elaboração dessa dissertação e bate-papos. Aos meus pais, pelas palavras de apoio e incentivo que formam o motor para seguir em frente com meus objetivos. À Ingrid, pelo apoio e companheirismo durante essa difícil temporada. Aos amigos de laboratório, Camila, Érika, Vanessa Pinheiro, Vanessa Sales, Francy, Rafael, Jéssica, Júlia e, especialmente, a Nathália pelo auxílio nos primeiros passos no laboratório e parceria na realização da FISH, e a Thaís, pelo companheirismo, diálogos e sugestões na etapa final. Aos amigos do laboratório de genética animal: Eduardo, Leila, Lucas, Marajó, Maelin, Masseo, Milena, KK e Ramon pelas conversas com garantia de muitos risos. Aos demais amigos: Maysa, Roberta, Lincoln e Ramon, pelos momentos de descontração, a Iracema, pelos ensinamentos durante a coleta, pela preocupação e pelos bate-papos madrugada a dentro, e ao Mário Nunes pela ajuda com o sequenciamento. Ao CNPq pelo bolsa fornecida. vi RESUMO Dentre os roedores sul-americanos, a família Cricetiadae contêm o maior número de representantes, distribuídos em uma única subfamília, Sigmodontinae. Oryzomyini é uma das sete tribos reconhecidas de Sigmodontinae, que por sua vez é composta por 27 gêneros, entre os quais o Oecomys, composto atualmente por 16 espécies. Esse táxon apresenta uma difícil classificação sistemática devido a pouca diferenciação morfológica e grande variabilidade cariotípica. Assim sendo, o presente trabalho teve como objetivo contribuir para a caracterização cromossômica e análise das relações filogenéticas entre as espécies do gênero Oecomys da Amazônia brasileira. Para isso, 30 indivíduos de quatro localidades distintas foram analisados. Três espécies foram reconhecidas: Oecomys auyantepui apresentou dois citótipos distinto, 2n=64, NFa=110 e 2n=66 NFa=114, apesar disto, as técnicas citogenéticas empregadas foram similares entre os citótipos. O bandeamento C evidenciou marcações centroméricas tênues em todos os cromossomos, a sonda de rDNA 5S evidenciou duas marcações; três marcações foram observadas pela coloração com nitrato de prata e 4 para a sonda de rDNA 18S. A FISH telomérica evidenciou uma marcação intersticial em ambos citótipos no cromossomo sexual X, indicando um possível rearranjo cromossômico. A análise molecular corrobora a similaridade citogenética, apenas um clado foi formado com distância intraespecífica de 1,41%, e com alto valor de suporte nodal. Para Oecomys bicolor não houve variação entre as técnicas citogenéticas empregadas, encontrando 2n=80 e NFa=142, a heterocromatina constitutiva se distribuiu pelos centrômeros, e se espalhou pelos braços curtos de alguns cromossomos, com marcações conspícuas. O nitrato de prata evidenciou 7 marcações, que representam as regiões organizadoras de nucléolo ativas, e a sonda de rDNA 18S 24 marcações, sugerindo uma possível duplicação e dispersão dessas sequências. A sonda de rDNA 5S apresentou 2 marcações. Pela análise molecular 3 grupos foram formados, com distância genética entre eles que varia de 7,12% a 13,44%, sugerindo representar um complexo de espécies. Oecomys rutilus apresentou 2n=54 e NFa=90, a distribuição da heterocromatina foi do tipo tênue com distribuição pelos centrômeros, porção pericentromérica e terminal. Quatro marcações foram observadas pela coloração com nitrato de prata, assim como pela sonda de rDNA 18S. O sítio ribossomal 5S apresentou apenas 2 marcações. Dois clados foram formados pela análise molecular, com alto suporte nodal, condizentes com distribuição geográfica entre os espécimes. A análise molecular sugere que Oecomys rex seja o táxon basal para o grupo. vii ABSTRACT Among South American rodents, the Cricetiadae family contain the largest number of representatives allotted in a single subfamily, Sigmodontinae. Oryzomyini is one of the seven tribes recognized as Sigmodontinae, which is composed by 27 genera, including Oecomys genus with 16 species. This taxon presents a difficult systematic classification due to limited morphological variability and great karyotype differentiation. Therefore, this study aims to contribute to the chromosomal characterization and analysis of phylogenetic relationships among species of the Oecomys genus from Brazilian Amazon. In this study, 30 individuals were analyzed in four different regions. Four species were recognized as Oecomys auyantepui with two distinct cytotypes, 2n=64, FN=110 and 2n=66, FN=114, despite this, cytogenetic techniques employed were similar between the cytotypes. C banding revealed centromeric faint markings in all chromosomes, the 5S rDNA probe showed two marks, three marks were observed by silver-staining, and 4 for the 18S rDNA probe. The telomeric FISH showed an interstitial marking on both cytotypes of the sex chromosome X, indicating a possible chromosomal rearrangement. Molecular cytogenetic analysis supports the similarity, only one clade was formed with intraspecific distance of 1,41%, and high value of nodal support; For Oecomys bicolor no variation among cytogenetic techniques employed, with 2n=80 and FN=142 , constitutive heterochromatin was distributed by centromeres and spread by short arms of some chromosomes with conspicuous markings. Silver-staining showed 7 tags, which represent the active nucleolus organizer regions, and 24 markings with 18S rDNA probe, suggesting a possible duplication and dispersion of these sequences. The 5S rDNA probe showed 2 tags. Molecular analysis of 3 groups were performed with genetic distance between them ranging from 7, 12% to 13,44%, suggesting a species complex; Oecomys rutilus had 2n=54 and FN=90, distribution of heterochromatin was the faint type distribution with the centromeres , pericentromeric portion and terminal. Four markers were observed by silver-staining, as well as the 18S rDNA probe. The site ribosomal 5S showed only two markings. Two clades were formed by molecular analysis with high nodal support consistent with a geographical distribution among the specimens. Oecomys rex was considered basally among all Oecomys species in molecular analysis. viii SUMÁRIO 1. INTRODUÇÃO ------------------------------------------------------------------------------------ 16 1.1 Roedores do gênero Oecomys ---------------------------------------------------------------------- 16 1.2 Evolução cariotípica no gênero Oecomys ----------------------------------------------------- 20 1.3 DNA barcoding e a identificação molecular dos táxons -------------------------------- 23 2. OBJETIVOS --------------------------------------------------------------------------------------- 26 2.1 Objetivo geral ----------------------------------------------------------------------------------------------- 26 2.2 Objetivos específicos ----------------------------------------------------------------------------------- 26 3. MATERIAL E MÉTODOS ----------------------------------------------------------------------
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