Os Peixes Formam Um Grupo Parafilético Surgido No Siluriano E

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Os Peixes Formam Um Grupo Parafilético Surgido No Siluriano E MARIA APARECIDA FERNANDES PADRÕES TEMPORAIS E GRAU DE DIVERSIFICAÇÃO CARIOTÍPICA EM ESPÉCIES ATLÂNTICAS DA FAMÍLIA ACANTHURIDAE (PERCIFORMES) Dissertação apresentada ao Programa de Pós-Graduação em Sistemática e Evolução da Universidade Federal do Rio Grande do Norte, em cumprimento às exigências para obtenção do título de Mestre em Sistemática e Evolução. Orientador: Prof. Dr. Wagner Franco Molina NATAL-RN 2015 1 Catalogação da Publicação na Fonte. UFRN / Biblioteca Setorial do Centro de Biociências Fernandes, Maria Aparecida. Padrões temporais e grau de diversificação cariotípica em espécies Atlânticas da família Acanthuridae (Perciformes) / Maria Aparecida Fernandes. – Natal, RN, 2015. 54 f.: il. Orientador: Prof. Dr. Wagner Franco Molina. Dissertação (Mestrado) – Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em Sistemática e Evolução. 1. Evolução cromossômica. – Dissertação. 2. Fusões cêntricas. – Dissertação. 3. Acanthuridae. – Dissertação . I. Molina, Wagner Franco II. Universidade Federal do Rio Grande do Norte. III. Título. RN/UF/BSE-CB CDU 575 2 MARIA APARECIDA FERNANDES PADRÕES TEMPORAIS E GRAU DE DIVERSIFICAÇÃO CARIOTÍPICA EM ESPÉCIES ATLÂNTICAS DA FAMÍLIA ACANTHURIDAE (PERCIFORMES) Dissertação apresentada ao Programa de Pós-Graduação em Sistemática e Evolução da Universidade Federal do Rio Grande do Norte, em cumprimento às exigências para obtenção do título de Mestre em Sistemática e Evolução. Área de concentração: Sistemática e Evolução Aprovada em 26/03/2015 BANCA EXAMINADORA: __________________________________________________________ Dr. Wagner Franco Molina Universidade Federal do Rio Grande Do Norte (Orientador) ___________________________________________________________ Dra. Sathyabama Chellappa Universidade Federal do Rio Grande do Norte ___________________________________________________________ Dr. Roberto Ferreira Artoni Universidade Estadual de Ponta Grossa 3 DEDICATÓRIA A meus pais Vilmar (In memoriam) e Inácia, por todo amor, conselhos e dedicação, e a meu esposo Leandro pelo incondicional apoio ao longo desses anos. A vocês todo meu amor e gratidão. 4 AGRADECIMENTOS Gostaria de agradecer especialmente aos meus pais, por todo esforço empenhado em minha educação. As minhas irmãs (Josivânia e Josenilda) e irmãos (Adriano, Mariano e João Paulo) pela maravilhosa convivência familiar, pelo carinho demonstrado e principalmente pela compreensão nos momentos difíceis! Ao meu esposo Leandro pelo companheirismo e por contribuir sempre de maneira incondicional para o meu crescimento pessoal e profissional. Ao professor Dr. Wagner Franco Molina, pelo apoio, orientação e confiança depositados durante a realização deste trabalho. Aos companheiros de luta do LGRM: Gideão, Karlla, Rafael, Rodrigo, Amanda, Paulo, Clóvis, Juliana, Inailson, Allyson, Josi, Roberta, Calado e Cris. Obrigada a todos pelas significativas contribuições seja nos procedimentos laboratoriais e/ou em campo, e pelos momentos de descontração durante nossos cafezinhos. Registro também meu agradecimento à minha querida professora Drª. Simone Almeida por todo apoio, disponibilidade e conselhos (acadêmicos e pessoais) desde os primeiros momentos da minha vida acadêmica. Agradeço também as professoras Drª Danielle Pereti, Danielly Alves e Drª Maisa Clari pelo apoio durante a graduação e a transição para o mestrado. À CAPES pela concessão da bolsa de estudos. À UFRN e ao Programa de Pós-Graduação em Sistemática e Evolução por me permitir concluir mais esta estapa de meu processo educacional. Ao CNPq pelo suporte financeiro (Processo No. 556793/2009-9), INCT “Ciências do Mar,” , FAPESB (no. APP0064/2011) e ICMBio/SISBIO (licenças 19135-1, 131360-1, e 27027-2) pela autorização de coleta de espécimes. 5 SUMÁRIO 1. INTRODUÇÃO 1.1. Família Acanthuridae 11 1.2. Filogenia e evolução da família Acanthuridae 12 1.3. Aspectos biológicos e taxonômicos do gênero Acanthurus 13 1.4. Citogenética como ferramenta para inferências evolutivas 16 2. OBJETIVOS 2.1. Objetivo geral 19 2.2. Objetivos específicos 19 3. MATERIAL E MÉTODOS 3.1. Material 20 3.2. Métodos 20 3.2.1. Obtenção de cromossomos mitóticos 20 3.2.2. Análises cromossômicas e montagem do cariótipo 20 3.2.3. Detecção de regiões organizadoras de nucléolo 21 3.2.4. Detecção de heterocromatina constitutiva 21 3.2.5. Dupla coloração com fluorocromos base-específicos 21 3.2.6. Hibridação in situ fluorescente FISH 22 3.2.6.1. Obtenção de sondas para hibridação 22 3.2.6.2. Hibridação cromossômica 22 4. CAPÍTULO I - Sequential Steps of Chromosomal Differentiation in 24 Atlantic Surgeonfishes: Evolutionary Inferences 5. CAPÍTULO II - Atlantic Surgeonfishes Bear Only Minor 37 Microstructural Changes in Highly Derived Karyotypes 6. CONCLUSÕES 48 7. REFERÊNCIAS 49 6 LISTA DE FIGURAS Parte introdutória Figura 1. (a) Hipótese filogenética para a família Acanthuridae, 13 modificado de Sorenson et al. (2013). (b) Relações entre espécies do gênero Acanthurus presentes no Atlântico, proposta por Bernal & Rocha (2011). Figura 2. Espécime de Acanthurus coeruleus. Em destaque indivíduo na 15 fase juvenil. Barra: 2 cm. Figura 3. Espécime de Acanthurus chirurgus. Barra: 2 cm. 15 Figura 4. Espécime de Acanthurus bahianus. Barra: 2 cm. 16 Capítulo 1 Figura 1. Mapa da América do Sul mostrando os pontos de coleta de 26 Acanthurus coeruleus (a), A. bahianus (b), e A. chirurgus (c) no estado do Rio Grande do Norte (1) e Bahia (2), nordeste do Brasil. Figura 2. Cariótipos de Acanthurus coeruleus ((a) e (b)) com 2푛 = 48, A. 28 bahianus ((c) e (d)) com 2푛 = 36, e A. chirurgus ((e) e (f) ) com 2푛 = 34, após coloração convencional com Giemsa ((a), (c), e (e)) e badamento-C ((b), (d) e (f)). Os cromossomos portadores de RONs, de cada espécie, após tratamento com nitrato de prata são apresentados em caixas (par 2 de A. coeruleus e par de 8 de A. bahianus e A. chirurgus). Figura 3. Idiogramas de conjuntos cromossômicos de Acanthurus 29 coeruleus (a), A. bahianus (b), e A. chirurgus (c) mostrando as características citogenéticas mais conspícuas divididas em caixas. Em (d), o cariótipo basal dos Perciformes (2푛 = 48a) e uma hipótese filogenética com base em rearranjos 7 cromossômicos sequênciais inferidos nas três espécies Acanthurus. Capítulo 2 Figura 1. Cariótipo e metáfases de A. coeruleus (A), A. bahianus (B) e A. 40 chirurgus (C) após a coloração Giemsa (esquerda), two-color FISH (centro) com DNAr 18S (vermelho) e 5S DNAr (sinais verdes) distribuição de sondas de (TTAGGG)n sequências (direita). Caixa, os cromossomos portadores de RONs são mostrados após a coloração de nitrato de prata (esquerda) e coloração com fluorocromos (centro). Barra = 5m. Figura 2. Idiograma de A. coeruleus (A), A. bahianus (B) e A. chirurgus 44 (C), combinando os resultados de NOR / localização de região rica em GC e mapeamento físico de ribossomal e sequências teloméricas. Os cromossomos homeólogos derivados de inversões pericêntricas (cromossomos sm-st) e fusões cêntricas (m-cromossomos) são destacados nas caixas. O asterisco no par 7 de A. chirurgus indica uma suposta autapomorfia (fusão em tandem) para esta espécie. 8 LISTA DE ABREVIATURAS 2n – Número diploide a – Acrocêntrico AFLP – Amplified fragment lenght polymorphism AgNO3 – Nitrato de prata Ag-RONs – Regiões organizadoras de nucléolo evidenciadas pela impregnação com nitrato de prata AT – Adenina e Timina Ba(OH)28H2O – Hidróxido de bário BC – Bandamento C CMA3 – Cromomicina A3 DAPI – 4`4’,6-diamidino-2-fenilindol DNA – Ácido desoxirribonucleico DNAr/rDNA – Ácido desoxirribonucleico ribossômico FISH – Fluorescence in situ hybridization GC – Guanina e Citosina HCl – Ácido clorídrico KCl – Cloreto de potássio LGRM – Laboratório de Genética de Recursos Marinhos m – Metacêntrico Ma – Milhões de anos MM - Mitramicina n – Número amostral NF – Número fundamental RN – Rio Grande do Norte RNAr/rRNA – Ácido ribonucleico ribossômico RONs – Regiões organizadoras de nucléolo sm – Submetacêntrico st – Subtelocêntrico UFRN – Universidade Federal do Rio Grande do Norte 9 RESUMO A família Acanthuridae é um grupo bastante representativo dentre os peixes marinhos e que desempenha um papel fundamental na dinâmica ecológica dos recifes de corais. Três espécies pertencentes ao gênero Acanthurus são comuns ao longo dos recifes costeiros do Atlântico Ocidental: A. coeruleus, A. bahianus e A. chirurgus. No presente estudo, são apresentados dados citogenéticos para estas três espécies de Acanthurus com base em métodos citogenéticos clássicos e no mapeamento de sequências ribossomais repetitivas, como DNAr 18S e 5S, além de sondas teloméricas com a finalidade de auxiliar na compreensão da carioevolução deste grupo. O padrão citogenético dessas espécies indica que as etapas sequenciais de rearranjos cromossômicos, que datam 19-5 milhões de anos atrás (Ma), são responsáveis por suas diferenças interespecíficas. Acanthurus coeruleus (2n=48; 2sm + 4st + 42a), A. bahianus (2n=36; 12m + 2sm + 4st + 18a) e A. chirurgus (2n=34; 12m + 2sm + 4st + 16a) compartilham um antigo conjunto de três pares cromossômicos originados através de inversões pericêntricas. Um conjunto de seis grandes pares metacêntricos formados por translocações Robertsonianas (Rb) encontrado em A. bahianus e A. chirurgus e uma suposta fusão em tandem presente em A. chirurgus são eventos mais recentes. A falta de sequências teloméricas intersticiais (ITS), apesar de várias fusões cêntricas em A. bahianus e A. chirurgus pode estar relacionada com o longo período de tempo após a sua ocorrência (estimado em 5 Ma). Além disso,
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