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Danillo Pinhal UNIVERSIDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS APLICAÇÃO DA GENÉTICA MOLECULAR NO MANEJO E CONSERVAÇÃO DE TUBARÕES TESE DE DOUTORADO Danillo Pinhal BOTUCATU - SP 2010 UNIVERSIDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS APLICAÇÃO DA GENÉTICA MOLECULAR NO MANEJO E CONSERVAÇÃO DE TUBARÕES Doutorando: Danillo Pinhal Orientador: Prof. Dr. Cesar Martins Co-orientador: Prof. Dr. Otto Bismarck Fazzano Gadig Tese apresentada ao Programa de Pós-Graduação do Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Doutor em Ciências Biológicas Genética BOTUCATU - SP 2010 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO DE AQUIS. E TRAT. DA INFORMAÇÃO DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CAMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSEMEIRE APARECIDA VICENTE Pinhal, Danillo. Aplicação da genética molecular no manejo e conservação de tubarões / Danillo Pinhal. - Botucatu, 2010 Tese (doutorado) - Instituto de Biociências de Botucatu, Universidade Estadual Paulista, 2010 Orientador: Cesar Martins Co-orientador: Otto Bismarck Fazzano Gadig Capes: 20204000 1. Genética animal. 2. Tubarão. 3. Pesca. Palavras-chave: Conservação; Estrutura populacional; Genética; Manejo da pesca; Marcadores moleculares; Rhizoprionodon; Sphyrna; Tubarões. Dedicatória Dedico esta nova e maior conquista, A Deus pela maravilhosa vida que a mim concedeu Aos meus pais, Pinhal e Alzira, por todo amor e carinho, por serem exemplos de caráter, perseverança e legítima fé. À minha irmãzinha querida, Mariane, a“maricota”, meu grande orgulho! Aos meus avós, Benedito e Angela (in memorian), Geraldo e Alzira, pelo exemplo de vida e por terem construído o alicerce de nossa grande família. À minha noiva Flavia Delella, pelo carinho e amor incondicionais, paciência, tolerância e fundamental incentivo. “Não sabendo que era impossível, ele foi lá e fez” (Jean Cocteau) Agradecimentos Agradeço à todas as instituições que contribuiram direta ou indiretamente para a realização desse trabalho. À Universidade Estadual Paulista – UNESP e ao Instituto de Biociências de Botucatu, pelos 10 anos de relacionamento. Muito Obrigado! Ao Programa de Pós-Graduação em Ciências Biológicas Genética e seus coordenadores. À FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo, pela bolsa de estudos e auxílio financeiros concedidos. Ao CNPq pela bolsa de estudos no início do projeto. Ao meu orientador-amigo Prof. Dr. Cesar Martins, por acreditar e amparar minhas idéias e aspirações, pelos ensinamentos diversos e esforços para a realização deste trabalho e contribuições ao meu futuro profissional. Ao Prof. Dr. Mahmood Shivji, pelas valiosas discussões nos “lab meetings” semanais, por ceder grande parte das amostras obtidas no exterior e fornecer toda a estrutura para a realização de experimentos em seu laboratório nos EUA. Aos grandes amigos do Laboratório de Genômica Integrativa, atuais e ex- integrantes, Andréia, Irani, Juliana, Guilherme Kbelo, Diogo, Sarah, Gilberto, Marcela, Bruno, Juliano, Carlitos, Rafael, Érica, Marcos e Pedro, por todos os momentos que passamos juntos nos últimos 6 anos!! À extinta Republica Bartira, minha família durante todo o mestrado e boa parte do doutorado, e aos amigos Renato, Adriano, Osni, Guilherme Kbelo, Juan, Ângelo, e agregados. Ao Prof. Dr. Demian Chapman pelo convite para coletas em alto mar e pela colaboração nos trabalhos. Aos Profs. Drs. Otto Gadig, Ricardo Garla, Alexandre Azevedo, e principalmente à Fabiana Daniela pela fundamental contribuição na obtenção de amostras. Ao Prof. Dr. Marcelo Vallinoto pela amizade e valiosas contribuições para o aprimoramento da análise dos dados. Aos amigos do IECOS na UFPA, especialmente ao Luis Fernando e Davidson pelas discussões e moradia durante os tempos de coleta no Pará. Aos amigos do Conservation Genetics Lab: Andrea Bernard, Rebeka Horn, Jennifer Hester, Lucy Howe; Naoko Kurata e Vince Richards. Especialmente à Ms. Christine Testerman pela amizade e por introduzir-me nos protocolos laboratoriais utilizados no exterior. Aos amigosda Nova Southeastern University, Florida, Estados Unidos: Andia, Atsushi, Yuko, Ekaterina, Ewelina, Crystal, Karita, Mae, Kerry, Michael Lehman, Bryan, Bruno Mourato, Dusty, Briggs, Jarrousy, Emily, Connor e James Brown pelos momentos de descontração e motivação proporcionados, bem como pelas caronas para o trabalho nos dias de chuva!. Em especial à Meghan e ao Maurício meus melhores amigos! Grande abraço! Aos amigos do Departamento de Morfologia e outros dispersos por todo o Instituto de Biociências que de alguma forma participaram deste trabalho Aos companheiros “botucudos”, Justulin, Dé, Robson, dentre outros, por todos os grandes momentos musicais e fins de semana compartilhados Aos amigos de Caraguatatuba, pelas festas e encontros de final de ano fundamentais para renovar minhas energias e seguir firme em frente. Às famílias Pinhal, Cruz e Delella por toda o carinho com que cuidam de mim. Aos amigos do Laboratório de Biologia e Genética de Peixes que contribuiram no início do desenvolvimento do trabalho. Aos funcionários e técnicos do Departamento de Morfologia da UNESP de Botucatu, especialmente à secretária Luciana e ao técnico Zé Eduardo, pela assistência e eficiência. Às empresas de pesca Produmar e Norte Pesca e seus funcionários Adriana, Amanda, Soni e Alzenir, pelo auxílio na obtenção de amostras no Rio Grande do Norte. Aos pescadores por possibilitarem a coleta de amostras em suas embarcações. Ao IBAMA pela concessão das licenças necessárias para a coleta, envio e recebimento de amostras do Brasil e exterior. SUMÁRIO página i. RESUMO .................................................................................................................... ix ii. ABSTRACT............................................................................................................... xi I. INTRODUÇÃO ......................................................................................................... 1 I.1. Biodiversidade e conservação ............................................................................ 1 I.2. Biologia e pesca de tubarões .............................................................................. 5 I.3. Os tubarões do gênero Rhizoprionodon ........................................................... 7 I.4. O tubarão-martelo Sphyrna lewini ................................................................... 11 I.5. Marcadores moleculares .................................................................................... 17 I.5.1. DNA mitocondrial ............................................................................................ 17 I.5.2. Microssatélites ................................................................................................. 19 I.5.3. Identificação molecular .................................................................................... 20 I.6. Estrutura Genética Populacional ..................................................................... 22 II. OBJETIVOS ............................................................................................................ 25 III. MATERIAL E MÉTODOS ................................................................................. 26 IV. RESULTADOS E DISCUSSÃO ......................................................................... 26 IV.1. Capítulo I Tracking the fin trade: Genetic stock identification in Western Atlantic scalloped hammerheads sharks (Sphyrna lewini) ............................................... 27 IV.2. Capítulo II Estrutura genética de populações do tubarão-martelo Sphyrna lewini no Atlantico Ocidental, utilizando marcadores moleculares de microssatélites e do DNA mitocondrial ................................................................................................ 47 IV.3. Capítulo III Cryptic hammerhead shark occurrence in South Atlantic: implications for management and conservation ............................................................................. 72 IV.4 Capítulo IV Multiplex PCR-based identification of globally distributed sharpnose sharks (Rhizoprionodon) for fisheries and trade monitoring ......................................... 89 V. REFERÊNCIAS BIBLIOGRÁFICAS ................................................................ 105 ix RESUMO Muitas espécies marinhas estão em risco de extinção devido à exploração excessiva e carência de manejo de nossos oceanos. Reduzir a intensidade do impacto das atividades antrópicas sobre o ambiente marinho é uma tarefa complexa, uma vez que grande parte da população mundial reside próximo às áreas costeiras, e por isso, freqüentemente depende dos recursos marinhos para sua subsistência e lazer. Essa dependência dos seres humanos em relação aos oceanos torna crucial o conhecimento dos ecossistemas marinhos para adequado manejo de seus recursos. Hoje em dia, a pesquisa nessa área é ainda bastante limitada, principalmente, devido às dificuldades de se trabalhar nos oceanos. Entretanto as análises de DNA estão contribuindo significativamente para o nosso conhecimento acerca da distribuição e saúde das populações de espécies marinhas criticamente ameaçadas. Dentre as espécies ameaçadas destaca-se o tubarão-martelo Sphyrna lewini cujas populações naturais estão atualmente em acelerada depleção devido à excessiva exploração pela pesca, principalmente para a comercialização de suas valorizadas nadadeiras nos mercados asiáticos. Esta espécie apresenta características relacionadas à sua disribuição e ao seu ciclo de vida que podem conduzir à estruturação
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