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Marcos André Machado Lima Teixeira Lançamento De Uma Biblioteca De Referência De DNA Barcodes Para Poliquetas De Estuários D

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Marcos André Machado Lima Teixeira Lançamento De Uma Biblioteca De Referência De DNA Barcodes Para Poliquetas De Estuários D Marcos André Machado Lima Teixeira Lançamento de uma biblioteca de referência de DNA barcodes para poliquetas de estuários de Portugal e do mar profundo da Peninsula Ibérica Tese de Mestrado Mestrado em Ecologia Trabalho realizado sob a supervisão do Professor Doutor Filipe Costa Trabalho realizado sob a orientação da Doutora Ascensão Ravara Outubro de 2013 DECLARAÇÃO Nome: Marcos André Machado Lima Teixeira Endereço eléctrónico: [email protected] Telefone: 917974620 Número do Bilhete de Identidade: 13243951 Título da Tese: Lançamento de uma biblioteca de referência de DNA barcodes para poliquetas de estuários de Portugal e do mar profundo da Peninsula Ibérica Orientador(es): Doutor Filipe Costa e Doutora Ascensão Ravara Ano de Conclusão: 2013 Designação do Mestrado: Mestrado em Ecologia De acordo com a legislação em vigor, não é permitida a reprodução de qualquer parte desta tese Universidade do Minho, ___/___/______ Assinatura: ________________________________________________ Agradecimentos Um agradecimento especial ao meu orientador, Doutor Filipe Oliveira Costa, não só pela oportunidade de realização de uma tese de mestrado dentro da temática do DNA barcoding, uma área que desde que me foi apresentada me despertou um enorme interesse, entusiasmo e motivação para aplicar todo o meu poder de trabalho, mas também pela paciência, companheirismo, disponibilização, apoio e conhecimento transmitidos ultimando na realização desta tese. Aos meus co-orientadores Sara Ferreira e Jorge Lobo por me ensinarem como trabalhar corretamente num laboratório, pela amizade e apoio demonstrado. Nunca esquecerei a ajuda prestada no meu primeiro passo para a vida profissional e espero que no futuro, possamos voltar a trabalhar juntos. À Doutora Ascensão Ravara por toda a disponibilidade e dedicação na passagem de conhecimento sobre a taxonomia de poliquetas. Gostava também de agradecer, aos meus pais e irmã por fazerem de mim o que sou hoje, que com muito sacrifício, foi possivel oferecerem-me a oportunidade de me licenciar e de obter o grau de mestre em Ecologia, possibilitando toda esta minha aventura que foi a vida académica na Universidade do Minho. A todos os meus colegas de laboratório (Mónica, Artur, Adriana, Soraia, Hugo, Nuno, Ana Sofia e Rita) por todos os momentos de sal e risada, apoio incansável, conselhos e discussões de trabalho, aumentando exponencialmente o meu rendimento, realçando a importância da convivência e do trabalho de grupo nos objectivos a atingir. Um forte e sincero agradecimento à minha turma de mestrado, por partilharem comigo esta viagem curta mas cheia de sucessos e de bons momentos para mais tarde recordar. Este trabalho foi financiado por Fundos FEDER através do Programa Operacional de Factores de Competitividade - COMPETE e por Fundos Nacionais através da FCT "Fundação para a Ciência e a Tecnologia (FCT)” no âmbito dos projetos FCOMP-01-0124-FEDER-015429 e PEst- C/BIA/UI4050/2011. A sequenciação no BIO (Biodiversity Institute of Ontario) foi financiado pelo iBOL (International Barcode of Life Project) através do Canadian Centre for DNA Barcoding, a partir do Ontario Genomics Institute (2008-OGI-ICI-03), Genome Canada, Ontario Ministry of Research and Innovation e pelo Natural Sciences and Engineering Research Council of Canada. iii Lançamento de uma biblioteca de referência de DNA barcodes para poliquetas de estuários de Portugal e do mar profundo da Peninsula Ibérica Resumo Os invertebrados bentónicos, como os poliquetas, são importantes indicadores de qualidade ambiental sendo também um elo relevante nas cadeias tróficas. A taxonomia, com base nos caracteres morfológicos desta classe é difícil, demorada, e muitas vezes requer um conhecimento especializado que nem sempre está disponível. Este trabalho pretende contribuir para a implementação de uma biblioteca de referência de DNA barcodes (citocromo oxidase I (COI)) de poliquetas de modo a melhorar e facilitar futuras identificações taxonómicas, podendo mais tarde, em conjunto com bibliotecas de outros organismos bentónicos, ser usada em estudos de monitorização, utilizando a sequenciação de segunda geração. Com recurso a DNA barcodes não publicados e gerados por equipas de projectos de investigação LusoMarBoL e BestBarcode, foram compiladas bibliotecas de poliquetas de duas comunidades distintas, nomeadamente de estuários e zonas costeiras de Portugal e do mar profundo da Península Ibérica. Em conjunto com os 26 DNA barcodes de poliquetas de estuários obtidos no âmbito deste estudo, a primeira biblioteca totalizou 31 espécies e 71 sequências, enquanto a segunda englobou 37 espécies e 66 DNA barcodes. Aos alinhamentos das sequências destas duas bibliotecas foram adicionadas sequências homólogas da mesma família ou género disponíveis em bases de dados públicas, por forma a permitir a sua análise conjunta. Os valores médios das divergências intra-específicas e congenéricas do conjunto dos DNA barcodes dos poliquetas de estuários foi de 0,52% (0 a 2,82%) e 20,53% (12,03 a 27,58%) respetivamente, enquanto para os poliquetas do mar profundo foram de 0,61% (0 a 2,63%) e 20,10% (7,36 a 31,77%) respetivamente. Estes valores estão dentro da gama de variação reportada em outros estudos de DNA barcodes de poliquetas. Nas árvores construídas pelo método Neighbour-joining, 79% e 73% das espécies agruparam-se em clados monofiléticos de baixa divergência, no caso dos poliquetas de estuário e do mar profundo, respetivamente. Paralelamente foram detetadas várias incongruências taxonómicas relevantes, bem como divergências intra-específicas relativamente elevadas, evidenciando a necessidade de uma revisão taxonómica em vários taxa. Palavras-chave: DNA barcodes, Poliquetas, COI-5P, Portugal, estuário, mar profundo, Península Ibérica v Launching a DNA barcodes reference library for estuarine polychaetes in Portugal and deep sea polychaetes in the Iberian Peninsula Abstract Marine benthic invertebrates, such as the polychaetes, are important environmental indicators and an important link in the marine trophic chains. The taxonomy based on morphological characteristics for this class is difficult, time consuming and often requires specialized knowledge that is not always available. This work aims to contribute to the implementation of a reference library of DNA barcodes (cytochrome c oxidase (COI)) of polychaetes in order to improve and facilitate future taxonomic identifications, and latter to be used in monitoring studies using the next generation sequencing., together with other benthic organisms libraries. Using the unpublished DNA barcodes, generated by the research teams from the projects LusoMarBoL and BestBarcode, libraries for two distinct polychaete communities were compiled, including estuaries and coastal areas of Portugal and the deep sea of the Iberian Peninsula. Together with the 26 DNA barcodes from estuarine polychaetes obtained in this study, the first library had 31 species and 71 sequences, while the second comprised 37 species and 66 DNA barcodes. Homologous sequences of the same family or genus searched in public databases were added to the sequence alignments of these two libraries, in order to enable their joint analysis. The average values of intraspecific and congeneric divergences for the estuarine polychaetes were 0,52% (0 to 2,82%) and 20,53% (12,03 to 27,58%) respectively, whereas for deep sea polychaetes were 0,61% (0 to 2,63%) and 20,10% (7,36 to 31,77%) respectively. These values are within the range of variation reported in other studies examining DNA barcodes of polychaetes. In the Neighbor-joining trees, 79% and 73% of the species grouped in monophyletic taxonomic congruent clades with low divergence, in the case of estuarine and deep sea polychaetes respectively. Concurrently, were also detected several relevant taxonomic ambiguities, as well as considerable genetic divergence, indicating the need for a taxonomic revision in several of the studied taxa. Keywords: DNA barcodes, polychaetes, COI-5P, Portugal, estuary, Iberian Peninsula, deep sea vii Índice Geral Agradecimentos .............................................................................................................................. iii Resumo ........................................................................................................................................... v Abstract .......................................................................................................................................... vii Índice de figuras ............................................................................................................................... xi Índice de tabelas ............................................................................................................................. xii Capítulo 1 Introdução geral 1.1. Motivação para novas abordagens na identificação de espécies ................................................. 3 1.2. DNA barcoding ...................................................................................................................... 7 1.3. Iniciativa Barcode of Life (BOLI) ............................................................................................ 12 1.4. Classe Polychaeta ................................................................................................................ 15 1.5. Objectivos gerais e estrutura da tese ..................................................................................... 19 Referências ...............................................................................................................................
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