Microbial Biodiversity in the southern Indian Ocean and Southern Ocean Flavia Flaviani Town Cape of Thesis presented for the degree of Doctor of Philosophy Department of Molecular and Cell Biology UniversityUniversity of Cape Town December 2016 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Plagiarism Declaration “This thesis/dissertation has been submitted to the Turnitin module (or equivalent similarity and originality checking software) and I confirm that my supervisor has seen my report and any concerns revealed by such have been resolved with my supervisor.” Name: Flavia Flaviani Student number: FLVFLA001 Signature: Signature removed Date: 07 December 2016 1 Certification by Supervisor In terms of paragraph GP7 of the regulations for the degree of Doctor of Philosophy at the University of Cape Town, I certify that I approve of the inclusion in this thesis of the material already published, or submitted for publication by the candidate Flavia Flaviani. Signature removed Signature Edward P. Rybicki, PhD Professor in Microbiology Director of the Biopharming Research Unit Department of Molecular and Cell Biology, Faculty of Science & Institute of Infectious Disease and Molecular Medicine University of Cape Town 2 Abstract The multi-phylotype and ecologically important community of microbes in aquatic environments ranges from the numerically dominant viruses to the diverse climate-change regulating phytoplankton. Recent advances in next generation sequencing are starting to reveal the true diversity and biological complexity of this previously invisible component of Earth’s hydrosphere. An increased awareness of this microbiome’s importance has led to the rise of microbial studies with marine environmental samples being collected and sequenced daily around the globe. Despite the rapid advancement in knowledge of marine microbial diversity, technical difficulties have constrained the ability to perform basin wide physical and chemical oceanographic assessments in tandem with microbiological screening with the majority of studies only looking at a single component of the microbial community. In this study the full microbial diversity, from viruses to protists, was characterised within the southern Indian Ocean and the Southern Ocean from a small volume of seawater collected using the same CTD equipment used by oceanographers. Throughout this study it will be demonstrated how this small volume is sufficient to describe the core microbial taxa in the marine environment. The application of a bespoke bioinformatics pipeline, integrated with sequencing replication, improved the description of the dominant core microbiome whilst removing OTUs present due to PCR and sequencing artefacts thereby improving the accurate description of rare phylotypes. Analyses confirmed the dominance of Cyanobacteria, Alphaproteobacteria and Gammaproteobacteria in the pelagic prokaryotic microbiome, while the Stramenopiles-Alveolata-Rhizaria (SAR) cluster dominates the eukaryotic microbiome. A decrease in the SAR community will be reported for the Southern Ocean with a concomitant increase in the haptophyte community. Whilst the virome confirmed the dominance of tailed phages and giant viruses across all stations, there was a significant variation in the caudoviruses and Nucleocytoplasmic Large DNA viruses (NCLDV) across defined biogeographical boundaries. The described method will allow the characterisation of the microbial biodiversity as well as future integration with oceanographic data with a much reduced sampling effort. The characterisation of the whole microbial community from a single water sample will improve the understanding of microbial interactions and represent a step towards in the inclusion of viruses into biogeochemical models. 3 Acknowledgments I would like to acknowledge my supervisor Prof. Ed Rybicki and co-supervisors Dr. Declan Schroeder and Dr. Maya Pfaff. For the guidance, support and the intellectual challenge you gave to me. I am thankful for the opportunity and the trust; it has been a journey that I will always cherish. A special thanks to Prof. Rybicki for being such an inspiration and a great mentor, for showing me my strength and allowing me to believe in myself as well as showing me the joy of communicating science to the world. For samples collections, statistical advices and substantial support I would like to acknowledge doctors Cecilia Balestreri, Jo Schroeder and Karen Lebret. I would like to acknowledge the University of Cape Town's ICTS High Performance Computing team for providing the computational infrastructure and special thanks to Andrew Lewis and Timothy Carr for the technical support. I am grateful to the Linux support provided by Alec Colebrook-Clark during my time at the Marine Biological Association, as well as the technical support provided by Nick Bloomer and Scott Middleton. For the help and support in the lab I would like to thank Madhu Chauhan, Shakiera Sattar and Tatiana Millard from the University of Cape Town; Matt Hall, Angela Ward and Claire Jasper from the Marine Biological Association of the UK for all your help and support during my time in both laboratories. This project would have not be possible without the support of the National Research Foundation (NRF) grant to Prof. Ed Rybicki (CPR20110717000020991) and the funding by FP7-OCEAN-2011 call, MicroB3 (grant number 287589) to Dr. Declan Schroeder. My time spent between UCT and the MBA has granted me the opportunity to meet so many amazing people. I am immensely grateful to Hayley Evers-King and Ben Loveday for the long hours chatting about my project, the oceanography conversations and for showing me how beautiful and special South Africa is, my time there would have not been the same without you. To my friends Aleyo Chabeda, Hazel Dickens, Megan Coates, Amy Betzelberger, Cecilia Balestreri, Karen Lebret, Jo Schroeder and Beatriz De Francisco thank you for being there for 4 me and listening to me whine while showing me that there is always a way. You were always able to cheer me up when I was down, dragging me out for a chat and some food. Thanks to Thomas and Rebecca Jackson, Stefan Simis, Marianne Kettunen, Robert Camp, Silvia Pardo, Aser Mata, Emily Mc Gregor and Silvana Mallor Hoya. You guys have been my surrogate family, some from day one some more recently, but you have all provided incredible moral support and chats in the time of difficulties, showing me what friendship is. It’s thanks to all of you that I have enjoyed both my time in South Africa and the UK, you filled my life with laughter, adventure, travels and love. To my family in Sardinia whose support has been constant despite the distance, and for reminding me that no matter where I was, home was always warmer. Final and enormous thanks to Matt Hall, for your support through these years, the long conversations and the help reading my drafts, this would have not been possible without you. Thank you for reminding me that I am worth it and that someday I will be able to take down that little monster that keeps telling me I am not good enough. You’ve always been there for me and I am looking forward to an easier time at your side. 5 Table of Contents Page Plagiarism Declaration ………………………………………………………………………..1 Certification by Supervisor ……………………………………………………………….......2 Abstract ……………………………………………………………………….……………... 3 Aknowledgments ……………………………………………………………….......................4 Contents ………………………………………………………………………………………6 List of Abbreviations and Acronims ………………………………………...........................10 List of Tables ...........................................................................................................................14 List of Figures .........................................................................................................................15 Chapter 1: Literature Review ..................................................................................................18 1.1 Introduction ...........................................................................................................18 1.2 A water world ........................................................................................................19 1.3 The importance of microbes in the oceans ............................................................22 1.4 The missing link ....................................................................................................25 1.5 Marine prokaryotes ...............................................................................................27 1.6 Marine microbial eukaryotes .................................................................................28 1.7 Marine viruses .......................................................................................................30 1.8 Sequencing technologies .......................................................................................31 1.9 Thesis outline ........................................................................................................35 Chapter 2: PCR amplification replicates and singleton removal in tag amplicon NGS project: a method for the removal of erroneous diversity ………………………………………........38