BIODIVERSITY, DISTRIBUTION AND EVOLUTION OF ENDOLITHIC MICROORGANISMS IN CORAL SKELETONS Vanessa Rossetto Marcelino ORCID 0000-0003-1755-0597 Doctor of Philosophy School of Biosciences The University of Melbourne Thesis submitted in total fulfillment of the requirements of the degree of Doctor of Philosophy December 2016 ABSTRACT Prokaryotic and eukaryotic microbes regulate key processes in reef ecosystems but very little is known about the biodiversity of microorganisms living inside coral skeletons (i.e. endolithic). Endolithic microalgae, for example, are among the main contributors of reef bioerosion and can facilitate coral survival during bleaching events, but their phylogenetic diversity, distribution and evolutionary origins are largely unknown. We developed a high- throughput sequencing procedure to assess the biodiversity of prokaryotic and eukaryotic microbes in coral skeletons. A surprisingly high biodiversity of green algae was found, including entirely new lineages that are distantly related to known genera. This technique was then applied to study the relative effects of niche specialisation and neutral processes on the spatial distribution of endolithic communities. The results indicated that stochastic processes and dispersal limitation create a high rate of bacterial species turnover within colonies, while niche specialisation explains most of the distribution of endolithic microbes at larger spatial scales. Finally, we studied whether signatures of an endolithic lifestyle could be observed in the chloroplast genome of a common endolithic alga. The results suggested that chloroplast genome streamlining and slow rates of molecular evolution are associated with the low light inherent of endolithic lifestyles. i DECLARATION This is to certify that: i. The thesis comprises only my original work towards the PhD except where indicate in the preface. ii. Due acknowledgment has been made in the text to all other material used. iii. The thesis is fewer than 100,000 words in length, exclusive of tables, bibliographies and appendices. ___________________ Vanessa R. Marcelino ii PREFACE The four data chapters of this thesis have been written for submission to refereed journals and involve collaborators. Chapter 2 has been published as: Marcelino VR, Verbruggen H (2016) Multi-marker metabarcoding of coral skeletons reveals a rich microbiome and diverse evolutionary origins of endolithic algae. Scientific Reports 6, 31508. Both authors conceptualized the study and collected the samples. VRM performed DNA extractions and library preparation. VRM and HV developed the analysis pipeline. VRM wrote the manuscript, HV edited. VRM contributed with ~85% of the work. Chapter 3 is being prepared for publication in collaboration with Kathleen M. Morrow, Madeleine van Oppen, David G. Bourne and Heroen Verbruggen. KMM and DGB were responsible for the samples collection. VRM performed DNA extractions, library preparation and analyses. VRM wrote the manuscript and received comments from all other authors. VRM contributed with ~ 70% of the work. Chapter 4 is being prepared for publication in collaboration with Eric Treml, Madeleine van Oppen and Heroen Verbruggen. VRM and HV conceptualized the study and collected the samples. VRM performed DNA extractions, library preparation and analyses. ET, MO and HV provided feedback and supervision for analyses. HV produced scripts for species accumulation curves. VRM wrote the manuscript and received feedback from HV. VRM contributed with ~90% of the work. Chapter 5 has been published as: Marcelino VR, Cremen MC, Jackson CJ, Larkum AA, Verbruggen H (2016) Evolutionary dynamics of chloroplast genomes in low light: a case study of the endolithic green alga Ostreobium quekettii. Genome Biol Evol 8, 2939-2951. VRM and HV conceptualized the study. CJ and CMC performed DNA extractions. VRM and CC performed genome assemblies and annotations. VRM performed the analyses. VRM wrote the manuscript and received comments from all other authors. VRM contributed with ~85% of the work. iii ACKNOWLEDGMENTS Back in 2012 Heroen Verbruggen suggested me to do a PhD project on the diversity of limestone-boring algae using high-throughput sequencing. I had never heard of boring algae before and doubted his unfounded expectation of finding several algal species inside coral skeletons. The idea was intriguing though, or perhaps I just wanted to prove him wrong, so I accepted the challenge. Well, he was right. I am immensely thankful to him for giving me the opportunity of pursuing this project, for being supportive about the directions and subprojects I have tried to pursue, and for being patient and still supportive when they failed. Heroen’s optimism, determination and passion for science are contagious and this project would not be possible without his excellent supervision. I am very thankful to Tom Schils (University of Guam) for sending me the very first coral skeletons samples I analysed and for supporting the boring algae idea from the start (still in 2012). I am also thankful to Tom’s student Adrian Kense for the interest and for sharing some good and bad library prep moments. In 2013 I started my PhD, and the first thing a PhD student in Australia needs is an advisory committee. I am very thankful to Jane Elith, Ed Newbigin and Mick Keough, their well-grounded opinion was extremely valuable and our discussions during the progress meetings were very educational for me. I am also very grateful to my co-supervisor Madeleine van Oppen for her insightful inputs during those discussions, but I am writing these acknowledgments in chronological order so I will get to Madeleine later. The second thing a PhD student need is samples and mine were underwater. I would like to thank Mel Tate and the Melbourne University Underwater Club for teaching me the first Open Water dive courses, and the Diveline crew for making me a dive master. The field work associated with this thesis was fantastic thanks to all the amazing people involved in it. My first field trip was in Western Australia and I would like to thank Seraphya Berrin, Heroen Verbruggen, Joana Ferreira Costa, Lambros Stravias and the personnel from Murdoch University Coral Bay Research Station. Then my first samples from the renowned Great Barrier Reef were collected on Keppel Islands thanks to Guillermo Diaz-Pulido (Griffith University), his student Carlos Del Monaco and the skipper Peter Williams. This was during my mid-PhD “why am I doing this?” crisis moment. Guillermo is one of the few scientists on Earth who has interest and experience with boring algae, his genuine excitement was a very important motivation to carry on with this project and I am very thankful for that. Maybe that was the best outcome of this field trip as I lost all the collected samples due to a bad storage buffer… Same fate for all deep sea samples kindly provided by Heather Spalding (University iv of Hawaii), thanks anyway Heather. The final large field excursion was carried out on Heron Island, and I am thankful to Chiela Cremen, Pilar Diaz-Tapia and Heroen Verbruggen, in addition to the helpful staff at Heron Island. On the way back we had a chance of passing by Keppels for re-collecting those lost samples, but the windy weather only allowed us to dive once. Oh well, I got some samples and the trip was fun anyway. A substantial part of my field work was supported by philanthropic funding, and I am very grateful to the Botany Foundation and the Holsworth Wildlife Research Endowment. Then it comes the molecular work, and dozens of people have helped me enormously. Before the Verbruggen lab physically existed I was hosted by the Systematics and the Malaria labs. I would like to thank Mike Bayly and Geoffrey McFadden for letting me use their facilities, and all their staff and students for welcoming and helping me around, including Erin Batty, Anton Cozijnsen and Vanessa Mollard. Special thanks to Todd McLay for helping with the library preparation issues, and there were a lot of issues. The staff from the sequencing facilities has also been very helpful, and I would like to thank Kym Pham (Centre for Translational Pathology), Matthew Tinning (Australian Genome Research Facility) and Stephen Wilcox (Walter and Eliza Hall Institute). I would like to thank Cassie Watts, Ouda Khammy and Kirsty for the countless favors. Last but not least, I would like to thank all past and present members of the Verbruggen lab, especially Joana Costa, Chiela Cremen, Lupita Bribiesca and Chris Jackson for their assistance with all sorts of stuff, for all the SWASIs we went through (and survived), and for making the everyday work more fun. Not only algae live inside coral skeletons and I thought it would be interesting to adventure into the world of prokaryotes, but they were so scarily unfamiliar to me. Luckily Madeleine van Oppen moved to Melbourne Uni in 2015 and accepted to be my co-supervisor, which was a boost of confidence to work on bacteria. I am very thankful to her for assisting with her extensive expertise, nice ideas and even moral support. I would also like to thank Raquel Peixoto (Universidade Federal do Rio de Janeiro) and the members of her lab for hosting me during an internship, which gave me a taste of what is like to work in a microbiology lab. I am also grateful to Kathy Morrow and David Bourne (AIMS) for providing samples and microbial ecology expertise. I am also thankful to the always-happy Joan and Asmira (Melbourne Uni) for making the university feel more like home. To Fran and Witold