LABYRINTHULOMYCETES DIVERSITY META-ANALYSIS by Jingwen Pan A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2016 © Jingwen Pan, 2016 Abstract Labyrinthulomycetes are a group of ubiquitous stramenopiles that inhabit a wide range of habitats and play important ecological roles as nutrient recyclers and sometimes disease causing agents. Even though they have had a long history of being studied, their diversity has not yet been fully explored. The lack of a comprehensive reference database with up-to-date phylogeny also hinders any pursuits in understanding the ecological distribution of this group. This study was designed with the purpose of constructing a curated reference database and a phylogenetic tree based on existing 18S rDNA data, and then using this database to uncover any hidden diversity and novelty among Labyrinthulomycetes and provide a reference guidance for future identification. Using the newly-created reference database, I also analyzed high-throughput environmental sequencing data from two databases. My results reveal extensive diversity within the Labyrinthulomycetes, and recover many previously unknown environmental sequences, greatly expanding our knowledge of the ecological distribution of this group. The high- throughput environmental sequencing data analysis also shows some of the newly identified environmental clades to be particularly abundant in the ocean. The phylogenetic framework I have provided in this study, together with the metadata I have compiled, will serve as a useful tool for future ecological and evolutionary studies of this widespread lineage. ii Preface This thesis is original and unpublished. It is based on work designed by J. del Campo and I. I conducted all the analysis and J. del Campo provided supervisory guidance. I prepared all figures and wrote the manuscript, and J. del Campo and P.J. Keeling revised it. iii Table of Contents Abstract .................................................................................................................................................... ii Preface .................................................................................................................................................... iii Table of Contents ................................................................................................................................. iv List of Tables .......................................................................................................................................... v List of Figures ........................................................................................................................................ vi Acknowledgements ........................................................................................................................... vii Chapter 1: Introduction ...................................................................................................................... 1 1.1 Labyrinthulomycetes Introduction ...................................................................................................................... 1 1.1.1 Labyrinthulomycetes Classification ............................................................................................................ 1 1.1.2 Labyrinthulomycetes Morphology and Physiology .............................................................................. 3 1.1.3 Labyrinthulomycetes Ecology and Lifestyle ............................................................................................ 3 1.1.4 Summary ................................................................................................................................................................. 5 1.2 High-throughput Environmental Sequences Studies .................................................................................... 5 1.3 Goals ................................................................................................................................................................................... 7 Chapter 2: Materials and Methods .................................................................................................. 9 2.1 Reference Phylogenetic Tree Construction ....................................................................................................... 9 2.2 Reference Database Annotation ........................................................................................................................... 10 2.3 Labyrinthulomycetes V9 Reads Database ....................................................................................................... 11 2.4 Abundance and Richness Distribution Patterns ........................................................................................... 12 Chapter 3: Results ............................................................................................................................. 13 3.1 Phylogeny of Labyrinthulomycetes .................................................................................................................... 13 3.2 Examining Abundance and Richness using GenBank Sanger Sequences ........................................... 14 3.2.1 Host Association ................................................................................................................................................. 15 3.3 Abundance and Richness using V9 Reads ........................................................................................................ 16 Chapter 4: Discussion ....................................................................................................................... 19 4.1 Phylogeny and Classification ................................................................................................................................. 19 4.2 Environmental Distribution of Labyrinthulomycetes ................................................................................ 22 Chapter 5: Conclusion and Future Directions .......................................................................... 24 References ........................................................................................................................................... 34 iv List of Tables Table 1: List of previously described environmental clades (Collado-Mercado et al. 2010) and the new clades they now belong to according to the present phylogenetic study .... 30 Table 2: Total abundance and richness for the three Labyrinthulomycetes databases ....... 32 v List of Figures Figure 1: Diversity of Labyrinthulomycetes inferred from a maximum likelihood (RAxML) phylogenetic tree constructed using 18S rDNA sequences. ............................................ 26 Figure 2: Diversity of Labyrinthulomycetes (full version) ...................................................... 27 Figure 3: Abundance and richness for major groups of Labyrinthulomycetes .................... 31 Figure 4: Heatmap showing the abundance distribution of major groups of Labyrinthulomycetes according to different environmental parameters ..................... 33 vi Acknowledgements I would like to thank many people who made this thesis possible. First, I would like to express my sincere gratitude to my supervisor Patrick Keeling for his support, guidance and patience on my study. I also thank the members of my committee, Brian Leander, James Berger and Rosie Redfield for their advice. I am also grateful to all members from the Keeling lab, the Leander lab and the Fast lab for their help and inspiration along the way, and for creating such a wonderful working environment. I am also thankful to faculty and staff of UBC, especially Veronica Oxtoby, for general help. I own particular thanks to Javier del Campo, whose expertise and guidance have helped me in all the time of research and in writing of this thesis, and for being patient with me during stressful moments. Finally, I wish to thank my parents and friends for their love and continuous support throughout my study and my life. They have walked through every joyful and difficult moment with me. Special thanks to Fen Liu and Cuiyu Lin for their daily amusement and advice on Excel from the later. vii Chapter 1: Introduction 1.1 Labyrinthulomycetes Introduction Labyrinthulomycetes are a group of unicellular eukaryotic microorganisms commonly found in marine environments (Raghukumar 2002). The slime-like appearance of most of its members has resulted in previous misplacement of this group within Fungi, Oomycetes (Stramenopiles) and amoebae. Observations of mitochondria with tubular cristae and their heterokont, biflagellate zoospore production, together with support from 18S rRNA gene phylogenies eventually led to the placement of this group within the Stramenopiles, together with the photosynthetic Ochrophytes (diatom, brown algae, etc) and other non-photosynthetic groups, like the plant pathogenic Oomycetes (Cavalier-Smith et al. 1994; Tsui et al. 2009). Within the stramenopiles, the Labyrinthulomycetes is a monophyletic group and is characterized by having a cell wall made of scales containing proteins and sulphated polysaccharides, as well as the production of an ectoplasmic network (EN), which is a membrane-bound, branched network secreted through a unique organelle called bothrosome (sagenogenetosome). 1.1.1 Labyrinthulomycetes Classification