Copyright by Matt Peter Ashworth 2013 The Dissertation Committee for Matt Peter Ashworth Certifies that this is the approved version of the following dissertation: Rock snot in the age of transcriptomes: Using a phylogenetic framework to identify genes involved in diatom extracellular polymeric substance-secretion pathways Committee: Edward C. Theriot, Supervisor Robert K. Jansen, Co-supervisor John W. La Claire David Cannatella Zengjian J. Chen Rock snot in the age of transcriptomes: Using a phylogenetic framework to identify genes involved in diatom extracellular polymeric substance- secretion pathways by Matt Peter Ashworth, B.S., M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December 2013 Dedication This dissertation is dedicated to my parents, Pat and Bob, and my wife Alison for their support through the process and for twice now not objecting to my desire to quit my job and go back to school to study molecular evolution in algae. Acknowledgements I would like to thank my fellow authors of the published versions of these dissertation chapters: Elizabeth Ruck, Teofil Nakov, Dwight Romanovicz. I would also like to acknowledge the help I received from my committee—in particular John La Claire for his assistance in developing the RNA extraction protocol for diatoms and interpretation of TEM images. I would also like to thank the following people for collection of live material or facilitating the collection of live material: Haj Allali, Andrew Alverson, Craig Aumack, Mariska Brady, Tom Frankovich, Michael Gruenstaeudl, Paul Hargraves, Sheryl Kanno, Daryl Lam, Elizabeth Ruck, Eric Salomaki, Maria Schefter and Anna Yu. I am also extremely grateful to the Texas Advanced Computing Center (TACC) for access to the RANGER and LONESTAR supercomputers and to Dhivya Arasappan, Tracey Ruhlman and Zhang Jin for assistance building and interpreting my transcriptome data. I must also acknowledge the efforts of the Marine Microbial Eukaryote Transcriptome Project for sequencing Cyclophora tenuis. I am deeply indebted to my advisors, Ed Theriot and Bob Jansen, for their support, friendship and demonstrated faith in my abilities. And a profound thanks to Chris Lobban—coauthor, unofficial advisor and friend—for his enthusiasm, efforts and advice in all things tropical and taxonomic where diatoms are concerned. This research was supported by an NSF grant to Ed Theriot (EF 0629410), the Blumberg Fellowship to Ed Theriot and the C.P. "Pete" Oliver Memorial Endowment for Genetic Research Award to Matt Ashworth. v Rock snot in the age of transcriptomes: Using a phylogenetic framework to identify genes involved in diatom extracellular polymeric substance- secretion pathways Matt Peter Ashworth, Ph.D. The University of Texas at Austin, 2013 Supervisors: Edward C. Theriot & Robert K. Jansen vi We are coming to understand that the ecological importance of diatoms is not limited to primary productivity, as many diatoms produce extracellular polymeric substances (EPS), which are vital components in algal and bacterial “biofilms.” While great effort has been made to chemically identify the types of molecules and polymers used to create and modify diatom EPS there is still much about the process we do not know. Rather than studying this process chemically, we have elected to search for the genes involved in EPS production and secretion. We assembled transcriptomes from three EPS-producing diatoms (Cyclophora tenuis, Lucanicum concatenatum, Thalassionema frauenfeldii) and two diatoms which do not (Astrosyne radiata, Thalassionema sp. ‘BlueH20’). In an attempt to limit the differences to EPS-related transcripts, the taxa were selected in a phylogenetic framework (which is also discussed in this dissertation), where EPS- producing taxa were closely-related to taxa which did not produce EPS (A. radiata, C. tenuis, L. concatenatum as one set, T. frauenfeldii and T. sp. ‘BlueH20’ as the other). The resulting pool of transcripts sorted for contigs which appeared in the EPS-producing taxa but not their closely-related non EPS-producing counterparts, and those contigs were then compared to two annotated diatom genomes and sorted by function, looking specifically for genes related to secretion, polysaccharide assembly or modification and carbohydrate metabolism. In the Thalassionema clade, 41 contigs with the aforementioned annotations were found, while 22 such contigs were found in the Cyclophora/Lucanicum/Astrosyne clade. These putative EPS-related markers are identified in this dissertation for further study on their function and evolution across diatoms. vii Table of Contents List of Tables ...........................................................................................................x List of Figures ....................................................................................................... xii Chapter 1: A revision of the genus Cyclophora and description of Astrosyne gen. nov. (Bacillariophyta), two genera with the pyrenoids contained within pseudosepta. ....................................................................................................1 Introduction .....................................................................................................1 Methods and materials ....................................................................................4 Electron Microscopy Preparation .................................................4 DNA Extraction, Sequencing and Phylogenetic Analysis ............5 Results .............................................................................................................8 Taxonomic revisions .....................................................................8 Cellular Ultrastructure ................................................................25 Phylogenetic analysis ..................................................................28 Discussion .....................................................................................................30 Taxonomic implications..............................................................30 Ultrastructural implications ........................................................31 Cyclophora and Astrosyne in the Diatom Phylogeny .................32 “Centric” Morphology in “Pennate” Diatoms ............................35 Chapter 2: Revisiting Ross and Sims (1971): Towards a molecular phylogeny of the Biddulphiaceae and Eupodiscaceae (Bacillariophyceae)..............................38 Introduction ...................................................................................................38 Methods and Materials ..................................................................................44 Microscopy Preparation ..............................................................44 DNA Extraction, Sequencing and Phylogenetic Analysis ..........45 Results and Discussion .................................................................................51 Phylogenetic Analysis .................................................................51 Hypothesis Testing......................................................................54 Ocellate taxa (Eupodiscaceae) ....................................................55 viii Pseudocellate taxa (Biddulphiaceae) ..........................................66 Conclusions ...................................................................................................70 Nomenclatural Amendments ........................................................................72 Chapter 3: Rock snot in the age of transcriptomes: Using a phylogenetic framework to identify genes involved in diatom extracellular polymeric substance-secretion pathways .......................................................................................................74 Introduction ...................................................................................................74 Methods and Materials ..................................................................................78 RNA extraction and Sequencing .................................................78 Transcriptome assembly and analysis .........................................79 Results ...........................................................................................................81 Thalassionema Clade ..................................................................82 Cyclophora Clade........................................................................86 Thalassionema Clade/Cyclophora Clade Overlap ......................87 Discussion .....................................................................................................88 Appendix ..............................................................................................................110 Taxa used in phylogenetic analysis, with locality, Theriot Lab specimen voucher ID (HK###) and GenBank accession numbers. Taxa marked with asterisks were used in Theriot et al (2010) but were misidentified in that manuscript (* = “Biddulphia alterans;” ** = “Isthmia enervis;” *** = “Coscinodiscus concinniformis”). .....................................................110 References ............................................................................................................121 ix List of Tables Table 1. Taxa and genes added to the Theriot et al 2011 data set for this paper. Locality indicates