ABSTRACT Title of Dissertation: THE ORIGIN OF THE DINOFLAGELLATE PLASTID Tsvetan Radoslavov Bachvaroff, Doctor of Philosophy 2004 Dissertation directed by: Associate Professor Charles F. Delwiche Department of Cell Biology and Molecular Genetics The peridinin pigmented dinoflagellate chloroplasts are the result of a secondary endosymbiotic event between a photosynthetic eukaryote and a dinoflagellate. Dinoflagellate chloroplast and nuclear evolution were independent before this endosymbiotic event. To reconstruct the evolution of the dinoflagellate chloroplast, phylogenies were constructed with a chloroplast gene psbB. The gene phylogeny should reflect the evolution of the chloroplast and indicate the plastid donor lineage. Gene sequences derived from the dinoflagellate chloroplast were extremely divergent but suggested that the plastid donor could have been a haptophyte. In an attempt to find better genes for analysis and to further understand gene transfer about 4900 randomly selected expressed genes were sequenced from two dinoflagellates, Lingulodinium polyedra and Amphidinium carterae. From these genes, thirty typically plastid-encoded genes were found, including eight otherwise known only from plastid genomes. Based on poly-A tails, gene families, and leader sequences these genes appear to be nuclear- encoded in dinoflagellates. This result suggests that dinoflagellate chloroplasts may have the smallest protein-coding potential yet known. These genes and the partially sequenced chloroplast genome of a haptophyte were used in a phylogenetic analysis. There is strong conflict between genes encoded in the chloroplast and those in the nucleus. The chloroplast genes suggest relationship between haptophyte and dinoflagellate plastids, while the nuclear-encoded genes suggest a relationship with heterokonts. Chromophyte plastid monophyly is supported by these data but the single origin of the chromophyte plastid from red algae does not mean that the host lineages are monophyletic. These results are consistent with at least two different scenarios: either dinoflagellates and haptophytes independently acquired a plastid from the heterokonts, or dinoflagellates acquired their plastids from haptophtyes, who in turn acquired their plastids from heterokonts. The evolutionary rate of the remaining plastid-encoded genes was compared with formerly plastid-encoded genes. These relative rate tests revealed strong incongruence between minicircle genes, formerly plastid-encoded genes and genes that were likely to have been acquired from the nucleus of the plastid donor lineage. THE ORIGIN OF THE DINOFLAGELLATE PLASTID by Tsvetan Radoslavov Bachvaroff Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2003 Advisory Committee: Professor Charles F. Delwiche, Chair/Advisor Dr. Kirk Apt Professor Elisabeth Gantt Professor Charles Mitter Professor Eugene Small Copyright by Tsvetan Radoslavov Bachvaroff 2004 ACKNOWLEGEMENTS The author would like to acknowledge the following individuals for their contributions to this document. Torstein Tengs taught me valuable laboratory techniques and performed many sequencing reactions on my behalf. He also made substantial intellectual contributions to my understanding of plastid evolution. Dr. David Oldach of the Institute for Human Virology generously loaned equipment and provided encouragement. Professor Robert Rowan of the University of Guam challenged me to find better data. The psbB sequences from haptophytes were obtained from Kamran Shalchian-Tabrizi in Professor Jakobsen’s lab at the University of Oslo, Norway. Kamran also contributed to my understanding of dinoflagellate nuclear phylogeny by generously sharing unpublished data. Three talented undergraduates participated in the EST data collection, Ernest Williams, Gregory Concepcion, and Carolyn Rogers. Greg also contributed substantially to the dinoflagellate EST database. A fourth undergraduate, Ali Tabatabai, worked on a dinoflagellate rubisco project not presented here. Finally, I would like to thank Maria Virginia Sanchez Puerta who has read and commented on many subunits of this document, and who has sequenced the organellar genomes of Emiliania. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS..............................................................................................ii LIST OF TABLES.............................................................................................................v LIST OF FIGURES..........................................................................................................vi CHAPTER I INTRODUCTION .................................................................................. 1 I.1. INTRODUCTION ...................................................................................................... 1 Features of dinoflagellates ...................................................................................... 2 The peridinin plastid ............................................................................................... 2 The other plastids of dinoflagellates ........................................................................ 4 I.2. SECONDARY ENDOSYMBIOSIS ................................................................................. 7 When did dinoflagellates acquire the peridinin plastid? .......................................... 8 Features of primitive dinoflagellates ....................................................................... 9 Eukaryotes related to dinoflagellates..................................................................... 10 How many plastid gains? ...................................................................................... 11 I.3. THE OTHER CHROMOPHYTE GROUPS...................................................................... 13 Chromophyte plastid vs. host phylogeny................................................................ 19 CHAPTER II DINOFLAGELLATE EXPRESSED SEQUENCE TAG DATA INDICATE MASSIVE TRANSFER OF CHLOROPLAST GENES TO THE NUCLEUS................................................................................................................... 23 II.1. ABSTRACT.......................................................................................................... 23 II.2. INTRODUCTION ................................................................................................... 24 II.3. RESULTS............................................................................................................. 26 Overview............................................................................................................... 26 Plastid-associated sequences................................................................................. 28 Signal peptides...................................................................................................... 33 Nucleus-to-nucleus gene transfer........................................................................... 33 Comparison to Plasmodium .................................................................................. 34 II.4. DISCUSSION........................................................................................................ 39 Overview............................................................................................................... 39 Chloroplast to Nucleus gene transfer..................................................................... 40 Nucleus-to-nucleus gene transfer........................................................................... 41 Cyanobacterial Genes and Biochemistry ............................................................... 44 Comparison with Plasmodium............................................................................... 45 Conclusions........................................................................................................... 46 II.5. MATERIALS AND METHODS................................................................................. 46 Library Construction............................................................................................. 46 Sequencing............................................................................................................ 48 Bioinformatics....................................................................................................... 48 CHAPTER III MULTI-GENE ANALYSES OF PLASTID RELATIONSHIPS WITHIN THE CHROMOPHYTE ALGAE .............................................................. 50 III.1. ABSTRACT ........................................................................................................ 50 III.2. INTRODUCTION.................................................................................................. 51 III.3. 2. RESULTS ....................................................................................................... 53 iii 2.1 Minicircle genes .............................................................................................. 53 III.2.2 Plastid genes from the dinoflagellate EST survey........................................ 60 2.3 Comparison of the minicircle dataset with the nuclear-encoded dataset .......... 62 III.4. 3. DISCUSSION................................................................................................... 71 3.1 Assessing monophyly of chromophyte plastids ................................................. 71 3.1 Minicircle