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EVOLUTION AND POPULATION GENETICS OF PROCHLOROCOCCUSMARINUS by Ena Urbach B.S. Molecular Biophysics and Biochemistry Yale University, 1980 Submitted in partial fulfillment of the requirements of the DEGREE OF DOCTOR OF PHILOSOPHY in Biology and Civil and Environmental Engineering at the Massachusetts Institute of Technology February 1995 @ Massachusetts Institute of Technology 1995 Signature of Author ,_ _ _ Departments of Biology and Civil and Environmental Engineering Certified by Sallie W. Chisholm, Professor BiQlogv and Civil and Environmental Engineering Accepted b) Departmental Committee on Graduate Studies Civil and Environmental Engineering Joseph M.Sussman Accepted b3 I A- gl 'I. )" tal Committee on Graduate Stuaies Departm Biology Jo-Ann Murray r..'I : i i EVOLUTION AND POPULATION GENETICS OF PROCHLOROCOCCUSMARINUS by Ena Urbach Submitted to the Departments of Biology and Civil and Environmental Engineering February, 1995, in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology and Civil and Environmental Engineering ABSTRACT Prochlorococcusmarinus is a tiny, photosynthetic marine prokaryote containing the unusual photosynthetic pigments divinyl chlorophylls a and b, and lacking phycobilisomes, the light harvesting apparatus found in most cyanobacteria. The unique pigments of this organism suggest a phylogenetic affinity with the Prochlorophyta, a recently described group of (monovinyl) chlorophyll a and b-containing prokaryotes which also lack phycobilisomes. The Prochlorophyta have been proposed as a monophyletic group sharing a common ancestry with "green" chloroplasts in algae and higher plants. Phylogenetic analysis of relationships among P. marinus, the prochlorophytes Prochloronsp. and Prochlorothrixhollandica, and other members of the cyanobacterial radiation indicates that the distribution of prochlorophytes within the cyanobacterial radiation is polyphyletic, and that none of the known chlorophyll b-containing prokaryotes is specifically related to chloroplasts. This result, inferred from 16S ribosomal RNA (rRNA) sequences, has been confirmed by a parallel, independent study using rpoC gene sequences. The polyphyletic distribution of prochlorophytes and green chloroplasts among the cyanobacteria implies that photosynthetic systems employing (monovinyl and divinyl) chlorophyll b arose several times during the evolution of photosynthetic organisms. However, since the publication of these results it has been pointed out that they are also consistent with the existence of an ancestral cyanobacterium containing both chlorophyll b and phycobilisomes, with subsequent loss of one or the other light harvesting system in all known surviving lineages. P. marinus is a major constituent of the photosynthetic picoplankton across wide regions of the tropical and subtropical open oceans, with cell densities of 105 cells/ml frequently encountered in both the Sargasso Sea and the Pacific. Cultured strains of this ecologically important organism isolated from Sargasso Sea, north Atlantic, Mediterranean and Pacific waters were found by phylogenetic analysis of 16S rRNA, psbB and petB and D sequences to belong to a single lineage within the cyanobacteria. P. marinus shares this lineage with strains of marine A Synechococcus, a planktonic cyanobacterium which also shares its open ocean habitat. An investigation into the genetic structure of P. marinus field populations in depth profiles from the Sargasso Sea and the Gulf Stream revealed a high degree of genetic heterogeneity within water samples, detected by partial sequencing of cloned PCR products containing portions of the single-copy genes petB and D and their intergenic region, amplified from flow cytometrically sorted cells. Populations within water samples contained a minumum of six alleles recovered from a sample of 23 clones. The presence of numerous additional alleles were implied by rarefaction analyses, which indicated that diversity was incompletely sampled in datasets containing 19 to 28 clones per water sample. Overlapping sets of alleles were recovered from the two water columns, from different depths within each water column and from flow cytometrically distinguishable subpopulations within water samples, suggesting that each of these populations drew its membership from a single gene pool. Thesis Subervisor: Prof. Sallie W. Chisholm Title: Professor of Biology and Civil and Environmental Engineering ACKNOWLEDGEMENTS I would like to thank my thesis advisor, Penny Chisholm, for allowing me an unusual degree of intellectual freedom in pursuing what I wanted to study. Penny's intellectual courage has allowed her to develop a laboratory with an extraordinary range of skills and interests. I would also like to thank the members of my thesis committee, past and present, who have helped me with sharp critical eyes and large amount of patience: Alan Grossman and Ethan Signer of the Biology Department, Bill Thilly of the Department of Civil and Environmental Engineering, Bruce Levin of the Zoology Departement at University of Massachusetts at Amherst (and currently at Emory University), John Waterbury of the Woods Hole Oceanographic Institution and Mitchel Sogin of the Center for Molecular Evolution at the Marine Biological Laboratories. Special thanks to Bill Thilly for his generous contributions of laboratory space, equipment and supplies and for his intellectual input and to Mitchel Sogin for generous contributions of his phylogenetic experitise and computer time. Over the years at MIT I have been fortunate to learn from a number of talented individuals who have been generous in sharing their technical expertise. Brian Binder, Phouthone Keohavong, Jasper Reese, Jason Seaman, Michael Way and Sheila Frankel have all served as both teacher and friend, and without them I would never have been able to complete this thesis. Samantha Roberts has been a great companion and source of information as the other "molecular person" in the Parsons lab. I would like to thank Cremilda Dias for her excellent technical help in obtaining four of the 16S ribosomal gene seuqneces used in Chapter Two. I have also had the good fortune to interact with the unflaggingly friendly and stimulating members of the Chisholm laboratory: Ginger Armbrust, Lana Arras, Kent Barres, Brian Binder, Jim Bowen, Jeff Dusenberry, Sheila Frankel, Mark Gerath, Karina Gin, Liz Mann and Lisa Moore. Finally, I would like to acknowledge the financial support provided by NSF (BSR-9020254) and by NIH through a training grant to the Department of Biology. TABLE OF CONTENTS Page A BSTRA CT ............................................ ................................................ 2 ACKNOWLEDGEMENTS ...................................................... 4 INTRODUCTION ...................................................................................... .. 11 R eferences .............................................. ...................................................... 16 CHAPTER ONE: MULTIPLE EVOLUTIONARY ORIGINS OF ........ 20 PROCHLOROPHYTES WITHIN THE CYANOBACTERIAL RADIATION CHAPTER TWO: PHYLOGENETIC RELATIONSHIPS AMONG ............ 25 CULTURED STRAINS OF PROCHLOROCOCCUSMARINUS ISOLATED FROM DIVERSE OCEANIC PROVINCES Abstract ...................................................... .................................................. 26 Introduction ................................................ ..................................................... 26 Background ......................................................................... ........................ 28 M ethods .................................. ............ .......... ................. 35 Results ............................................................................................................... 37 Discussion .................................................... ................................................ 58 References ................................................... ................................................ 61 CHAPTER THREE: GENETIC DIVERSITY IN NATURAL ..................... 67 POPULATIONS OF PROCHLOROCOCCUS MARINUS IN THE SARGASSO SEA AND THE GULF STREAM Abstract ...................................................... .................................................. 68 Introduction ................................................... ............................................... 68 M ethods ............................................................... .............. ................ 73 Results .......................................................................... ................................ 82 Discussion........................................................................................................ 108 References ................................................ 113 CHAPTER FOUR: EPILOGUE: RESPONSES IN THE LITERATURE ... 116 TO THE PUBLICATION OF CHAPTER ONE: MULTIPLE ORIGINS OF PROCHLOROPHYTES WITHIN THE CYANOBACTERIAL RADIATION References ....................................................................................................... 120 Page APPENDIX I: PRELIMINARY ANALYSIS OF GENETIC ............... 122 DIVERSITY IN GULF STREAM POPULATIONS OF PROCHLOROCOCCUSMARINUS BY DIRECT SEQUENCING OF PCR PRODUCTS AMPLIFIED FROM FLOW CYTOMETRICALLY SORTED CELLS Reference ......................................................................................................... 129 APPENDIX II: JUSTIFICATION FOR THE • 3.5% SEQUENCE ......130 DIFFERENCE CRITERION FOR

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