YOU CANNOT HIDE YOUR LICHENIZE: INFIDELITY IN THE LEPTOGIUM CYANESCENS SPECIES COMPLEX AND ITS ASSOCIATED NOSTOC By BARRY SAUL KAMINSKY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016 © 2016 Barry Kaminsky To everyone who has helped me along this journey ACKNOWLEDGMENTS Many people have helped me throughout my time as a graduate student. First and foremost, I thank Stuart McDaniel, my advisor, for his tireless enthusiasm and guidance. I also thank the members of the McDaniel lab, especially labmates Sarah Carey and Leslie Kollar. I also thank Adam Payton and Emily Woodruff for all of their patience and advice. Jared Miller helped with the fieldwork. Jacob Landis helped with the ancestral state reconstructions. My collaborators, Roger Rosentreter and Rick and Jean Seavey made helpful suggestions to refine ideas. I also thank the members of my committee, Matthew Smith, and Pamela Soltis for providing lab resources and suggestions throughout the thesis process. Their support greatly shaped how I approached science and research. Lastly I thank my family for their support throughout my entire time in graduate school. Permits were issued by the Florida Department of Environmental Protection (permit # 11051410), Everglades National Park (permit # EVER-00378), and Ordway- Swisher Biological Station (permit # OSR-14-10). Funding for the research was supported by the Thad Owens Memorial Fund (Ordway-Swisher Biological Station), and the Michael May Interdisciplinary Fund (University of Florida Biology Department). 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 SYMBIONT SHARING IN THE LEPTOGIUM CYANESCENS COMPLEX IN FLORIDA ................................................................................................................ 15 Methods .................................................................................................................. 17 Collection Localities and Sampling ................................................................... 17 DNA Extraction and Sequencing ...................................................................... 19 Tests for Sexual Reproduction ......................................................................... 20 Phylogenetic Analyses ..................................................................................... 20 Tests for Symbiont Switching ........................................................................... 22 Results .................................................................................................................... 22 Mycobiont Phylogenetic Analyses .................................................................... 23 Recombination ................................................................................................. 24 Nostoc Phylogeny of Florida Specimens .......................................................... 24 Symbiont Switching between Florida Populations ............................................ 24 Mycobiont Switching of Photobiont on a Global Scale ..................................... 25 Discussion .............................................................................................................. 26 High Specificity in Leptogium sp. A .................................................................. 26 Mycobiont Specificity amongst Leptogium sp. C and Leptogium sp. D ............ 27 Coevolution ...................................................................................................... 28 3 TAXONOMIC REVISION OF THE LEPTOGIUM CYANESCENS SPECIES COMPLEX IN FLORIDA ......................................................................................... 41 Methods .................................................................................................................. 44 Collection Methods ........................................................................................... 44 Phylogenetic Work ........................................................................................... 44 Morphological Work .......................................................................................... 45 Results .................................................................................................................... 46 Phylogenetic Analyses ..................................................................................... 46 Morphology ....................................................................................................... 47 5 Discussion .............................................................................................................. 47 Molecular Evidence for Cryptic Fungal Diversity .............................................. 47 Morphological Investigations and Their Impact on Taxonomy .......................... 48 Taxonomic Diversity ......................................................................................... 50 The Putative Species .............................................................................................. 50 Leptogium cf. arsenei ....................................................................................... 50 Description ................................................................................................. 50 Ecology and distribution ............................................................................. 51 Differentiation ............................................................................................. 51 Nomenclatural investigations ..................................................................... 51 Leptogium austroamericanum .......................................................................... 52 Description ................................................................................................. 52 Ecology and distribution ............................................................................. 53 Differentiation ............................................................................................. 53 Nomenclatural investigation ....................................................................... 53 Leptogium cyanescens ..................................................................................... 54 Description ................................................................................................. 54 Ecology and distribution ............................................................................. 54 Key characteristics ..................................................................................... 54 Nomenclature investigation ........................................................................ 55 Leptogium cf. denticulatum ............................................................................... 55 Description ................................................................................................. 55 Ecology and distribution ............................................................................. 55 Differentiation ............................................................................................. 55 Nomenclatural investigations ..................................................................... 56 APPENDIX: GENBANK RECORDS FOR NOSTOC PHYLOGENY ............................. 63 LIST OF REFERENCES ............................................................................................... 66 BIOGRAPHICAL SKETCH ............................................................................................ 71 6 LIST OF TABLES Table page 2-1 Names of localities, GPS and vegetation community of the eight localities included in analyses ........................................................................................... 30 2-2 Primers utilized for PCR amplification of mycobiont and photobiont markers. .... 31 2-3 List of all Sanger sequences utilized in analyses................................................ 32 2-4 Tests for recombination and location of breakpoints in the four mycobiont clades using the Four Gamete Test and Single Breakpoint analyses ................. 35 3-1 List of GenBank records included in the fungal gene MCM7 phylogeny ............. 57 A-1 GenBank rbcLX sequences of the cyanobacterial photobiont of free-living and lichenized Nostoc ........................................................................................ 63 7 LIST OF FIGURES Figure page 2-1 Phylogeny of the individual mycobiont genes ..................................................... 36 2-2 Phylogenetic tree for the concatenated fungal gene dataset .............................. 37 2-3 Frequency of the 4 mycobiont clades by locality ................................................ 38 2-4 A concatenated phylogeny of the Nostoc associated