ECOLOGY AND METABOLIC ADAPTATIONS OF BARK BEETLE-ASSOCIATED FUNGI By YIN-TSE HUANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2019 © 2019 Yin-Tse Huang To family and friends ACKNOWLEDGMENTS I would like to thank my advisor and committee chair Jiri Hulcr for guidance and support. I would also like to thank my committee members Jason Smith, Jeffrey Rollins, Matthew Smith, and Miroslav Kolařík for their time and feedback. My research work cannot be done without the help from my lab members: A. Simon Ernstsons, Allan Gonzalez, Andrew Johnson, Caroline Storer, Craig Bateman, Demian Gómez, Jackson Landers, James Skelton, Michelle Jusino, and You Li. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .....................................................................................................4 LIST OF TABLES ................................................................................................................8 LIST OF FIGURES .............................................................................................................9 ABSTRACT ...................................................................................................................... 11 CHAPTER 1 INTRODUCTION ....................................................................................................... 13 2 TWO NEW GEOSMITHIA SPECIES IN G. PALLIDA SPECIES COMPLEX FROM BARK BEETLES IN EASTERN USA ............................................................ 15 Introduction to Chapter 2 ........................................................................................... 15 Materials and Methods for Chapter 2 ....................................................................... 17 Sampling, Isolating, and Preserving of Fungal Isolates ..................................... 17 Studies of Morphology and Growth Rate ........................................................... 18 DNA Extraction, Amplification and Sequencing ................................................. 19 DNA Sequences Analyses ................................................................................. 20 Results for Chapter 2 ................................................................................................ 21 Culturing Data of Geosmithia ............................................................................. 21 DNA Sequence Data and Phylogenetic Analysis............................................... 22 Taxonomy .................................................................................................................. 23 Geosmithia brunnea Y.T. Huang, M. Kolařík & J. Hulcr, sp. nov. ..................... 23 Geosmithia proliferans Y.T. Huang, M. Kolařík, M.T. Kasson & J. Hulcr, sp. nov. .................................................................................................................. 25 Discussion for Chapter 2 ........................................................................................... 27 3 GEOSMITHIA SPECIES IN SOUTHEASTERN USA AND THEIR AFFINITY TO BEETLE VECTORS AND TREE HOSTS ................................................................. 54 Introduction to Chapter 3 ........................................................................................... 54 Materials and Methods for Chapter 3 ....................................................................... 58 Sampling and Isolation ....................................................................................... 58 DNA Extraction, Amplification and Sequencing ................................................. 59 DNA Sequences Analyses ................................................................................. 60 Community Analysis ........................................................................................... 61 Results for Chapter 3 ................................................................................................ 62 Identification of Geosmithia Species and Their Occurrence on Trees and Beetles ............................................................................................................. 62 Community Analysis ........................................................................................... 63 Discussion for Chapter 3 ........................................................................................... 65 5 TCD Causal Agents ............................................................................................ 65 Beetle Vector and Tree Host Affinities with Geosmithia spp. ............................ 66 Conclusion for Chapter 3 .......................................................................................... 69 4 MULTIPLE EVOLUTIONARY ORIGINS LEAD TO DIVERSITY IN THE METABOLIC PROFILES OF AMBROSIA FUNGI .................................................... 92 Introduction to Chapter 4 ........................................................................................... 92 Materials and Methods for Chapter 4 ....................................................................... 96 Fungal Isolates ................................................................................................... 96 Phenotype Microarrays (PM) Study ................................................................... 96 Phylogenetic Distances among Fungal Species ................................................ 98 Statistical Analyses ............................................................................................. 99 Convergence of Carbon Assimilation Test ......................................................... 99 Metabolic Niche Breadth Test ............................................................................ 99 Do Ambrosia Fungi Share One or A Few Preferred Carbon Sources? ........... 100 How Much Is Carbon Assimilation Explained by Phylogenetic Origin Versus Symbiotic Habit?............................................................................................ 101 Results for Chapter 4 .............................................................................................. 102 Convergence of Carbon Assimilation Test ....................................................... 102 Metabolic Niche Breadth Test .......................................................................... 103 Do Ambrosia Fungi Share One or A Few Preferred Carbon Sources? ........... 103 How Much Is Carbon Assimilation Explained by Phylogenetic Origin Versus Lifestyle?........................................................................................................ 104 Discussion for Chapter 4 ......................................................................................... 105 Suitability of Method ......................................................................................... 106 Carbon Assimilation in Relation to Fungal Phylogeny and Lifestyle ............... 106 Metabolic Breadth of Ambrosia and Non-Ambrosia Fungi .............................. 108 Conclusion for Chapter 4 ........................................................................................ 111 5 LIPIDS AND SMALL METABOLITES PROVISIONED BY AMBROSIA FUNGI TO SYMBIOTIC BEETLES ARE PHYLOGENY-DEPENDENT, NOT CONVERGENT ....................................................................................................... 121 Introduction to Chapter 5 ......................................................................................... 121 Materials and Methods for Chapter 5 ..................................................................... 124 Fungal Isolates ................................................................................................. 124 Fungal Inoculation and Sampling ..................................................................... 124 Liquid Chromatography-Tandem Mass Spectrometry ..................................... 126 Statistical Analysis ............................................................................................ 127 Convergence of Overall Metabolite Profile Test .............................................. 128 Convergence of Spore Metabolite Profile Test ................................................ 128 Do Ambrosia Fungi Synthesize A Specific Type of Molecules? ...................... 129 Results for Chapter 5 .............................................................................................. 130 Convergence of the Overall Metabolome......................................................... 130 Convergence of Spore Metabolites .................................................................. 131 Do Ambrosia Fungi Synthesize or Lack Unique Compounds?........................ 131 6 Discussion for Chapter 5 ......................................................................................... 133 Conserved Metabolome of Each Ambrosia Fungus ........................................ 133 Specific Compounds in Ambrosia Tissues ....................................................... 135 Conclusion for Chapter 5 ........................................................................................ 137 6 CONCLUSIONS ...................................................................................................... 144 LIST OF REFERENCES ................................................................................................ 145 BIOGRAPHICAL SKETCH ............................................................................................ 164 7 LIST OF TABLES