INTEGRATED CONSERVATION OF FLORIDA ORCHIDACEAE IN THE GENERA Habenaria AND Spiranthes: MODEL ORCHID CONSERVATION SYSTEMS FOR THE AMERICAS By SCOTT L. STEWART 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 2007 1 © 2007 Scott L. Stewart 2 To all those who have a true passion in life 3 ACKNOWLEDGMENTS I thank Dr. Michael Kane for his support, patience, dedication, and encouragement during my research, and for the many conversations about plants and more. I also thank Dr. Charles Guy, Dr. Thomas Sheehan, Dr. Doria Gordon, and Dr. James Kimbrough for serving on my supervisory committee. The members of my supervisory committee have shown me what it means to be true professionals and academicians. Much of this work could never have been undertaken without a great deal of help from many people, including Paul Martin Brown (Ocala, Florida), Dr. Lawrence Zettler (Illinois College), Larry Richardson (USFWS-FPNWR), James Durwachter (USFWS-FPNWR), Layne Hamilton (USFWS-FPNWR), and Ben Nottingham (USFWS-FPNWR). Their guidance and assistance made many experiments and field research sessions possible. I also thank the fellow graduate students with whom I have worked over the years: Carmen Valero-Aracama, Philip Kauth, Daniela Dutra, Timothy Johnson, and Xiliu Shen. Brainstorming sessions, research conversations, and field work would have not been possible without their assistance. I thank the Socash family (Brooskville, Florida) for generously allowing me to use their population of Habenaria macroceratitis as a research site, as well as the Rayonier Corporation (Starke, Florida) for allowing me access to the population of Spiranthes floridana. The City of Brooksville (Florida), Marjorie Harris Carr Cross Florida Greenway, National Fish and Wildlife Foundation, U.S. Fish and Wildlife Service, Florida Division of Forestry, Florida Division of Environmental Protection, Florida Division of Plant Industry, and the San Diego County Orchid Society all contributed to the completion of this work. The assistance of Ginger Clark and the University of Florida ICBR Genetic Analysis laboratory with the preparation and analysis of AFLP data is greatly appreciated. 4 I also thank my parents, Duane and Victoria Stewart; and my sister, Leslie Stewart, for their unwavering support of my educational goals despite not fully understanding what I was doing. Finally, I thank my wife, Angela O’Donnell, for her unflinching love, support, grudging interest, and immense degree of patience during my work. Without her love and understanding none of this would have been possible. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................10 ABSTRACT...................................................................................................................................13 CHAPTERS 1 LITERATURE REVIEW.......................................................................................................14 Introduction.............................................................................................................................14 Study Rationale.......................................................................................................................15 Conservation Ethic and Modes........................................................................................15 Integrated Orchid Conservation ......................................................................................19 Plants of Interest .....................................................................................................................20 Selection Criteria.............................................................................................................20 Plants of Study.................................................................................................................21 Habenaria macroceratitis ........................................................................................21 Spiranthes floridana.................................................................................................22 Spiranthes brevilabris ..............................................................................................23 Spiranthes cernua Deep South race .........................................................................24 Overview of Pertinent Literature............................................................................................24 Orchid Ecology................................................................................................................24 Orchid-Mycobiont Association .......................................................................................27 History and background ...........................................................................................27 Infection and digestion.............................................................................................28 Photomycotrophic balance .......................................................................................30 Orchid mycobiont taxonomy....................................................................................31 Orchid Propagation and Culture......................................................................................35 Asymbiotic seed culture...........................................................................................35 Symbiotic seed culture .............................................................................................40 Orchid Pollination Biology..............................................................................................45 Molecular Genetics and Genetic Diversity in Orchids....................................................50 2 POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Habenaria macroceratitis.........................................................................................................................63 Introduction.............................................................................................................................63 Materials and Methods ...........................................................................................................65 Study Sites.......................................................................................................................65 Plant Demography...........................................................................................................65 Pollinator Observations ...................................................................................................66 6 Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination .................67 Sampling, DNA Extraction and Amplified Fragment Length Polymorphism (AFLP) .........................................................................................................................70 Results.....................................................................................................................................72 Study Sites.......................................................................................................................72 Plant Demography...........................................................................................................72 Pollinator Observations ...................................................................................................73 Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination .................74 AFLP Data.......................................................................................................................75 Discussion...............................................................................................................................75 Implications for Integrated Conservation Planning................................................................83 3 SEED CULTURE AND IN VITRO SEEDLING DEVELOPMENT OF Habenaria macroceratitis.........................................................................................................................96 Introduction.............................................................................................................................96 Materials and Methods ...........................................................................................................99 Asymbiotic Seed Germination ........................................................................................99 Seed source and sterilization....................................................................................99 Asymbiotic media survey.........................................................................................99 Effects of carbohydrate source on asymbiotic seed germination...........................100 Effects of exogenous cytokinins on asymbiotic seed germination ........................101 Effects of photoperiod on asymbiotic seed germination........................................101 Effects of photoperiod on in vitro seedling development ......................................102 Symbiotic Seed Germination.........................................................................................102