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Ann Arbor, MI 48106 COLONIZATION OF SEAGRASS LEAVES; A MODEL BIOLOGICAL SYSTEM FOR THE STUDY OF RECRUITMENT IN A MARINE ENVIRONMENT A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANICAL SCIENCES (BOTANY) AUGUST 1993 By Teena Michael-Taxis Dissertation Committee: Celia M. smith, Chairperson Isabella A. Abbott Kent W. Bridges Harry Yamamoto Robert Kinzie III Copyright by Teena Michael-Taxis All Rights Reserved iii ------------------- ACKNOWLEDGEMENTS I deeply appreciate each committee member including the late Dr. Sanford Siegel for his or her role in the development of this resea~ch. The insights and efforts of Dr. Celia smith in particular as well as Drs. Isabella Abbott, Kim Bridges, Robert Kinzie III and Harry Yamamoto were invaluable in preparation of the text and extending questions that furthers the research. Tina Corvalo and the Biological Electron Microscope Facility assisted in many aspects of the ultrastructural analysis. This research was supported by the Department of Botany, University of Hawaii, the U.S. Office of Naval Research Grant No. N00014-90-J-1932 and Hawaii Natural Energy Institute, University of Hawaii, Honolulu, Hawaii. I extend special appreciation to Rick Hanna for his multifaceted role in development of this dissertation. I gratefully acknowledge the support of Harriet Matsumoto, Dr. Gerry Carr, Gerry Ochicubo and Dora Tsuha from the Botany Department. I sincerely thank my friends and family, Jan Taketa, Sue Douglas, Phillip Moravchic, In Sun Kim, Chris Omeara, Jane Dawson, Marie Bruegman, Naomi Phillips, Luis Vega and my mother and daughter, Milledge and Teale for their patience, help and challenges. iv ABSTRACT Leaves of the Hawaiian seagrass, Halophila hawaiiana Doty and stone are a base for diverse epiphyte communities. The leaves were considered a model system for the study of patterns in colonization. The anatomy of the leaves, as substrate for colonization, was documented prior to investigating patterns and processes of colonization along a gradient of wave exposure. Ultrastructural assessment of mature leaves revealed details of epidermal, ground and vascular tissues that extends our knowledge of the genus. Cell wall ingrowths with invaginated plasmalemma characterized the epidermal cells and were most elaborately developed in the upper leaf surfaces. Chloroplasts, mitochondria, endoplasmic reticulum and dictyosomes are commonly associated with the ingrowth regions. structures indicative of symplastic and apoplastic systems were detected. Mature, twelve day old leaves showed ultrastructural modifications of when colonized by specific epiphytes. These modifications included: 1) distinctive elaborations of the cell wall ingrowths and abundant secretory organelles when colonized by crustose coralline algae, 2) disruption of the fibrillar cell wall, osmiophilic droplets, vesiculate membrane-bound structures and altered ingrowth regions as well as reduced numbers of chloroplasts and mitochondria when colonized by specific bacteria. The v distribution of these epiphytes followed a wave exposure gradient. Colonization by filamentous red algae, cyanobacteria and bacteria in microcolonies occurred in both sites and did not alter the leaf ultrastructure. Distinctive polysaccharides were observed at the microbial cell surfaces; these molecules may provide adhesion between host and epiphytes. A possible mechanism(s) driving patterns in colonization and recruitment was examined via novel use of specific lectins as probes for newly emergent seagrass leaves and an artificial substrate. Distributions and identities of several naturally occurring glycoconjugates were resolved in films that formed between one and three days on glass slides. In contrast, no film nor glycoconjugate sites were visible on surfaces of young seagrass leaves. The spatial and chemical heterogeneity of settlement cues on substrates may generate microscale patterns in "lock and key mechanism(s)". Surface glycoconjugates appear to provide a cue for recognition by settling stages that may provide an initial step that influences ultimate community features. VI TABLE OF CONTENTS Acknowledgements ••......••••••••••••••••••••••••••••••.••. i v Abstract v List of Tables xi List of Figures xii List of Abbreviations xviii Chapter 1: Literature Review••••••••.••••••.••••••••••.•.•.1 Background 1 Introduction to Plant community Ecology•.•..•..•.••.••• 2 Hawaiian Seagrass Biology, Taxonomy and Ecology•.•...•. 6 Seagrass Leaves as Host Substrates•••.••••••••••••••••• 9 Epiphyte Communities••••••••••••.••••••••••••••••••••• 13 Epiphyte Influences on Hosts••••••••••••.••.•.••.•.•.• 17 Establishment of Seagrass and Epiphyte Associations•.• 19 Forces of Attachment..•....•................•......... 25 Surface Properties and Modifying Films••.••.••••••..•• 28 Surface Texture 29 Surface Tension 30 Hydrophobic Surface Properties.•••.•...•..•.••.•.•.• 31 Surface Charge 32 Surface Modifications•.•••.•••••••••••.•.••....•.... 33 Glycoprotein-mediated Cell Surface Interactions•.... 34 Focus on Halophila hawaiiana as Host for Epiphytes...• 37 Literature Cited 38 Chapter 2: Leaf Ultrastructure of the Hawaiian Seagrass, Halophila hawaiiana Doty and Stone.... 56 Abstract 56 vii Introduction 57 systematics 57 Anatomy •.••••••••••••••••••••••.•••••.••••.•••••••.••• 60 Materials and Methods. • ••••••••••••••••••• 63 Collection of Halophila hawaiiana. ........ 63 Sample Preparation for Transmission Electron Microscopy . 64 Observations by Light Microscopy......•.••.•......•... 65 Results 66 The Epidermis 67 Ground Tissue 69 Vascular Tissue 70 Discussion 73 Conclusions. 82 Literature cited. •••.• 102 Chapter 3: Ultrastructure of Seagrass and Epiphyte Interfaces from Wave Exposed and Sheltered Subtidal Habitats••••••..•••...••..•.••••...... 107 Abstract 107 Introduction. .108 Epiphytes... • 109 The Host and Epiphyte Interface••......••...•....•... 111 Environmental Influences on Settlement of Epiphytes 114 Materials and Methods....•..•......................•... 116 Sampling and site.. ................... 116 Sample Preparation for Electron Microscopy 116 viii Epiphyte Population Counts..••••••••.•.••••••••••.••. 118 Results••• .120 Part A. Technical and Qualitative Evaluation of Epiphytes and the Host••••••••••••••••••••.•••••• .120 Fixation Results••••• .120 A Contrast of Seagrass and Epiphyte Populations/Site••••••••••••••••.••• • ••• 122 Part B. Qualitative Observations on Epiphytized Seagrass Leaves ••.•..•••••.•.•••.•••............. .125 The Epiphytes. .125 The Host and Epiphyte Interface. .127 Discussion•• .......................................... .132 Conclusions. .145 Literature cited. .173 Chapter 4: Lectins Probe Molecular Films in Biofouling: Characterization of Early Films on Living and Non-living Surfaces•••.••••••• .182 Abstract••••• ......................................... .182 Introduction. ................................ .183 Materials and Methods. .186 Laboratory Films•.•• .186 Natural Films on Glass. .187 Natural Films on a Living Surface. .187 Application of Lectins••••.••••••• .187 Microscopy. .189 Results•••••• .189 single Lectin Studies in Artificial Films. .189 ix Lectin Studies of One and Three-day Natural Films on Glass Slides . • •• 190 Lectin Studies of a Newly Emergent Living Substrate 192 Discussion•• ...................................... • •••• 193 Conclusions. • •••• 197 Literature cited. • .203 Chapter 5: Synthesis. •• 208 x LIST OF TABLES Table Page 3.1. Preservation of Eukaryotic and Prokaryotic Cells and Cell Products•.••••.••••••••••••••••. 147 3.2. A Profile of Halophila hawaiiana and its Epiphytes; Number of Epiphyte Cells/Host cell. N = 100 host cells 148 3.3. A Profile of Halophila hawaiiana Surface Cell Components;