Chemical ecology and phenotype in lichens: examining some environmental factors that influence variability, species assemblages and chemical compounds By Mohanad Talal Zraik A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biological Sciences University of Manitoba Winnipeg, Manitoba Canada Copyright © 2018 by Mohanad Talal Zraik ABSTRACT The general goal of this thesis was to explore the chemical ecology and macromorphological variability in lichens that colonize soils in five locations in Manitoba with respect to the carbon-nutrient balance hypothesis (CNBH). The CNB hypothesize states that when a plant or lichen is growing in nutrient-poor conditions, the excess carbon may be shunted into biosynthesis of carbon-based secondary metabolites such as polyketides. Three specific goals were further examined in four chapters to investigate:1) the distribution of secondary metabolites and species assemblages with respect to soil characteristics (Ch. 2), which resulted in 2) a report on Cladonia magyarica and C. humilis in Manitoba, describing the habitat, and providing morphological and chemical comparisons to distinguish five species (Ch. 3), 3) the phenotype in lichen species showing plasticity with respect to soil type, temperature and precipitation (Ch. 4), and 4) the effect of temperature and moisture on the quantity of three secondary metabolites (atranorin, fumarprotocetraric acid, and usnic acid) produced by three lichen species (Ch. 5). Field collections of lichens, soil features and environmental data were made in five locations. Secondary metabolites were determined using Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Morphological and fecundity characteristics were podetium height, cup diameter, squamule diameter, apothecium diameter, number of apothecia on cup and presence of apothecia. Soil characteristics were pH, organic matter, sand content, and sand grain shapes. The results showed that usnic acid and atranorin supported the CNB hypothesis. Species as Cladonia magyarica and C. humulis were reported for the first time in Manitoba. The CNB hypothesis was not supported for all lichen species, which may have been complicated by variability in biotic and abiotic conditions. Temperature and moisture affected the stability of secondary metabolites and partial degradation i occurred. This thesis forms a foundation for further studies on the relationship between secondary metabolites with biotic and abiotic factors. ii ACKNOWLEDGEMENTS I would like to thank first and foremost my advisor Dr. Michele Piercey-Normore. Thank-you very much Michele for accepting me for the project in your lab. Her patient guidance, understanding and support were integral for the completion of this project. Working with you was a privilege and I could not have asked for a better supervisor. I would like to thank my thesis committee of Dr. Jim Roth, Dr. Georg Hausner and Dr. John Sorensen for their guidance and advice regarding my thesis and during the candidacy exam and annual meetings. Thank-you Dr. Sorensen and your lab personnel for letting me work, and use your lab for a very long time to finish my HPLC analysis, and for all your guidance and valuable comments. My committee members were much appreciated and they taught me a lot about writing and data analysis. I would like to thank my labmates from the former Buller Lab 509; special thanks to Jennifer Doering and Chris Deduke. Their friendship, help and laughter made work enjoyable. Lab discussions taught me how to better analyze and interpret scientific papers. I would like to thank Dr. Tom Booth. He taught me a lot about efficient field work and data collection while being able to enjoy being outdoors. Thank-you Dr. John Markham, Dr. Bruce Ford, Diana Sawatzky and Dr. Sylvia Renault for providing your labs to finish my study. I would like to thank the Biological Sciences faculty and office staff, particularly Maureen Foster, Jaime Stringer and Sylvia Lapointe. In addition, I thank the Faculty of Science, Faculty of Graduate Studies, University of Manitoba Graduate Student Association, Canadian Botanical Association, and National Science and Engineering Research Council (NSERC) for financial support offered throughout my project. iii Finally, I would like to thank my family: my beautiful wife Iman, my three angels Shamel, Hamza, Yussif, and my parents, who are overseas and I have not seen them for seven years, but I feel their support. Their love and support really helped me complete this project. iv DEDICATION To all the innocent people who were killed in my country, Syria, many of whom were friends and relatives; to you I dedicate this thesis. v Thesis, the publication status of chapters, and the role of each coauthor in each chapter Chapter Author Contributions of each author Article status 1 Mohanad Zraik MZ reviewed the literature, organized the Not to be chapter, and wrote the chapter. MPN edited published the chapter. 2 Mohanad Zraik MZ collected the material, performed Published in statistical analysis of the data and wrote the Botany 96(4): Tom Booth chapter. TB helped with field work, 267-279 Michele D. Piercey- discussed results and recommended Normore literature. MPN generated the original idea with me, guided the process and edited the chapter. 3 Mohanad Zraik MZ conducted the experiment, collected the Published in data, performed the analysis of the data and Evansia Tom Booth wrote the chapter. TB helped with field 33(3):136-144 Michele D. Piercey- work, discussed results and recommended Normore literature. MPN generated the original idea with me, guided the process and edited the chapter. 4 Mohanad Zraik MZ collected the material, performed HPLC Submitted to analysis and statistical analysis of the data the journal John Sorensen and wrote the chapter. JS helped with HPLC vi Michele D. Piercey- analysis. MPN generated the original idea Oecologia Normore with MZ, guided the process and edited the chapter 5 Mohanad Zraik MZ conducted the experiment, collected the To be data, performed the analysis of the data and submitted John Sorensen wrote the chapter. JS Reviewed, discussed Michele D. Piercey- the results and recommended literature. Normore MPN generated the original idea with me, guided the process and edited the chapter 6 Mohanad Zraik Final Discussion and Conclusions were Not to be written by MZ and edited by MPN. published vii TABLE OF CONTENTS ABSTRACT............................................................................................................................i ACKNOWLEDGEMENTS..................................................................................................iii DEDECATION......................................................................................................................v THESIS, THE PUBLICATION STATUS OF CHAPTERS AND THE ROLE OF EACH COAUTHOR IN EACH CHAPTER....................................................................................vi TABLE OF CONTENTS......................................................................................................viii LIST OF TABLES................................................................................................................xiv LIST OF FIGURES ............................................................................................................xviii CHAPTER 1: GENERAL INTRODUCTION AND LITERATURE REVIEW 1.1 General Introduction....................................................................................................1 1.2 Thesis goals and objectives...........................................................................................7 1.3 Literature review...........................................................................................................9 1.3.1. Lichen classification and taxonomy............................................................. 9 1.3.2. Soil and geology in Manitoba....................................................................... 11 1.3.3. Chemical ecology in lichens..........................................................................14 1.3.4. Lichen morphology and growth forms..........................................................14 1.3.5. Secondary metabolites...................................................................................17 1.3.6. Carbon-nutrient balance hypothesis.............................................................21 viii 1.3.7. Water availability and phenotypic plasticity................................................22 1.3.8. Techniques for studying lichen secondary metabolites...............................24 CHAPTER 2: RELATIONSHIP BETWEEN LICHEN SPECIES COMPOSITION, SECONDARY METABOLITES AND SOIL PH, ORGANIC MATTER, AND GRAIN CHARACTERISTICS IN MANITOBA 2.1 Abstract.......................................................................................................................27 2.2 Introduction................................................................................................................ 28 2.3 Materials and Methods.............................................................................................. 31 2.3.1. Experimental design and sample collection...................................................31 2.3.2. Soil characteristics.........................................................................................34 2.3.3. Statistical analyses.........................................................................................35