An investigation of the spatial distribution and inference of dispersal method in the semi-aquatic lichenised green alga Diplosphaera chodatii around Payuk Lake, Manitoba by Jennifer A. Doering A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of MASTER OF SCIENCE Department of Biological Sciences University of Manitoba Winnipeg, Manitoba, Canada Copyright © 2017 by Jennifer A. Doering ABSTRACT The spatial distribution of haplotypes within the lichenised green alga Diplosphaera chodatii was investigated by collecting specimens from the inflows, lake, and outflow of Payuk Lake, Manitoba. Rock scrapings were cultured showing the presence of free-living Di. chodatii and seven additional algal taxa on rocks near the lichen thallus. The Internal Transcribed Spacer of ribosomal DNA (ITS rDNA) and β-actin genes were sequenced for phylogenetic and haplotype network analyses showing similarity to one another, but neither corresponded to geographic location suggesting high levels of gene flow. Haplotype variation was investigated with respect to four landscape hypotheses to infer the mode of dispersal, which supported the Wind (ITS) and Hydrology (β-actin) hypotheses. This is the first study to provide evidence that Di. chodatii is dispersed by wind and water, and free-living Di. chodatii may aid in the potential establishment of the lichen, Dermatocarpon luridum, and other semi-aquatic lichens within this ecosystem. ii ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Michele Piercey-Normore, for her many years of guidance and mentorship throughout this project and the many years spent in her lab. I also thank my committee members, Drs. Jim Roth and Georg Hausner, for their constructive feedback of both the project and thesis. Special thanks is given to Kyle Fontaine, whose previous research and enthusiasm fuelled the motivation of this project, and for assistance with the microscopy. I would like to give special thanks to Dr. Yolanda Wiersma and the Landscape Ecology and Spatial Analysis lab (Emma LeClerc, Emilie Kissler, Matt McWilliams, Andrew Roberts, Rachel Winsor) at Memorial University for their invaluable mentorship of advanced GIS analyses and techniques. Furthermore, thank you to Mr. Mac Pitcher for an exhilarating excursion along Salmonier Line to experience the beauty and mystique of coastal Newfoundland lichens. Thank you to the staff at the Roses Heritage Inn for their hospitality during my stay. Many thanks are given to Mohanad Zraik, Dr. Tom Booth, and Kam Chhokker for their valuable assistance in both the field and lab. Thank you to Mr. Andre Dufresne for additional help with the microscopy. I would also like to acknowledge the assistance of Dr. David Walker and Mr. Jason Northage for the use of the GIS software at the University of Manitoba. Thank you to Mr. Garry Lux (Government of Manitoba) for providing the digital elevation data of Payuk Lake/Cranberry Portage. Many thanks are given to my lab mates, both past and present, for their companionship. Thank you also to my family for their endless support. Funding was provided by Natural Science and Engineering Research Council of Canada (NSERC CGS-M), The University of Manitoba (TSMA, Dalgarno Award), and Nature Manitoba through scholarships, and through a NSERC Discovery Grant award to Dr. Piercey-Normore. iii DEDICATION Maria, a child is one of the most beautiful gifts the world has to give. You have filled my life with happiness and my heart with love; to you I dedicate this thesis. iv “There is a low mist in the woods – It is a good day to study lichens.” ~ Henry David Thoreau, 1851 v TABLE OF CONTENTS Abstract ........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iii Dedication ...................................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................ vii List of Figures ................................................................................................................................ ix CHAPTER 1 : Introduction ............................................................................................................ 1 1.1 Overview ............................................................................................................................... 1 1.2 Literature Review .................................................................................................................. 7 CHAPTER 2 : Materials and Methods ......................................................................................... 37 2.1 Overview ............................................................................................................................. 37 2.2 Field Collections ................................................................................................................. 37 2.3 Algal Culturing .................................................................................................................... 42 2.4 DNA extraction, PCR, and sequencing ............................................................................... 43 2.5 GIS Analyses ....................................................................................................................... 53 2.6 Data Analysis ...................................................................................................................... 56 CHAPTER 3 : Results .................................................................................................................. 60 3.1 Photobiont ITS rDNA Phylogeny ....................................................................................... 60 3.2 Photobiont β-actin Phylogeny ............................................................................................. 69 3.3 Combined ITS/β-actin Phylogeny ....................................................................................... 70 3.4 Haplotype Networks ............................................................................................................ 74 3.5 Haplotype Distribution ........................................................................................................ 79 3.6 Hydrology and Gene Flow .................................................................................................. 87 3.7 Algal Morphological Diversity ......................................................................................... 100 CHAPTER 4 : Discussion ........................................................................................................... 105 4.1 Population structure is present despite high gene flow ..................................................... 105 4.2 Total algal genetic variation was higher than expected .................................................... 108 4.3 Free-living Diplosphaera chodatii may contribute to high levels of genetic variation .... 110 4.4 Phylogenetic incongruence may be a result of high genetic variation .............................. 113 4.5 Haplotype networks and phylogenies did not correspond to geographic location ............ 117 CHAPTER 5 : Conclusions and Future Directions..................................................................... 124 5.1 Conclusions ....................................................................................................................... 124 5.2 Future Directions ............................................................................................................... 127 Literature Cited ........................................................................................................................... 130 Appendix A: Environmental parameters collected at collection sites in Payuk Lake ................ 147 Appendix B: Recipe for Bold’s Basal Medium (BBM) for green algae..................................... 154 Appendix C: Workflow of hydrological modelling and spatial analyses in the LESA lab during a travel study to St. John’s, Newfoundland (April 2015) .............................................................. 155 vi LIST OF TABLES Table 2.1. Internal transcribed spacer (ITS) region of rDNA and β-actin primers used for polymerase chain reaction (PCR) and DNA sequencing. ............................................................. 46 Table 2.2. Collection sites, photobiont samples, and sequence accession numbers used in this study. Collection sites 1-34 and from Whiteshell Provincial Park (WS) were sampled in triplicate from this study. Collection sites with a letter before the number were taken from Fontaine et al. (2013). Photobiont samples D (Dermatocarpon luridum from Payuk Lake) and WS (De. luridum from Whiteshell) were collected and sequenced in this study, photobiont samples with P (from Payuk Lake) and MC (Mistik Creek) were collected from Fontaine et al. (2013). Latitude and longitude are included. GenBank Accession Numbers are indicated and a dash refers to samples that were not sequenced. Collection site numbers correspond with