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ANALYSIS of MERCURY CONTENT of LICHENS in NOVA SCOTIA: POTENTIAL USE AS PASSIVE AIR SAMPLERS by Cardy Hallett Saunders Thesis Su

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ANALYSIS OF MERCURY CONTENT OF LICHENS IN NOVA SCOTIA: POTENTIAL USE AS PASSIVE AIR SAMPLERS By Cardy Hallett Saunders Thesis submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours in Biology Acadia University May 2017 © Copyright by Cardy Hallett Saunders, 2017 ii This thesis by Cardy Hallett Saunders is accepted in its present form by the Department of Biology as satisfying the thesis requirements for the degree of Bachelor of Science with Honours Approved by the Thesis Supervisors _______________________________ ___________________ Dr. Allison Walker Date _______________________________ ___________________ Dr. Nelson O’Driscoll Date Approved by the Head of the Department _______________________________ ___________________ Dr. Brian Wilson Date Approved by the Honours Committee _______________________________ ___________________ Dr. Jun Yang Date iii I, CARDY HALLETT SAUNDERS, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in paper, microform, or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis. ________________________________ Signature of Author ________________________________ Date iv v DEDICATION This thesis is dedicated to Col. Spon Thursby for keeping me insane for the duration of this adventure. Col. Spon Thursby is a multi-faceted quasi-existential entity comprising my partner, family, and close friends; my long-suffering honours advisors, corresponding authors, academic professors, and lab-mates; and of course The Goons. A heartfelt and sincere thank-you to everyone involved for keeping me on track, hoisting my spirits up, retaining confidence in me, pressing my nose to the grindstone, and just generally putting up with me for the whole duration. This thesis would not be here if it was not for you all. Thank you. vi ACKNOWLEDGEMENTS The author would like to express his profound thanks to all those who assisted and advised with this study. Drs. Allison Walker and Nelson O’Driscoll are of course owed thanks for their dedication and tenacity in overseeing and contributing to this project, despite having numerous other duties. Thanks are also owed to Drs. Mark Mallory and Dave Shutler who provided advice on data visualization, interpolation, and also served as sounding boards for certain aspects of data manipulation and statistical interpretations. Robert Cameron, provincial lichenologist to the Nova Scotia government, provided much assistance with sample collection, advice on data visualization, and was indispensible for the production of interpolated maps, as he alone had a working copy of ArcGIS at his disposal. Deep thanks are owed to Frances Anderson, lichenologist extraordinaire at the Nova Scotia Museum, who instilled in me a sense of awe for lichens of all types, and provided essential training in the identification of different species thereof. In addition to the persons above named, this study would lack most or all of its data, were it not for a band of intrepid research assistants who assisted in sample collection, preparation, analysis, and data interpretation. Dr. David Kristie is himself owed thanks, as is Phyllis Essex-Fraser from his lab. From the O’Driscoll lab: Rachel Clarke, Sara Klapstein, Jocelyn Kickbush, and Kelly Stevens. From the Walker lab: Brent Robicheau, Alexander Young, Sarah Adams, Adele Bunbury-Blanchette, Sarah Fancy, and Tyler D’Entremont. Ruth Newell is also owed thanks for her input, encouragement, and for allowing countless boxes of samples to clutter up her herbarium during the collection and preparation phases of the project. Erika Holland is also owed thanks for her encouragement both in and out of the lab environment. This work was supported by an Arthur Irving Academy of the Environment Research Grant to A. Walker and N. O’Driscoll. Thanks are thus owed to the trustees of the Arthur Irving Academy of the Environment. vii TABLE OF CONTENTS APPROVAL PAGE ........................................................................................................ iii PERMISSION FOR DUPLICATION .......................................................................... iv DEDICATION ................................................................................................................ vi ACKOWLEDGEMENTS ............................................................................................. vii TABLE OF CONTENTS ............................................................................................. viii ABSTRACT ...................................................................................................................... x INTRODUCTION ........................................................................................................... 1 1.1 Mercury ................................................................................................ 1 1.2 Air Pollution in Eastern Canada .......................................................... 5 1.3 Mercury Adsorption to Surfaces .......................................................... 6 1.4 Lichens ................................................................................................. 8 1.5 Lichens as Passive Air Samplers ....................................................... 10 1.6 DNA Barcoding ................................................................................. 12 1.7 Study Objectives and Hypotheses ...................................................... 13 MATERIALS AND METHODS .................................................................................. 14 2.1 Field Sample Collection and Preparation .......................................... 14 2.2 Mercury Analysis ............................................................................... 15 2.3 Data Analysis ..................................................................................... 17 2.4 DNA Extraction and Sequencing ....................................................... 18 2.5 Phylogenetic Tree Construction ......................................................... 20 RESULTS ....................................................................................................................... 22 viii 3.1 Mercury Concentrations ..................................................................... 22 3.2 DNA Barcoding and Phylogenetics ................................................... 29 DISCUSSION ................................................................................................................. 32 4.1 Mercury Concentration Data Distribution ......................................... 32 4.2 Mercury Concentration in Usnea vs. Hypogymnia Lichens .............. 32 4.3 Spatial Hotspots for Mercury Based on Lichen Data ........................ 35 4.4 Phylogenetic Analysis of Nova Scotia Lichens ................................. 36 4.5 Final Notes and Future Research Topics ........................................... 38 LITERATURE CITED ................................................................................................. 42 APPENDICES ................................................................................................................ 52 Appendix 1 ........................................................................................................... 52 Appendix 2 ........................................................................................................... 60 FIGURES ............................................................................................................................. Figure 1. Location of ITS primers on fungal rDNA ............................................ 12 Figure 2. Frequency distribution of all lichen sample mercury concentrations ... 22 Figure 3. Frequency distribution of all Hypogymnia mercury concentrations .... 23 Figure 4. Frequency distribution of all Usnea mercury concentrations ............... 24 Figure 5. Plot of Usnea vs Hypogymnia mercury concentrations per location ... 25 Figure 6. GIS plot of sample locations throughout Nova Scotia ......................... 26 Figure 7. Kernel-density interpolation of all lichen mercury concentrations ...... 27 Figure 8. Kernel-density interpolation of Hypogymnia mercury concentrations 28 Figure 9. Kernel-density interpolation of Usnea mercury concentrations ........... 28 Figure 10. Neighbor-joining phylogenetic tree of ITS rDNA barcodes .............. 29 ix ABSTRACT Mercury is a global pollutant present in a wide range of ecosystems. Atmospheric transport of elemental mercury gas enables this substance to be deposited far from its source. Quantifying mercury in biological indicator species, such as lichens, provides information about where mercury pollution may be an issue. Close to three hundred samples of Hypogymnia sp. and Usnea sp. mainly, but 11 lichen species in total were collected from trees throughout mainland Nova Scotia and analyzed for total mercury using thermal degradation - atomic absorbance analysis. The resulting concentrations and geolocations were mapped using QGIS 2.12 and ArcGIS 10.4 to produce kernel density analysis hotspot maps. From these maps, it was possible to discern areas of higher mercury concentrations and trends across Nova Scotia, Canada. The presence of high concentration features in areas with known mercury problems such as Kejimkujik National Park and National Historic Site suggested that lichens may be useful indicators of regional atmospheric mercury trends. 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