SALTMARSH SEDIMENT FUNGAL COMMUNITIES AND ARBUSCULAR MYCORRHIZAL FUNGI IN SPOROBOLUS PUMILUS (ROTH) (POACEAE) (SPARTINA PATENS) OF THE MINAS BASIN, NOVA SCOTIA; IDENTIFICATION, ABUNDANCE AND ROLE IN RESTORATION by TYLER WADE D’ENTREMONT BScH Acadia University 2017 Thesis submitted in partial fulfillment of the requirements for the Degree of Master of Science (Biology) Acadia University Fall Convocation 2019 © by TYLER WADE D’ENTREMONT, 2019 This thesis by Tyler Wade d'Entremont was defended successfully in an oral examination on 24 June 2019. The examining committee for the thesis was: _____________________________________ Dr. Mary Sweatman, Chair _____________________________________ Dr. Jeremy Lundholm, External Reader _____________________________________ Dr. Mark Mallory, Internal Reader _____________________________________ Dr. Allison Walker, Supervisor _____________________________________ Dr. Juan Carlos López-Gutiérrez, Supervisor _____________________________________ Dr. Rodger Evans, Head/Director This thesis is accepted in its present form by the Division of Research and Graduate Studies as satisfying the thesis requirements for the degree Master of Science (Biology). ..................................................................... ii I, Tyler Wade d’Entremont, grant permission to the University Librarian at Acadia University to archive, preserve, reproduce, loan or distribute copies of my thesis in microform, paper, or electronic formats on a non-profit basis. I undertake to submit my thesis, through my University, to Library and Archives Canada and to allow them to archive, preserve, reproduce, convert into any format, and to make available in print or online to the public for non-profit purposes. I, however, retain the copyright in my thesis. _________________________________ Author _________________________________ Supervisor _________________________________ Supervisor _________________________________ Date iii Table of Contents LIST OF TABLES ...................................................................................................................................... VI LIST OF FIGURES ................................................................................................................................... VII ABSTRACT .................................................................................................................................................. X ACKNOWLEDGEMENTS ....................................................................................................................... XI PRELUDE .................................................................................................................................................. XII CHAPTER 1: SALTMARSH SEDIMENT FUNGAL COMMUNITIES DIFFER BASED ON SALTMARSH ZONATION AND LOCATION ......................................................................................... 1 1.1 INTRODUCTION .................................................................................................................... 1 1.1.1 Saltmarsh status ....................................................................................................1 1.1.2 Fungi in saltmarshes .............................................................................................2 1.1.3 Environmental DNA ..............................................................................................2 1.1.4 Objectives ..............................................................................................................3 1.2 MATERIALS AND METHODS ................................................................................................. 4 1.2.1 Study areas ............................................................................................................4 1.2.2 Sample collection ..................................................................................................5 1.2.3 DNA extraction ......................................................................................................6 1.2.4 Analysis of fungal ITS2 metaamplicon sequence data ..........................................6 1.3 RESULTS .............................................................................................................................. 7 1.3.1 Fungal species composition in Minas Basin, Nova Scotia saltmarshes ................7 1.3.2 Species richness among Minas Basin saltmarsh sites and sediment zones ...........8 1.3.3 Comparison of species composition in saltmarshes of the Minas Basin ...............9 1.4 DISCUSSION ....................................................................................................................... 10 1.4.1 Sediment fungal diversity in saltmarshes of the Minas Basin, Nova Scotia ........ 10 1.4.2 Fungal species richness differs by location and sediment zone .......................... 13 1.4.3 Species composition is dependent on the location and sediment collection zone 13 1.4.4 Concluding remarks ............................................................................................ 14 CHAPTER 2: AMF ARE UBIQUITOUS THROUGHOUT SALTMARSHES OF THE MINAS BASIN, NOVA SCOTIA ........................................................................................................................................... 15 2.1 INTRODUCTION .................................................................................................................. 15 2.1.1 Saltmarshes ......................................................................................................... 15 2.1.2 Arbuscular mycorrhizal fungi ............................................................................. 17 2.1.3 Identification of fungal symbionts ....................................................................... 18 2.1.4 Quantification of AMF colonization .................................................................... 19 2.1.5 Objectives ............................................................................................................ 20 2.2 MATERIALS AND METHODS ............................................................................................... 20 2.2.1 Study Areas .......................................................................................................... 20 2.2.2 Core collection .................................................................................................... 20 2.2.3 Sample preparation ............................................................................................. 22 2.2.4 DNA extraction .................................................................................................... 22 2.2.5 Nested polymerase chain reaction ...................................................................... 22 2.2.6 Agarose gel extraction for amplicon purification ............................................... 23 2.2.7 PCR of isolated DNA amplicons from gel extraction .......................................... 23 2.2.8 Phylogenetic analysis of DNA sequences ............................................................ 24 2.2.9 Sediment chemical characterization.................................................................... 25 iv 2.2.10 Staining of mycorrhizae and root colonization assessment................................. 25 2.2.11 Statistical analysis ............................................................................................... 26 2.3 RESULTS ............................................................................................................................ 26 2.3.1 Fungal rDNA analysis......................................................................................... 26 2.3.2 Sediment chemical characterization ................................................................... 28 2.3.3 Interannual AMF colonization of Sporobolus pumilus roots at the study sites ... 29 2.3.4 Seasonal AMF colonization of Sporobolus pumilus roots .................................. 30 2.3.5 Analysis of site-specific AMF colonization ......................................................... 31 2.4 DISCUSSION ....................................................................................................................... 32 2.4.1 AMF diversity in Minas Basin, Nova Scotia saltmarshes ................................... 32 2.4.2 Saltmarsh sediment chemical characterization at three sites in the Minas Basin, NS 34 2.4.3 AMF colonization by year ................................................................................... 35 2.4.4 AMF colonization based on collection period..................................................... 35 2.4.5 AMF colonization differs by collection site ......................................................... 36 2.4.6 Concluding remarks ............................................................................................ 37 CHAPTER 3: INOCULATION OF RHIZOME DERIVED SPOROBOLUS PUMILUS WITH FUNNELIFORMIS GEOSPORUM MAY BE ESSENTIAL FOR SALTMARSH RESTORATION SUCCESS ..................................................................................................................................................... 39 3.1 INTRODUCTION .................................................................................................................. 39 3.1.1 Restoration efforts ............................................................................................... 39 3.1.2 Objectives ...........................................................................................................