ECTOMYCORRHIZAL FUNGI OF WHITEBARK PINE (PINUS ALBICAULIS) IN THE NORTHERN GREATER YELLOWSTONE ECOSYSTEM by Katherine Rose Mohatt A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant Sciences MONTANA STATE UNIVERSITY Bozeman, Montana April 2006 © COPYRIGHT by Katherine Rose Mohatt 2006 All Rights Reserved ii APPROVAL of a thesis submitted by Katherine Rose Mohatt This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. Cathy L. Cripps Approval for the Department of Plant Sciences and Plant Pathology Dr. John E. Sherwood Approved for the Division of Graduate Education Dr. Joseph Fedock iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Montana State University, I agree that the Library shall make it available to borrowers under the rules of the Library. If I have indicated my intention to copyright this thesis by including a copyright notice page, copying is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation from or reproduction of this thesis in whole or in parts may be granted only by the copyright holder. Katherine R. Mohatt April, 2006 iv ACKNOWLEDGEMENTS I would like to thank my graduate advisor Dr. Cathy Cripps for fungal taxonomic expertise, endless editing, and support, and the members of my graduate committee, Dr. Matthew Lavin for assistance with phylogenetic analysis and Dr. Sharon Eversman for support and assistance. My thanks also goes to Andy Hogg, Alan Dyer, Thamir Al- Niemi, Kathi Trujillo, and Jeff Cameron for molecular technique expertise, Dr. Cliff Montagne for soils information, Don Bachman for field logistics and avalanche path expertise, Tim Sieple for help with R, and Paul Trusty, Danial Keeler, Leslie Eddington, Mara Saccoccia, and Rubyn (canine) for assistance in the field. I would also like to acknowledge Dr. Robert Garrott, Colleen Wisinski, Silvia Murcia, and Kelley Proffitt for their constructive critiques of this work. Finally, I would like to thank my family for their love and support. v TABLE OF CONTENTS 1. LITERATURE REVIEW .............................................................................................1 Whitebark Pine..............................................................................................................1 Restoration of Whitebark Pine Forests ...................................................................5 Mycorrhizae ..................................................................................................................7 Components of the Ectomycorrhizal Association....................................................9 Ecology of Ectomycorrhizae....................................................................................9 Identification of Ectomycorrhizal Fungi................................................................12 Restoration Using Mycorrhizal Fungi ...................................................................15 Whitebark Pine Mycorrhizae .................................................................................16 Research Objectives....................................................................................................18 2. A SURVEY OF ECTOMYCORRHIZAL FUNGI FRUITING IN PINUS ALBICAULIS FORESTS .......................................................20 Introduction.................................................................................................................20 Methods.......................................................................................................................25 Study Area .............................................................................................................25 Sporocarp Collection and Identification ................................................................34 Analysis of Ectomycorrhizal Communities...........................................................36 Results.........................................................................................................................37 Dichotomous Keys to ECM Species in Pinus albicaulis forests ..........................40 Key A: Hypogeous and Secotioid Ectomycorrhizal fungi in Northern Greater Yellowstone Ecosystem Pinus albicaulis forests ..................................................40 Key B: Epigeous Ectomycorrhizal Boletales, Russulales, and Light-Spored Agaricales in NGYE Pinus albicaulis forests........................................................41 Key C: Cortinariales (Agaricales) in NGYE Pinus albicaulis forests...................43 Descriptions of ECM Species in Pinus albicaulis forests .....................................44 Discussion...................................................................................................................76 3. ECTOMYCORRHIZAL FUNGI ASSOCIATED WITH PINUS ALBICAULIS SEEDLING ROOTS................................................................83 Introduction.................................................................................................................83 Methods.......................................................................................................................88 Sample Collection and Morphotyping....................................................................88 DNA Extraction ......................................................................................................92 PCR Amplification and Sequencing .......................................................................93 Analysis of ITS Sequences .....................................................................................95 Results.........................................................................................................................96 Discussion.................................................................................................................103 vi TABLE OF CONTENTS CONTINUED 4. A COMPARISON OF ECTOMYCORRHIZAL FUNGI ON PINUS ALBICAULIS SEEDLINGS IN MATURE FORESTS AND ADJACENT AVALANCHE PATHS..............................................................109 Introduction...............................................................................................................109 Methods.....................................................................................................................113 Study Area ...........................................................................................................113 Experimental Design and Seedling Collection ....................................................114 ECM Community Assessment and Identification................................................116 Analysis of ECM Fungi in Paths vs. Canopy ......................................................116 Results.......................................................................................................................117 Discussion.................................................................................................................125 SUMMARY TABLE OF ALL ECM SPECIES.......................................................133 LITURATURE CITED.............................................................................................135 APPENDICES ..........................................................................................................151 APPENDIX A: SPOROCARP COLLECTIONS OF ECTOMYCORRHIZAL FUNGI FROM PINUS ALBICAULIS FORESTS.....................................................152 APPENDIX B: PINUS ALBICAULIS SEEDLINGS COLLECTED IN MATURE FORESTS..................................................................156 APPENDIX C: ECTOMYCORRHIZAL FUNGAL MORPHOTYPES ON PINUS ALBICAULIS SEEDLINGS FROM SCOTCH BONNET MOUNTAIN, N-GYE ..160 vii LIST OF TABLES Table Page 2.1 Species of ectomycorrhizal fungi previously recorded in Pinus albicaulis forests.........................................................................24 2.2 Ectomycorrhizal sporocarp survey collection dates and sampling effort ...............................................................................38 2.3 Fruiting structures of ectomycorrhizal species collected in the Northern Greater Yellowstone Ecosystem ..................39 2.4 Dice’s similarity coefficients for each possible pairwise comparison of sites.................................................................75 3.1 Sporocarps used in molecular analysis .................................................88 3.2 Collection dates and locations of Pinus albicaulis seedlings examined ...............................................................................90 3.3 Ectomycorrhizal taxa identified on the roots of Pinus albicaulis seedlings .......................................................99 3.4 Morphotypes of ectomycorrhizae on Pinus albicaulis seedlings by site ..................................................................................100 4.1 Locations and age data for Pinus albicaulis seedlings collected on Scotch Bonnet Mountain ................................................115 4.2 Morphotypes of ectomycorrhizae on Pinus albicaulis seedlings collected on Scotch Bonnet Mountain ................................118 4.3 Taxonomic identity of ectomycorrhizal