SYSTEMATICS OF LACTARIUS IN THE ROCKY MOUNTAIN ALPINE ZONE by Edward Gilman Barge 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 2015 ©COPYRIGHT by Edward Gilman Barge 2015 All Rights Reserved ii ACKNOWLEDGMENTS I would like to thank my major professor Dr. Cathy Cripps for taking me in and teaching me so much about mycology and life in general. Her dedication to her students is a rare and precious thing; she has had an extremely positive influence on all of us. I would like to thank Dr. Todd Osmundson for taking time out of his busy schedule to teach me and help me with molecular work. Thank you to members of my thesis committee, Dr. Matt Lavin and Carol Johnson. Thank you to friends and family that have assisted with various aspects of this project. My father, Marcy Barge, helped out immensely with field work, and spent countless hours searching for small, inedible mushrooms in alpine tundra landscapes. Don Bachman gave directions and often transportation to alpine sites; his knowledge and steady demeanor are an inspiration. While I conducted field work in Southwestern Colorado, my aunt and uncle Barbara and John Nolan provided lodging, warm meals and support. Thank you to mycologists and herbaria that supplied additional collections for this project. Dr. Ellen Larsson in Sweden, Dr. Pierre-Arthur Moreau in France, Jukka Vauras in Finland, and Dr. Ursula Peintner in Austria provided crucial European type locality collections. Dr. Egon Horak provided additional Rocky Mountain alpine collections. I gratefully acknowledge funding support from Montana Institute on Ecosystems, and Dr. John Sherwood of the Plant Science and Plant Pathology Department. Lastly, thank you to my wonderful girlfriend, Charissa Bujak, for being a loving partner throughout my graduate career. iii TABLE OF CONTENTS 1. LITERATURE REVIEW AND INTRODUCTION TO THE GENUS LACTARIUS ........................ 1 What is the Arctic-Alpine Zone? ...................................................................................... 1 Climate .................................................................................................................... 2 Soils ......................................................................................................................... 4 Microbial Activity .................................................................................................... 7 Flora ........................................................................................................................ 8 Rocky Mountain Alpine Zone ....................................................................................... 12 Ectomycorrhizal Plants of the Rocky Mountain Alpine Zone ............................... 16 Salix arctica Pall. ....................................................................................... 17 Salix reticulata L. (=Salix Nivalis Hook) ..................................................... 18 Salix planifolia Pursh. ................................................................................ 18 Salix glauca L. ............................................................................................ 18 Other Salix species .................................................................................... 19 Betula glandulosa Michx. .......................................................................... 19 Dryas octopetala L. ................................................................................... 19 Bistorta vivipara (L.) Delarbre. .................................................................. 19 Kobresia myosuroides (Vill.) Fiori. ............................................................. 20 Ectomycorrhizal Fungi ................................................................................................... 20 Species Concepts Used for Fungi .................................................................................. 22 The Molecular Era ......................................................................................................... 24 Arctic-Alpine Ectomycorrhizal Fungi ............................................................................. 26 Traditional Studies ................................................................................................ 27 Molecular Ecology, Systematics and Biogeography of Arctic-Alpine Ectomycorrhizal Fungi ......................................... 28 Macrofungi of the Rocky Mountain Alpine Zone .......................................................... 34 The Genus Lactarius ...................................................................................................... 37 Ecology and Host Associations .............................................................................. 40 European Studies .................................................................................................. 41 North American Studies ........................................................................................ 42 Macroscopic and Microscopic Characters ............................................................ 43 Infrageneric Classification ..................................................................................... 45 Molecular Phylogenetic Studies ........................................................................... 47 Lactarius in Arctic-Alpine Habitats ........................................................................ 51 Rocky Mountain Alpine Lactarius ......................................................................... 59 2. SYSTEMATICS OF LACTARIUS IN THE ROCKY MOUNTAIN ALPINE ZONE ...................... 61 Introduction ................................................................................................................... 61 iv TABLE OF CONTENTS – CONTINUED Materials and Methods ................................................................................................. 66 Study Sites ............................................................................................................. 66 Taxon Sampling and Processing ............................................................................ 68 Morphological Descriptions .................................................................................. 71 DNA Extraction ...................................................................................................... 73 PCR Amplification of the ITS Region ..................................................................... 85 PCR Amplification of the Region Between Conserved Domains 6 and 7 of the rpb2 Gene ..................................................... 86 Visualization, Purification and Quantification of PCR Products ........................... 87 DNA Sequencing.................................................................................................... 87 Rational for Multiple Phylogenetic Analyses ........................................................ 89 Broad Phylogenetic Analysis ..................................................................... 89 Phylogenetic Analyses of Clades A-C ........................................................ 90 Sequence Alignment and Phylogenetic Analyses ................................................. 92 Results .......................................................................................................................... 95 Broad Phylogenetic Analysis ................................................................................. 95 Selection of Backbone Clades (A, B, C) ..................................................... 99 MegaBLAST Results for Select Rocky Mountain Alpine Specimens .................... 100 Phylogenetic Analyses of Clades A-C .................................................................. 103 Phylogenetic Analysis of Clade A ........................................................................ 103 Clade A, Summary ................................................................................... 104 Lactarius lanceolatus: Combined Analyses ............................................ 105 Lactarius lanceolatus: ITS Analyses ........................................................ 106 Lactarius lanceolatus: rpb2 Analyses ..................................................... 106 Clade A, Rational for Nomenclatural Assignments ................................. 111 Clade A, Other Taxa ................................................................................ 113 Phylogenetic Analysis of Clade B ........................................................................ 114 Clade B, Summary ................................................................................... 115 Lactarius nanus: Combined Analyses..................................................... 115 Lactarius nanus: ITS Analyses ................................................................ 115 Lactarius nanus: rpb2 Analyses .............................................................. 118 Lactarius glyciosmus: Combined Analyses ............................................. 118 Lactarius glyciosmus: ITS Analyses ........................................................ 119 Lactarius glyciosmus: rpb2 Analyses ...................................................... 119 Clade B, Other Taxa ................................................................................. 119 Phylogenetic Analysis of Clade C .......................................................................