CHARACTERIZATION AND SEASONAL ECOLOGY OF ECTOMYCORRHIZAE ASSOCIATED WITH SITKA ALDER AND LODGEPOLE PINE FROM NATURALLY REGENERATING YOUNG AND MATURE SPRUCE FORESTS IN THE SUB-BOREAL SPRUCE ZONE OF BRITISH COLUMBIA BY ANIKO M. VARGA B.Sc., THE UNIVERSITY OF BRITISH COLUMBIA, 1 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE, NATURAL RESOURCE MANAGEMENT THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA ©ANIKO M . VARGA THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA AUGUST 1998 ALL RIGHTS RESERVED . THIS WORK MAY NOT BE REPRODUCED IN WHOLE OR IN PART, BY PHOTOCOPY OR OTHER MEANS, WITHOUT THE PERMISSION OF THE AUTHOR . UNIVERSITY OF NORTHERN BRITISH COLUMBIA ABSTRACT CHARACTERIZATION AND SEASONAL ECOLOGY OF ECTOMYCORRHIZAE ASSOCIATED WITH SITKA ALDER AND LODGEPOLE PINE FROM NATURALLY REGENERATING YOUNG AND MATURE FORESTS IN THE SUB-BOREAL SPRUCE ZONE OF BRITISH COLUMBIA BYANIKO M. VARGA Prior to this thesis project, our understanding of the influence of stand age and season on in vivo ectomycorrhizal communities of Sitka alder growing with Lodgepole pine within the Sub-Boreal Spruce biogeoclimatic zone was limited. The magnitude of ectomycorrhizal associates present on Sitka alder and Lodgepole pine was not known. In addition, it was of ecological interest to assess if the two host species, common to early-successional, post-harvest forest communities, were directly linked via common ectomycorrhizal fungi with reference to ecosystem interactions and possible nutrient translocation. Stemming from this general lack of knowledge, the purpose of this thesis project was to strengthen our ecological understanding of these two ectomycorrhizal communities. Two complementary methodologies were implemented in order to assess the ectomycorrhizal communities. Traditional morphological assessments offered one perspective of the mycorrhizal associates and permitted morphological description and mycobiont identification when possible, as well as richness, abundance, diversity, and evenness comparisons with respect to host, stand age, and season. It was found that mycorrhizal richness was greatest for Lodgepole pine. Seral and seasonal effects were shown to significantly impact certain Sitka alder and Lodgepole pine ectomycorrhizae. Seral and seasonal effects also significantly impacted alder ectomycorrhizal diversity and evenness ii assessments, however, no similar impact was observed for pine. Several possible genera of fungal symbionts were identified that may link Sitka alder and Lodgepole pine. The resolution of ectomycorrhizal taxa comprising the belowground communities were improved over morphological assessments through the use of molecular techniques, specifically PCR-RFLP analyses, the second technique applied . Molecular datasets further confirmed some morpholog_ical assessments and defined morphological descriptions allowing for identification of some mycobionts mostly to the genus level, but also to the species level, as well as determining inter- and intraspecific variation of the int!'lrnal transcribed spacer region of rONA within certain morphotypes. Two mycobiont genera (Cortinarius and Lactarius) were identified on both hosts which suggest the possibility of direct mycorrhizal linkage. In this study, molecular diversity assessments, when used in conjunction with defined morphological techniques (such as morphotyping), offered a complementary synopsis of the diversity and dynamics of ectomycorrhizal communities. Taxa were distinguished to a finer taxonomic level and could be identified using PCR-RFLP analysis while the abundance of ectomycorrhizal morphotypes was best described using morphological techniques. The research indicated that stand age and season can significantly influence alder and pine ectomycorrhizae. iii TABLE OF CONTENTS Abstract. ........ .... ....... ...... .. .. ......... ...... ......... ............ .. .................. ..................... ............ .ii List of Tables .......... ..... ....... ............ ..... .... .... .... ... ...... ... ................. ... ........ ... .................. vii List of Figures .................. ..... .... ... ............ x xi Acknowledgement ..... ..... ............ xii Dedication ....... ............ 00 00 00 • • • ••• • • •• •• • •••• 00 •• • •• •• • 00 00 . .. 00 • . . •• 00 . • 00 00 .... 00 . 00 . . ... 00 00 00.00 .. .. 00 .............. .. .. ...... ........ 00 •••• 00 .. . • 00 .. .00 . 00 ••••• 00 00 00 ..... 00 . 00 00 00 . .. 00.00 00 00.00 ...................... ....... . ..... . ... . ..... 00 00 00 . . . 00 •• ..... •••••••• INTRODUCTION ................................................................................................................................. 1 Rationale ............ .. .. ..... .......... .... ... ........ ......... ............... .................... ...... .... ....... ......................... 1 Research Objectives and Hypotheses ........................................... .. .. .............. .. .... .. ................. 3 1 Literature review ............................................................................................................................ 4 1.1 Definitions of symbiosis and mycorrhizae ............. ............... .. .... ............................ .. .......... 4 1 .2 Functions of mycorrhizae ........ ....... ........... ... .. ..... ............. .. .. .. ................ ........ .. .. ................ 4 1.3 Classes of mycorrhizae and segregation by habitat.. ............. .......................... .. .. .... .... .. .. .. 5 1.4 Ectomycorrhizae ............. ............... .. ..... .. .......... ... ..... .. ... ...... .. .. .... ... .. ............. .. ........ ..... ..... . 6 1.4.1 Structure ......... ............ ... .. ................ ....... ............. ............ ... ......... .... .......................... 6 1.4.2 Characterization and identification .. ........ .... .. .... ........ .... .. ................ .. .. .. .................... 7 1.4.3 Hosts and fungi .. ................ .. .... ................................ ................... .. ............................ 8 1.5 Taxonomy and ecology of Sitka alder ................................................................................ 8 1.6 Ecology of lodgepole pine ................ .......................................... .. .. .. .............. .... .. ............ 11 1.7 Specificity and receptivity .......................... ........... ................ .. ......... .. ............ ................... 11 1.7.1 A/nusspp. as mycorrhizal hosts ...................... ....................................................... 12 1.7.2 Pinus spp. as mycorrhizal hosts ................................................ ............................. 15 1.7.3 Mycorrhizal linkages between hosts .......... .. ........... .. .. .... .... .... .... .......... .. ................ 15 1.8 Mycorrhizal fungi succession theory: 'early' and 'late' stage fung i .... .................... .. .... .. .. 18 1.8.1 Factors influencing succession and presence of ectomycorrhizal fungi .. .. .... ........ . 19 1.9 Succession of ectomycorrhizae ...... .. ................ .. .............................................................. 20 1.9.1 Alnus spp. hosts ........ .... .. .. .... .. ............ .. .... .. ............................................................ 20 1.9.2 Pinus spp . hosts .......................... .. ..... .. ................................................................... 21 1.10 Diversity indices ............................................... .. ...... .. ........................................ .. .. ... ... ... 22 1. 10.1 Simpson diversity and evenness indices .............................................................. 23 1.10.2 Shannon diversity and evenness indices ...... .. .................... .. .... .. .... ...... ................ 24 1.10.3 Mcintosh diversity and evenness indices .... .... .. .... ............................................... 24 1.11 Use of statistical analysis in ectomycorrhizal community studies .................................. 25 1.12 PCR-Restriction Fragment Length Polymorphism (RFLP) analyses .. ........................... 27 1.13 References ......................................................... .. .. .... .. ...... .. .................................. .. ..... . 29 iv 2. Characterization and seasonal ecology of ectomycorrhizae associated with Sitka alder and Lodgepole pine from naturally regenerating young and mature forests in the SubBoreal Spruce zone of British Columbia. Morphological characterization ....................... 35 Abstract ........................................................... ............................................................ ......... ... 35 2.1 Introduction ............... ............. ..... ............................. ......................................................... 36 2.2 Material and Methods .................. ..... ............. ................................................................... 38 2.2.1 Site designations: the Sub-boreal spruce biogeoclimatic zone ............................. 38 2.2.2 Site location and description ........................................... ....... ........................... ...... 39 2.2.3 Field ectomycorrhizae sampling and collection ........................ .............................. 43 2.2.4 Ectomycorrhizae sub-sampling ................................................................ ............... 44 2.2.5 Morphological characterization ............................................................ .. ...... ........... 44 2.2.6 Terminology ............................................................................. ............................... 45 2.2.7 Statistical analysis ................................................................................ .. ................. 45 2.3 Results .......................... .............. ..... ............ .................. ............. .. .... ............................ .... 46 2.3.1 Morphological characterization .................... ........................................................... 46 2.3.2 Ectomycorrhizae frequency and abundance for alder and pine ............ ............ ..... 49 2.3.3 Morphotype richness, diversity, and evenness ........... .. ...... .................................... 56 2.4 Discussion ................................................. .................. ............................. .... .................... 60 2.4.1 Morphological characterization ...... ......... ................ ....................................... ......... 60 2.4.1 .1 Sitka alder ectomycorrhizae .................................... ...... .... .................................. 60 2.4.1 .2 Lodgepole pine ectomycorrhizae ........ ......................................... .. ................. ..... 62 2.4.1 .3 Ectomycorrhizal linkage ............................................ ....... ................. ................... 65 2.4.2 Abundance, frequency, and colonization ................................................................ 65 2.4.2.1 Sitka alder ectomycorrhizae .......... .............................. ........................................ 65 2.4.2.2 Lodgepole pine ectomycorrhizae ......... ........... .................. .................... ............ ... 68 2.4.3 Morphotype richness, diversity, and evenness ... .. ...... ............................................ 71 2.5 References ............................................................... ........................................................ 75 3. Characterization and seasonal ecology of ectomycorrhizae associated with Sitka alder and Lodgepole pine from naturally regenerating young and mature forests in the SubBoreal Spruce zone of British Columbia. PCR-RFLP Analyses .......................................... 83 Abstr.act ................................................... .... ............................... ............... ......... ...... ............... 83 3.1 Introduction .......... ........ ... ................. ..................................... .... .................... ........... ......... 84 3.2 Material and methods ................. .. .. ..... ................... .... ...... ...................... .... ........... ...... ..... 86 3.2.1 Ectomycorrhizae sub-sampling ........ .. ................... ........ ..... ..... ....... ... .................... .. 86 3.2.2 Molecular characterization of ectomycorrhizae .................. ...... ............. ..... ............ 86 v 3.2.3 Sporocarp survey ............ ...................... ............... ................. ............................ ...... 89 3.2.4 Software analysis of mycorrhizal RFLP patterns ..... ....... ........... .. .. ............ ..... .... .... 90 3.3 Results ................. ... .... ............................. .... ... ... ...... .................................. ... .... ....... .. .. ... .. 93 3.3.1 PCR and digestion success rates and optimal protocols ..... ... ................ ........ ........ 93 3.3.2 Molecular characterization of Sitka alder ectomycorrhizae ...... ..... ..... ... ... .... .... ..... 96 3.3.3 Molecular characterization of lodgepole pine ectomycorrhizae ... ......... .......... ....... . 99 3.3.4 Summary of molecular diversity assessment ... ... .... ............ ... .................. .... ........ 109 3.3.5 Co-occurrence or replacement of morphotype DNA with other mycorrhizal DNA 111 3.3.6 Characterization of non-target DNA ............. ..... ............... ............ .......... .. ............ . 111 3.3.7 Sporocarp survey of study sites .......... ... ..... ..... ..... ......... .. ..... .. ......... ... .................. 114 3.3.8 Ectomycorrhizallinkages conferred from molecular dataset comparisons ......... .. 114 3.4 Discussion ........ ......... ...................................... ................................ ..... .... ...... ................ 115 3.4.1 Optimal protocols, success rates, and competitive PCR ...... .... ........... ... .............. 115 3.4.2 Molecular characterization of Sitka alder ectomycorrhizae ............ ... .. ...... ...... ..... 116 3.4 .3 Molecular characterization of lodgepole pine ectomycorrhizae ...... ... ... ... ............. 120 3.4.4 Ectomycorrhizal linkages inferred from molecular dataset comparisons : intraspecific variation .......... .......................... ... ........................ .................. ........ ...... . 124 3.4 .5 Succession of fungi on root tip ................................... .... .......... . ! . . . . . ..• . ••••••• . •..•.•.•.. 125 3.4.6 Characterization of non-target DNA .. .......... ............ ............. .......... .. .. .... ... ............ 125 3.4.7 Conclusions .... ........ ........ ....................................... .. ............. ............ ............... ...... 126 3.4.8 Improvements ........................... .... ......... .................... ... .... ................. ............... .. .. 127 3.5 References ............ ............................ ...... ... .... ...................................................... .... ...... 130 APPENDIX 1: SITKA ALDER MORPHOTYPE DESCRIPTIONS .................................................. 133 APPENDIX 2: LODGEPOLE PINE MORPHOTYPE DESCRIPTIONS .......................................... 146 APPENDIX 3: SITKA ALDER ABUNDANCE DATA ..................................................................... 174 APPENDIX 4: LODGEPOLE PINE ABUNDANCE DATA ............................................................. 176 APPENDIX 5: LISTING OF ECTOMYCORRHIZAL SPOROCARP SAMPLES ............................ 179 APPENDIX 6: LISTING OF SAPROPHYTIC SPOROCARP SAMPLES ....................................... 182 APPENDIX 7: BANDING TOPOLOGIES OF ALU I, HINF I, AND RSA I ENDONUCLEASES FOR SUSPECTED ECTOMYCORRHIZAL SPOROCARPS COLLECTED AT THE BOBTAIL STUDY SITE ............................................................................................................................. 185 APPENDIX 8: BANDING TOPOLOGIES OF ALU I, HINF I, AND RSA I ENDONUCLEASES FOR SUSPECTED NON-ECTOMYCORRHIZAL SPOROCARPS COLLECTED AT THE BOBTAIL STUDY SITE ............................................................................................................................. 189 APPENDIX 9: UNROOTED RADIAL CLADOGRAM FROM SITKA ALDER AND LODGEPOLE PINE RFLP PATTERNS ........................................................................................................... 193 APPENDIX 10: SELECTED MICROGRAPHS OF SITKA ALDER AND LODGEPOLE PINE MORPHOTYPES ..................................................................................................................... 195 vi LIST OF TABLES Page Number Table 1 Latin binom ial equivalents of Alnus species located in British Columbia ... ...... .. ... ............ 9 Table 2 Putative ectomycorrh izal fung i for the genus Alnus ............ .......................... .............. .... . 13 Table 3 Putative ectomycorrhizal fungi for Pinus contorta var. latifolia and P. banksiana ....... .... 16 Table 4 List of suspected genera of alder and pine ectomycorrhizal mycobionts from the literature ... .... ..... ........ .......................................... ........ ..................... ........................... 48 Table 5 Number of plants colonized by morphotype, number with <5%, mean abundance (± standard error (SE)) for Sitka alder from 4 sites and 2 stand ages (young and mature) sampled in June and September ....... ........ ................................. .......... ........ 50 Table 6 Number of plants (plants or combined mats) colonized by morphotype, number with <5%, mean abundance (± standard error (SE)) for lodgepole pine from 4 sites and 2 stand ages (young and mature) sampled in June ....... ...................... ............... 51 Table 7 Number of plants (plants or combined mats) colonized by morphotype, number with <5% , mean abundance (± standard error (SE)) for lodgepole pine from 4 sites and 2 stand ages (young and mature) sampled in September .......... .............. .... .. .... 52 Table 8 Statistical summation (3-way ANOVA) of main effects (mean (x), standard deviation (SO), and probability, a=0.05) and interactions effects of Sitka alder morphotype abundance. N= 35 .......................... ....................... .......................... ..... ................ .. ... 53 Table 9 Statistical summation (3-way ANOVA) of main effects (mean (x), standard deviation (SO), and probability, a=0.05) and interactions effects of lodgepole pine morphotype abundance. N= 39 . ...................... ... ... .. ..... .... ......... .. ........ ... .............. .... . 55 Table 10 Total mycorrhizal colonization (mean% colonization of total live tips) for Alder and Pine with respect to stand age and season . Statistical analysis using t-test (two sample assuming unequal variances) within each column ; a=0.05 ........ .......... ......... 56 Table 11 Total mycorrhizal colon ization (mean % colonization of total live tips) with respect to stand age between hosts .... .... ....... ....... .... ..... ..... .... ... ...... .................. ........ .... ..... .... 56 Table 12 Total mycorrhizal colonization (mean % colonization of total live tips) with respect to season between hosts .... .. ... ......... .... ...... .... ...... ........ ........ .. ..... ............ ....... ............ 56 Table 13 Morphotype richness (mean seedling richness per block) for alder and pine with respect to transect, stand age, and season . Statistical analysis using 3-way AN OVA for alder and pine and t-test (two sample assuming unequal variances) between alder and pine; a=0.05 .. ................. ...... ..... ............. ...... ..... ...... ........ .... ... ..... . 57 vii Table 14 Diversity and evenness statistical summation (3-way ANOVA for each indices) showing main effects {0 and p values) and interactions for transect, stand age, and season for Sitka alder morphotypes. N=35; a=0.05 ................................ .......... 59 Table 15 Diversity and evenness statistical summation (3-way ANOVA for each indices) showing main effects {0 and p values) and interactions for transect, stand age, and season for lodgepole pine morphotypes. N=39; a=0.05 .............. ...................... 59 Table 16 Molecular results for Sitka alder ectomycorrhizae showing morphotype, number of tips sampled (N), amplification success rates (%, actual number in brackets), doublets (%, actual number in brackets), and digestion success rates (%, actual number in brackets). Optimal and attempted protocols (re-extraction, annealing temperature ( 0 C), Taq concentration) are presented . ... .......... ......... .. ....................... . 94 Table 17 Molecular results for lodgepole pine ectomycorrhizae showing morphotype, number of tips sampled (N), amplification success rates (%, actual number in brackets), doublets(%, actual number in brackets), and digestion success rates (%, actual number in brackets). Optimal and attempted protocols (re-extraction, annealing temperature ( 0 C), Taq concentration) are presented ........................... ............ .. ........ 95 Table 18 Banding topologies of 3 taxa for Morphotype A 1 resulting from 3 endonucleases (Aiu I, Hinf I, and Rsa I) .............................. ..... ................ ........ .. .. ............. ................... 96 Table 19 Banding topologies of 4 taxa from Morphotype A2 .......... .............................................. 97 Table 20 Banding topologies of 2 taxa from Morphotype A3 ... ...... ............... ..................... .... ...... . 98 Table 21 Banding topologies of 1 taxon from Morphotype A4 ............................................ ..... ..... 98 Table 22 Banding topologies of 1 taxon from Morphotype A5 ............ ....... ........ ........................... 99 Table 23 Banding topologies of Cenococcum geophilum-like from Morphotype P1 ............... ... 100 Table 24 Banding topologies of Pi/oderma fa/lax-like from Morphotype P2 ..... ........... ............... 101 Table 25 Banding topologies of Piloderma fa/lax-like from Morphotype P12 ................. .... ........ 101 Table 26 Banding topologies of Suil/us/Rhizopogon-like Tuberculate from Morphotype P3 ...... 102 Table 27 Banding topologies of Suil/us/Rhizopogon-like Single from Morphotype P4 ............... 102 Table 28 Banding topologies of 1 taxon from Morphotype P5 .... ............... ................................. 103 Table 29 Banding topologies of 2 taxa from Morphotype P8 .... .. ............... .. ...... ......................... 104 Table 30 Banding topologies of 2 taxa from Morphotype P11 ........ .... ....... .. .... ........... ............ .... 105 Table 31 Banding topologies of 2 taxa from Morphotype P6 ......... ..... ......... .... .. ... .... ..... ........... .. 105 Table 32 Banding topologies of 1 intraspecific taxon from Morphotype P7 .. .,...... .. ......... .......... 106 Table 33 Banding topologies of taxa from Morphotype P9 .. ..... ... ... .. ......... .. .. .... ...... .. ....... ....... ... 107 Table 34 Banding topologies of 3 taxa from Morphotype P10 ... .................... ..... ........................ 108 Table 35 Banding topologies of one or two taxa from Morphotype P13 ..... .... .......... .. ................ 108 Table 36 Distribution of Sitka alder ectomycorrhizae showing closest identified mycobionts from databases, topology name, and presence in site(s) ................ ....... ................. 109 viii Table 37 Distribution of lodgepole pine ectomycorrhizae showing closest identified mycobionts from databases, topology name, and presence in site(s) ........ .. ....... ..... .. ....... .... .... . 110 Table 38 Clade 1 banding topologies representing non-target DNA ..... ......... ..................... ... :...... 112 Table 39 Clade 2 banding topologies representing non-target DNA ................ ......... ................ .... 113 Table 40 Clade 3 banding topologies representing non-target DNA ·····································: ······· 113 ix LIST OF FIGURES Number Page Figure 1 Two mechanisms causing inter- and intra-specific variation in PCR-RFLP Analysis ... .. 28 Figure 2 Location of research site in relation to Prince George and Highway 16 (from Sanborn et al. 1997) ... ... .... ... .... ................. .... ....... ... ...... ... ... ............. ....... .............. .... . 40 Figure 3 Young Sitka alder (grey brush understory) and lodgepole pine regenerating stand within the SBSdw3 .......................................................................... .................. .......... 41 Figure 4 Mature Sitka alder (grey brush understory) and lodgepole pine regenerating stand within the SBSdw3 ............................................................................ ................ ....... ... 41 Figure 5 Sampling of pine roots from forest floor. Yellow arrows indicate location of mats cut relative to host tree .... .. ................. ......... ........................................ .. .. ..... ... .............. ... 42 Figure 6 Alder root burl. Scale bar is 30 em ........ .. ........ ........ .... ............................ .................. .. ... 42 2 Figure 7 Ectomycorrhizae sub-sampling . Grids are 1 cm .. .............. . ........ .. ................................ 42 X ACKNOWLEDGEMENTS I would like to thank those involved directly and indirectly with my thesis project. Dr. Hugues Massicotte aimed me through voluminous research . I will always be obliged that he accepted me as his first graduate student, readying me for further academics. Dr. Keith Egger always had an addendum to make me cogitate. Dr. Paul Sanborn was eager to transport me to his research site and offer abstraction . Thanks to Dr. Art Fredeen for being the plant ecophysiologist on my committee. Research assistant Linda Tackaberry always offered constructive criticism . I also thank Drs. Alan Ewert, Lito Arocena, Mike Walters, Bruno Zumbo, Hugues Massicotte, Art Fredeen, and Keith Egger. Their classes offered a stimulating break from sometimes monotonous research exercises. Thanks to NRES secretaries Judy Armstrong, Yvonne Smith, and Paula Poirier; they always know the room number, who is around the building, and where your paycheque is. Thanks to my mom , Mamie Varga, and my dad, Laszlo Varga, who still support me in schooling after all these years. Thanks to my extended family Auntie Beth Stanga, Auntie Ellen and Uncle Jim Togyi, Uncle Milan Stanga, and cousins John Togyi, Mike Togyi, lmre Togyi, and Zane Stanga, who seemed to be around whenever needed. Probably most importantly, much appreciation to all my friends during the last 3 years (!) while in resplendent (?) Prince George. Listed in particular order (hah!): Quentin 'Farrell Hampden Baldwin Ill' Baldwin, Laine 'Polyester' Cotton , Ed 'Eduardo Staffon~· Stafford, Keith 'Junior' Williams, Nadine 'Nerdine' Gray, Brendan 'Big B' Murphy, Thierry Claval, William (Bill) Scherk, Melinda (Millie) Hirt, Mark 'Didgereedoo' Thompson, Stevan 'the hair' Springer, Anne Marie Macisaac, Jelmer Weird Wiersma, Chris 'Doc' Johnson, Roy Rea, Michelle Oster, Kevin Driscoll, Kristen Parker and Dawn Hoffert. Friends keep life in perspective. And more fun. And tell you where to go (for vacation). Also thanks to Fyodor Dostoyevsky, Aldous Huxley, Henry David Thoreau, Forsythe P. (Jughead) Jones, Sid Barrett, David Gilmore, Leonard Cohen, Miles Davis, Thelonius Monk, and Ani DiFranco, to mention a few. Frolic also makes life more fun . Tra -Ia- Ia-Ia, man . xi DEDICATION I would like to dedicate this thesis to my grandparents, Mary Moren, William Herbert Richmond , Anna Varga, and Jozsef Varga, whom I will never forget. xii INTRODUCTION RATIONALE The health and productivity of a forest ecosystem are directly related to the condition of the forest soil. Physical and chemical properties of the soil regulate the population dynamics of forest soil organisms which are key to essential biological processes such as organic decomposition and nutrient cycling. A mature forest soil is composed of a diverse array of microorganisms participating in these processes. However, harvested areas are becoming common components of the BC interior's forest landscape. Such harvesting practices add to the ecosystem complexity through anthropomorphic disturbance impacting these processes. For instance, in the Sub-boreal spruce biogeoclimatic zone, mature mixed lodgepole pine and spruce stands are sometimes replaced by mixed Sitka alder and lodgepole pine over- and understory communities. These changes in vegetation may alter the belowground ecosystem, however, our understanding of the influence of stand age and season on ectomycorrhizal communities of Sitka alder and lodgepole pine within the Sub-Boreal spruce biogeoclimatic zone is practically non-existent. The opening of the canopy following harvest will influence light and water availability and temperature (Kimmins 1997). Consequently, the understory vegetation may change from a mossy cover to one composed mostly of grasses and shrubs. These changes in vegetation will affect the soil and as result, the habitat it provides for soil organisms will be modified, further impacting the remaining vegetation of the site. Secondary succession following timber harvest such as clearcutting is well documented with respect to above-ground dynamics (Kimmins 1997). However, our knowledge of the dynam ics of soil organisms following disturbance is limited, especially the fungal component. Fungi are a major part of the terrestrial ecosystem acting as mutual symbionts, endophytes, pathogens, and saprophytes as well as components of the belowground foodweb. A large group of fungi form ectomycorrhizae, a mutually beneficial symbiotic relationship existing between the roots of hundreds of plant species and specialized fungi (Harley and Smith 1983). They have been documented as being essential to the healthy growth of trees worldwide (Read 1991 ). Ectomycorrhizal associations are ubiquitous and are believed to be critical to successful tree growth and enhanced seedling establishment by facilitating nutrient uptake of phosphorus, nitrogen, and water (Allen 1991; Harley and Smith 1983). Ectomycorrhizae and their fungal mycelium contribute to nutrient cycling, a biological process significant to the overall forest health, resilience, and community stability (Kimmins 1997) by transporting cations and anions (especially immobile molecules such as phosphates), nutrients, organic materials, and water to the host plant from the soil. A major attribute of a large contingent of ectomycorrhizal fungi is their capacity to form symbiotic relationships with several species of trees belonging to different genera (Molina et al. 1992). Plants sharing common fungal symbionts may benefit from this fungal linkage through the exchange of nutrients and metabolites. Experimental evidence has shown nutrients to pass interspecifically as well as intraspecifically between plants (Read 1995; Simard et al. 1997c). Interspecific linkages are particularly significant to this thesis research as one of the host species examined is Sitka alder. Members of the genus Alnus are capable of nitrogen fixation (Farrar 1995) whereby actinomycetes in root nodules fix atmospheric nitrogen into organic forms. Consequently, alder plays an important role in soil and site fertility where nitrogen is often limiting. Laboratory experiments between alder and pine species have demonstrated significant nitrogen translocation between the two interspecific host species (Arnebrant et al. 1993). If a common mycobiont exists between Sitka alder and lodgepole pine, this could impact on current competitive theories that have been applied to post-harvest alder and pine communities. Currently prescribed eradication programs physically eliminate alder from the site through brushing. However, if alder plays a role in fertilizing sites and if regenerating conifers benefit via nitrogen translocation through mycorrhizal linkages, this prescription may not be necessary nor advantageous. The first step in testing this hypothesis is to examine the ectomycorrhizae of Sitka alder and lodgepole pine. 2 RESEARCH OBJECTIVES AND HYPOTHESES Using morphological and molecular methods, this thesis examined the seasonal ecology of ectomycorrhizae associated with Sitka alder and lodgepole pine from both naturally regenerating young and mature Sub-boreal stands following timber harvest by focusing on 6 objectives and associated hypotheses. Chapter 1 includes a literature review of key concepts related to the objectives. Chapter 2 describes morphological methods used to assess the seasonal ecology of ectomycorrhizae associated with Sitka alder and lodgepole pine from both naturally regenerating young and mature Sub-boreal stands following timber harvest. The first objective was to provide a morphological assessment of the ectomycorrhizal types (herein called morphotypes) and to determine the level for fungal linkage. The two working hypotheses were that only a limited number of morphotypes would be found on Sitka alder whereas numerous morphotypes would be found on lodgepole pine and that there would be no mycorrhizal linkage between the two hosts. The second objective was to estimate the abundance of the morphotypes on each host and determine the effect of stand age and season on these estimates. The working hypothesis was that there would be no difference in relative abundance of morphotypes with respect to stand age and season for each host. The third objective was to assess and compare morphotype richness (host receptivity), diversity, and evenness for alder and pine based on morphological data for both stand age and season using Shannon, Simpson, and Mcintosh indices. The working hypothesis was that there would be no differences in any of these values. In Chapter 3, molecular methods were used to examine three objectives. The first objective was to provide RFLP topologies for each morphotype (including interspecific and intraspecific variation when present). The second objective was to identify mycobionts involved in the symbioses through cross-referencing of RFLP topologies between ectomycorrhizae and sporocarps (including interspecific and intraspecific variation). The third objective was to assess the level of ectomycorrhizal linkage through RFLP topology comparisons. The working hypotheses were that only a limited number of morphotypes would be found on Sitka alder whereas numerous ectomycorrhizal types would be found on lodgepole pine, that the morphological and molecular assessments are similar, and that ectomycorrhizal linkages do not exist between Sitka alder and lodgepole pine. 3 ·~ 1. Literature Review 1.1 DEFINITIONS OF SYMBIOSIS AND MYCORRHIZAE In any forest, thousands of organisms coexist and interact. A common type of interaCtion occurring between organisms includes symbiosis, defined as a long-lasting or permanent relationship {Ahmadjian and Paracer 1986) usually composed of two or more physiologically different organisms acting together as part of a single super-organism (Harley and Smith 1983). Partners in such relationships may be affected differently: when both benefit, it is called mutualism, when one or both are harmed, it is termed parasitism, or when one or both remain unaffected, the term commensalism applies (Ahmadjian and Paracer 1986). The roots of higher plants and an array of soil fungi have developed mutually beneficial symbiotic relationships and are called mycorrhizae (Harley and Smith 1983). 1.2 FUNCTIONS OF MYCORRHIZAE For several decades, mycorrhizae have been documented as being essential to the healthy growth of trees, especially where soil nutrients are limited or environmental conditions are extreme (Molina and Trappe 1982a). Bilateral exchange of nutrients between the plant {the autotroph) and its associated fungal partner(s) {the heterotroph(s)) is the most emphasized function of mycorrhizae . Typically, carbon is transferred from autotroph to heterotroph and nutrients or mineral elements are transferred from the heterotroph to the autotroph (Harley and Smith 1983). This exchange of photosynthates, metabolites, nutrients (or mineral elements), and water occurs at the interface between the two symbionts established during the formation of the mycorrhizae (Bonfante-Fasolo and Scannerini 1992). 4 The obvious benefit for the fungi is a gain of carbohydrates from the plant. Autotrophs furnish the mycobiont with vital energy-rich photosynthates; literature suggests that as much as 20% of the total carbon assimilated may be transferred in ectomycorrhizal systems (Finlay and Soderstrom 1992). Vogt et al. (1982) estimated that 70-80% of Pacific Silver fir (Abies amabi/is) net primary productivity went to support mycorrhizal symbionts . Similar values have been estimated for Douglas-fir (Fogel and Hunt 1983). In exchange for photosynthates, fungal symbionts take-up and transport cations and anions (especially immobile molecules such as phosphates), nutrients, organic materials, and water to the host plant via Miller and Allen 1992; Yanai et al. 1995) which hyphae and mycelium (Harley and Smith 1983; ultimately improve the ability of plants to fix carbon by increasing the flow of nutrients to photosynthetically active leaves (Vogt et al. 1991 ). The extra matrical phase of mycorrhizae (mycelium and rhizomorphs) extends through the soil and thus acts as an exploratory organ which functions mainly in the uptake of water and nutrients (Miller and Allen 1992). Some additional benefits from ectomycorrhizal fungi may be in the form of hormone production which promotes branching of plant feeder roots thereby increasing the area of absorbing root surface and exchange zone between fungus and plant (Molina and Trappe 1982b}. Other reported benefits include lengthening of root life, protection against soil pathogens, increased resistance to drought, tolerance to heavy metals as well as resistance to increased soils temperature, soil toxins, and extreme pH (Molina and Trappe 1982b; Harley and Smith 1983). 1.3 CLASSES OF MYCORRHIZAE AND SEGREGATION BY HABITAT Several classes of mycorrhizae are currently recognized (Harley and Smith 1983; Allen et al. 1995) and classified with respect to specialized structures that form in the roots . These structures include vesicles, arbuscules, intracellular hyphal coils as well as other key characteristics including the 5 presence of a fungal sheath (mantle) and Hartig net (Harley and Smith 1983). The seven main classes of mycorrhizae are: arbuscular, arbutoid, ectomycorrhiza, ectendomycorrhiza, ericoid, monotropoid, and orchidaceous . Each class involves specific groups of plant and fungal species (Molina et al. 1992). Generally, the classes of mycorrhizae are distinct in their distribution with respect to ecological and environmental conditions (Read 1991 ). Arbuscular mycorrhizae are almost universally present in all ecosystems of the world (Harley and Smith 1983) and will dominate in herbaceous and woody plant communities on mineral soil at lower latitudes (Read 1991 ). Ericoid mycorrhizae (specific to members of the Ericales) are adapted to acidic soils with limited nutrients in both northern and southern hemispheres (Harley and Smith 1983) and to mor humus soils at higher latitudes and altitudes (Read 1991 ). Ectomycorrhizae also occur worldwide at intermediate altitudes and latitudes and are adapted to soils in temperate forests where surface litter accumulates, where moisture is usually not limited (Read 1991; Allen et al. 1995), and where seasonal changes and flushes of nutrients occur in the soil (Vogt et al. 1991 ). In temperate forests of North America, several important genera of the families Pinaceae, Betulaceae, and Fagaceae commonly form ectomycorrhiza (Molina et al. 1992). 1.4 ECTOMYCORRHIZAE 1.4.1 STRUCTURE Ectomycorrhizae are characterized by their structural features . Hyphae surround the root to form a mantle or sheath and emanate outwards to penetrate the substrate (Harley and Smith 1983). Hyphae also penetrate between root cells to form the Hartig net. For angiosperms, the Hartig net generally forms only in the outer layer of cells (epidermis) whereas deeper formation involving the cortical layer (to the endodermal cells) occurs in gymnosperms. Hyphal coils, vesicles and haustoria are all absent. The extramatrical phase of ectomycorrhizal fungi (epigeous or hypogeous) extensively colonize the soil, often forming visible persistent mats in the upper soil and humus (Molina et al. 1992). 6 1.4.2 CHARACTERIZATION AND IDENTIFICATION Traditionally, ectomycorrhizae have been morphologically characterized by describing a variety of macroscopic and microscopic features (Trappe 1965; Zak 1973). Recently, Goodman et al. (1996), Agerer (1987-1995), pnd lngleby et al. (1990) have refined these protocols. Ectomycorrhizae are usually characterized (and sometimes identified) on the basis of morphology, anatomy, .ultrastructure, and chemical testing (Agerer 1987-1995). Characteristics used to distinguish ectomycorrhizae include mycorrhizae shape and color, mantle features, rhizomorphs, emanating hyphae, Hartig net and cystidia (Agerer 1987-1995). However, the identification of the ectomycorrhizal mycobiont based solely on morphology is difficult and often considered tentative because of the number and similarities of potential types of ectomycorrhizae. Direct identification of the mycobiont include hyphal tracings whereby rhizomorphs and mycelium are traced from the fruiting body to the root tip (Agerer 1987-1995), but this procedure is difficult in field and laboratory settings leading to uncertain conclusions . Indirect identifications rely on pure and pot culture syntheses, however, a majority of ectomycorrhizal fungi are not easily cultured axenically, making pure culture approaches impossible for some ectomycorrhizal taxa . Because of these difficulties, molecular techniques are increasingly being used to help identify the species of ectomycorrhizal fungi. Molecular techniques, such as restriction fragment length polymorphism (RFLP), are used to characterize and identify species of mycorrhizal fungi (Bruns 1995; Egger 1995; Erland 1995; Gardes and Bruns 1993a). The internal transcribed spacer region (ITS) of the rRNA gene is commonly targeted to differentiate interspecific fungal variation by using different primers (Egger 1995; Gardes et al. 1991; Bruns et al. 1991 ). Many researchers have recently used RFLP analysis to study ectomycorrhizal community dynamics (Karen and Nylund 1997; Kernaghan et al. 1997; Pritsch et al. 1997b; Gardes and Bruns 1996; Karen and Nylund 1996; Nylund et al. 1995). 7 1.4.3 HOSTS AND FUNGI Ectomycorrhizal fungi are associated with several plant families includ ing the Pinaceae, Fagaceae, Betulaceae, and Salicaceae (Molina and Trappe, 1982b). In BC, many species are ectomycorrhizal including conifers such as Pinus contorta, P. banksiana, Picea mariana , Pseudotsuga menziesii, and Abies lasiocarpa and angiosperms such as Alnus spp., Betula papyrifera, and Populus tremuloides . Fungi forming ectomycorrhizae belong to the Basidiomycotina, Ascomycotina, and Zygomycotina Subphyla. Molina et al. (1992) provides a worldwide estimate of 5400 species of ectomycorrhizal fung i. The best known and probably most important fungal species involved in ectomycorrhizal formation are the basidiomycetes (Read 1995). 1.5 TAXONOMY AND ECOLOGY OF SITKA ALDER Worldwide, there are 30 species of alder {the Alnus genus, a member of the Betulaceae). Of those, eight are found in Canada, of which A. viridis ssp. sinuata (Sitka alder), A. rubra (Red alder), A. incana ssp . rugosa (Speckled alder) and A. incana ssp. tenuifolia (Mountain alder) are found in BC (Farrar 1995). There are literature discrepancies in the taxonomy of Alnus species; Alnus species and subspecies classifications differ between authorities (see Table 1). Throughout this thesis, Sitka alder is referred to as A. viridis ssp. sinuata (following Farrar 1995). Sitka alder is found from the Pacific coast to the western slopes of the Rockies, from southwest and central Alaska and the Yukon to northwest California and central Montana and is common along streams and lakes, in valley bottoms, along avalanche tracks, talus, and moraines (Farrar 1995). It is a thicket-forming shrub with several gray to light-gray stems . Maximum height and diameter are 915m and 0.2m respectively. 8 Table 1 Latin binomial equivalents of Alnus species located in British Columbia Common Name Latin Binomials following Reference Farrar (1995) A. viridis ssp. sinuata (Regel) A. Love & D. Love =A. sinuata (Regel) Rydb Brayshaw (1996) Alnus crispa (Aiton) Pursh 1 ssp. sinuata (Regel) Hulten =A. sinuata Regel =A. sitchensis (Regel) Sargent Green Alder A. viridis ssp. crispa (Ait.) Turrill =A. crispa (Ait.) Pursh A. crispa (Aiton) Pursh ssp. 1 crispa Red Alder A. rubra Bong. =A. oregona Nutt. Mountain, Thin leaf Alder A. incana ssp. tenuifolia (Nuff.) Breit. =A. tenuifo/ia Nutt. Speckled, Tag, Gray, Hoary Alder Siberian ~ Hazel ~ A. incana ssp. rugosa (Du Roi) =A. rugosa (Du Roi) Spreng. A. rubra Bongard var. rubra =A. rubra var. pinnatisecta Starker =A. oregona Nuttall A. tenuifolia Nuttall var. occidentalis (Dippel) Callier ex Schneider =A. tenuifo/ia var. virescens (Watson) Callier ex Schneider, =A. tenuifolia var. ferrugineotomentosa Brayshaw =A. incana var. virescens Watson =A. tenuifo/ia var. purpusii Koehne in Sched. A. rugosa (Du Roi) Sprengel Sitka Alder A. viridis ssp. fruticosa (Rupr.) Nym. A. serrulata (Ait.) Willd. =A. incana var. serrulata (Ait) Boivin A. glutinosa (L.) Gaertn . Furlow (1979) A. viridis ssp. sinuata (Regel) Love & Love =A. sinuata (Regel) Rydb =A. crispa ssp . sinuata (Regel) Hulten . =A. crispa var. sinuata (Regel) Breitung =A. sitchensis (Regel) Sargent A. viridis ssp . crispa (Aiton) Turrill =A. viridis var. crispa (Aiton) House equivalent not provided equivalent not provided European equivalent not provided A. glutinosa (L.) Gaertner =A. vulgaris (Hill) Black ~ . . ,, Hybnd1zat1on can occur w1th overlapping ranges (Brayshaw 1996). Spec1es not found 1n BC . 9 Literature suggests that alder root systems are shallow. When prostrate Alnus sp. branches touch the soil and/or become submerged, adventitious rooting occurs (Furlow 1979; Wilson et al. 1985). In addition , epicormic shoot development is common among Green alder (A. viridis ssp. crispa (Aiton) Turrill) (Wilson et al. 1985), whereby a shoot develops from a dormant or adventitious bud on the belowground root mass in response to the opening of the canopy and will grow into a new sucker (Dunster and Dunster 1996). This type of clonal reproduction makes the estimation of the age of Green alder thickets difficult as older stems will die and rot (Wilson et al. 1985). Also, ramets (clonal reproduction) from larger parent plants occur, evident from existing older shoot and root material (Wilson et al. 1985). They also showed that depending on disturbance regime, different Green alder from Alaska stands will vary proportionally in age, a reflection of epicormic shoot development where stems on the same plant will vary in age. The plant will be older than the oldest stem , potentially of unlimited age, depending on disturbance regime through epicormic shoot development (Wilson et al. 1985). On average, Sitka alder was oldest in alpine tundra (114y of age), 48y along river cuts , 36y in moist tundra, and 33y in flood plains with the estimated longevity to be 200, 100, 50, and 50y, respectively, depending on disturbance regime (Wilson et al. 1985). Sitka alder and the entire genus are noted pioneer species following landslides, glacial retreat, and logging where nitrogen levels are low. The success as pioneering species is due to the ability of the genus to form actinorhiza with nitrogen-fixing actinomycetes which fix atmospheric nitrogen into organic forms in root nodules. Sitka alder (A. sinuata (Regel) Rydb.; Binkley 1986) in BC has been reported to fix 20-65 kg of nitrogen ha- 1 y" 1 . Alder leaves also contain high levels of nitrogen and return nitrogen to the soil when they are shed (Farrar 1995). As a result, Sitka alder has been noted to affect soil properties . For example, the pH can become very acidic underneath alder bushes; Wurtz (1995) found that Alaskan Sitka alder soils had lower pH, lower phosphorus, and less potassium versus soils lacking alder and that the nutrient supplying capacity of alder soils was related to total N. Mitchell (1968) found very acidic soils (pH of 3.3) under Sitka alder in Alaska. Binkley et al. (1984) found that 10 the soil N availability index was 3-fold greater under alder from Nanaimo, BC . In contrast, A. rubra was found to increase the availability of P and not affect pH in Oregon soils (Giardina et al. 1995). 1.6 ECOLOGY OF LODGEPOLE PINE Lodgepole pine (Pinus contorta Dougl. ex Loud . var. latifolia Engelm .) is a member of the Pinaceae and is distributed throughout the northern hemisphere. Of the 95 species of pine, nine are native to Canada, six native to BC (Farrar 1995). Lodgepole pine is found throughout the interior of BC, north into the central Yukon and southeast Alaska regions, and south along the Pacific coast to southern California (Farrar 1995). The tree has brown to gray bark less than 2cm th ick and is medium sized with average height and diameter of 30m and 0.6m respectively (Farrar 1995). Root systems consist of a taproot with many spreading lateral roots with vertical sinkers (Farrar 1995). Evergreen needles are 3-?cm long, in bundles of 2, and stiff, sharply pointed . Seed cones begin to be produced at 5-10 years of maturity with good seed crops occurring every 1-3 years (Farrar 1995). Cones are in clusters and rema in on the tree for 10-20 years. As the cones are serotinous, they will commonly open with heat from wildfire , consequently, most stands in BC are of fire origin with a maximum age of 200 years . 1.7 SPECIFICITY AND RECEPTIVITY Specificity refers to the number of host genera with which the fungus can associate and form ectomycorrh izae. Receptivity refers to the acceptance level of the host in relation to ectomycorrhizal fungi. A given species of fungus may be able to form ectomycorrhizal relationships with a group of host species (broad host range) and can be considered to exhibit low specificity. Most ectomycorrhizal fungi have a broad host range in that they are able to form mycorrhizae with many species of plant hosts and most ectomycorrhizal host plants are broadly receptive and recognize many species of mycobionts (Molina et al. 1992). 11 Some genera of plants associate with a very low number of fungi and are cons idered poorly receptive (Alnus) while others such as Douglas-fir and pine associate with hundreds of fungi and are very receptive . Examples of broad host range fungi include Laccaria /accata , Cenococcum geophilum, and Paxillus involutus (Godbout and Fortin 1983). In contrast, a fungal species may form ectomycorrhizae with only one genus of plant as observed with Alpova diplophloeus on alder species (genus-specific or narrow host range) and can be considered to exhibit high specificity. The apparent ecological consequence of being a genus-specific fungus is that intergeneric host bridges might not exist between pine and alder. Conversely, intergeneric linkages would be more likely when ectomycorrhizal fungi are broad host ranging and able to form mycorrhizae with theoretically several host genera . In a review of specificity in mycorrhizal symbiosis, Molina et al. (1992) list ectomycorrhizal fungi known to associate with only one host genus . The list was based primarily on sporocarps fruiting in the vicinity of host plants , and includes the genus Alnus. Some of the fungi thought to be associated with alder exhibit high specificity and narrow host range while other fungi exhibit broader host ranges (Table 2). 1 .7.1 ALNUS SPP. AS MYCORRHIZAL HOSTS As noted above, host plants such as Alnus sp. show a low degree of receptivity by associating with few fungal species (Miller et al. 1991 ). Our recent compilation of fungi known to associate with Alnus species (Table 2) illustrates this limited receptivity with primarily narrow host range fungi. Worldwide, about 50 ectomycorrhizal types have been described in association with Alnus spp. (Pritsch et al. 1997a) from laboratory or greenhouse synthesis experiments, confirming again the very low receptivity of alder towards ectomycorrhizal fung i. 12 Table 2 Putative ectomycorrhizal fungi for the genus Alnus Ectomycorrhizal Fungi Host A. viridis {Chaix.) D.C., A. incana (L.) Loc Ob" FB Swiss 21 species including : Alpova diploph/oeus, National Cortinarius atropusil/us, Lactarius obscuratus, Park Naucoria escharoides, Paxillus filamentosus, Russula a/netorum. USA FB Gyrodon, Alnico/a, Russula, Lactarius, Alnus spp . Cenococcum FB Alnico/a, lnocybe, Hydrocybe, Phlegmacium, not Alnus spp. Russula, Lactarius provided r-A -=-.-r u--:b-ra - - - - - t --=3-=o-s_p_e_c""" ie-s--;in- c--:1-ud --:ci=n-g-: - A-=-:-lp_o_v_a-;-h-c....,.in_n_a_m_o_m - eu _s_,----lrN ;-;-.~ ont A. viridis Hymenogaster alnico/a, Lactarius obscuratus, two lnocybe spp . 3 unknown morphotypes A. rubra Lactarius obscuratus A. rubra Alpova diplophloeus, Paxillus involutus, Astraeus pteridis, Scleroderma hypogaeum, Laccaria /accata (weak) Alpova diploph/oeus, Astraeus pteridis, Paxil/us involutus, Pisolithus tinctorius (weak) Alpova dip/oph/oeus, Astraeus pteridis, Paxil/us involutus Alpova diplophloeus, Astraeus pteridis (weak), Paxil/us involutus Alnico/a, Alpova, Hydrocybe, lnocybe, Laccaria, Lactarius, Ph/egmacium, Russula A. glutinosa A. incana A. sinuata A. rhombifolia A. crispa, A. rugosa var. americana A. crispa, A. rugosa var. americana A. diploph/oeus, Cenococcum geophilum, Cortinarius cf. subporphyropus, Hebeloma crustuliniforme, Laccaria laccata, Leccinum holopus, L. subleucophaeum, Paxillus involutus, Pisolithus tinctorius, Scleroderma citrinum Ca/ocybe fa/lax Alnus crispa Alnion glutinosae, A/no-Padion A. crispa A. tenuifolia I New Zealand M t ~ a e - :t: -a--:1-. w. M : : I - i 1 PC Mejstrik and Benecke 1969 Froidevaux 1973 Molina 1979 i Pacific Northwest see above PC Molina 1981 PC Molina 19811 see above PC Molina 1981 / Eastern Canada FB Eastern Canada PC Godbout and Fortin 1983 Godbout and Fortin 1983 Alaska FB Oregon Pacific Northwest Europe, North America FB · Alaska FB A. diploph/oeus, Hebeloma cf. crustuliniforme, Alaska M 13 Trappe 1962 Horak 1963 1968 Amanita, Cortinarius, Ga/erina, Gyrodon, Hebeloma, lnocybe, Lactarius, Naucoria, Paxillus, Rickenella, Russula, Tricholoma, Xerocomus Russula subarctica and R. alnicrispae Cortinarius cf. saturninus, Paxillus filamentosus, Gyrodon cf. lividus Reference Favre 1960 I ! l Brunner and ; Miller 1988 1 Bujakiewicz 1989 Brunner 1989 Brunner et al. 1990 Table 2 Putative ectomycorrhizal fung i for the genus Alnus (continued) Loc. Host ! Ectomycorrhizal Fungi A. rubra Oregon , I A. diplophloeus, Thelephora terrestris, Lactarius obscuratus, Cortinarius bibu/us, Wash ing1 ! Laccaria /accata, Hebe/oma crustuliniforme, ton, N. ! Paxillus involutus, 4 unknown morphotypes California I Alaska A. tenuifolia I 57 species including: Hebeloma cf. (=A. incana ssp . j crustuliniforme, Alpova diplophloeus , and tenuifolia) I Clitocybe cf. catervata Alaska A. crispa 1 95 species including: Russula alnicrispae, R. (=A. viridis ssp . subarctica, Plicatura nivea, Clitocybe crispa) A. rubra Alnus spp. subalutacea T. terrestris, A. diplophloeus, Lactarius obscuratus Gyrodon lividus, G. monticola, Alpova diploph/oeus, Cortinarius alnetorum, C. alneus, C. dilutus, Naucoria alnetorum, N. escharoides, N. striatula, Hymenogaster alnico/a, Paxillus filamentosus, Lactarius obscuratus, L. pusillus, Russula alnetorum P. invo/utus A. g/utinosa I A viridis (Chaix) I 6 morphotypes including: Lactarius DC. I I obscuratus, Alnico/a escharoides (hyphal Reference Miller et al. 1991 Ob." M ! I i I' i ! FB FB Oregon M global FB . Brunner et al. 1992 ----1 Brunner et al. 1992 A. incana A. sinuata A. glutinosa A. glutinosa Alnus viridis i I Sweden PC Italy M 1 PC 5 unknown morphotypes including: A/pova Alaska FB,M 14 morphotypes including: Russula, Germany M Germany RF Switzerland FB unidentified taxa 20 species including: Lactarius obscuratus, I L. /epidotus, L. a/pinus, Alpova diplophloeus, I Russula alnetorum, Cortinarius he/vel/oides, I C. badiovestitus, C. bilulus, C. atropusillus, i C. (Phlegmacium) alnobetulae, C. j (Phlegmacium) kuehneri, C. (Myxacium) , pluvius, C. cedriolens, Alnico/a j submelinoides, A. luteolofibrillosa , A. subconspersa, A. suavis, A. paludosa, lnocybe obscurobadia , /. mixtilis. 1 1992 Mol ina et al. 1992 l ' ' ! ' I Ekblad and Huss-Danell 1995 Helmet al. 1996 Pritsch et al. 1997a Pritsch et al. 1997b Senn-lrlet 1997 3 Locat1on of study or sampling. Observation method . Ectomycorrh1zal fung1 on alder were observed either on the root via morphotyping in vivo (M), on the root from pure culture synthesis (PC), or via PCRIRFLP (RF) analysis . Possible associations obtained by fruiting body assays are noted with (FB). Weak associations are< 5% of roots infected . 3 Personal communication 14 ! Arnebrant et I i al. 1993 i Airaud i et al. i I 1993 I L, '' I Sweden Lactarius, Naucoria, Cortinarius 16 taxa including : Lactarius obscuratus, L. omphaliformis, L. lilacinus, Russula pumila, Naucoria escharoides, N. subconspersa, I Cortinarius cf. alneus, C. cf. helvelloides, 8 I I' P. involutus diplophloeus !I I I connections between fruiting body and tip); I i I ! -·-..J Miller et al. ! I Lactarius a/pinus, Paxil/us invo/utus (morphology), two unidentified morphotypes I I i i I i I I ! i I I ' 1.7.2 PINUS SPP . AS MYCORRHIZAL HOSTS Potentially, members of the Pinaceae and Pinus in particular are very receptive and will form ectomycorrhizal associations with a wide range of mycobionts, numbering up to an estimated 2000 species (Trappe 1962; Molina et al. 1992) of which many belong to the Sub-Phylum Basidiomycotina (Table 3). Because of the great amount of literature existing on Pinus sp., we have restricted our compilation to P. contorta and P. banksiana found primarily in Western Canada and the Pacific Northwest. Included in these associations are species exhibiting broad host range, T. terrestris (Miller et al. 1991 ), as well as species associated almost exclusively with Pinaceae, such as Sui/Ius sp . and Rhizopogon sp. (Molina 1979). 1.7.3 MYCORRHIZAL LINKAGES BETWEEN HOSTS Past studies have examined mycorrhizal linkages between various species of host plants including Alnus, Pinus, and others. Miller et al. (1992) examined fungal symbionts and ectomycorrhizal types of Douglas-fir and red alder from six distinct Oregon forest soils using a bioassay procedure. A total of 12 ectomycorrhizal types were found and of these, only Thelephora terrestris was shared between the two hosts . Theoretical calculations examining linkages between Pinus sp . and Pseudotsuga sp . (Molina et al. 1992) estimate 2000 fungal associations exist for both hosts and , of these, 1800 (72%) compatible species overlap, most of these fungal species having broad host ranges . 15 Table 3 Putative ectomycorrhizal fungi for the Pinus contorta var. latifolia and P. banksiana Host P. contorta var. latifolia P. contorta P. contorta P. banksiana P. banksiana P. banksiana P. contorta var. latifolia P. contorta var. latifolia P. banksiana P. banksiana P. contorta P. contorta var. latifolia Loc Pacific Northwest Ob"' FB Reference Trappe 1962 not given FB Harvey et al. 1976 Pacific Northwest PC Molina and Trappe 1982a N. Alberta M Danielson 1984 N. Alberta PC Danielson 1984 N. Alberta N. England M Danielson et al. 1984 Dighton and Mason 1985 Ectomycorrhizal Fungi Amanita muscaria, A. pantherina, A. vaginata, Cenococcum graniforme, Cortinarius croceofo/ius, Gomphidius rutilus, G. smithii, G. vinicolor, Hygrophorus gliocyclus, Laccaria laccata, Lactarius deliciosus, Leccinum aurantiacum, Rhizopogon luteolus, Russula de/ica, Sui/Ius bovinus, S. granulatus, S. piperatus, S. ruber, S. subaureus, S. subluteus, S. tomentosus, Tricholoma flavovirens Lactarius rufus, Laccaria sp., lnocybe longicystis, Cortinarius sp., Russula emetica, Amanita sp., Paxillus involutus, Clitocybe sp. Amanita muscaria, Boletus edulis, Laccaria laccata and Lactarius de/iciosus, Paxillus involutus, Rhizopogon fuscorubens, Suillus brevipes, Tricholoma .flavovirens, Astraeus pteridis, Cenococcum geophilum, Cortinarius pistorius, Gastroboletus subalpinus, Hysterangium separabile, Leccinum manzanitae, Melanogaster intermedius, Piso/ithus tinctorius, Rhizopogon cokeri, R. occidentalis, Scleroderma hypogaeum, Sui/Ius brevipes, S. cavipes, S. grevillei, S. lakei Cenococcum geophilum, Elaphomyces muricatus, E. granulatus, Lactarius paradoxus, Tricholoma flavovirens, Suil/us tomentosus Tricholoma flavovirens, T. pessundatum, T. ze/leri, Sui/Ius flavovirens, S. albidipes, Cenococcum geophilum, Laccaria proxima, Scleroderma macrorhizon, Astraeus hygrometricus, Lactarius paradoxus, Coltricia perennis, Bankera fuligineoalba Tricholoma flavovirens, Cenococcum geophilum, Laccaria proxima Lactarius rufus, Laccaria sp., lnocybe longicystis, Cortinarius sp., Russula emetica, Paxillus involutus Amanita muscaria, Rhizopogon roseolus, Paxillus involutus, Suillus granulatus, S. bovinus, S. luteus not given PC Read et al. 1985 Inoculated with : E-strain, Hebeloma sp., Thelephora terrestris, Laccaria proxima, Cenococcum geophilum, Pisolithus tinctorius, Astraeus hygrometricus, Lactarius paradoxus, Sphaerospore/la brunnea. After 3 years outplanted : E-strain, Tuber sp., Suil/us-like spp., MRA, an unidentified basidiomycete ( 17 types total) E-strain, Cenococcum sp., MRA, Amphinema sp., Suil/us sp., Thelephora sp., Hebeloma sp., Tomente/la sp. T. terrestris, one unidentified pink isolate N. Alberta M Danielson and Visser 1989 N. Alberta Sweden M PC Boletus, Pulveroboletus, Suil/us, Cortinarius, Dermocybe, lnocybe spp. not given FB, M Danielson 1991 Finlay et al. 1992 Molina et al. 1992 16 FB Table 3 Putative ectomycorrhizal fungi for Pinus contorta var. latifolia and P. banksiana s (continued) Ob"' Reference Host Ectomycorrhizal Fungi Loc P. contorta Amphinema or Clitocybe spp. Alberta PC Zelmer and Currah 1995 P. Cenococcum geophi/um, Cortinarius/Dermocybe, E- N.E. M Visser 1995 banksiana stain, Hebeloma sp., Hydnellum peckii, Alberta Hygrophorus sp. , Lactarius deliciosus, MRA, Piloderma byssinum, Russula spp. (6 types), Sui/Ius brevipes, S. tomentosus, Sui/Ius sp., Tomentella spp. (4 types), Tricholoma spp. (3 types), 12 unknown types 1 "L , Locat1on . Locat1on of study or sampling . Observed. Ectomycorrh1zal fung1 on alder were observed either on the root via morphotyping in vivo (M), on the root from pure culture synthesis (PC), or via PCRIRFLP (RF) analysis. Possible associations obtained by fruiting body assays are noted with (FB). Weak associations are < 5 % of roots infected. 3 1dentification was only speculative as the cultures resembled the hyphal morphology of Amphinema and Clitocybe spp. ;j Arnebrant et al. (1993) directly examined functional linkages between A. glutinosa and Pinus contorta seedlings inoculated with Paxil/us involutus grown in sealed observation chambers and fed left in growth chambers. After a 7d incubation period, between 5-15% of the 15 15 N2 , then N recovered was found in the pine seedlings, suggesting a fully functional ectomycorrhizal association connected the two species (Arnebrant et al. 1993}. Ekblad and Huss-Danell (1995) did a similar experiment involving A. incana and P. sylvestris linked via Paxil/us involutus and found that wh ile some fixed N (applied in solution as 15 15 NH 4 N0 3 ) was found in pine, it was not significantly different from controls. Recently, a study by Simard et al. (1997c) examined the transfer of labeled carbon between ectomycorrhizal Pseudotsuga menziesii and Betula papyrifera and non-ectomycorrhizal, arbuscular mycorrhizal Thuja plicata in the field . Reciprocal isotope labeling showed bi-directional 13 C and 14 C transfer (applied as 13 C0 2 and 14 C0 2 respectively) between the ectomycorrhizal hosts. Since T. plicata absorbed only small amounts of the isotopes, the significant transfer occurring between P. menziesii and B. papyrifera was suggested to occur primarily through a direct ectomycorrhizal hyphal pathway (Simard et al. 1997c). 17 1.8 MYCORRHIZAL FUNGI SUCCESSION THEORY: 'EARLY' AND 'LATE' STAGE FUNGI There is no single unifying theory clarifying succession of ectomycorrhizal fungi during stand development. Many researchers have tried to elucidate the succession of fungi during stand development or following disturbance. Dighton and Mason (1985) suggested that in northern temperate forests, succession to ecosystem climax communities includes first colonization of herbaceous plants and shrubs by arbuscule mycorrhizal-forming fungi followed by ectomycorrhizalforming fungi associated with conifers and hardwoods. They postulate that the mechanism influencing changes in mycorrhizal dynamics is related to the carbohydrate supply from the tree and increased recalcitrance of forest floor organic matter (Dighton and Mason 1985). Dighton and Mason (1985) offered a secondary succession theory involving types of ectomycorrhizal fungi present in different stages of stand development. Early stage fungi are defined as being first present on a site and are characterized by: a low host-supplied carbohydrate demand, ability to obtain available nutrients from inorganic pools, competing primarily by rapid mycelial growth (r-selected}, having competitive interactions with saprophytic fungi, and exhibition of rapid growth on simple media (Dighton and Mason 1985). In contrast, late stage fungi possess a high host-derived carbohydrate demand, obtain available nutrients from organic pools, compete primarily by production of mycelial strands (k-selection), have a synergistic association with saprophytic fungi if the mycorrhizal fungus is not able to breakdown organics or a competitive association with saprophytic fungi if mycorrhizal fungi are also decomposers, and are difficult to culture on complex growth media (containing sugars and vitamins) (Dighton and Mason 1985). In addition, Bruns (1995) noted differences in seral establishment of early and late stage fungi: early stage fungi are established via spores rather than via mycelial growth. Late stage fungi are usually stress tolerant, good competitors, require high N availability, and invest in extramatrical mycelia and strands (Bruns 1995). The enzymatic ability of fungi to digest protein for N sources may also correspond to early and late stage fungi (Abuzinadah and Read 1986). Early stage fungi present in mineral soil lack the enzymatic ability to digest proteins; 18 proteolytic and peptidolytic enzymes are present in late stage fungi where the presence of proteins in humus is high. Typical examples of early stage fungal genera include : Laccaria, Hebeloma, Thelephora (Deacon and Fleming 1992; Abuzinadah and Read 1986), Mycelium radicis atrovirens complex and E-Strain I and II (Simard et al. 1997a). Examples of late stage fungi genera include: Lactarius, Amphinema, Russula, Amanita, and Cortinarius (Deacon and Fleming 1992; Abuzinadah and Read 1986). Other genera have characteristics of both early and late stage fungi, such as Rhizopogon (Molina et al. 1992). Although the concepts of early and late stage fungi has been used in the literature, they have not been universally accepted. The early-late stage concept has been criticized by Termorshuizen (1991) in that it only applies to the first 10-20 years of first rotation stands. Molina et al. (1992) also note that in natural forest ecosystems affected by periodic disturbances, this simplified model of early and late stage fungi is inadequate, however, they do not suggest another theory. 1.8.1 FACTORS INFLUENCING SUCCESSION AND PRESENCE OF ECTOMYCORRHIZAL FUNGI Literature suggests that fungal succession occurs with stand development as qualitative and quantitative biotic and abiotic changes proceed which impact fungal diversity (Dighton et al. 1986; Dighton and Mason 1985). Several biotic factors are postulated to influence secondary succession : competitive interactions between fungi, interactions between host and fungi (Molina et al. 1992), rhizosphere microbes and mycorrhizal development (Garbaye and Bowen 1989), presence of ectomycorrhizal plants remaining on the site (Danielson and Visser 1989), seral host tree characteristics (age structure and size, nutrient availability, carbohydrate supply) (Dighton and Mason 1985; Vogt et al. 1992), and seral mycorrhizal fungal characteristics (carbohydrate demand , competitive ability, competition with saprophytic fungi) (Dighton and Mason 1985). In addition , Termorshuizen (1991) noted the following factors as influencing ectomycorrhizal fungi : 19 different distributions of photosynthates to the root and shoot, increased internal recycling of nutrients as the trees ages, allelopathic affects of litter and/or plants, and competition for nutrients with saprophytic fungi and/or plants. Among abiotic factors documented that influence secondary succession, one can find the severity of disturbance such as silvicultural practices and treatments (Vogt et al. 1992), soil properties such as development, pH, fertility, organic content, and moisture content (Danielson and Visser 1989; Molina et al. 1992; Dighton and Mason 1985; Vogt et al. 1992), environmental factors such as climate (Dighton and Mason 1985; Vogt et al. 1992), season and geographical location (Vogt et al. 1992), nutrient availability and type and quality of substrate (Vogt et al. 1992), and anthropogenic factors such as acid rain and air pollution (Vogt et al. 1992). Rhizosphere microbes are also dependent on soil type and soil-plant interactions which influence development of ectomycorrhizal associations (Garbaye and Bowen 1989). Danielson and Visser (1989) suggest that soil types and the ability of ectomycorrhizal fungi to colonize different soil types may have a more important role than competitive replacement in determining the sequence of ectomycorrhizal colonization following disturbance. 1.9 SUCCESSION OF ECTOMYCORRHIZAE 1.9.1 ALNUS SPP. HOSTS Minimal information exists on primary or secondary succession of ectomycorrhizae associated with alder. A recent study by Helm et al. (1996) examined Sitka alder ectomycorrhizal communities through a chronosequence on deglaciated land at Exit Glacier, Alaska. Five morphotypes were found on Alnus sinuata. All five morphotypes were found in most stages. An Alpova diploph/oeus-like and an unidentified morphotype were dominant (Helm et al. 1996). 20 1.9.2 PINUS SPP . HOSTS More information is available on the succession of ectomycorrhizal fungi on Pinus species during stand development. Dighton and Mason (1985), observing fruiting bodies under lodgepole pine stands in England (based on height classes ranging from 3.0- 7.6m) planted on peat, found that diversity of mycorrhizal fungi decreases with canopy closure. Under small trees, Lactarius rufus was common. With canopy closure, Laccaria sp., lnocybe longicystis , and Cortinarius sp. were common . Paxillus involutus and Galerina sp. were also found before canopy closure where Russula emetica became common . Based on sporocarps, they suggested a "Laccariallnocybe!Hebeloma" complex for young stands and a "Cortinarius/Russula!Amanita" complex for older stands. This sequence was suggested to be modified by soil type and environmental factors. Termorshuizen and Schaffers (1987) examined P. sylvestris stands in the Netherlands and found 5-10 year-old stands colonized primarily by Laccaria proxima , lnocybe brevispora, I. umbrina, and Suillus bovinus while 50-80 year-old stands possessed Lactarius hepaticus and Russula emetica. Danielson (1991) found Thelephora terrestris and E-strain common on 1-4y old container grown P. banksiana seedlings while Suillus sp . was abundant at 1Oy of age. Tomentella, Amphinema byssoides, and Mycelium radicis atrovirens were minor components across all seedling ages. Visser (1995) studied ectomycorrhizal fungi on Pinus banksiana from 6, 41, 65, and 122 year-old regenerated stands following wildfire and found that Russula morphotypes were common in older stands and absent in younger stands. Other species seemed to occur equally throughout the forest ages, which is not reflective of any process of species succession . Successional data based on aboveground fruiting structures may reflect poorly on belowground ectomycorrhizal community dynamics and care must be taken when extrapolating sporocarp data onto ectomycorrhizal community data. Based on the few studies reported in the literature, the congruence between above- and belowground ectomycorrhizal dynamics is uncertain . In mature Jack pine stands, Danielson (1984) found that the most common sporocarps were Suillus tomentosus and Russula sp . even though he determined that only 5% of ectomycorrhizae were characteristic of Suillus species . Older P. sylvestris stands in Sweden exhibited few aboveground Suillus bovinus sporocarps even 21 though their belowground genet size had increased relative to younger stands (Dahlberg and Stenlid 1984). However, Visser (1995) found that ectomycorrhizal tips on regenerated Pinus banksiana stands greatly reflected Russula sp. aboveground sporocarp abundance . Gardes and Bruns (1996) described three scenarios from their study of Californian stands of Bishops Pine (P. muricata) : firstly , some species sych as Russula xerampelina and Amanita francheti were well represented above- and belowground. Secondly, some common sporocarps, such as Sui/Ius pungens, were rarely found as ectomycorrhizae tips. Thirdly, some common ectomycorrhizal fungi found on tips, such as Russula amoenoleus and thelephoroid and boletoid types, were poorly or under represented aboveground . This is an indication that, among species, resource allocation differs with respect to production of fru itbodies versus ectomycorrhizae (Gardes and Bruns 1996) and that caution is necessary when inferring from either of these variables to the mycorrhizal community as a whole. 1.10 DIVERSITY INDICES Mycorrhizal species diversity and its effect on the ecological health of forest ecosystems is still poorly understood (Bruns 1995). structures among the Understanding species diversity in a community (and niche and guild species) should reveal much about the functional significance of ectomycorrhizae (Bruns 1995). The diversity of a community depends on both species richness and evenness (equitability) (Began et al. 1990; Bruns 1995). Few studies have examined the ectomycorrhizal fungi species' richness (the number of mycorrhizal types) or diversity in communities (Bruns 1995). In pine monocultures , the number of ectomycorrhizal fungi (based on sporocarp observations) varies from 13 to 34 while the diversity in mixed natural forests varies from 23 to 34 species (Bruns 1995). However, evenness is almost .totally unknown in ectomycorrhizal systems because individuals are seldom identified (Bruns 1995). Simard et al. (1997b) examined the effects of soil trenching on ectomycorrhizae associated with Douglas-fir and paper birch in mature forests . Treatment comparisons were made with respect to 22 mean richness, diversity, evenness and redundancy using Simpson, Shannon, and Mcintosh indices and it was observed that diversity was highest in untrenched treatments. 1.1 0.1 SIMPSON DIVERSITY AND EVENNESS INDICES Numerical indices are used to interpret species composition in a community. Measures of community structure take into account the abundance patterns and the species richness, for example Simpson's Index (D): where p i is the proportion of individuals that contributes to the total in the sample for the i th species and s is the total number of species in the community (Began et al. 1990). Using this formula , as D increases, diversity decreases. To avoid this confusion, D is usually expressed as 1-D or 1/D. For comparison to Simard et al. (1997b}, we will express D as 1-D. Using these relationships, Simpson evenness or equitability (E) can also be determined from the Simpson's Diversity index: E =D ~ where s is the total number of species in a community. The Simpson's index reflects dominance because it weights the most abundant species more heavily than the rare species (Barbour et al. 1987). Its use is advantageous as lt is not likely to vary much from sample to sample as it is the rare species that tend to change from site to site (Barbour et al.1987). 23 1.10.2 SHANNON DIVERSITY AND EVENNESS INDICES The Shannon Diversity Index (D ') is another index used to interpret species composition : i=l where P; is the proportion of individuals that contributes to the total in the sample for the i th species and s is the total number of species in the community (Began et al. 1990). From this, the Shannon evenness index (E ') is calculated : H E' =- lns where s is the total number of species in the community (Began et al. 1990). Relative to the Simpson index, the Shannon-Weaver is more sensitive to changes in the abundance of rare species (Magurran 1988; Simard et al. 1997b). Care must be taken when interpreting Simpson and Shannon-Weaver index values since D and D • increase with equitability and for a given equitability, D and D • also increase with richness . A species-rich but inequitable community has a lower index that one that is less-species rich but highly equitable (Began et al. 1990). 1.10 .3 MCINTOSH DIVERSITY AND EVENNESS INDICES Mcintosh (1967) proposed that the Euclidean distance of the community from the origin could be used to measure diversity (Magurran 1988). Simard et al. (1997b) found that the Mcintosh Index (expressed as the U distance) is more sensitive to changes in species ectomycorrhizal fungal community abundance than either of the Shannon or Simpson Indices. The distance U is expressed as : 24 U= /In;2 where n; is the number of individuals of each species (Magurran 1988). Simard et al. (1997b) used this distance to describe diversity of Douglas-fir seedlings and for comparison purposes, we will also report this distan,ce. In order to calculate evenness, Mcintosh's Dominance Diversity (D · ') must be calculated which is independent of N (the total number of individuals) (Magurran 1988): N-U D " = ---== N-JN· From D ", Mcintosh evenness (E"} can be calculated: N-U E" =--- N- -N fs where N is the total number of individuals (=:En;) and s is the number of species . 1.11 USE OF STATISTICAL ANALYSIS IN ECTOMYCORRHIZAL COMMUNITY STUDIES Early studies described ectomycorrhizal communities by noting presence or absence of morphotypes and did not attempt to describe abundance of these types statistically (Danielson and Visser 1989; Danielson and Pruden 1989). Later research emphasized the biological importance of detecting changes in relative abundance of morphotypes across treatments. Early studies used Analysis of Variance (ANOVA) to describe abundance dynamics, however, considerations of the model and assumptions were not met or discussed. Distributions of the data were not analyzed to examine possible skewness of data (with data transformation when necessary) and models were not evaluated properly (Danielson 1991 ). Visser (1995) attempted to statistically analyze abundance data but found that the high degree of variation in the abundance data did not allow for any statistical analysis. 25 Most recent publications have used parametric tests such as ANOVA to detect differences in abundance across treatments, however, techniques used vary between researchers , making comparisons between studies difficult. Investigators have also neglected to check model aptness . Karen and Nylund (1996 , 1997) used 2-way ANOVA and Tukey's studentized range test on their arcsin(squareroot)-transformed abundance data to statistically compare communities between treatments. They attempted to meet model assumptions and evaluated the model aptness . Simard et al. (1997a) used a similar approach with a 2x2 factorial ANOVA, however, they made no mention of the distribution of the data. Simard et al. (1997b) used a 1-way randomized complete block design ANOVA to detect differences between some of the abundant morphotypes. Only 7 of 20 types were analyzed due to low abundance and frequency (problems with the model aptness). Ursie et al. (1997) used a three-level nested ANOVA to examine mycorrhizal colonization and diversity. However, different transformations on the data were performed which still did not completely normalize their skewed data , complicating interpretations of the results. If treatment effects are minimal or confounded , differences in commun ities may not be detected with one-way ANOVA (Visser et al. 1998). These studies show that ANOVA modeling of ectomycorrhizal abundance data can be useful for those morphotypes that exhibit some minimal level of abundance (which is highly dependent on the experimental design). The model's aptness at describing common types at some low level of abundance is good , as shown through model evaluations including residual analys is, normal plots of residuals , and plots of residuals against predictors. However, the model cannot detect differences in rare species in low abundance (too much error with in groups to detect error between groups) due to too many zero cells counts or minimal difference exh ibited between the experimental treatments . Advanced statistical tests can be used to examine such abundance data (such as Zero cell count analysis), but these techn iques are still in the developmental stages and are not within the scope of this thesis (Bruno Zumbo, personal commun ication). 26 1.12 PCR-RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ANALYSES PCR-RFLP analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rONA) is a commonly used technique to characterize ectomycorrhizae and their communities (Egger 1995). The ITS regions of the rONA tends to be conserved among fungi and is highly variable between species but less variable within a species making it suitable to distinguish species necessary in community studies (Egger 1995). Methods include direct extraction of DNA from root tips and sporocarps, PCR amplification (Mullis and Faloona 1987) of a target region of DNA (ITS of rONA) (Gardes et al. 1991 }, digestion of the product using endonucleases, separation of fragments via gel electrophoresis, and analysis of band topologies. Fragment length polymorphisms occur mainly between and within a species, termed interspecific and intraspecific variation , respectively. Mechanisms causing both inter- and intra-specific variation in the ITS-1 and ITS-2 regions include : 1) insertions and deletions and 2) one or more point mutation(s) in the endonuclease recognition site (see Figure 1) (see Karen and Nylund 1996; Henrion et al. 1992). Insertions or deletions will increase or decrease the fragment size relative to other samples. If fragments are initially small and deletions occur, bands may not be visualized electrophoretically, and an entire band fragment may be lost. Karen et al. ( 1997) found insertions and deletions of some taxa to be greater than the methodological error (on average 5-15bp in length). With respect to point mutations, if a new enzyme binding site is created , the product will be cut into two smaller pieces and the number of fragments will be increased by one. If a recognition site is removed , one larger fragment will be present, whose sum mass is equal to the two smaller fragments (see Figure 1). The two mechanisms may occur at the same time, complicating interpretation of both inter- and intraspecific band topologies . 27 ___I_L Theoretical Interspecific Digestion of Product: 8 1) Insertions and 3 1 (base pairs x1 00) Deletions I_ _ _I_L _ _ _ l_l_ _ _ l_l _ _ _I 9 9 3 1 3 7 7 2 ---=----1_1_ 9 4 3 1 3 0 --::---1_1 2 7 2 0 2) Point Mutation(s) in Restriction Enzyme Site Addition of Site Removal of Site _ _l_l_l_ _ ___ I_ __I_I_L (see below) ___ I__ 6 2 5 3 1 3 11 3 1 8 U(Addition and Removal of 2 Sites) U(Gel Electrophoresis) __I_L 5 3 _I_ 5 1 _I 1 7 1 4 I_ 4 U(Low Amount of Product) 3 Figure 1 Two mechanisms causing inter- and intra-specific variation in PCR-RFLP Analysis . 28 1.13 REFERENCES AGERER, R. 1987-1995. Color atlas of ectomycorrhizae. Schwabisch Gmund, Germany. EinhornVerlag Eduard Dietenberger ABUZINADAH, R.A., READ, D.J. 1986. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. Ill. 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Methods for studying species composition of mycorrhizal fungal communities in ecological studies and environmental monitoring. In Biotechnology of ectomycorrhizae. Stocchi, V., Bonfante, P., Nuti, M. (Eds.). Plenum Press, New York. pp 229-240. PRITSCH, K., MUNCH, J.C., BUSCOT, F. 1997a. Morphological and anatomical characterisation of black alder Alnus glutinosa (L.) Gaertn. ectomycorrhizas. Mycorrhiza 7: 201-216 PRITSCH, K., BOYLE, H., MUNCH, J.C., BUSCOT, F. 1997b. Characterization and identification of black alder ectomycorrhizas by PCRIRFLP analyses of the rDNA internal transcribed spacer (ITS) . New Phytol. 137: 357-369 READ, D.J. 1991. Mycorrhizas in ecosystems. Experientia 47: 376-391 READ, D.J. 1995. Ectomycorrhizas in the ecosystem: Structural, functional, and community aspects . In Biotechnology of ectomycorrhizae: molecular approaches. Stocchi, V., Bonfante, P., Nuti, M. (Eds .). Plenum Press, New York. pp 1-23 READ, D.J., FRANCIS, R., FINLAY, R.D. 1985. Mycorrhizal mycelium and nutrient cycling in plant communities . In Ecological Interactions in Soil - plants, microbes, and plants . Blackwell Scientific Publications, Oxford . pp 193-217 SIMARD, S.W., MOLINA, R., SMITH, J.E., PERRY, D.A., JONES, M.J. 1997a. Shared compatibility of ectomycorrhizae on Pseudotsuga menziesii and Betula papyrifera seedlings grown in mixture in soils from southern British Columbia. Can. J. For. Res. 27: 331-342 SIMARD, S.W., PERRY, D.A., SMITH, J., AND MOLINA, R. 1997b. Effects of soil trenching on occurrence of ectomycorrhizas on Pseudotsuga menziesii seedlings grown in mature forests of Betula papyrifera and Pseudotsuga menziesii. New Phytol. 136: 327-340 SIMARD, S.W., PERRY, D.A., JONES, M.D., MYROLD, D.O., DURALL, D.M., MOLINA, R. 1997c. Net transfer of carbon between ectomycorrhizal tree species in the field . Nature 388 : 579-582 TERMORSHUIZEN, A.J. 1991 . Succession of mycorrhizal fungi in stands of Pinus sylvestris in the Netherlands. Journal of Vegetation Science 2: 555-564 TERMORSHUIZEN, A.J., SCHAFFERS, A.P. 1987. Occurrence of carpophores of ectomycorrhizal fungi in selected stands of Pinus sylvestris in the Netherlands in relation to stand vitality and air pollution . Plant and Soil104: 209-217 33 I TRAPPE, J.M. 1965. Tuberculate mycorrhizae of Douglas-fir. Forest Science 11: 27-32 TRAPPE, J.M. 1962. Fungus associates of ectotrophic mycorrhizae. Bot. Rev. 38: 538-606 URSIC, M., PETERSON, R.L., HUSBAND, B. 1997. Relative abundance of mycorrhizal fungi on Pinus strobus seedlings affected by root rot in a southern Ontario nursery. Can . J. For. Res. 27: 54-62 VISSER, S. 1995. ECM fungal succession in jack pine stands following wildfire. New Phytol. 129: 389-401 VISSER, S., MAYNARD, D., DANIELSON, R.M. 1998. Response of ectomycorrhizae and arbuscular mycorrhizal fungi to clear-cutting and the application of chipped aspen wood in a mixedwood site in Alberta, Canada. Applied Soil Ecology 7: 257-269 VOGT, K.A., BLOOMFIELD, J., AMMIRATI, J.F., AMMIRATI, S.R. 1992. Sporocarp production by basidiomycetes with emphasis on forest ecosystems. In The fungal community, its organization and role in the ecosystem. Carroll, G.C., Wicklow, D.T. (Eds.). Marcel Dekker, New York. pp 563581 VOGT, K.A., PUBLICOVER, D.A., VOGT, D.J. 1991 . A critique of the role of ectomycorrhizae in forest ecology. Agriculture, Ecosystems and Environment 35: 171-190 VOGT, K.A., GRIER, C.C., MEIER, C.E., EDMONDS, R.L. 1982. Mycorrhizal role in net primary production and nutrient cycling in Abies amabilis (Dougl.) ecosystem in Western Washington. Ecology 63: 370-380 WILSON, B.F., PATTERSON Ill, W.A., O'KEEFE, J.F. 1985. Longevity and persistence of alder west of the tree line on the Seward Peninsula, Alaska. Can . J. Bot. 63: 1870-1875 WURTZ, T.L. 1995. Understory alder in three boreal forests of Alaska: local distribution and effects on soil fertility. Can . J . For. Res. 25: 987-996 YANAI, R.D., FAHEY, T.J., MILLER, S.L. 1995. Efficiency of nutrient acquisition by fine roots and mycorrhizae. In Resource physiology of conifers: acquisition, allocation, and utilization. Smith W.K., Hinckley T.M. (Eds.). Academic Press, California. pp 75-103 ZAK, B. 1973. Classification of Ectomycorrhiza. In Ectomycorrhizae -Their ecology and physiology. Marks G.C., Kozlowski T.T. (Eds.). Academic Press, New York. pp 43-78 ZELMER, C.D., CURRAH, R.S. 1995. Evidence for a fungal liaison between Corallorhiza trifida (Orchidaceae) and Pinus contorta (Pinaceae). Can . J . Bot. 73: 862-866 I I 34 2. Characterization and seasonal ecology of ectomycorrhizae associated with Sitka alder and Lodgepole pine from naturally regenerating young and mature forests in the Sub-Boreal Spruce zone of British Columbia. Morphological characterization ABSTRACT Following timber harvest in the Sub-Boreal Spruce Zone of the central interior of British Columbia, Sitka alder and lodgepole pine often regenerate to form mixed stands. Alder contributes to site fertility through nitrogen fixation, however, current site prescriptions include brushing of alder from the site to lower competition for other resources. If ectomycorrhizal linkages exist between pine and alder, eradication programs may be unnecessary. To explore the influence of season and stand age on the ecology of ectomycorrhizae in these mixed stands as well as possible fungal linkages, ectomycorrhizae were sampled in June and September from naturally regenerating 10 and 120 year old stands and characterized morphologically. Four alder morphotypes were characterized in the spring sampling with an additional one in the fall. Ten pine morphotypes were characterized in the spring with three additional ones in the fall. There were significant differences in abundance with respect to stand age and season of some alder and pine morphotypes. Alder showed greater total percent colonization in September with no significant difference with respect to stand age. Pine exhibited significantly greater percent colonization than alder with respect to stand age and season. Mycobionts forming similar morphotypes between the two hosts included Cortinarius, Hebeloma, and Laccaria genera. Morphotype richness was greatest in pine. Model sensitivities produced differing trends in diversity and evenness values between hosts. There were significant differences in diversity and evenness in alder with respect to stand age and season wh ile there were no significant differences in pine. Evenness of ectomycorrhizal communities was comparable between the two hosts. Eight genera and two unknowns and 16 genera and two unknowns are suspected mycobionts of alder and pine, respectively, which show minimal ectomycorrhizal overlap or linkage. I 35 2.1 INTRODUCTION Increasingly, harvested areas and young regenerating stands are becoming common components of the BC interior's forest landscape. Within regions of the Sub-Boreal Spruce biogeoclimatic Zone, mature mixed lodgepole pine and hybrid spruce stands are sometimes replaced by mixed Sitka alder and lodgepole pi'ne under- and overstory communities. Our understanding of such aboveground secondary succession following timber harvest in temperate forests is fairly well documented (Kimmins 1997). However, knowledge of the dynamics of soil organisms following such disturbances is limited, especially the fungal and ectomycorrhizal component (Vogt et al. 1991 ). Members of the genus Alnus are capable of nitrogen fixation: actinomycetes in root nodules fix atmospheric nitrogen into organic forms . Because of this capacity, alder plays an important role in soil and site fertility where nitrogen is limited. If a common mycobiont between alder and lodgepole pine exists and transfer is plausible, this could impact current competitive theories that have been applied to post-harvest alder and pine communities whereby eradication prescriptions eliminate alder from the site. Experimental evidence has shown that nutrients pass intraspecifically as well as interspecifically via ectomycorrhizal bridges . Simard et al. (1997c) demonstrated bi-directional carbon flow occurring between field Betula papyrifera and Pseudotsuga menziesii in the field. Arnebrant et al. (1993) directly examined in vitro functional linkages between A. glutinosa and P. contorta inoculated with Paxillus involutus and found that between 5-15% of the 15 N recovered was in pine seedling tissues, suggesting a fully fUnctional ectomycorrhizal connection between the two species. Ekblad and Huss-Danell (1995) examined A. incana and P. sylvestris linked via Paxillus involutus and found that while some fixed N was found in the pine tissues , it was not statistically significant. 36 The first step in testing the linkage hypothesis is to examine the ectomycorrhizae of Sitka alder and lodgepole pine. Characterization and identification of ectomycorrhizae associated with Alnus have traditionally been done through pure culture synthesis (Ekblad and Huss-Danell 1995; Arnebrant et al. 1993; Miller et al. 1991; Godbout and Fortin 1983; Molina 1981, 1979). Other studies have characterized and attempted to identify field-sampled ectomycorrhizal tips on alder (Pritsch et al. 1997a, 1997b; Helm et al. 1996; Airaudi et al. 1993; Miller et al. 1991, 1992; Brunner et al. 1990; Froidevaux 1973; Mejstrik and Benecke 1969). Pinus contorta ectomycorrhizae have also been characterized through pure culture synthesis (Finlay et al. 1992; Molina and Trappe 1982a) and field sampling (Visser 1995; Zelmer and Currah 1995; Danielson 1991; Read et al. 1985; Danielson 1984). Rarely, however, have the host genera Alnus and Pinus been studied together to compare their respective ectomycorrhizal communities and determine the possibilities of fungal linkages in the field. Host plants such as Alnus sp. show a low degree of receptivity. Alder recognizes few fungal species with which it forms mycorrhizae (Miller et al. 1991 ). About 50 ectomycorrhizal morphotypes have been described worldwide in association with Alnus sp. (summarized by Pritsch et al. 1997a) from laboratory or greenhouse experiments. Potentially, members of the Pinaceae and Pinus, in particular, will form ectomycorrhizal associations with a wide range of fungal symbionts, numbering up to an estimated 2000 (Trappe 1962; Molina et al. 1992). An advantage of many ectomycorrhizal fungi is their capacity to form symbiotic relationships with several genera and species of trees (Molina et al. 1992). Plants sharing common fungal symbionts (physical linkage) may benefit through the exchange of nutrients and metabolites. However, because alder exhibits low receptivity, the level of linkage with other host species is presumed to be low. Miller et al. (1992) examined fungal symbionts and ectomycorrhizal types of Pseudotsuga menziesii and Alnus rubra from six Oregon forest soils using a bioassay procedure and found that, out of a total of 12 ectomycorrhizal types, only Thelephora terrestris was shared between the two hosts. 37 This chapter describes the seasonal ecology of ectomycorrhizae associated with Sitka alder and lodgepole pine from naturally regenerating young and mature Sub-boreal stands following timber harvest. Our objectives were to provide a morphological assessment of the ectomycorrh izal types (herein called morphotypes) found on both species and determine the level for fungal linkages ; to estimate the abundance of the these morphotypes on each host and determine the effect of stand age and season on these estimates; and to assess and compare ectomycorrh izal type richness (host receptivity), diversity, and evenness for alder and pine based on morphological data for both stand age and season using Shannon , Simpson , and Mcintosh indices. 2.2 MATERIAL AND METHODS 2.2.1 SITE DES IGNATIONS : THE SUB-BOREAL SPRUCE BIOGEOCLIMATIC ZONE The research sites are located within naturally regenerating young (post-winter harvested) and mature SBSdw3 stands. The SBS biogeoclimatic zone dominates the montane zone of the central interior of British Columbia from the Nechako and Fraser plateaus, the Fraser Basin, and extends into the mountainous regions on its western , northern, and eastern borders. It is located between -51 °30' to -59°N latitudes and occurs from valley bottoms to 11 00-1300m in elevation (Meidinger et al. 1991 ). Specifically, the SBSdw3 stretches westward from Prince George to the Nechako River, northwest to Stuart and lnzana Lakes, and farther westward to the west ends of Stuart and Trembleur Lakes (Delong et al. 1993). The mean elevation is 750 to 1100m. Coniferous forests dominate the SBS and climax trees species include hybrid white spruce (Picea englemannii x glauca) and subalpine fir (Abies /asiocarpa) . In drier parts of the zone, lodgepole pine is a common seral pioneer species (Meidinger et al. 1991 ). Douglas-fir is another longer lived seral species present in this zone. As the climate of the SBS is continental, seasonal conditions are extreme. Temperatures vary greatly; winters are severe and snowy while summers are warm , moist and short. Mean annual temperature of the SBS ranges from 1.7-5.0°C. On average, the temperature is below ooc 4-5 months of the year 38 and above 10°C for 2-5 months. Mean annual precipitation varies from 415-1650mm of which 25-50% is snow (Meidinger et al. 1991 ). Specifically, the SBSdw3 is warmer than other subzones and precipitation is also less with winter snowpacks accumulating to a depth of 2m. Factors limiting growth include drought and frost. Soils within the SBS are primarily luvisolic, Podzolic, and Brunisolic, and moraine and a ~t n deposits are abundant (Delong et al. 1993). Moraine deposits are associated with Gray luvisolic soils such as Brunisolic Gray luvisols while lacustrine deposits include silts to heavy clay and Gray luvisolic soils (Delong et al. 1993). Associated with regions of glaciofluvial materials are gravelly sand and Dystric Brunisols (Delong et al. 1993). At the study sites, soil textures are loam , with approximately 40% coarse fragments (Sanborn et al. 1997). 2.2.2 SITE LOCATION AND DESCRIPTION YOUNG REGENERATING STANDS Two openings (site 1 and 2), situated in block 1 (048) and 2 (049), were selected as regenerating stands at km 4.2 and 4.5 of the Bobtaii-Berta (800) Rd . in the Vanderhoof Forest District at approximately 123°44' Wand 53°41' N (Figures 2 and 3). These openings are -50ha in size, were winter logged in 1987, and left to naturally regenerate lodgepole pine from the existing seed bank. Sitka alder has primarily resprouted from existing burls (with the exception of a few new seedlings growing on exposed mineral soil) and exhibit a density of -3850 (2200-5200) clumps/ha (Sanborn et al. 1997). As most alder plants have regenerated from existing burls, their age is probably comparable to those plants in the mature stands while new plants range in age from 0-10 years . The overstory consists of both naturally regenerating lodgepole pine a maximum of -10 years old and Sitka alder (Figure 3). Some hybrid spruce are also regenerating on the site. Understory shrubs and herbaceous plants include Rubus idaeus, Clintonia uniffora, Arnica /atifolia , Orthilia secunda, Epilobium angustifolium, various species from the Asteraceae, mosses including P/eurozium schreberi, Ptilium crista-castrensis, and Rhytidiopsis robusta, and grasses such as Calamagrostis rubescens . Woody debris and charcoal were present on these sites. The aspect is predominantly west, with slopes ranging from 5-20% and a mean elevation of approximately 1030m. 39 1 Prince Forest : : , George District , Forest District Vanderhoof / I I / 0 5 ;....""'""'== 10 15 =-'--====' Scale in kilometres Figure 2 Location of research site in relation to Prince George and Highway 16 (from Sanborn et al. 1997). 40 Figure 3 Young Sitka alder (grey brush understory) and lodgepole pine regenerating stand within the SBSdw3 Figure 4 Mature Sitka alder (grey brush understory) and lodgepole pine regenerating stand within the SBSdw3. 41 Figure 5 Sampling of pine roots (contained in mats) from forest floor. Yellow arrows indicate location of mats cut relative to host tree. '• Figure 6 Alder root burl from young stand showing regrowth. Scale bar is 30cm. 2 Figure 7 Ectomycorrhizae sub-sampling showing 1 cm grids. 42 MATURE REGENERATING STANDS Two openings (site 3 and 4), situated alongside block 1 (048) and 2 (049), were also located at -km 4.2 and -km 4.5 of the Bobtail Berta (800) Forest Service Road. These mature stands are -100-140 years old, fire regenerated, and composed primarily of lodgepole pine overstory with some hybrid spruce and an old Sitka alder understory. The alder most likely regenerated at the same time as pine and was in poor vigor as shading is extensive (Figure 4 ). Vaccinium membranaceum, Rubus pedatus, and Comus canadensis dominate the understory on these sites with a ground cover of mosses such as Pleurozium schreberi and Pfilium crista-castrensis. Fallen trees in various decay classes contribute to a high woody debris presence. Charcoal is also common . 2.2.3 FIELD ECTOMYCORRHIZAE SAMPLING AND COLLECTION In June 1996, a 50m transect was placed in each of the two young sites as well as the two mature sites for a total of 4 transects. Each transect was placed arbitrarily where uniformity of alder-pine density, aspect, slope, mean elevation, exposure, and soil type were displayed. Sampling occurred twice: once in June 1996 and once in September 1996 in each of the 4 sites. In the regenerating stands, trees were selected with the following criteria: lodgepole pine and Sitka alder were 0.6-1.0 m in height and in good vigor (not chlorotic, diseased , nor parasitized). In the mature stands, trees were selected with the following criteria: lodgepole pine trees were mature (-70-120 years of age) and in good vigor and alder shrubs were vigorous with -5 living aboveground stems per clump. On all 4 sites, 10 trees each of alder and pine were sampled at 5m intervals for a total of 80. Half were harvested from each site in June (at 0, 10, 20 , 30, and 40m markings) and the remaining in September (at the 5, 15, 25, 35, 45m markings). Root systems were collected as follows for young stands: the pine and alder root systems were excavated to a radius of -20cm around the stem and a depth of -1 0-15cm to the mineral soil and were bagged . In the mature stands, root systems were collected as follows: pine roots were obtained by cutting three 30x30cm mats from the forest floor at a radius of-1m from the trunk, 120° to each other, and at a depth to the mineral soil (Figure 5). Alder roots and burls (Figure 6) were collected as in 43 young stands. All roots and mats were collected with soil, bagged, and kept cool at soc prior to morphotyping . Following processing, only 35 alder and 39 pine samples were considered satisfactory for morphological characterization . 2.2.4 ECTOMYCORRHIZAE SUB-SAMPLING Root systems and mats were soaked in tap water for 5-12 hours to loosen soil and roots, facilitating root recovery . Using frequent changes of water, soil was removed by agitating root clumps and pulling apart roots and attached soil. Pine and alder roots were gently cleaned of debris while in water to min imize stripping of fine roots . All roots attached to pine stems, alder burls and pine roots from mats (identified by dichotomously-branched tips) were collected . The number of recovered fine roots varied between samples. All roots from each sample were placed in water in trays over a 1cm 2 numbered grid (Figure 7). Two hundred live root tips were sub-sampled using a random number table for a final total of 7000 alder and 7800 pine tips. Tips chosen had characteristic healthy, light-colored apices , were turgid, and had an intact stele. 2.2.5 MORPHOLOGICAL CHARACTERIZATION Each set of 200 root tips was segregated into 3 categories: 1) mycorrhizal , 2) non-mycorrhizal , and 3) other (tips too immature with minimal mantle development to characterize). Whole mounts and squashes of root tips were used to morphologically and anatomically characterize the different morphotypes using protocols from lngleby et al. (1990a), Agerer (1987-1995), and Goodman et al. (1996) . The following characters were used to describe ectomycorrhizae (as per Goodman et al. 1996): morphology of ectomycorrhizal system , dimensions of ectomycorrhizae, texture of ectomycorrhizae , color, and anatomy of mantle in plan view. Other characters included mantle hyphae, mycelial strand hyphae, emanating hyphae, anatomy of mycelial strands in plan view, and cystidia, if present. An identity or type description of mycobiont was determined for each ectomycorrhizal type. 44 2.2.6. TERMINOLOGY MORPHOTYPE FREQUENCY, ABUNDANCE, RICHNESS, DIVERSITY, AND EVENNESS The frequency of occurrence (number of seedlings colonized by a morphotype) was calculated for each host, keeping transect, stand age, and season separate. The abundance (percent or proportion of roots colonized) was calculated for each morphotype per seedling . Mean abundance for each morphotype was also calculated for each host, keeping transect, stand age, and season separate. Morphotype richness was calculated by summing the number of morphotypes per seedling and dividing by the number of seedlings, keeping transect, stand age, and season separate . Three diversity and evenness indices were compared to describe the ectomycorrhizal community: the Simpson, the Shannon, and the Mcintosh U distance measure (Magurran 1988; Simard et al. 1997b). Each morphotype and the 'other' category were each scored as a 'species' for richness and diversity calculations. Each tip count in each morphotype and the 'other' category was scored as an individual for evenness calculations. 2.2.7 STATISTICAL ANALYSIS All abundance data were transformed to (arcsine( fa)) to satisfy ANOVA model assumptions on data distribution (Sakal and Rohlf 1981). This transformation was best able to normalize skewed data (a x2like distribution). Other transformations, fa, logarithm, natural log, and logit transformations (data not shown), were less effective. The main effects of transect, season, and stand age and associated 4 interactions on the abundance of alder and pine morphotypes were compared using 3-way ANOVA. The 3-way ANOVA including the independent transect variable was compared to the 2-way ANOVA models without the transect variable with no significant differences despite different degrees of freedom (degrees of freedom increased by four to 31 and 35 for 2-way alder and pine models respectively) . Three-way model values including the transect variable were reported to illustrate the behavior of the theorized replicated transects located in different openings (Bruno Zumbo, personal communication). Tukey honest significant difference (HSD) test were used for post hoc analyses . 45 Total ectomycorrhizal colonization for season , stand age, and host were compared using 2-Sample ttests (assuming unequal variances). Pooled variances were not used to avoid serious error. Diversity and evenness index values were compared using 3-way ANOVA indices produced similar results (see above). (Magurran 1988). Two-way AN OVA analysis of the No transformation of index values was necessary Statistical analyses were done with a significance level (a) of 0.05 using STATISTICA® for Windows, Release 5.1 B, ©1984-1996 StatSoft, Inc. All ANOVA models were evaluated (Bruno Zumbo, personal communication) by examining plots of residuals versus fits , residuals versus predictors, box-plots of standardized residuals against the predictors, histograms of residuals, and normal plots of residuals (data not shown). 2.3 RESULTS 2.3.1 MORPHOLOGICAL CHARACTERIZATION Using the morphological and anatomical features mentioned, five and thirteen ectomycorrhizal morphotypes were characterized for alder and pine, respectively. The following are keys for determination of Sitka alder and lodgepole pine morphotypes. A 1 to A5 refer to alder morphotypes (complete descriptions are in Appendix 1) and P1 -P13 refer to pine morphotypes (Appendix 2). 46 Key for Determination of Ectomycorrhizae on Alnus viridis ssp. sinuata Regel at Bobtail Site Mycorrhizae dark brown or black 2 Mycorrhizae with cystidia ~ A3 2* Mycorrhizae without cystidia ~ AS 1* Mycorrhizae not dark brown or black 3 Emanating hyphae (EH) present 4 White mycorrhizae ~ A1 4* Grey mycorrhizae ~ A2 3* EH absent ~ A4 Key for Determination of Ectomycorrhizae on Pinus contorta Dougl. ex Loud var. latifo/ia Englem. at Bobtail Site EH with clamps 2 Rhizomorphs present 3 Rhizomorphs loose-undifferentiated ~ P6 3* Rhizomorphs smooth undifferentiated to slightly differentiated ~ PS 2* Rhizomorphs lacking 4 White mantle of cottony texture ~ PS 4* Tan to orange mantle, transparent ~ P13 1* EH without clamps 5 Mycorrhizae and EH black to brown 6 EH 4(3-5)).lm wide ~ P1 6* EH 1.5(1 .5-2 .5))-lm wide ~ P9 5* Mycorrhizae and EH not black to brown 7 Rhizomorphs present 8 Rhizomorphs loose undifferentiated ~ P2 8* Rhizomorphs highly differentiated 9 Yellow mycorrhizae; dichotomous to coralloid ~ P7 9* Tan to pink to brown mycorrhizae 10 Tuberculate to subtuberculate morphology ~ P3 10* Dichotomous morphology ~ P4 7* Rhizomorphs lacking 11 Cystidia present 12 Bottle-shaped straight neck ~ P11 12* Bristly-like awl ~ P10 11 Cystidia lacking ~ P12 2.3.1.1 ECTOMYCORRHIZAL LINKAGE BETWEEN HOSTS Comparisons of our morphotypes with published morphotype descriptions show some similarities between emanating hyphae, inner and outer mantle features, and mycelial strand type with identified and non-identified mycobionts. Based on morphology and a synoptic approach, 8 genera and two unknowns are possible alder mycobionts and 16 genera and two unknowns are suspected pine 47 mycobionts (Table 4). Some genera are present on both hosts, such as Cortinarius, Hebeloma , and Laccaria and are possible mycobionts between alder (morphotype A 1) and pine (morphotypes P5 and P8) (Table 4 ). Table 4 List of suspected genera of alder and pine ectomycorrhizal mycobionts from the literature M* Alder A1 A2 A3 A4 A5 Pine P1 Suspected Mycobiont 3 7 1 Agerer and Treu 1993; M iller et al. 1991; 1997a 2 3 3 1 1 2 3 Cortinarius 2 ' , Gyrodon , 2 2 Hebeloma , Laccaria , 3 3 Lactarius , Naucoria , 2 Paxillus (cf. involutus) Cortinarius 2' , Gyrodon , 3 2 Laccaria , Lactarius , 3 Naucoria , 2 Paxillus (cf. involutus) 3 Russula , unknown Type 3 (Russula-like) and 4 (Naucoria-like) 2 Russula 3 , unknown Type 3 2 (Russula-like) unknown Type 2 and 4 (Naucoria-like) 2 Cenococcum geophilum P2 P3 P4 P5 Piloderma fa/lax Sui/Ius-like Sui/Ius-like 1 2 Cortinarius , Hebeloma , 3 4 5 lnocybe , Laccaria , Paxillus involutus P6 P7 Amphinema byssoides 6 Boletinus , Dermocybe 7, 8 Rhizopogon P8 Cortinarius , Hebeloma , 3 4 5 lnocybe , Laccaria , Paxillus (cf. involutus) Mycelium radicis atrovirens Tuber 9 10 Russula , Chroogomphus , 11 Tuber P9 P10 P11 References 1 P12 Piloderma-like P13 Unknown *M: Morphotype 2 Pritsch et al. Agerer and Treu 1993; Miller et al . 1991 ; Pritsch et al . 1997a 2 Miller et al. 1991 ; Pritsch et al. 1997a 3 2 Miller et al. 1991 ; Pritsch et al. 1997a 2 Milleretal.1991 3 Agerer and Gronbach 1988; Danielson 1991 ; Harniman and Durall 1996; lngleby et al. 1990c; Simard et al. 1997 a, b Brand 1991 a; Goodman and Trofymow 1996 Danielson 1991 ; Goodman 1996; Treu 1990a, b, c, 1993 Danielson 1991 ; Goodman 1996; Treu 1990a, b, c, 1993 1 Cuvelier and Agerer 1991 ; Brand 1992a,b; Agerer 1988a, b, 1989, 1990b. 2Danielson 1991 ; Simard et al. 1997b; 3 Treu 1990e. Beenken 1996a, b, c; lngleby et al. 1990f. 4 Brand 1988a; lngleby et al. 1990f; Simard et al. 1997b. 5 Agerer and Gronbach 1989; lngleby et al. 1990f. Danielson 1991 ; lngleby et al. 1990b; Weiss 1989 6 Agerer and Gronbach 1990; Treu 1990d, 7Agerer and Uhl 8 1989; Uhl and Agerer 1988. Agerer 1996; Simard et al. 1997b; Molina and Trappe 1994; Uhl 1988 (see P5) Dan ielson 1991 ; lngleby et al. 1990d; Simard et al. 1997b Blaschke 1988; lngleby et al. 1990e; Simard et al. 1997b 9 R. fellea (Brand 1988b), R. mairei(Brand 1991b), and R. 1 ochroleuca (Agerer 1987). °Chroogomphus helveticus 11 (Agerer 1990a). Tuber aestivum (Rauscher et al. 1996a), and Tuber sp . (lngleby et al. 1990e; Simard et al. 1997b ). Brand 1991 a; Goodman and Trofymow 1996 48 2.3.2 ECTOMYCORRHIZAE FREQUENCY AND ABUNDANCE FOR ALDER AND PINE Frequency and abundance data are shown in Table 5 and Appendix 3 for alder and Tables 6 and 7 and Appendix 4 for pine. For alder, A 1 and A2 were the most abundant morphotypes across transects, stand age, and season . Mean abundance varied between 23 .1 and 85.4% (A 1) and between 0.6 to 55.7% (A2) {Table 5). Alder morphotypes A3 to A5 were all less abundant with mean abundance values between 0 and 15.5% (A3), 0 and 13.4% (A4), and 0-1.8% (A5) (Table 5). A5 was only found in one mature stand in the fall sampling whereas all others (A 1-A4) were variously found at both young and mature alder sites in June and September (A3 missing in June sampling from the mature site). Non-mycorrhizal tips were more abundant in the spring (28-42 .9%) than fall (4 .5-22.9%) (Table 5). For pine samples, P2, P8, and P1 0 were the most abundant morphotypes across transects, stand age, and season ; mean abundance varied between 0-42%, 0-41.4%, and 1.6-35% respectively. P1 and P7 morphotypes had intermediate abundance with mean values between 0.4-18.1% {P1) and 0.030 .1% (P7) {Tables 6 and 7). The remaining 8 morphotypes were less abundant across all variables with most mean abundance values less than 10%. P1 to P3 and P6-P12 (a total of 10) were found on both young and mature pine in both June and September (P11 was absent on young pine sampled in September). Morphotypes P4, P5, and P13 were seen on both young and mature pine but only in fall samples (Tables 6, 7, and Appendix 4). Mean abundance of P2 varied bimodally between young (012.1%) and mature (28 .0-42%) sites. Non-mycorrhizal roots did not vary greatly between sites, stand age, and season (0.7-8.5% mean abundance). 49 Table 5 Number of plants colonized by morphotype, number with <5%, mean abundance (±standard error (SE)) for Sitka alder from 4 sites and 2 stand ages (young and mature) sampled in June and September Morphotype No. plants colonized A1 A2 5 2 3 1 Mean No. plants No. plants No. plants colonized colonized abundance colonized (%) (SE) of with 0.1-5% with 0.1-5% colonization colonization morphotype per transect June Sampling Site 1: Young (n-5) 32.4 (10.0) 11.9(10.7) 15.5 (9 .0) 1 0.8 (0.8) 4 2 Mean abundance (%) (SE) of morphotype per transect Site 2: Mature(n=4) 37.8 (9 .3) 2 11 .9 (6 .9) A3 A4 A5 NonMycorrhizal Other 5 30.9 (7.1) 4 42 .9 (6 .3) 4 4 A1 4 8.4 (3 .9) 2 Site 3: Mature (n-4) 43.6 (8 .9) 6.3 (6.3) 5 5 6.4 (0 .9) Site 4: Mature (n-5) 1 23 .1 (8 .2) 37 .2 (6.4) 2 2.9 (2.2) 2 2.3 (1.4) 4 28.0 (29) 5 32.4 (8.4) A2 A3 A4 A5 NonMycorrhizal Other A1 A2 A3 A4 A5 NonMycorrhizal Other A1 A2 A3 A4. A5 NonMycorrhizal Other 4 5 5 0 0 0 5 5 5 3 1 2 4 5 5 1.0(1 .0) 2 19.1 (10.6) 4 September Sampling Site 1: Young (n-5) 30 .7 (11 .5) 1 5 37 .7 (7.4) 5 2 2 22.9 (8.5) 5 8.7 (2.7) 2 Site 3: Mature (n=5) 57 .5 (13 .6) 17.6 (12 .9) 0.1 (0.1) 13.4 (10.4) 4 1.8 (0.7) 3 4.5 (1 .6) 4 2 5.1 (1 .1} 50 3 5.1(1 .8) Site 2: Mature (n-5) 31 .3 (7 .7) 55.7 (7.5) 1 1.9(1 .5) 2 0.8 (0.5) 3 6.0 (2.0) 2 1 2 4.3 (1 .7) Site 4: Mature (n=5) 85.4 (0 .5) 0.6 (0 .6) 2 9.5 (1 .8) 2 4.5 (0 .6) \ Table 6 Number of plants (plant or combined mats) colonized by morphotype , number with <5% , mean abundance (±standard error (SE)) for lodgepole pine from 4 sites and 2 stand ages (young and mature) sampled in June Morphotype No. plants colonized P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 NonMycorrhizal Other P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 NonMycorrhizal Other 1 2 5 No. plants colonized with 0.1-5% colonization Mean No. plants No. plants abundance colonized colonized with 0.1-5% (%) (SE) of morphotype colonization per transect June Sampling Site 1: Young (n=5) 1 0.4 (0.4) 1 2.1 (1.7) 1 9.4 (4.3) 1 3 5 1 2 3 2 1 1 5 3 0.4 (0.4) 15.7 (10.6) 41.4 (7.4) 0.9 (0 .9) 1.6 (1 .1) 3.3 (2.1) 3.8 (2.4) 1 2 Site 2: Young (n=4) 4.2 (4.2) 12.1 (12 .1) 3.7 (3 .1) 1 4 1 2 2 8.7 (4.9) 4 2 4 5 5 2 12.3 (5.3) Site 3: Mature (n-5) 8.1 (2 .0) 1 30.8 (9.9) 1.8 (1.6) 2 1 3 1 5 2.2 (1 .8) 0.9 (0.9) 6.9 (4.6) 0.8 (0.8) 35.0 (12.5) 5 5 5 3 4 5 2 1.1 (1 .1) 2 2 4 2 4 2 2 3.3 (0 .9) 5 9.1(1 .8) 5 51 Mean abundance (%) (SE) of morphotype per transect 4.5 (4.5) 40.5 (9.6) 6.4 (6.4) 1.7(1 .1) 10.1 (10.1) 8.6 (2 .8) 8.2 (2 .5) Site 4: Mature (n=5) 1 9.5 (4.1) 42 .0 (8 .9) 2 1.3 (0.9) 2 1 2 2 3 1.0 (0.6) 5.4 (3 .5) 9.0 (2 .6) 2.7 (1.9) 10.8 (4.9) 1.1 (1 .0) 3.0 (1 .9) 4.5 (1 .6) 9.7 (1 .3) Table 7 Number of plants (plant or combined mats) colonized by morphotype, number with <5% , mean abundance (±standard error (SE)) for lodgepole pine from 4 sites and 2 stand ages (young and mature) sampled in September Morphotype P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 Nonmycorrhizal Other P1 P2 P3 P4 P5 P6 P7 P8 pg P10 P11 P12 P13 Non. mycorrhizal Other No. seedlings colonized 3 3 1 No. seedlings colonized with 0.1-5% colonization No. Mean No. seedlings abundance seedlings colonized (%) (SE) of colonized morphotype with 0.1-5% per transect colonization Fall Sampling Site 1: Young (n=5) 1 5.4 (3.2) 2 7.6 (5.4) 6.5 (6 .5) 4 2 2 4 30.1 (9.5) 15.7 (13.3) 3.0 (1.9) 11 .5 (3 .5) 2 1 5 6.4 (4.2) 2.1 (2 .1) 5.3 (1.2) 2 Mean abundance (%) (SE) of morphotype per transect 5 3 2 4 1 1 Site 2: Young (n=5) 11 .3 (2 .3) 5.3 (2 .8) 3.5 (2 .1) 10.3 (3 .0) 0.7 (0 .7) 1.7 (1 .7) 3 2 3 16.9 (7.8) 5.9 (4.0) 12.0 (5.3) 4 1.1 (1 .1) 14.4 (14.4) 3.9 (1 .5) 3 3 5 6.4 (2 .1) Site 3: Mature (n-5) 18.1 (7.8) 28 .0 (5.8) 1 2 2 1 1.5(1 .5) 2.7 (1.9) 7.7 (5.2) 3.2 (3.2) 1 2 1 1 2 4 1 4 4.4 (4.4) 18.4(11 .6) 0.7 (0.7) 1.0(1.0) 7.8 (7 .2) 1.9 (0 .6) 1 3 3 9.6 (9 .6) 0.7 (0.3) 5 3 4.6 (0.6) 5 4 3.8 (0 .5) 5 52 4 5 1 1 13.2 (3 .6) Site 4: Mature (n=5) 13.8 (4.9) 28 .6 (4.0) 0.2 (0.2) 0.3 (0 .3) 3 1 3 1 4 1 6.8 (3 .0) 4.3 (4.3) 5.4 (2 .7) 1.3 (1 .3) 21 .6 (7 .8 3.5 (3.5) 5 i ! Three-way ANOVA analysis showed significant differences in abundance of alder morphotypes with respect to age and season (main effects) but no difference between transects (sites) (Table 8) . Morphotype A 1 was significantly more abundant in the mature stands in September but not in June while morphotype A2 was significantly more abundant in young stands. showed no signi,ficant difference in abundance. A3 and A4 morphotypes Residual analysis shows limitations of ANOVA modeling for morphotype AS (low sample size). The 'Non-mycorrhizal' category was significantly greater in June for both young and mature alder. Significant stand age*season interactions occurred for morphotypes A1 and A2 (p=0.011 and 0.001 , respectively). Significant transect*stand age*season interactions occurred for morphotypes A2, A3, and the non-mycorrhizal category (p= 0.022, 0.047, and 0.048, respectively}. Table 8 Statistical summation (3-way ANOVA) of main effects (mean (x}, standard deviation (SO), and probability, a=0.05) and interactions effects of Sitka alder morphotype abundance. N= 35. M* Transect (T} Block 1 x (SO) Block 2 x (SO) p value A1 40 .5 (0 .36) 44 .0 (0.49) 0.721 10.3 (0.39) A2 19.2 (0.49) 0.203 A3 0.6 (0.10) 0(0 .13) 0.237 A4 1.0 (0.15} 0.3 (0.20) 0.423 A5 0.1 (0 .01) 0 (0.01) 0.012 Non19.1 (0.12) mycorrhizal 20 .1 {0 .17} 0.831 7.3 (0 .15) Other 4.4 (0 .20) 0.304 M*: Morphotype MAIN EFFECTS Season (S) Age (A) Young x (SO) June x (SO) Mature x (SO) Sept. x (SO) p value p value 32.4 (0.38) 31 .5 (0 .36) 53.9 (0.49) 52 .8 (0.49) 0.031 0.048 22.4 (0 .39) 9.4 (0.42) 8.1 (0.53) 20 .5 (0 .51) 0.045 0.116 0.6(0 .11) 0.9 (0.10) 0(0 .13) 0.1 (0.13) 0.063 0.297 0.1 (0.15} 0.6 (0.16) 1.3 (0 .13) 0.5 (0.19) 0.974 0.206 0 (0 .01) 0 (0 .01) 0.1 (0.01) 0.1 (0.01) 0.012 0.012 23 .1 (0 .12) 33.5{0 .13) 16.3 (0 .17) 8.9(0 .16) 0.000 0. 128 6.7 (0 .15} 5.4 (0 .15) 4.9 (0 .19) 6.1 (0 .20) 0.531 0.798 53 INTERACTIONS T*S A*S T*A*S p value p value p value p value 0.854 0.152 0.011 0.100 0.966 0. 116 0.001 0.022 0.297 0.063 0.237 0.047 0. 179 0.467 0.801 0.273 0.012 0.012 0.012 0.012 0.234 0.183 0.795 0.048 0.684 0.927 0.660 0.232 T*A Three-way ANOVA showed significant differences in abundance of pine morphotypes with respect to block, age, and season (main effects) (Table 9). Morphotype P1 was significantly more abundant in the mature stands than in the young stands and in September than in June. Morphotypes P2, P6, P1 0 were more abundant in mature stands in both seasons (p<0.05) . Morphotypes P3 and P8 (both in June) and P4 (in September) were significantly more abundant in young stands. P7 showed the only significant block effect (p<0.05); it was most abundant in block 1 and showed a significant transect*stand age interaction (p<0.007) . P4 showed significant stand age*season interaction (p<0.01}. All other morphotypes showed no significant main effects or interactions . Non-mycorrhizal roots were significantly greater in young stands over mature stands sampled in June than in September. When examining total root colonization for alder and pine, differences were seen with respect to stand age and season as well as between hosts (Tables 10, 11, 12). Mycorrhizal colonization for alder in young and mature stands was similar; pooled values (young and mature) were significantly greater in September (p<0.0001) than in June (Table 10). Total colonization of pine was greater for mature than young stands in September (p=0.007) while no difference was seen in June (Table 10). Between host comparisons of total colonization (pooled) shows greater mycorrhizal colonization by pine in both young and mature stands (Table 11 ); as well as by pine in both June and September (p=0.05) (Table 12). 54 Table 9 Statistical summation (3-way ANOVA) of main effects (mean ( x), standard deviation (SO), and probability, a=0.05) and interactions effects of lodgepole pine morphotype abundance. N= 39 . M* P1 Transect (T) Block 1 x (SO) Block 2 x (SO) p value 4.3 (0.18) 6.6 (0.19) 0.398 P2 8.7 (0 .20) 15.5(0.21) P3 0.7 (0.07) 0.7 (0.08) P4 0.4 (0.07) 0.6 (0.07) P5 0.3 (0.07) 0 (0.07) P6 0.5 (0.10) 0.7 (0 .10) P7 5.6 (0.26) 0.8 (0.27) 0.049 8.0 (0 .29) 12.9 (0 .30) P8 0. 115 0.969 0.686 0.211 MAIN EFFECTS Age (A) Young x (SO) Mature x (SO) p value 2.6 (0.19) 9.1 (0.18) 0.022 1.3 (0 .21) 31 .5 (0 .20) 0.000 1.6 (0 .08) 0.2 (0.07) 0.037 1.5 (0 .07) 0 (0.07) 0.010 0.2 (0.07) 0.1 (0.07) 0.714 0.863 0.0 (0.10) 1.8(0 .10) 0.017 5.4 (0.27) 0.9 (0.26) 0.301 22.5 (0 .30) 2.5 (0.29) 0.000 1.2 (0.15) 0.5 (0.14) P9 0.5 (0.14) 1.2 (0.15) P10 10.2 (0.33) 7.1 (0 .35) P11 0.500 0.062 0.496 INTERACTIONS T*S A*S T*A*S p value p value p value p value 0.273 0.698 0.335 0.740 0.587 0.845 0.865 0.484 0.865 0.080 0.369 0.158 0.401 0.686 0.010 0.401 0.086 0.899 0.211 0.714 0.899 0. 149 0.859 0.436 0.269 0.845 0.736 19.9 (0.30) 3.6 (0.29) 0.007 0.156 0.668 0.420 0.534 0.573 0.111 0.961 0.654 0.592 0.489 0.707 0.787 0.460 0.579 0.209 0.083 0. 129 0.222 0.746 0.768 0.061 0.849 0.865 0.140 0.574 0.260 0.502 0.061 0.635 . 0.669 0.882 0.635 0.930 0.222 0.766 0.937 0.575 0.274 0. 131 0.107 Season (S) June x (SO) Sept. x (SO) p value 2.9 (0 .19) 8.6(0.18) 0.041 14.1 (0.21) 9.9 (0.20) 0.324 1.8 (0.08) 0.1 (0.07) 0.017 0 (0.07) 2.0 (0.07) 0.001 0.0 (0.07) 0.4 (0 .07) 0.2 (0.10) 1.2 (0.10) 2.3 (0.27) 3.1 (0 .26) 0.001 0.6 (0.15) 1.1 (0.14) 0.2 (0.11) 0.6 (0 .11) 3.5 (0.35) 15.8 (0 .33) 0.012 0.6 (0.11) 0.2 (0.11) 0.8 (0 .11) 0.1 (0 .11) P12 0.9 (0 .09) 0.2 (0.09) 0.7 (0 .09) 0.3 (0 .09) 0.5 (0.09) 0.4 (0.09) P13 0.3 (0.27) 0.8 (0 .29) 0.5 (0.29) 0.6 (0 .27) 0 (0.29) 2.1 (0.27) Nonmycorrhizal 3.8 (0 .05) 3.3 (0 .05) Other 7.2 (0 .05) 8.0 (0 .05) 5.6 (0.05) 2.0 (0.05) 0.003 8.9 (0 .05) 6.4 (0.05) 0.134 5.3 (0.05) 2.1 (0 .05) 0.009 9.2 (0.05) 6.2 (0.05) 0.509 0.420 0.197 0.669 0.627 0.655 M*: Morphotype 0.538 0.445 0.882 7.0 (0.35) 10.4 (0.33) 0.074 55 T*A Table 10 Total mycorrhizal colonization (mean %colonization of total live tips) for Alder and Pine with respect to stand age and season . Statistical analysis using t-test (two sample assuming unequal variances) within each column ; a=0.05 Young Mature P(T ~ t) two-tail Pooled Age P(T ~ t) one-tail ALDER June September % (SE) % (SE) 63.64 (4.96) n=9 85.56 (4.97) n=10 69.54 (4.62) n=9 94.07 (1.48) n=7 0.3972 0.1293 66.59 (3.37) n=18 89.06 (3.10) n=17 <0.0001 PINE June September % (SE) % (SE) 91.33 (2 .82) n=9 95.37 (0.94) n=1 0 96.10 (0.89) n=1 0 98.70 (0.37) n=10 0.138 0.007 n/a n/a Table 11 Total mycorrhizal colonization (mean% colonization of total live tips) with respect to stand age between hosts. Alder Pine P(T ~ t) two-tail STAND AGE (Pooled) Summer Fall % (SE) % (SE) 66.59 (3 .37) n=18 89 .06 (3.10) n=17 93.84 (1.48) n=19 97 .04 (0.62) n=19 <0.0001 <0.0001 Table 12 Total mycorrh izal colonization (mean %colonization of total live tips) with respect to season between hosts. Alder Pine P(T ~ t) two-tail SEASON (Pooled) Young Mature % (SE) % (SE) 75.18 (4.28) n=19 80.27 (4.08) n=16 93.46 (1.46) n=19 97.40 (0.56) n=19 <0.001 <0.001 2.3.3 MORPHOTYPE RICHNESS, DIVERSITY, AND EVENNESS Between the two hosts, nearly 3 times as many morphotypes were characterized for pine (13 morphotypes) as for alder (5 morphotypes). Three-way ANOVA showed no significant main effects (transect, stand age, and season) on morphotype richness for either host; richness did not significantly vary between blocks, stand ages, nor seasons . Two-sample t-testing showed significantly 56 greater morphotype richness for pine (mean richness 5.512 ± 0.31SE) over alder (mean richness 3.019 ± 0.19SE) (p0.05) were observed {data not shown) Of the three models used, diversity values were greatest in the Mcintosh, second in the Shannon , and lowest in the Simpson for both hosts {Tables 14 and 15). No significant differences in diversity or evenness indices were found between blocks for either alder or pine {Tables 14 and 15). Both Simpson and Shannon indices showed significantly greater diversity in young alder stands sampled in June while the Mcintosh index showed higher diversity for mature alder stands sampled in September (Table 14). The mean ± SE (range) of alder ectomycorrhizal diversity was 0.55 ± 0.23 (0 .23-0.72), 1.00 ± 0.04 (0.51-1.38), 66 .11 ± 1.60 (52 .78-87.57), respectively, for the Simpson, Shannon, and Mcintosh indices. The mean ± SE (range) of pine ectomycorrhizal diversity was 0.70 ± 0.02 (0 .370.86), 1.46 ± 0.05 (0.83-2.04), and 54.43 ± 1.57 (36 .93-79 .69), respectively, for the Simpson , Shannon , and Mcintosh indices. Alder showed significant stand age*season interactions for diversity across ihdices {Table 14). No significant main effects or interactions using any diversity index were found for pine (Table 15). Pine diversity values were higher than alder for both the Simpson , and the Shannon indices , but not for the Mcintosh . 57 Evenness values increased from the Simpson to the Shannon to the Mcintosh for both hosts (Tables 14 and 15). The mean± SE (range) of mean alder evenness was 0.08 ± 0.001 (0 .033-0 .103), 0.51 ± 0.02 (0 .26-0 .71 ), and 0.54 ± 0.03 (0.20-0.76), respectively, for the Simpson , Shannon , and Mcintosh indices. The mean± SE (range) of pine evenness was 0.046 ± 0.001 (0 .024-0 .058), 0.54 ± 0.02 (0.310.75), and 0.64 ± 0.02 (0.29-0.89) respectively. Young alder stands had greater evenness values than mature stands and June alder samples showed higher evenness values than September samples for all indices (p "' . ~ :a ~~ 1:' ~~~ H lj ~ ~~ "' ':! 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U1 :I ....()" CD Ill cCi" c ~ Ill APPENDIX 9: UN ROOTED RADIAL CLADOGRAM FROM SITKA ALDER AND LODGEPOLE PINE RFLP PATTERNS 193 .. 'I I I - : I' ' 1 , , I' : Pil