INFORMATION TO USERS This manuscript h a s b een rep ro d u ced from the microfilm m aster. UMI films the text directly from the original or copy submitted. Thus, som e th esis a n d dissertation copies a re in typew riter face, while others may be from any ty p e of com puter printer. T he quality of th is re p ro d u c tio n is d e p e n d e n t upon th e qu ality o f th e c o p y subm itted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, a n d im proper alignment can adversely affect reproduction. In the unlikely event that th e a u th o r did not send UMI a com plete m anuscript and there are missing p a g e s, th e s e will b e noted. Also, if unauthorized copyright material had to b e rem oved, a n o te will indicate the deletion. Oversize materials (e.g., m ap s, draw ings, charts) are rep ro d u ced by sectioning the original, beginning a t th e u p p e r left-hand corner an d continuing from left to right in equal sectio n s with sm all overlaps. ProQ uest Information a n d Learning 300 North Z eeb R oad, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMI* NOTE TO USERS This reproduction is the best copy availabie. UMI’ THE ECTOMYCORRHIZAL ASSOCIATIONS OF LARIX lARICINA (DU ROI) (TAMARACK) K. KOCH AND BETULA GLANDULOSA MICHAUX (SCRUB BIRCH) SEEDLINGS IN PEATLANDS OF CENTRAL BRITISH COLUMBIA by Jennifer M. Catherail B.Sc., University of Northern British Columbia, 2001 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in NATURAL RESOURCES AND ENVIRONMENTAL STUDIES © Jennifer M. Catherail, 2004 THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA April, 2004 1^1 Library and Archives Canada Bibliothèque et Archives Canada Published Heritage Branch Direction du Patrimoine de l'édition 395 W ellington Street Ottawa ON K 1A 0N 4 Canada 395, rue W ellington Ottawa O N K 1A O N 4 Canada 0-494-04677-5 Your file Votre référence ISBN: 0-596-00283-1 Our file Notre référence ISBN: 0-596-00283-1 NOTICE; The author has granted a non­ exclusive license allowing Library and Archives Canada to reproduce, publish, archive, preserve, conserve, communicate to the public by telecommunication or on the Internet, loan, distribute and sell theses worldwide, for commercial or non­ commercial purposes, in microform, paper, electronic and/or any other formats. AVIS: L'auteur a accordé une licence non exclusive permettant à la Bibliothèque et Archives Canada de reproduire, publier, archiver, sauvegarder, conserver, transmettre au public par télécommunication ou par l'Internet, prêter, distribuer et vendre des thèses partout dans le monde, à des fins commerciales ou autres, sur support microforme, papier, électronique et/ou autres formats. The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. L'auteur conserve la propriété du droit d'auteur et des droits moraux qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation. In compliance with the Canadian Privacy Act some supporting forms may have been removed from this thesis. Conformément à la loi canadienne sur la protection de la vie privée, quelques formulaires secondaires ont été enlevés de cette thèse. While these forms may be included in the document page count, their removal does not represent any loss of content from the thesis. Bien que ces formulaires aient inclus dans la pagination, il n'y aura aucun contenu manquant. Canada ABSTRACT Peatlands are habitats where peat accumulation exceeds decomposition, resulting in poorly drained, nutrient-poor and acidic soils. Tamarack {Lxirix lariciiia, family Pinaceae), a deciduous conifer, and scrub birch {Betula glandulosa, family Betulaceae), a low-lying deciduous shrub, are two plant species well adapted to the cold climates and short growing seasons of central British Columbia and generally able to tolerate the wet, poorly drained soils of peatlands. Ectomycorrhizas are mutualistic associations formed between plant roots and symbiotic fungi; ectomycorrhizal fungi that facilitate nutrient acquisition and water uptake in exchange for host carbon, may play an important role in the survival of these species. This study characterized tamarack and scrub birch ectomycorrhizas in three different peatland habitats using microscopy) and molecular analysis (PCR-RFLP) morphological methods. (light Ectomycorrhizal morphotypes and corresponding genotypes (fragment patterns) are described and ideas of host and peatland site specificity are explored. Results suggest that ectomycorrhizal colonization in peatland habitats may be similar to that for other hosts in other habitat types. Both morphology and molecular results indicate a high potential for ectomycorrhizal fungal linkages between hosts. This study presents the first published information on ectomycorrhizal associations of scrub birch. TABLE OF CONTENTS Abstract................................... ii Table of contents........................................................................................................................... iii List of Tables.................................................................................................................................. vi List of Figures...............................................................................................................................viii Acknowledgements........................................................................................................................ ix In tro d u ctio n .................................................................................................................................... 1 L iteratu re Review Wetlands of British Columbia...................................................................................................... 5 Ecology and descriptions......................................................................................................... 5 Mycorrhizal symbiosis................................................................................................................... 7 Definition and structure............................................................................................................7 Functions and benefits........................................................................................ 8 Fungal mycelial networks.......................................................................................................10 Mycorrhizas in wetland systems........................................................................................... 11 Ectomycorrhizal diversity.......................................................................................................12 Methods for measuring ectomycorrhizal diversity.................................................................. 13 Sporocarp surveys and seedling sampling...........................................................................13 Microscopy and ectomycorrhizal characterization............................................................ 14 Molecular techniques.............................................................................................................. 15 Measures of ectomycorrhizal diversity................................................................................ 17 Tamarack {Larix laricina)................. 19 Distribution and ecology.........................................................................................................19 Identified fungal symbionts...................................................................................................21 Scrub birch {Betula glandulosa).................................................................................................22 Distribution and ecology........................................................................................................ 22 Identified fungal symbionts...................................................................................................24 Literature cited...............................................................................................................................25 M orphological ch aracterizatio n o f ectom ycorrhizal associations o f Larix laricina (Du Roi) (tam arack) K. Koch an d Betula glandulosa M ichaux (scrub birch) in peatlands of central B ritish C olum bia Abstract........................... ;..............................................................................................................36 Introduction............................................ ;......................................................................................37 M ethods...........................................................................................................................................40 Site descriptions.......................................................................................................................40 Seedling sarhpling regim e...................................................................................................... 44 Vegetation plot analysis and sporocarp sampling.............................................................. 44 Morphological characterization of ectomycorrhizas.........................................................45 Statistical analysis of morphological data........................................................................... 46 Results.............................................................................................................................................47 Ectomycorrhizal richness, frequency, and abundance...................................................... 47 Scrub birch {Betula glandulosa)..................................................................................... 51 111 Tamarack {Larix laricina) ectomycorrhizas..................................................................53 Ectomycorrhizal community diversity................................................................................. 58 Discussion...................................................................................................................................... 60 Ectomycorrhizal frequency and abundance........................................................................ 60 Tamarack (Larix laricina)................................................................................................ 63 Scrub birch (Betula glandulosa)........................................................................... 65 Morphotype frequency and abundance by peatland site type...........................................68 Potential for shared fungal symbionts.................................................................................. 68 Ectomycorrhizal diversity...................................................................................................... 70 Literature cited................................................................................................................. 73 M olecular analysis of ectom ycorrhizal associations of Larix laricina (Du Roi) (tam arack) K. Koch and Betula glandulosa M ichaux (scrub birch) in peatlands of central B ritish C olum bia Abstract...........................................................................................................................................77 Introduction....................................................................................................................................78 M ethods.......................................................................................................................................... 80 Ectomycorrhizal root tip selection and DNA extraction.................................................. 81 DNA amplification and restriction endonuclease digestion............................................82 Molecular analysis................................................................................................................. 83 Results.............................................................................................................................................86 Amplification and digestion success rates......................................................................... 86 Phylogenetic analysis of ectomycorrhizal root tips.......................................................... 86 Genotype distribution within peatland site types.............................................................. 90 Ectomycorrhizal fragment pattern comparison between tamarack and scrub birch...97 Ectomycorrhizal fragment pattern comparison with black spruce (Picea mariana)...91 Phylogenetic analysis of sporocarps................................................................................. 101 Molecular diversity within ectomycorrhizal morphotypes............................................105 Peatland site type effects on ectomycorrhizal diversity ........................................ 106 Discussion.................................................................................................................................... 107 Ectomycorrhizal genotypes, host specificity, and site distribution..............................107 Ectomycorrhizas: Intraspecific variation....................................................................... I l l Potential linkages between tamarack, scrub birch, and black spruce......................... 114 Sporocarp and ectomycorrhizal genotype comparison................................................. 115 Challenges with genotype classification..........................................................................117 Literature Cited........................................................................................................................... 118 C onclusions.................................................................................................................................121 A ppendix I. Map of study area showing approximate locations (indicated by rectangle) of the six peatland sites in the Prince George Forest District in central British Columbia...................................................................................................................................... 123 A ppendix II. Plant species list of vegetation growing within four 1 m x 1 m plots in each of the Mix (scrub birch-tamarack-black spruce), BsLt (scrub birch-tamarack), and ■Bs (scrub birch) peatland site types......................................................................................... 124 IV Appendix III. Descriptions of tamarack (Lt) and scrub birch (Bs) ectomycorrhizal morphotypes from Mix (scrub birch-tamarack-black spruce), BsLt (scrub birchtamarack), and Bs (scrub birch) peatland sitetypes...............................................................125 Appendix IV. Unrooted phylogram generated from restriction fragment patterns of tamarack ectomycorrhizal morphotypes. Phylogram shows the relationship between morphotypes and peatland site types.......................................................................................133 Appendix V. Unrooted phylogram generated from restriction fragment patterns of scrub birch ectomycorrhizal morphotypes. Phylogram shows the relationship between morphotypes and peatland site types....................................................................................... 134 LIST OF TABLES Table 1.1. Identified mycorrhizal symbionts of Larix laricina, L. decidua, and L. occidentalis.....................................................................................................................................23 Table 1.2. Identified mycorrhizal symbionts o f Betula glandulosa, B. pendula and B. nana................................................................................................................................................25 Table 2.1. Summary of replicate peatland sites and number of plants sampled for two hosts, tamarack and scrub birch..................................................................................................41 Table 2.2. Mean number of ectomycorrhizal morphotypes (SE in parentheses) for tamarack and scrub birch seedlings growing in three peatland site types; Bs (birch dominated), BsLt (scrub birch-tamarack), and Mix (scrub birch-tamarack-black spruce).............................................................................................................................................48 Table 2.3. Site effect, percent abundance (mean ±SE in parentheses) and frequency of occurrence (%) of ectomycorrhizal morphotypes of scrub birch growing in three peatland site types..........................................................................................................................................52 Table 2.4. Site effect, percent abundance (mean ±SE in parentheses) and frequency of occurrence (%) of ectomycorrhizal morphotypes of tamarack growing in two peatland site types..........................................................................................................................................54 Table 2.5. Two-way ANOVA showing site (BsLt and Mix), host (scrub birch and tamarack) and interaction effects based on mean percent abundance of 15 shared ectomycorrhizal morphotypes (a = 0.05, df = 1, 4 2 ).............................................................. 56 Table 2.6. One-way ANOVA showing site (BsLt and Mix) differences for percent abundance (mean ±SE) of 15 shared ectomycorrhizal morphotypes (a = 0.05, df = 1, 4 4 )................................................................................................................................................... 57 Table 2.7. One-way ANOVA showing host (tamarack and scrub birch) differences for percent abundance (mean ±SE) of 15 shared ectomycorrhizal morphotypes (a = 0.05, df = 1 ,4 4 )...........................................................................................................................................58 Table 2.8. One-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) comparing peatland site types for scrub birch (a = 0.05, df = 2, 31)................................................................................................................................................... 59 Table 2.9. One-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) comparing peatland site types for tamarack (a = 0.05, df = 1, 22)................................................................................................................................................... 59 VI Table 2.10. Two-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) showing comparison between peatland site types (BsLt and Mix), host (tamarack and scmb birch), and interaction effects (a = 0.05, df = 1,42)........60 Table 2.11. One-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) for combined host species showing comparison between two peatland site types (a = 0.05, df = 1, 44).................................................................................. 60 Table 3.1. Summary of ectomycorrhizal root tip DNA amplification (PCR) and digestion (RFLP) success rates (%) from tamarack and scrub birch seedlings.................................... 87 Table 3.2. Approximate fragment sizes of the amplified ITS region for ectomycorrhizas from tamarack (Lt) and scrub birch (Bs) seedlings occurring in three peatland site types (scrub birch dominated (B), scrub birch and tamarack (L), and scrub birch, tamarack, and black spruce (M )).......................................................................................................................... 92 Table 3.3. Approximate fragment sizes of the amplified ITS region of ectomycorrhizas that were potentially shared between hosts (scrub birch (Bs), tamarack (Lt), and black spruce (Sb))....................................................................................................................................99 Table 3.4. Approximate fragment sizes (bp) of the amplified ITS region for sporocarps collected in Mix, BsLt and Bs peatland site types.................................................................102 Table 3.5. Approximate fragment sizes (bp) of the amplified ITS region for sporocarps and for closest ectomycorrhizal match. Samples originated from the Mix, BsLt, and Bs peatland site types....................................................................................................................... 104 Table 3.6. Phi diversity values for commonly occurring and shared (those found on both host species) ectomycorrhizal morphotypes on tamarack and scrub birch........................106 Table 3.7. Two-way ANOVA showing site (BsLt and Mix), host (tamarack and scrub birch), and interaction effects based on Phi values for ectomycorrhizal genotypes (a = 0.05) 107 Table 3.8. One-way ANOVA showing Phi diversity values (mean ±SE) for ectomycorrhizal genotypes originating from tamarack and scrub birch from three peatland site types (a = 0.05)................ :.................................................................................. 107 vu LIST OF FIGURES Figure 2.1. Photographs showing the three peatland site types in central BC selected for this study, local vegetation, and fungi. A) Bs peatland site type of scrub birch. (B) BsLt peatland site type of scrub birch and tamarack. (C) Mix peatland site type of scrub birch, tamarack and black spruce. (D) Sphagnum covered hummock in peatland with tamarack seedling. (E) scrub birch {Betula glandulosa). (F) buckbean {Menyanthes trifoliata). (G) larch suillus {Suillus grevillei)....................................................................................................43 Figure 2.2. Bar graph showing comparison of ECM morphotype abundance between Mix (scrub birch-tamarack-black spruce), BsLt (scrub birch-tamarack) and Bs (scrub birch) peatland site types for both tamarack and scrub birch hosts ........................... 49 Figure 2.3. Photographs showing ectomycorrhizal morphotypes from tamarack and scrub birch. A, B, C, D, E, and F ectomycorrhizas on scrub birch, and G, H, and I ectomycorrhizas on tamarack. (A) Tomentella-Wke 2 outer mantle (OM). (B) Tomentella-\ike 2 ectomycorrhizal root tip. (C) E-strain OM with enlarged hyphal cells. (D) Lactarius ectomycorrhiza. (E) Lactarius OM with laticifers. (F) Lactarius root showing crystal-like deposits. (G) Suillus 2 OM. (H) TomentellaAike 1 ectomycorrhizal root tip. (I) Tamentella-Wke 1 O M ................................................... 50 vm ACKNOWLEDGEMENTS I would like to thank many individuals who provided both guidance and encouragement throughout this project. My supervisor, Dr. Hugues Massicotte, opened my eyes to the forest around me, especially the underground world beneath my feet. His confidence in my abilities as both a student, and as a researcher, supported me throughout both my undergraduate and graduate degrees. Dr. Keith Egger, Dr. Art Fredeen, and Dr. Chris Hawkins, have shared their extensive knowledge in molecular biology, tree physiology, and resource management, both as instructors, and as committee members. Many of my fellow lab-mates offered their shoulder to lean on, as well as an ear to listen, throughout this project, especially Susan Robertson who helped to pave the way into the world of peatland ecosystems and provided invaluable support and information. I would also like to thank Linda Tackaberry for her morphotyping expertise, seedling washing patience, and editorial skills. I am also grateful to Cameron Grose, for his patience, knowledge, and help during the memorable months using genetic software, as well as Carmen Holshuh, for her peatland photographs. I want to express my thanks to Brad Eckford, for tolerating the use of the word “ectomycorrhiza” during dinner conversation and for his help during the field season. Finally, I would have never had the courage to pursue this academic path without the never-ending support and encouragement of my parents, Roy and Sandra Catherail. In addition, this study would not have been possible without the financial support provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) through a research grant to Dr. Massicotte, as well as the University of Northern British Columbia, with travel grants, laboratory facilities, and field vehicles. ix . INTRODUCTION The wetland ecosystems of the interior of British Columbia present a challenging environment for many of the plants that occupy them. Efficient conservation and acquisition of nutrients, as well as tolerance of fluctuating water tables, may be advantageous traits for plant species in order to survive, grow, and reproduce in these often nutrient deficient, poorly drained environments. Peatlands, specifically bogs and fens, form in cool climate areas with stable, high water tables that promote peat formation and bryophyte cover (MacKenzie and Moran, 2003). Peat is derived from partially decomposed mosses (e.g. Sphagnum spp.) and sedges, resulting in an acidic environment. Peatlands occur in all biogeoclimatic zones in British Columbia, with the exception of the Bunchgrass/Ponderosa Pine (BG/PP) zone. They are especially common in the Boreal White and Black spruce/Spruce-Willow-Birch (BWBS/SWB), Interior Cedar-Hemlock (ICH), and Sub-Boreal Pine-Spruce/Sub-Boreal Spruce (SBPS/SBS) zones (Delong et al., 1991; Hope et al., 1991; Meidinger et al., 1991). Wetlands can be sensitive to anthropogenic disturbance that can result in permanent conversion to a different wetland type or an upland ecosystem. Road construction can cause water run-off to be channeled into peatlands, or impede wetland drainage, thereby influencing the hydrodynamics of the system (MacKenzie and Moran, 2003). Browsing of vegetation by livestock, as well as selective cutting has altered the structure of forested wetlands in Sweden (Segerstrom, 1997). Harvesting of trees in forested wetlands can cause paludification, a rise in the water table due to conversion of mineral soil to peatland (Paavilainen and Paivanen, 1995), and make seedling 1 regeneration difficult. As a result, the subsequent drainage of these flooded areas, in order to increase soil aeration and create favorable conditions for tree establishment, has been investigated (Rothwell et al., 1996; Roy et al., 1999). It has also been suggested that continued harvesting activity could create new wetland types not indigenous to the regional area, or disrupt the successional stages required to produce the original wetland community (Gale et al., 1998). Ectomycorrhizas are mutualistic associations between symbiotic fungi and plant roots. Ectomycorrhizal fungi facilitate nutrient acquisition and water uptake in exchange for carbon from the host plant. Mycelial networks of underground fungal hyphae can link different host plants that share common fungal symbionts (Bjorkman, 1960; Finlay and Read, 1986; Dahlberg and Stenlid, 1990; Simard et al., 1997b; McKendrick et al., 2000). The concept of mycelial networks is particularly relevant in regards to nutrient poor environments (e.g. fens and bogs) where carbon and nutrients can be exchanged across resource gradients (Tilman et al., 1996; Simard et al., 1997b). It has been established that many wetland plant species are mycorrhizal, however, the ecological role that symbiotic fungi play in wetland ecosystems has been relatively unexplored. Although the literature suggests that mycorrhizal fungi are important in nutrient poor, ground-water fed ecosystems (Turner et al., 2000) and that they may be an important mechanism in wetland rehabilitation following anthropogenic disturbance (Turner and Friese, 1998), more research into the mycorrhizal associations of wetland plants is still required to fully understand the relationship between these unique ecosystems and symbiotic fungi. The occurrence of mycelial networks, or shared mycorrhizal symbionts between different host species, and their possible function in wetland environments, is largely unknown. Tamarack is a unique deciduous conifer that is able to tolerate the conditions occurring in peatland environments. It is able to grow at a faster rate (Strong and LaRoi, 1983), conserve more foliar nutrients (Tyrell and Boemer, 1987), utilize a higher amount of available N (MacDonald and Lieffers, 1990), and be less affected by flooded conditions than its counterpart black spruce (Islam and MacDonald, 2003). One hypothesis for the success of tamarack in peatland ecosystems is attributed to its efficient genus-specific mutualistic ectomycorrhizal associations (Tyrell and Boemer, 1987). Scrub birch is a low-lying shrub that is often found growing with tamarack in these environments. Even less is known about the associated ectomycorrhizal fungal symbionts of this peatland species. However, several studies have investigated the ectomycorrhizal relationships of the more northern swamp birch {Betula nana L.) (Miller, 1982), as well as upland Betula spp., such as paper birch {Betula papyrifera Marsh.) (Simard et al., 1997a and 1997b; Jones et al., 1997) and European white birch {Betula pendula Roth) (Miller, 1982; Feugy et al., 1999; Blaudez et al., 2001). It is possible that ectomycoiThizal fungi play an important role in the survival and growth of scrub birch, as well as tamarack, growing in these wetland ecosystems. This project was established to examine the mycorrhizal associations of two plant species, tamarack and scrub birch, growing in three habitats i) scmb birch dominated, ii) tamarack-scmb birch, and iii) mixed tamarack-scrub birch-black spruce peatland site types. The specific objectives of this research project were to use a combination of morphological (light microscopy) and molecular analysis (polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP)) to: 1. Characterize and identify the fungal symbionts that associate with tamarack and scrub birch host species in peatland sites, 2. Determine the abundance and diversity of the fungal symbionts associating with tamarack and scrub birch on the sites, 3. Assess differences in the ectomycorrhizal community occurring between the two host species as well as amongst the three different peatland site types, 4. Determine if the potential for fungal linkages exists between tamarack and scrub birch in these peatlands, 5. Determine if possible fungal linkages exist between these two host species and a third host, black spruce {Picea mariana), that co-occurs in the Mix peatland site type, using molecular data derived from a study by Robertson (2003). LITERATURE REVIEW Wetlands of British Columbia Ecology and descriptions Wetlands have been defined as “areas where soils are water-saturated for a sufficient length of time such that excess water and resulting low soil oxygen levels are principal determinants of vegetation and soil development” (MacKenzie and Moran, 2003). Many different types of ecosystems, such as fens, bogs, and swamps, are included in this definition. Water table attributes such as pH, annual fluctuation levels, and carbon concentration can influence the plant species distribution within these environments (Girardin et al., 2001). Composition of vegetation may also reflect regional geographic variations (Warner and Rubec, 1997). The high water table and poorly aerated soils of wetlands can make growing conditions difficult even for flood-tolerant vegetation. Poor growth rate and decreased rooting depth are characteristics of coniferous trees in wetland ecosystems (Lieffers and Rothwell, 1986). Peatland ecosystems, specifically fens and bogs, are of particular interest since they support the species under investigation: Betula glandulosa Michaux (=B. nana) (scrub birch) and Larix laricina (Du Roi) K. Koch (tamarack). A fen, as described by M eidinger and Pojar (1991), is a non-tidal wetland that is fed water from belowground sources, and receives minerotrophic runoff from surrounding upland mineral soils. Fens are relatively higher in nutrients and lower in acidity, compared to the more acidic, nutrient-poor bog (Warner and Rubec, 1997). Moderately decomposed peat accumulates to more than 40 cm within the organic layer of the Mesisol and Humisol soils, which maintain a high mineral content in the rooting zone (Meidinger and Pojar, 1991; MacKenzie and Moran, 2003). Fens are the most common wetland class in British Columbia, especially within the poorly drained basins of the Boreal Black and White Spruce (BWBS), Spruce Willow Birch (SWB), Interior Douglasfir (IDF), Sub-Boreal Pine-Spruce (SBPS) and Sub-Boreal Spruce (SBS) biogeoclimatic zones. Associated non-ericaceous shrub and plant species include scrub birch, Betula pumila (swamp birch), Carex spp. (sedges), Eqidsetum arvense (common horsetail), and Platanthera dilatata (white bog-orchid). Picea mariana (black spruce), P. glauca (white spruce), and tamarack are the characteristic tree species within the BWBS biogeoclimatic zone (Meidinger and Pojar, 1991). However, a more recent wetland classification describes fen ecosystems as peatlands dominated by sedges and brown mosses (e.g. Tomenthypnum), with high water tables limiting the establishment of tall shrub and tree species (MacKenzie and Moran, 2003). Bogs are nutrient-poor, acidic. Sphagnum-dominated ecosystems characterized by woody vegetation, such as conifers and ericaceous plants (MacKenzie and Moran, 2003). These wetlands are often raised or level with their immediate environment, which makes the minerotrophic run-off and nutrient-rich groundwater from the surrounding soils less available to the rooting zone (Meidinger and Pojar, 1991). Bogs are most common in the BWBS, SWB, SBPS, and SBS biogeoclimatic zones in British Columbia. Fibrisol, Mesisol, or Humisol soils, with upper layers of poorly decomposed peat moss, support slow-growing black spruce, tamarack, Pinus contorta (lodgepole pine), and scrub birch plant communities. The sparse dwarf shrub and herb layer consists of the ericaceous Vaccinium oxycoccos (bog-cranberry), Andromeda polifolia (bog-rosemary), and Kalmia microphylla (western bog-laurel), as well as Carex spp. (sedges), Drosera spp. (sundews), and Menyanthes trifoliata (buckbean) (MacKenzie and Moran, 2003). Mycorrhizal symbiosis Definition and structure Many plants and fungi form beneficial relationships that result in mutualistic symbioses which serve to increase both partner’s fitness within their natural environment. The association between a fungus and the roots of a plant is termed ‘mycorrhiza’ (Smith and Read, 1997). There are seven different categories of mycorrhizal associations that are defined according to the morphological and anatomical characteristics that they exhibit, as well as to the plant and fungal partners involved in the relationships. The present study examines ectomycorrhizas; however, other categories of mycorrhizas that might be of interest in peatland ecosystems include arbuscular (AM), ericoid, and ectendomycorrhizas. Ectomycorrhiza refers to the category commonly formed between basidiomycete or ascomycete fungi, and gymnosperm and angiosperm plant species, or more specifically, coniferous and deciduous trees. Ectomycorrhizal roots are typically colonized by fungi that form an outer mantle of fungal hyphae, as well as a Hartig net (an intercellular network of hyphae that surrounds the root cells up to the endodermis in gymnosperms, and up to the exodermis in angiosperms) (Molina et al., 1992). Arbuscular mycorrhizas are formed between many plant species (including the majority of angiosperm families), as well as some mosses and lycopods, and members of the order Glomales (zygomycete fungi). They are distinctly characterized by the presence of highly branched arbuscules (formed within cortical root cells), and, in some species, intraradical vesicles (enlarged lipid-filled portions of hyphae formed within or between cortical cells) (Smith and Read, 1997; Peterson et al., in press). Typical wetland AM plants include members of the grasses (Poaceae), sedges (Cyperaceae), and willows (Salix) (Turner and Friese, 1998; Miller, 1999; Turner et al., 2000; Marshall and Pattullo, 1981). Ericoid mycorrhizas are named by the association with host plants involved in this symbiosis: the order Ericales, which includes many peatland plants such as Labrador tea {Ledum groenlandiciim), bog cranberry {Vaccinium oxycoccos), and bog-rosemary {Andromeda polifolia). This category of mycorrhizas is characterized by the formation of narrow diameter "hair roots” by the host plant, whose root epidermal cells are colonized by fungi that produce unique, highly branched, hyphal complexes (Peterson et al., in press). Ectendomycorrhizas, a variant of ectomycorrhizas (Egger and Fortin, 1988), form primarily between Pinus and Larix host species, and E-strain {Wilcoxina spp.) ascomycete fungi (Yu et al., 2001). These mycorrhizas exhibit morphological characteristics similar to ectomycorrhizas, with the exception of intracellular hyphae that penetrate the cortical root cells (Laiho, 1965; Mikola, 1965; Yu et al., 2001). Ectomycorrhizas form the main type of symbiosis found on both tamarack and scrub birch tree species and are the main focus of this thesis. Functions and benefits It is well known that the fungal associates in mycorrhizal relationships facilitate the uptake of water (Dosskey et ah, 1990; Bending and Read, 1995; Smith and Read, 1997) and nutrients to the host plant from soil (Harley and Smith, 1983; Perez-Moreno 8 and Read, 2000); however, mycorrhizas can also participate in the biological control against pathogenic root fungi and soil-home diseases (Duchesne, 1994; SchelkJe and Peterson, 1996; Ursic et al., 1997; Morin et al. 1999). Some mycorrhizal fungi can also degrade persistant organic soil pollutants (Meharg and Caimey, 2000; Meharg and Caimey, 2002), as well as withstand a range of environmental stresses (Anderson 1988; Kendrick, 1992; Colpaert and van Tichelen, 1994). Mycorrhizal fungi have been shown to aid in nitrogen transformation from protein sources (Abuzinadah and Read, 1986; Li and Hung, 1987; Li et al., 1992), as well as from simple organic forms (reviewed in Leake and Read, 1997). Some mycorrhizal fungi can produce proteolytic enzymes that exploit N and P, which are important determinants of plant growth, from substrates in their natural environment (Read, 1991; Smith and Read, 1997; Read and Perez-Moreno, 2003). It was once thought that two distinctly separate groups of soil fungi existed: saprophytic decomposers that broke down organic substrates into usable forms, and mutualists, that associated with plant roots and absorbed mineral nutrient ions (Hibbett et al., 2000). However, molecular research has revealed that some inconspicuously fruiting ectomycorrhizal fungi can exhibit decomposer capabilities when also in the mycorrhizal state (Koljalg et al., 2000). Genetic study of the phylogeny of ectomycorrhizal fungi has resulted in some uncertainty with respect to the distinction between these two fungal groups (Hibbett et al., 2000), as well as to our full understanding of the role of mycorrhizal fungi in this complex system. Fungal mycelial networks In addition to their impact on water and nutrient acquisition by the host plant, mycorrhizal fungi may also link different host plant species, or plants of the same species, via underground networks of fungal hyphae (Bjorkman, 1960; Finlay and Read, 1986; Dahlberg and Stenlid, 1990; Simard et al., 1997b; McKendrick et al., 2000). Plants that share common fungal symbionts may have the ability to tap into this functional pathway. Trees colonized by the same symbionts may have similar capabilities to capture soil nutrients, by connected mycelia, thereby possibly reducing competition for resources (Finlay, 1989; Horton and Bruns, 1998). Plant-to-plant nutrient transfer could be vital in nutrient poor or shaded environments where hyphal pathways may allow the transport of carbon and nutrients across resource gradients between host species (Tilman et al., 1996; Simard et al., 1997). However, the structure and function of ectomycorrhizal communities, as well as the potential for interplant linkages in an ecosystem, is complex and not fully understood (Molina et al., 1992). The guild concept (Perry et al., 1989) describes the shared fungal linkages between ectomycorrhizal host species as strengthening ecosystem resiliency by contributing to its “mutual aid and the promotion of common interests”. In terms of nutrient cycling within an ecosystem, it has been hypothesized that host species that share common symbionts may cycle nutrients among themselves, thereby excluding other host species that associate with different fungal partners (Newman, 1988). With respect to tamarack and scrub birch, the identity and linkage associations with mycorrhizal fungi have not yet been studied. 10 M ycorrhizas in wetland ecosystem s Plants growing in wetland ecosystems were once thought to be non-mycorrhizal (Powell, 1975). Instead of forming a mycorrhizal relationship, plants might increase root length in order to acquire more nutrients, a function possibly hampered in poorly aerated flooded soils (Powell, 1975; Coutts and Phillipson, 1978; Mosse et al., 1981). A more recent analysis by Turner and Friese (1998) stressed that it cannot be assumed that wetland plant species are non-mycorrhizal simply because their roots are submerged under water. Recent studies have shown that many wetland plant species are, in fact, mycorrhizal. Numerous species of aquatic grasses, sedges, and herbaceous plants growing in wetland environments often have AM associations (Stevens and Peterson, 1996; Turner and Friese, 1998; Miller, 1999; Turner et al., 2000). Marshall and Pattullo (1981) reported that willows were found to be ectomycorrhizal in a fen ecosystem. With respect to many shrub species and conifers, little is known about their mycorrhizal habits in wetland ecosystems. It has been suggested that ectomycorrhizas associated with trees and woody shrubs in these wet environments may be able to exist, in part, due to soil aeration caused by seasonal fluctuations of the water table (Meyer, 1974); oxygen deficiency has been suggested as a limiting factor to mycorrhizal fungal formation (Stenstrom, 1991). Turner et al. (2000) suggest that mycorrhizas may have an important role in reduced nutrient and ground-water driven communities where colonized roots have been found to be more numerous. 11 Ectom ycorrhizal diversity Ectomycorrhizal community diversity can be simply defined as the measure of species richness, the number of different species found in the community, and community evenness, the relative abundance of each of those species within the community (Magurran, 1988). The belowground diversity of ectomycorrhizal fungi is thought to be directly influenced by the type of forest community, successional stages within a given forest community, as well as the distinctive microhabitats that encompass a forest landscape (Amaranthus, 1998). Host receptivity refers to the range of fungal species with which a host plant associates (Molina et al., 1992). The diversity of mycorrhizal fungi associating with a given host can range from high (e.g. approximately 2,000 fungal species may associate with Pseudotsuga menziesii (Douglas-fir) (Trappe, 1977)), to low (e.g. Alnus, which has about 20 fungal associates) (Molina et al., 1992). Likewise, the level of specificity exhibited by ectomycorrhizal fungi in associating with a given host species can range from broad to narrow. For example, Suillus grevillei and Boletinus cavipes demonstrate a narrow specificity with members of the genus Larix, and appear to preferentially “choose” to associate with that genus (Finlay, 1989), whereas Cenococcum associates with most known ectomycorrhizal hosts (Molina et al., 1992). Given this specificity concept, vital maintaining plant host species diversity may be to supporting ectomycorrhizal fungi diversity, especially for fungi with apparently narrow host ranges (Massicotte et al., 1999). 12 M ethods for m easuring ectom ycorrhizal diversity Sporocarp surveys and seedling sampling Most ectomycorrhizal fungi at some point in their life cycle produce reproductive fruiting bodies, known as sporocarps. Fmiting can occur aboveground (epigeous) or belowground (hypogeous) and is believed to be closely related to environmental conditions present at the site, such as soil temperature and moisture (Godbout and Fortin, 1990). Sporocarp surveys (hypogeous sporocarp collections may be included) have traditionally been used to assess ectomycorrhizal diversity; with this method, fruiting bodies may be identified to species using standard taxonomic approaches (Sakakibara et al., 2002). An important advantage of sporocarp surveys is that one can collect samples throughout several growing seasons. Sporocarp surveys allow for minimal interference within the study site, an important criterion for long-term monitoring projects. However, it is now widely accepted that the production of sporocarps is not always an accurate reflection of ectomycorrhizal species richness belowground (Mehmann et al., 1995; Gardes and Bruns, 1996; Dahlberg, 1997; Dahlberg, 2001). As well, not all sporocarps represent fungal species that are ectomycorrhizal; some may instead be saprophytic in nature. More recently, sporocarp surveys have been combined with other sampling methods in order to more accurately estimate fungal diversity (Bradbury et al., 1998). Ectomycorrhizal fungi can fruit sporadically at a specific site or remain as microscopic, undetected components in the soil, such as spores or sclerotia (Taylor, 2002). As well, sporocarp production varies both temporally and spatially, due to an array of different external factors (Watling, 1995). Some ectomycorrhizal fungi never appear to reproduce sexually and exist primarily in a vegetative state (e.g. Cenococcum geophilum), or the 13 sporocarps fruit belowground and are difficult to detect, or are resupinate in nature (Jonsson et al. 1999; Stendell et al. 1999; Taylor and Bruns 1999; Peter et al. 2001a). Some suggest the presence of a species is best assessed by its presence in its vegetative state (Luoma 1991; Horton 2002). One of the most common ways used to assess ectomycorrhizal community diversity is by direct sampling (also referred to as field bioassays) of ectomycorrhizal root tips from planted or naturally regenerated seedlings. Entire seedlings can be removed with the surrounding soil in order to keep root systems relatively intact, and a sub-sample (or all) of the roots are examined for ectomycorrhizas. When whole seedling destructive sampling is not desirable, such as in regenerating clearcuts where stocking standards must be met, partial collection of lateral roots can also be conducted (Jones et al., 2002). In addition, root coring using cylindrical soil corers (Peter et al., 2001a) is often done in habitats where one host plant dominates, or when host roots can be easily identified (e.g. Pinus spp.), or when molecular analysis can be used to separate the different host species (Horton and Bruns, 1998). Compared to sporocarp surveys, which may repeatedly sample specimens over several seasons, seedling or root core sampling may occur only once or twice during a study, often due to time constraints or other determining factors (Horton and Bruns, 2001). Microscopy and ectomycorrhlza characterization Morphological classification of mycorrhizal root tips (morphotyping) using dissecting and compound microscopy is a common approach for family, genus and species identification. Although accurate characterization of ectomycorrhizas takes time 14 to leam (Dahlberg, 2001), macroscopic characteristics of ectomycorrhizas, such as shape, texture and colour, as well as microscopic features such as the presence of emanating hyphae, mantle, and rhizomorphs, can all aid in fungal identification (Agerer, 1987-2000; Ingleby et al., 1990; Goodman et al., 1996). Fungal diversity can be accurately assessed using detailed morphological descriptions and this assessment can provide a valuable basis for further molecular investigations (Horton, 2002). Nevertheless, morphotyping can sometimes be subjective and, if performed incorrectly, can lead to identification problems (Peter et al., 2001a). In some instances, it is not always possible to accurately group or distinguish all ectomycorrhizas whether from the same, or from different, fungal species (Sakakibara et al., 2002). To use morphotyping to its maximum benefit and to overcome some of the above limitations, morphological characterization of mycorrhizal root tips is often combined with molecular analysis techniques (Varga, 1998; Horton and Bruns, 1998; Hagerman et al., 1999; Jonsson et al., 1999; Mah et al., 2001; Robertson et al., 2003). Molecular techniques Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), common molecular analysis techniques, have advanced the study of ectomycorrhizal community assessment through the identification of fungal symbionts and their genotypes. PCR is able to amplify specific regions of the fungal ribosomal genes and spacers through the development of universal and fungal specific primers (White et al., 1990; Cullings and Bruns, 1992; Gardes and Bruns, 1993; Egger, 1995). The target region of the nuclear encoded ribosomal DNA (rDNA) ranges from the 3’ end 15 of the 18S small subunit, to the 5 ’ end of the 28S large subunit, including both internal transcribed spacer (ITSl and ITS2) regions. The ITS regions (moderately conserved regions) reveal species-specific variability allowing for the discrimination of closely related species, and the large and small subunit regions act as sites for primer design (Egger, 1995; Horton and Bruns, 2001). However, PCR analysis alone is not sufficient for the detection of genotypes or to distinguish between closely related species (Egger, 1995). RFLP analysis, with the aid of restriction endonucleases, allows for the digestion of the amplified target region into fragments of variable sizes. Resulting fragment patterns reveal small size differences that enable the researcher to separate closely related fungal species (Egger, 1995; Mehmann et al., 1995; Gardes and Bruns, 1996; Horton and Bruns, 1998), and to identify these through comparison to established RFLP ectomycorrhizal root tip and sporocarp databases. This method is cost effective and useful for distinguishing between different ectomycorrhizal fungal species from root tip samples (Horton, 2002); however, identification is still not always possible for several reasons. RFLP databases tend to be primarily composed of commonly observed sporocarps, which may not account for the fungal species that do not fruit frequently or not at all (Jonsson et al. 1999; Stendell et al. 1999; Taylor and Bruns 1999; Horton and Bruns, 2001; Peter et al. 2001b). In addition, size estimates for fragment patterns, protocols, and restriction endonucleases can vary between research labs and may hinder comparisons; intraspecific variation within fungal species can also occur across large geographic scales (Kârén et al., 1997; Methven et al., 2000). In some cases, ITS-RFLP data offers limited taxonomic information for identification to the species or species 16 group level, information that DNA sequencing analysis, if used, might provide (Horton, 2002 ). Measurements of ectomycorrhizal diversity Numerous diversity indices are often used to assess the level of ecological complexity within and between communities (Magurran, 1988). Methods for calculating ectomycorrhizal diversity for host species or between sites can include measures of species richness, frequency of occurrence, and proportional abundance (percent); the resulting means and standard errors can be compared by analysis of variance (ANOVA) (Magurran, 1988). This study includes five indices that were selected to measure the ectomycorrhizal diversity of the fungal symbionts associated with tamarack and scrub birch: the M argalef (species richness). Shannon, Shannon Evenness, Simpson (species dominance), and Phi (molecular diversity) indices. Species richness (a measure of the number of the species found) was calculated using the Margalef index. It is calculated as follows: Dmg= (S-l)/ln A where S = number of species, and N = total number of individuals summed over all S species (Magurran, 1988). The Shannon (//') and Shannon Evenness (E) diversity indices are based on proportional abundance of each species, as well as on species richness (the number of species). These indices place increased emphasis on species richness; with respect to mycorrhizas, this includes rare fungal species. The indices are calculated: H ’ = -YPi In pi 17 . E = H'l\nS where pi = proportion of individuals found in the /th species, and where S = number of species. As the ectomycorrhizal diversity increases, the Shannon index values increase (values usually range from 1.5-3.4) as well. The Shannon Evenness index values range from 0-1.0, with 1.0 meaning all species are equally abundant. These indices assume that species are randomly sampled from an infinite population and that all species are represented. Failure to include all species is considered to be a common source of error when using these indices (Magurran, 1988). The Simpson index (D) is also calculated using the proportional abundance of the each species, as well as the number of species identified. However, the index emphasizes the most abundant (dominant) species, and is often expressed as a reciprocal (1/D) value so that higher values represent increased diversity (Magurran, 1988). Since the index is weighted towards the more abundant species, it is less sensitive to species richness or rare species. The Simpson index is calculated as follows:— D = X (« , («,-1))/ { N{ N- \ ) ) where n, = number of individuals in the ,th species and N = the total number of individuals (Magurran, 1988). The Phi index (O) was developed by Egger (Baldwin, 1999) to specifically assess molecular diversity within a community. This index uses pairwise distances (in contrast to proportional abundance data often used to calculate traditional diversity indices) for each sample, with more distantly related samples being separated by greater phylogenetic distance (Khetmalas et al., 2002). D ice’s index matrices are calculated from RFLP fragment patterns to estimate the similarity between samples; then the average squared 18 distance is calculated for the entire data matrix. Values range from 0 (identical fragment patterns) to 1 (no fragments shared between any pairs) (Mah et al„ 2001). The Phi index is calculated as follows; Z4 Z n-i 1=1 7=1 For a data matrix with i - j rows and columns, the pairwise distances {d) for each sample were squared, summed, then divided by n-i to give an average squared distance for each column, where n equals the total number of samples in the matrix. As with all other indices, resulting mean Phi values can be compared using an ANOVA; an increase in the Phi value implies greater diversity. Tamarack {Larix laricind) Distribution and ecology Uniquely characterized by deciduous needles, the genus Larix (family Pinaceae) is well adapted to the cold climates and short growing seasons typical of the boreal, montane and subalpine forests of the northern hemisphere (LePage, 1995). Three of the ten tree species in this genus are endemic to Canada and North America: Larix occidentalis Nutt, (western larch), Larix lyallii Pari, (alpine larch), and Larix laricina (Du Roi) K. Koch (tamarack) (Farrar, 1995). Tamarack, also known as eastern larch, is the widest ranging conifer species in North America; it occurs in every province and territory in Canada, as well as Alaska (Johnston, 1990). The species can generally tolerate most soil conditions, such as wet, organic Sphagnum peat found in lowland bogs, muskegs, or 19 fens, as well as well-drained, mineral soils found on upland northern slopes (Johnston, 1990; Farrar, 1995). In northern British Columbia, tamarack is commonly found within the BWBS biogeoclimatic zone (Delong et al., 1991), often occurring in mixed stands with black spruce and scrub birch in the wet, nutrient poor Sb-Tamarack site series association (Krestov et al., 2000). Tamarack is considered rare in the Sub Boreal Spruce (SBS) zone (Meidinger et al., 1991; Beaudry et al., 1999), but it can be locally common within the Tamarack - Water sedge - Fen moss (W b 06) Bog Site Association (MacKenzie and Moran, 2003). Tamarack exhibits several interesting physiological adaptations in response to its harsh growing conditions. High water tables, poor soil aeration, low nutrient availability and the cold substrate of fen and bog environments result in extremely slow growth rates (Payandeh, 1973; Lieffers and Rothwell, 1986, 1987; MacDonald and Lieffers, 1990), however, tamarack may still grow at a faster rate than black spruce (Strong and LaRoi, 1983). Tyrell and Boemer (1987) investigated how tamarack conserves foliar nutrients as a mechanism to persist in peatland environments without the evergreen habit that is exhibited by its counterpart, black spruce. They suggested that the efficient genus- specific mycorrhizal associations unique to tamarack may enable the tree to uptake a greater amount of nutrients than black spruce. This, when combined with a higher foliar nitrogen resorption, as well as a higher photosynthetic rate than black spruce, allows it to remain productive in bog environments. Further evidence of the benefits of this specific ectomycorrhizal relationship was demonstrated by Samson and Fortin (1986); they determined that the fungi previously identified as being L arâ-specific (e.g. Suillus grevillei) in field conditions, showed faster and better mycorrhizal development (e.g. 20 extensive extramatrical hyphal networks) in vitro. As well, Suillus grevillei is consistently associated with tamarack in its full habitat distribution range, including wet, boggy areas (Samson and Fortin, 1986). MacDonald and Lieffers (1990) also reported differences between tamarack and black spruce in their ability to utilize nitrogen; they found that tamarack was more effective in utilizing improved nutrient conditions following peatland drainage. Simulated flooding in a greenhouse caused reduced root hydraulic conductance, net assimilation rate, and stomatal conductance in both tamarack and black spruce seedlings; however, tamarack was less affected than black spruce in all measurements (Islam and MacDonald, 2003). It was also noted that tamarack showed no visible flooding damage symptoms, such as necrotic needles and electrolyte leakage as experienced by black spruce. Chakravarty and Chatarpaul (1990) reported that, in an in vitro tamarack study, inoculated seedlings with mycorrhizal fungi performed better than non-mycorrhizal seedlings in nutrient limited environments. Identified fungal symbionts Early studies describing the mycorrhizal associations for the genus Larix include those by McDougal (1914), Melin (1922), and Hammerlund (1923); these pioneer studies led others to attempt to identify the numerous fungal symbionts (Table 1.1). How (1940, 1941, 1942) completed detailed studies on L. decidua, including studies on its fungal associates and its specialized relationship with the fungus Boletus elegans. The fungal species Suillus grevillei and 5. cavipes have been reported to be highly specific to Larix spp. as are several other fungal species that exhibit a narrow host preference (Molina et 21 al., 1992). Roots of tamarack sampled from the field have also indicated the possibility of an ectendomycorrhizal association, though the fungal species remained unidentified (Malloch and Malloch, 1981). Samson and Fortin (1986) also assessed fungal symbionts of tamarack by inoculating plantlets with different isolated fungi; they reported that 91 isolates belonging to 25 fungal species formed ectomycorrhizae with tamarack seedlings. Table 1.1 summarizes the reported mycorrhizal associations for three Larix species. Scrub birch (Betula glandulosa) Description and ecology As its common name implies, scrub birch {Betula glandulosa) is a low lying spreading shrub that can reach two metres in height in both wetland and upland areas of British Columbia (Mackinnon et al., 1992). Within the northern half of the province, scrub birch is commonly found at low elevations in wetlands with black spruce, tamarack and lodgepole pine (Pinus contorta var. latifolia). Swamp birch (Betula nana) is a commonly misidentified species found in similar habitats but is a more northern and Eurasian species (Brayshaw, 1996). Some confusion can arise since swamp birch is also referred to as Betida piunila (dwarf birch) or Betula glandulosa var. glandulifera within various tree identification guides. 22 T able 1.1. Identified mycorrhizal symbionts of Larix laricina, L. decidua, and L. occidentalis. Fungal associate Amanita muscaria A. rubescens Astraeiis pteridis Boletus elegans B. viscidus B. edulis Cenococcum spp. E-strain Fuscoboletinus aeruginascens F. paluster F. spectabilis F. grisellus F. glandulosus F. ochraceoroseus Hebeloma spp. Laccaria laccata L. amethystea L. bicolor Lactarius deliciosus Leccinum holopus var. americanus Melanogaster intennedius Paxillus involutus Pisolithus tinctorius Rhizopogon rubescens R. vinicolor Scleroderma hypogaeum Sphaerosporella brunnea Suillus grevillei S. cavipes S. lakei Tlielepliora terrestris Triclioloma pessundatum T. vaccinum T. flavovirens Truncocolumella citrina L. laricina L. decidua L occidentalis .G ,3 .4 .4 .G ,6 ,9 1 .G 1 I _ .1 ,1 ,5 1 .1 .G I _ 1 1 .G .G .G 1 .G .G 8 . 1 ^1 .G #G *G ,1 ,5 , I _ 1 .G #G Bouchard (1 986), ®Molina and Trappe (1982), ^Munzenberger et al. (1995), ^Danielson (1984), ’Laiho (1965), '“Thormann et al. (1999). 23 Scrub birch is primarily found in the Spruce-Willow-Birch (SWB) biogeoclimatic zone, the most northerly subalpine zone of British Columbia (Pojar and Stewart, 1991). Within this zone, scrub birch grows on dry to wet, moderately well-drained upland soils in open forests and woodlands of the White spruce-Grey-leaved willow-Scrub birch site association, as well as in moderately rich, shrubby fens within the Barclay’s willowScrub birch-Water sedge site association (Pojar and Stewart, 1991). The recently published guide to the wetland areas of interior British Columbia (Mackenzie and Moran, 2003) lists scrub birch as occurring mainly within the Scrub birch-Water sedge (WF02) and Scrub birch-Buckbean-Shore sedge (WF07) Fen Site Associations. A very wet, nutrient-medium Sb-Swamp birch site series association in the SBS (Sub-boreal Spruce) biogeoclimatic zone is tentatively identified by Krestov et al. (2000). Identified fungal symbionts The mycorrhizal associations of these small birches, scrub birch in particular, have been largely uninvestigated. However, one study in the subalpine tundra of Alaska examined swamp birch roots from the field and identified 12 species of ectomycorrhizal fungi (Miller, 1982). Numerous studies have recently explored the relationship between some of the larger Betula spp. and their fungal symbionts, including Paxillus involutus (Blaudez et al., 1998; Jordy et al., 1998; Feugy et al., 1999; Perez-Moreno and Read, 2000; Blaudez et al., 2001). It is important to note that most of these studies involve Betula spp. that grow in distinctly different (mostly well drained) habitats. Table 1.2 summarizes the ectomycorrhizal fungal symbionts of three Betula species. 24 T able 1.2. The identified mycorrhizal symbionts of B. glandulosa, B. pendula and B. nana. Fungal Associate Amanita inaurata A. pantlierina A. vaginata Boletus edidis Hebeloma piisillum H. cylindrosponun Lactarius musteus L. uvidus Leccinum scabrum Hygrophorus chrysodon H. conicus Paxillus involutus Russula emetica R. obscura B. glandulosa B. pendula B. nana #' ‘Miller, 1982; ^Blaudez et al., 1998; ^Feugy, 1999 LIT E R A T U R E CITED Abuzinadah, R.A., and Read, D. 1986. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. I: Utilization of peptides and proteins by ectomycorrhizal fungi. New Phytol. 103: 481-493. Agerer, R. 1987-2000. Colour Atlas of Ectomycorrhizae. Eihom-Verlag Eduard Dietenberger, Schwabisch Gmiind, Germany. Amaranthus, M.P. 1998. The importance and conservation of ectomycorrhizal fungal diversity in forest ecosystems: Lessons from Europe and the Pacific Northwest. PNW-GTR-431, 1-15. Forest Service, United States Department of Agriculture, Pacific Northwest Research Station. Anderson, A.J. 1988. 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Mycorrhiza, 11: 167-177. 35 Morphological characterization o f ectomycorrhizal associations of Larix laricina (Du Roi) (tamarack) K. Koch and Betula glandulosa Michaux (scrub birch) in peatlands of central British Columbia. ABSTRACT Peatland habitats accumulate peat in lowland areas, resulting in poorly drained, moderately acidic, and nutrient deficient soils. In these ecosystems, tamarack and scrub birch are often found growing in close proximity in central British Columbia. Morphological methods (light microscopy) were used to characterize the ectomycorrhizas of these two host species in three peatland site types (scrub birch-tamarack-black spruce (Mix), scrub birch-tamarack (BsLt), and scrub birch (Bs) only), and to determine differences in ectomycorrhizal community structure and diversity between hosts and peatland site types, as well as the potential for host-fungal linkages. A total of 30 morphotypes were described from 24 tamarack and 36 scrub birch seedlings; 17 common morphotypes were found on both hosts. MRA, Thelephoraceae 1 and Tomentella-Wke. 2 found on scrub birch, and Suillus 2 and Cenococcum found on tamarack, were the most frequent morphotypes. Lactarius and Suillus also showed some host specificity. Some morphotypes exhibited site specificity (e.g. the three Thelephoraceae spp. (tamarack) in the Mix site, and cotton orange and Tomentella-hke 1 (scrub birch) in the Bs and BsLt sites); many morphotypes were found in all site types. Although ectomycorrhizal abundance varied between hosts for some morphotypes, no overall difference in ectomycorrhizal diversity was seen between hosts. However, ectomycorrhizal diversity was highest in the Mix sites for both hosts compared to the BsLt sites (Margalef, Shannon, and Simpson indices) (a = 0.05). Overall, ectomycorrhizal colonization of tamarack and scrub birch showed a high potential for fungal linkages in these peatland habitats. 36 INTRODUCTION Peatlands form in cool climates where water input exceeds evaporation, and where deep formations of peat (poorly decomposed mosses and sedges) accumulate due to stagnant high water tables and slow decomposition rates (MacKenzie and Moran, 2003). In British Columbia, peatlands can be divided into two site classes; the Bog Wetland Class (Wb) which, with its highly acidic, nutrient and oxygen poor soils, supports ericaceous shrubs and coniferous trees, and the Fen Wetland Class (Wf) that is dominated by sedges and non-ericaceous shrubs (e.g. scrub birch {Betiila glandulosa)) which grow in less acidic, minerotrophic soils (MacKenzie and Moran, 2003). In peatland ecosystems, growth of flood tolerant vegetation such as tamarack {Larix laricina) and black spruce {Picea mariana), is often stunted and slow (Payandeh, 1973; Lieffers and Rothwell, 1986 and 1987; MacDonald and Lief fers, 1990). Recent investigations into some of the mechanisms for the survival and growth of trees in these systems have shown that tamarack is more resistant to flooding damage (Islam and MacDonald, 2003), and conserves foliar nutrients more efficiently than black spruce (Tyre 11 and Boemer, 1987). However, few studies have explored the possible role of specialized plant-fungal relationships (ectomycorrhizas) in peatland environments. Ectomycorrhizal fungi have developed a symbiotic relationship with plant roots; these symbioses facilitate the uptake of water and nutrients by the fungi in exchange for carbon from the host plant (Harley and Smith, 1983). Ectomycorrhizal fungi play an important role in forest communities where they provide protection to roots from soil pathogens and diseases (Duchesne, 1994; Schelkle and Peterson, 1996; Ursic et al., 1997; ■Morin et al., 1999), aid in nutrient cycling (Smith and Read, 1997), and can increase host 37 plant tolerance to environmental stress (Anderson 1988; Colpaert and Van Tichelen, 1994). Most interestingly, fungal symbionts can be shared by different plant species, as well as by neighboring plants of the same species, and these fungi can translocate nutrients between hosts, thereby linking hosts through underground mycelial networks of fungal hyphae (Bjorkman, 1960; Finlay and Read, 1986; Dahl berg and Stenlid, 1990; Simard et al., 1997b; McKendrick et al., 2000). These fungal linkages allow nutrients to be cycled between hosts, and such interactions may positively impact and reduce competition for soil resources (Newman, 1988; Finlay, 1989; Horton and Bruns, 1998). Many wetland plants, such as woody plants (Thormann et al., 1999), willows (Marshall and Pattullo, 1981), and some aquatic grasses, sedges and herbaceous plants (Turner and Friese, 1998; Miller, 1999; Turner et al., 2000) are mycorrhizal. These symbiotic associations may be important to the trees and shrubs that occur in peatland ecosystems where wet, poorly aerated soils may impede plant growth and root formation (Lieffers and Rothwell, 1986). It has been hypothesized that genus-specific ectomycorrhizal fungi may enable tamarack roots to take up a greater amount of nutrients compared to other wetland species, thereby increasing the survival and growth rate of this species (Tyrell and Boemer, 1987). Mycelial networks could also allow the transport of carbon across resource gradients between host species in nutrient poor environments (Tilman et al., 1996; Simard et al., 1997b), such as fens and bogs. Jones et al. (1997) determined (through morphological investigation) that a high potential for carbon or nutrient transfer through hyphal linkages exists between paper birch {Betula papyriferd) and Douglas-fir (Pseudotsuga menziesii). However, the potential for, and the role of. 38 ectomycorrhizas and mycelial networks in peatland ecosystems has not been documented. Tamarack and scrub birch are common plant species in certain fen and bog site associations in British Columbia. Several studies have attempted to identify some of the fungal symbionts associated with tamarack; these are mostly from in vitro inoculation trials (Samson and Fortin, 1986; Molina and Trappe, 1982; Danielson, 1984; LeTacon, 1986). Interestingly, the genus Larix has been found to associate with several genusspecific fungi {Siiilhis grevillei and S. cavipes) (Molina et al., 1992) and some Larix species have been shown to be ectendomycorrhizal with E-strain fungi (Laiho, 1965; Malloch and Malloch, 1981; Danielson, 1984). Less is known about the mycorrhizal associations of scrub birch; however, the fungal symbionts identified for swamp birch {Betula nano) growing in the subalpine tundra of Alaska included Amanita, Lactarius, Russula species, as well as several other genera (Miller, 1982). Little is known about the ectomycorrhizal communities associating with tamarack and scrub birch in natural peatland ecosystems in British Columbia. A main objective of this study was to use morphological techniques to characterize the ectomycorrhizal associations of tamarack and scrub birch growing in three different peatland site types in the central interior of British Columbia. The three peatland site types included i) scrub birch dominated, ii) scrub birch-tamarack, and iii) mixed scrub birch-tamarack-black spruce site types. The second objective was to assess differences in the abundance and diversity of the ectomycorrhizal communities associating with the two host species, as well as between site types, and to determine the 39 potential for fungal linkages, through shared ectomycorrhizal symbionts, between tamarack and scrub birch. METHODS Site descriptions Seedlings were sampled in three peatland areas within the dry, warm subzone variant of the Sub-boreal Spruce (SBSdwS) biogeoclimatic zone, specifically in the Norman Lake area (approximately 40 km west of Prince George) in central British Columbia, Canada (map of study area shown in Appendix I). Ranging in latitude from 5 r 30’ to 59° N, this zone is characterized by cold, snowy winters and warm, short summers (Meidinger et al., 1991). Scrub birch is found primarily in peatland systems within the SBS zone, most commonly within the Scrub birch - W ater sedge (W f 02) and Scrub birch - Buckbean - Shore sedge (W f 07) Fen Site Associations (MacKenzie and Moran, 2003). Tamarack is considered rare within the SBS zone (Beaudry et al., 1999), but it can be locally common within the Tamarack - Water sedge - Fen moss (W b 06) Bog Site Association (MacKenzie and Moran, 2003). Three peatland site types were selected for study: scrub birch dominated (Bs), scrub birch and tamarack dominated (BsLt), and scrub birch, tamarack, and black spruce (Mix) (Figure 2.1). Two replicate sites were located for each peatland site type, for a total of six sampling sites. Boundaries of each site were determined by changes in the surrounding topography and vegetation. Sites were located a distance (>25 m) from 40 access roads to minimize airborne particulate matter, run-off, and other disturbance effects. Table 2.1. Summary of replicate peatland sites and number of plants sampled for two hosts, tamarack and scrub birch. Site type* Tamarack Scrub birch Bsl - 6 Bs2 - 6 BsLt I 6 BsLt2 M ixl 6 6 6 6 Mix2 Total 6 24 6 36 6 *Bs (scrub birch), BsLt (scrub birch and tamarack), M ix (scrub birch, tamarack, and black spruce). Note: Ail sites were located near the Norman Lake Road w est o f Prince George, access from Highway 16. The Bs peatland site type was characterized by scrub birch and Salix spp. (willow) as the dominant shrub species, with sporadic and disparate Pinus contorta Dougl. Ex Loud. var. latifolia Engelm. (lodgepole pine) and occasional Picea mariana (Mill.) B.S.P. (black spruce) trees in < 1% of the site area. This site type also consisted of several dwarf shrubs, such as Vacciniiim oxycoccos L. MacM. (bog cranberry), Andromeda polifolia L. (bog-rosemary). Ledum groenlandicum Oeder (Labrador tea), and Rubiis arcticus L. (dwarf nagoonberry). Flowering herbaceous plants were absent from this site type, with the exception of Potentilla palustris (L.) Scop, (marsh cinquefoil). Carex rostrata Stokes and C. interior L.H. Bailey (beaked and inland sedge), as well as Triglochin maritimum L. (sea side arrow grass), were common sedge and grass species. The moss layer consisted of Aulacomnium palustre (Hedw;) (Schwaegr) (glow moss). Sphagnum spp. (peat moss), and Tomenthypnum nitens (Hedw.) Loeske (golden fuzzy fen moss). 41 The BsLt wetland site type was dominated by tamarack and scrub birch, as well as willow species; however, the only dwarf shrub present was Labrador tea. Sporadic lodgepole pine and black spruce trees occurred in < 1% of the site area. The presence of two orchid species, Platanthera dilatata (Pursh) Lindl. ex Beck and P. hyperborea (L.) Lindley (white and northern green bog orchids), were unique to this site type. Other flowering herbaceous plants included marsh cinquefoil, Galium spp. (bedstraw), and Pyrola asarifolia Michx. (pink wintergreen). All the sedge and grass species listed in the Bs site type were also found in the BsLt site type, with the addition of Ecjuisetiim hyemale L. (scouring rush). The moss layer consisted of glow moss, golden fuzzy fen moss and Mnium spp. (leafy moss), with a notable reduction in the amount of Sphagnum spp. The third wetland site type. Mix, consisted of a dominant mixture of black spruce, tamarack and scrub birch. Dwarf shrubs included bog cranberry, bog-rosemary, Kalmia microphylla (bog-laurel), Labrador tea and dwarf nagoonberry. Petasites sagittatus (Banks x Pursh) A. Gray (arrow-leaved coltsfoot), Menyanthes trifoliata L. (buckbean), Mitella nuda L. (common mitre wort) and Drosera rotundifolia L. (round-leaved sundew) were unique herbaceous plants to this site type; Mix sites also contained bedstraw, white bog orchid, marsh cinquefoil, and pink wintergreen. Many of the common grass and sedge species on the other sites were also found here, such as beaked sedge, narrow­ leaved cotton grass, scouring rush and sea-side arrow grass. Glow moss, peat moss and golden fuzzy fen moss were common in the moss layer, along with Campylium stellatiim (Hedw.) Jens, (golden star moss). Figure 2.1 shows images of the three peatland site types, as well as plants and fungi found on those sites. 42 m Figure 2.1. Photographs showing the three peatland site types in central BC selected for this study, local vegetation, and fungi. A) Bs peatland site type of scrub birch. (B) BsLt peatland site type of scrub birch and tamarack. (C) Mix peatland site type of scrub birch, tamarack and black spruce. (D) Sphagnum covered hummock in peatland with tamarack seedling. (E) scrub birch {Betula glandulosa). (F) buckbean {Menyanthes trifoliata). (G) larch suillus {Suillus grevillei). 43 Seedling sampling regime Harvesting of entire plants with root systems occurred during the last week of July (2002) and the first week in August using a simple random sampling technique. In the interior of each site, a 50 x 50 m plot was established. In the BsLt and Mix sites, each tamarack seedling (between 15-30 cm in height) was flagged and numbered. Using a random number table, six tamarack seedlings were selected from each site. Due to the large number of birch plants present within all the sites, a 1 x 1 m grid sampling system was established in which each grid square was assigned a number. A birch plant (between 15-30 cm in height) was harvested if it was growing within a grid square (grid squares were chosen using a random number table). Six scrub birch plants were selected from each site. Tamarack plants that appeared to be layered or attached to older “parent” trees were eliminated from the selection process. Plants were harvested using a pruning saw (to cut through the peat moss and surrounding roots); organic matter was removed with each root system to minimize root disturbance. Plants were placed into 7 L plant pots, double bagged in plastic bags, and stored at 5°C until processing. During root assessment, several tamarack seedlings had few root tips and appeared to be layered seedlings. These seedlings were replaced in mid-September in an effort to assess only single seedlings. Vegetation plot analysis and sporocarp sampling To document vegetation growing on peatland sites, each site was divided into four quadrants and, within each, a representative I x I m vegetation plot was established. Bryophytes, herbaceous plants, shrubs and trees were identified and recorded (Appendix 44 II). All tree species that did not fall within the I plots were visually assessed throughout the entire site. Epigeous sporocarps were collected during the summer months within all six sites. Sporocarp samples were collected throughout each entire site, placed in paper bags, and transported to the laboratory for identification. Sporocarp characteristics, such as shape, colour, size, and odour, as well as spore features, were described. Samples were identified to the closest family, genus, or species, which ever was possible. Sporocarp tissue (approximately 0.5 x 0.5 mm) was collected from the pileus and spore producing area and stored in sterile 1.5 ml microtubes at -20°C for later molecular analysis. Sporocarps were then dehydrated and kept as reference material. Morphological characterization o f ectomycorrhizas All extraneous soil and organic matter (moss, herbaceous material, etc.) was gently removed from each root system through sequential soaking and rinsing with water. Shoots were removed and the remaining roots were cut into 2 cm lengths and placed on a numbered 1 cm^ grid for random sampling. Two-hundred root tips were randomly selected for microscopic characterization (Massicotte et al., 1994; Durai I et. al., 1999). A total of 60 plants were assessed; 24 tamarack and 36 scrub birch. Ectomycorrhizal root tips were characterized using light microscopy following methods described by Ingleby et al. (1990), Massicotte et al. (1999), Agerer (1987-2000), Goodman et al. (1996), and Mah et al. (2001). Characteristics such as branching pattern, tip shape, colour, and texture, as well as inner and outer mantle patterns, depth of mantle and presence of a Hartig net were described. 45 The presence and type of cystidia. emanating hyphae, and rhizomorphs were determined. Each different type of ectomycorrhiza was tested for a reaction to 5% KOH. Representative permanent slides were made for some of the morphotypes. Characterized ectomycorrhizas were classified into morphotypes and given a family, genus or species name; if this was not possible, morphotypes were assigned a descriptive name based on their morphological features. To document certain morphotypes, photographs were taken with an automatic exposure camera (PM -1OAK) attached to a compound (Olympus BX-50) or dissecting (Olympus SZ-40) microscope using Ektachrome 160T tungsten professional colour reversal film. The total number of morphotypes, as well as the number of root tips exhibiting each morphotype, was determined for each seedling. Statistical analysis of morphological data Morphotype descriptions were reviewed prior to data analysis; this resulted in merging several morphotypes that could not be separated by descriptive characteristics alone. The number of ectomycorrhizal morphotypes and their proportional abundance (percent of each morphotype) on the root system were calculated for each seedling. The seedling values were used to determine frequency of occurrence and morphotype mean abundance for each peatland site type. For each host, tamarack and scrub birch, a one­ way ANOVA (Statistica version 6.1, 2002, StatSoft, Inc.) using morphotype abundance data, was used to assess differences between the peatland site types in which each host occurred (a = 0.05). On the sites where the two hosts co-occurred, site type and host differences based on morphotype abundance were determined by a two-way ANOVA (a 46 = 0.05). The post hoc Fisher’s Least Significant Difference (LSD) test (a = 0.05) was used to test mean comparisons. To assess peatland site type diversity, the Margalef index (measure of species richness), the Shannon and Shannon evenness diversity index (considers both species richness and evenness), and the Simpson index (which places more weight on those morphotypes that are most abundant) were used (Magurran, 1988). Diversity values where calculated for each seedling based on the proportional abundance of each ectomycorrhizal morphotype and the number of morphotypes per seedling. These values were used to calculate diversity indices. For each host, a one-way ANOVA was used to determine peatland site types effects on diversity. On sites where the two hosts co­ occurred, a two-way ANOVA was used to determine site type and host effects on diversity (a = 0.05). The Fisher’s Least Significant Difference test (a = 0.05) was used to test mean comparisons. RESULTS Ectomycorrhiza morphotype richness, frequency and abundance A total of 30 ectomycorrhizal morphotypes (excluding the lightly colonized category) were characterized from 11,600 root tips on 58 seedlings (Figure 2.2). Of these, 24 morphotypes were described from 34 scrub birch seedlings (two seedlings were eliminated due to very low root tip numbers), and 23 morphotypes were described from 24 tamarack seedlings. Seventeen of the 30 morphotypes were common on both host 47 species, seven were unique to scrub birch and six were unique to tamarack. Complete morphological descriptions of these morphotypes are presented in Appendix HI and several images detailing distinct features are shown in Fig. 2.3. The mean number of morphotypes for each host, within each peatland site type, is presented in Table 2.2. The number of morphotypes varied significantly between site types for scrub birch (p = 0.051), but not for tamarack (p = 0.06); for both host species, the greatest number of morphotypes occuned on seedlings from the Mix site (scrub birch, tamarack, and black spruce). The BsLt sites (scrub birch and tamarack) exhibited the lowest morphotype richness for both hosts. T able 2.2. Mean number of ectomycorrhizal morphotypes (SE in parenthesis) for tamarack and scrub birch seedlings growing in three peatland site types: Bs (birch dominated), BsLt (scrub birch-tamarack), and Mix (scrub birch-tamarack-black spruce). F P B sLt Mix Tamarack 3.934 0.060 4.5 (0.4) 5.6 (0.4) Bs - Scrub birch 3.288 0.051 3.6 (0.4)b 5.5 (0.7)a 4.2 (0.4)a Host Morphotype richness values were tested using a one-w ay A N O V A for peatland site types ( a = 0.05) (tamarack d f = 1,22) (birch d f = 2,31). Note: Within rows, means follow ed by the sam e letter are not significantly different. Lightly colonized root tips (those lacking distinguishable mantle features) represented 1.3% (n = 85) of all roots sampled for scrub birch; these occurred on seedlings more frequently in the Mix peatland site type (27.3% of seedlings), compared to the Bs (8.3%) and BsLt (0.0%) sites (Table 2.3). In contrast, 25.9% (n = 1241) of all tamarack roots were described as lightly colonized; these also occurred more frequently on seedlings in the Mix peatland site type (83.3% of seedlings), compared to the BsLt (50.0%) site (Table 2.4). 48 a yeDowsteDate BsLt peatland site type woolfy brown Mix peatland site type white M ed □ white clanp P Bs peatland site type P tn TonEnteDa-ïkE2 TontnteDadike I a ThelqjhoraceæS 0 Thelq*oraceae2 Thelephoraceae 1 SuiDus2 SuiDusl silver white Kussulaceae D Russula MRA lightly cokmiæd cm i lactarius HHrdona granular brown e-stMi crystal net brown I eotton-orange □ a ! co%e brown Gnococcum i P brown snD0th2 a brown snnothl brown sOveiy brovtfi ink clanp tamarack scrub birch brown clarrp 1 blackcystidia Anphenhra □ 0 10 20 30 % abundance 40 K) 23 % abundance 30 40 0 20 30 40 % abundance Figure 2.2. Comparison o f ectomycorrhizal morphotype abundance o f tamarack and scrub birch between the BsLt, Mix, and Bs peatland site types. M y - .'? f F igure 2.3. Photographs showing ectomycorrhizal morphotypes from tamarack and scrub birch. A, B, C, D, E, and F ectomycorrhizas on scrub birch, and G, H, and I ectomycorrhizas on tamarack. (A) Tomentella-Wke. 2 outer mantle (OM). (B) Tomentella-\\k& 2 ectomycorrhizal root tip. (C) E-strain OM with enlarged hyphal cells. (D ) Lactarius ectomycorrhiza. (E) L actarius OM with laticifers. (F) Lactarius root showing crystal-like deposits. (G) Suillus 2 OM (H) Toineiitella-like 1 ectomycorrhizal root tip. (I) Tom entella-U kc 1 OM. 50 Scrub birch {Betula glandulosa) ectom ycorrhizas Of the 24 morphotypes characterized from scrub birch, 20 morphotypes were found in the Mix peatland site type, 14 morphotypes in the BsLt site type, and 16 morphotypes in the Bs site type. Seven morphotypes were common to all three peatland site types, and two morphotypes were unique to each of the site types. The 13 most commonly occurring ectomycorrhizal morphotypes (found on four or more seedlings) belonged to the family Thelephoraceae, or to the genera Lactarius, Tomentella, Cenococciim, and MRA (Table 2.3). Four morphotypes (brown inky clamp, granular brown, brown smooth 2, and woolly brown) could not be assigned to a family. Mycelium radicis atrovirens was the most frequently occum ng morphotype; it was found on 41.2% of all scrub birch seedlings and in all the site types. Other frequently occurring morphotypes, Thelephoraceae 2 (38.2% of seedlings) and brown inky clamp (29.5%) were absent from the BsLt sites; Tomentella-Wke 2 (38.2%), brown smooth 2 (32.4%) and granular brown (26.5%) were absent from the Bs sites. Interestingly, Cenococciim (20.6%), Lactarius (38.2%), Thelephoraceae 1 (29.5%), Thelephoraceae 3 (23.5%), and woolly brown (14.7%) were present in all site types (Table 2.3). 51 Table 2.3. Site effect, percent abundance (mean ±SE in parenthesis) and frequency of occurrence (%) of ectomycorrhizal morphotypes of scrub birch growing in three peatland M ix (n = l l ) B sL t Bs (n = l l ) (n = 12) M orphotype F P A b u n dan ce Freq A b u n dan ce Freq A b u n dan ce Freq A m phinem a 1.116 0.340 1.4 (0.8) 27.3 0.0 (0.0) 0.0 0.7 (0.7) 8.3 black cystidia 1.049 0.363 1.0 (1.0) 9.1 0 .0 (0.0) 0.0 0.0 (0.0) 0.0 brown inky clamp 1.845 0.175 16.4 (6.7) 45.5 0 .0 (0.0) 0.0 14.6 (8.8) 41.7 brown sm ooth 1 1.572 0.224 0 .0 (0.0) 0.0 0 .0 (0.0) 0.0 3.2 (2.4) 16.7 brown sm ooth 2 4 .2 9 0 0.023 10.6 (6.3)afc 36.4 2 4 .6 (7.9)0 63.6 0 .0 (0.0)6 0.0 C enococciim 0.636 0.536 9 .0 (5.4) 27.3 4.9 (4.6) 18.2 2.4 (2.1) 16.7 cotton orange 2.314 0.116 0 .0 (0.0) 0.0 0.0 (0.0) 0.0 2.4 (1.5) 25.0 crystal net brown 1.049 0.363 0.2 (0.2) 9.1 0.0 (0.0) 0.0 0.0 (0.0) 0.0 0.0 E-strain 1.046 0.363 0 .0 (0.0) 0.0 0.4 (0.4) 9.1 0.0 (0.0) granular brown 2.653 0.086 5.7 (2.5) 45.5 2.8 (1.8) 3&4 0.0 (0.0) 0.0 Lactarius 5.251 0.011 8.4 (4.9)6 36.4 3 6 .6 (1 2 .4 )0 54.5 4.6 (2.5)6 25.0 MRA 8.406 0.001 1.3 (1.0)6 27.3 3.5 (2.0)6 27.3 26.7 (7.8)a 66.7 9.1 1.2 (1.0) 18.2 0.0 (0.0) 0.0 R ussula 0.716 0.496 0.6 (0.6) Russulaceae 0.501 0.611 0 .0 (0.0) 0.0 0.5 (0.5) 9.1 0.7 (0.7) 8.3 silver white 0.517 0.602 0.7 (0.5) 18.2 0.8 (0.8) 9.1 0.1 (0.1) 8.3 Thelephoraceae 1 11.26 0.000* 3.9 (2.6)6 27.3 0.2 (0.2)6 9.1 24.2 (5.9)0 75.0 Thelephoraceae 2 3.070 0.061 7.3 (3.8) 45.5 0.0 (0.0) 0.0 13.1 (5.1) 41.7 Thelephoraceae 3 2 .9 1 0 0.069 5 j# 4 ) 36.4 0.5 (0.5) 9.1 0.9 (0.7) 25.0 Tom entella-Vike 1 2.227 0.125 0 .0 (0.0) 0.0 0.2 (0.2) 18.2 0.0 (0.0) 0.0 Tom entella-U ke 2 6.100 0.006 7 .4 (3.4)fl6 45.5 22.8 (7.7)0 72.7 0 .0 (0.0)6 0.0 white clamp 0.779 0.467 8.6 (8.6) 9.1 0 .0 (0.0) 0.0 2.4 (2.0) 16.7 white felted 0.609 0.550 1.6 (1.4) 18.2 0 .0 (0.0) 0.0 2.8 (2.6) 16.7 w oolly brown 3.068 0.061 6 .6 (3.3) 36.4 1 .0 (1 .1 ) 9.1 0.2 (1.7) 8.3 yellow stellate 1.081 0.352 0.2 (0.2) 9.1 0.0 (0.0) 0.0 0.8 (0.6) 16.7 lightly colonized 2.574 0.092 3.4 (1.9) 27.3 0.0 (0.0) 0.0 0.5 (0.5) 8.3 * = 0.0001 Abundance values were assessed using a one-w ay A N O V A to test for site differences ( a = 0.05). Fisher’s Least Significant D ifference (LSD ) test was used to test mean com parisons. Across each row, means follow ed be the same letter are not significantly different. Significant differences in the abundance of some morphotypes occurred between the three peatland site types (Table 2.3). Mycelium radicis atrovirens (p = 0.001) and Thelephoraceae 1 (p = 0.0001) were most abundant in the Bs site type and least abundant 52 in the Mix and BsLt site types, respectively. In contrast, brown smooth 2 (p = 0.023), Lactarius (p = 0.011), and Tomentella-Yike 2 (p = 0.006) were most abundant in the BsLt site type, and least in the Bs site type. Several other morphotypes occurred in some peatland site types, but not in others. Granular brown was abundant in the Mix and BsLt site type, but absent in the Bs site type, and Thelephoraceae 2 and brown inky clamp were frequently identified in the Mix and Bs site types, and absent in the BsLt site type. The remaining less common or rarely occurring morphotypes (found on less than 4 seedlings) tended to be found in only one or two of the peatland site types. Tamarack (Larix laricina) ectomycorrhizas Of the 23 morphotypes characterized from tamarack, 21 morphotypes were found in the Mix peatland site type, and 16 morphotypes in the BsLt site type. The 13 most common morphotypes (occurring on four or more seedlings) on tamarack included ectomycorrhizas in the genera Suillus, Amphinema, Tomentella, MRA, and Cenococcum (Table 2.4), as well as several morphotypes that could not be assigned to a family or genus (brown silvery, woolly brown, brown smooth 1 and crystal net brown). All commonly occurring morphotypes were found in both the Mix peatland site type as well as the BsLt site type; however, Suillus 2 and Cenococcum were identified most frequently on all tamarack seedlings (58% and 38%, respectively). Crystal-net brown, brown silvery, MRA, and Tomentella-Wke, 2 were more abundant (although not significant) in the BsLt site type, than the Mix site type. In contrast, Suillus 2 (p = 0.041), Cenococcum, woolly brown, and Amphinema were more abundant in the Mix site type compared to the 53 BsLt site type. The remaining less common, or rarely seen morphotypes were mostly described from the Mix site type (Table 2.4). Table 2.4. Site effect, percent abundance (mean ±SE in parentheses) and frequency of occurrence (%) of ectomycorrhizal morphotypes of tamarack growing in two peatland M ix B sL t (n = 12) (n = 12) M orp h otyp e F A m phinem a 3.415 brown clamp 1.114 brown silvery 3.289 0.083 brown sm ooth I 0.489 0.492 brown sm ooth 2 1.000 0.328 2.7 (2.7) 8.3 0 .0 (0.0) 0.0 C enococcum 0.153 0.699 7.1 (2.7) 50.0 10.2 (7.3) 25.0 coffee brown 1.000 0.328 0 .0 (0.0) 0.0 2.1 (2.1) 8.3 crystal net-brown 2.202 0.152 0.8 (0.5) 16.7 8.9 (5.5) 41.7 E-strain 1.836 0.189 1.3 (0.9) 16.7 5.8 (3.2) 25.0 granular brown 0 .000 0.989 2.8 (1.7) 25.0 2 .8 (2.7) 16.7 H ebelom a-like 2 .156 0.156 1.0 (0.6) 25.0 0 .0 (0.0) 0.0 Lactarius 1.000 0.328 0 .0 (0.0) 0 .0 0.5 (0.5) 8.3 M RA 1.697 0.206 4.1 (2.7) 25.0 14.1 (7.1) 33.3 R ussula 0.654 0.427 0.8 (0.6) 25.0 2.7 (2.3) 25.0 Suillus 1 1.231 0.298 3.1 (3.1) 8.3 0 .0 (0.0) 0.0 P A b u n dan ce Freq A b u n dan ce 0.078 5.3 (2.4) 41.7 0.7 (0.66) 8.3 0.303 4.2 (4.0) 16.7 0 .0 (0.0) 0.0 0 .0 (0.0) 8.3 10.4 (5.7) 41.7 1.9 (1.9) 8.3 4.5 (3.1) 33.3 Freq Suillus 2 4.732 0.041 22.6 (7.3)a 66.7 6.0 (2.2)6 50.0 Thelephoraceae 1 3.211 0.087 0.4 (0.3) 25.0 0 .0 (0.0) 0.0 Thelephoraceae 2 2.163 0.156 2.2 (1.5) 16.7 0 .0 (0.0) 0.0 Thelephoraceae 3 3.564 0.072 0.5 (0.2) 25.0 0 .0 (0.0) 0.0 Tom entella-W ke 1 0.216 0.647 0.8 (0.6) 25.0 1.2 (0.6) 33.3 Tom entella-U ke 2 1.904 0.181 1.0 (0.7) 16.7 5.5 (3.2) 41.7 w oolly brown 2.805 0.108 5.2 (2.8) 33.3 0.5 (0.5) 8.3 yellow stellate 1.000 0.328 0.5 (0.5) 8.3 0.0 (0.0) 0.0 lightly colonized 0.375 0.546 3 1 .7 (8 .3 ) 83.3 24.3 (8.9) 50.0 Abundance values were assessed using a one-w ay A N O V A to test for site differences ( a = 0.05). Amongst the 17 shared morphotypes between tamarack and scrub birch, five of these were identified as commonly occurring on both host species (i.e. Cenococcum, granular brown, MRA, Tomentella-XDno, 2, and woolly brown). Four others were only 54 common on tamarack {Amphinema, brown smooth 1, crystal net brown, and Tomentellalike 1), and five were only common on scrub birch {Lactarius, Thelephoraceae 1, Thelephoraceae 2, Thelephoraceae 3, and brown smooth 2); three were uncommon, or rare, for both host species {Russula, E-strain, and yellow stellate). Interestingly, the most abundant morphotype found on tamarack, Suillus 2, was never found on any of the scrub birch seedlings. All shared morphotypes were present on both hosts in at least one of the two peatland site types in which they co-occurred (with the exception of brown smooth 1 that was found only on scrub birch in the Bs site type). The majority of shared morphotypes were found on tamarack and scrub birch in the Mix site type. Table 2.5 shows the site, host, and interaction effects for the percent abundance of 15 shared morphotypes between tamarack and scrub birch. Several morphotypes had significant site and host differences. Amphinema (p = 0.034), Thelephoraceae 2 (p = 0.025), and woolly brown (p = 0.025) morphotypes were significantly more abundant in the Mix site type, than in the BsLt site type. Thelephoraceae 3 was also more abundant in the Mix site type, but the difference was not significant (Table 2.6). Tomentella-Vike 2 (p = 0.044) was the only shared morphotype significantly more abundant in the BsLt site type; Lactarius was also more abundant in this site, although not significant (p = 0.069) (Table 2.6). 55 Table 2.5. Two-way ANOVA showing site (BsLt and Mix), host (scrub birch and tamarack) and interaction effects based on mean percent abundance of 15 shared ectomycorrhizal morphotypes (a = 0.05, df = 1, 42). Site Effect Host Effect Host*Site F P F P F P Amphinema brown smooth 2 Cenococcum crystal net brown E-strain granular brown Lactarius MRA Russula Thelephoraceae 1 Thelephoraceae 2 Thelephoraceae 3 Tomentella-Wke 1 4.786 1.125 0.010 1.923 2.816 0.381 5.068 2.145 0.789 2.816 5.795 4.101 0.481 0.034 0.295 0.923 0.173 0.101 0.540 0.030 0.150 0.380 0.101 0.021 0.049 0.492 2.814 9.382 0.103 2.717 2.136 0.394 12.300 2.609 0.376 2.136 1.632 4.299 3.699 0.101 0.004 0.750 0.107 0.152 0.534 0.001 0.114 0.543 0.152 0.208 0.044 0.061 1.435 Z478 0.451 2.100 1.729 0.407 4.749 0.886 0.261 1.729 1.632 2.914 0.033 0.238 0.123 0.505 0.155 0.196 0.527 0.035 0.352 0.612 0.196 0.208 0.095 0.857 Tomentella-Wke 2 5.194 0.028 7.332 0.010 1.553 0.220 woolly brown 5.220 0.027 0.191 0.665 0.032 0.859 Morphotype Note: M ean percent abundance were tested using a 2-w ay A N O V A . Brown smooth 1 was not included in the analysis since the morphotype only occurred in the Bs peatland site type and yellow stellate was not included due to low abundance values. With respect to host differences, brown smooth 2 (p = 0.004), Lactarius (p = 0.002), Tomentella-Wke 2 (p = 0.013), and Thelephoraceae 3 (p = 0.055) morphotypes were all significantly more abundant on scrub birch compared to tamarack when host abundance values were pooled for peatland sites types (Table 2.7). Tomentella-Wke 1 was also more abundant on tamarack (p = 0.057) compared to scrub birch. One interaction effect was observed for Lactarius (p = 0.035) (Table 2.5); this was possibly due to its dominance in the BsLt peatland site type and on scrub birch, since it was only detected on one tamarack seedling. 56 Table 2.6. One-way ANOVA showing site (BsLt and Mix) differences for percent abundance (mean ±SE) of 15 shared ectomycorrhizal morphotypes (a = 0.05, df = 1, 44). Morphotype P Amphinema brown smooth 2 0.034 0.374 Cenococcum crystal net brown 0.945 BsLt 0.340 (0.3) 11.758 (4.8) Mix 3.430 (1.4) 6.498 (3.4) 8.014 (2.9) 0.167 7.651 (4.4) 4.633 (2.9) 0.475 (0.3) E-strain 0.169 3.178(1.7) 0.685 (0.5) granular brown 0.552 2.836(1.6) 4.164(1.5) Lactarius 0.069 0.145 17.731 (6.9) 4.034 (2.5) 9.015 (3.9) 2.778 (1.5) Thelephoraceae 1 0.360 0.119 1.938 (1.3) 0.086 (0.1) 0.698 (0.4) 2.087 (1.3) Thelephoraceae 2 Thelephoraceae 3 0.025 0.072 0.000 (0.0) 4.648 (2.0) 0.250 (0.3) 3.028 (1.5) Tomentella-Uke 1 0.494 0.732 (0.4) 0.413 (0.3) Tomentella-Wke 2 0.044 13.795 (4.4) 4.046 (1.8) woolly brown 0.025 0.776 (0.6) 5.841 (2.1) MRA Russula Note; Brown sm ooth L was not included in the analysis since this morphotype only occurred in the Bs peatland site type and yellow stellate was not included in analysis due to abundance values. 57 Table 2.7. One-way ANOVA showing host (tamarack and scrub birch) differences for percent abundance (mean ±SE) of 15 shared ectomycorrhizal morphotypes (a = 0.05, df = 1,44). Morphotype P Scrub birch Tamarack Amphinema 0.117 0.682 (0.4) 2.992 (1.3) brown smooth 2 0.004 17.600 (5.4) 1.361 (1.4) Cenococcum 0.745 6.942 (3.5) 8.649 (3.8) crystal net brown 0.115 0.088 (0.1) 4.815(2.8) E-strain 0.675 3.816(0.2) granular brown 0.528 4.237(1.6) 2.844(1.7) 2.825 (1.6) Lactarius MRA 0.002 22.503 (7.2) 2.406 (1.1) 0.229 (0.2) 9.096 (3.9) 0.882 (0.6) 1.718 (1.2) 0.224 (0.1) Russula Thelephoraceae 1 0.118 0.539 0.162 Thelephoraceae 2 0.233 2.027 (1.3) 3.642 (2.0) Thelephoraceae 3 0.055 3.179(1.6) 1.117(0.8) 0.226 (0.1) Tomentella-hke 1 0.057 0.116(0.1) 0.991 (0.4) Tomentella-like 2 0.013 15.111 (4.5) 3.246(1.7) woolly brown 0.675 3.816(1.8) 2.844 (1.5) Note: Brown sm ooth 1 was not included in the analysis since this morphotype only occurred in the Bs peatland site type and yellow stellate was not included in analysis due to abundance values. Ectomycorrhizal community diversity According to all diversity indices, ectomycorrhizal community diversity was highest in the Mix sites for both host species. For scrub birch, ectomycorrhizal diversity decreased from the Mix, to the Bs sites, with the lowest diversity occurring in the BsLt peatland site type (Table 2.8). The Simpson index showed significant differences (p = 0.020) between the peatland site types for this host species; the Shannon index also showed strong differences, although these were not significant. No significant differences in ectomycorrhizal diversity were detected between peatland site types for 58 tamarack, although all indices suggested that diversity was greater in the Mix compared to the BsLt peatland site types (Table 2.9). Table 2.8. One-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) comparing peatland site types for scrub birch (a = 0.05, df = 2, 31). Diversity Index F P Bs BsLt Mix Margalef Shannon Evenness Shannon 2.301 0.403 3.108 0.117 0.672 0.059 0.602 (0.085) 0.718 (0.075) 1.046 (0.120) 0.489 (0.057) 0.644 (0.077) 0.764 (0.094) 0.777 (0.129) 0.737 (0.077) 1.209(0.157) Simpson 4.446 0.020 2.774 (0.285)o6 1.932(0.185)6 3.265 (0.43l)a D iversity values are means (±SE in parentheses). Fisher’s Least Significant D ifference (LSD ) test was used to determine where significant differences between means occurred. M eans follow ed be the same letter are not significantly different. Table 2.9. One-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) comparing peatland site types for tamarack (a = 0.05, df = 1, 22). Diversity Index F P BsLt Mix Margalef Shannon Evenness Shannon Simpson Z235 1.829 0.208 0.166 0.206 0.658 0.813 (0.115) 0.698 (0.071) 1.138 (0.136) 0.157 0.700 0.594 (0.088) 0.635 (0.087) 0.896 (0.137) 2.396 (0.330) 3.497 (0.633) Diversity values are means (±SE in parentheses). When ectomycorrhizal diversity indices were assessed for scrub birch and tamarack on sites where they co-occurred, ANOVA showed significant site effects (Table 2.10). Species richness (Margalef Index, p = 0.029), as well as the Shannon (p = 0.021) and Simpson Indices (p = 0.011), indicated greater diversity in the Mix compared to the BsLt peatland site type (Table 2.11). Shannon Evenness values were similar between site types. No significant host or interaction effects were detected (Table 2.10). 59 Table 2.10. Two-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) showing comparison between peatland site types (BsLt and Mix), host (tamarack and scrub birch), and interaction effects (a = 0.05, df = 1, 42). Site Effect Host effect Host*Site Diversity Index F P F F F F M argalef Shannon Evenness Shannon 6.802 0.780 7.119 I.7I9 0.401 1.040 0.197 0.530 0.314 0.186 0.315 1.168 0.668 0.577 0.286 Simpson 7.272 0.013 0.382 0.011 0.010 1.037 0.314 0.156 0.695 Table 2.11. One-way ANOVA for diversity indices (Margalef, Shannon Evenness, Shannon, and Simpson) for combined host species showing comparison between two peatland site types, (a = 0.05, df = 1, 44) Diversity Index F F BsLt Mix Margalef Shannon Evenness Shannon 5.128 1.217 5.769 0.029 0.276 0.021 0.769 (0.084) 0.724(0.051) 1.149 (0.102) Simpson 7.015 0.011 0.544 (0.053) 0.639 (0.057) 0.832 (0.084) 2.174 (0.196) 3.308 (0.381) Diversity values are means (±SE in parentheses) and include values for both tamarack and scrub birch. DISCUSSION Ectomycorrhizal morphotype frequency and abundance This study presents some of the first information available on ectomycorrhizal colonization for scrub birch in peatland ecosystems. It also extends our knowledge on tamarack mycorrhizal associations, as well as on potential fungal linkages in peatland sites. Overall, 30 ectomycorrhizal morphotypes were characterized from the two host species, with 23 and 24 morphotypes found on tamarack and scrub birch, respectively. In similar studies investigating multiple host species, it appears that ectomycorrhizal 60 species richness can vary considerably. For example, Kranabetter et al. (1999) examined three different conifer seedling species (lodgepole pine (Pinus contorta var. latifolia), white spruce (Picea glauca) and subalpine fir (Abies lasiocarpa)) planted on the edges of forest gaps, and found 74 morphotypes, with an average of 52 morphotypes per host species. In contrast, an investigation into the fungal symbionts of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) revealed only 11 morphotypes for those two hosts, with seven morphotypes found on both Douglas-fir and paper birch (Simard et al., 1997a). Jones et al. (1997), also studying Douglas-fir and paper birch, identified 43 morphotypes on the two host species three years after outplanting; 26 ectomycorrhizal morphotypes were described on paper birch seedlings, and 32 morphotypes on Douglas-fir seedlings. Interestingly, the number of morphotypes described by Jones et al. (1999) for paper birch is similar to the number found on scrub birch in our study. Studies investigating single ectomycorrhizal host species also show variation in the number of morphotypes identified. Robertson (2003) described 33 morphotypes on naturally regenerating black spruce (Picea mariana) seedlings growing in both peatland and upland habitats. Mah et al. (2001) reported similar species richness, with 24 morphotypes occurring on naturally regenerating and planted hybrid spnjce (Picea glauca X engelmannii) seedlings in disturbed (cut and burned), as well as undisturbed. Sub-boreal Spruce habitats. When non-mycorrhizal hybrid spruce seedlings were out- planted onto a cut block, 15 distinct morphotypes were identified within one year of planting (Hagerman et al., 1999). Japanese larch (Larix kaempferi) seedlings that were harvested from a naturally regenerated volcano in Japan exhibited 12 different 61 ectomycorrhizal morphotypes (Yang et al., 1998). Interestingly, prior to the catastrophic eruption disturbance, the volcano was dominated by an Erman birch {Betula ennanii) forest. Reasons for the differences in ectomycorrhizal richness amongst studies could be due to the differences in seedling age, in sample size or intensity, and in host receptivity to fungal species, as well as variation in environmental conditions across the sampling sites (Robertson, 2003). Numbers of characterized ectomycorrhizas and species richness values presented in this study for tamarack or scrub birch growing in peatland environments generally agree with those described by Robertson (2003), Mah et al. (2001) and Jones et al. (1997) for other host species growing in a variety of different habitats in British Columbia. One of the most abundant and frequently occurring groups of ectomycorrhizal roots was the lightly colonized; some lightly colonized roots occurred on 67% of all tamarack seedlings, but only 15% of scrub birch seedlings. This was especially so for tamarack seedlings in both peatland site types. This group represented a large portion of the ectomycorrhizal community, especially for tamarack, that could not be identified. Many of these roots most likely were weakly colonized examples of the already identified morphotypes, but they could not be distinguished morphologically. Some roots may have been colonized by ectomycorrhizal fungi that were not identified in this study. Robertson (2003) also reported a large portion (66.7%) of black spruce seedlings, harvested from wetland and upland sites, to have some level of non-mycorrhizal or lightly colonized roots. Mah et al. (2001) found lightly colonized roots on almost all hybrid spruce seedlings growing in disturbed and mature forest sites, with approximately 18% of all root tips sampled to be poorly colonized. 62 Tamarack {Larix lancina) morphotype frequency and abundance Many of the 23 ectomycorrhizal morphotypes described on tamarack might be described as intermediate to broad host ranging fungi (Molina et al., 1991). They included such genera as Ampliinema, Cenococcum, E-strain, members of the Russulaceae (Russula), and Thelephoraceae (including Tomentella). Some of these fungi were often relatively abundant on tamarack and many have been described on other host species. Robertson (2003) and Mah et al. (2001) identified ectomycorrhizas in these fungal genera/families on black spruce seedlings growing in wetland and upland sites, as well as on hybrid spruce seedlings in disturbed and mature sites, respectively. Jones et al. (1997), in a greenhouse and field bioassay study, and Simard et al. (1997a), in a soil bioassay greenhouse study, also described many of these fungi on paper birch growing in single species monoculture, or in mixed species dual culture, with Douglas-fir. These intermediate or broad host ranging fungal species have the potential to not only contribute substantially to ectomycorrhizal functioning, but also to linkages within forest ecosystems (Massicotte et al., 1999). Other studies have investigated the ectomycorrhizal fungal symbionts of tamarack, as well as other Larix spp., growing in different habitat types. Cenococcum, Estrain, Hebeloma, Suillus, and Thelepliora were all reported to occur on tamarack, European larch (Larix decidua), and/or western larch (Larix occidentalis) (Laiho, 1965; Malloch and Malloch, 1981; Molina and Trappe, 1982; LeTacon and Bouchard, 1986; Samson and Fortin, 1986; Thormann et al., 1999). These genera (or closely related members) were identified on tamarack roots from our study. 63 The most abundant and frequently occurring morphotype for tamarack was Suillus 2, followed by Cenococcum. Suillus 2 was identified on 66.7% of all tamarack seedlings, and this rhizomorphic morphotype represented 22.6% of the entire ectomycorrhizal community for tamarack in the Mix peatland site type. Although this morphotype was also found on many seedlings in the BsLt site type, it was never as abundant. The genus Suillus is known to have a narrower host range, and prefers to associate with members of the Pinaceae, including Pinus and Pseudotsuga, as well as Larix spp. (Molina et al, 1992). For example, Suillus grevillei was found to be highly specific to western larch {Larix occidentalis) (Melin, 1922; M olina and Trappe, 1982), whereas S. cavipes often associates with European larch (Larix eurolepis) (Finlay, 1989). Sidllus 2 was a dominant ectomycorrhizal fungi in the Mix peatland site type, which included black spruce as an ectomycorrhizal host species. Even though Suillus primarily associates with Pinus and Larix spp., it has been documented that black spruce can form ectomycorrhizas with some species, such as 5. granulatus (Browning and Whitney, 1991) and S. cavipes (Stein et al., 1990) following inoculation. However, Suillus was not identified on black spruce in these Mix sites (Robertson, 2003), nor on scrub birch seedlings in any of the peatland site types. In addition, the literature does not report this genus on any other birch species. Black spruce may play a greater role in the abundance and frequency of other ectomycorrhizal fungal species occurring in these site types. Another frequently occurring morphotype unique to tamarack was brown silvery; this ectomycorrhiza was almost exclusively retrieved from the BsLt peatland site type. The identity of this ectomycorrhiza remains unknown and, although it had no clamps and no rhizomorphs, we cannot exclude it from the Basidiomycetes. Brown clamp, coffee 64 brown, and Hebeloma-Vike ectomycorrhiza were also primarily found to associate with tamarack and were often found on only one site type. Even though some of these morphotypes could not be identified to the family or genus level, and were only found in small numbers, they still contributed to the ectomycorrhizal species richness for the peatland site types. Scrub birch {Betula glandulosa) morphotype frequency and abundance This study was able to characterize 24 ectomycorrhizal morphotypes for scrub birch; many could also be considered to have intermediate to broad host specificity and included such fungi as Amphinema, Cenococcum, E-strain, MR A, Lactarius, numerous species in the Thelephoraceae (including Tomentella), as well as several Russulaceae (including Russula). Many of these also occurred on tamarack, and most occurred in all or two of the peatland site types. Robertson (2003) described many of these fungi as also occurring on black spruce seedlings growing in wetland and upland habitats. However, there were seven morphotypes that were unique to scrub birch (black cystidia, cotton orange, silver white, white clamp, white felted, brown inky clamp, and a Russulaceae), as well as a Lactarius (one exception on tamarack). Although most of these morphotypes were infrequent, two types (brown inky clamp and Lactarius) occurred both frequently and abundantly. Brown inky clamp shared some morphological features with Lactarius, but laticifers were never observed and emanating hyphae were wider and clamped. The genus Lactarius is generally considered to have a narrow to intermediate host range, with approximately a quarter of the species associating with a broad array of ectomycorrhizal hosts; these include members of the Pinaceae (i.e. Picea, 65 Pinus and Larix) and Betulaceae (i.e. Betula and Alnus) (Molina et al., 1992). Interestingly, very few tamarack roots were colonized by Lactarius in all the peatland site types, even though the host genus is known to associate with these fungi. There were also some similarities between ectomycorrhizal fungal species identified on other Betula spp. and fungi identified on scrub birch. Jones et al. (1997) and Simard et al. (1997a) reported numerous fungal genera {Amphinema (only identified by Jones et al. (1997)), Cenococcum, E-strain, Hebeloma, Lactarius, MRA, Russula (only identified by Jones et al. (1997)), and Thelephora) on paper birch seedlings; fungi in all of these genera were also identified on scrub birch from our study. Miller (1982) investigated the ectomycorrhizal symbionts associated with swamp birch {Betula nana) growing in the sub-alpine tundra of Alaska; three of the fungal genera {Hebeloma, Lactarius, Russula) identified in his study were found to associate with scrub birch as well. Although these two Betula species share similar growth forms (i.e. low-lying shrub) and habitat requirements (i.e. wetlands such as fens and bogs), only three out of the five fungal genera identified as associating with swamp birch were found on scrub birch in our study. The differences may be partly attributed to the fact that Miller (1982) identified ectomycorrhizal fungi from sporocarps fruiting near the host and assumed these to be ectomycorrhizal with swamp birch. However, characterization was not performed on the birch roots and sporocarp occurrence is not always an accurate measure of ectomycorrhizal species richness belowground (Mehmann et al., 1995; Gardes and Bruns, 1996; Dahlberg, 1997; Dahlberg, 2001). Scrub birch seedlings in the BsLt and Bs peatland site types had fewer ectomycorrhizal morphotypes, but several species dominated each of the two peatland 66 site types. Three morphotypes dominated scrub birch seedlings in the BsLt site type, Lactarius, brown smooth 2, and Tomentella-Vike 2. Interestingly, Robertson (2003) identified two Lactarius morphotypes on black spruce growing in the same Mix peatland sites with tamarack and scrub birch. Lactarius was also present on scrub birch in the Mix and Bs site types, but it did not dominate those sites. The Lactarius morphotype identified on scrub birch was the most abundant species in the BsLt site, suggesting a high level of host specificity on this site. In the Bs peatland site type, two other morphotypes, MRA and Thelephoraceae 1, dominated scrub birch. No single morphotype appeared to dominate scrub birch or tamarack in all three peatland site types in which each host occurred. Robertson (2003) found that many Thelephoraceae and Tomentella morphotypes on black spruce were predominantly identified in the wetland compared to the upland habitats. In addition, MRA was found on one third of all her wetland black spruce seedlings. Fewer potential ectomycorrhizal host species were present in the BsLt site (scrub birch and tamarack), and the Bs site was solely composed of birch (with a negligible component of black spruce, but generally coniferous species were absent). The observed decrease in the number of ectomycorrhizal morphotypes (species richness) on these two sites may be closely associated with the reduction of host species. As well, the frequency and abundance of several morphotypes seemed to greatly increase when fewer fungal species were present on the scrub birch seedling root systems. This may account for a small decrease in evenness in these sites when compared to the Mix site type. Jones et al. (1997) found that when paper birch and Douglas-fir were planted in mixtures, evenness values for the ectomycorrhizal types present on the roots of the two hosts, increased. 67 Morphotype frequency and abundance by peatland site type Tamarack and scrub birch both exhibited the highest number of morphotypes in the Mix peatland site type, when compared to the other site types in which they occurred. Host species planted in mixture have been reported to influence the frequency, abundance, and the proportion of ectomycorrhizas associating with the co-occurring species (Simard et al., 1997a; Massicotte et al., 1999). Jones et al. (1997) determined that when paper birch and Douglas-fir were planted together, an increase occurred in the abundance of the minor morphotypes on Douglas-fir. It is possible that with the increase of ectomycorrhizal host species (i.e. black spruce) in the Mix site, tamarack and scrub birch had the potential to associate with a wider array of fungal species that may not have been present in the sites with fewer hosts. Robertson (2003) found the ectomycorrhizal species diversity was greater (though not significant) on black spruce growing in the Mix site type than in pure black spruce wetland habitats. Potential for sbared ectomycorrhizal fungal symbionts Over half of the morphotypes (53.3%) characterized in this study were found on both tamarack and scrub birch, and have the potential for forming fungal linkages for carbon transfer between host species. The majority of morphotypes that were shared between the hosts were found on seedlings growing in both the Mix (scrub birchtamarack-black spruce) peatland site type and the BsLt (scrub birch-tamarack) site type, suggesting that these peatland sites have a good possibility of supporting fungal linkages between the two hosts. 68 A number of studies have investigated the potential for shared ectomycorrhizal fungal species between different host species using isotope tracers and morphological characterization techniques. Most notably, Simard et al. (1997b) used gaseous, pulse labeled, C'^ and C*”*, to demonstrate the bi-directional carbon transfer between paper birch and shaded Douglas-fir seedlings via shared fungal symbionts. This study and others (Bjorkman, 1960; Finlay and Read, 1986; Finlay, 1989; Dahlberg and Stenlid, 1990; McKendrick et al., 2000) provide additional evidence to support the hypothesis that common mycorrhizal symbionts associated with different host species can form hyphal linkages, or mycelial networks, for the transport of carbon between plants. Ectomycorrhiza characterization is a commonly used indirect method for establishing the potential for mycelial networks between host species; although it may not provide as conclusive evidence as the tracer technique, morphotype characterization can determine if two or more hosts are able to form mycorrhizal associations with the same fungal species. Prior to the use of isotope tracers, Simard et al. (1997) characterized seven morphotypes, out of a total of 11 identified fungal species, shared between paper birch and Douglas-fir (Simard et al., 1997). Jones et al. (1997) reported that five of the six most common morphotypes found on out-planted paper birch and Douglas-fir seedlings were shared between the hosts. In our study, six morphotypes belonged to those that occurred frequently on both hosts. Nine others, although shared, were often disproportionately more abundant (or occurred more often) on one of the two host. For example, brown smooth 2, Lactarius, and Tomentella-Vike 2 had much higher abundance values on scrub birch compared to tamarack. Kranabetter et al. (1999) investigated multiple host species (lodgepole pine, white spruce, and subalpine fir) seedlings planted 69 on mature-forest edges, and determined that 47% of the ectomycorrhizal community colonized all three conifer species. In a bioassay study examining the ectomycorrhizas from plants grown in mixed-pot cultures, Massicotte et al. (1999) reported that 14 morphotypes, from a total of 18 identified, were found to associate with two or more host species; hosts included grand fir {Abies grandis), tanoak {Lithocarpus densiflora), ponderosa pine {Pinus ponderosa), Douglas-fir, and madrone {Arbutus menziesn). The present study, and those cited, all suggest that shared mycorrhizal fungi may be the normal situation, rather than the exception, in many forest ecosystems, including peatlands. Ectomycorrhizal diversity Overall, for the peatland site types examined in this study, ectomycorrhizal diversity was always greatest in the Mix sites, compared to the BsLt and Bs peatland site types for tamarack and scrub birch. This difference was significant for diversity indices (except the Shannon evenness) when values for host species were pooled. For separate hosts, the Mix site type was also the most diverse, but differences were only significant for scrub birch (Simpson Index). With respect to scrub birch, ectomycorrhizal diversity decreased from the Mix to the Bs, with the BsLt peatland site type having the lowest. The Bs and BsLt sites were similar, in that they both had fewer fungal species, with several that appeared to dominate each of these habitats. In the bioassay study by Massicotte et al. (1999), similar numbers of morphotypes were retrieved from both monoculture treatments (host species growing in single culture), as well as mixed (four hosts per pot) species cultures; however, in most cases, more 70 morphotypes were identified from .the mature stands (more potential ectomycorrhizal hosts) compared to clearcut sites. Results for ectomycorrhizal community diversity on black spruce support the findings in this study; Robertson (2003) found higher ectomycorrhizal diversity in the tamarack-black spruce mix wetland habitat compared to the black spruce dominated wetland sites. Since the peatland site types generally did not differ in soil or moisture regimes, differences in fungal species richness are most likely due to variations in the vegetation and ectomycorrhizal host species composition. Dwarf shrub and grass species varied across the three peatland site types. The BsLt site type contained one ericaceous plant species, compared to five species in the other two site types. Although Poaceae spp. were observed in all the peatland site types, grasses were particularly common in the Bs sites. Perhaps the absence or presence of AM grasses and/or ericoid shrubs has also influenced the level of ectomycorrhizal diversity within the peatland site types. Neighboring plants have been reported to influence the frequency of occurrence and abundance of mycorrhizal development (Simard et al., 1997a; Jones et al., 1997). More ectomycorrhizal host species were available for colonization in the Mix site, which could account for the higher number of fungal species; or perhaps a greater fungal inoculum potential existed in this site type and allowed for the establishment of more host species. Van der Heijden et al. (1998) suggested that AM fungi species composition and diversity below-ground, may have the potential to determine plant biodiversity above­ ground, in a natural ecosystem. However, given that black spruce exhibited approximately 19 morphotypes in this site type, it is more likely that the additional host species contributed more potential fungal symbionts for tamarack and scrub birch. The 71 addition of black spruce to the mixture of tamarack and scrub birch appears to have increased the possibility of potential linkages via shared fungi between these two hosts; this supports the concept of companion plants influencing the ability of ectomycorrhizal fungi to colonize neighboring plants (Molina et al., 1992; Massicotte et al., 1994). Although site type appeared to have a significant effect on the ectomycorrhizal diversity between the Mix and BsLt peatland site types, diversity between the two hosts did not appear to differ. Similar numbers of morphotypes were identified for both tamarack and scrub birch and, for both hosts, these showed a decrease from the Mix to the BsLt habitats (Bs sites having intermediate values for scrub birch). Morphological analysis of tamarack and scrub birch ectomycorrhizas resulted in the characterization of 30 morphotypes. Some morphotypes were found on both hosts, suggesting a high potential for shared fungal linkages, whereas others were unique to either tamarack or scrub birch. Both hosts appear to be equally receptive to a wide range of ectomycorrhizal fungi. In addition, several morphotypes were site-specific, as well as more abundant in certain peatland site types. Ectomycorrhizal diversity was highest in the Mix peatland site type for both hosts; however, for scrub birch, the Bs sites were more diverse compared to the BsLt site type. Our results indicate that these peatland environments appear to be similar to upland terrestrial forest ecosystems in regards to ectomycorrhizal abundance, frequency and diversity. 72 LITERATURE CITED Anderson, A.J. 1988. Mycorrhizae: Host specificity and recognition. Phytopathol. 78; 375-378. Bjorkman, E. 1960. Monotropa hypopithys L.: An epiparasite on tree roots. Physiol. Plantarum, 13: 308-327. Browning, M.H.R. and Whitney, R.D. 1990. 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Morphological and molecular assessment of ectomycorrhizal communities associating with black spruce {Picea mariana (Mill.) BSP) in wetland and upland forests in central British Columbia. M.Sc. thesis. University of Northern British Columbia, Prince George. Samson, J., and Fortin, J.A. 1986. Ectomycorrhizal fungi of Larix laricina and the interspecific and intraspecific variation in response to temperature. Can. J. Bot. 64: 3020-3028. Schelkle, M., and Peterson, R.L. 1996. Suppression of common root pathogens by helper bacteria and ectomycorrhizal fungi in vitro. Mycorrhiza, 6: 1861-1870. Simard, S.W., Perry, D.A., Jones, M.D., Myrold, D.D., Durall, D.M., and Molina, R. 1997. Net transfer of carbon between ectomycorrhizal tree species in the field. Nature, 388: 579-582. 75 Smith, S.E., and Read, D.J. 1997. Mycorrhizal symbiosis (2"‘*éd.). Academic Press, Cambridge. Stein, A., Fortin, J.A., and Vallee, G. 1990. Enhanced rooting of Picea mariana cuttings by ectomycorrhizal fungi. Can. J. Bot. 68: 468-470. Thormann, M.N., Currah, R.S., and Bayley, S.E. 1999. The mycorrhizal status of the dominant vegetation along a peatland gradient in southern boreal Alberta, Canada. Wetlands, 19: 438-450. Tilman, D., Wedin, D., and Knops, J. 1996. Productivity and sustainability influence biodiversity in grassland ecosystems. Nature, 379: 718-720. Turner, S.D., and Friese, C.F. 1998. Plant-mycorrhizal community dynamics associated with a moisture gradient within a rehabilitated prairie fen. Restor. Ecol. 6: 44-51. Turner, S.D., Amon, J.P., Schneble, R.M., and Friese, C.F. 2000. Mycorrhizal fungi associated with plants in ground-water fed wetlands. Wetlands, 20: 200-204. Tyrell, L.E., and Boemer, R.E.J. 1987. Larix laricina and Picea mariana: Relationships among leaf life-span, foliar nutrient patterns, nutrient conservation, and growth efficiency. Can. J. Bot. 65: 1570-1577. Ursic, M., Peterson, R.L., and Husband, B. 1997. Relative abundance of mycorrhizal fungi and frequency of root rot in Pinus strobus seedlings in a southern Ontario nursery. Can. J. For. Res. 27: 54-62. Van der.Heijden, M.G.A., Klironomos, J.N., Ursic, M., Moutoglis, D., Streitwolf-Engel, T, Boiler, T., Wiemken, A., and Sanders, I.R. 1998. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature, 396: 69-72. Yang, G., Cha, J.Y., Shibuya, M., Yajima, T., and Takahashi, K. 1998. The occurrence and diversity of ectomycorrhizas of Larix kaempferi seedlings on a volcanic mountain in Japan. Mycol. Res. 102: 1503-1508. 76 Molecular analysis of ectomycorrhizal associations of Larix laricina (Du Roi) (tamarack) K. Koch and Betula glandulosa Michaux (scrub birch) in peatlands of central British Columbia. ABSTRACT Tamarack and scrub birch are ectomycorrhizal hosts often found growing in the wet, nutrient poor, peatland ecosystems of British Columbia. Fungal linkages can allow for carbon and nutrient transfer between hosts that share the same symbionts. Molecular analysis (PCR-RFLP) of 326 tamarack and 360 scrub birch root tips was used to assess genetic diversity of ectomycorrhizal fungi associating with tamarack and scrub birch in three peatland site types (scrub birch-tamarack-black spruce (Mix), scrub birch-tamarack (BsLt), and scrub birch (Bs) only) in central BC, and to determine the potential for fungal linkages between the two hosts. Twenty-six of 30 described morphotypes (plus the lightly colonized) generated fragment patterns that were classified into 69 distinct genotypes (38 for tamarack, and 43 for scrub birch). Suillus 2 on tamarack and Lactarius on scrub birch appeared host specific and each contained five genotypes; many morphotypes had two or more genotypes. Twelve genotypes from 10 morphotypes were shared between the hosts. One genotype each, belonging to silver white, Suillus 2, and Lactarius (plus brown silvery and yellow stellate) matched sporocarp fragment patterns for Cortinarius, Hebeloma, and Hygrocybe, respectively. More genotypes were on both hosts in the Mix compared to the BsLt sites; BsLt and Bs sites contained similar numbers for scrub birch. However, site differences in molecular diversity were not significant as measured by the Phi index. Similarities between scrub birch and tamarack genotypes and several sporocarps, suggest a high probability for fungal linkages in these peatland ecosystems. 77 INTRODUCTION Ectomycorrhizal fungi are an integral part of a forest ecosystem; they serve as symbiotic partners in mutualistic relationships with the roots of many gymnosperm and angiosperm species (Smith and Read, 1997; Amaranthus, 1998). These fungi provide nutrients, such as nitrogen and phosphorous, and water to host plants in exchange for fixed carbon. The resultant underground network of hyphae can serve as linkages for the movement of nutrients and carbon between the same or different host plant species that share the same fungal symbionts (Bjorkman, 1960; Finlay and Read, 1986; Dahlberg and Stenlid, 1990; Simard et al., 1997b; McKendrick et al., 2000). Considering the possibility that emanating hyphae from numerous different ectomycorrhizal fungi can travel through the rhizosphere, and contact and colonize roots from neighboring trees and shrubs (Read, 1987), it has been hypothesized that plant-to-plant nutrient transfer could be a common occurrence in ecosystems (Newman, 1988). Although many questions still remain concerning the relationship between ectomycorrhizal fungi and plant community structure, advances in molecular research have provided a crucial step towards the identification of the fungal species involved in these complex systems (Egger, 1995; Horton and Bruns, 2001). The amplification of minute quantities of ribosomal DNA from colonized roots, using the polymerase chain reaction (PCR) technique, followed by restriction fragment length polymorphism (RFLP) analysis, allows for species identification through compaiison of restriction fragment patterns to those existing in reference databases (Egger, 1995; Horton and Bruns, 2001; Mah et al., 2001; Bruns and Bidartondo, 2002; Robertson, 2003). When combined with 78 morphological characterization of ectomycorrhizas, molecular analysis of fungal DNA can be a powerful tool to separate fungal taxa, as well as to determine genotypic variation within taxa (Horton and Bruns, 2002; Sakakibara et al., 2002). Several recent studies that have used molecular methods to describe the ectomycorrhizal composition and diversity of seedlings growing under different treatment regimes include Hagerman et al. (1999), Baldwin (1999), Mah et al. (2001), and Robertson (2003). Horton and Bruns (1998) investigated ectomycorrhizal fungi of Douglas-fir {Pseudotsuga menziesii) and bishop pine {Pinus muricata) and used molecular methods to determine potential hyphal linkages between the two host species. They concluded that since the two hosts were found to associate with a majority of the same fungal symbionts in the study site, that these two host species may have been connected by fungal mycelia and perhaps shared similar capabilities for resource acquisition. Many plants growing in nutrient-poor and water-saturated peatland ecosystems seem able to withstand a wide range of environmental and physiological stresses (MacKenzie and Moran, 2003). Tamarack {Larix laricina) and scrub birch {Betula glandulosa), two ectomycorrhizal hosts that often occur together, also appear to be able to tolerate the conditions of peatland environments. The extent of the ectomycorrhizal colonization in peatlands for these two hosts, and whether this facilitates adaptation to such environments, is largely unknown. With respect to the genetic composition of the ectomycorrhizal fungal community associated with these tamarack and scrub birch in peatlands, this has not been investigated at the molecular level. Recently however, Robertson (2003) used molecular techniques to determine the genotypic variation of the 79 fungal symbionts of black spruce {Picea mariana), another commonly occurring peatland species. The study described the number of fungal genotypes associated with black spruce growing in both peatland and upland sites, and provided insight into the ectomycorrhizal community in these environments. The first objective of this study was to use PCR-RFLP analysis to describe and compare the molecular diversity and genotypic variation of the ectomycorrhizal fungi associating with tamarack and scrub birch growing in three different peatland site types in central British Columbia. The three peatland site types included i) scrub birch dominated, ii) mixed scrub birch-tamarack, and iii) mixed scrub birch-tamarack-black spruce. The second objective was to determine, using the genotypic information, the potential for fungal linkages between tamarack and scrub birch in these peatland ecosystems. In addition, my goal was to compare the fungal genotypes identified for these two hosts with the results from a previous study on black spruce that were sampled from the same mixed scrub birch-tamarack-black spruce peatland sites, to determine the potential for further linkages. METHODS Ectomycorrhizal root selection and DNA extraction From the 200 roots tips per seedling sampled for morphological assessment, a proportional number of tips (10%) of each mycorrhizal morphotype (a total of 20 tips per seedling) were selected for molecular analysis (Mah et al., 2001; Robertson, 2003). Individual root tips were stored in 1.5 ml microtubes at -20°C until processed. A modified Zolan and Pukkila (1986) hexadecyltrimethyl ammonium bromide (CTAB) 80 protocol was used to extract the fungal DNA from the mycorrhizal root tips, as well as from sporocarp samples (Baldwin and Egger, 1996). Using glass micromortars and micropestles, individual frozen root tips were crushed cold (-20°C) in 350 ml CTAB extraction buffer (5 M NaCl (Sigma), 1 M Tris-HCL (pH 8) (Invitrogen)), 0.5 M ethylenediaminetetraacetic acid (EDTA) (Invitrogen), 10% CTAB, and 0.2% (3- mercaptoethanol (Sigma), transferred into sterile 1.5 ml microtubes, and incubated at 60°C in a water bath heat block (VWR Scientific) for 45-60 min. Tubes were removed from the heat block, 350 pL of a chloroform (BDH): isoamyl alcohol (24:1) solution (Fisher Chemicals) was added to each, and briefly voitexed. Tubes were then centrifuged (Hermle, Mandel Scientific Co. Ltd.) at 13000 x g for 10 min at room temperature. The top aqueous layer was transferred to a new sterile microtube and 350 pL of cold (-10°C) absolute isopropanol (BDH) was added. The solutions were mixed by inverting the microtubes several times over 1 min, and then placed in a -10°C freezer overnight. Prior to a second centrifugation at 13000 x g for 10 min, the tubes were again inverted several times. The aqueous phase was poured off and the remaining DNA pellet was washed twice with 175 pL of cold (-10°C) 70% ethanol then centrifuged at 13000 x g for 3 min. The tubes were left to dehydrate overnight in a dessicator, and then the dried pellet was resuspended in 50 pL of Tris-EDTA buffer and stored at -20°C. DNA amplification and restriction endonuclease digestion The extracted DNA samples were subjected to the polymerase chain reaction (PCR) in order to amplify an approximate 1,100 bp fragment of nuclear-encoded ribosomal DNA (rDNA) gene repeat. The fungal specific primer, NL6Bmun (CAA GCG 81 TTT CGC TTT CAA CA) (Egger, 1995), and the universal primer, IT Sl (TCC OTA GOT GAA CCT GCG G) (White et al., 1990), were used to amplify the target region (3' end of the 18S small subunit to the 5' end of the 28S large subunit rDNA gene, including both internal transcribed spacer (ITSl and ITS2) regions). A single PCR reaction master mix consisted of 16.5 pL millipore H 2O, 2.9 pL lOX PCR buffer (Invitrogen), 2.9 pL dNTP (Invitrogen) mixture (containing equal amounts of 100 pM dATP, dCTP, dGTP and dTTP), 2.3 pL MgClz (25 pM) (Invitrogen), 1.2 pL of each primer (10 pM) (Gibco BRL), and 3.0 pL Taq DNA polymerase (Gibco BRL). While working on ice, 27 pL of PCR master mix was added to a 0.2 ml microtube containing 3 pL of either a 1:10 dilution of ectomycorrhizal DNA or a 1:50 dilution of sporocarp DNA. Tubes were placed in a PTC-100™ Programmable Thermal Controller (MJ Research, Inc.) and underwent the following program: dénaturation at 94°C for 30 s and 93°C for 35 s, annealing at 50-52”C for 53 s and extension at 72°C for 5 min. Following amplification, 5 pL of PCR product was mixed with 1.8 pL of lOX xylene cyanole loading buffer (Sigma). A 150 ml 0.7% agarose gel (0.7 g agarose in 100 ml of Tris-borate (TBE) buffer) (Gibco BRL), containing 11 pL of ethidium bromide (fluorescent stain for DNA visualization), was submerged in a gel box of TBE buffer. In the first well of the agarose gel, 5 pL of Hind III DNA ladder (Invitrogen) was loaded; 4 pL of each DNA sample were loaded in subsequent wells. Gels were run at approximately 90-110 mV for 35-45 min. Once complete, gels were visualized under UV light using a Gel Print 2000i photodocumentation system (Bio/Can Scientific), photographed, Mitsubishi thermal paper (K65H Mitsubishi Electronic Corp.). and printed on Samples that did not amplify (or produced faint bands) were re-amplified in an attempt to improve resolution. 82 Samples that appeared to contain DNA from more than one fungal species (double bands) were eliminated from the analysis. The resulting PCR product was cleaved at specific sites using three restriction endonucleases for restriction fragment length polymorphism (RFLP) analysis; Alul (AGCT), HinU. (GANTC), and Rsal (GTAC) (Invitrogen). While working on ice, 6.3 pL millipore H^O, 0.8 pL of either lOx React® 1 or React® 2 assay buffer (Invitrogen), 0.5 pL of one of the three restriction enzymes, and 7 pL of PCR product was added to a 0.2 mL microtube. This procedure was repeated for each restriction enzyme. Tubes were incubated in a 3 T C oven for 5 h or overnight. Following incubation, a 2.5% L.M.P (low melting point) (Invitrogen) agarose gel (1 g agarose and 1.5 g L.M.P. agarose in 100 ml lOX TBE) containing 11 pL of ethidium bromide (lOmg/mL) was submerged in a gel box containing TBE. To each digestion microtube, 4 pL of loading buffer (bromophenol blue and glycerol) (Sigma) was added. The L ‘, 15“’, and 30“’ wells contained 5 pL of Ikb ladder (Invitrogen); remaining wells contained the digestion samples. Gels ran at 90-100 mV for 2.5 to 3 h, and were then visualized under UV light, photographed, and images saved to disk using the BioPhotonics Gel Print 2000i system. Partial and incomplete digests were removed from the data set and were not re-digested. Molecular analysis RFLP gel images were imported into Gene Profiler, Version 4.05 (Scanalytics, Inc.), a genotyping and DNA fragment analysis software. Individual restriction fragments were selected and their bp size calibrated against the Ikb ladder standards (1018, 514, 356, 344, 298, 220, 201, 154, 134, and 75 bp fragments) using the Desmile 83 calibration method with log piecewise linear curve-fitting. Fragments of 75 bp or less were not included in the analysis. Once all fragments were marked, fragment patterns for individual samples (keeping the two hosts separated) were imported and sorted into both seedling and morphotype databases created in Database Manager, Version 4.05 (Scanalytics, Inc.). Sporocarp fragment patterns were compiled into a separate fungal database. Pairwise comparisons of all band patterns were made for each database; a 5% match tolerance was set to obtain fragment pattern similarity values for every sample pair and for each restriction enzyme. The neighbor-joining/unweighted pair-group method with arithmetic means (UPGMA) option in PHYLIP (Phylogeny Inference Package), Version 3.573c, (Felsenstein, J., University of Washington) was used to perform UPGMA cluster analysis on the resulting similarity matrix. To examine host species and site ectomycorrhizal community structure, individual ectomycorrhizal morphotype databases were merged to create an all-inclusive morphotype database for each host species. The sporocarp database was merged with each tamarack and birch all-inclusive ectomycorrhizal morphotype database to determine if sporocarp fragment patterns matched with ectomycorrhizal fragment patterns. Resulting phylograms were viewed in TreeView, Version Win 3.2 (1998, Roderick DM Page). The D ice’s index (Dice, 1945) was used to match pairs of ectomycorrhizal root tip band patterns and to create a distance matrix for each pair of samples in order to calculate Phi Index values for an estimation of genetic diversity within each ectomycorrhizal morphotype, and between peatland site types for each host species (Mah et al., 2001; Khetmalas et al., 2002; Robertson, 2003). 84 The fragment pattern base pair sizes were imported into a Microsoft® Excel 2000, Version 9.0, spreadsheet to aid in the classification of genotypes, as well as possible identification of lightly colonized morphotypes. Fragment patterns were sorted by morphotype, and then grouped into genotypes based on their molecular weights (5% tolerance of similarity), as well as their position in the neighbor-joining phylogram. Matching fragment patterns were averaged for each genotype for each host species; patterns for both hosts were compared to determine shared genotypes. Fragment patterns within morphotypes that did not match any of the determined genotypes, as well as those within the lightly colonized morphotype group, were compared to all other fragment patterns to determine their placement and possible identification. Morphotype RFLP databases, and all related matrices, were modified according to the above changes. To determine genetic diversity. Phi index values were calculated from fragment patterns from individual seedlings for the commonly occurring ectomycorrhizal morphotypes, as well as for those morphotypes shared by both host species, and for each peatland site type. The index values range from 0-1, where a higher Phi value implies greater diversity. For the four BsLt and Mix sites where the two hosts co-occurred, a two-way ANOVA was used to test effects of peatland site type and host species on genetic diversity (a = 0.05). A one-way ANOVA (Statistica version 6.1, 2002, StatSoft, Inc.) was used to compare the genetic diversity between site types in which each host occurred (a = 0.05). Mean comparisons were tested using Fisher’s Least Significant Difference (LSD) test (a = 0.05). 85 RESULTS Amplification and digestion success rates From a total of 1048 ectomycorrhizal root tips, 686 (65%) were successfully amplified and digested (Table 3.1). This represented 326 (76%) of all tamarack root tips and 360 (58%) of all scrub birch root tips. The morphotypes brown smooth 1 and brown smooth 2 on tamarack, and woolly brown and lightly colonized tips on scrub birch roots exhibited the lowest amplification success rates (Table 3.1). In addition. E-strain (one tip) and Russulaceae (two tips) on scrub birch did not amplify. In contrast, the woolly brown and lightly colonized roots on tamarack had a high amplification success rate even though the lightly colonized root tips lacked a developed mantle. Table 3.1 shows a summary of the root tip sample size for each morphotype and the success rate for DNA amplification and digestion. Phylogenetic analysis of ectomycorrhizal root tips Successful rDNA amplification and digestion of tamarack and scrub birch ectomycorrhizal root tips yielded fragment patterns that were used to determine differences between and within morphotypes, and to identify genotypes. In total, 69 distinct genotypes were generated from 26 of the 30 morphotypes (plus the lightly colonized group) that were described (Chapter 2) for tamarack and scrub birch. 86 T able 3.1. Summary of ectomycorrhizal root tip DNA amplification (PCR) and digestion (RFLP) success rates (%) from tamarack and scrub birch seedlings. (n) Morphotype Tamarack Amplification Digestion rate (%)** rate (%) 14 11 26 13 7 21 6 12 14 14 3 1 34 9 - 929 63.6 88.5 38.5 42.9 85.7 83.3 100.0 64.3 100.0 100.0 100.0 61.8 100.0 929 63.6 8A6 23.1 42.9 71.4 83.3 100.0 64.3 100.0 100.0 100.0 47.1 100.0 - - - - - 8 69 4 5 3 7 30 87.5 84.1 75.0 100.0 66.7 100.0 80.0 75.0 826 25.0 100.0 66.7 85.7 70.0 - - - (n) rate (%)** rate (%) 5 3 51 7 68 35 8 15 1 15 80.0 66.7 78.4 42.9 82.4 85.7 75.0 100.0 0.0 86.7 80.0 66.7 - - 85 52 2 2 5 84.7 65.4 100.0 0.0 100.0 49.0 0.0 622 65.7 37.5 66.7 0.0 86.7 56.5 61.5 100.0 0.0 100.0 - - - - - - 69.5 74.5 71.4 50.0 69.6 85.2 90.9 21.4 100.0 22.2 62.7 528 57.1 50.0 49.3 66.7 90.9 21.4 100.0 222 68.1* 5&5* Arnphinema black cystidia brown clamp brown inky clamp brown silvery brown smooth 1 brown smooth 2 Cenococcum coffee brown cotton orange crystal net brown E-strain granular brown Hebeloma-Vike Lactarius MRA Russula Russulaceae silver white Siiillus 1 Suillus 2 Thelephoraceae 1 Thelephoraceae 2 Thelephoraceae 3 Tomentella-like 1 Tomentella-Wkt 2 white clamp white felted woolly brown yellow stellate lightly colonized - - - 13 2 101 92.3 100.0 90.1 84.6 100.0 82.2 59 51 21 4 69 27 11 14 2 9 Total/mean* 427 83.2* 76.9* 621 - **includes ectom ycorrhizal root tips which exhibited ectomycorrhizal root tips that exhibited double bands. 87 weakly Scrub birch Amplification Digestion am plified bands - and excludes From the 23 morphotypes (plus the lightly colonized group) characterized for tamarack, 38 genotypes (fragment patterns) were generated. Six uncommon morphotypes occurring on tamarack produced poor fragment patterns that could not be used in the analysis. These included brown smooth 1, brown smooth 2, coffee brown, Lactarius, Thelephoraceae 1, and Hebeloma-like. One to five patterns were identified within each morphotype and those generally varied in one or more restriction endonucleases (Table 3.2). In some cases, such as Arnphinema, variation only occurred in one or two fragments. Five morphotypes (excluding the lightly colonized category) were unique to tamarack: Suillus 2 (five genotypes), brown silvery (three genotypes), Estrain (two genotypes), Suillus 1 (one genotype) and brown clamp (one genotype). It should be noted that one sample of E-strain did occur on scrub birch, but it did not amplify. The remaining morphotypes that were found on tamarack each contained one or more genotypes that were shared with scrub birch; however, not all fragment patterns in these morphotypes were common to both hosts. Fragment patterns for the lightly colonized root tips (84 tips of those successfully amplified and digested for tamarack) were compared to established genotypes. Of these, 35 matched patterns for Suillus 2, ten were placed with crystal net brown, five were placed in Tomentella-Yxks. 2, and three were placed with other morphotypes. The remaining 16 were sorted into the three lightly colonized genotypes; 15 could not be placed and remained as unknowns. Twenty-one of the 24 scrub birch morphotypes produced 43 genotypes. Within each morphotype, fragment patterns varied from one to five, with the most genotypes occurring in Lactarius (five) (Table 3.2). Interestingly, the morphotypes Suillus 2 and 88 Lactarius, unique to tamarack and scrub birch, respectively (with the exception of Lactarius one root tip on tamarack that did not produce a fragment pattern), had the most genotypes and were the most dominant morphotypes found on the two hosts. Six morphotypes that were only found on scrub birch produced the following numbers of genotypes: black cystidia (one), brown inky clamp (three), cotton orange (one), silver white (one), white felted (two) and white clamp (one). Three other morphotypes that were also found on tamarack in small numbers only produced fragment patterns for scrub birch: brown smooth 2 (three genotypes), Lactarius (five genotypes), and Thelephoraceae 1 (two genotypes). The remaining scrub birch morphotypes generated patterns of which some, for each morphotype, were shared with tamarack. When genotypes were compared at the 5% tolerance level, some that belonged to different morphotypes had very similar fragment patterns (Table 3.2). For example, brown inky clamp (genotype 2) matched white clamp (genotype 1), E-strain (genotype 1) matched MRA (genotype 1), Tomentella-like 2 (genotype 1), yellow stellate (genotype 1), and Thelephoraceae 2 (genotype 3) shared similar fragment patterns, as did brown silvery (genotype 2), Lactarius (genotype 2), and yellow stellate (genotype 3). No attempt was made to merge or re-assign these genotypes (Table 3.2). Phylogenetic trees based on the restriction fragment patterns from ectomycorrhizal root tips were created for each host species; these aided in the classification of genotypes (Appendix IV and V). With the exception of Suillus 2, groups within the tamarack phylogenetic tree were not well defined, since the branch clusters often contained more than one morphotype (Appendix IV). Suillus 2 consisted of five genotypes, four of which formed distinct groups in the first half of the tree. Suillus 1, 89 Russula, brown stringy and woolly brown did not share their branches with any other morphotype. Some clusters included genotypes from several different morphotypes, whereas others contained only one morphotype. For scrub birch, Thelephoraceae 2 formed several very tight branches that separated from the rest of the samples (Appendix V). Neither MRA nor Cenococcum grouped with other morphotypes compared to crystal net brown, white clamp and brown inky clamp genotypes that clustered together on the same branches. Interestingly, several Thelephoraceae 2 samples grouped with Lactarius. Genotype distribution within peatland site types With respect to genotypes that were successfully generated from tamarack root tips, 27 occurred within the Mix (scrub birch-tamarack-black spruce) peatland site type compared to 22 in the BsLt (scrub birch-tamarack) site type; 11 genotypes were present in both peatland site types (Table 3.2). Within ectomycorrhizal morphotypes that were found in both peatland site types, the genotypic distribution sometimes varied. Almost one third of the morphotypes (Suillus 2, Cenococcum, granular brown, Tomentella-Vike 1, Tomentella-like 2, and woolly brown) exhibited one or two genotypes that occurred in both the BsLt and Mix site types. In some cases, one or two additional genotypes within these morphotypes were site specific (Table 3.2). Three morphotypes (MRA, crystal net brown, and E-strain), although found in both site types, produced genotypes that were site-specific, that is to say, each genotype was only identified from one or the other site type, never both. Although 12 genotypes belonging to eight morphotypes (brown silvery. Russula, Amphinema, brown clamp, Thelephoraceae 2, Thelephoraceae 3, Suillus 1, and yellow stellate) appeared to show some specificity to one or the other peatland site type. 90 these often belonged to morphotypes that were either only found in one site type, or to morphotypes for which root tips on the corresponding site type failed to produce fragment patterns (Table 3.2). With respect to scrub birch genotypes, almost twice as many fragment patterns were identified in the Mix site type (33), compared to the BsLt (18) or Bs (17) sites (Table 3.2). Only four genotypes were present in all three peatland site types; Cenococcum (genotype 1), Lactarius (genotype 5), and Tomentella-Vike. 2 (genotype 1 and 2). Although five morphotypes {Cenococcum, Lactarius, Thelephoraceae 2, Tomentella-like 2, and yellow stellate) occurred in all peatland site types, they produced genotypes that occurred mostly in one, or in a combination of two of the site types. Ten morphotypes generated genotypes that only occurred in two of the three peatland types; some of these genotypes were found in both site types and others in only one of the two site types. The remaining six morphotypes, and their genotypes, were site-specific, only occurring within one peatland site type. As with tamarack, some genotype-peatland specificity was due to morphotypes occurring only in one site type, or to a loss of fragment patterns during PCR/RFLP analysis (Table 3.2). 91 T able 3.2. Approximate fragment sizes of the amplified ITS region for ectomycorrhizas from tamarack (Lt) and scrub birch (Bs) seedlings occurring in three peatland site types (scrub birch dominated (B), scrub birch and tamarack (L), and scrub birch, tamarack, and black spruce (M)). M orp h o ty p es H ost U n d igested A p p ro x im ate F ragm en t S izes (bp) L t. Bs (n) S ize (bp) genotype 1 M M ,B 13 840 585 190 110 330 280 165 genotype 2 M 4 845 350 190 110 325 290 155 2 855 410 190 80 320 220 155 2 915 670 42 0 350 300 285 180 165 105 430 150 1000 an d G en otyp es Alu\ Hinfi Rsa\ Arnphinem a 150 825 210 780 180 black cystidia M genotype 1 130 980 brown clamp genotype 1 M 1000 brown inky clamp genotype 1 M 8 830 355 190 150 130 “genotype 2 M ,B 9 925 400 245 190 115 295 220 165 genotype 3 B 4 765 245 175 150 90 415 240 180 130 335 165 145 345 255 115 385 600 200 100 395 350 165 105 440 200 110 165 130 160 105 170 110 brown silvery genotype 1 L 815 300 225 185 ‘’genotype 2 L 820 355 185 150 genotype 3 L 890 390 200 185 140 340 165 150 125 865 115 brown sm ooth 2 genotype 1 M,L 11 975 390 225 190 115 305 215 165 150 1020 genotype 2 L 10 955 700 190 135 115 350 250 160 150 565 genotype 3 L 3 750 560 185 150 115 315 180 165 100 410 92 250 Morphotypes and Genotypes Undigested Host Approximate Fragment Sizes (bp) Aliil Size (bp) Lt Bs (n) M ,L M ,L, B 19 770 410 150 110 280 165 genotype 2 M 5 750 450 160 120 285 170 130 940 genotype 3 M,L 6 755 405 150 115 345 240 145 870 3 950 380 185 135 335 205 170 950 605 185 130 345 220 170 295 220 165 Hinn Rsal C enococcum genotype 1 genotype 4 M ,L 110 125 95 155 85 135 890 1030 cotton orange B genotype 1 865 175 crystal net brown genotype 1 M 21 885 405 245 185 genotype 2 L 3 940 355 185 150 genotype 3 L 6 985 540 210 165 M 105 110 150 885 1005 42 0 200 160 365 295 300 195 150 115 90 420 350 310 175 155 120 985 220 175 150 130 980 granular brown genotype 1 M 3 1020 400 215 165 genotype 2 M 4 780 410 190 90 M ,L 90 genotype 3 M ,L 15 915 395 190 130 340 280 170 155 975 genotype 4 M 4 985 805 190 115 295 240 165 150 1000 "^genotype 1 M 3 935 355 260 185 515 175 155 135 735 180 genotype 2 L 5 930 45 0 190 150 300 270 205 125 345 270 E-strain 93 240 105 Host Morphotypes and Genotypes Lt Undigested Bs (n) Size (bp) A p p ro xim ate F ragm en t S izes (bp) Alul H /«fl Rsal Lactarius genotype 1 L 5 1010 345 230 160 115 475 275 195 195 800 ’’genotype 2 M ,L 17 900 355 185 150 120 95 345 260 170 100 435 205 genotype 3 M,L 10 800 350 185 150 130 110 285 175 160 100 410 355 genotype 4 M 3 99 0 430 275 185 115 355 320 170 155 965 genotype 5 M,L, B 32 1070 525 280 185 110 390 350 170 150 1045 940 550 250 185 340 195 165 130 235 160 lightly colonized genotype 1 L 10 genotype 2 M 3 750 655 130 115 440 215 165 355 180 genotype 3 M 3 965 660 350 115 415 275 185 7 10 190 “'genotype 1 M 3 975 360 265 190 510 160 150 135 775 185 genotype 2 L 115 335 240 125 115 825 445 165 450 250 480 265 85 9 20 M RA 115 85 9 1035 725 185 genotype 3 B 20 880 575 180 110 415 295 genotype 4 B,M 6 855 575 135 115 655 340 genotype 1 M,L 910 415 180 145 29 0 270 210 315 290 M 935 400 190 145 330 285 165 765 175 940 795 190 115 22 0 195 170 195 R ussula silver white genotype 1 S u illu s 1 genotype 1 M 94 105 1030 255 115 Morphotypes în o ty p es Host Lt Undigested Bs (n) S ize (bp) Approximate Fragment Sizes (bp) A lu l H in fl « sa l Suillus 2 genotype 1 M 10 1050 350 250 185 145 115 415 285 230 165 1005 genotype 2 L,M 8 92 0 345 250 185 160 110 410 255 165 145 960 genotype 3 L,M 6 905 350 250 150 110 270 230 180 165 1015 genotype 4 L,M 36 91 0 350 245 185 110 415 165 145 genotype 5 L,M 30 875 515 185 110 240 205 165 320 220 150 135 100 935 185 795 180 845 180 Thelephoraceae 1 genotype 1 B 3 885 395 190 155 110 'genotype 2 B,M 26 1045 495 285 190 110 365 345 165 150 1025 360 345 165 150 1065 95 Thelephoraceae 2 B 10 1055 490 285 190 115 M ,B 16 930 385 190 150 115 95 325 220 160 140 ‘‘genotype 3 M ,L 5 1010 405 195 155 110 95 350 330 170 160 890 genotype 4 M 4 900 465 225 190 110 90 1005 2 1080 650 350 M,L 935 385 185 120 M 870 350 190 120 820 365 185 'genotype 1 genotype 2 genotype 5 M M 115 890 310 160 145 715 285 260 110 350 320 165 150 1015 110 350 325 170 155 845 345 315 170 155 930 980 Thelephoraceae 3 genotype 1 M genotype 2 Tomentella-Vike. 1 genotype 1 M ,L 95 175 Host Morphotypes and Genotypes Lt U n d igested Bs Approximate Fragment Sizes (bp) Alul (n ) S ize (bp) 25 885 395 185 120 8 78 0 40 0 175 135 6 93 0 395 190 140 Hinn Rsal Tom entella -like 2 ‘‘genotype 1 genotype 2 MX MX MX, B M,L, B genotype 3 M M,L genotype 4 L 3 785 580 190 155 genotype 5 L 5 855 770 195 120 95 335 210 165 235 185 85 350 300 115 320 150 1005 160 140 905 155 135 860 185 170 120 325 225 130 115 690 90 175 435 white felted genotype 1 M 3 3 875 365 185 130 115 350 295 165 150 425 340 genotype 2 M 3 72 0 53 0 21 0 145 115 220 205 170 95 200 160 M 3 15 930 405 245 190 110 300 220 170 155 990 M 13 795 280 250 190 350 180 150 325 225 170 155 355 335 165 150 585 335 245 160 105 425 105 white clamp “genotype 1 w oolly brown genotype 1 MX 990 yellow stellate ‘‘genotype 1 L,B 4 1040 395 190 155 genotype 2 M 2 855 585 205 190 2 880 355 185 150 ’’genotype 3 M 115 125 95 110 Genotypes that shared similar fragment patterns betw een morphotypes are indicated by the sam e letter (a,b,c,d, or e). 96 1020 275 190 155 95 Ectomycorrhizal fragment pattern comparison between tamarack and scrub birch In addition to genotypes that were similar within (or amongst) morphotypes, some fragment patterns of tamarack and scrub birch ectomycorrhizal genotypes, when compared at an approximate 5% tolerance level, were also similar. In total, 12 genotypes belonging to 10 morphotypes appeared to occur on both host species. Restriction fragment patterns for genotypes that occurred on both hosts were averaged and the resultant fragment patterns appear in Table 3.2. They include: Amphinema (genotype 1), Cenococcum (genotype I), crystal net brown (genotype I), granular brown (genotype 3), Russula (genotype 1), Thelephoraceae 2 (genotype 2), Thelephoraceae 3 (genotype 1), woolly brown (genotype I), Tomentella-\\k.& 1 (genotype 1), and Tomentella-Wke 2 (genotypes 1, 2, and 3). Ectomycorrhizal fragment pattern comparisons with black spruce (Picea mariana) A study by Robertson (2003) examined black spruce ectomycorrhizas in two of the same peatland sites as the present study (“T” black spruce-tamarack wetland sites (Robertson, 2003) = “M ix” scrub birch-tamarack-black spruce peatland sites). By assessing the database information on ectomycorrhizal fragment patterns from her study, black spruce genotypes were compared to those for tamarack and scrub birch. Interestingly, scrub birch shared eight genotypes with black spruce in these sites, whereas tamarack shared only two fragment patterns (Table 3.3). Only one of these genotypes was found on all three hosts (woolly brown, from tamarack and scrub birch, matched an Amphinema identified on black spruce). Some of these fragment patterns contain one or two fragments that varied between host species; however, given that individual fragment 97 pattern selection can be subjective, and that standards can vary between users and between experiments, these genotypes were considered to be very similar. 98 Table 3.3. Approximate fragment sizes of the amplified ITS region of ectomycorrhizas that were potentially shared between hosts (scrub birch (Bs), tamarack (Lt), and black spruce (Sb)). Host Shared Undigested Morphotype Cenococcum Species Sitef Sb Approximate Fragment Sizes (bp) Hinn A liil 440 150 110 80 275 165 130 100 920 750 450 160 120 285 170 130 940 370 170 155 350 180 150 1085 990 Size (bp) 790 R sal Cenococcum' Bs Amphinema woolly brown' Sb Bs/Lt + + 920 795 275 280 240 250 Tomentella-Mke 3' granular brown" Sb Bs + + 875 780 415 410 185 120 110 190 90 90 220 190 165 150 220 175 150 130 980 980 T h e le p h o r a c e a e -iik e l ' Sb 950 420 185 150 110 95 Thelephoraceae l ' Bs 885 395 190 155 110 95 320 225 165 150 320 220 150 855 175 845 180 Amphinema Sb 940 365 235 150 125 100 335 285 165 110 775 175 silver white' Bs 935 400 190 145 110 330 285 165 155 765 175 Amphinema Sb 940 365 190 140 110 325 295 165 155 780 175 white felted’ Bs 875 365 185 130 115 350 295 165 150 425 340 Russulaceae 2 Sb 950 690 190 110 335 290 165 150 555 brown smooth 2" Bs 955 700 190 350 250 160 150 565 250 185 175 110 190 135 115 99 85 195 175 170 110 Host Shared Undigested Site Amphinema' Species Sb Lt Lactarius 2" Lactarius^ M orphotype Russulaceae l ' Approximate Fragment Sizes (bp) 370 A lu l 195 110 320 H in a 290 165 150 980 845 350 190 110 325 290 155 780 180 980 1070 520 525 190 115 85 280 185 110 340 390 320 350 165 Size (bp) 860 + ■ Sb + Bs + 155 170 150 R sa l 1055 1045 * = denotes genotype, t = indicates ectom ycorrhiza(s) cam e from a host in the shared M ix peatland site type (no + means the root tips originated from another site type. Note: The first morphotype in each pair is alw ays from the study on black spruce, Robertson (2003) with perm ission. T he second morphotype is from the present study. 100 Phylogenetic analysis of sporocarps Results from successful amplification and digestion of sporocarp samples are presented in Table 3.4. Species in the same genus, whose identity was uncertain, were given a “group” number. Approximately twice as many sporocarps/genotypes were identified for the Mix peatland site type (13), compared to the BsLt (6) and Bs (6) site types. In total, 19 restriction patterns were generated from 13 genera/families (n = 35) (Table 3.4). Although this likely represents only a small sub-sample of the potential sporocarps for these sites, when ectomycorrhiza and sporocarp fragment patterns were compared, several genotypes were determined to be very similar (Table 3.5). Matching patterns included silver white (genotype 1) on scrub birch and the fungus Cortinarius (group 1), as well as Suillus 2 (genotype 4) on tamarack and the fungus Hebelorna (group 1). In addition, a larger group of three morphotypes {Lactarius (genotype 2) on scrub birch, brown silvery (genotype 2) and yellow stellate (genotype 3) both on tamarack were all similar to the fungus Hygrocybe (group 1). 101 Table 3.4. Approximate fragment sizes (bp) of the amplified ITS region for sporocarps collected in Mix, BsLt and Bs peatland site types. Site Type Sporocarp Amanita vaginata Bs BsLt (n) 1 Size (bp) + 1 900 + + 8 5 845 + + Mix + Chroogomphus vinicolor Cortinarius spp. group 1 + + group 2 Entolomataceae Fuscoboletinus Undigested + A lu l 395 200 140 120 365 320 185 110 940 365 645 2 935 1 spectabilis Hebelorna sp. Hygrocybe spp. group 1 745 Approximate Fragment Sizes (bp) 1 1 1 190 120 175 340 165 150 795 875 175 175 855 175 110 350 170 155 290 190 145 100 970 525 195 125 390 345 165 630 235 195 915 335 245 185 170 825 350 140 150 1010 870 105 110 345 270 165 340 305 205 430 305 355 345 165 345 170 150 1 1025 830 540 285 530 185 190 145 110 group 1 1 1030 630 210 150 125 group 2 2 1025 140 Leccimim sp. Russula emetica 1 Russula emetica 2 1 1065 2 960 975 605 205 550 230 440 275 470 285 110 R sal 345 165 150 190 Laccaria laccata 915 185 985 group 3 925 355 165 110 145 150 195 375 280 group 2 95 H in tt 295 160 175 165 805 175 150 1045 685 175 Lactarius spp. 9 102 175 190 195 125 125 580 370 160 595 335 170 905 680 405 800 230 120 125 315 235 165 325 255 160 870 145 145 875 115 535 430 175 Site Type S porocarp Scutellinia Bs B sLt Undigested M ix + Approximate Fragment Sizes (bp) A lu l Hinn 190 120 480 335 160 785 190 120 785 195 125 290 235 165 605 215 165 (n) 1 Size (bp) 980 715 2 1 965 1030 R sal 940 sciitellata Suillus spp. group 1 group 2 + + +, indicates that sporocarps were collected from that site type. 103 105 1010 1005 Table 3.5. Approximate fragment sizes (bp) of the amplified ITS region for sporocarps and for closest ectomycorrhiza match. Samples originated from the Mix, BsLt, and Bs peatland site types. Site Type$ Morphotype Undigested Approximate Fragment Sizes (bp) A lu l silver white*' Bs Cortinarius spp.' 190 145 110 85 365 190 145 120 95 910 916 350 335 245 245 185 185 110 170 110 415 345 150 120 95 345 260 150 150 125 110 140 105 335 245 345 255 345 270 935 + + + + + + + 900 355 + 880 820 355 355 185 185 185 + 827 350 195 Suillus 2"* Hebelorna^ Lt Lactarius^ yellow stellate^ Bs Lt + brown silvery" Lt + Hygrocybe spp.' 400 H infl 330 285 165 155 340 165 152 165 165 R sal 765 175 795 175 145 150 935 870 185 175 170 100 160 105 165 105 435 205 160 425 440 190 200 155 160 165 430 305 175 *, superscript number denotes genotype (morphotype) or group (sporocarp) number, t = indicates host from which ectom ycorrhizas originated (Lt = tamarack, B s = scrub birch), t = indicates on which site type ectomycorrhiza(s)/sporocarp(s) were found. 104 Molecular diversity within ectomycorrhizal morphotypes Phi diversity values derived from the restriction fragment patterns for 17 commonly occurring and/or shared (found on both host species) ectomycorrhizal morphotypes are presented in Table 3.6. Values ranged between 0.002 (low intraspecific diversity) to 0.550 (high intraspecific diversity) and were not always similar for the same morphotype on the two hosts. On tamarack, Russula, Tomentella-Mke I, and Thelephoraceae 3 morphotypes each had only one genotype and exhibited the lowest Phi diversity values. Thelephoraceae 2 had the highest diversity value although it had only two genotypes and represented a small sample size. The next highest values were for crystal net brown, followed by Tomentella-Wke 2, each of which had three genotypes. Interestingly, Suillus 2, which had five genotypes, had an intermediate diversity value when compared to all other morphotypes. For scrub birch. Russula and Tomentella-Y\ke I morphotypes also had the lowest diversity values, as well as crystal net brown, each with one genotype. Lactarius (with five genotypes) had the highest Phi diversity values for this host, followed by the brown inky clamp (three genotypes) and MRA (two genotypes) morphotypes. When Phi diversity values were pooled for the shared morphotypes. Russula, Tomentella-Wke. 1, and Amphinema had the lowest value, compared to MRA, Thelephoraceae 2, and crystal net brown that exhibited the highest diversity. 105 Table 3.6. Phi diversity values for commonly occurring and shared (those found on both host species) ectomycorrhizal morphotypes on tamarack and scrub birch. Shared! B irch T am arack M orp h otyp e (n) genotypes* Phi (n) gen otyp es Phi (n) gen otyp es Phi A m p h in em a 13 2 0.131 4 1 0.157 17 2 0.137 brown inky clamp - - - 19 3 0.358 - - - brown silvery 18 3 0.220 - - - - - - brown smooth 2 - - - 19 3 0.309 - - - C enococcum 12 2 0.278 23 3 0.208 35 4 0.237 crystal net brown 20 3 0.355 10 1 0.010 30 3 0.291 E-strain 7 2 0.332 - - - - - - granular brown 15 2 0.182 11 3 0.207 26 4 0.199 5 0.361 Lactarius - - - 67 - - - MRA 12 2 0.153 26 2 0.311 38 4 0.515 R ussula 5 1 0.002 3 1 0.002 8 1 0.016 Suillus 2 84 5 0.225 - - - - - - Thelephoraceae I - - - 30 2 0.138 - - - Thelephoraceae 2 5 2 0.550 30 4 0.276 35 5 0.331 Thelephoraceae 3 2 1 0.137 12 2 0.294 14 2 0.282 T om entella-like 1 5 1 0.093 2 1 0.028 7 1 0.131 Tom entella-like 2 20 4 0.341 28 3 0 .1 9 0 48 5 0.286 t = pooled Phi values for those morphotypes on both tamarack and scrub birch, n = number o f root tips successfully am plified and used to calculate Phi value, * = number o f genotypes identified for each ectom ycorrhizal morphotype Note: lower Phi values suggest lower intraspecific diversity in that morphotype Peatland site type effects on ectomycorrhizal diversity In terms of genotypic diversity as measured by the Phi index, a two-way ANOVA showed no significant differences between peatland site types or between the two host species (Table 3.7). However, for both tamarack and scrub birch, mean Phi values were higher (although not significant) in the BsLt peatland site types, compared to the Mix site type with the highest diversity values for scrub birch within the Bs site type (Table 3.8). 106 Table 3.7. Two-way ANOVA showing site (BsLt and Mix), host (tamarack and scrub birch), and interaction effects based on Phi values for ectomycorrhizal genotypes (a = 0.05). Diversity index Phi Site Effect Host Effect Host*Site F P F P F P 1.857 0.245 0.274 0.628 0.0003 0.986 Table 3.8. One-way ANOVA showing Phi diversity values (mean ±SE) for ectomycorrhizal genotypes originating from tamarack and scrub birch from three peatland site types (a = 0.05). Site Host Species Tamarack Birch F 3.583 P 0.199 Bs 0.727 0.553 - BsLt 0.460 (0.043) Mix 0.376 (0.013) 0.474 (0.069) 0.428 (0.059) 0.341 (0.102) DISCUSSION Ectomycorrhizal genotypes, host speciHcity, and site distribution Overall, 69 distinct genotypes were identified from 26 successfully amplified morphotypes in this study for tamarack and scrub birch. This included 38 genotypes from 17 ectomycorrhizas on tamarack, and 43 genotypes from the 21 ectomycorrhizas on scrub birch. The number of genotypes identified are similar to those reported by Robertson (2003) and Sakakibara et al. (2002), who identified 65 genotypes from 29 ectomycorrhizal morphotypes on black spruce, and 26 genotypes form 11 morphotypes characterized on Douglas-fir, respectively. Mah et al. (2001) characterized 46 genotypes from 24 ectomycorrhizal morphotypes on hybrid spruce. 107 Some genotypes within a morphotype showed variation in only one of the restriction endonucleases, while other genotypes varied in two or more; genotypes of Lactarius show examples of both occurrences. Horton (2002) and Sakakibara et al. (2002) also report a similar range in genetic variation within their identified ectomycorrhizal morphotypes. Differences in the amount of genotypic variation in our study compare favourably to those by Mah et al. (2001) and Robertson (2003), but it is perhaps higher than that found in other studies, such as the one by Hagerman et al. (1999). Reported differences could be due to the number of seedlings studied or to the number of root tips analyzed in each of the studies. For example, our sample size resulted in 34 scrub birch compared to 24 tamarack seedlings, and the study by Robertson (2003) examined 45 black spruce seedlings. The number of root tips successfully digested for molecular analysis by Hagerman et al. (1999) was 38 compared to 686 in the present study, and to approximately 1276 by Mah et al. (2001). Some of the fungal genotypes belonging to morphotypes in our study appeared to be host and/or site specific. For example, three tamarack morphotypes (MRA, E-strain, and crystal net brown) produced genotypes that were found on both the BsLt and Mix site types, but individual genotypes were specific to one or the other peatland site type. Eleven tamarack genotypes were found on both site types; these included most Suillus 2, all Cenococcum, and several Tomentella genotypes. The remaining 26 genotypes for tamarack were found in only one of the two peatland site types; genotypes showing the greatest specificity belonged to brown silvery (only in the BsLt sites) and some Thelephoraceae (Mix sites). With respect to scrub birch, some genotypes within five morphotypes were found in all three of the peatland site types; at least one fragment 108 pattern in many of the remaining morphotypes occurred in two of the three peatland site types. A few genotypes mostly from rarely found morphotypes were often recorded from only one site (e.g. black cystidia and silver white). Robertson (2003) found that 54 of the 65 genotypes were retrieved from only one of three sites; however, some genotypes from Cenococcum, MRA, Russulaceae, Cortinariaceae, and E-strain ectomycorrhizas occurred on all three site types. Studies by Gehring et al. (1998), Jonsson et al. (1999), Mah et al. (2001), Sakakibara et al. (2002) also support this trend. The distribution of the numbers of genotypes in these sites for both tamarack and scrub birch was always highest in the Mix sites, followed by the BsLt site type, and finally the Bs site type (for scrub birch). In total, 50 genotypes were identified from the Mix peatland site type, compared to 34 from the BsLt sites. Only 17 genotypes were described from the Bs sites; this lower number might be due in part to one host instead of two being examined on this site. Although Robertson (2003) also described similar patterns of an uneven distribution of genotypes across sites (i.e. genotypes occurring in all sites vs in two sites vs only one), she reported equal numbers of genotypes (approximately 30) in each of the three habitats. Interestingly, Robertson (2003) identified numerous genotypes belonging to fungi in the family Thelephoraceae, and in the genera Tomentella and Lactarius, from her two wetland sites. These were also genotypes that often occurred in the present study. The decrease in genotypic variation on scrub birch in the BsLt (absence of black spruce) and Bs (absence of black spruce and tamarack) peatland site types could be due to fewer woody host species occurring on these sites. Although one might also expect to see a difference between the BsLt and Bs sites, but this was not observed. 109 When we examined the pooled results for fragment patterns for both hosts, the number of genotypes on the Mix and BsLt site types reflected the number of successfully amplified morphotypes. Twenty-four morphotypes (not including the lightly colonized group) generated 48 genotypes on the Mix site, decreasing to 16 morphotypes and 33 genotypes, respectively, for the BsLt site type. The Bs site had the least number of morphotypes (12) and, compared to the other two site types, had proportionately fewer genotypes (only 17). This decrease in genotypes may indicate that a lower ectomycorrhizal host diversity or a change in the plant community may be influencing the level of intraspecific variation expressed in the ITS region, resulting in a decrease in the number of genotypes exhibited by a given number of fungal species. Robertson (2003) suggested that genotype differences within and between sites might be due to localized heterogeneity in soil characteristics, site features, and vegetation composition in the peatland environments. Despite numerous examples of genotypic variation described between hosts and sites, when the genetic diversity between peatland site types was compared using the Phi index, no significant differences in diversity were detected. In fact, the Bs and BsLt peatland site types resulted in higher Phi values than the Mix site type. Mah et al. (2001) and Robertson (2003) also did not find significant differences when genotypic diversity was compared between sites using the Phi index. However, Robertson (2003) did find that Phi values were highest for genotypic diversity within the Mix peatland site type compared to the black spruce dominated wetland sites. 110 Ectomycorrhizas: Intraspecific variation Intraspecifie variation for the 26 morphotypes that generated fragment patterns varied between one and five genotypes. Combining results for both hosts (tamarack and scrub birch), 17 morphotypes plus the lightly colonized each had more than one genotype, and seven of these contained four or more genotypes. Suillus 2 and Lactarius ectomycorrhizas showed the most intraspecific variation expressed on a single host species, with five genotypes each. Thelephoraceae 2 (five), MRA (four), Tomentella-Vike 2 (four), granular brown (four) and Cenococcum (four) had the most genotypes expressed present on both host species. In most cases, an ectomycorrhizal morphotype had one or two dominant genotypes (representing higher numbers of ectomycorrhizal roots), with the remaining genotypes containing fewer samples in number and being more evenly distributed. Sakakibara et al. (2002) and Mah et al. (2002) also noted that some of the morphotypes that exhibited more than one fragment pattern tended to have a dominant pattern and other less frequently occurring patterns. The remaining nine morphotypes in our study expressed little variation, with only one genotype each. Some of these morphotypes were considered to be rare types, and were usually only represented by a few ectomycorrhizal root tip samples. The majority of morphotypes identified by Hagerman et al. (1999) exhibited only one RFLP pattern; however, this low genetic variability may have been due to the small sample size (38 roots representing 10 morphotypes) collected for molecular analysis. Genotypic variation in ectomycorrhizal species has been investigated in other studies. Robertson (2003) detected several morphotypes with large intraspecific variation (6-7 genotypes), including Amphinema, Cortinariaceae, and Russulaceae species. Mah et 111 al. (2001) identified multiple genotypes in the morphotypes of Amphinema and MRA, and Horton (2002) yielded multiple RFLP fragment patterns in Laccaria, Tricholoma, and Lactarius species. Even though our study sampled fewer seedlings for each host species compared to the above-mentioned studies, more morphotypes containing four or more genotypes were detected. Several reasons may partially explain differences in genotypic numbers for morphotypes. Fragment selection during RFLP analysis is somewhat subjective and selection protocols may vary among laboratories (e.g. fragment patterns can be manually marked by hand (Sakakibara et al., 2002), or one can utilize the software such as GeneProfiler for fragment pattern selection (Mah et al., 2001; Robertson, 2003). High intraspecific variation within the ITS region of some ectomycorrhizal morphotypes does exist; Horton (2002), Gardes et al. (1991) and Kârén et al. (1997) have attributed multiple fragment patterns to intraspecific RFLP polymorphisms within ectomycorrhizal fungi. In addition, several studies have suggested that some variation in RFLP fragment patterns may be due to differences in morphological characterization and the selection of ectomycorrhizal root tips; misidentification or selection might lead to genotypes within a morphotype that were perhaps actually from a different fungal species. M olecular diversity within ectomycorrhizal morphotypes, for each host species as measured by the Phi Index, indicated that Thelephoraceae 2 on tamarack, and Lactarius and brown inky clamp on scrub birch, had the most intraspecific diversity. Interestingly, Thelephoraceae 2 had only two genotypes and a Phi value of 0.550, compared to Suillus 2 that had five genotypes and a Phi value of 0.225. This provides an example of how molecular diversity, as measured by the Phi Index, does not necessarily increase for a 112 al. (2001) identified multiple genotypes in the morphotypes of Amphinema and MRA, and Horton (2002) yielded multiple RFLP fragment patterns in Laccaria, Tricholoma, and Lactarius species. Even though our study sampled fewer seedlings for each host species compared to the above-mentioned studies, more morphotypes containing four or more genotypes were detected. Several reasons may partially explain differences in genotypic numbers for morphotypes. Fragment selection during RFLP analysis is somewhat subjective and selection protocols may vary among laboratories (e.g. fragment patterns can be manually marked by hand (Sakakibara et al., 2002), or one can utilize the software such as GeneProfiler for fragment pattern selection (Mah et al., 2001; Robertson, 2003). High intraspecific variation within the ITS region of some ectomycorrhizal morphotypes does exist; Horton (2002), Gardes et al. (1991) and Kârén et al. (1997) have attributed multiple fragment patterns to intraspecific RFLP polymorphisms within ectomycorrhizal fungi. In addition, several studies have suggested that some variation in RFLP fragment patterns may be due to differences in morphological characterization and the selection of ectomycorrhizal root tips; misidentification or selection might lead to genotypes within a morphotype that were perhaps actually from a different fungal species. Molecular diversity within ectomycorrhizal morphotypes, for each host species as measured by the Phi Index, indicated that Thelephoraceae 2 on tamarack, and Lactarius and brown inky clamp on scrub birch, had the most intraspecific diversity. Interestingly, Thelephoraceae 2 had only two genotypes and a Phi value of 0.550, compared to Suillus 2 that had five genotypes and a Phi value of 0.225. This provides an example of how molecular diversity, as measured by the Phi Index, does not necessarily increase for a 112 morphotype with an increase in the number of genotypes. This is because the Phi Index does not calculate diversity based on proportional abundance; instead, it uses pairwise distances between ectomycorrhizal samples. A morphotype, such as Suillus 2, that consists of five genotypes that have very similar fragments patterns can have a low Phi index value. Genotypes that share few fragments, such as in Thelephoraceae 2, even though they may comprise fewer distinct genotypes, can have a higher Phi index value. The Phi index measures the average squared distance in the data matrix, not the proportional abundance of the different genotypes. The morphotypes with only one genotype (e.g. Russula and Tomentella-Yike 1 on both host species) had low within morphotype diversity (low Phi values), suggesting a high level of similarity between the samples that comprised the morphotype. When Phi values of morphotypes that were shared by both tamarack and scrub birch were combined, molecular diversity within each morphotype was highest for MRA, Thelephoraceae 2, and crystal net brown, compared to low diversity within Tomentellalike 1 and Russula. Robertson (2003) reported high within morphotype diversity for the black spruce morphotypes Tomentella-like. 1 (not necessarily the same morphotype as described in the present study), Thelephoraceae 4, and MRA 1, and low Phi values for Piloderma and cottony halo. Compared to our results, Mah et al. (2001) had relatively low within morphotype diversity for all commonly occurring morphotypes found on hybrid spruce; however, the greatest intraspecific diversity according to the Phi index was also for an MRA morphotype. 113 Potential linkages between tamarack, scrub birch and black spruce ectomycorrhizas The results from this study suggest that there is a very high potential for fungal linkages between tamarack and scrub birch in these peatland site types. Twelve fragment patterns (almost one fifth of all genotypes), representing 10 morphotypes (33%), were identified on both tamarack and scrub birch. This included genotypes for Amphinema, Cenococcum, Russula, several Thelephoraceae spp. and Tomentella spp., as well as crystal net brown, granular brown and woolly brown morphotypes. In a molecular study investigating Douglas-fir and bishop pine, Horton and Bruns (1998) reported that 12 out of 16 (75%) fungal species were shared between the two hosts. Some of the commonly shared fungi in their study, which took place in a mixed forest ecosystem along the California coastline, included Tomentella, Russula, Amanita, and Cenococcum spp. O f the 12 genotypes that were shared between tamarack and scrub birch in our study, 10 were found on both hosts in the Mix peatland site type, six were found on both hosts in the BsLt site type, and four were found in both hosts in both the Mix and BsLt sites. Several genotypes also occurred on scrub birch in the Bs site type. Greater numbers of shared genotypes occurred in the Mix peatland site type; this suggests that an increase in host species in the Mix sites, with three potential ectomycorrhizal hosts compared to two in the BsLt, and one in the Bs sites, may play an important role in the establishment of - potential linkages. It is interesting to note that several of these genotypes were not frequently observed, and only represented by a few samples. This may mean that less frequent genotypes may also be important in forming fungal linkages, or it that these genotypes were simply under-sampled. The genetic variation that we 114 identified as representing the potential for fungal linkages could actually be greater had we been able to increase the seedling and/or root tip sample size. Time constraints in processing larger sample sizes precluded this in this study. When tamarack and scrub birch ectomycorrhizal fragment patterns were compared to the black spruce fragment patterns identified by Robertson (2003), nine genotypes were identified as being highly similar between the host species. Interestingly, scrub birch and black spruce shared more fragment patterns compared to tamarack which matched only two out of nine black spruce genotypes. The genotypes included the ectomycoiThizal morphotypes Cenococcum, Lactarius, and Amphinema, as well as several members of the Thelephoraceae 1 and Russulaceae. Genotypes also belonged to the unidentified ectomycorrhizal morphotypes granular brown, white felted, silver white, brown smooth 2, and woolly brown, some of which had morphological features similar to these identified ectomycorrhizas. Since both tamarack and black spruce shared more fungal symbionts with scrub birch than with each other, scrub birch may be the major common link between host species in these peatland ecosystems. In the Mix site type, where three hosts occurred, more genotypes were identified on scrub birch than for tamarack or for the other peatland site types. Robertson (2003) successfully identified 30 genotypes for black spruce on the same Mix peatland site type. Of the nine genotypes found on black spruce that were considered to be shared fragment patterns, four come from sites others than the Mix site type. Sporocarp and ectomycorrhiza genotype comparison Of the 19 fragment patterns identified from sporocarp samples, four genotypes were similar to ectomycorrhizal fragment patterns. Although not a large sample, it is 115 interesting considering that only a small percentage of ectomycorrhizal fungi produce sporocarps (Gardes and Bruns, 1996), and that sporocarp sampling only occurred over one summer season. In addition, several studies suggest that there is a poor correlation between sporocarp and ectomycorrhizas occuirence (Gardes and Bruns, 1996; Kârén et al., 1997; Robertson, 2003). Nevertheless, Dahl berg et al. (1997) reported that several ectomycorrhizal species characterized on Norway spruce roots in a Swedish old-growth forest were identified using a sporocarp RFLP database composed of fungal species found within the study site. Horton and Bruns (1998) also successfully identified over half of their ectomycorrhizal fungal species using RFLP fragment patterns from voucher sporocarp specimens. In two cases in the present study, the fungal sporocarp and ectomycorrhizal morphotype were present in the same peatland site type. Lactarius, brown silvery and yellow stellate ectomycorrhizas all shared one genotype with a Hygrocybe species, suggesting that these genotypes might belong to the same fungal genus. Fragment patterns of one genotype belonging to Suillus 2 were similar to the fungus Hebeloma, even though the morphological characteristics between the ectomycorrhiza and fungi varied. Two bands differed between the fragment patterns and it remains unclear whether this Suillus genotype was actually a Hebeloma species. Although the morphotype silver white (one genotype) and the fungus Cortinarius did not co-occur on the same site type, their shared morphological features, as well as their similar fragment patterns increase the likelihood that this morphotype could be a species of Cortinarius. 116 Challenges with genotype classification Lightly colonized root tips represented a large portion (25.7%) of all tamarack roots characterized. However, most (63.1%) of these were successfully placed in several of the established genotypes. Mah et al. (2001) matched five lightly colonized genotypes with other morphotypes identified in that study. Root tips characterized as lightly colonized were often brown with weakly developed mantles. The remaining roots (36.9%) that could not be placed with an established genotype formed three distinct genotypes referred to as lightly colonized and remain as unidentified morphotypes. Five genotypes that belonged to different morphotypes had matching fragment patterns. Jonsson et al. (1999) also found that some RFLP-taxa (genotypes or fragment patterns) were detected in more than one morphotype, and Mah et al. (2001), identified several identical genotypes from different morphotypes. In this study, some of these matching genotypes belonged to the different host species. These genotypes most likely represent mis-characterized samples of ectomycorrhizal root tips that were sorted into the wrong morphotype during initial classification. The question remains as to which of the two (or three) morphotypes these samples belong. No attempt was made to re-classify these genotypes. The molecular analysis of tamarack and scrub birch ectomycorrhizas identified numerous genotypes, some of which exhibited both host and peatland site type preferences. In addition, intraspecific variation was observed within most morphotypes, with up to five ectomycorrhizas. genotypes being expressed for several commonly occurring Most importantly, this study has provided strong evidence for the 117 existence of potential fungal linkages between both tamarack and scrub birch, as well as with black spruce, in these peatland sites. LITE R A T U R E C ITED Amaranthus, M.P. 1998. The importance and conservation of ectomycorrhizal fungal diversity in forest ecosystems: Lessons from Europe and the Pacific Northwest. PNW -GTR-43I. Forest Service, United States Department of Agriculture, Pacific Northwest Research Station, pp. 1-15 Baldwin, Q.F. 1999. 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Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas-fir {Pseudotsuga menziesii) and bishop pine (Pinus muricata). New Phytol. 139: 331-339 Horton, T.R., and Bruns, T.D. 2001. The molecular revolution in ectomycorrhizal ecology: Peeking into the black box. Mol. Ecol. 10: 1855-1871. Horton, T.R. 2002. Molecular approaches to ectomycorrhizal diversity studies: Variation in ITS at a local scale. Plant and Soil, 244: 29-39. Jonsson, L., Dahlberg, A., Nilsson, M.-C., Kârén, O., and Zackrisson, O. 1999. Continuity of ectomycorrhizal fungi in self-regenerating boreal Pinus sylvestris forests studied by comparing mycobiont diversity on seedlings and mature trees. New Phytol. 142: 151-162. Kârén, O., Hogberg, N., Dahlberg, L., Jonsson, L., and Nylund, J.-E. 1997. Inter- and intraspecific variation of the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis (rDNA ITS variation in ectomycorrhizal fungi). New Phytol. 136: 313-325. Khetmalas, M.B., Egger, K.N., Massicotte, H.B., Tackaberry, L.E., and Clapperton, M.J. 2002. Bacterial diversity associated with subalpine fir {Abies lasiocarpa) ectomycorrhizae following wildfire and salvage logging in central British Columbia. Can. J. Microbiol. 48: 611-625. 119 MacKenzie, W.H., and Moran, J R. 2003. The Wetlands of British Columbia: A field guide to Identification of Wetlands and Related Ecosystems in British Columbia. Land Management Handbook, BC Ministry of Forests, Research Branch. Mah, K., Tackaberry, L.E., Egger, K.N., and Massicotte H.B. 2001. The impacts of broadcast burning after clearcutting on the diversity of ectomycorrhizal fungi associated with hybrid spruce seedlings in central British Columbia. Can. J. For. Res. 31: 224-235. McKendrick, S.L., Leake, R., and Read, D.J. 2000, Symbiotic germination and development of myco-heterotrophic plants in nature: Transfer of carbon from ectomycorrhizal Salix repens and Betula pendula to the orchid Corallorhiza trifida through shared hyphal connections. New Phytol. 145: 539-548. Newman, E.I. 1988. Mycorrhizal links between plants: Their functioning and ecological significance. Adv. Ecol. Res. 18: 243-270. Read, D.J. 1987. Development and function of mycorrhizal hyphae in soil. In: Mycorrhizae in the next decade: practical applications and research priorities. Sylvia, D.M., Hung, L.L., and Graham, J.H. eds. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, pp. 178-180. Robertson, S. J. 2003. Morphological and molecular assessment of ectomycorrhizal communities associating with black spruce {Picea mariana (Mill.) BSP) in wetland and upland forests in central British Columbia. M.Sc. thesis. University of Northern British Columbia, Prince George. Sakakibara, S.M., Jones, M.D., Gillespie, M., Hagerman, S.M., Forrest, M.E., Simard, S.W., and Durall, D.M. 2002. A comparison of ectomycorrhiza identification based on morphotyping and PCR-RFLP analysis. Mycol. Res. 106: 868-878. Simard, S.W „ Perry, D.A., Jones, M.D., Myrold, D.D., Durall, D.M., and Molina, R. 1997b. Net transfer of carbon between ectomycorrhizal tree species in the field. Nature, 388: 579-582. Smith, S.E., and Read, D.J. 1997. Mycorrhizal symbiosis (2"^ ed.). Academic Press, Cambridge. Zolan, M.E., and Pukkila, P.J. 1986. Inheritance of DNA méthylation in Coprinus cinereus. Mol. Cell. Biol. 6: 195-200. 120 CO NCLUSIO N Peatlands, also referred to as bogs or fens, are unique ecosystems in British Columbia. Although we have extensive information on the plant communities associated with peatlands, we known little about the ectomycorrhizal status, in particular for tamarack and scrub birch in these habitats. This study investigated the ectomycorrhizal associations of these two hosts growing in peatland environments of central British Columbia, and addressed several questions concerning below-ground, ectomycorrhizal communities in peatland habitats. Through morphological and molecular analysis, we determined that the ectomycorrhizal abundance and diversity, even though peatlands are often described as poorly drained, nutrient-poor environments, did not appear to differ noticeably compared to the literature for upland forest ecosystems. More importantly, it appears that there is a high potential for fungal linkages between tamarack, scrub birch, and black spruce in these systems. Morphological characterization described 30 ectomycorrhizas on tamarack and scrub birch roots, some of which exhibited host and/or site specificity. Ectomycorrhizal diversity was highest (as measured by the Margalef, Shannon, and Simpson indices) in the peatland site type that contained three potential host species (scrub birch, tamarack, and black spruce) compared to the sites that consisted of only one or two ectomycorrhizal hosts. Molecular analysis of the ectomycorrhizas identified numerous genotypes that reflected high intraspecific variation within some morphotypes, especially for those morphotypes which occurred in high abundance. Although more fungal genotypes were found in sites with three ectomycorrhizal hosts, compared to two or one host sites. 121 molecular diversity, according to the Phi index, was highest in the sites with only one potential ectomycorrhizal host, and lowest in the site type with three host species. This difference was not significant and most likely reflects the fact that genotypes on those sites, although fewer in numbers compared to the Mix site type, may have been separated by greater branch distance on the phylogenetic tree. Both morphological and molecular analyses ectomycorrhizal fungi were found on both host species. determined that numerous However, the molecular investigation into the genetic composition of these ectomycorrhizas provided strong supporting evidence for fungal linkages in these environments. Shared ectomycorrhizal fungi between tamarack and scrub birch, as well as with black spruce, may be part of a complex underground system of mycelial networks. The transfer of carbon or nutrients between different host species, facilitated by symbiotic fungi, especially in these wet, nutrient-poor habitats, may be vital for the survival and growth of many peatland plant species. 122 Appendix I. Map of study area showing approximate locations (indicated by rectangle) of the six peatland sites in the Prince George Forest District in central British Columbia. N ess J w um p L ake Lake Saxton L ake CITY C rossing C o tb L a ke J ISLE PIERRE PRINCE F3 — 1/ GEORGE L ake ^ B td n L ake veyara Lf ke L it tie B o b ta ii L ake fiu td a / \S T O N E R L ake B o b fo // L ake S /!e s/c f ----- sà Source: Province o f British Columbia, Ministry o f Forests, Prince George Forest District Recreation map) 123 A ppendix IL Plant species list of vegetation growing within four 1 m x 1 m plots in each of the Mix (scrub birch-tamarack-black spruce), BsLt (scrub birch-tamarack), and S ite T ype L atin nam e C om m on nam e B sl B s2 B sL tl B sL t2 M ix l M ix2 • e trees/shru bs Picea m ariana black spruce L arix laricina tamarack • # • • # # P inus contorta lodgepole pine • B etula g land u lo sa scrub birch • • • • • Salix spp. w illow • • • • • V accinium oxycoccos bog cranberry • • • A ndrom eda po lifo lia bog-rosemary • • # R ubus piibescen s trailing raspberry • • K alm ia m icrophylla bog-laurel d w a rf shrub s Ledum groen la n d icu m R ubus arcticu s • • labrador tea • • • dwarf nagoonberry • # • w ildflow ers e P etasites sag itta tu s arrow-leaved coltsfoot P latanthera dilatata white bog orchid • P latantliera hyperborea northern green bog orchid • M itella niida comm on mitrewort P otentilla p a lu stris marsh cinquefoil G alium spp. bedstraw M enyantlies trifoliata buckbean P yrola asarifolia pink wintergreen D rosera rotundifolia round-leaved sundew • * • • • • • • • # • # e • sed ges/oth ers beaked sedge • • • # C arex interior inland sedge * • • e E riophorum a n g ustifolium narrow-leaved cotton grass • • • e • E quisetum spp. horsetail • # • Triglochin m aritim iun sea-side arrow grass • • C arex rostrata grass • A ulacom nium p a lu stre glow moss • S phagnum spp. peat moss • • M nium spp. leafy m oss • • Poaceae spp. • • • • • # # # * m osses/lichen s Tom enthypnum nitens # # e # # 9 # golden fuzzy fen moss • • Surveys were conducted in four 1 x I m plots within each site replicate. Presence o f vegetation is indicated by '« 124 Appendix III. Descriptions of tamarack (Lt) and scrub birch (Bs) ectomycorrhizal morphotypes from Mix (scrub birch-tamarackblack spruce), BsLt (scrub birch-tamarack), and Bs (scrub birch) peatland site types. Morphotype (Host) Amphinema (Lt and Bs) Macroscopic Features orange-brown, cottony, unbranched straight tips (0.2 mm wide x 0.4 mm long) Microscopic Features outer mantle (OM)/inner mantle (IM) net synenchyma to non-interlocking irregular synenchyma, mantle -2 0 pm thick Emanating Hyphae yellow emanating hyphae (EH), highly branched, ornamented, 3-3.5 pm wide; clamps Rhizomorphs yellow, loose undifferentiated; hyphae finely verrucose with clamps black cystidia (Bs) black, short spiny, straight tips with monopodial pinnate branching (0.25 mm wide X 1 mm long) OM regular synenchyma, IM net synenchyma (cells 5-10 pm wide), mantle 4050 pm thick brown cystidia, bottle­ shaped, bent n e c k ,3-5 pm wide x 10-30 pm long; few septa with no clamps not observed brown clamp (Lt) brown, smooth, unbranched straight tips (0.25 mm wide x 1.5 mm long) OM net prosenchyma (cells 3-5 pm wide), IM net synenchyma (cells 1-2 pm wide), mantle -2 0 pm thick EH yellow-orange, smooth, up to 2 pm wide; clamps not observed brown inky clamp (Bs) white with brown netlike overlay, smooth, straight tips with monopodial pinnate branching (0.25 mm wide X 1.5 mm long) OM net prosenchyma (cells 3-4.5 pm wide), IM net synenchyma (cells 2-3 pm wide), stains rust in KOH, mantle 15-30 pm thick hyaline EH, smooth, 34 pm wide; clamps white, loose, undifferentiated, up to 150 pm wide; hyphae 5-6 pm wide with rounded clamps 125 Morphotype (Host) brown smooth 1 (Lt and Bs) Macroscopic Features brown, smooth, unbranched, straight tips Microscopic Features OM net prosenchyma to interlocking irregular synenchyma (cells 2-3 pm wide), IM net synenchyma (cells 22.5 pm wide), mantle -3 0 pm thick Emanating Hyphae not observed Rhizomorphs not observed brown smooth 2 (Lt and Bs) mottled yellow brown, smooth, unbranched beaded tips OM net synenchyma (cells 1.5-2 pm wide), mantle 15-20 pm thick hyaline EH, smooth, -1 pm wide; no clamps observed not observed brown silvery (Lt) brown, silvery, unbranched straight tips (0.5 mm wide x 0.5-1 mm long) OM felt prosenchyma (cells -1 pm wide), IM net synenchyma, mantle -2 0 pm thick hyaline EH, smooth, 0.5-2 pm wide; no clamps observed not observed Cenococcum (Lt and Bs) black, woolly, unbranched straight to beaded tips (0.5 mm wide X 0.5 mm long) OM net synenchyma with typical stellate pattern (cells 2-5 pm wide) dark brown EH, thick walled, mostly smooth, 2-5 pm wide; no clamps observed not observed coffee brown (Lt) dark brown, shiny, straight tips with monopodial pinnate branching OM net prosenchyma (cells 4-5 pm wide), IM net synenchyma (cells 1-2 pm wide) yellow EH, smooth, 2-3 pm wide; clamps not observed 126 Morphotype (Host) cotton orange (Bs) Macroscopic Features orange-brown, cottony, unbranched straight tips (0.5 mm wide x 0.75 mm long) Microscopic Features OM obscured by EH, IM interlocking irregular synenchyma, mantle -2 0 pm thick Emanating Hyphae EH pale yellow, highly branched, smooth, thin walled, 5 pm wide; clamps Rhizomorphs not observed crystal net brown (Lt and Bs) brown, smooth to felty, un branched straight tips (0.25 mm wide x 1.5 mm long) OM net synenchyma (cells 1-2 pm wide), mantle -3 0 pm thick yellow EH, highly branched, net-like in appearance, verrucose, 2-3 pm wide; no clamps observed not observed E-strain (Lt and Bs) brown to dark brown, smooth, shiny, unbranched straight tips (0.5 mm wide x 2 mm long) OM net prosenchyma (cells 5-10 pm wide), IM net synenchyma (cells somewhat angular in appearance), mantle -2 0 pm thick not observed not observed granular brown (Lt and Bs) yellow to dark brown, grainy appearance, unbranched straight tips OM regular synenchyma (cells 5-7 pm wide) EH yellow to dark brown, sometimes verrucose, thick walled, 5-7 pm wide; clamps occasionally observed not observed 127 Morphotype (Host) Hebeloma-Wke (Lt) Macroscopic Features brown often with white at base, silvery, cottony, unbranched straight tips Microscopic Features OM/IM obscured by EH Emanating Hyphae hyaline EH, smooth to verrucose, 5-5.5 pm wide; clamps Rhizomorphs white, undifferentiated; hyphae smooth to verrucose; clamps Lactarius (Lt and Bs) yellow to light brown, smooth, straight to beaded tips with monopodial pinnate branching (0.5 mm wide X 2 mm long) OM net synenchyma (cells 4-5 pm wide) with possible crystals, laticifers (4-6 pm wide, 40-100 pm long), producing rust colour when squashed, mantle -2 0 pm thick pale yellow EH, smooth, -1 pm wide; fine septa with no clamps observed yellow, slightly differentiated, hyphae 3-8 pm wide; no clamps observed MRA (Lt and Bs) brown-black, grainy, shiny, unbranched straight tips (0.2 mm wide X 0.2 mm long) OM net prosenchyma (cells 3-4 pm wide), IM non-interlocking irregular synenchyma (4-5 pm wide), mantle 10-25 pm thick dark yellow EH, smooth, 3-4.5 pm wide; no clamps observed not observed Russula (Lt and Bs) brown, spiny, unbranched, straight tips (0.5 mm wide x 4 mm long) OM interlocking irregular synenchyma (cells 2-7 pm wide), IM net synenchyma (cells 2-3 pm wide), mantle 20-40 pm thick cystidia hyaline to pale yellow, smooth, two types a) awl-shaped (25 pm wide x 110-150 pm long) and b) flask shaped with apical knob (3-4 pm wide x 100-130 pm long); no clamps observed not observed 128 M orphotype (Host) Russulaceae (Bs) M acroscopic Features E m anating H yphae R hizom orphs hyaline EH, smooth, 23 pm wide; no clamps observed; EH not observed on all tips not observed silver white (Bs) white to yellow, cottony, unbranched straight tips (0.25 mm wide X 1 mm long) OM elongated interlocking irregular synenchyma, mantle 15-20 pm thick hyaline EH, highly branched, finely verrucose, -3 pm wide; clamps not observed Suillus 1 (Lt) patchy yellow and white, silvery, stringy, straight tips with monopodial pinnate branching (0.5 mm wide X 1 mm long) OM net prosenchyma (cells 2-3 pm wide), yellow crystals (20-25 pm wide) deposited on mantle, mantle -3 0 pm thick hyaline EH, 3-4 pm wide; no clamps observed; reddish purple amorphous crystals ornament EH yellow, differentiated (central core), up to 40 pm wide, hyphae 2-10 pm wide, ornamented with reddish violet crystals Suillus 2 (Lt) brown, felt-like, straight tips with monopodial pinnate branching (0.5 mm wide X 4 mm long) OM felt to net prosenchyma (cells 2.53 pm wide), IM net synenchyma (spiral­ shaped cells 1-2.5 pm wide), mantle 15-20 pm thick EH dark yellow to olive, verrucose, 2-4 pm wide; no clamps observed rust, undifferentiated to slightly differentiated, compact, 70-90 pm wide; hyphae ornamented with rusty amorphous crystals (-2 pm wide X 15 pm long) light brown, smooth, unbranched straight tips (0.2 mm wide x 1.5 mm long) M icroscopic Features OM non-interlocking irregular synenchyma, IM net synenchyma (cells 3-5 pm wide), produces an orange colour when squashed 129 M orphotype (Host) M acroscopic Features white to beige, smooth, straight tips with monopodial pinnate branching (0.25 mm wide X 1.5 mm long) M icroscopic Features OM interlocking irregular synenchyma (cells 4-10 pm wide), mantle -2 0 pm thick Em anating H yphae not observed Thelephoraceae 2 (Lt and Bs) black with reflective metallic bronze colour, grainy, straight to beaded tips (0.25 mm wide X 1 mm long) OM interlocking irregular synenchyma (cells 1.5-2 pm wide); stains blue-green in KOH, mantle -5 0 pm thick EH dark brown, thick walled (-2 pm), 3-5 pm wide, smooth, no clamps observed not observed Thelephoraceae 3 (Lt and Bs) olive yellow, grainy, unbranched straight tips (0.5 mm wide x 0.75 mm long) OM regular synenchyma (cells 5-10 pm wide), IM net synenchyma (cells 2-3 pm wide), mantle -3 0 pm thick yellow EH, smooth, 22.5 pm wide; clamps yellow, loose undifferentiated, 3040 pm wide; hyphae with clamps Tomentella-Wke. 1 (Lt and Bs) yellow-brown, sparsely spiny, unbranched straight tips (0.5 mm wide X 0.75 mm long) OM rounded non­ interlocking irregular synenchyma (cells 5-10 pm wide), mantle -2 0 pm thick cystidia yellow, smooth, 2-5 pm wide x -5 0 pm long, awl-like, thick-walled, clamped at base yellow, undifferentiated, up to 30 pm wide, hyphae verrucose; clamps Thelephoraceae 1 (Lt and Bs) 130 R hizom orphs not observed M orphotype (Host) M acroscopic Features M icroscopic Features Tomentella-Wke. 2 (Lt and Bs) black, grainy to rough, unbranched straight tips (0.25 mm wide x 1 mm long) OM interlocking to non-interlocking irregular synenchyma, no KOH reaction, mantle 20-30 pm thick Em anating H yphae dark brown EH, thick walled ( up to I pm), -5 pm wide; clamps occasionally observed R hizom orphs white clamp (Bs) white with black netlike appearance, smooth, straight tips with monopodial pinnate branching (0.2 mm wide x 0.3 mm long) OM net prosenchyma (cells 4-5 pm wide), yellow ornaments on surface noticeable when squashed, mantle -2 0 pm thick hyaline EH, smooth, short, - 2 pm wide x 10 pm long; clamps not observed white felted (Bs) white, felt-like, unbranched, straight tips (0.25 mm wide x 1 mm long) OM felt prosenchyma (cells 1-1.5 pm wide), IM net synenchyma (cells 2-3 pm wide), mantle -2 0 pm thick hyaline EH, verrucose, 1-1.5 pm wide; no clamps observed not observed woolly brown (Lt and Bs) brown, woolly to cottony, unbranched straight tips (-0.25 mm wide X 0.5-1 mm long) OM elongated interlocking irregular net synenchyma (cells -3 pm wide), IM net synenchyma (cells 4-5 pm wide), stains rustyellow in KOH, mantle -2 0 pm thick EH yellow-orange, smooth, 4-5 pm wide, forming a hyphal fan; clamps yellow, loose undifferentiated, strands 30-35 pm wide; hyphae with clamps 131 not observed Morphotype (Host) yellow stellate (Lt and Bs) Macroscopic Features Microscopic Features Emanating Hyphae Rhizomorphs dark yellow, smooth, OM net synenchyma hyaline EH, smooth, not observed unbranched tortuous with stellate pattern fine septa, 1-2 pm tips (0.5 mm wide x 3 (cells 1-2 pm wide), wide; no clamps mm long) IM net synenchyma observed ____________________ (cells ~1 pm wide)__________________________________________ 132 Appendix IV. Unrooted phylogram generated from restriction fragment patterns o f tamarack ectomycorrhizal morphotypes. Phylogram shows the relationship between morphotypes and peatland site types. lightly colonized \ 1 , Scrub birch-tamarack site AI Scrub birch-tamarackblack spruce site Suillus 2 (genotype 4) M -V M AL M Suillus 2 (genotype 3) AJ Suillus 2 (genotype 1) Ai A! M iM A.i - lAJ Amphinema Tomentella-WkQ 2 AI Ai" ------------------^ A l ^ M tr -Ai -r -A! Ai Ai A Ai Suillus 1 -Al granular brown Suillus 2 (genotype 5) AI (genotype 5) M -d l Suillus 1 -M granular brown M Tonieiitella-Uke 2 crystal net brown 1^1. ' crystal net brown brown stringy M yellow stellate I I 1--------- lightly colonized MRA Tomentella-Uke 2 -d . Russula -CE -bM M JsJL M Toinentella-Wke 2 Cenococcum M Tomentella-Wke 1 granular brown L Tomentella-kkQ 2 granular brown M -c = ^ -Q M — M M lightly colonized M M M M -r 1. Jcrystal net brown ----------crystal net brown i. brown stringy M yellow stellate I lightly colonized I MRA Tomentella-Wke. 2 I -Ll. iZ . Russula M -V M M Tomentella-Wke 2 Cenococcum Tonieiitella-V\ke 1 granular brown M M M Tomentella-V\ke 2 granular brown -Q — ■M M M M lightly colonized M M M % \i M woolly brown M brown stringy A p pendix V. Unrooted phylogram generated from restriction fragm ent patterns o f scrub birch ectomycorrhizal morphotypes. Phylogram shows the relationship between morphotypes and peatland site types. B = scrub birch site 5. = tamarack and scrub : birch site M = tamarack, scrub birch, i and black spruce mix site Thelephoraceae 1 Thelephoraceae 2 MRA Lactarhis Lcictciriiis brown ink clamp brown ink clamp brown smooth 2 Thelephoraceae 2 Thelephoraceae 2 y e l l o w stellate Cenococcum i~~] Thelephoraceae crystal net brown white clamp brown inky clamp J granular brown Tomentella-Vikc 2 brown smooth 2 granular brown Toinentella-WkQ 2 2] Lactariii.s J y e llo w s te iia ie Cenococcum Thelephoraceae crystal net brown white clamp brown inky clamp J granular brown Tomentella-Wke 2 brown smooth 2 granular brown Tomentella-Wke. 2 21 Lactarius Lactarius Thelephoraceae 2 Lactarius Thelephoraceae 2