MORPHOLOGICAL AND MOLECULAR ASSESSMENT OF ECTOMYCORRHIZAL
COMMUNITIES ASSOCIATING WITH BLACK SPRUCE (PICEA MARIANA (MILL.) BSP) IN
WETLAND AND UPLAND FORESTS IN CENTRAL BRITISH COLUMBIA
by
Susan Robertson
B.Sc., The University of Calgary, 1990

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
NATURAL RESOURCES AND ENVIRONMENTAL STUDIES

© Susan Robertson, 2003
THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA
June 2003

All rights reserved. This work may not be
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APPROVAL
Name:

Susan Robertson

Degree:

Master of Science

Thesis Title:

MORPHOLOGICAL AND MOLECULAR ASSESSMENT OF
ECTOMYCORRHIZAL COMMUNITIES ASSOCIATING W ITH BLACK
SPRUCE {PICEA MARIANA (MILL.) BSP) IN WETLAND AND UPLAND
FORESTS IN CENTRAL BRITISH COLUMBIA

Examining Committee:

Chair: Dr. Robert Tait
Dean of Graduate Studies
UNBC

f! Hugues M&s^cotte
Associate Professor, F o r e s ^ Program
UNBC

Comntttfee M em ber P y P a u l Sanborn
Associate ProfessorTForestry Program
UNBC

Committee Member: Dr. Arthur Fredeen
Associate Professgy, Forestry Program
UNBC

Coj^fmttee Member: Dr. Keith Egger
ifessor, Biology Program
UNBC

Committee Menroér: Craig DelOT
Landscape Ecologist, Ministry ^ f b r e s t s
Northern Interior Forest Region (Prince George, BC)

External Examiner: Dr. Michael Rutherford
Associate Professor, Environmental Science & Engineering Programs
UNBC

Date Approved:

ABSTRACT
Ectomycorrhizal (ECM) symbioses form one of the primary systems for nutrient and energy
exchange in northern coniferous forests. ECM communities consist of a diverse array of
fungal species that exhibit variable patterns of distribution and abundance, depending on
host plant species, soil properties, and environmental factors. Using morphological
(microscopy) and molecular (PGR-RFLP) techniques, this study describes ECM community
composition and diversity of black spruce across its habitat range in BC’s central interior.
Habitats included pure black spruce and mixed spruce - tamarack wetlands and black
spruce - pine upland forests. Black spruce was found to form ECM with a diverse
community of fungal species, several of which were limited to one or two habitats. Although
ECM community composition varied between habitats, the total number of types
(morphological and molecular) did not. This study emphasizes the importance of sampling
across a habitat range to describe ECM communities associated with a particular host
species.

TABLE OF CONTENTS
Abstract..................................................................................................................................................ii
Table of Contents................................................................................................................................iii
List of Tables........................................................................................................................................ v
List of Figures...................................................................................................................................... vi
Acknowledgements........................................................................................................................... vii
Introduction........................................................................................................................................ 1
Literature R e vie w ..............................................................................................................................5
Ectomycorrhizae.................................................................................................................................. 5
Definition, classification and structure.........................................................................................5
Functions and benefits of symbioses..........................................................................................6
ECM fungal communities....................................................................................................................9
Composition and diversity.............................................................................................................9
Factors that alter diversity in ECM communities......................................................................10
Host specificity and receptivity..............................................................................................10
Environmental gradients......................................................................................................... 13
Disturbance and other factors............................................................................................... 15
Methods of studying ECM communities.........................................................................................16
Morphological techniques (microscopy)................................................................................... 16
Molecular techniques (PCR-RFLP)........................................................................................... 17
Measuring ECM community structure and diversity...............................................................18
Black spruce as an ECM host......................................................................................................... 21
Distribution.....................................................................................................................................21
Habitat preferences.....................................................................................................................22
Morphology and growth.............................................................................................................. 23
ECM fungal associates............................................................................................................... 26
References......................................................................................................................................... 29
Morphological Characterization of Ectomycorrhizal Communities Associating with
Black Spruce {Picea mariana) in Wetland and Upland Forests In the Central
Interior of B C ....................................................................................................................................37
Abstract...............................................................................................................................................37
Introduction........................................................................................................................................ 38
Methods and Materials.....................................................................................................................41
Site descriptions.......................................................................................................................... 41
Seedling sampling and vegetation and soil analyses............................................................ 47
Fungal sporocarp sampling........................................................................................................ 48
Seedling harvest and ECM characterization...........................................................................49
Analysis of morphological d a ta ..................................................................................................50
Results................................................................................................................................................ 51
Seedling and site characteristics............................................................................................... 51
ECM morphotype occurrence, frequency and abundance.................................................... 56
ECM community diversity...........................................................................................................62
Discussion.......................................................................................................................................... 62

ECM morphotype occurrence and abundance........................................................................62
ECM community structure and diversity...................................................................................72
References......................................................................................................................................... 78
Molecular Characterization of Ectomycorrhizai Communities Associating with
Biack Spruce {Picea mariana) in Wetiand and Upland Forests in the Centrai
interior of B C ....................................................................................................................................84
Abstract...............................................................................................................................................84
introduction........................................................................................................................................ 85
Methods and Materiais.....................................................................................................................87
ECM sample collection for molecular analysis and DNA extraction.....................................87
DNA amplification and restriction endonuclease digestion................................................... 88
Analysis of molecular data......................................................................................................... 90
DNA extraction, amplification and restriction endonuclease digestion................................ 92
Cluster analysis of fragment patterns for ECM morphotypes...............................................93
Molecular diversity within ECM morphotypes.......................................................................... 99
Habitat effects on ECM genotype distribution and diversity.................................................. 99
Discussion........................................................................................................................................ 101
Molecular diversity: genotype and ECM community variation across habitats................ 101
Molecular diversity within ECM morphotypes........................................................................104
Identification of ECM Species..................................................................................................108
References....................................................................................................................................... 109
Conclusions....................................................................................................................................112
Appendix I. Types of vegetation compared between sites in spruce - tamarack wetland
(T), spruce-dominated wetiand (W) and spruce - pine upland forest (U) habitats in central
BC...................................................................................................................................................... 116
Appendix II. Descriptions of black spruce ECM morphotypes from black spruce tamarack wetland (T), black spruce-dominated wetland (W) and black spruce - pine
upland forest (U) habitats in central BC....................................................................................... 118
Appendix III. Unrooted neighbor joining tree generated from restriction fragment patterns
of black spruce ECM morphotypes. The tree shows the relationships between ECM
morphotypes, genotypes and habitat of origin (spruce - tamarack wetiand [T], sprucedominated wetland [W] and spruce - pine upland forest [U] habitats) in central BC............125

IV

LIST OF TABLES
Table 2.1. Locations, biogeocllmatlc ecological classification (BEC) (DeLong etal.
1994) and site characteristics of the 9 study sites (T = black spruce - tamarack
wetland sites; W = black spruce-dominated wetland sites; U = black spruce - pine
upland forest sites).................................................................................................................... 42
Table 2.2. ANOVA comparisons of mean % carbon, % nitrogen, C:N ratio, available
phosphorus and pH between habitats based on combined samples from each site
(n=3).............................................................................................................................................53
Table 2.3. Treatment effects, percent abundance (mean ± SB) and frequency of
occurrence (%) for ECM morphotypes of black spruce growing in three habitats.
ECM morphotypes are presented in order of decreasing overall frequency rank........... 58
Table 2.4. ANOVA comparisons of diversity indices (Margalef, Shannon, Shannon
evenness and Simpson) between habitats based on calculations for each seedling
(n=15)...........................................................................................................................................62
Table 3.1. Habitat (black spruce - tamarack (T) wetlands, black spruce-dominated (W)
wetlands, and black spruce - pine (U) upland forests) and approximate fragment
sizes (bp) of the amplified ITS region for black spruce ECM morphotypes and
genotypes....................................................................................................................................95
Table 3.2. Diversity values (Shannon, Simpson and Phi) for 14 ECM commonly
occurring on regenerating black spruce in three habitats....................................................99
Table 3.3. Mean diversity values (Shannon, Simpson and Phi indices) for molecular
genotypes of ECM from three black spruce habitats......................................................... 100

LIST OF FIGURES
Figure 2.1. Map (left) of British Columbia showing the study area (orange square) in the
BBS biogeoclimatic zone in central BC (shading indicates the provincial range of
black spruce). Map (right) showing approximate locations of 9 black spruce study
sites ( I = black spruce - tamarack wetland sites; W = black spruce-dominated
wetland sites; U = black spruce - pine upland forest sites) in the Prince George
Forest District............................................................................................................................. 43
Figure 2.2. Photographs showing examples of three black spruce habitats in central
BC: A. mixed black spruce - tamarack wetland (T) habitat; B, black sprucedominated wetland (W) habitat; C, black spruce - lodgepole pine upland forest (U)
habitat.......................................................................................................................................... 46
Figure 2.3. Bar graph showing mean (±SE) number of ECM morphotypes compared to
mean (±SE) number of potential ECM host vegetation (within 0.5 m of seedlings) for
each site (black spruce - tamarack wetlands [T sites], black spruce-dominated
wetlands [W sites] and black spruce - pine upland forests [U sites]). ECM
morphotype bars labelled with the same letter are not significantly different...................54
Figure 2.4. ECM morphotypes described on black spruce from three habitats in central
BC. A - Lactarius 1 ; B - Cortinariaceae 1; C - Piloderma', D - Thelephoraceae-like
4; E - Tomentella] F - Tomentella-Wke 1 (outer mantle); G - MRA 1 (outer mantle); H
- Amphinema (outer mantle with emanating hyphae).......................................................... 55
Figure 2.5. Log-transformed rank-abundance plot of the overall ECM fungal community
of black spruce. The morphotype abundance rank order, beginning with
Cenococcum, corresponds to the order on the y-axis of Figure 2.7..................................59
Figure 2.6. Comparison of rank-abundance of ECM morphotype communities of black
spruce in three habitats (black spruce - tamarack wetlands [T sites], black sprucedominated wetlands [W sites] and black spruce - pine upland forests [U sites]),
using log abundance plotted against ranked morphotype abundance in each habitat...59
Figure 2.7. Comparison of ECM morphotype abundance (proportion of the community
represented by each morphotype per habitat) between three black spruce habitats.
ECM morphotypes are ranked in order of overall decreasing abundance from
bottom to top (excluding the non-mycorrhizal type)............................................................. 61

VI

ACKNOWLEDGEMENTS
I would like to express my thanks to several people for their support and guidance
throughout this study. I especially thank my supervisor, Dr. Hugues Massicotte, who
contributed hours of encouragement and discussion and kept me from straying too far from
my study objectives. Committee members, Dr. Keith Egger, Dr. Paul Sanborn, Dr. Art
Fredeen and Craig DeLong, provided expertise on molecular, soil, vegetation and field
sampling issues. I particularly thank Dr. Egger for keeping me on track while Dr. Massicotte
was away on sabbatical.

I am greatly indebted to Jen Catherall who not only provided invaluable field and lab
assistance, but also acted as a sounding board for thesis and general life matters. Her
excellent advice to always carry banana bread as well as her expertise in spotting edible
mushrooms and berries always made trips to field sites enjoyable despite the mosquitoes.
Kei Fujimura and Susan Gibson helped with molecular issues and made lab 403 a fun place
to work. Cam Grose helped with computer problems and trouble-shooting on updated
phylogenetic software. I am very grateful to Linda Tackaberry for initial training in
morphological techniques, as well as for editing my thesis and helping me see the bigger
picture amongst all the details. I also thank Julie Deslippe for her fascination with all things
microbial and for engaging me in hours of interesting discussion, microbial and otherwise.
Finally, special thanks to Andy Kendrick and Misha for their constant love and support and
for reminding me about life on the outside.

In addition, this study would not have been possible without financial support provided by
the Natural Science and Engineering Research Council of Canada (NSERC) through a
research grant to Dr. Massicotte. Also, the University of Northern British Columbia provided
travel grants, field vehicles and laboratory facilities.
vii

Introduction
Biodiversity is the term adopted to explain all aspects of biological diversity, including
species richness (taxonomic diversity), ecosystem complexity (functional diversity) and
genetic variation (genetic diversity) (Zak and Visser 1996). The United Nations Conference
on Environment and Development at Rio de Janeiro (1992) recognized the planet’s
dependence on gene, species, population and ecosystem diversity and the serious threats
that biodiversity declines pose to human development (Andrén and Balandreau 1999). High
diversity has been related to healthy ecosystem function and may provide the means for
ecosystems to adapt to changes in environmental conditions such as global climate change,
fire, outbreaks of insect or pathogen attacks, or forestry practices.

Soil probably harbors most of the undiscovered biodiversity on earth and the microbial
communities within the rhizosphere (zone surrounding plant roots) account for much of the
diversity of northern coniferous forests (Amaranthus 1998; Tiedje et al. 1999). In these
forests, most plants belonging to the families Pinaceae, Fagaceae and Betulaceae normally
form mutualistic associations with diverse communities of filamentous fungi known as
ectomycorrhizal fungi (Kendrick 1992). The nature of these symbioses is the transfer of
poorly accessible, inorganic nutrients (nitrogen and phosphorus) from fungi to plants in
exchange for photosynthetically derived, energy-rich carbon compounds to fuel fungal
metabolic processes and growth (Harley and Smith 1983; Allen 1991). Ectomycorrhizae
(ECM) are essential components of forest ecosystems because of their central role in plant
growth and survival, biogeochemical cycling, soil structure, forest food webs, and buffering
capacity against environmental stress (Colpaert and van Tichelen 1994; Amaranthus 1998).

Because of their ubiquitous nature and vital importance to plant and soil biology, the focus of
recent research has been to describe ECM communities at the taxonomic and genetic levels
1

to attempt to gain some understanding of functional diversity. There are two major reasons
why it is important to understand which fungi are able to form ECM with which plants. First,
fungi vary in their ability to form ECM or enhance plant nutrient uptake when grown with
different plant species and under different environmental conditions (Molina etal. 1992b;
Gehring et al. 1998). Second, species diversity of fungi appears to be high when host plant
species diversity is relatively low (Bruns 1995; Gehring etal. 1998). These contributions
have advanced our understanding of how ECM communities can vary across different
habitats and geographic locations, as well as change in response to environmental changes
at scales varying from large disturbance events to local differences in soil characteristics
(Haug and Oberwinkler 1987). Applications of such information might include improvements
to forestry practices, such as avoiding soil compaction or avoiding the removal of all major
réfugia for fungal inoculum (Jones et al. 2003). Ecosystem recovery programs (such as
silviculture or phytoremediation) may also benefit by planting seedlings inoculated with
ecologically adapted ECM fungi.

ECM communities can be described by the number of species present (richness), the
relative abundance (evenness) of each species, and the physiological role that each species
plays in the environment and in interaction(s) with other species (Tiedje etal. 1999).
Several recent studies have used a combination of morphological and molecular techniques
to describe ECM communities in natural ecosystems. The use of light microscopy to directly
examine ECM roots allows certain morphological features to be used to taxonomically group
ECM into families, genera or species (Agerer 1987-2002; Ingleby etal. 1990; Goodman et
al. 1996); microscopy has the advantage of being relatively inexpensive and enables one to
assess large numbers of roots in a short period of time (Taylor et al. 2000). Molecular
(PCR-RFLP) analysis has the advantage of being largely independent of environmental
effects and of being able to improve upon the resolution of ECM identification through

comparisons to reference databases (Egger 1995; Horton and Bruns 2001). In a
comparison of methods for assessing diversity of hybrid spruce ECM, Mah etal. (2001)
found that morphotyping may have underestimated ECM diversity, but identified rare types
that did not amplify properly during the PCR analysis. Studies that incorporate both
morphological and molecular data provide better descriptions and interpretations of diversity
than those that focus on just one approach (Moritz and Hillis 1996).

This study describes the community structure and diversity of ECM associated with naturally
regenerating black spruce {Picea mariana (Mill.) BSP) across its habitat range in the subboreal spruce (SBS) zone of BC’s central interior using both morphological (light
microscopy) and molecular (PCR-RFLP) techniques. The specific objectives of this study
were:

1. To describe the ECM associated with naturally regenerating black spruce seedlings in
the Sub-Boreal Spruce (SBS) zone of central BC using morphological techniques (light
microscopy).

2. To compare the structure and diversity of black spruce ECM communities between three
habitats, including both black spruce-dominated and black spruce - tamarack {Larix
iaricina (Du Roi) K. Koch) wetland forests, as well as black spruce - lodgepole pine
{Pinus contorta Dougl. ex Loud. var. iatifoiia ) upland forests.

3. To describe and compare the molecular diversity (using PCR-RFLP analysis) of ECM
communities associating with naturally regenerating black spruce seedlings across the
three habitats and within each characterized fungal morphotype.

4. To confirm the characterization and improve on the resolution of identified ECM
morphotypes by comparing restriction fragment profiles generated from a representative
sub-sample of roots from the same morphotype. To also compare these fragment
patterns to those in reference databases and descriptions published in the literature.

Literature Review
ECTOMYCORRHiZAE
Definition, classification and structure
Mycorrhizae are mutualistic symbioses (based on a bidirectional exchange of nutrients)
between fungi and plant roots (Smith and Read 1997). There are seven currently
recognized groups of mycorrhizae, including the vesicular-arbuscular, ectomycorrhizal,
ectendomycorrhizal, ericoid, arbutoid, monotropoid and orchid types. These groups are
distinguished based on the fungal and plant taxa involved, as well as on differences in
structure and physiology of the associations (Allen 1991 ; Molina et al. 1992b; Smith and
Read 1997; Ursic and Peterson 1997). All but the ectomycorrhizae exhibit intracellular
colonization of the root cortical cells to some extent and, except for the intermediate
ectendomycorrhizal type, were formerly classified as endomycorrhizae.

In ectomycorrhizae (ECM), fungal hyphae (the main fungal structures) form a sheath
(mantle) around the absorptive areas of growing root tips, which changes fine root
morphology (increased thickness, root bifurcation and clustering) for more efficient nutrient
transfer between fungus and plant host (Read 1997; Smith and Read 1997). From the
mantle, inwardly growing hyphae penetrate the root and grow between the epidermal and
cortical cells, producing an intricate netlike structure (the interface of nutrient exchange)
known as the Hartig net (Smith and Read 1997). In basidiomycetes, hyphal strands
contiguous with the mantle extend into the surrounding soil where they join with the
extensive networks of soil mycelia and form essential pathways for nutrient and water
transport and connections with fungal fruiting bodies (sporocarps) (Smith and Read 1997).
ECM are characteristic of forest trees and shrubs growing in areas where nitrogen (N) and
phosphorus (P) availability is seasonal or intermittent and tend to inhabit the acidic litter
layers near the soil surface (Allen 1991; Brundrett 1991; Isaac 1992; Kendrick 1992). ECM

fungal colonization has been related to soil moisture, nutrients, organic matter, and pH, with
ECM fungi preferring moist but well-drained soils with high organic matter content, low N
and P availability, and acidic pH (McAfee and Fortin 1989; Thormann etal. 1999).

Functions and benefits o f symbioses
ECM symbioses between plants and fungi play some central roles in important ecosystem
processes and functions (Brundrett 1991; Read 1991; Cornelissen etal. 2001). The
structure of ECM not only increases the root absorbing area for greater efficiency of carbon
and nutrient exchange between symbionts, but also increases the root area in contact with
the soil (Rygiewicz and Andersen 1994). Nitrogen (and to a lesser degree P) is the most
important determinant of plant growth and productivity in northern forests (Smith and Read
1997). Ammonium (NH 4^) is the predominant form of N available to plants, but when soils
are relatively acidic, cold, or poorly aerated, the rate of ammonification is generally low, so
most N is present in organically-bound forms (Smith and Read 1997). Through increased
root absorptive area and soil volume explored, ECM plants have greater access to
previously inaccessible sources of inorganic N (N H /) and P in the soil. Colonized roots
may also have different uptake properties (such as a lower Michaelis constant [Km]), as
uncolonized roots do not absorb P as efficiently (Tinker 1984).

Recent molecular studies have revealed that some fungal species previously regarded (in
evolutionary terms) as decomposers of woody debris are both frequent and abundant
components of ECM communities (Hibbett et al. 2000; Kôijalg et al. 2000). These fungi
possess proteolytic enzymes involved in the decomposition of organic material and may
provide host plants with access to both simple and complex organic forms of N and P (Read
1991 ; Smith and Read 1997; Read and Perez-Moreno 2003). Future research is required to

assess the roles of ECM fungi capable of degrading recalcitrant compounds in nutrient
cycling in forest ecosystems.

The benefits of ECM symbioses to plant hosts have been reported in numerous studies.
ECM plants have been shown to exhibit increased growth and survival (Gagnon etal. 1988),
enhanced rooting (Stein etal. 1990), and greater resistance to soil-borne diseases (Morin et
al. 1999) when compared to non-mycorrhizal plants. Furthermore, the plants require less
fertilizer and appear to be better able to withstand environmental pollution, stress, and
transplant shock than non-mycorrhizal plants (Anderson 1988; Kendrick 1992; Colpaert and
van Tichelen 1994). Some plants are unable to become established or grow normally
without an appropriate fungal partner (Harley and Smith 1983). Different species of ECM
fungi exhibit phenotypic variation with respect to nutrient transfer and uptake, storage
capacity, and promotion of host growth (Haug and Oberwinkler 1987; Bruns 1995).
Therefore, it is likely that, for a plant to receive the maximum benefits of ECM symbiosis, it
must be associated with the best fungal partner for the environmental conditions present
(Haug and Oberwinkler 1987). From bioassay experiments using PInus sylvestris L. and
Betula pendula Roth, Jonsson et al. (2001) recently reported that the effects of ECM
symbiosis on host productivity depend on context-specific factors including plant host
species, fungal species richness and composition, and soil nutrient regime.

Growing root tips release considerable amounts of photosynthetically-derived carbon (C)
into the soil, which plays an important role in soil aggregation, nutrient availability and
uptake, and microorganism nutrition (Darrah 1991). ECM fungi require C for sustaining
existing fungal biomass (on root tips and in the soil) and for producing new fungal biomass,
and acquire most of their C from their plant partners (Harley and Smith 1983; Soderstrom
1992). Cornelissen etal. (2001) suggest that ECM strategies are linked with low ecosystem

c turnover based on intermediate seedling relative growth rates, high foliar N and P content,
and intermediate to poor litter decomposition rates as compared to different types of plants
harboring ericoid and arbuscular mycorrhizae. Rygiewicz and Andersen (1994) revealed a
reduction in overall C retention due to increased C in the roots and higher rates of
belowground respiration in a mycorrhizal coniferous seedling (ponderosa pine and
Hebeloma crustuliniforme) microcosm experiment. The key role of ECM in 0 cycling
(particularly in the positive feedback loop between plant growth rate, leaf and litter quality,
and decomposition rate) may have important repercussions for the 0 gains and losses of
ecosystems and thus for the 0 budget at regional and global scales (Read 1991 ;
Cornelissen etal. 2001).

Nutrients are not only exchanged in one plant - one fungus systems, but have been shown
to be translocated through soil mycelial networks between the same and different species of
plants and fungi (Finlay and Read 1986a, b; Simard etal. 1997). In growth chamber
experiments, Finlay and Read (1986b) found that P accumulated in mycorrhizal roots of one
plant before being transported to shoots of other plants connected through the mycelial
network. The distance of translocation was limited only by the size of the growth chamber.
It was evident from this study that the plant’s investment of C as fuel for fungal metabolic
processes provided the potential for exploitation of the soil P resources (Finlay and Read
1986b). Radiolabelled C-transfer has been demonstrated between Sitka spruce {Picea
sitchensis (Bong.) Carr.) and pine species {Pinus contorta Dougl. ex Loud, and P. sylvestris)
in microcosm experiments (Finlay and Read 1986a) and between paper birch {Betula
papyrifera Marsh.) and Douglas-fir {Pseudotsuga menziesii (Mirb.) Franc.) in the field
(Simard etal. 1997). Similarities in ECM communities of lodgepole pine {Pinus contorta var.
Iatifoiia), white spruce {Picea giauca (Moench) Voss.) and subalpine fir {Abies iasiocarpa

(Hook.) Nutt.) may also indicate potential for hyphal linkages between different plant species
(Kranabetter et al. 1999).

ECM FUNGAL COMMUNITIES
Composition and diversity
Communities are assemblages of species within a defined area and are described in terms
of the species present (composition and richness) and their relative abundance (Egger
1995). At least 5000-6000 species of fungi are involved in ectomycorrhizal associations,
representing about 10% of all known soil fungi (Molina etal. 1992b; Smith and Read 1997).
Most ECM fungi are members of the Basidiomycota, with some representation within the
Ascomycota and Zygomycota (Smith and Read 1997). Molecular dating places the
basidiomycetes at about 125 million years old, which is about four times younger than the
zygomycetes (that form vesicular-arbuscular mycorrhizae) that appear to have originated
with land plants (Hibbett et al. 2000). Phylogenetic analyses reveal that ECM fungi are not a
monophyletic group (i.e. originate from several independent lineages), and symbiosis with
plants has been convergently derived (and perhaps lost) many times over millions of years
(Bruns 1995; Hibbett etal. 2000). Some taxa are closely related to wood-decaying
saprobes, some are thought to have wood-rotting ancestors, and some are related to other
saprobictaxa (Tanesaka etal. 1993; Hibbett etal. 2000). Evolutionarily, this variation in
ability to degrade wood could have made possible spéciation to avoid competition between
closely related species that would otherwise use the same resources and occupy the same
niche (Tanesaka etal. 1993).

Community diversity is the variation in species assemblages within a community, and can be
high (many species with relatively even abundance) or low (few species with relatively
uneven abundance) (Bruns 1995; Miller 1995). Diversity results from resource partitioning.

disturbance, competition, or interactions with other organisms. It is important to understand
factors influencing diversity on a local scale because niche and guild structure among
different fungal species may provide information furthering the understanding of the
functional significance of ECM diversity (Bruns 1995). ECM fungal communities appear to
exhibit high diversity, even within small areas where factors that tend to alter diversity (e.g.
host specificity, regional soil differences, climatic differences, and large-scale disturbances)
are fairly constant (Bruns 1995; Gehring etal. 1998). This high ECM fungal diversity may
provide individual trees and entire forests with a range of strategies for efficient functioning
in an array of plant-soil systems (Amaranthus 1998). There is still much remaining to be
investigated in this area of research.

In field surveys involving various coniferous hosts, species richness (number of ECM types
described) has been reported to vary from less than 10 to greater than 50 (Danielson and
Pruden 1989; Bradbury 1998; Bradbury etal. 1998; Flynn etal. 1998; Arocena etal. 1999;
Hagerman etal. 1999a; Kranabetter etal. 1999; Mah etal. 2001). Bruns (1995) found a
range of 20-35 fungal species for several small sites with homogeneous environmental
conditions and occupied by a single host species. Studies generally report ECM community
structures consisting of few very abundant fungal species and many less abundant or rare
species (Kranabetter etal. 1999; Taylor etal. 2000; Kranabetter and Friesen 2002).

Factors that alter diversity in ECM communities
Host specificitv and receotivitv
The composition of ECM communities may include several types of ECM fungi, depending
on the specificity of the host and the fungus (Egger 1995). ECM fungi form associations
with about 30 families of plants, including many important timber species within the
Pinaceae, Betulaceae, Fagaceae and Dipterocarpaceae (Smith and Read 1997). Often,
10

specific host characteristics determine the presence and abundance of fungal species
(Samson and Fortin 1986). Host specificity refers to the range of plants with which fungal
species can form functional ECM, and varies from narrow (genus- or family-specific) to
broad (several different orders or classes) (Molina et al. 1992a, b). Many fungal genera
show relatively even distribution along the host range spectrum. Host receptivity refers to
the range of fungi with which a host plant can form functional ECM, varying from those
plants receptive to few to those receptive to many fungal symbionts (Molina et al. 1992b).
Host receptivity is difficult to measure, although measurements of the fungal diversity
associated with a particular host may indirectly reflect this receptivity.

Host-specific fungi provide biological mechanisms to partition soil resources and provide
nutrients to specific plant species, but are restricted by the ecological tolerances of their
hosts (Brundrett 1991 ; Molina et al. 1992b). For example, Suillus grevlllei is consistently
associated with tamarack over diverse environmental conditions ranging from bogs to weiidrained sandy sites (Samson and Fortin 1986). Although host-specific fungi show strongly
specialized relationships with specific plants in nature, they have been induced to associate
with other genera in pure culture (Molina etal. 1992b). Samson and Fortin (1986) grew
tamarack seeds in sterile vermiculite and inoculated plantlets with fungi cultured from
sporocarps collected beneath tamarack or black spruce stands. ECM fungi previously
identified as sporocarp-specific to Larix species under field conditions showed faster and
better ECM development with this host, indicating some degree of host specialization. Eight
of the 12 fungi collected in the vicinity of black spruce also formed ECM with tamarack.
Massicotte et al. (1994) reported that some Rhizopogon species that were restricted to one
host in monoculture were able to extend their host range to a nearby (companion) plant in
dual culture. This study shows, at least in pure culture and pot experiments, that the
presence of companion plants appears to influence the ability of some less host-specific
11

fungi to colonize neighboring plants by first developing on a primary host, and then
spreading onto a secondary host (Walker 1987; Massicotte et al. 1994; Massicotte etal.
1999). Walker (1987) suggested that pines and larches may be more easily colonized than
spruces, and that whereas some fungi may be unable to establish symbioses with spruces
alone, they may succeed if pines or larches are present to provide a source of inoculum (the
spruce mixture effect). In natural systems, ecological specificity may be more important
than host specificity, as companion plants may influence the ability of ECM fungi to colonize
neighboring plants (Massicotte etal. 1999). Fungal mycelia extending from ectomycorrhizal
fine roots of mature trees are thought to be an important source of inoculum available to
regenerating or outplanted seedlings (Finlay and Read 1986a; Hagerman etal. 1999). The
inoculum potential of a fungal mycelium may depend on whether it is linked to a compatible
host (Massicotte etal. 1994).

Overlaps in host compatibility (i.e. lack of host specificity) permit fungi to connect inter- and
intra-specific combinations of host plants through a common mycelial network (Finlay and
Read 1986a; Molina etal. 1992a, b; Read 1997; Simard et al. 1997). These mycorrhizal
guilds are groups of plants and fungi sharing compatibilities and forming a functional unit in
space and time (Massicotte et al. 1999). Linkages play a key role in mycorrhizal community
structure and dynamics as they are important for nutrient flow via mycorrhizal mycelia. It
has been suggested that hosts such as red alder {Alnus rubra Bong.) and Douglas-fir, which
often form pure stands early in their succession, tend to form specific associations with ECM
fungi, while species such as western hemlock [Tsuga heterophylla (Raf.) Sarg.), which
grows in the shaded understory of other trees, tend to form non-specific interactions (Kropp
and Trappe 1982).

12

Environmental gradients
Host plant community composition may not accurately reflect fungal community composition,
especially across sites with heterogeneous soil conditions (Gehring etal. 1998).
Environmental gradients help explain patterns of fungal species distribution across
landscapes (O’Dell at al. 1999). For example, the growth responses of different ECM fungi
vary with soil moisture content, and both drought and waterlogging can be limiting factors to
ECM formation (Stenstrom 1991). In waterlogged soils, oxygen deficiency is expected to
slow or prevent ECM formation due to inhibition of fungal aerobic metabolic processes
(Tinker 1984; Walker 1987; Stenstrom 1991). The accompanying changes in soil chemistry
and redox potential also may favor the accumulation of compounds toxic to fungi (Isaac
1992). In vitro experiments to examine the effect of brief flooding on mycorrhizal formation
were conducted by Stenstrom (1991). She found that different groups of ECM fungi varied
in their susceptibility to flooding, and also that waterlogging was not associated with fungal
oxygen deficiency, possibly because many forest fungi have hydrophobic mycelia, which
may provide air pockets in the soil. The effect of dry soil conditions on ECM fungi is not well
understood. In greenhouse and field experiments with Norway spruce {Picea abies (L.)
Karst.), Feil etal. (1988) found that drought conditions did not completely inhibit ECM
growth, but resulted in an increased branching density of very fine roots, thereby providing
contact with greater volumes of soil. This may be an adaptation to water stress that allows
enhanced uptake of water from dry soils via ECM.

Nitrogen availability may also be a major factor in structuring ECM fungal communities.
Lilleskov et al. (2002) recently described a shift in ECM fungal community composition from
taxa specialized for N uptake under low N conditions, to taxa specialized for high overall
nutrient uptake, and finally to taxa specialized for P uptake under high N, low P, and
acidified conditions. Taylor etal. (2000) also reported shifts in species composition of ECM
13

communities in response to increased N input in European spruce and beech forests. The
consequences of any changes in ECM fungal community structure for ecosystem function
and plant nutrition depend on how community function changes as community structure
changes.

Few (if any) studies have attempted to relate ECM fungal distribution to environmental
gradients independent of plant hosts (O’Dell et al. 1999). Thus, the effects of environmental
gradients on ECM fungal communities are difficult to assess in natural soils because
gradients may limit the distribution of the host (Gehring at al. 1998; O’Dell at al. 1999). In
ECM fungal sporocarp surveys, Nantel and Neumann (1992) found mushrooms associated
with a particular tree species were only found with that species over part of its range.
Similarly, O’Dell at al. (1999) found some ECM fungal sporocarps under hemlock were
restricted to the dry end of the soil moisture gradient (e.g. Cortinarlus olympianus and
Russula bravlpas), while other fungi were restricted to the wet end of the gradient (e.g.
Amanita constricta and Boletus mirabilis). The maximum number of species was found
midway along the soil moisture gradient. Although sporocarp abundance does not directly
represent the abundance of ECM belowground (Egger 1995; Mehmann etal. 1995; Gardes
and Bruns 1996), both studies demonstrate the differences in soil moisture tolerances of
different fungal species. In response to long-term atmospheric N deposition in Alaska,
Lilleskov at al. (2002) found a drastic decline in ECM fungal abundance and diversity.
Taylor etal. (2000) also reported decreased ECM community diversity with increased N
input in Europe. This response occurred rapidly in sporocarp communities and more slowly
in belowground ECM communities. Through direct examination of ECM root tips. Walker
(1987) described differences in Sitka spruce ECM community diversity that corresponded to
changes in soil conditions and nutrient availability. Similarly, Gehring etal. (1998) found
variable patterns of ECM fungal species occurrence and abundance on Pinyon pine {Pinus
14

edulis Engelm.) roots across environmental gradients ranging from very dry, nutrient-poor
soils to sandy loam soils in Arizona, but no difference in fungal species richiness. Tfiey also
found that each tree was dominated by a single fungal species, but that the same species
did not dominate on all trees. From the results presented in these studies, it appears that
ECM fungal community composition depends on the individual tolerances of individual
species for specific site conditions (Doudrick etal. 1990). Plants with access to a broad
diversity of ECM fungi are probably colonized by those best suited to the range of soil
conditions present and may be more capable of adapting to changes in the environment
(Amaranthus 1998; Durall et al. 1999).

Disturbance and other factors
The role of disturbance in maintaining ECM fungal diversity is to open up patches for
colonization (Bruns 1995). Changes associated with disturbance events may decrease C
input to fungi, modify the age and species of plants present on a site, remove or displace the
forest floor, lose large overstory trees that can change the physical environment of the soil,
and alter the composition of soil microflora and fauna communities (Jones et al. 2003).
These changes might select for different communities of ECM fungi. There are many recent
studies that focus on comparing ECM fungal community diversity between sites differing in
some form of disturbance (Bradbury 1998; Bradbury etal. 1998; Baldwin 1999; Durall etal.
1999; Hagerman et al. 1999; Kranabetter et al. 1999; Stendell et al. 1999; Byrd et al. 2000;
Mah etal. 2001; Kranabetter and Friesen 2002; Jones etal. 2003). These studies usually
found decreased fungal species richness and a shift in abundance for many species
following disturbance events such as fire or forestry practices. Very recently, Jones et al.
(2003) concluded that the major impact of forestry practices on ECM fungal communities is
a shift in species composition rather than a reduction in the percentage of roots colonized.

15

Numerous other biotic and abiotic factors may influence ECM fungal community composition
and diversity on very localized scales. This is due to the high heterogeneity of the soii
environment, which can be partitioned into multiple niches, each with its own unique
combination of nutrient source, moisture level, physical and chemical properties, and
particle size distribution (Egger 1995; Gehring etal. 1998). Local climate, topography, and
aspect may aiso alter fungal communities. Some authors suggest that changes in
community composition and diversity occur as forest stands mature, and there is evidence
for replacement competition in ECM fungi (Bruns 1995). Fungal community composition
and diversity appear to shift throughout forest succession, until fungi specialized to the
climax host and conditions dominate the community (Doudrick etal. 1990; Brundrett 1991).

METHODS OF STUDYING ECM COMMUNITIES
Morphological techniques (microscopy)
It is generally agreed that attempts to isolate and culture ECM directly from root tips are of
iimited value to community studies because many ECM fungi will not grow or may exhibit
altered characteristics under experimental conditions in the laboratory. Instead, the use of
light microscopy to directly examine ECM roots allows certain morphological features to be
used to taxonomically group ECM into families, genera, or species (Agerer 1987-2002;
Ingleby etal. 1990; Goodman etal. 1996). Morphological characterization (morphotyping)
of ECM has the advantage that it is reiatively inexpensive and enables large numbers of
roots to be assessed in a short period of time (Taylor et al. 2000). Although some
morphological features such as color and ramification may vary under different
environmental conditions, other features such as mantle characteristics and hyphai
associations do not change considerably (Haug and Oberwinkler 1987; Bruns and Gardes
1993; Egger 1995). However, the taxonomic distinction between simiiar morphotypes is

16

sometimes difficult, and whereas some fungi may be identified to species, others may be
identified only to genus, family, or larger grouping.

Molecular techniques (PCR-RFLP)
The most important methodological advance in ECM community studies is probably the use
of the polymerase chain reaction (PCR) to amplify target sequences of DNA (Horton and
Bruns 2001). The DNA most commonly targeted in these studies is the nuclear-encoded
ribosomal DNA (rDNA), which is present in high copy number and consists of highly
conserved coding regions (the ribosomal small subunit (18S) and large subunit (28S) genes)
as well as noncoding (internal transcribed spacer (ITS)) regions between genes (Bruns and
Gardes 1993; Egger 1995; Horton and Bruns 2001). The conserved sequences are suitable
targets for universal and fungal-specific oligonucleotide primer pairs that are used to
selectively amplify specific regions of the more variable ITS regions (Egger 1995; Horton
and Bruns 2001).

Restriction fragment length polymorphism (RFLP), the use of restriction endonucleases to
digest the amplified rDNA at specific target sequences, is often used in conjunction with
PGR (Egger 1995). Sequence differences in ITS DNA are usually the result of insertions or
deletions causing length variation (Horton and Bruns 2001). Thus, enzymatic digestion of
ITS DNA with just a few restriction endonucleases produces DNA fragments of varying sizes
(providing there is no overlap in the specific DNA sequences where cleavage occurs) that
may be used to distinguish between closely related species (Egger 1995; Kraigher et al.
1995; Mehmann et al. 1995). Comparisons of ECM and sporocarp restriction fragments
may facilitate fungal identification as sporocarps are generally much easier to identify than
root-associated ECM fungi. The RFLP patterns from ECM do not always agree with
sporocarp patterns (Gardes and Bruns 1996; Dahlberg et a i 1997), but closely related fungi
17

can often be grouped together based on the presence or absence of ITS fragments (Kârén
et al. 1997). Providing the same protocols, primers and restriction endonucleases are used,
databases of restriction fragment patterns allow comparisons of ECM fungal communities
across different studies (Mah etal. 2001).

Molecular techniques have the advantages that they are largely independent of
environmental effects and improve the resolution of ECM identification. Whereas
microscopic examination of ECM is suitable for sorting fungi into families and genera based
on differences in morphological characteristics, molecular analyses (PCR-RFLP) potentially
allow identification of fungal species, although identification is limited by the availability of
reference databases (Egger 1995; Horton and Bruns 2001). Recent ECM community
studies have employed a combination of morphological and molecular methods. In a
comparison of methods for assessing diversity of hybrid spruce ECM, Mah etal. (2001)
found that morphotyping may have underestimated ECM diversity, but identified rare types
that did not amplify properly during the PCR. According to Moritz and Hillis (1996), studies
that incorporate both morphological and molecular data provide better descriptions and
interpretations of diversity than those that focus on just one approach for estimating
phylogeny. Phylogenetic analysis of the ITS-RFLPs within morphotypes revealed that
morphological classification was useful for grouping ECM formed by the same fungal genera
and families for many, but not all, fungi (Sakakibara et al. 2002).

Measuring ECM com m unity structure and diversity
Community structure can be compared between habitats through analysis of species
abundance patterns (^-diversity) within communities (Taylor etal. 2000). Rank-abundance
curves (plot of arithmetic rank of species versus log abundance) of ECM types are generally
straight lines, indicating the dominance of a few fungal types (Taylor et al. 2000). Less

18

diverse and more uneven distributions of morphotypes (broken stick distribution) have also
been sometimes observed (Kranabetter and Friesen 2002). These curves are thought to
result from situations where availability of a single prevailing ecological resource equally
constrains all species present in the community (Taylor et al. 2000).

Species richness measures can be expressed as simple counts of individuals, proportions of
total populations, or averages per sampling unit. Community diversity (species richness
and/or abundance) is often compared between different treatments or studies by calculating
non-parametric diversity indices and comparing the means by analysis of variance (ANOVA)
(Magurran 1988; Krebs 1989). Information theory indices (e.g. Margalef, Shannon,
Shannon Evenness indices) attempt to measure the amount of order in a system by
assuming that all individuals are randomly sampled from an infinitely large population, and
that all species are represented in a sample (Krebs 1989). The Margalef Index is a measure
of species richness and is calculated by dividing one less than the number of species (S) by
the natural log of the number of individuals (n), or (S-1 / In n) (Magurran 1988). The
Shannon Index equally emphasizes both richness and abundance (evenness) by
determining the relative contribution of each species to the community (Taylor at ai. 2000).
It is calculated from - I p, (In pj, where p, is the proportion of individuals in the i‘*^species
(Magurran 1988). Dividing the Shannon Index by the natural log of the number of species
(S) gives the ratio of observed to maximum diversity, and is a measure of species evenness
(Shannon Evenness Index) (Magurran 1988). Because the Shannon and Shannon
Evenness indices are based on proportional abundances of all morphotypes present
(heterogeneity indices), they are sensitive to the presence of rare ECM types. For each of
these measures, the diversity index increases with an increase in number of morphotypes.
Dominance measures (e.g. Simpson Index) are heterogeneity measures that are weighted
towards the more abundant species in a sample. The Simpson Index, which suggests that

19

diversity is inversely related to the probability that two samples picked at random belong to
the same morphotype, is calculated from I p,^, and is usually expressed as its reciprocal
value (Magurran 1988).

Genetic diversity of various populations and communities of fungi may also be compared
across different treatments and studies, as long as the same molecular methods were used.
Any of the previously described non-parametric diversity indices may be calculated for
molecularly-derived data by replacing proportional abundance of morphotypes with
proportional abundance of genotypes (defined by similarities in restriction fragment patterns
or sequences). Some suggest that these analytical approaches to fungal diversity may be
of limited value in community studies as they employ sampling approaches and diversity
indices that were designed for unitary organisms (Zak and Visser 1996). In particular, the
abundance component of diversity may be lost due to loss of samples during the various
phases of the molecular procedures.

The Phi (cp) Index was recently derived to attempt to resolve issues regarding the use of
proportional abundance in calculating ECM diversity measures (Mah etal. 2001). This
index is based on pairwise distances (obtained from the Dice index distance matrices) of
ECM restriction fragment patterns and ranges from zero (identical fragment patterns
between pairs of samples) to 1 (no fragments shared between pairs of samples).
In a data matrix with i rows and j columns, the pairwise distances (d) for individual root tips
are squared, summed, and divided by one less than the total number of samples (n-1 ). To
calculate the Phi Index, the sum of these average squared distances for each column is
divided by the total number of samples (n):
n

n

O = Z [Z d^ij / n-1] / n
\=^ 1=1

where I = j = n

20

As with the non-parametric diversity index values, an increase in Phi values implies greater
diversity.

BLACK SPRUCE AS AN ECM HOST
Distribution
Black spruce is the most widely distributed tree species of the Canadian boreal forest. Its
range extends from Alaska through all the provinces and territories of Canada, and reaches
south into the United States below eastern Manitoba (Farrar 1995). In British Columbia, its
range is limited to the northern regions of the province where it is common north of the
Fraser River, and occurs in patches southward to just north of Quesnel (Brayshaw 1996).
Its western limit is Seely Lake, between Smithers and Terrace, and it extends past McBride
to the east. It is found in the Boreal White and Black Spruce (BWBS), Spruce-Willow-Birch
(SWB), Sub-Boreal Spruce (SBS), Sub-Boreal Pine - Spruce (SBPS), Interior CedarHemlock (ICH) and Engelmann spruce - subalpine fir (ESSF) biogeoclimatic zones
(Meidinger etal. 1991; Brayshaw 1996; C. DeLong, pers. comm.).

In the central interior of BC, black spruce occurs in the SBS zone, which includes the
Nechako and Fraser plateaus and Fraser Basin from valley bottoms to 1100-1300 m
elevation (Meidinger etal. 1991). The “typical” SBS subzone is SBSmk (moist, cold), which
ranges from Prince George to Fort St. James to Nation Lakes to the Williston Reservoir
(Meidinger et al. 1991 ). The climate of the SBS zone is characterized by severe, snowy
winters, warm, moist and short summers, and moderate annual precipitation. Forests are
broadly transitional between the Douglas-fir and pine - spruce forests to the south and
southwest, and the boreal forests to the north (Meidinger etal. 1991).

The commercial range of black spruce is considerably less than its geographic range
(Viereck and Johnston 1990). It is one of the major timber crop species in eastern Canada
21

as its long wood fibres make It Ideal for pulp and paper production. In BC, black spruce Is
considered undesirable and Is not selectively harvested (Krestov et al. 2000).

Habitat preferences
Black spruce Is typically found In cold, poorly drained, nutrient-poor habitats, and generally
Increases In abundance with Increasing latitude and decreasing soil drainage (Argus et al.
1992; Krestov etal. 2000). The most productive growth of black spruce occurs In subhygric
(where water Is removed slowly so that soil remains moist for a significant portion of the
growing season) and submesic (where water Is removed rapidly In relation to supply and Is
available for only short times following precipitation) soils (Krajina et al. 1982; Meidinger et
al. 1991). Substrates are usually wet organic soils, but black spruce also grows on deep
humus, clays, loam, sand, coarse till, boulder pavements and shallow soil mantles over
bedrock (Viereck and Johnston 1990; Meidinger et al. 1991 ; Krestov et al. 2000). It
commonly occurs In acidic boreal bogs and fens, as well as In neutral or alkaline swamps,
where It tends to establish on hummock-llke mounds of decaying wood material. This
remarkable ability to grow In a range of soil conditions makes black spruce an Ideal host
species for studying ECM community variation across an ecological gradient.

In central BC, black spruce typically grows In Sphagnum -dominated wetlands, In pure
stands, or with tamarack {Larix larlcina), a species at the southwestern edge of Its range In
the central Interior (Krajina etal. 1982; Meidinger etal. 1991; Farrar 1995; Krestov etal.
2000). Tamarack Is a rare species In the SBS that occurs In only a few fens and swamps of
the Nechako, Chllako and Blackwater drainages (Krajina etal. 1982; Meidinger etal. 1991;
Krestov etal. 2000). Wetland soils are In the Organic order and vegetation communities
Include sedge marshes, shrub fens, black spruce and hybrid white spruce fens and swamps,
and black spruce - Sphagnum bogs (Meidinger etal. 1991; Soil Classification Working
22

Group 1998). Associated vegetation commonly includes scrub birch {Betula glandulosa var.
glandulosa Michx.), Labrador tea {Ledum groenlandicum Oeder), and willow (Salix) species
(Krestov at al. 2000).

Black spruce is also found in mid-seral upland habitats, associated with lodgepole pine
{Pinus contorta var. latifolia), interior hybrid spruce {Picea glauca (Moench) Voss x
engelmannii Parry ex Engelm.) and trembling aspen {Populus tremuloides Michx.) (Krajina
et al. 1982; Meidinger et al. 1991 ; Farrar 1995; Krestov et al. 2000). Upland forest soils are
primarily Luvisols, Podzols, Brunisols and Gleysols (Meidinger etal. 1991), and sites are
dominated by conifers, mosses, lichens and dwarf woody plants, with reindeer lichen
{Cladina species), lingonberry {Vacclnium vltls-idaea L.), highbush cranberry ( V/burnum
edule (Michx.) Raf.), soopolallie {Shepherdia canadensis (L.) Nutt.), and common mitrewort
{MItella nuda L.) representing dominant plant associations (Krestov et al. 2000). These
plant associations generally Indicate low to medium productivity communities developed on
nutrient-poor, water-deficient sites. However, soil moisture conditions may actually vary
from water surplus (after snowmelt and summer precipitation events) to water deficit (in late
spring), depending on local topography, soil texture and structure, and drainage constraints
posed by the presence of a root-restricting clay soil horizon near the soil surface (Krestov et
al. 2000).

Morphology and growth
Black spruce is usually a small tree with a characteristic narrow crown (which is often
deformed near the top) of branches that turn down and out (Brayshaw 1996). It is a shadetolerant species with modest spatial requirements (Krajina etal. 1982). The needles are dull
gray-green, 8-15 cm long, straight and blunt, and are densely set along dark orange or
brown twigs that have many short brown hairs. The seed cones are ovoid, 2-3 cm long and
23

purple-brown at maturity (Farrar 1995). It has a shallow rooting system, an adaptation to
permafrost, as It can grow on ground solidly frozen for 5-8 months of the year (Krajina etal.
1982). Some roots may penetrate to a depth of 60 cm, but most spread laterally at the
moss-humus Interface so that the bulk of root biomass Is In the top 20 cm of soil. With the
rapid accumulation of the organic layer each year, younger adventitious roots grow from the
main stem (Viereck and Johnston 1990). Roots function as an anchor for aerial shoots as
well as storage organs for nutrients or water.

Black spruce reaches Its final developmental stage at 10-15 years, and possesses the ability
to restore Its structure and Integrity as an adaptive response to Increased physiological
needs or as a reaction to environmental stress (Begin and Flllon 1999). This ability
accounts for the high plasticity of the species, which can maintain reproductive sustainability
In the absence of cone or seed production and grow In spite of harsh climatic conditions.
Over Its range and In different habitats, black spruce varies In height growth rate and other
phenologlcal traits. For example. In northwestern Ontario, It was observed that black spruce
trees In bogs were slow-growing and stunted, while better drained soils of upland sites
supported trees that were more robust (Parker etal. 1983). To determine whether local site
differences corresponded to morphological or chemical differentiation In mature black
spruce, Parker etal. (1983) compared 17 morphological characters of cones, needles and
twigs from the crowns of trees from three sites differing In level of soil drainage. Significant
differences In mean measurements were detected for only one characteristic (cone scale
concavity), leading them to conclude that ecotypic differentiation Is not well developed In
black spruce In response to soil moisture level. This was surprising considering the variety
of moisture regimes, soil types, levels and types of competition, and gross differences In
black spruce phenotypes corresponding to the three habitat types. Although differences
were not detected between treatments, there was high variation In characteristics within
24

sites with poor drainage (Parker et al. 1983). In a comparison of water relations in black
spruce seedlings of upland versus wetland origin, Zine El Abidine etal. (1994, 1995) found
no consistent differences, again indicating a lack of ecotypic variation for this species.
Though black spruce is very sensitive to drought, Zine El Abidine et al. (1994) demonstrated
that seedlings can be preconditioned for increased drought resistance. However, the
physiological effects of the preconditioning are variable, and response is not cumulative with
successive drought episodes (Zine El Abidine etal. 1994, 1995).

In wetlands, plant growth is extremely slow due to high water tables, poor aeration, cold
substrates and low nutrient availability (Lieffers and Rothwell 1987a; Macdonald and Yin
1999; Mugasha etal. 1999). High water tables decrease root growth and nutrient availability
(N assimilation) in the rhizosphere (root zone), and lower oxygen levels can result in root
asphyxiation and rotting (Roy et al. 1999). Anaerobic conditions may also favor microbial
activities that produce nitrites and other compounds toxic to the plant (Isaac 1992).
Whereas these conditions tend to limit root growth and survival of most woody plant
species, black spruce Is fairly tolerant of flooding and poor aeration (Lieffers and Rothwell
1987b; Steele et al. 1997; Roy et al. 1999). It tends to avoid total submergence by
establishing on hummocks of decaying wood material and Sphagnum and producing small
volumes of shallow roots that spread laterally (in the top 5-20 cm) at the moss-humus
interface (Lieffers and Rothwell 1987b; Conlin and Lieffers 1993; Steele etal. 1997; Roy et
al. 1999). The lateral roots of mature trees may extend as far as 10 m from the taproot, and
interweave with the roots of neighboring trees for anchorage (Krajina et al. 1982). Both
growth rate and rooting depth have been strongly correlated with depth of the water table
and soil temperature, with deeper penetration (to 60 cm) and increased growth observed in
drier and warmer peatlands (Lieffers and Rothwell 1987b; Conlin and Lieffers 1993). Cold
and wet conditions also decrease the rate of microbial decomposition, which leads to rapid
25

accumulation of organic material around slow-growing seedlings. Vegetative layering (the
rooting of attached branches) is often the primary means of regeneration in organic soils,
where accumulation of mosses cover the lower branches of slow growing seedings or
saplings (Viereck and Johnston 1990). Where conditions are favorable for layering and
unfavorable for seed germination and survival, layering can result in an extending clump of
trees all derived from one seedling (Farrar 1995).

Growth on upland sites is comparable to white spruce and on nutrient-rich soils, the two
species compete (Krajina et al. 1982; Farrar 1995). Black spruce is well adapted to acidic
mor humus forms (containing unincorporated organic material) in forests with a high
accumulation of spruce litter (Krajina etal. 1982). The supply of calcium and magnesium is
low in these habitats, indicating it may not require as high a supply as white or Engelmann
spruces. Greater quantities of calcium, magnesium and nitrates appear to function
adversely on the growth of black spruce, as it exhibits stunted growth in krummholz form on
limestone and dolomitic substrata (where white spruce grows well). It has a greater
dependency on the NH ^ form of N as evidenced by poor growth with even the presence of
a small quantity of nitrate (Krajina et al. 1982).

ECM fungal associates
Although there have been few attempts to describe the ECM fungal community associated
with black spruce, studies examining the mycorrhizal status of this host have reported that
the majority of field-collected black spruce roots are indeed mycorrhizal (Malloch and
Malloch 1981; McAfee and Fortin 1986; Browning etal. 1991; Baldwin 1999; Thormann et
al. 1999). Field surveys In Ontario, Quebec, and Alberta have identified Cenococcum
geophilum as a common ECM symbiont, but further characterization of other black spruce
ECM has not been attempted (Malloch and Malloch 1981 ; McAfee and Fortin 1986;
26

Thormann etal. 1999). Summerbell (1989) cultured several ECM fungi from black spruce
roots collected from different habitats in Ontario and found the most abundant to be the
common, broad host range fungus, Mycelium radiais atrovirens (MRA). Thormann et al.
(1999) also found MRA on black spruce roots from northern Alberta fens.

Scales and Peterson (1991) described non-mycorrhizal black spruce (growing in pouches)
first order lateral roots as slender, with rounded apices and many long, straight root hairs.
Ectomycorrhizal roots exhibited at least one continuous layer of mantle tissue and one or
more discontinuous layers of vacuolate hyphae surrounding the root. Several fungi have
been reported to form ECM with black spruce under laboratory or greenhouse conditions. In
pot and growth pouch experiments, ECM were formed with Hebeloma cyllndrosporum,
Laccaria bicolor, L. proxima, PIsolithus tinctorlus, Tricholoma pessundatum, and Wilcoxina
species (E-strain fungi) (Thomson etal. 1989; Browning and Whitney 1991; Scales and
Peterson 1991). In aseptic culture, Doudrick etal. (1990) found Cenococcum, L. bicolor, L.
laccata, Rhizopogon sp. and Suillus cavipes cultured from sporocarps formed ECM with
black spruce. From this study, the authors concluded that fungal associations with young
black spruce on mineral soils appear to have a broad host range and thus a local inoculum
source (i.e. other hosts). Browning and Whitney (1991) described black spruce ECM formed
upon inoculation with L. bicolor, L. proxima and P. tinctorlus, but found that C. geophilum
and Suillus granulatusiormed very few ECM. Recently, Piercy etal. (2002) described the
occurrence of intracellular hyphae of the inoperculate discomycetes Hymenoscyphus erlcae
and Variable White Taxon (which usually form erlcoid mycorrhizae) in aseptic culture with
black spruce seedlings. In bioassays, Gagnon etal. (1988) demonstrated increased growth
and survival of black spruce seedlings grown in containers with low N after inoculation with
L. bicolor, and Stein etal. (1990) documented enhanced rooting (increased rooting percent,
number of adventitious roots per cutting, and mean root length) of cuttings after inoculation
27

with L bicolor or S. cavipes. Morin etal. (1999) found the ECM fungi Paxillus Involutus,
Hebeloma cyllndrosporum and Tricholoma species inhibited Cyllndrocladlum floridanum, the
pathogen causing root rot and severe mortality in many conifers including black spruce.
There are no known studies that have attempted to describe ECM community structure and
diversity on this host, nor to compare black spruce ECM communities across different
habitats.

28

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36

Morphological Characterization of Ectomycorrhizai Communities Associating
with Black Spruce (Picea mariana) in Wetland and Upland Forests in the
Centrai Interior of BC

ABSTRACT
To compare ectomycorrhizal (ECM) community composition and diversity across black
spruce habitats in central BC, 15 naturally regenerating black spruce seedlings were
randomly sampled from three sites within each of three habitats, including black sprucedominated wetland forests, mixed black spruce - tamarack wetland forests, and mixed black
spruce - lodgepole pine upland forests. Two hundred root tips per seedling were
characterized using morphological techniques (light microscopy). A total of 33 distinct ECM
morphotypes were described and 14 morphotypes were found on three or more seedlings.
Some morphotypes (e.g. Cenococcum, MRA, Amphinema, Russulaceae 2 and
Cortinariaceae 1 and 2) were found in all habitats, whereas others (e.g. Piloderma,
Tomentella and Russulaceae 1) were associated with specific habitats. Seedlings from the
black spruce-dominated wetlands had a significantly greater proportion of non-mycorrhizai
root tips as compared to the mixed species habitats. Morphotype abundance varied
significantly between habitats for six of the 14 most frequently occurring morphotypes, but
the total number of ECM types described per habitat did not. In general, ECM community
diversity was greatest in the black spruce - pine uplands and lowest in the black sprucedominated wetlands, with significant differences between habitats detected for only those
measures emphasizing species richness (i.e. Margalef and Shannon indices). Variation in
ECM community composition and diversity may be due to differences in soil moisture and
nutrient availability (between the wetland and upland habitats) or alternate inoculum sources
(between the pure and mixed species stands).

37

INTRODUCTION
In northern coniferous forests, the roots of most woody plants are colonized by a diverse
group of filamentous fungi that form mutualistic symbioses known as ectomycorrhizae
(ECM) (Harley and Smith 1983). The nature of this association is such that inaccessible
pools of nutrients (N and P) and water are supplied to the plant through their fungal
partners, while energy-rich carbon compounds that support fungal metabolism are supplied
to the fungi via their plant hosts (Harley and Smith 1983). These symbioses are vitally
important to forest health and ecosystem function as they mediate nutrient and water
uptake, protect roots from pathogens and environmental stress, and maintain soil structure
and food webs (Brundrett 1991; Colpaert and van Tichelen 1994; Amaranthus 1998; Morin
etal. 1999). ECM communities often exhibit high diversity (species richness) even when
plant species diversity is low (Bruns 1995). The variation in ECM community diversity
across landscapes has been widely reported, and depends on preferences (or tolerances) of
individual fungal species for the plant hosts present, soil conditions (e.g. moisture and
nutrient regimes, organic content, temperature, etc.), disturbance patterns, and other sitespecific ecological and environmental factors (Doudrick etal. 1990; Brundrett 1991; Bruns
1995). It has been proposed that plants with access to a diverse array of fungi are most
likely colonized by those best suited to the range of conditions present, and that these plants
may be better able to adapt to changes in environmental conditions (Haug and Oberwinkler
1987; Amaranthus 1998; Durall etal. 1999). Because of their ubiquitous nature and
importance to plant and soil biology, there has been growing interest in characterizing ECM
in natural systems in order to try to understand the functional significance of individual
fungal species.

The focus of many recent studies has been to document variation in ECM communities
(species composition and diversity) between disturbed (e.g. by fire, forestry practices, etc.)
38

and control sites within similar forest types (Bradbury 1998; Bradbury etal. 1998; Byrd etal.
1999; Hagerman etal. 1999; Kranabetter etal. 1999; Stendell etal. 1999; Mah etal. 2001).
Fewer studies have examined ECM communities in undisturbed forests, where patterns of
both plant and fungal species distribution across landscapes are at least partly determined
by environmental (particularly moisture) gradients (Gehring etal. 1998; O’Dell etal. 1999).
Rarely do studies attempt to relate ECM distribution to environmental gradients independent
of hosts (O’Dell etal. 1999). For example, Gehring etal. (1998) reported a change in ECM
community composition associated with pinyon pine {Pinus edulis) across a moisture
gradient (xeric to mesic), but no significant decrease in the overall number of species
(species richness) found at different sites. Black spruce (Picea mariana (Mill.) BSP), a
species with broad tolerances for soil moisture conditions (subhygric to submesic), offers an
excellent opportunity to examine variation in ECM communities over an environmental
gradient.

For most woody plant species, growth and survival are limited in water-saturated soils due to
poor aeration, decreased nutrient availability (N assimilation), and changes in soil chemistry
and microbial metabolic processes that mobilize potentially toxic compounds in the
rhizosphere (Tinker 1984; Isaac 1992; Roy etal. 1999). Wetland species such as black
spruce are well adapted to these conditions, establishing on hummocks of woody debris and
producing small volumes of shallow roots that spread laterally (in the top 5-20 cm) at the
moss-humus interface (Lieffers and Rothwell 1987; Conlin and Lieffers 1993; Steele etal.
1997; Roy et al. 1999). Rooting depth is limited by the depth of the water table and soil
temperature, with deeper penetration (to 60 cm) and increased growth observed in drier and
warmer peatlands (Lieffers and Rothwell 1987; Conlin and Lieffers 1993). In these organic
soils, low dissolved oxygen content and differences in soil chemistry may inhibit ECM
formation by limiting fungal aerobic metabolic processes (Tinker 1984; Walker 1987;
39

Stenstrom 1991 ). However, studies conducted in vitro on Inoculated jack pine {Pinus
sylvestris) seedlings showed that different species of ECM fungi varied in their susceptibility
to periodic flooding (Stenstrom 1991). Hebeioma, Laccaria and Thelephora were tolerant of
water saturated soil conditions, while Suilius species were very sensitive to even brief
periods of flooding conditions. Despite challenges presented to ECM formation in wetland
habitats, all woody plant species surveyed in bogs and fens in northern Alberta were found
to be mycorrhizal (Thormann etal. 1999). Little more is known of ECM communities in
northern wetland habitats.

Plant communities associated with black spruce in mid-seral upland forests in BC are
indicators of nutrient-poor, water-deficient sites (Krestov et ai. 2000). These habitats
generally have sandy loam soils, often with a root-restricting clay horizon near the soil
surface that perches the water table during wet periods of the year, leading to seasonal
cycles of moisture surplus and deficit (Krestov etal. 2000). Several studies have reported a
lack of ecotypic variation in black spruce morphological features or water relations due to
improved edaphic conditions in upland forests, even though upland trees often appear more
robust than their wetland counterparts (Parker etal. 1983; Zine El Abidine etal. 1994,
1995). Although black spruce tolerates periods of flooding, it is sensitive to drought
conditions. Preconditioning of containerized black spruce seedlings for increased drought
resistance (higher photosynthetic and gas exchange activities) was achieved by Zine El
Abidine etal. (1994), but the response was not cumulative with successive drought
episodes. In greenhouse and field experiments with Norway spruce (Picea abies), Feil etal.
(1988) noted an increased branching density of very fine roots initiated by drought, an
adaptation to water stress allowing uptake of water from dry soil via ECM.

40

Few studies have been undertaken to investigate ECM symbioses of black spruce. Through
field surveys, the ectomycorrhizal status of naturally regenerating black spruce has been
confirmed in northeastern Ontario (Malloch and Malloch 1981) and northern Alberta
(Thormann etal. 1999), and of outplanted black spruce seedlings in Quebec (McAfee and
Fortin 1989). Studies, in which containerized black spruce seedlings were inoculated with
ECM fungi, have reported increased growth and survival of ECM seedlings grown with low
nitrogen (Gagnon etal. 1988), enhanced rooting of cuttings (Stein etal. 1990), and inhibition
of root rot pathogen infections that are responsible for severe mortality in many conifers
(Morin et al. 1999). There are no known studies that have attempted to describe the ECM
associated with black spruce or to compare ECM communities in different habitats.

This study’s first objective was to use morphological techniques (light microscopy) to
describe ECM associating with naturally regenerating black spruce seedlings across its
habitat range in central BC. In this region, black spruce habitats include both black sprucedominated and black spruce - tamarack wetland forests, as well as black spruce lodgepole pine upland forests. The second objective of this study was to compare the
structure and diversity of ECM communities between the three habitats and to attempt to
relate differences to specific habitat characteristics.

METHODS AND MATERIALS
Site descriptions
The study area was located in the Sub-Boreal Spruce (SBS) biogeoclimatic zone in the
central interior of British Columbia. This area ranges from 51° 30’ to 59° N latitude and 660
to 1140 m in elevation (Meidinger etal. 1991; DeLong and Fahlman 1996). The climate of
the SBS is characterized by severe and snowy winters, warm, moist and short summers,
and moderate annual precipitation (Meidinger etal. 1991). Potential field sites were
41

identified in the Prince George Forest District by studying Forest Cover (1:20,000) maps
(1993) and consulting Forest Service personnel. Subsequently, following site
reconnaissance, selection resulted in three (“replicate”) sites within each of three forest
habitats (black spruce-dominated wetlands, mixed black spruce - tamarack wetlands and
mixed black spruce - lodgepole pine upland forests) (Figure 2.1). “Replicate” sites were
similar in composition of the dominant vegetation and soil properties. All sites contained
mature black spruce trees and an abundance of naturally regenerating black spruce
seedlings ranging from approximately 15-30 cm in height (estimated to be 10-15 years in
age) and lacked visible indications of recent disturbance (such as fire, logging, windthrow
and roads). The location and general characteristics of these sites are presented in Table
2.1. Photographs showing examples of each black spruce habitat are presented in Figure
2 .2 .
Table 2.1. Locations, biogeoclimatic ecological classification (EEC) (DeLong et a i 1994)
and site characteristics of the 9 study sites (T = black spruce - tamarack wetland sites; W =
black spruce-dominated wetland sites; U = black spruce - pine upland forest sites).
Site

Location

PC*
Mapsheet

UTM*
East

UTM *
North

BEC

Site
Series

Stand
Age

# Flagged
Seedlings

T1
T2
T3

Norman Lake Rd (6 km)
Norman Lake Rd (8.4 km)
Hwy 16 W (across from
Tamarack Lake)
Teardrop Rd (km 204)
Teardrop Rd (km 203)
Teardrop Rd (1.5 km W
from km 207)
Teardrop Rd (km 211)
Teardrop Rd (km 401)
Teardrop Rd (km 202)

93G 084
93G 084
93G 084

476580
477890
476740

5965300
5963700
5968800

SBSdw3
SBSdw3
SBSdw3

10
10
10

-50 y
-75 y
90-95 y

36
64
90

93J 025
93J 025
93J 025

497460
497580
494260

6008200
5997200
5999900

SBSmkI
SBSmkI
SBSmkI

10
10
10

—60 y
-60 y
80-90 y

74
21
37

93J 025
93J 025
93J 025

496230
494540
497870

6003800
6008200
5996300

SBSmkI
SBSmkI
SBSmkI

6
6
6

-60 y
-60 y
-70 y

21
21
46

W1
W2
W3
U1
U2
U3

* geographic locations by Forest Cover mapsheet and universal transverse mercator (UTM)
coordinates (approximate) taken from within each plot with a Garmin eTREX GPS unit.

42

ï
UfT'-'—^
4 * 1 t i|V ^ /t
i

WZlTv^OS

B uxrn; MKtwacaiPAPK

ymirr.

IMH

CITY f m
o>

'

'"P.MRL

■

^STI

I.A L I

1

-ifin O ^E l

K iiiir r r h : " .

Figure 2.1. Map (left) of British Columbia showing the study area (orange square) In the
SBS biogeoclimatic zone in central BC (shading indicates the provincial range of black
spruce). Map (right) showing approximate locations of 9 black spruce study sites (T = black
spruce - tamarack wetland sites; W = black spruce-dominated wetland sites; U = black
spruce - pine upland forest sites) in the Prince George Forest District.

The black spruce - tamarack wetland (T) sites were located near Highway 16, about 40 km
west of Prince George. They occurred in fens dominated by black spruce and tamarack in
the dry, warm (SBSdwS) subzone variant of the SBS. The shrub understory consisted
mainly of scrub birch, with common occurrences of willow (Sa//x species), labrador tea and
dwarf nagoonberry {Rubus arcticus L.) on some sites. Herbs present included pink
wintergreen {Pyrola asarifolia Michx.), marsh cinquefoil {Potentilla palustris (L.) Scop.),
common mitrewort, palmate coltsfoot {Petasites palmatus (Ait) A. Gray), buckbean
{Menyanthes trifoliata L.), and round-leaved sundew {Drosera rotundifolia L.). The
microtopography of the T sites consisted of raised mounds (hummocks) of partially
decomposed moss covering woody debris amongst depressions (hollows) that were often
below the level of the water table. Sphagnum moss covered most of the open areas, with
43

other mosses and lichens (Cladina and Peltigera species) observed on drier hummocks and
woody debris. Grasses and sedges were present on sites T2 and T3, and Equisetum
species were observed in the wetter areas of all T sites. Soils were Typic Humisols, a sub­
group of the Organic soils composed of organic material (derived mainly from mosses) in an
advanced stage of decomposition to a depth of greater than 160 cm (Soil Classification
Working Group 1998).

The black spruce-dominated wetland (W) sites were located along the Teardrop Forest
Road (about 60 km northwest of Prince George) in the moist, cool (SBSmkI) subzone
variant. These fens were dominated by mature black spruce stands with an understory of
scrub birch, willow, labrador tea, bog cranberry {Vaccinium oxycoccos L. MacM.) and bog
rosemary {Andromeda polifolia L.). Herb species included pink wintergreen, white bog
orchid {Platanthera dilatata (Pursh) Lindl. ex Beck), single delight {Moneses uniflora),
northern twayblade {Listera borealis Morong.), marsh cinquefoil, common mitrewort, palmate
coltsfoot, and buckbean. Like the T sites, the W sites were characterized by rolling
hummock-hollow microtopography covered with Sphagnum moss (and other species of
mosses and lichens on the warmer, drier hummocks) and organic soils that were also
classified as Typic Humisols.

The upland forest (U) sites were also located in the moist, cool (SBSmkI ) subzone variant
along the Teardrop Forest Road. These sites were dominated by mature black spruce and
lodgepole pine, sometimes with a small component of hybrid white spruce, subalpine fir, or
Sitka alder {Ainus crispa var. sinuata (Regel.) A. & D. Love). A wide diversity of shrubs and
herbs formed the understory of the U-sites: species included prickly rose {Rosa acicuiaris
Lindl.), pink spirea {Spiraea douglasiissg. menziesii Hook.), black twinberry {Lonicera
invoiucrata (Richards.) Banks exSpreng.), highbush cranberry ( V/bumum eduie (Michx.)
44

Raf.), twinflower {Linnaea borealis L.), dwarf blueberry {Vaccinium caespitcsum Michx.),
trailing raspberry {Rubus pubescens Raf.), kinnikinnick {Arctcstaphyics uva-ursi L. Spreng.),
round-leaved rein-orchid {Platanthera crbiouiata (Pursh) Lindl.), rattlesnake plantain
{Gccdyera cbicngifciia Raf.), one-sided wintergreen {Orthiiia secunda (L.) House), pink
wintergreen, and bunchberry {Ccrnus canadensis L.). Mosses (including red-stemmed
feathermoss {Pieurczium schreberi), step moss {Hyicccmium spiendens), knight’s plume
{Ptiiium crista-castrensis), and electrified cat’s tail moss {Rhytidiadeiphus triquetrus)) and
lichens (such as Peltigera, Ciadcnia, and Cladina species) formed the forest floor. Upland
soils were Orthic Gray Luvisols, which characteristically develop in well to imperfectly
drained sites with sandy loam to clay soils, under boreal or mixed forests, in mild to very
cold climates (Soil Classification Working Group 1998).

45

4

Figure 2.2. Photographs showing examples of three black spruce habitats in central BC: A,
mixed black spruce - tamarack wetland (T) habitat; B, black spruce-dominated wetland (W)
habitat; 0, black spruce - lodgepole pine upland forest (U) habitat.

46

Seedling sampling and vegetation and soil analyses
Plots (50 X 50 m^) were established at each site (except for one smaller upland (U1 ) site cn
which a 30 X 35

plot was laid cut). Plots were located at least 10 m inside site

boundaries to decrease edge effects. All regenerating (no obvious signs of vegetative
layering) black spruce seedlings ranging from 15-30 cm in height were located within each
plot. These seedlings were flagged, numbered and approximately plotted on site maps.
When necessary, cross sections of needles were examined with a hand lens to distinguish
black spruce seedlings from hybrid spruce seedlings, based on differences in needle
morphology (Weng and Jackson 2000).

Seedlings were selected for harvest following a simple random sampling design, which
assumed that aii flagged seedlings were equal in terms of environmental influences, and
that each had an equal chance to be sampled. Using a random number table, five seedlings
were randomly selected from each site, for a total of 45 seedlings. Details of the specific
location (e.g. hummock - hollow microtopography) and rooting substrate were recorded for
each selected seedling. In addition, lists of vegetation were compiled for all nine sites
(Appendix i), as well as vegetation occurring within a radius of 0.5 m^ of each selected
seedling (45).

Organic soil samples were collected from several locations within each wetland plot and
combined in plastic bags for classification according to the Soil Classification Working Group
(1998). Soil pits (2-3 per site) were excavated on the upland sites, and the forest floor and
mineral horizons were described and classified according to Green etal. (1993) and the Soil
Classification Working Group (1998). Forest floor and mineral soil (collected from each A
and B horizon) samples were analyzed for texture (clay, silt and sand content), as well as

47

total carbon and nitrogen content, extractable P (Bray P1 method), and pH (Kaira and
Maynard 1991). AI! dried soil samples were stored in plastic bags at 22°C until analyses.

Measuring the depth of rust formation on steel rods has been used to determine the depth of
the water table (or depth of the aerobic zone) over an extended period of time (Carnell and
Anderson 1986; Bridgham etal. 1991; Thormann etal. 2000). The method is based on the
theory that iron will rust rapidly In aerated zones, but not in the saturated, non-aerated or
reduced zone in poorly drained soils (McKee 1978). Steel welding rods (100 cm x 6 mm)
were sanded to remove the copper coating and sets of three rods were pounded into the soil
near each of three selected seedlings in each plot. Rod placement mimicked the
topographical position (i.e. hummock, hollow, level ground, etc.) of the nearby selected
seedling. All rods were placed in the soil July 17-19, 2001, and pulled sequentially at
approximately monthly Intervals (August 22-23, September 17-18, and October 18-19) over
three months. Just prior to removal, a mark was etched into each rod to indicate the level of
soil surface. Rods were gently cleaned of soil and Sphagnum, and the depth of rusting
(determined as the lowest point of the obvious, heaviest rust zone) was measured from the
substrate surface line. All rods were measured immediately upon removal from the soil
substrate.

Fungal sporocarp sampling
During July and August of 2000 and 2001, fungal sporocarps were collected from all study
sites. The mushrooms were described (data not included) and samples were taken from the
spore-producing tissues (i.e. gills, pores, teeth, etc.) of each sporocarp and stored in
microcentrifuge tubes at -20°C for DNA analysis. The sporocarps were then dried and
stored at room temperature.

48

Seedling harvest and ECM characterization
Seedlings were harvested between July 26 to August 2, 2001, when optimal fine root growth
and ECM development was expected to occur. On the wetland sites, a saw was used to
facilitate cutting through the peat surrounding the entire root systems. Seedlings were
placed into 7 L plant pots, double-bagged in heavy-duty garbage bags, and stored at 4°C
until processing.

Prior to each examination, the root system was gently washed free of soil (and Sphagnum)
with tap water. The location of the root collar (the boundary between root and stem tissue)
was determined by hand sectioning the taproot or stem just below root emergence points,
and examining these sections under the dissecting microscope. Stem tissue was
recognized by the presence of distinct vascular bundles or secondary xylem and central
pith, while the presence of central xylem indicated root tissue (Raven et al. 1986). Seedling
height was measured from the root collar and the age of each seedling was determined by
counting growth rings using a cross section of stem just above the root collar. At this time,
seedlings were identified as either true (regenerating from seed) or layered (regenerating
from adventitious roots) seedlings. The presence of stem tissue in cross sections below the
root collar was accepted as evidence of a layered seedling. Root quality was described as
either good (most roots appeared healthy and robust) or poor (many roots appeared black
and withered) and the abundance (high, medium or low) of roots was also noted.

Cleaned roots were floated in a large tray of water overlaying a grid of 1 cm^ cells. Using a
random number table to select grid cell numbers, 1 cm lengths of roots were removed from
the tray until approximately 200 root tips had been selected. Root tips (200 per seedling)
were described using bright field light microscopy following standard techniques of Agerer
(1987-2002), Ingleby etal. (1990) and Goodman etal. (1996). One unbranched root tip was
49

considered to be one mycorrhiza and only root tips that appeared healthy and robust were
examined. ECM were initially described using a dissecting microscope (9-40 x
magnification) and classified according to color, texture, lustre, dimensions, tip shape,
branching pattern, and presence or absence of rhizomorphs (mycelial strands). Root
squash mounts were prepared and examined under a compound microscope (100-1000 x
magnification). Descriptions of mantle features, emanating hyphae, rhizomorphs, and other
distinguishing features were recorded and used to further categorize the different ECM
morphotypes. Root tips that appeared uncolonized or partially colonized (due to the lack of
a well-developed mantle) were categorized as non-mycorrhizal. When possible, preliminary
identifications to families or genera were assigned based on similarities in features to
published descriptions (Agerer 1987-2002; Ingleby etal. 1990; Goodman etal. 1996), but
when identification was not possible, a descriptive name was assigned. Some morphotypes
were photographed using an automatic exposure (PM-10AK) camera attached to a
dissecting (Olympus BX-50) or compound (Olympus SZ-40) microscope, using Ektachrome
160T tungsten professional color reversal film.

Analysis o f morphological data
Several seedling features (e.g. seedling height, root collar diameter, age, root quality, root
abundance, etc.) and site characteristics (e.g. depth of the aerobic zone, microtopography,
ECM vegetation, etc.) were compared qualitatively. Soil nutrient content (C, N and P) and
pH were compared between habitats (and between different soil horizons in the uplands)
using a one-way ANOVA (SYSTAT version 8.0, 1998, SPSS Inc.) to determine significant
differences (a=0.05). Mean comparisons were tested using the Fisher’s Least Significant
Difference (LSD) test (a=0.05).

50

Prior to data analysis, several morphotypes were merged based on descriptions that were
very similar. The ECM frequency (proportion of seedlings with each morphotype) and
abundance (proportion of the entire community represented by each morphotype) were
calculated from the total sample and within each habitat type. The relative abundance of
each morphotype was plotted against species rank order to visualize the overall community
structure and to compare community structure between habitats (Taylor et al. 2000). Mean
ECM abundance values (calculated per seedling and averaged within each habitat) were
compared between habitats using a one-way ANOVA to determine significant differences
(a=0.05). Mean comparisons were tested using the Fisher’s LSD test (a=0.05).

ECM community diversity was measured by means of several nonparametric heterogeneity
indices, including the Margalef (measure of species richness). Shannon (measure of
species richness and relative abundance). Shannon Evenness (measure of relative
abundance) and Simpson (measure of relative abundance weighted towards more the
abundant types) indices (Magurran 1988; Krebs 1989). For each index, an increase in value
indicates greater diversity. Diversity indices were calculated for each seedling and
averaged within habitats. Habitat means were compared using a one-way ANOVA to
determine significant differences (a=0.05) and the post-hoc Fisher’s LSD test (a=0.05).

RESULTS
Seedling and site characteristics
Seedling height ranged from 15 to 54.2 cm, with a mean value of 31.5 cm. Seedling age
ranged from seven to 30 years with an average of 14.3 years. Seedling height and diameter
at the root collar generally increased with seedling age, but this relationship was not always
consistent. No relationship was found between seedling height or root collar diameter and
the extent or diversity of ECM colonization or habitat of origin.

51

Rooting substrate(s) included moss and silty loam soils (upland sites), moss and peat
(wetland sites), and coarse woody debris (CWD), which was present on all sites. Based on
the qualitative appearance of root systems (good or poor), root quality was always described
as good when seedlings were rooted in CWD, but there was no relationship with root quality
for the other substrate types. Root quality did not appear to be related to storage time as
poor root quality was observed on seedlings processed early just as frequently as on
seedlings stored for up to four months. No relationship was identified between root quality
and the number of morphotypes found on each seedling. Root quality appeared to be
related to the topographical position of the seedling’s location on the site. In general, those
seedlings growing in hollow depressions or on level ground to low rises had a greater
proportion of poor quality roots (54%) than seedlings harvested from hummocks of any size
(20 %).

The depth of the aerobic zone generally increased during the period of root growth and ECM
establishment, but these results were not consistently observed at every location of rod
placement. The level of the water table with respect to the roots of any seedling depended
on the microtopographical position of that seedling (i.e. relative water table depth was
greater for seedlings established on hummocks as compared to level ground or
depressions). On most sites, the water table depth generally fluctuated between 10 and 30
cm; depth was greater on site US, where fluctuations between 75 and 90 cm were observed.
No relationships were found between relative depth of the water table and the number of
ECM morphotypes described on a seedling.

Soil nutrient content (C, N and P) varied significantly between the wetland (both T and W)
and upland habitats (Table 2.2). Total C and N content was greater (p=0.000 and 0.001,
respectively) in the wetlands than the upland habitats. Within upland sites, C and N levels in

52

the forest floor (LFH) were approximately twice the levels in the A horizon; both nutrients
were at least 10 times greater in the A horizon than in the B horizons. The C:N ratio was
greater (p=0.036) in the upland forest floors than in the wetland soils. The content of
available P was also greater (p=0.025) in forest floor soils than in wetland soils, as well as
when compared to mineral soil horizons. All soil samples were acidic (pH ranged from 3.8
to 6.9) and there were no significant differences between the wetland and forest floor soils.
Acidity decreased with depth in the upland soil profile; the B horizon was significantly more
alkaline than all other horizons but the T sites.
Table 2.2. ANOVA comparisons of mean % carbon, % nitrogen, C:N ratio, available
phosphorus and pH between habitats based on combined samples from each site (n=3).
Soil Analysis

Treatment Effect Tamarack - Spruce
F
P
Wetland

Black Spruce
Wetland

Pine - Spruce
Upland

% Total Carbon
% Total Nitrogen
C:N ratio
Phosphorus (ppm)
pH

88.067
23.882
6.088
7.268
2.709

48.05 (1.87)a
2.24 (0.13)a
21.51 (0.46)a
1.37 (1.36)a
4.92 (0.46)

24.80 (1.16)b
0.80 (0.16)b
33.31 (5.54)6
191.63 (70.70)6
4.58 (0.34)

0.000
0.001
0.036
0.025
0.145

44.58 (0.70)a
2.60 (0.26)a
17.46 (1.58)a
0.61 (0.60)a
5.67 (0.12)

* Upland values are from analysis of the forest floor soils only (not A or B horizons).
The F statistic is the ratio of variance in sample means to variance within groups. The P
value is the significance level (a=0.05). Values for nutrient content and pH are means with
standard error in parentheses. Means followed by the same letter are not significantly
different.

Even though only seedlings thought to be naturally regenerating (true seedlings) were
selected for harvest, almost half (44.4%) of the harvested seedlings were found to originate
from branches of trees that had developed underground adventitious roots and formed new
seedlings (layered seedlings). The qualitative attributes of root abundance (high, medium or
low) and root quality (good or poor) were compared between layered and true seedlings. A

greater proportion of true seedlings seemed to exhibit medium or high root abundance
(92%) when compared to layered seedlings (70%). No relationship was found between root
quality and regeneration type. However, a comparison between the mean number of

53

morphotypes found on 25 true seedlings (5.16 ± 0.24) and the number occurring on 20
layered seedlings (4.35 ± 0.29) indicated a significantly greater number of morphotypes on
the true seedlings, regardless of habitat (p=0.037).

An average of 4.8 (range of two to eight) ECM morphotypes was identified per seedling.
The mean number of morphotypes varied significantly (p=0.005) between habitats, with a
greater number of morphotypes occurring in the two mixed species habitats (mean of 5.2
morphotypes in each of the T and U habitats) compared to the black spruce-dominated
habitat (mean of 4.0 morphotypes). The number of potential ECM host plants present in the
vicinity (within 1 m^) of harvested black spruce seedlings ranged from zero to four, with an
average of 1.82 per seedling. No significant differences were found in the number of
potential ECM hosts between sites or habitats. Figure 2.3 shows the lack of relationship
between the mean number of morphotypes and potential ECM host plants on each site.

8

□ ECM morphotypes

7

□ potential ECM hosts
6

*

5
0)
S i

I

z

4

il

3 2
1

0

T1

T2

T3

W1

W2

W3

U1

U2

U3

Site

Figure 2.3. Bar graph showing mean (±SE) number of ECM morphotypes compared to
mean (+SE) number of potential ECM host vegetation (within 0.5 m of seedlings) for each
site (black spruce - tamarack wetlands [T sites], black spruce-dominated wetlands [W sites]
and black spruce - pine upland forests [U sites]). ECM morphotype bars labelled with the
same letter are not significantly different.

54

f r '^ v

*

;i:

'.'4*'« ft'
.4^
£ f._ ;B > »

Figure 2.4. ECM morphotypes described on black spruce from three habitats in central BC.
A - Lactarius 1; B - Cortinariaceae 1; C - Piloderma-, D - Thelephoraceae-like 4; E Tomentella', F - Tomentella-Wke 1 (outer mantle); G - MRA 1 (outer mantle); H - Amphinema
(outer mantle with emanating hyphae).

55

'2 i

ECM morphotype occurrence, frequency and abundance
A total of 33 ECM morphotypes were described from 8858 root tips. Photographs of some
morphotypes are presented in Figure 2.4, and complete morphological descriptions are
included in Appendix II. Based on morphology, 27 ECM morphotypes were most likely
basidiomycetes, three ECM types were probably ascomycetes, and three remain uncertain.
Twenty-seven morphotypes were assigned to families including Thelephoraceae,
Cortinariaceae and Russulaceae, or genera such as Cenococcum, MRA, Amphinema,
Lactarius, Tomenteiia, Piloderma and Hebeloma. Six morphotypes could not be assigned
with confidence to any taxonomic group. Overall, 85% of all root tips examined were
ectomycorrhizal. Those tips (n=1337) that appeared uncolonized or only partially colonized
(lacked developed mantle) were classified into a separate non-mycorrhizal group. Nonmycorrhizal tips were found on 66.7% of seedlings, including seedlings from each habitat
and from all sites. The frequency of seedlings with non-mycorrhizal root tips was greater in
the W sites (93.3%) than the T (60.0%) or U sites (46.7%) (Table 2.3).

Nineteen morphotypes were found on three or fewer seedlings (<7%) and were defined as
rare. Of the 14 more commonly identified morphotypes, eight were found in all three black
spruce habitats, and accounted for 51 % of all root tips. Overall, Cenococcum occurred the
most often (68.9% of all seedlings) of any morphotype (Table 2.3). It was found on 80% of
seedlings from both the W and U sites, but on only 46.7% of seedlings from the T sites.
Cenococcum was completely absent from one site (T1), but occurred on 90% of seedlings
from T2 and T3. The second most commonly encountered morphotype overall was
Cortinariaceae 2 (55.6%), which was almost twice as frequentiy identified in the U sites
(80%) than in either of the wetland habitats. Russulaceae 2, MRA 1, Amphinema and
Cortinariaceae 1 (31.1 to 37.8%) were all found most often on seedlings from the U sites.
Thelephoraceae-like 1 and Tomenteiia-Wke 1 types were more frequently found on seedlings
56

from the T sites than on seedlings from the W or U sites. Interestingly, Thelephoraceae-like
1 was found on 80% of the seedlings from T 1, the site lacking Cenococcum.

Six of the most commonly identified morphotypes appeared to be absent from one or more
habitats. Lactarius 1 ECM were found on seedlings from both wetland habitats but not from
the upland habitats and occurred much more often in the T sites (31.1% of seedlings).
Three morphotypes were found on seedlings from both the W and U habitats:
Thelephoraceae 1 and orange 1 were more frequently described from the U sites and
Thelephoraceae-like 4 was more frequently described from W sites. Of the morphotypes
described from a single habitat (total of 14), most were considered rare and only two were
found on more than three seedlings. Tomentella (13.3% of all seedlings) was found only in
the T habitat (p=0.011) and Piloderma (15.6% of all seedlings) was found in only the U
habitats (p=0.001). Several morphotypes exhibited <6% overall frequency, but were fairly
common within a single habitat. These included Lactarius 3, Russulaceae 1 and cottony
halo types, which occurred on 20% of seedlings from the U, T, and W sites, respectively.

The average community similarity, based on ECM morphotype composition, was 27%
between all three black spruce habitats. The T sites shared 36% of ECM morphotypes with
each of the W and U sites; 42% of morphotypes were described from both the W and U
sites.

57

Table 2.3. Treatment effects, percent abundance (mean ± SE) and frequency of occurrence
(%) for ECM morphotypes of black spruce growing in three habitats. ECM morphotypes are
presented in order of decreasing overall frequency rank.
Treatment
Effect
ECM Morphotype

Cenococcum
Cortinariaceae 2
Russulaceae 2
MRA 1

Amphinema
Lactarius 1
Cortinariaceae 1
Thelephoraceae-like 1
Tomentelia-Wke 1

Piioderma
Thelephoraceae-like 4

Tomentella
cottony gold-brown
Thelephoraceae 2
Lactarius 3
cottony halo
Russulaceae 1
Thelephoraceae-like 2
Russulaceae 4
Tomentelia-Wke 3
Thelephoraceae-like 3
brown 1
Thelephoraceae 3
Lactarius 2
creamy rhizomorphic
clamp
orange 1
Cortinariaceae 3
Russulaceae 3

Black Spruce
Wetland (W)

Black Spruce - Pine
Upland Forest (U)

F

P

Abundance

Freq

Abundance

Freq

Abundance

Freq

0.966
1.515
4.295
1.599
1.130
3.293
0.413
3.165
3.567
9.099
4.013
4.987
1.591
1.292

0.389
0.232
0.020
0.214
0.333
0.047
0.664
0.052
0.037
0.001
0.025
0.011
0.216
0.285

10.2 (4.6)
4.9 (3.3)
4.3 (7.1 )a
2.2 (1.4)
10.1 (7.9)
15.7 (6.6)a
5.6 (3.3)
7.0 (3.4)
5.4 (2.6)a
0.0 (O.O)a
0.0 (O.O)a
7.9 (3.5)a
2.6 (1.8)
0.0 (0.0)
0.0 (0.0)
0.0 (O.O)a
4.1 (2.7)
2.2 (1.5)
0.0 (0.0)
1.6 (0.9)
0.5 (0.4)
0.3 (0.3)
0.3 (0.3)
3.1 (2.8)
2.0 (2.0)

46.7
40.0
33.3
33.3
33.3
66.7
20.0
46.7
33.3
0.0
0.0
40.0
13.3
0.0
0.0
0.0
20.0
13.3
0.0
20.0
13.3
6.7
6.7
13.3
6.7

18.2 (5.9)
3.9 (1.6)
3.0 (1.8)a
2.2 (1.2)
4.0 (2.4)
6.0 (3.7)ab
2.4 (1.9)
1.1 (1.1)
0.5 (0.5)b
0.0 (O.O)a
11.7 (5.8)b
0.0 (O.O)b
0.7 (0.5)
0.7 (0.7)
0.0 (0.0)
6.0 (3.3)b
0.0 (0.0)
0.0 (0.0)
0.8 (0.5)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
3.7 (3.7)
0.0 (0.0)
0.0 (0.0)

80.0
46.7
26.7
33.3
20.0
26.7
33.3
13.3
6.7
0.0
33.3
0.0
13.3
6.7
0.0
20.0
0.0
0.0
13.3
0.0
0.0
6.7
6.7
0.0
0.0

10.7 (2.3)
10.9 (3.8)
21.8 (6.8)6
6.1 (2.5)
12.1 (4.2)
0.0 (0.0)6
4.2 (2.0)
0.3 (0.3)
0.3 (0.3)6
7.0 (2.3)6
0.1 (0.1 )a
0.0 (0.0)6
0.0 (0.0)
0.7 (0.7)
6.6 (5.2)
0.0 (O.O)a
0.0 (0.0)
1.6 (1.6)
1.7 (1.7)
0.0 (0.0)
1.1 (1.1)
1.0 (1.0)
0.0 (0.0)
0.0 (0.0)
0.8 (0.8)

80.0
80.0
53.3
53.3
46.7
0.0
40.0
6.7
13.3
46.7
6.7
0.0
0.0
20.0
20.0
0.0
0.0
6.7
6.7
0.0
6.7
6.7
0.0
0.0
6.7

0.0 (0.0)
0.0 (0.0)
2.2 (2.2)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
5.2 (1.7)a

0.0
0.0
6.7
0.0
0.0
0.0
0.0
6.7
60.0

1.5 (1.5)
2.8 (2.8)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.7 (0.7)
0.3 (0.3)
0.0 (0.0)
30.0 (5.7)b

6.7
6.7
0.0
0.0
0.0
6.7
6.7
0.0
93.3

0.6 (0.6)
0.0 (0.0)
0.0 (0.0)
0.8 (0.8)
0.8 (0.8)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
10.0 (4.1 )a

6.7
0.0
0.0
6.7
6.7
0.0
0.0
0.0
46.7

1.594 0.215
3.343 0.045

MRA 2
brown 3

Hebeloma
Tomenteiia-Wke 2
Thelephoraceae 1
non-mycorrhizal

Black Spruce Tamarack Wetland (T)

9.785 0.000

The F statistic is the ratio of variance in sample means to variance within groups. The P
value is the significance level (a=0.05). Mean abundance values (calculated per seedling
[n=15]) were tested using a one-way ANOVA for each habitat. Post-hoc Fisher’s LSD tests
were used to determine where significant differences occurred. Means followed by the
same letter within rows are not significantly different.

58

In general, morphotypes that occurred most frequently were also more abundant on root
systems (greater proportion of total root tips). Cenococcum and Russulaceae 2 each
accounted for greater than 10% overall ECM abundance (Table 2.3), whereas nine
morphotypes each represented between 2-5% of the overall ECM community and 19
morphotypes accounted for less than 2% each. The rank-abundance curve is shown as a
straight line (Figure 2.5).

3.5
3

g
<0

2.5

2

s.
p

1.5

I
%

1
0.5

0
0

5

10

15

20

30

25

35

Morphotype Abundance Rank

Figure 2.5. Log-transformed rank-abundance plot of the overall ECM fungal community of
black spruce. The morphotype abundance rank order, beginning with Cenococcum,
corresponds to the order on the y-axis of Figure 2.7.
■ jr— T

2 .5

Sites

■s— W S ite s
U Sites

</)

I O

P

I'
*

0 .5

0

5

10

15

20

M orphotype A bundance Rank

Figure 2.6. Comparison of rank-abundance of ECM morphotype communities of black
spruce in three habitats (black spruce - tamarack wetlands [T sites], black sprucedominated wetlands [W sites] and black spruce - pine upland forests [U sites]), using log
abundance plotted against ranked morphotype abundance in each habitat.
59

The relative abundance of ECM morphotypes is shown for all three habitats in Figure 2.5
and compared between habitats in Figure 2.6. For six of the 14 most frequently described
morphotypes, the mean morphotype abundance (calculated for each seedling and averaged
for each habitat type) varied significantly between habitats (Table 2.3). Russulaceae 2 was
significantly more abundant (p=0.020) on roots from the U seedlings than from the W
seedlings. This morphotype was found in all three U sites and was exceptionally abundant
on Site US (ranging from 48.5 to 73.1% on four of five seedlings). The abundance of
Russulaceae 2 was much lower in the T sites compared to the U sites, but not significantly
so. The ECM abundance for Tomentella-Wke 1 was also significantly different between
habitats (p=0.037); it was more abundant in the tamarack wetlands than in either of the
other habitats. Similarly, Lactarius 1 abundance was significantly greater (p=0.047) in the
tamarack wetlands than in the upland habitat (where it was not detected) and greater (but
not significant) than in the spruce-dominated wetlands. Thelephoraceae-like 4 abundance
was higher in the spruce-dominated wetlands than either of the mixed species habitats
(p=0.025). Interestingly, the non-mycorrhizal root tips showed the same trend in the W
habitat (p=0.000). The cottony halo ECM, a rare morphotype, was described only from W
sites (p=0.045). Two frequently occurring morphotypes {Tomentella and Piloderma) were
each found in single habitat types (p=0.011 and p=0.001, respectively). Within habitat sites,
Tomentella was more broadly distributed than Piloderma.

Although ECM frequency varied between habitat types for some of the more commonly
occurring morphotypes such as Cenococcum, Cortinariaceae 1, Cortinariaceae 2,
Amphinema and MRA 1, mean abundance values did not vary significantly. Although
Cenococcum appeared to be more abundant in the W sites than the other two habitats, it
was locally abundant (60%) on one or more seedlings from each different habitat.
Amphinema was less abundant in the spruce-dominated wetlands, but had higher within60

habitat variability (especially in the T sites). Cortinariaceae 2 and MRA 1 were both more
abundant in U sites than either wetland site, however exceptions of high local abundance
did occur for both of these ECM on several seedlings on T sites. Cortinariaceae 1 was quite
evenly abundant across habitats.

non-m ycorrhizal
T h e le p h o ra c e a e 1
T o rnenfe//a-llke 2
H e b e lo m a
brown 1
T h e le p h o ra c e a e -lik e 3
Tom entella-W ke 3

I Black Spruce -

□ B lack Spruce

Tam arack

□ Black Spruce -

W etlan d Habitat

Pine Upland
Forest Habitat

W etlan d Habitat

T h e ie p h o ra c e a e 2
orange 1
R u ssu laceae 4
cream y rhizom orphic clam p
brown 3
MRA 2
R u ssu laceae 3
C o rtinariaceae 3
Lactarius 2
cottony goid-brown
T h e ie p h o ra c e a e 3
T h e ie p h o ra c e a e -iik e 2
R u ssu iaceae 1
cottony haio
Tom entella-W ke 1
Lactarius 3
Piloderm a
Tom entella
T h e le p h o ra c e a e -iik e 1
MRA 1
T h e ie p h o ra c e a e -lik e 4
C o rtinariaceae 1
C o rtinariaceae 2
Lactarius 1
A m ph inem a
R u ssu laceae 2
C enococcum
30

% Abundance

% Abundance

0

10

20

% Abundance

Figure 2.7. Comparison of ECM morphotype abundance (proportion of the community
represented by each morphotype per habitat) between three black spruce habitats. ECM
morphotypes are ranked in order of overall decreasing abundance from bottom to top
(excluding the non-mycorrhizal type).

61

30

ECM community diversity
In general, ECM community diversity was greatest in the black spruce - pine upland forests
and least in the spruce-dominated wetland, with intermediate diversity in the spruce tamarack wetland habitat. Species richness (Margalef Index) varied significantly (p=0.038)
between the black spruce-dominated wetlands and both mixed habitats, but not between the
black spruce - tamarack wetland and the black spruce - pine upland habitats. Mean
Shannon diversity values were significantly different (p=0.028) between the black spruce pine upland and black spruce-dominated wetland habitats only. Shannon evenness and
Simpson indices did not detect significant differences in diversity between habitats, but
supported the same trends.
Table 2.4. ANOVA comparisons of diversity indices (Margalef, Shannon, Shannon
evenness and Simpson) between habitats based on calculations for each seedling (n=15).
Diversity Index
Margalef
Shannon
Shannon Evenness
Simpson

Treatment Effect
P
F
3.527
0.038
0.028
3.899
1.740
0.188
2.949
0.063

Tamarack-Spruce
Wetland
0.802 (0.071 )a
1.188 (0.085)ab
0.727 (0.038)
2.903 (0.217)

Black Spruce
Wetland
0.616 (0.059)5
0.950 (O.IIO)a
0.677 (0.055)
2.456 (0.268)

Pine - Spruce
Upland
0.814 (0.045)a
1.296 (0.070)5
0.795 (0.040)
3.292 (0.243)

Values for indices are means with standard error in parentheses. Means followed by the
same letter are not significantly different.

DISCUSSION
ECM morphotype occurrence and abundance
Overall, 33 ECM morphotypes were characterized from the roots of naturally regenerating
black spruce (Picea mariana) seedlings. Although others have previously confirmed the
ectomycorrhizal status of black spruce in natural habitats (Malloch and Malloch 1981 ;
Thormann et ai. 1999), this is the first study to report on the composition and diversity of
ECM fungal communities associating with this host. In similar studies involving other plant
hosts, the number of ECM morphotypes (species richness) varies widely. For example, in

62

central BC, Mah etal. (2001) described 24 ECM morphotypes on naturally regenerating
hybrid spruce {Picea glauca x engelmannii) seedlings, while Kranabetter et al. (1999)
reported an average of 52 morphotypes (total of 74 morphotypes) on white spruce {Picea
glauca), subalpine fir {Abies iasiocarpa), and lodgepole pine {Pinus contorta var. latifolia)
seedlings two years after outplanting. Flynn et al. (1998) described 13 morphotypes on
naturally regenerating Sitka spruce seedlings {Picea sitchensis) harvested from Scottish
plantation forests, and in southwestern Alberta cutblocks, Bradbury (1998) described 14
ECM morphotypes on two year-old regenerating lodgepole pine seedlings. On mature
hosts, Danielson and Pruden (1989) described eight morphotypes on the roots of urban blue
and white spruce {Picea glauca and P. pungens Engelm., respectively) in Calgary.
Bradbury etal. (1998) found 20 morphotypes on lodgepole pine in southwestern Alberta and
Arocena etal. (1999) found 18 morphotypes on subalpine fir in central BC. The morphotype
richness reported here is within the range of 20-35 fungal species reported by Bruns (1995)
for several small sites occupied by single host tree species and with homogeneous
environmental conditions. The variation in reported values between studies may be
attributable to differences in host receptivity, inoculum potential at different sites, soil and
environmental factors, sampling intensity, as well as in differences in individual techniques
of those doing the descriptive analysis. Overall, 85% of all sampled root tips were
ectomycorrhizal, which agrees with results from a similar study in Scotland that found about
80% of naturally regenerating Sitka spruce {Picea sitchensis) root tips were mycorrhizal
(Flynn etal. 1998).

The relatively high number of morphotypes found in this study may be partly explained by
differences in the composition of ECM communities in the three habitats. The total number
of ECM morphotypes present in each habitat was almost equal: 19 morphotypes were found
in the spruce-dominated wetlands (including 12 of the 14 most frequently occurring types)
63

compared to 20 types found in each of the mixed species habitats (including 11 of the 14
most frequently occurring types in each). Based on ECM morphotype composition, the
average community similarity was 27% between all three black spruce habitats. Gehring et
al. (1998) reported a change in ECM community composition associated with pinyon pine
{Pinus edulis) across a moisture gradient (xeric to mesic), but no significant decrease in the
overall number of species found at different sites. These findings emphasize the importance
of sampling across the ecological range of a host species in order to completely describe its
ECM fungal associates.

The majority of ECM morphotypes described in our study were basidiomycetes (-30 ) with
only a few ascomycetes identified. However, ascomycete morphotype abundance
(predominantly Cenococcum and MRA 1 ECM) accounted for between 17 and 19% of the
entire community. In contrast, Danielson and Pruden (1989) found 31% of urban blue or
white spruce ECM were basidiomycetes and 47% were ascomycetes. One reason for this
difference may be that soils in the urban spruce study were dry and alkaline, as compared to
the moist, acidic soils found on our sites. Gehring etal. (1998) also reported a greater
proportion of ascomycetes to basidiomycetes associated with pinyon pine roots in xeric sites
in Arizona. Strand-forming fungi (basidiomycetes) accounted for 60% of the ECM
morphotypes described on lodgepole pine, white spruce and subalpine fir in mesic forests in
northwestern BC (Kranabetter etal. 1999).

When all ECM morphotypes were compared, the occurrence, frequency and abundance of
Cenococcum were higher than for any other ECM morphotype described. This morphotype
was easily recognized by its black color, stellate outer mantle pattern, and large-diameter,
undamped emanating hyphae 5-6 pm in width (Agerer 1987-2002). Cenococcum is often
considered a cosmopolitan fungus that has a wide host range and broad environmental
64

tolerances. It does not appear to be negatively affected by high water levels and is also
able to survive in dry soils (Goodman and Trofymow 1998; Thormann etal. 1999). Malloch
and Malloch (1981) found that Cenococcum vjas frequently associated with black spruce
roots (16 of 30 root segments) collected from upland forest sites in northeastern Ontario; in
northern Alberta, Thormann etal. (1999) also found Cenococcum on most black spruce
roots from bogs and fens. In other studies, Cenococcum was the most frequently occurring
ECM fungus found on lodgepole pine, white spruce and subalpine fir (Kranabetter et al.
1999) and western hemlock (Kranabetter and Friesen 2002) in mixed forests in
northwestern BC. It has been found on roots of tamarack and willow in fens of varying
nutrient status (Thormann etal. 1999). Van der Heijden etal. (1999) also describe
Cenococcum on willow roots in calcareous-wet, acidic-dry and acidic-wet sites, but not from
calcareous dry sites in dune ecosystems in Holland. Several authors suggest that
Cenococcum seems to prefer moist, acidic soils of forests and wetlands, where it occupies
the upper organic soil layers (Arocena etal. 1999; Taylor 2002).

In our study, Cenococcum abundance was higher in the black spruce-dominated wetlands
than in either of the other habitats, but this difference was not significant. It was most
frequently found in both the black spruce-dominated wetland and upland habitats compared
to the black spruce - tamarack wetlands. Interestingly, several other studies did not detect
Cenococcum or found it in low abundance. For example, Cenococcum accounted for only
2% of the overall ECM fungal community of urban blue and white spruce in Calgary, Alberta
(Danielson and Pruden 1989), and it was not found on Sitka spruce roots in Scottish
plantations (Flynn et al. 1998). In our study, it was not detected on seedlings from one site
in the T habitat despite similarities in vegetation, soil and stand age compared to the other T
sites. In fact, a subsequent study on tamarack and scrub birch roots growing on this same
site has described Cenococcum on 50% of seedlings of each species, although overall
65

abundance was low (J. Catherall, pars. comm.). One reason that it may not have been
detected on black spruce may have been due to the small number of seedlings sampled (5
from this site). Alternatively, there may be some competitive interactions between
Cenococcum and other ECM fungi associating with black spruce on this site (e.g. members
of the Thelephoraceae that appeared to dominate this site).

Mycelium Radicis Atrovirens {MRA) 1, the other abundant ascomycete characterized in our
study, has been described as a common, broad host range, root-associated fungus by some
(Summerbell 1989), but as a pathogenic fungus by others (De la Bastide and Kendrick
1990). It has been previously described on black spruce growing in northern Alberta fens
(Thormann etal. 1999), as well as on numerous other hosts including lodgepole pine
(Bradbury 1998; Durall etal. 1999; Kranabetter etal. 1999), hybrid spruce (Mah etal. 2001),
white spruce (Kranabetter etal. 1999), Sitka and Norway spruce (Flynn etal. 1998),
subalpine fir (Kranabetter etal. 1999), and Douglas-fir (Sakakibara etal. 2002). Here, MRA
1 was found on one third of all seedlings from the wetland habitats (although at low
abundance), and on over half of the seedlings from the upland sites (at approximately three
times the abundance level). This ECM type appears to be a habitat-generalist fungus,
perhaps with a greater ability to compete with other fungal species for roots in upland as
compared to wetland soils.

The family Russulaceae was represented by at least five morphotypes, including three in the
genus Lactarius. These five morphotypes were yellow-orange to orange-brown in color,
with mainly smooth textures and outer mantles of net to interlocking synenchyma. Hyphae
of the other two Russulaceae types were usually undamped and were often arranged in
parallel sheets on the mantle surface. The Russulaceae 2 type was the second most
abundant morphotype overall and was described from all three habitats. The three

66

Lactarius types had clampless hyphae, including larger laticifers, and hyphae filled with
granular contents.

The Russulaceae include both narrow and broad host range ECM fungal species, including
several that associate with members of the Pinaceae (including the genera Picea, Larix and
Pinus) (Molina et al. 1992). Members of the Russulaceae have not been previously
described associating with black spruce, although Lactarius and Russula species have been
found on lodgepole pine (Bradbury 1998; Bradbury etal. 1998), ponderosa pine (Massicotte
etal. 1999; Stendell etal. 1999), white spruce (Lilleskov etal. 2002), Sitka spruce (Flynn et
al. 1998), Norway spruce (Agerer 1987-2002), Douglas fir (Massicotte etal. 1999),
kinnikinnick (Sakakibara et al. 2002), and willow (Van der Heijden et al. 1999). In an In vitro
study using black spruce seedlings, Doudrick etal. (1990) noted Lactarius deliciosus,
Lactarius rufus and a third Lactarius species formed only weak hyphal mantles and no
Hartig nets on seedling roots when grown in culture. In contrast, we found that the Lactarius
morphotypes formed robust mantles and labyrinthine Hartig nets on black spruce roots from
field sites. It has been suggested that ECM morphology following lab syntheses may differ
from those ECM of hosts growing in soil where environmental influences and multiple
organisms interact (Massicotte et al. 1999). Lactarius sporocarps were collected from our
field sites, including Lactarius deliciosus, which has been described as having wide
environmental tolerances and a lack of substrate specificity (Goodman and Trofymow 1998).
Lactarius rubrilacteus, although not identified from our sporocarp survey, has also been
found frequently in both mineral and organic soils (Goodman and Trofymow 1998).

The three Lactarius morphotypes identified in our study appear to exhibit habitat
preferences. Lactarius 1, the most frequently occurring morphotype of the three, was
significantly more abundant in the spruce - tamarack wetlands than in the upland habitats
67

(where it was not found). It also occurred in the spruce-dominated wetlands, but at low
abundance. Lactarius 3 and Lactarius 2 were found in single habitats (upland and sprucedominated wetland, respectively), but in relatively low abundance. Differences in dominant
ECM host plants present in the mixed species habitats and soil moisture and nutrient
content may account for the habitat specificity observed in these morphotypes. The
Lactarius species identified from white spruce along a soil nitrogen gradient in Alaska
occurred at low abundance at low N sites, but substantially increased in abundance on high
N sites (Lilleskov et al. 2002). In our study, it is likely that differences in soil moisture played
a greater role than nutrient availability in the occurrence of Lactarius 1, as it occurred in both
wetland habitats. The occurrence of Lactarius 3 in only upland sites may be a result of
fungal preference for drier habitats with a lower content of organic matter, and possibly
increased nutrient levels. Alternatively, Lactarius 3 may be a broad host ranging fungus that
forms symbiotic associations with other upland species such as lodgepole pine, which may
provide a source of inoculum to regenerating black spruce seedlings.

Eleven morphotypes were classified as Thelephoraceae, including Tomentella and other
unidentified genera. According to Agerer et ai. (1995), mantles of irregular to regular
synenchyma (pseudoparenchyma) and the consistent presence of hyphal clamps are known
only from Tomenteiia. Here, in cases when clamp connections were not observed,
morphotypes were classified as Thelephoraceae-like. The Tomentella morphotype
described in our study closely matches that of an uncommon variant of the “Tomentella-Wke
with cystidia” morphotype described by Danielson and Pruden (1989) on urban blue and
white spruce in Calgary. The color, mantle features and presence of cystidia of the
Tomentella-Wke 1 type are consistent with dark-mantled types of Tomentella (Agerer 19872002; Lilleskov et ai. 2002), which have been described from black spruce peatlands in
northern Alberta (Thormann et al. 1999). However, the lack of clamp connections prevented
68

classification of this ECM type into the Tomentella genus. Except for the lack of clamp
connections, Thelephoraceae-like 1 resembles the “Picelrhlza nigra” morphotype described
by Agerer etal. (1995).

The Thelephoraceae are known mycobionts of conifers and may be a widespread and
important component of ECM communities (Danielson and Pruden 1989; Bradbury 1998;
Bradbury etal. 1998; Durall etal. 1999; Koljaig etal. 2000; Mah etal. 2001). Several
species of Thelephora have been found to form ECM with little host restriction (Molina et al.
1992). Species from several genera of Thelephoraceae have been shown to form ECM with
Norway spruce, including Hydnellum peckll (Agerer et al. 1995). Interestingly, the sporocarp
for this species was collected from upland sites in our study. In addition, Thormann et al.
(1999) described a Tomentella species associated with willow roots from Alberta peatlands.

Three frequently occurring Thelephoraceae and Thelephoraceae-like morphotypes
appeared to exhibit habitat specificity, occurring predominantly in the wetland habitats
compared to the upland sites. A Tomentella species was identified only in the spruce tamarack wetlands; the Tomenfe/Za-like types also occurred most abundantly in the wetland
habitats. Thelephoraceae-like 4 morphotype was found both more often and more
abundantly in the spruce-dominated wetlands. Thelephoraceae-like 1 was most often
described from the spruce - tamarack wetlands, but did occur in both of the other habitats.
Many tomentelloid fungi form inconspicuous resupinate fruiting bodies on the underside of
dead plants, wood and soil debris, making them difficult to find during sporocarp surveys
(Agerer 1995; Koljaig 1996; Agerer and Bougher 2001). This fruiting habit also led many to
functionally classify this group of fungi as decomposers (Read and Perez-Moreno 2003).
Hibbett et al. (2000) suggest the occurrence of occasional reversals of ECM fungi to a freeliving saprotrophic lifestyle, particularly in the thelephoroid group. Perhaps this ability gives

69

them a competitive advantage over non-degrading ECM fungal morphotypes in nutrient-poor
habitats.

The Cortinariaceae are a large family of woodland fungi, many of which are mycorrhizal
(Arora 1986). Genera of this family contain species exhibiting the entire spectrum of narrow
to broad host specificity (Molina et al. 1992). ECM of Cortinarius have been described on
lodgepole pine (Bradbury 1998; Kranabetter etal. 1999), white spruce (Kranabetter etal.
1999; Lilleskov etal. 2002), Sitka spruce (Flynn etal. 1998), subalpine fir (Kranabetter etal.
1999), and willow (van der Heijden etal. 1999). Hebeloma has been described on hybrid
spruce by Mah et al. (2001 ) and white spruce by Lilleskov et al. (2002), as well as other host
species. In our study, four morphotypes were characterized as belonging to the
Cortinariaceae family. Both the Cortinariaceae 1 and 2 types were among the most
abundant ECM morphotypes and occurred frequently in all three habitats. The
Cortinariaceae 3 and Hebeloma types occurred only in the spruce-dominated wetland
habitat, but with very low abundance. Although our study shows a lack of habitat specificity
by the Cortinariaceae types, Lilleskov et al. (2002) found that Cortinarius species
associating with white spruce roots were confined to sites at the low end of a nitrogen
deposition gradient in Alaska. In contrast, Hebeloma was found on sites with an
intermediate supply of inorganic N. Like Piloderma, the Cortinariaceae are mat-forming
fungi that may be specialized for nitrogen uptake under N-limiting conditions. Because all
habitats in our study were presumed to be N-limiting, no habitat specificity was observed
here.

Piloderma is a well-known ECM fungus with worldwide distribution and a broad host range
(Harley and Smith 1983; Molina etal. 1992). In central BC, it has been described on hybrid
spruce and subalpine fir (Arocena et al. 1999; Arocena et al. 2001 ). Smith et al. (2000)
70

reported that the presence of Piloderma is associated with forest stand age, based on their
more frequent observations in old-growth than in younger stands of Douglas-fir. The
occurrence of Piloderma has been strongly related to the percent cover of coarse woody
debris (CWD) in decay class 5 (Goodman and Trofymow 1998; Smith etal. 2000), but it has
also been described from acidic humus and at the boundary between the forest floor and
mineral soils (Agerer 1987-2002; Arocena etal. 1999). In our study, the Piloderma
morphotype was identified only from the upland sites, on all seedlings from site U3 (which
were all rooted in CWD) and on one seedling from site U2. This species resembled
Piloderma fallax {=croceum), which has been characterized by having pale to bright yellow,
clampless emanating hyphae (often with needle-like crystals on the surface) and being
rhizomorphic (Agerer 1987-2002; Arocena et al. 2001).

The habitat specificity of Piloderma observed in this study (and others) may provide some
insight into its functional role in ecosystem processes such as nutrient cycling. Lilleskov et
al. (2002) hypothesized that Piloderma (as well as Cortinarius and Russula) species are
adapted to relatively acidic, N-poor soil conditions and appear to be specialized in efficient
uptake of N. Some have suggested that Piloderma and other mat-forming fungi may
increase nutrient availability to host trees partly by enzymatic degradation of the forest floor
and soil organic matter (Griffiths and Caldwell 1992). Arocena etal. (1999) described higher
exchangeable Ca^^, Mg^"^ and

values in soils dominated by Piloderma as compared to

MRA, suggesting differences in biological activities of these two fungi. Soils with high
amounts of exchangeable cations are considered fertile soils, which are able to supply trees
with essential nutrients (Arocena etal. 1999).

The ECM fungus Amphlnema was the third most abundant morphotype detected in our
study and occurred frequently (20-47%) in each of the three habitats. Amphlnema
71

(byssoides) is known to form ECM with many hosts (Molina etal. 1992) and has been
reported as a major colonizer of urban blue and white spruce (Danielson and Pruden 1989),
white spruce (Kranabetter etal. 1999; Lilleskov etal. 2002), hybrid spruce (Mah etal. 2001),
lodgepole pine (Durall etal. 1999; Kranabetter etal. 1999), subalpine fir (Kranabetter etal.
1999), and Dougias-fir (Sakakibara etal. 2002). Our results agree with others in support of
a lack of habitat specificity exhibited by this species. Despite the widespread distribution
and broad host range of this fungus, little is known of its biology. In their N deposition
gradient study, Lilleskov et al. (2002) found that Amphlnema byssoides was present on sites
where soil N content varied from low to intermediate levels. They suggest that Amphlnema
occurrence and abundance may be limited by soil acidification or nutrient imbalances rather
than by the N content of the soil. This may reflect a role of this fungus in increasing the
availability of nutrients other than N for plants (Lilleskov etal. 2002).

Several morphotypes remained unidentified following morphological characterization of
black spruce ECM. The cottony halo type was the most abundant of the unidentified
morphotypes and was described from the W habitat only. This type was distinguished by
the organized arrangement of emanating hyphae in an interwoven halo loosely encircling
the ECM root tip. No match could be located in published descriptions of ECM (Agerer
1987-2002; Ingleby et al. 1990; Goodman et al. 1996) to suggest the identity of this fungus.
Other unidentified fungi occurred quite infrequently and it is possible that complete
morphological descriptions (including an appreciation for the variation within a morphotype)
may not have been obtained.

ECM community structure and diversity
From comparisons using several diversity indices, ECM community diversity for black
spruce was generali greater in the black spruce - pine upland forests than in the mixed
72

black spruce - tamarack wetlands, which was, In turn, greater than In the black sprucedominated wetlands. Both the Shannon and Margalef diversity Indices are measures of
species richness within communities, but the Shannon Index places equal emphasis on the
richness and relative abundance of community members (Magurran 1988; Krebs 1989).
Using these two measures, differences detected were significant between the upland and
spruce-dominated wetland habitats. The same community diversity trends were supported
when diversity was assessed using the Simpson Index, which places emphasis on the ECM
types occurring In greater abundance. Neither the Simpson nor the Shannon Evenness
measures were able to detect significant differences between habitats. With respect to
black spruce, rare ECM types (those with low abundance) probably accounted for much of
the diversity detected.

Greater community similarity (based on composition of different ECM morphotypes) was
observed between the black spruce-dominated wetland and black spruce - pine upland
habitats than between either of these habitats when compared to the black spruce tamarack habitat. This may suggest that the composition of ECM fungal communities Is
Influenced by the presence of tamarack, a potential host known to form ECM with several
narrow to broad host ranging fungal genera (Molina etal. 1992). The Importance of
alternate ECM hosts as sources of Inoculum Is further supported by our observation that
higher proportions of colonized roots were described from both the mixed species habitats
(T and U) as compared to the black spruce-dominated habitat (W). Alternate ECM hosts
(companion plants) can Influence the ability of ECM fungi to colonize neighboring plants and
may also Influence the proportion of ECM that develop (Molina et al. 1992; Massicotte et al.
1999). Walker (1987) hypothesized that some ECM fungi that are unable to form symbioses
with spruce alone may succeed In mixed spruce forests where pine or larch species are

73

present. In our study, both tamarack and lodgepole pine may have contributed to biack
spruce ECM community composition as sources of inocuium for newiy growing roots.

ECM morphotype richness (calculated per seedling and averaged per habitat) was also
greater in habitats that had a greater number of dominant host species (T and U). There
were 11 potentiai ectomycorrhizal host plants near seedlings harvested for this study. No
differences in ECM morphotype diversity were related to differences in number of host
species present on a site, nor within the 0.5 m radius piots surrounding seediings seiected
for harvest. Any influence of companion piants on ECM diversity may have been due to the
biomass of underground roots interacting with the seediing roots. Thus, it may not be the
number of potentiai ECM host species present, but the density of coionized roots acting as
inoculum potential. A large proportion of these may have come from other iarge host
species on the sites (i.e. tamarack and pine). Similarities in species composition of ECM
coionizing naturaiiy regenerating pine seediings and mature trees found in Swedish boreai
forests by Jonsson et al. (1999) may refiect the importance of iarge root systems as a
source of inocuium for regenerating seedlings. The presence of companion species may
expiain at ieast part of the greater ECM community diversity found in the mixed species sites
compared to the black spruce-dominated sites.

The trend showing greater diversity (significantly greater when compared to the sprucedominated wetland habitat) in the upland habitat than in either wetiand habitat might aiso be
partiy explained by differences in soil conditions on upland sites. In water-saturated, organic
soiis (such as those present on both the T and W sites), oxygen deficiency is expected to
siow or prevent ECM formation due to inhibition of fungal aerobic metabolic processes and
inability to meet the oxygen demand of the soii myceiial network (Tinker 1984; Walker 1987;
Stenstrom 1991). W e observed a significantly greater proportion of non-mycorrhizai roots
74

from W sites compared to U sites, and intermediate colonization levels from the T sites
(which were very similar to W sites with respect to soil characteristics). In vitro experiments
conducted by Stenstrom (1991) revealed variation in the susceptibility to flooding between
different ECM fungi, and also that waterlogging was not associated with fungal oxygen
deficiency. This could be because many forest fungi have hydrophobic mycelium, which
may provide air pockets in the soil (Stenstrom 1991). Our observations of a generally
increasing depth of the aerobic zone during the period of root growth and ECM
establishment, as well as the propensity of black spruce seedlings to establish on
hummocks in the wetland habitats, indicate that oxygen deficiency was unlikely to be a
major factor in the composition of ECM communities on wetland black spruce.

Differences in soil nutrient availability may also influence the structure and diversity of ECM
communities in different habitats. The methods used in our study to determine C and N
content did not distinguish between inorganic and organic sources of these nutrients.
Although greater amounts of C and N were found in wetland soils compared to upland soils,
much of this may have been in inaccessible, organic forms due to low rates of
decomposition in these cold, wet habitats (Thormann etal. 1999). All habitats were likely Nlimited, as the highest N content found on any site was 2.6%. In habitats where
mineralization rates are slow, natural selection is thought to favor associations with fungal
symbionts that are physiologically equipped to capture nutrients from organic sources (Read
and Perez-Moreno 2003). Cortinarius, Hebeloma, Piloderma, Russula, Lactarius,
Amphlnema, and Tomentella species have all been associated with relatively acidic, N-poor
soil conditions (Lilleskov et al. 2002). Very low levels of available P were found in wetland
soils as compared to upland soils. The availability of P may be less important than
availability of N in ectomycorrhizal systems (Smith and Read 1997), but it is expected that

75

ECM fungi in P-limited habitats would also be equipped to maximize assimilation of P for
their plant partners.

In our study, the relative abundance of ECM morphotypes showed the expected dominance
of a few ECM types, with many other morphotypes found in progressively lower abundances
(Krebs 1989; Horton and Bruns 2001 ; Taylor 2002). Overall, five morphotypes
{Cenococcum, Russulaceae 2, Amphinema, Lactarius 1 and Cortinariaceae 2) were found
with greater than 5% abundance, and nine additionai morphotypes were found with greater
than 2% abundance. All but Lactarius 1 were identified in all three habitats. The ability of
certain ECM fungal species to dominate the root systems of host trees appears to be a
widespread phenomenon, possibly due to competitive interactions between different species
of fungi (Flynn etal. 1998; Kranabetter etal. 1999; Stendell etal. 1999; Taylor et ai. 2000).
Some suggest that the total number of species comprising a community may not be as
important as the dominant species present because of perceived high levels of functional
redundancy that may exist in soil microbial communities (Andrén and Balandreau 1999).
When the relative abundance of ECM fungi comprising communities within each of the three
habitats (p-diversity) was examined, we again observed the same pattern of dominance by a
few ECM types. These results indicate that differences in relative abundance of ECM fungal
species do not account for differences in community diversity between habitats.

With respect to our ability to measure richness and abundance, there is a relationship
between sampling effort (number of tips sampled), probability of detection, and the relative
abundance of species (percentage of root tips colonized) present (Taylor 2002). Compared
to other similar assessments, the number of root tips examined in this study is believed to
have been high enough to detect most ECM types. In a recent review, Horton and Bruns
(2001) emphasized that the distribution of many ECM species is clustered, and that most
76

species occur In <10% of samples. This may explain why numbers of morphotypes
described on individual seedlings are much lower than the overall number of morphotypes
described on a site or within a specific habitat. One explanation for this clustered
distribution of individual species may be due to the patchy distribution of suitable habitat
features, such as the presence of CWD consistently associated with Piloderma (Bradbury at
al. 1998; Smith et al. 2000). The correlation between specific habitat features and the
occurrence and abundance of ECM fungi is a critical step towards developing functional
hypotheses of the importance of individual species to ecosystem dynamics (Smith et al.
2000).

77

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83

Molecular Characterization of Ectomycorrhizal Communities Associating with
Black Spruce {Picea mariana) in Wetland and Upland Forests
In the Central Interior of BC

ABSTRACT
Ectomycorrhizal (ECM) fungal communities are composed of many genetic individuals,
between which exist variations in their ability to colonize different host plants, to promote
plant growth and to adapt to abiotic environmental factors. To examine the genetic diversity
of ECM communities associating with naturally regenerating black spruce seedlings across
three distinct habitats in central BC and to assess differences within characterized fungal
morphotypes, a subsample of 888 root tips were subjected to molecular (PCR-RFLP)
analysis. A total of 65 genotypes were delimited from 29 ECM morphotypes, with
approximately 28 genotypes found in each of the three habitats. Within previously
described ECM morphotypes, low diversity (one or two genotypes) was observed for
Piloderma, Lactarius 3 and Cenococcum\ higher diversity was observed for Amphinema,
Russulaceae 1, Cortinariaceae 2, MRA 1, Tomentella-Wke 1 and Thelephoraceae-like 4.
Molecular diversity of ECM communities did not vary between habitats; however, analysis
revealed habitat-specificity of some genotypes within morphotypes described from all three
habitats. Whereas ECM morphotypes such as Cenococcum, MRA 1 and Cortinariaceae 1
consisted of genotypes that appeared to be evenly distributed across the habitats,
genotypes for Amphinema, Cortinariaceae 2, and Russulaceae 1 and 2 had more uneven
patterns of distribution, occurring variously in one, two or all three of the habitats. Results
may indicate ecotypic adaptation of ECM in response to heterogeneous environmental
conditions.

84

INTRODUCTION
The ubiquitous, mutualistic symbioses between the roots of woody plants and
ectomycorrhizal fungi are essential components of northern forest ecosystems (Smith and
Read 1997; Amaranthus 1998). Ectomycorrhizae (ECM) mediate nutrient and water uptake
in their piant hosts in return for energy-rich carbon compounds that support fungal
metabolism and exchange resources with other members of the plant community through a
common myceliai network (Smith and Read 1997; Simard etal. 1997; Amaranthus 1998).
ECM fungal species exist as populations of many genetic individuals, between which there
exists phenotypic variation in the abiiity to colonize different genotypes of host plant,
promote plant growth and adapt to abiotic environmental factors (Bruns 1995; Jonsson of al.
2001). These fungi form diverse communities, even in forest stands with reiatively
homogenous soii conditions and dominated by a single host species (Bruns 1995; Horton
and Bruns 2001). The functional role of high ECM diversity is still unclear, but it has been
suggested that plants with access to a greater diversity of fungi may be better abie to adapt
to environmentai changes and stress (Haug and Oberwinkler 1987; Amaranthus 1998;
Durall etal. 1999). High ECM diversity may be especially important when considering the
ability of entire ecosystems to respond to changing environmental conditions at regional and
global scales (Prosser 2002).

Research questions related to ECM community structure and diversity rely on the abiiity to
distinguish individual fungal species (Egger 1995). The use of the polymerase chain
reaction (PCR) to amplify target sequences of rDNA is probably the most important
methodological advance in the study of ECM communities (Horton and Bruns 2001).
Subsequent enzymatic digestion of the internal transcribed spacer (ITS) region of rDNA with
just a few restriction endonucleases produces sufficiently variable restriction fragment length
polymorphism (RFLP) patterns that can distinguish between closely related species (Egger

85

1995; Kraigher etal. 1995; Mehmann etal. 1995). Based on differences In morphological
characteristics, microscopic examination of ECM is suitable for sorting fungi into families
genera, and in some cases, species; molecular analysis (PCR-RFLP) potentially allows
identification of most fungal species through comparisons to reference databases of
restriction fragment patterns (Egger 1995; Horton and Bruns 2001 ; Mah et al. 2001 ;
Sakakibara et al. 2002).

The composition and diversity of ECM communities have been compared at the molecular
level in several recent studies. Mah etal. (2001) found that the genetic composition of ECM
communities of hybrid spruce {Picea glauca x engelmannii) seedlings varied between
undisturbed, clear-cut, and cut and burned sites, but that diversity did not. Baldwin (1999)
also found no difference in ECM molecular diversity for regenerating black spruce {Picea
mariana) seedlings from clear-cut and cut and burned sites in paper birch {Betula
papyrifera) - black spruce forests. In contrast, Byrd et at. (2000) reported lower ECM fungal
species richness from soil core samples collected from clear-cut as compared to
undisturbed sites. Across gradients of N deposition in Alaska and Europe, changes in ECM
community composition of white spruce, Norway spruce {Picea ables (L.) Karst.) and beech
{Fagus syivatica L.) were accompanied by a strong decline in species richness as soil N
availability increased (Taylor etal. 2000; Lilleskov etal. 2002). To our knowledge,
molecular studies to compare ECM communities associated with a single host species
across soil moisture gradients have not been conducted.

The primary objective of this study was to describe and compare the molecular diversity
(using PCR-RFLP) of ECM communities associating with naturally regenerating black
spruce seedlings across habitats and within characterized fungal morphotypes. The
habitats examined consisted of two wetland forest types (black spruce-dominated and mixed

86

black spruce - tamarack sites) and an upland forest type (black spruce - lodgepole pine
sites) located In the Sub-Boreal Spruce (SBS) biogeoclimatic zone of the central interior of
BC (Meidinger etal. 1991). Soils were generally cold and nutrient-poor, with moisture
content varying from saturated (subhygric) to seasonally dry (Roy et ai. 1999; Krestov et al.

2000 ).

The second objective of this study was to confirm the characterization of ECM morphotypes
by comparing restriction fragment profiles generated from representative sub-samples of
roots from the same morphotypes previously described from these habitats. Whereas
morphological features such as color, texture, and branching pattern of ECM may vary with
changing environmental conditions, molecular approaches to describing ECM communities
are largely independent of environmental effects (Egger 1995). It was anticipated that the
molecular analysis would improve the resolution of ECM identification to species through
comparisons to sporocarp and root tip databases as well as to descriptions published in the
literature.

METHODS AND MATERIALS
ECM sample collection for molecular analysis and DNA extraction
Black spruce seedlings were randomly sampled from nine sites within three habitat types in
the SBS zone near Prince George, BC as described in the previous chapter (see previous
chapter for the detailed sampling protocol and site descriptions). Following morphological
assessment of 200 root tips per seedling, a proportionally representative sample of 20 root
tips (10% of each morphotype) was saved from each seedling for molecular analysis.
Individual samples were placed in 1.5 mL Eppendorf tubes and stored at -20°C until
processed for comparative molecular analysis.

87

DNA was extracted from ectomycorrhizal root tips using a modified Zolan and Pukkila (1986)
protocol. Approximately 2 mm of the apical end of each frozen root tip was placed into a
cold glass micromortar and crushed with the corresponding micropestel. To each
micromortar, 350 pL of 2X hexadecyl trimethyl ammonium bromide (CTAB) buffer with 0.2%
(3-mercaptoethanol (Sigma Chemical Co.) was added, and the tips were further crushed until
tissue was evenly suspended in the solution. The content of each micromortar was then
transferred to a 1.5 mL Eppendorf tube, and incubated in a heat block (VWR Scientific) at
60°C for 45-60 min. After heating, 350 pL of chloroform: isoamyl alcohol (24:1 solution) was
added to each tube and the tubes were centrifuged at 13000 x g for 10 min. at room
temperature. The aqueous layer was removed to a new Eppendorf tube to which 350 pL of
cold (-20°C) absolute isopropanol was added. The tubes were gently inverted repeatedly for
1 min. and then placed in the -10°C freezer for 5-10 min. After cooling, the tubes were
inverted several more times, then centrifuged at 13000 x g for 10 min. at room temperature.
The aqueous phase was poured off and the pellet was washed twice with 175 pL of cold
70% ethanol followed by centrifugation at 13000 x g for 3 min. The DNA pellet was dried
overnight in a desiccator and resuspended the following morning in 50 pL Tris-EDTA
(ethylenediaminetetraacetic acid) buffer. DNA extracts were stored in the freezer at -40°C.

DNA amplification and restriction endonuciease digestion
Enzymatic amplification of the fungal ITS region of fungal rDNA was conducted using the
universal oligonucleotide primer ITS1 (TCC GTA GGT GAA OCT GOG G) (White etal.
1991) and the fungal-specific primer NL6Bmun (GAA GOG TTT COG TTT GAA GA) (Egger
1995). A general PGR master mix was prepared that contained 17.2 pL millipore water, 3
pL 10X PGR buffer, 3 pL 2X dNTP’s (2 mM each of dATP, dTTP, dGTP, and cGTP), 2.4 pL
IVIgGIg (25pM), 1.2pL ITS1 (10 pM), 1.2 pL NL6Bmun (10 pM), and 0.14 pL Platinum Taq
88

DNA polymerase (Invitrogen). An aliquot of 27 pL PCR master mix was added each 0.2 pL
reaction tube along with 3 pL DNA extract. DNA extracts were gently thawed in a 37°C oven
for 15-20 min. prior to use. Reaction tubes were then placed in a PTC-100™ programmable
thermal controller (MJ Research, Inc.), programmed to the following settings: 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,
beginning at 30 s and increasing by 5 s per cycle for 35 cycles. Following DNA
amplification, 5 pL aliqots of PCR products were added to 3 pL aliquots of 6X loading buffer
and loaded into wells of a 0.7% agarose gel (0.7 g agarose in 100 mL of 10X Tris-borate
(TBE) buffer) submerged in a gel box of TBE buffer. Ethidium bromide fluorescent stain was
added to each gel (20 pL for new gels; 6pL for reused gels) for DNA visualization. The DNA
ladder {Hind\\\) was loaded into the first well of each row and the gel was run at 90-110 mV
for approximately 45 min. Subsequently, gels were removed to a UV box to view the
amplified DNA bands and to photograph using the Gel Print 20001 photographic system
(BioPhotonic Corp.). Photographs were printed on Mitsubishi thermal paper (K65H
Mitsubishi Electronic Corp.).

Three restriction endonucleases, Alu I (AGCT), Hinf\ (GANTC), and Rsa I (GTAC), were
used in this study. To 7.4 pL of enzyme solution (5 pL millipore water, 2 pL buffer, and 0.4
pL of one of Alu I, Hinf\, or Rsa I) in 0.2 mL microcentrifuge tubes, 7.4 pL PCR product was
added. The tubes were incubated in a 37°C oven for a minimum of 5 h. Following
incubation, the tubes were quickly centrifuged to 800 x g to remove condensation from the
caps, and then 4 pL of 6X loading buffer was added to each tube. Digested DNA was run
with 1 kb DNA ladder (Invitrogen) on high resolution 2.5% agarose (1 g agarose (Sigma
Chemical Co.) plus 1.5 g low melting point agarose (Invitrogen) in 100 mL 10X TBE buffer)
gels containing ethidium bromide for visualization. The gels were run at -1 1 5 mV for
approximately 3 h. Gels were removed to a UV box to view the DNA fragment patterns and
89

to photograph. Digital images were saved on disks using the Gel Print 20001 photographic
system (BioPhotonic Corp.).

Analysis o f m olecular data
RFLP gel images were analyzed using RFLPscan, Version 3.12 (Scanalytics). Individual
DNA fragments were marked and their sizes (base pairs) calibrated against the 1 kb
standards (1018, 514, 356, 344, 298, 220, 201,154, 134, and 75 bp fragments) using the
Desmile calibration method with log piecewise linear curve-fitting. Fragments less than 80
base pairs were excluded from analysis to reduce the possibility of primer dimer products
being included. Fragment patterns for individual samples were imported into both individual
morphotype and seedling databases in RFLPscan Database, Version 3.12 (Scanalytics).
Fragment patterns obtained from digestion of sporocarp samples (collected from field sites
as described in the previous chapter) were imported into a separate database for
comparisons.

Within each RFLPscan database, pairwise comparisons of ail band patterns (matched at a
5% threshhold) were compiled for each of the three enzymes. Values ranged from 0 to 1 for
each enzyme, and from 0 to 3 for each sample, with 3 indicating identical samples. Cluster
analysis using the neighbor joining method of the similarity matrix was performed using
PHYLIP (Phylogeny Inference Package), Version 3.573c (Felsenstein, J., University of
Washington). Resulting neighbor joining trees were viewed in TreeView, Version Win 3.2
(1998 Roderick DM Page). Individual databases were merged into one large database
consisting of all fragment patterns in order to compare patterns for the entire ECM
community. Where fragment patterns matched and when ECM morphotypes had similar
characteristics, two or more groups were merged into a single genotype or morphotype.
The database of all fragment patterns was compared with a reference database (K. Egger,
90

unpublished) as well as the sporocarp database. From these comparisons, we attempted to
confirm the morphological classification and taxonomic naming of ECM morphotypes, as
well as to identify unknowns based on clustering with known types on the neighbor joining
tree. Pairwise matching of all samples were also conducted using Dice’s index ([2 x the
number of common bands] / [2 x the number of common bands plus the number of
polymorphic bands]) and converted to distance matrices (1 - Dice’s index) to calculate the
Phi (0 ) Index (Mah etal. 2001). An increase in Phi values implies greater diversity.

The fragment pattern data were imported into a Microsoft Excel 97 (version SR-1)
spreadsheet. Using the neighbor joining tree as a guide, samples were first sorted into
morphotypes, then into genotypes. Genotypes were defined as sets of fragment patterns
that, when compared to each other, were within the 5% threshold of similarity for all three
enzyme digests. To detect root tips that were possibly mis-classified during morphological
assessment, contaminants that may have been amplified during PCR, or incomplete
digestions, morphotype and genotype groups were cross-referenced by seedling, site and
habitat. Samples determined as mis-classified were renamed. Partially digested or
potentially contaminated samples were removed from the total sample. Average fragment
sizes were calculated for each genotype.

Shannon, Simpson and Phi indices were used to assess genetic diversity between the
different habitats (data pooled for sites) as well as morphotype variation. The Shannon and
Simpson indices, which were calculated using proportional abundance of each ECM
genotype, excluded all partially digested or potentially contaminated samples. The Phi
Index, which was calculated from Dice distance matrices, included all successfully digested
root tips. All three indices were compared between habitats using one-way ANOVA

91

(SYSTAT version 8.0, 1998, SPSS Inc.) to determine significant differences (a=0.05). Mean
comparisons were tested using Fisfier’s Least Significant Difference (LSD) test (a=0.05).

RESULTS
DNA extraction, amplification and restriction endonuclease digestion
Based on tfie original sample of the 888 root tips, 454 (51%) yielded fragment patterns for
the molecular analysis. The success rate from the 755 colonized tips was 60%. Nonmycorrhizal tips accounted for 133 of the original sample, of which 32 (24%) actually
amplified and produced DNA fragment patterns. The remaining samples included those that
were either lost or contaminated during the DNA extraction procedure, did not amplify
properly to yield visible bands, produced doublet fragment patterns during the PCR
procedure, or produced very faint bands or extra fragments after restriction endonuclease
digestion.

The success rate of producing fragment patterns appeared to vary with morphotype. In
general, >75% of Amphinema and Piloderma types produced patterns compared to only 3748% for the Cortinariaceae and Thelephoraceae -like types (Table 3.1). Both Cenococcum
and MRA 1 had moderate success rates (50% and 74%, respectively). Of non-mycorrhizal
tips that produced patterns, over 80% matched patterns of Cenococcum (genotypes 1 and
2), MRA 1 (genotypes 2 and 3), Amphinema (genotype 3), Russulaceae 1 (genotype 1),
Russulaceae 2 (genotypes 1 and 6) and Thelephoraceae-like 2 (genotype 2). In each case,
the corresponding morphotype was previously described on the seedling or site with which
the non-mycorrhizal tip was associated.

92

Cluster analysis o f fragment patterns for ECM morphotypes
Cluster analysis of fragment patterns for all ECM sampled for molecular assessment
generally confirmed the morphological classification. ECM classified as the same
morphotype tended to cluster together on branches of the neighbor joining tree (Appendix
III). Comparisons of these ECM patterns with reference databases containing patterns from
ECM root tips from previous studies often confirmed the taxonomic naming of morphotypes
(phylograms not included). For example, reference samples of Amphinema, Piloderma,
Cenococcum and MRA 1 were consistently grouped within clades representing those
morphotypes described in our study. In one case, a group of non-mycorrhizal samples
clustered with a reference sample of E-strain, allowing tentative identification of these ECM
as a morphotype not described in this study.

Comparison of patterns from ECM root tips with patterns generated from sporocarps
collected from field sites generally did not aid in identification of morphotypes. The
sporocarp patterns usually grouped on their own branches rather than within morphotype
clades. However, there were two exceptions to this. Lactahus deiiciosus (Fries) S.F. Gray
grouped within the Lactahus 1 genotype 1 clade and Lactahus torminosus (Schaeff. ex Fr.)
Gray grouped within the Lactahus 3 genotype 1 clade.

Most morphotypes consisted of several genotypes; the average fragment lengths for all
genotypes are presented in Table 3.1. ECM morphotypes having the most genotypes
included Amphinema (six), Cortinariaceae 2 (six), and Russulaceae 2 (seven).
Cenococcum (two), Thelephoraceae-like 1 (one), Piloderma (one) and Lactahus 3 (one) had
the fewest number of genotypes. In some cases, delimitation of genotypes was based on
fragment size variation in only one of the three enzyme digests.

For example, Amphinema

genotypes 1, 2 and 3 varied by only the Alu I and genotype 4 differed from genotype 1 in
93

patterns generated by only the Rsa I enzyme. The single genotype of Lactarius 3 arguably
varied from Lactarius 1 genotype 1 in only the Hinf\ pattern, as both profiles showed
fragments that were not digested by the Rsa I enzyme, even though length variation was
observed. For some ECM genotypes, differences were observed in two of the enzyme
profiles. For example, with respect to Russulaceae 1, genotypes 1 and 2 showed very
similar fragment patterns with the Hinf\ enzyme, but different patterns with the Alu I and Rsa
I enzymes. Most genotypes differed from one another in all three enzyme profiles. This was
especially true when genotypes were compared between morphotypes.

In several instances, the similarity in fragment patterns allowed genotypes and, sometimes,
entire morphotypes to be merged. Examples include a Russulaceae 2 genotype that was
found to match Lactarius 1 genotype 3, and the single cottony gold-brown genotype, which
matched Amphinema genotype 6. In each case, there were also similarities in the
morphological descriptions and habitat occurrence, which supported merging the genotypes.

Interestingly, when multiple genotypes occurred within a morphotype, a single genotype
often appeared to dominate. For example, Cenococcum genotype 1 accounted for 83% of
all Cenococcum samples (total of two genotypes), MRA 1 genotype 2 represented 59%
(total of three genotypes), and Russulaceae 2 genotype 1 made up 46% (total of seven
genotypes) of all samples. Within other morphotypes such as Amphinema (total of six
genotypes), a more even abundance of genotypes was observed.

94

Table 3.1. Habitat (black spruce - tamarack (T) wetlands, black spruce-dominated (W) wetlands, and black spruce - pine (U) upland
forests) and approximate fragment sizes (bp) of the amplified ITS region for black spruce ECM morphotypes and genotypes.
ECM Morphotypes Habitat
No.
Undigested
and Genotypes
T W u samples size (bp) Alu\

Approximate fragment sizes (bp)

Hinf\

Rsa I

Cenococcum
genotype 1
genotype 2

+

+
+

+

45
8

790
915

440
400

150
240

110 80
150 115

275
435

165 130
290 165

3
14
6

870
855
935

640
650
655

145
150
150

120
110
115

385
445
440

210 145
250 160
185 165

7
2

770
860
920

365
445
425

175
230
190

160 110
190 110
130 115

280
285
350

165 150
165 155
165 150

690
740
710
530
600
620
370

190
190
190
310
260
185
195

110
115
115
150 110
150
150 110
110

335
335
335
295
365
360
320

290
295
290
200
185
345
290

165 150
170 155
165 150
165 140
165 145
160 145
165 150

555
610
775
465
995
975
980

100

920
950

MRA1
genotype 1
genotype 2
genotype 3
Russulaceae 1
genotype 1
genotype 2
genotype 3
Russulaceae 2
genotype 1
genotype 2
genotype 3
genotype 4
genotype 5
genotype 6
genotype 7
Russulaceae 3
genotype 1
Russulaceae 4
genotype 1
Lactarius 1
genotype 1
genotype 2
genotype 3

+
+

+
+

+
+
+

+
+
+

+

+

5

730 175
835 175
125

+
+
+

26

950

4
4
12

1000

+
+

1

5

+

3

980
980
955
1015
860

1

1040

590

190

170 110

360

285

170

155

615 300 175

4

1040

465

285

190 115

415

310

170

155

1015

+

21

3
11

520
430
470

290
245
280

190 110
190 115
185 105

415
325
335

350
215
285

165
165
170

150
145
155

1015

+

1070
950
1000

+

+

+
+
+
+

120

790 180
630 170
790 190

95

195 175
205 185
200
200 165

1020

555 465

105

ECM Morphotypes Habitat
No.
Undigested
T W u samples size (bp) Alu\
and Genotypes

Approximate fragment sizes (bp)

Hinfl

Rsa 1

Lactarius 2
+
genotype 1
+
genotype 2
Lactarius 3
genotype 1
Thelephoraceae 3
genotype 1
Thelephoraceae-like 1
+
genotype 1
genotype 2
Thelephoraceae-like 2
genotype 1
+
genotype 2
Thelephoraceae-like 3
+
genotype 1
Thelephoraceae-like 4
genotype 1
genotype 2
genotype 3
Tomentella 1
+
genotype 1
+
genotype 2
+
genotype 3
Tomentella-Wke 1
+
genotype 1
+
genotype 2

3
3

950
980

510
520

190
190

110
115

85

350
340

330
320

170
165

155
155

16

1070

515

285

185 110

350

315

165

150 100 1040

+

4

980

405

230

220 120

340

185

130

90

625 410

+

8
4

950
950

420
430

185
185

150 110
150 95

320
350

225
300

165
165

150
150

855
1025

4
3

1010
1000

560
525

190
235

155 110
180 110

320
330

260 170
255 165

150
150

1

965

425

185

150 120

360

320

165

150

1025

7
3
3

1000
900
965

475
395
465

280
260
245

185 125
185 110
185 110

325
320
315

280
215
295

170
155
160

150
145
145

545 455
580 180
975

6
2

1000
1000
990

370
480
430

190
380
190

125 105
185
150 115

95

325
315
360

265 165
285 235
320 165

155 110 945 180
120
1025
155
790 205

965
1010

435
525

230
250

185 120 110
190 160 115

315
365

190 165
170 155

150

1

1

875

415

185

120 110

220

190 165

150

4-

4-

+
+
+

1
6

95

90

935
1055

85

+

96

90

175

175

840 185
830 210

990
910

Tomentella-Wke 3
genotype 1

100

980

185

160

ECM Morphotypes Habitat
and Genotypes
T W U
Cortinariaceae 1
genotype 1
genotype 2
genotype 3
Cortinariaceae 2
genotype 1
genotype 2
genotype 3
genotype 4
genotype 5
genotype 6
Cortinariaceae 3
genotype 1
Hebeloma
genotype 1
Piloderma
genotype 1
Amphinema
genotype 1
genotype 2
genotype 3
genotype 4
genotype 5
genotype 6
cottony halo
genotype 1
genotype 2
genotype 3

+

+

+

+

+

+
+

+
+
+
+

+
+

+

+

+
+

+

No.
Undigested
mpies size (bp) A lu\

Approximate fragment sizes (bp)
Hinf\
Rsa i

6
9
2

1060
1040
1040

670
620
605

185
185
185

145 110
145 110
145 110

370
360
355

340
345
345

140 120
165 155
165 150

935
850
1060

175
175

7
11
2
5
3
2

955
940
1045
1075
1035
985

430
355
735
440
440
450

185
235
225
330
190
185

145 115 80
185 150 130

290 170 155
170 150
260 165 150
300 170 155
350 165 150
305 160 150

785
910
835
1090
905
1030

175
175
225

220 185
150 110
145 110

335
360
370
340
370
335

10

930

665

165

115

320

290

165

155

520

395

2

895

360

240

180 135

335

275

130

840

195

17

920

365

260

190 110

315

180 165

155

850

175

16
14
10
5
10
6

900
950
940
950
940
920

365
575
365
460
365
275

190
185
235
363
190
240

140
110
150
160
140
185

295
290
285
285
285
170

165 155
165 155
165 110
165 150
160 145
155

780
790
775
945
920
1085

175
175
175

115
175 110

325
325
335
315
320
370

6
8
2

1040
1055
1000

425
430
395

250
255
260

190 115 105
190 130 95
185 110 100

345
355
320

330
330
220

165 155
165 150
165 155

1035
885
85 625

97

90
85

110
85
125 100

165

180

180

175
200

175

Undigested
ECM Morphotypes Habitat
No.
and Genotypes
T W U samples size (bp) Alu I
creamy rhizomorphic clamped
+
genotype 1
+
genotype 2
orange 1
genotype 1
+
brown 1
+
genotype 1
brown 3
+
genotype 1
E-strain
+ + +
genotype 1

Approximate fragment sizes (bp)
Hinf\
Rsa 1

5
4

940
950

595
430

190
185

110
145 110

2

940

430

185

150 125 115

2

900

590

420

2

1040

365

295

185 125

6

830

360

255

180 105

90

95

295
295

240
230

165
165

155 85 985
145 115 1025

220

170

140

115

1070

215

180 165

150

985

370

335

165

150

1030

325

175

140

950

DNA fragments (bp) from amplification of fungal rDNA using the ITS1 and NLGBmun primers and digestion with the restriction
endonucleases Alu I, Hinf\ and Rsa I.

98

Molecular diversity within ECM morphotypes
Table 3.2 shows Shannon, Simpson and Phi diversity index values for 14 oommonly occurring
ECM morphotypes producing fragment patterns in this study. All three diversity indices
identified Piloderma, Lactarius 3 and Cenococcum as the morphotypes exhibiting the lowest
molecular diversity. Both Piloderma and Lactarius 3 samples (described from the upland sites
only) generated only one fragment pattern each; Cenococcum had two genotypes. The Phi
index value was also low for the cottony halo morphotype. Amphinema, Cortinariaceae 2 and
Russulaceae 2 (Shannon and Simpson) and Tomenfe/Za-like 1, Thelephoraceae-like 4 and MRA
1 (Phi) resulted in the highest index values. All three diversity indices suggested that Lactarius
1, Cortinariaceae 1 and Tomentella exhibited intermediate diversity values.
Table 3.2. Diversity values (Shannon, Simpson and Phi) for 14 ECM commonly occurring on
regenerating black spruce in three habitats.
ECM
Morphotype
Cenococcum
MRA 1
Russulaceae 2
Lactarius 1
Lactarius 3
Thelephoraceae-like 1
Thelephoraceae-like 4
Tomentella 1
Tomenfe//a-like 1
Cortinariaceae 1
Cortinariaceae 2
Piloderma
Amphinema
cottony halo

Habitat
No. of
genotypes T W U
2
3
7
3
1
2
3
3
2
3
6
1
6
3

+
+
+
+

+
+
+
+

+

+
+

+
+
+
+

+
+
+
+
+

+
+
+
+

+
+
+
+

Shannon Simpson

%

success

n

50.5
74.2
65.1
50.0
80.0
48.0
37.1
37.5
36.8
44.7
48.4
77.3
80.3
94.1

54
32
56
37
16
12
13
9
7
18
34
18
61
16

0.42
0.92
1.70
0.73
0.00
0.64
1.01
0.85
0.41
0.96
1.60
0.00
1.71
0.97

1.35
2.32
4.33
1.73
1.00
1.94
2.89
2.25
1.40
2.62
4.78
1.00
5.61
2.73

Phi
0.121
0.367
0.247
0.239
0.140
0.177
0.380
0.214
0.413
0.214
0.211
0.048
0.217
0.112

Habitat effects on ECM genotype distribution and diversity
With respect to molecular diversity, there were no significant differences between mixed black
spruce - tamarack wetland, spruce-dominated wetland, and mixed black spruce - lodgepole
pine upland habitats, as measured by the Shannon, Simpson and Phi indices (Table 3.3). The

99

Simpson index suggested possibly greater diversity in the W sites; in contrast, Phi values were
highest for the T and U habitats. Shannon values were similar across all habitats.
Table 3.3. Mean diversity values (Shannon, Simpson and Phi indices) for molecular genotypes
of ECM from three black spruce habitats.
Diversity Index
Shannon
Simpson
Phi

Treatment Effect Tam arack-Spruce
F
P
Wetland
0.075
1.727
1.436

0.928
0.256
0.309

2.300 (0.070)
9.513(0.877)
0.363 (0.035)

Black Spruce
Wetland

Pine - Spruce
Upland

2.297 (0.130)
11.300 (1.857)
0.307 (0.027)

2.247 (0.117)
7.670 (1.227)
0.378 (0.031)

Values for indices are means with standard error in parentheses. Means were tested using a
one-way ANOVA (a=0.05) for each habitat.

Genotype occurrence across habitats (Table 3.1) and between sites revealed distribution
patterns that were not obvious following morphotype characterization. Some ECM morphotypes
such as Cenococcum, MRA 1 and Cortinariaceae 1 were described morphologically as
occurring in all three habitats. Genotypes identified for each of these ECM also appeared to
have a fairly even distribution across the habitats. In contrast, genotypes for other ECM that
also occurred in all three habitats (morphotypes such as Amphinema, Cortinariaceae 2, and
Russulaceae 1 and 2) had more uneven patterns of distribution. Amphinema genotypes were
limited to one (for five genotypes) or two (for one genotype) habitats, with approximately two
genotypes in each habitat. Cortinariaceae 2 had two genotypes in two of three habitats, with
the remainder (four) in single habitats. One Russulaceae 1 genotype was in all three habitats;
two genotypes only occurred in T sites. With respect to Russulaceae 2, six of eight genotypes
only occurred in U sites, and one genotype each was in each wetland habitat.

Other habitat distribution patterns were observed when ECM genotypes were considered within
larger taxonomic groups. For the Cortinariaceae group, the number of genotypes occurring in
all habitats was fairly even. In contrast, more Lactarius genotypes were in the T habitat (four)

100

then in either of the W (two) or U (one) habitats. Interestingly, one half of Russulaceae
genotypes occurred in the U habitat (eight), with the remainder divided fairly evenly between the
wetland habitats. The majority (88%) of genotypes in the group comprised of Thelephoraceae,
Tomentella and brown types were in the wetland habitats, with twice as many in the T sites.

DISCUSSION
Molecular diversity: genotype and ECM community variation across habitats
Sixty-five individual genotypes were delimited from the 29 ECM morphotypes successfully
amplified and digested in our study. This order of magnitude is in line with findings of others
using similar methods. In BO, Mah et a i (2001) detected 22 genotypes for the eight most
common ECM morphotypes described on hybrid spruce and Sakakibara et a i (2002)
documented 26 genotypes for 11 morphotypes of Douglas-fir. Mehmann et a i (1995) found 23
genotypes within 18 morphotypes on Norway spruce in Switzerland. Not all fungal genotypes
were detected in all habitats sampled and this has also been previously corroborated by several
authors (Gehring et a i 1998; Jonsson et a i 1999; Mah et a i 2001; Sakakibara et a i 2002). Six
genotypes were found in all three black spruce habitats, six were identified in two habitats, and
53 occurred in single habitats only. Approximately 28 genotypes were found in each of the
three habitats.

Our results suggest that, although some genotypes (within morphotypes) were habitatgeneralists, several genotypes were habitat-specialized. Dominant genotypes for Cenococcum,
MRA 1, and Cortinariaceae 1 were found in all three habitats; however, one genotype for each
of these ECM occurred in low numbers in only one habitat. All genotypes for Amphinema,
Cortinariaceae 2, and all Russulaceae (with one exception) occurred in only one or two habitats.
Mah et a i (2001 ) also detected site-specific and seedling type-specific genotypes for MRA,

101

Amphinema and Tuber on hybrid spruce roots from naturally regenerating or planted seedlings
from mature, olear-out, or out and burned sites. Sakakibara et al. (2002) provide evidence for a
habitat-specific genotype of Cenococcum on Douglas-fir seedlings in addition to a habitatgeneralist Cenococcum genotype found in both forest types from which they sampled. Based
on RFLP profiles, Byrd et al. (2000) identified 106 ECM species for lodgepole pine, of which
only 10 species were found in both clear-cut and undisturbed sites. Koljaig etal. (2000) also
described a range of site-specificities for tomentelloid genotypes in Swedish boreal forests
based on ITS sequences. Few studies have directly examined the spatial distribution of ECM
genotypes in different habitats, and incidental distribution information is often lost in studies
where the number of samples subjected to molecular analysis is low. For example, Kernaghan
(2001) described Amphinema byssoldes, Cenococcum geophllum, and Tomentella species as
being ubiquitous in temperate ectotrophic plant communities, and as forming symbioses with a
wide variety of hosts in two high-elevation sites in the Alberta Rockies. Their small molecular
sample size, though suitable for confirming the identity of several ECM through comparisons to
a database of sporocarp RFLPs, was not large enough to detect variation in genotypes between
sites.

Based on comparisons of diversity index values (Shannon, Simpson and Phi), there was no
significant difference in molecular diversity between mixed black spruce - tamarack wetland,
black spruce-dominated wetland and black spruce - lodgepole pine upland forest habitats.
Similar to Gehring et al. (1998), our study found that species (genotype) richness of the ECM
fungal community remained fairly constant across environmental gradients ranging from
extreme (i.e. very wet in our study) to fairly moderate. One explanation may be that fungal
symbionts receive most of their energy from their plant hosts, and therefore may be buffered
against the environmental extremes that their plant associates experience (Gehring et al. 1998).

102

Although our original sampling for molecular analysis was proportional to ECM morphotype
occurrence, variable amplification and digestion success rates most likely affected the resulting
relative abundance of genotypes and subsequent analysis.

The Shannon index values indicated close similarities in genotype richness and evenness
between habitats. The Simpson index (which emphasizes dominant genotypes) showed a trend
of decreasing diversity from the black spruce-dominated wetlands, to the black spruce tamarack wetlands, to the black spruce - pine uplands; this trend was opposite to the results for
morphotype diversity measurements. The dependence of the Shannon and Simpson indices on
defining genotype proportional abundance presents an inherent problem with using these
indices to measure the molecular diversity of the ECM community. The Phi Index, which does
not rely on proportional abundance for genotypes (Mah et al. 2001 ), resulted in diversity values
that exhibited similar trends compared to those observed for morphological diversity (i.e. greater
diversity in uplands and mixed wetlands compared to black spruce-dominated wetlands).

Because ECM fungal species exist as populations of many genetic individuals, there is,
theoretically, some phenotypic variation in the ability of these species to colonize host plants,
promote plant growth, and adapt to changing environmental factors (Molina etal. 1992; Gehring
et al. 1998). Relatively little Is known about the levels of phenotypic variation under different
environmental conditions (Egger 1995). On large geographic scales, the ITS region is expected
to show intraspecific variation because it evolves rapidly and populations of a species can
become reproductively isolated (Horton 2002). However, based on a small sporocarp sample,
Kârén etal. (1997) reported that the Intraspecific variation in the ITS region was negligible at
local scales. If some intraspecific variation represents ecotypic adaptation to environmental
pressures, it seems probable that variation in the ITS region could occur at different geographic

103

scales ranging from regional landscapes to localized heterogeneity In edaphic conditions
(Calrney 1999). In a study that assessed pinyon pine ECM along soil moisture and nutrient
gradients In Arizona, Gehring etal. (1998) demonstrated a link between ECM community
composition (shifts In fungal genera and species between sites) and soil type In ablotlcally
stressed sites.

Ablotlcally stressed habitats, as defined by Gehring etal. (1998), are those at the extreme ends
of environmental gradients of soil moisture, nutrients, temperature, etc. In our study, watersaturated soils may present challenges to at least some ECM fungi In their ability to thrive and
form functional symbioses (Stenstrom 1991; Roy etal. 1999). In addition, saturated soils may
exert selective pressure on the ITS region, resulting In genotypic differences. Localized
heterogeneity In other soil characteristics (e.g. nutrient availability, temperature, pH, etc.) and
microtopography, as well as relative position to other major ECM vegetation, may account for
genotype differences within sites, although no correlations with specific habitat attributes were
detected In the present study. Assessing the distribution of genotypes and how this might relate
to the ecological roles of ECM fungi would be Interesting to explore In future studies; this would
require extensive multl-lsolate screening to determine the extent of ecotypic adaptation In ECM
fungi (Calrney 1999).

Molecular diversity within ECM morphotypes
Variation In the number of fragment patterns within morphotypes ranged from low for types such
as Piloderma, Lactarius 3, and Cenococcum to high for Amphinema, MRA 1, Cortinariaceae 2
and Russulaceae 2. The number of genotypes varied greatly, depending on morphotype. Mah
et al. (2001) also found large differences In patterns within some morphotypes; In their study,
Cenococcum exhibited a single genotype compared to six genotypes for Amphinema. In

104

contrast, Sakakibara et al. (2002) attempted to sample ECM to increase chances of detecting
variation but found very little in the ITS region for the morphotypes they examined. One
possible reason for this difference is that there may be little variation in the ITS region of some
Douglas-fir ECM. Minor differences in analysis techniques, as well as decisions made during
morphotyping (e.g. whether to classify morphotypes together or to separate them) and
assigning fragment patterns to genotypes, may all account for differences identified within
morphotypes. In addition, genotypes were defined by similarities in fragment patterns within a
5% tolerance limit in our study. Others have used a 6% tolerance limit using the same analysis
software (Scanalytics) as used in our study (Mah et ai. 2001), or set an approximate tolerance
limit based on visual pattern comparisons as the method of analysis. Viaud et al. (2000)
suggest that each step in a molecular analysis (i.e. sampling, DNA extraction, PGR and RFLP)
can be a source of bias which may lead to a distorted view of what is happening in the natural
system.

Some of the genotypic diversity within morphotypes in our study likely resulted from the
relatively large number of samples processed. Sakakibara et al. (2002) found that 8 of 11
morphotypes each had one dominant set of fragment patterns; these dominant patterns
represented 80-100% of all 227 root tips generating patterns. Situations in which one genotype
dominated within a morphotype were also found in our study as well as in those of Gehring et al.
(1998) and Mah etal. (2001); however, within some morphotypes, a more even abundance of
several genotypes was observed. Horton (2002) examined several sporocarps for each fungal
species from a 10 km^ area and reported that some species had multiple genotypes with one
dominating, while some had multiple genotypes without any genotype dominating (e.g. Lactarius
deiiciosus). In this example, most of the fragment patterns varied in only one of three enzymes,
suggesting limited variations in ITS sequence.

105

The Shannon and Simpson Indices assessed ECM molecular diversity as highest for
Amphinema, Cortinariaceae 2 and Russulaceae 2 morphotypes as compared to Phi index
values that were also high but less than values for MRA 1, Thelephoraceae-like 4 and
Tomentella-Wke 1. With respect to Thelephoraceae-like 4 and Tomentella-Wke 1, relatively low
sample sizes and very low amplification and digestion success rates may have contributed to
the higher Phi values. For MRA 1, the neighbor joining tree shows many small variations (within
the 5% tolerance) in fragment patterns grouped under three genotypes of MRA 1. These
differences might increase the pairwise distances resulting in greater Phi diversity as compared
to Shannon and Simpson diversity, where the number of genotypes and their relative
abundance were emphasized in the calculations. All diversity indices showed agreement for the
middle values, suggesting some level of congruence among indices. Intermediate diversity was
observed for Tomentella, Lactarius 1 and Cortinariaceae 1.

The lowest diversity, indicated by all three diversity indices, was in Piloderma and Lactarius 3,
two habitat-specific morphotypes found only in upland habitats, and Cenococcum, a generalist
found in all three habitats with one genotype found only in black spruce-dominated wetlands.
Fragment patterns of Piloderma ECM (found strictly associated with coarse woody debris in this
study) were similar to those of Piloderma genotype 2 (Mah et al. 2001 ), Piloderma III and P.
bysslnum IV (Sakakibara etal. 2002). Multiple genotypes for Piloderma have been described in
the other studies, but not ours. Lactarius 3 was tentatively identified as L. tormlnosis through
comparison to a sporocarp reference database. Lactarius species have been shown to exhibit
intraspecific variation in other habitats (Horton 2002).

106

Most investigators who have examined the ITS region have detected intraspecific variation in
some ECM morphotypes or species. Assessing fragment patterns from cultured Cenococcum
geophllum isolates from geographically-diverse origins, LoBuglio etal. (1991) reported a high
level of rDNA variation in 71 isolates; Farmer and Sylvia (1998) also detected intraspecific
genetic variation in the fungi Cenococcum geophllum and PIsolithus arhizus. From ITS
sequences of cultured organisms, Aanen etal. (2001) described variation in Hebeloma velutipes
and Manian etal. (2001) found heterogeneity among common European Suillus isolates.
Further evidence for multiple genotypic species within morphologically-defined fungal species
have been shown for Cortinarius rotundisporus and Hebeloma species (Sawyer et al. 1999) as
well as Lactarius deiiciosus and Inocybe lacera (Horton 2002). In our study, the highest
molecular variation was observed in the less well-defined morphotypes (i.e. Cortinariaceae and
Russulaceae); these grouped in polyphyletic clusters of genotypes on the neighbor joining tree
and may actually represent several species. In taxa such as Laccarla, Inocybe, and
Cortinariaceae, there appears to be a higher probability of observing intraspecific genetic
variation (Kârén et al. 1997; Horton 2002). This was true for the Cortinariaceae types described
in our study.

Interestingly, the genetic heterogeneity of Cenococcum rDNA reported from culture studies by
LoBuglio etal. (1991) and Farmer and Sylvia (1998) was not observed in the field-based studies
described by Mah etal. (2001), Sakakibara etal. (2002) and ours. Besides the use of cultured
isolates versus field-collected root tips and different PCR primers, an important distinction
between these studies was the geographic origin of fungal rDNA. Fungi used in the studies by
LoBuglio etal. (1991) and Farmer and Sylvia (1998) originated from broad geographic regions
in the eastern USA and Europe, while the three latter studies were conducted in temperate
forests in the interior of BC. Comparison between fragment patterns of the five Cenococcum

107

genotypes from BC suggests potential overlap in two patterns: genotype 1 (our study) and
Cenococcu/n-like I (Sakakibara et al. 2002). This similarity is based on patterns from two
restriction enzymes {Alu I and Hinf\) that also produced nearly identical patterns in
Cenococcum genotype 1 (Mah et al. 2001); the latter genotype differs from our dominant
Cenococcum genotype in the Rsa I enzyme pattern. Gehring etal. (1998) suggest that
restriction endonuclease digestion with two enzymes may produce fragment patterns that
distinguish among genera, and frequently species within a genus. Different species were also
distinguished by Gardes and Bruns (1996) using two restriction enzymes. Whether or not the
Cenococcum genotypes described in Mah etal. (2001), Sakakibara etal. (2002) and our study
are of the same species, there appears to be some variation in the Cenococcum ITS region
across three different host species within relatively similar forest types of interior BC.

Identification o f ECM Species
Comparisons between fragment patterns for ECM described in our study and reference
databases compiled from previously described ECM root tips and sporocarps generally
supported the morphological classification of ECM to fungal genera and families. In most
cases, these comparisons did not provide higher resolution for identification of fungal species
and, with the possible exception of two Lactarius species, failed to definitively identify
unidentified ECM morphotypes. Others have reported poor correlation between sporocarp and
ECM fragment patterns (Gardes and Bruns 1996; Kârén etal. 1997; Horton and Bruns 2001;
Horton 2002), but closely related fungi can be grouped together on the basis of the presence or
absence of ITS fragments (Kârén etal. 1997).

108

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Ill

Conclusions
Our study was the first to describe the structure and diversity cf ECM communities associating
with black spruce, an important tree species of the Canadian boreal forest. Across its habitat
range in the SBS zone of central BC, this species hosted a diverse community of rootassociated ECM fungi consisting of a total of 33 morphotypes and 65 genotypes (from 29
morphotypes). The composition of ECM communities varied in the different habitats (overall
similarity of 27%), but the total number of species described in each (richness) did not. The
number of morphotypes detected in any one of the habitats (black spruce-dominated wetlands,
mixed black spruce - tamarack wetlands, and black spruce - lodgepole pine upland forests)
represented approximately two thirds of the overall ECM community; approximately 43% of all
fungal genotypes were found in each habitat. Results emphasize the importance of sampling
across an ecological range to more completely describe ECM communities associated with a
particular host species.

The communities described in our study consisted of many ECM taxa, including Cenococcum,
MRA, Russulaceae, Lactarius, Thelephoraceae, Tomentella, Cortinariaceae, Hebeloma,
Piloderma and Amphinema. ECM morphotypes, and in some cases, fungal genotypes,
resembled those described on other hosts in similar studies. Many of these taxa have
previously been associated with nitrogen-limited, acidic forest soils. The molecular analysis
revealed varying degrees of genetic diversity (in the ITS region of fungal rDNA) within the
previously described ECM morphotypes. Piloderma, Cenococcum, and Lactarius 3 types
showed low intraspecific diversity whereas Amphinema, MRA, Russulaceae 2, Cortinariaceae 2,
Thelephoraceae-like 4 and Tomentella-Wke 1 types exhibited high diversity. These latter types
grouped In polyphyletic clusters of genotypes on the neighbor joining tree and may actually
represent several species.

112

Evidence supporting habitat specificity by certain taxa was obtained from both the
morphological and molecular analyses. The ECM morphotypes Tomentella and Russulaceae 1
were exclusively described from the mixed black spruce - tamarack wetlands; both Piloderma
and Lactarius 3 were identified from only the upland forest sites. In fact, Piloderma exhibited
even greater habitat specificity, as it was consistently associated with seedlings growing in
coarse woody debris. In general, fungi within the Thelephoraceae tended to be associated with
the wetland habitats whereas the different Russulaceae morphotypes were often associated
with either wetland or upland habitats. The molecular analysis provided greater resolution of
habitat specificity trends, particularly among the habitat-generalist morphotypes. The
distribution of Cenococcum, MRA and Cortinariaceae 1 morphotypes and genotypes was fairly
consistant across ail three habitats; however, genotypes of Amphinema, Cortinariaceae 2 and
Russulaceae 2 showed uneven patterns of distribution between habitats. This may reflect
context-specific selective pressure leading to variation in the fungal ITS region due to local
differences in soil characteristics such as moisture, nutrient and organic content. To determine
specific ecological functions of different fungi forming ECM symbioses with various tree hosts,
further studies should be conducted over ecological gradients to identify specific habitat
attributes that are consistently associated with certain types of ECM fungi.

ECM community diversity, as assessed by morphological analysis, was highest in the black
spruce - pine upland forest habitat and lowest in the black spruce-dominated wetland habitat,
with intermediate diversity in the black spruce - tamarack wetland habitat. Higher diversity in
both the mixed forest habitats is most likely due to the presence of large, dominant companion
plants that may provide a source of fungal inoculum for growing black spruce roots. Through
the spruce mixture effect, alternate hosts such as larch and pine species may facilitate fungal
colonization of black spruce roots by first allowing establishment of some fungal species on their

113

root systems. The higher ECM diversity observed in the upland forest habitat as compared to
the wetland habitats might be due to soil conditions that are less water-saturated for most of the
growing season.

Habitat diversity differences (using ECM morphotype data) were significant when measures
emphasizing species richness (Margalef and Shannon indices) were used. No significant
differences in ECM diversity between habitats were found following the molecular analysis.
These results may indicate that differences in community diversity between habitats were
mainly attributable to the presence of rare fungal types occurring at low frequency and
abundance. These rare types comprised a small proportion of the molecular sample; in
addition, losses during the molecular procedures may have under-represented these
morphotypes in the molecular diversity calculations. Similar community structure (dominance
by a few ECM morphotypes) was observed in all three habitats, reinforcing that differences in
diversity appear to be mainly attributable to differences in morphotype richness (particularly the
contribution of the less frequently occurring types) between habitats rather than to their relative
abundance. Future studies should be designed to examine the contributions of these rare
species to ecosystem processes since they are a widely reported component of ECM fungal
communities and appear to account for the differences in community diversity described in our
study.

Although all indices used to measure diversity between habitats in the morphology study
supported the same trend, this was not the case for the molecular diversity values. Only the Phi
Index supported the trends (highest diversity in the upland forests; lowest diversity in the
spruce-dominated wetlands) observed In the morphological analysis. Because the Shannon
and Simpson indices depend on proportional abundance of genotypes, calculation errors may

114

be introduced due to low sample sizes or losses during the experimental procedures.
Heterogeneity indices as measures of molecular diversity should be used with caution.

Our understanding of the community structure and diversity of black spruce ECM benefited by
using a combination of morphological and molecular techniques. Whereas the morphological
study captured contributions to diversity by less abundant ECM types, the molecular study
revealed patterns of genetic diversity and habitat use at greater resolutions than the
morphological study. Although fungal identification (as related to taxonomic placement) was
generally not improved by the molecular analysis, verification of morphological classification
was achieved in most cases.

115

A ppendix I. Types of vegetation compared between sites in spruce - tamarack wetland (T),

spruce-dominated wetland (W) and spruce - pine upland forest (U) habitats in central BC.
COMMON NAME

LATIN NAME

T1 12 T3 W1 W2 W3 U1 U2 U3

black spruce *
tamarack *
lodgepole pine *
subalpine fir *
scrub birch *
willow *
Sitka alder *
western mountain ash
prickly rose
northern black currant
black gooseberry
highbush-cranberry
thimbleberry
bog-laurel
Labrador tea
black huckleberry
hardback
saskatoon
black twinberry

Picea mariana (P. Mill.) B.S.P.
Larix iaricina (Du Rol) K. Koch
Pinus contorta Dougl. ex Loud. var. latifoiia
Abies iasiocarpa (Hook) Nutt.
Betuia giandulosa var. glanduiosa Michx.
Salix sp.
Alnus crispa var. sinuata Regel.
Sorbus scopulina Greene
Rosa acicularis Lindl.
Ribes hudsonianum Richard, in Frank.
Ribes lacustre (Persoon) Poiret in Larm.
Viburnum edule (Michx.) Raf.
Rubus parvifiorus Nutt.
Kaimia polifolia (Wang.) var. microphylla
Ledum groeniandicum Oeder
Vaccinium membranaceum Doug. Ex Hook.
Spiraea douglasii ssp. menziesii Hook.
Amelanchier alnifolia Nutt.
Lonicera involucrata (Richards.) Banks ex.

X
X

X
X

X
X

X

X

X

X
X

X
X

X

X
X

X
X

X

X

X

X

X

X
X

X
X

X
X
X
X

X
X

X

X

X

X
X
X

X
X
X

X

X
X

X

X
X
X
X

X

X

X
X
X

X

X
X

X

X

X

X

X

Spreng
kinnikinnick *
twinflower
bog cranberry
dwarf blueberry
llngonberry
bog-rosemary
prince's-pine
dwarf nagoonberry
trailing raspberry
rosy pussytoes
yarrow
heart-leaved arnica
palmate coltsfoot
clasping twistedstalk
queen's cup

Arctostaphyios uva-ursi{L.) Spreng.
Linnaea borealis L.
Vaccinium oxycoccos (L.) MacM.
Vaccinium caespitosum Michx.
Vaccinium vitis-idaea L.
Andromeda polifolia L.
Chimaphiia umbellata (L.) Bart. ssp.
occidentaiis (Rydb.) Huit.
Rubus arcticus L.
Rubus pubescens Raf.
Antennaria rosea Greene
Achillea millefolium L.
Arnica cordifolia Hook.
Petasites palmatus (Alt.) A. Gray
Streptopus amplexifoiius (L.) DC.
Ciintonia unifiera (Menzies ex J.A. & J.H.

X
X
X

X

X

X

X
X

X
X

X

X

X
X

X
X
X
X
X
X

X

X
X
X

X

X
X
X
X

Shult.) Kunth
yellow coralroot
round-leaved reln-orchid
white bog-orchid
northern twayblade
rattlesnake-plantain

Corallorhiza trifida Châtelain
Platanthera orbicuiata (Pursh) Lindl.
Platanthera dilatata (Pursh) Lindl. ex Beck
Listera borealis Morong
Goodyera oblongifolia Raf.

116

X
X

X
X
X
X

X

COMMON NAME

LATIN NAME

11 12 13 W1 W2 W3 U1 U2 U3

common mitrewort
Mitella nuda L.
Tiarella trifoliata L. var. trifoliata
three-leaved foamf lower
marsh cinquefoil
Potentilla palustris (L.) Scop.
wild strawberry
Fragaria virginiana Duchesne
Moneses uniflora (L.) A. Gray
single delight *
one-sided wintergreen *
Orthilia secunda (L.) House
pink wintergreen *
Pyrola asarifolia Michx.
common red paintbrush
Castilleja mlniata Dougl. ex Hook
cow-wheat
Melampyrum lineare Desr. var. iineare
Geocaulon livldum (Richards.) Fern.
false toad-flax
Epiloblum
angustifollum L.
fireweed
Cornus canadensis L.
bunchberry
Aralia nudicaulis L.
wild sarsaparilla
buckbean
Menyanthes trifoliata L.
Equisetum sp.
horsetail
Carex sp.
sedge
Poa sp.
grass
Lycopodium obscurum
ground-pine
ground-cedar
Lycopodium complanatum
red-stemmed feathermoss Pleurozium schreberi
Hylocomium splendens
step moss
Ptilium crista-castrensis
knight's plume moss
electrified cat's-tail moss Rhytidiadelphus triquetrus
Polytrichum juniperinum
juniper haircap moss
common leafy moss
Plagiomnium medium
Aulacomnium palustre
glow moss
golden fuzzy fen moss
Tomenthypnum nitens
golden ragged moss
Brachytheclum salebrosum
Sphagnum capillaceum
common red sphagnum
lungwort
Lobarla pulmonaria
dog lichen
Peltlgera canlna
freckled lichen
Peltigera aphthosa
pixie cup lichen
Cladonia sp.
reindeer lichen
Cladina sp.

Plants that are potential ECM hosts.

117

X
X

X

X

X

X

X

X

X
X
X

X
X
X

X

X

X
X

X
X
X
X

X

X
X
X
X
X
X
X

X
X
X
X

X
X

X
X
X
X

X
X
X

X
X

X
X

X
X

X

X

X

X

X

X

X

X

X

X
X
X
X
X

X
X
X
X
X

X

X
X
X
X

X

X

X

X

X

X

X
X
X

X
X
X

X
X

X

X
X
X

X

X
X

X

X

X
X

X
X

A ppendix II. Descriptions of black spruce ECM morphotypes from black spruce - tamarack wetland (T), black spruce-dominated

wetland (W) and black spruce - pine upland forest (U) habitats in central BO.
ECM Morphotype

Morphological Features

Mantle Features

Emanating Hyphal Features

Rhizomorphs

Cenococcum

black, grainy to woolly,
sometimes slightly reflective,
unbranched tips (0.5-2 mm)

outer mantle (OM) /inner mantle
(IM) net synenchyma (20-25 pm
thick); stellate pattern visible at
100 X on less woolly types; cells
3-6 pm wide

dark brown to black, thickwalled, septate emanating
hyphae (EH) (5-6 pm wide),
sometimes verrucose; no
clamps

none

MRA^

brown-black, velvety,
unbranched tip; often whitish
or hyaline at tip

OM felt prosenchyma; IM net
synenchyma

dark brown, verrucose, thicknone
walled EH (2-3 pm wide); no
clamps; septate H-anastomosis
without clamps

MRA 2

brown-black, smooth, slightly OM felt prosenchyma; IM net
reflective, unbranched tip
synenchyma; mantle stains pink
with KOH

EH rarely observed, or hyaline none
or with dark pigmentation,
verrucose, thick-walled EH (2-3
pm wide); no clamps; septate
H-anastomosis without clamps

Russulaceae 1

creamy or pale grey to
OM net to irregular (interlocking)
orange, smooth to slightly
synenchyma; IM net
cottony; straight, unbranched synenchyma; sometimes parallel
or monopodial pinnate tip (~1- cells on mantle surface of
2 mm)
variable widths (2-7 pm)

hyaline to dark grey, very
none
branched EH (2-3 pm wide);
septa appear close together (414 pm wide); no clamps;
sometimes yellow granular cell
contents

Russulaceae 2

pale yellow-orange, smooth,
velvety or cottony, usually
unbranched or monopodial
pinnate tip (0.5-2 mm);
sometimes dark mottling on
basal half;

OM felt prosenchyma towards net few to many branched, hyaline, yellow, smooth,
verrucose EH (1-3 pm wide);
undifferentiated
synenchyma; IM net
synenchyma; yellow deposits on clamps present on some, but
rhizomorphs (-20 pm
OM stain green or pink with KOH; not all samples
wide) sometimes present
perhaps half of surface mantle
(not always)
cells with granular contents;
mantle -10-16 pm wide

Russulaceae 3

white to pale orange, smooth
to slightly cottony,
unbranched, club-shaped tip
(-0.5 mm)

very thin mantle of net
prosenchyma

118

variable width (2-3 and -5 pm),
hyaline, branched EH with
clamps

uncertain

ECM Morphotype

Morphological Features

Mantle Features

Emanating Hyphal Features

Rhizomorphs

Russulaceae 4

yellow-orange to orangebrown, smooth to velvety,
straight to bent, unbranched
tip (~1 mm)

OM/IM net synenchyma

few, indeterminant, hyaline EH yellow to brown,
(2-3 pm wide) with large
differentiated (hyphae 2-5
clamps; septate H-anastomosis pm wide) rhizomorphs
with clamps
(-40 pm wide)
sometimes present (not
always)

Lactarius 1

frosty orange-brown, smooth, OM net to irregular (interlocking)
straight, monopodial pinnate synenchyma; IM irregular
tip, sometimes darker at the
synenchyma
base; rhizomorphs present

few short, thick (5-8 pm wide), sometimes rhizomorphs
branched, hyaline (pale yellow), (-70 pm wide) consisting
septate EH; no clamps; laticifer of loose, undifferentiated
hyphae (7-8 pm wide) with pale hyphae; attached at a
yellow cell contents
restricted point

Lactarius 2

yellow-orange to pale orange, OM/IM irregular (interlocking)
smooth or velvety to slightly
synenchyma; thin mantle (-14-18
cottony, straight to clubpm thick)
shaped, unbranched tip (<1 -4
mm)

many branched, hyaline EH (2- none
3 pm wide); clamps uncertain;
wide laticifer hyphae on mantle
surface (5-6 pm wide); Hanatomosis; sometimes
dolipores appear visible on
either side of septa

Lactarius 3

golden-beige, smooth,
tortuous tip (3-4 mm)

OM net to irregular (interlocking) very few, branched, hyaline EH none
synenchyma; IM net
of variable widths (2-5 pm); no
synenchyma; cell widths (3-6 pm) clamps; granular cell contents;
with some elongated cells (6-8
H-anatomosis
pm)

119

ECM Morphotype

Morphological Features

Mantle Features

Emanating Hyphal Features

Thelophoraceae 1

orange-brown, velvety, club- OM regular synenchyma; IM net occasionally branched, hyaline, none
shaped, unbranched tip (~1.5 synenchyma; angular cells 10-15 septate EH (3-4 pm wide) with
mm)
pm wide
clamps; awl-shaped,
sometimes branched cystidia
(45-65 pm long), sometimes
with basal clamp

Thelephoraceae 2

pale orange to orange-brown
(rusty-tawny), smooth to
cottony, monopodial pinnate
to pyramidal tip

OM net prosenchyma to wards
net synenchyma; IM net
synenchyma; very thin mantle
(-5-6 pm)

Thelephoraceae 3

pale greenish-yellow, yellow
or orange-brown, velvety to
slightly cottony, club-shaped,
unbranched tip (-1-2 mm)

OM irregular (non-interlocking) to indeterminant, branched,
regular synenchyma; IM net
hyaline EH (2-3 pm wide) with
synenchyma; sometimes oliveclamps; aseptate Hgreen deposits on mantle surface anastomosis; yellow granular
cell contents

none

Thelephoraceae-like 1 black, grainy to felty, straight dark black OM with irregular (non- hyaline to pale yellow, bent,
to club-shaped tip (0.5-3 mm) interlocking) synenchyma (cells 6- branched, septate EH (4-6 pm
8 pm wide; 16-22 pm long); IM
wide); no clamps
net synenchyma; with uneven
black mottling on mantle surface
that stains greenish-black with
KOH

none

Thelephoraceae-like 2

brown rhizomorphs with
verrucose hyphae;
consists of differentiated
hyphae (25-35 pm thick)

hyaline, bent, finely verrucose none
EH (3-4 pm wide) with granular
cell contents; no clamps;
septate H-anastomosis;
sometimes dolipores appear
visible on either side of septa

OM irregular (non-interlocking)
hyaline to brown, branched,
dark brown to brown-black,
smooth to slightly woolly or
synenchyma; IM net
sometimes verrucose, variable
stringy, unbranched, bent tip synenchyma; -1 2 pm thick;
width (2-3 and 4-5 pm) EH; no
(-2 mm); rhizomorphs present uneven black mottling on mantle clamps; sometimes bottlesurface that stains greenish-black shaped or awl-shaped cystidia
with KOH
(2-3 pm wide)

120

Rhizomorphs

ECM Morphotype

Morphological Features

Mantle Features

Thelephoraceae-like 3 yellow-orange-brown, smooth, OM irregular (non-interlocking) to
straight, monopodial pinnate regular synenchyma (cells 5-1 Op
tip (0.5-2 mm)
wide; 10-15p long); IM net
synenchyma; mantle -15-20 pm
thick

Emanating Hyphai Features

Rhizomorphs

rare to few branched, thickwalled, hyaline EH (2-3 pm
wide), sometimes with clamps
(not at every septa); awlshaped, sometimes branched
cystidia (50-60 pm long) with
variable basal cell shape and
retraction septa 2-3 pm wide;
sometimes a basal clamp

none

none

Thelephoraceae-like 4

yellow-orange, smooth,
straight to club-shaped,
unbranched tip (-1-3 mm)

OM regular synenchyma; IM net few hyaline EH (2-4 pm wide);
no clamps; awl-shaped.
synenchyma; perhaps half of
surface mantle cells with granular unbranched cystidia 2-4 pm
contents
wide and 20-100 pm long

Tomentella

golden-yellow to goldenbrown, smooth to grainy,
unbranched or monopodial
pinnate tip (-1-2 mm)

OM regular to irregular (non­
interlocking) synenchyma; IM net
synenchyma; clumps of round
deposits on mantle surface;
mantle -1 4 pm thick

Tomenfe//a-like 1

rusty-dark brown, grainy,
OM regular to irregular (non­
slightly reflective, straight.
interlocking) synenchyma; IM net
unbranched tip (-1 mm); often synenchyma; OM cell widths 2-5
with dark (black) mottling
pm

Tomentella-Wke 2

yellowish-brown to brownblack, velvety to cottony,
unbranched, club-shaped tip
(-1 mm)

OM net to irregular (possibly
bent, branched, hyaline EH (4- none
interlocking) synenchyma; IM net 6 pm wide); variable lengths
with clamps
synenchyma

Tomentella-Wke 3

olive-brown-black; smooth to
velvety, straight to bent,
monopodial pinnate tip (1-5
mm); usually hyaline at tip

few, short (50-80 pm wide),
OM irregular (possibly non­
interlocking) synenchyma; IM net highly branched, hyaline to
synenchyma; thin mantle (-8 pm) brown EH (2-4p wide) with
with uneven dark brown mottling clamps

121

few branched, hyaline EH (3.5- none
4 pm wide) with large clamps;
possibly tapering cystidia
(uncertain)
few to many, hyaline to brown,
branched, sometimes
verrucose EH (2-3 pm wide);
no clamps; possibly tapering
cystidia (uncertain)

none

none

Emanating Hyphai Features

Rhizomorphs

ECM Morphotype

Morphological Features

Mantle Features

brown 1

chocolate-brown, smooth to
velvety, straight, unbranched
tip (-1-2.5 mm)

OM/IM net to irregular
very few, finely verrucose,
(Interlocking) synenchyma; thin
hyaline EH (1-2 pm wide); no
mantle (-8 pm) with uneven dark clamps
brown mottling

brown 3

chocolate-brown, smooth to
cottony, straight to bent,
unbranched tip (2-4 mm)

OM felt to net prosenchyma; IM
net synenchyma; OM cells 1-2
pm wide; thin mantle (-14 pm)

orange 1

frosty coppery-brown, smooth, OM net to irregular synenchyma;
straight to club-shaped,
mounds of brown, rounded cells
usually unbranched tip
on OM; IM irregular synenchyma

few branched, hyaline EH (1 -3
pm wide) with clamps

none

Cortinariaceae 2

white, reflective, irregularlybranched, tortuous, cottony
tip; rhizomorphs present

very thin or incomplete mantle of
net prosenchyma (-10 pm thick)

variable width (1.5-5 pm),
hyaline, kinked, branching EH;
large clamps; septate Hanastomosls without clamps

often white rhizomorphs
(-40 pm wide) consisting
of loose, undifferentiated
hyphae

Cortinariaceae 3

yellow-orange to orangebrown, smooth to velvety,
straight, unbranched tip (-1
mm)

OM net to irregular (interlocking)
synenchyma; IM net
synenchyma; mantle -1 0 pm
thick

few, kinked, branched,
verrucose hyaline (sometimes
yellow) EH (2-3 pm wide);
possibly with clamps; Hanastomosis with clamps, but
not all septa

none

Hebeloma

white, short (-1 mm), clubshaped, smooth, velvety or
cottony tip

OM net prosenchyma; IM net
synenchyma

thin (1-3 pm), hyaline,
branching EH

none

122

none

hyaline to pale brown EH (1 -2 sometimes thick rope-like
pm wide) without clamps; some rhizomorphs
<100 pm long

ECM Morphotype

Morphological Features

Mantle Features

Emanating Hyphal Features

Rhizomorphs

PUoderma

creamy pale yellow to bright
lemon yellow, unbranched,
often bent to tortuous, woolly
tip (1-5 mm): rhizomorphs
present

OM/IM of net prosenchyma
towards net synenchyma;
granular material on mantle
surface stain purple with KOH

profuse hyaline, highly
verrucose, branching EH (~3
pm) without clamps; septate Hanastomosis without clamps;
verrucose and resinous
deposits (yellow) and needle­
like crystals on EH

broadly attached, thick
(50-130 pm), yellow
(sometimes white-purple)
rhizomorphs consisting of
loose to smooth,
undifferentiated hyphae

Amphinema

pale yellow to yellow-orange,
cottony, straight, unbranched
or monopodial pinnate tip;
rhizomorphs present

OM/IM net prosenchyma towards profuse hyaline (yellow tinge), sometimes golden-yellow
net synenchyma, -14 pm thick;
branched, curved, verrucose
rhizomorphs (50-100 pm
difficult to see mantle through EH EH (2-4 pm wide) with clamps; wide) consisting of loose,
granular cell contents
undifferentiated or slightly
differentiated hyphae

cottony halo

OM irregular (possibly non­
profuse, long, curved, hyaline
creamy-white, yellowish or
orange, straight, usually
interlocking) synenchyma; IM net (yellowish tinge), highly
unbranched tip with cottony;
synenchyma; thin mantle ( 10 pm verrucose EH (1-3 pm wide)
short EH form a "halo" around thick)
with clamps (nat all septa) and
tip
granular cell contents; septate
H-anastomosis with clamps

none

cream rhizomorphic
clamped

creamy to pale yellow,
monopodial pinnate, straight,
cottony tip; sometimes
reflective; rhizomorphs
present

rhizomorphs consisting of
loose, undifferentiated
hyphae (not always seen)

thin mantle of net to irregular
synenchyma (cells 5-6 pm wide)

123

variable (2-4 pm), hyaline,
branching EH with clamps;
aseptate H-anastomosis

ECM Morphotype

Morphological Features

Mantle Features

cottony gold-brown

light golden-yellow to coppery- OM/IM net synenchyma; thin
indeterminent, branched, bent, none
brown, smooth to cottony,
mantle (8-10 pm thick); possibly verrucose EH; hyaline
straight or slightly bent,
with oval crystals on mantle
(yellowish tinge) EH (2-3 pm
unbranched or monopodial
surface; mantle and EH stain pink wide) with clamps; septate Hpinnate tip (0.5-2.5 mm)
with KOH
anastomosis with clamps

124

Emanating Hyphal Features

Rhizomorphs

Appendix III Unrooted neighbor joining tree generated from restriction fragment patterns of
black spruce ECM morphotypes. The tree shows the relationships between ECM
morphotypes, genotypes and habitat of origin (spruce - tamarack wetland [T], sprucedominated wetland [W] and spruce - pine upland forest [U] habitats) in central BC.

Lactarius 1 (g1)

Lactarius 3

Lactarius 2 {g^ 12)

Thelephoraceae-like 4 (g3)
Tomente//a-llke 2 (g1)
Cenococcurr) (g1)

E-strain (g1)

125

Cenococcum (g2)
MRA 1 (82)
Amphinema (g3)

MRA 1 (g2)
MRA 1 (g3)

Amphinema (g5)
MRA 1 (g2)

Hebeioma (g1)

Cortinariaceae 1 (g1)
---------------------- 1

Russuiaceae 2 (g4)

—

Russulaceae 2 (g1)

-1 — 1

^

MRA 1 (g1)
Lactarius 1 (g1)

__ 1------------------------------ C L f.

brown 1 (g1)
Cortinariaceae 2 (g5)
cottony halo (g2)

Amphinema (g6)

Cortinariaceae 1 (g3)
Russuiaceae 2 (g6)
Cortinariaceae 1 (g2)

Cortinariaceae 2 (g 1/2)

Russulaceae 1 (g3)
Thelephoraceae 3 (g1)

126

Thelephoraceae-like 2 (g1)
Tomentella (g1)

Cottony halo (g1)

Tomentella (g3)

Russulaceae 2 (g6)
Thelephoraceae-like 1 (g2))
Cream rhizomorphic clamp (g2)
Thelephoraceae-like 4 (g1)
Lactarius 1 (g2)
Cottony halo (g3)
Thelephoraceae-like 4 (g2)
Tomentella-Wke (g1)

Russulaceae 3 (g1)
Russulaceae 1 (g1)

Cream rhizomorphic clamp (g1)
Amphinema (g2)

Amphinema (g1)

PUoderma (g1)

127

Russulaceae 2 (g7)
Amphinema (g4)
Cortinariaceae 1 (g1)
Amphinema (g5)
Russulaceae 2 (g2)
Russulaceae 2 (g3)
Russulaceae 2 (g 1)

Cortinariaceae 3 (g1)

Lactarius 1 (g3)

Thelephoraceae-like 4 (g1)
Cortinariaceae 2 (g1)

Russulaceae 1 (g2)
Cortinariaceae 2 (g4)

Black spruce - tamarack wetland habitat
^

Black spruce-dominanted wetland habitat
Black spruce - lodgepole pine upland forest habitat

128