ECTOMYCORRHIZAL FUNGAL COMMUNITIES OF DOUGLAS-FIR ON
DIVERSE SOIL LITHOLOGIES OF CENTRAL BRITISH COLUMBIA

by

Kirsten M. Thompson
B. Sc. Trinity Western University, 2011

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
NATURAL RESOURCE AND ENVIRONMENTAL STUDIES (BIOLOGY)

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
April 2015

© Kirsten M. Thompson, 2015

i

UMI Number: 1526492

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ECTOMYCORRHIZAL FUNGAL COMMUNITIES OF DOUGLAS-FIR ON DIVERSE
SOIL LITHOLOGIES OF CENTRAL BRITISH COLUMBIA
Abstract:
Ectomycorrhizal (ECM) fungi are symbiotic partners o f most conifers and improve
host health by increasing access to nutrients and water in return for photosynthates. ECM
fungi have been demonstrated to ameliorate the effects o f some harsh soil chemical
conditions on plants. Douglas-fir (Pseudotsuga menziesii var. glauca (Beissn.) Franco) is
found on and off extreme bedrock-derived soils in central British Columbia. The objectives
o f this study were to describe the soils and Douglas-fir forests found on the diverse
lithologies o f the Fort St. James area and to assess seedling health and ECM fungal
communities o f Douglas-fir grown on these soils by morphological and molecular means.
Fifteen ECM morphotypes, 12 basidiomycetes and 3 ascomycetes were identified with
Tuber anniae (Ascomycota) unique to ultramafic soils. Three morphotypes (E-strain,
Cenococcum geophilum, and Rhizopogon cf. villosulus) were ubiquitous on all sites and no
connection between parent material and ECM communities was established.

Table of Contents:
Abstract:..................................................................................................................................ii
Table o f Contents:................................................................................................................iii
List o f T ables:...................................................................................................................... vi
List o f Figures:................................................................................................................... viii
Acknowledgements:
1.

Introduction and literature review............................................................................................ 1
1.1.

Mycorrhizal fungi................................................................................................................2

1.2.

Arbuscular and orchid mycorrhizas: individual guilds................................................ 3

1.3.

Ericoid, arbutoid, ectendo and monotropoid mycorrhizas: shared guilds.................. 4

1.4.

Ectomycorrhizas..................................................................................................................5

1.4.1.

Structure and function.................................................................................................5

1.4.2.

Ecosystem impact and im portance.......................................................................... 7

1.4.3.

Development o f modern methods o f ECM identification................................... 8

1.5.

Ecological influence o f soil parent material................................................................. 10

1.5.1.

Pedogenesis in central British Colum bia.............................................................. 10

1.5.2.

Nutrient mobility within soils................................................................................. 11

1.6.

Douglas-fir in Central British Columbia...................................................................... 13

1.6.1.

Habitat and range.....................................................................................................13

1.6.2.

ECM associates..........................................................................................................14

1.7.

2.

xi i

Current research into ECM on extreme parent m aterial............................................ 14

1.7.1.

Mycorrhizal fungi o f serpentine and ultramafic-derived soils......................... 15

1.7.2.

Mycorrhizal fungi o f calcareous soils...................................................................18

1.8.

Objectives o f study........................................................................................................... 21

1.9.

References..........................................................................................................................22

Fort St. James: Soils and forest ecosystems related to diverse lithologies...................... 30
2.1.

Description o f bedrock origins....................................................................................... 30

2.1.1.

Ultramafic bedrock, serpentine rocks and so ils................................................... 30

2.1.2.

Above and below-ground effects o f ultramafic and serpentine bedrock

2.1.3.

Calcareous bedrock and soils.................................................................................. 33

2.1.4.

Above and below-ground effects o f calcareous bedrock................................... 33

2.2.

31

History o f glaciation......................................................................................................... 34

2.2.1.

Glacial parent material and soils.............................................................................34

2.3.

Sub-Boreal Spruce zone biogeoclimatic zone............................................................... 35

2.3.1.

Moisture and temperature regim e......................................................................... 35

2.3.2.

Plant community structure.......................................................................................36

2.3.3.

Douglas-fir in the Fort St. James area....................................................................37

2.4.

Methods o f soil collection...............................................................................................39

2.4.1.

Site selection.............................................................................................................. 39

2.4.2.

Transect layout and soil collection........................................................................ 40

2.4.3.

Soil pits and horizon description............................................................................41

2.4.4.

Soils preparation and analysis................................................................................. 41

2.4.5.

Statistical analysis o f soil d ata................................................................................42

2.5.

Methods o f vegetation and forest survey..................................................................... 42

2.5.1.

Site characteristics.................................................................................................... 42

2.5.2.

Host stand characteristics.........................................................................................43

2.5.3.

Statistical analysis o f forest inventory d a ta ......................................................... 43

2.6.

Soil analysis......................................................................................................................44

2.6.1.
2.7.

Soil chemical and texture d ata................................................................................ 44

Forest overstory and understory d ata............................................................................ 51

2.7.1.

Douglas-fir stand age, basal area, and understory data........................................ 51

2.7.2.

Evidence o f fire......................................................................................................... 57

2.8.

Discussion o f field data....................................................................................................58

2.8.1.

Soil properties............................................................................................................58

2.8.2.

Forest conditions....................................................................................................... 58

2.8.3.

The use o f sites o f the same parent material as replicates................................. 59

2.9.

Sum m ary............................................................................................................................ 60

2.10.

R eferences.....................................................................................................................62

3. Characterization o f Pseudotsuga menziesii var. glauca ectomycorrhizal fungi
communities on ultramafic and calcareous soils in central British Columbia......................67
3.1.

Douglas-fir fungal partners.............................................................................................67

3.1.1.

Douglas-fir and ECM in the fie ld .......................................................................... 67

3.1.2.

Douglas-fir and ECM in greenhouse conditions........................................

3.2.

68

Methods o f greenhouse b ioassay................................................................................... 69

3.2.1.

Seed preparation........................................................................................................69

3.2.2.

Seedling growth conditions.....................................................................................70

3.2.3.

Seedling growth and biom ass................................................................................. 72

3.2.4.

Collection o f seedling foliar samples for chemical analysis.............................. 72

3.2.5.

Mycorrhizal colonization......................................................................................... 72

3.2.6.

Morphology analysis.................................................................................................73

3.2.7.

Statistical analysis o f biomass and morphology d a ta ......................................... 75

3.3.

3.3.1.

DNA extraction..........................................................................................................76

3.3.2.

DNA amplification.................................................................................................... 77

3.3.3.

DNA sequencing........................................................................................................79

3.4.

Experimental results......................................................................................................... 79

3.4.1.

Growth and biomass o f seedlings...........................................................................79

3.4.2.

Foliar metal content o f seedlings............................................................................84

3.4.3.

ECM abundance and frequency.............................................................................. 86

3.4.4.

Diversity indices and rank abundance curves.......................................................94

3.4.5.

Two-way cluster analysis....................................................................................... 102

3.4.6.

Confirmation o f identity using D N A ................................................................... 105

3.5.

4.

Methods o f molecular an aly sis.......................................................................................76

D iscussion........................................................................................................................ 106

3.5.1.

Impacts on seedlings grown on ultramafic, calcareous, and glacial soils.... 106

3.5.2.

Diversity o f morphotypes and community structure.........................................108

3.5.3.

DNA sequence enhanced identification............................................................... 110

3.5.4.

Complications and limits to greenhouse studies.................................................112

3.6.

Sum m ary...........................................................................................................................114

3.7.

References.........................................................................................................................115

Conclusions.............................................................................................................................120
Appendices......................................................................................................................... 123
Appendix 1: Soil profile descriptions and analytical d ata.........................................123
Appendix 2: Elemental analyses o f soil composites used in greenhouse experiment
............................................................................................................................................. 142
Appendix 3: Morphotype descriptions..........................................................................144

List of Tables:
Table 2.6.1.1: Soil texture data from each sample location.......................................................50
Table 2.7.1.1: Age cohorts for Douglas-fir on each study site.................................................. 51
Table 2.7.1.2: Selected vegetation survey results, trees ( '+ ’ indicates presence).................. 52
Table 2.7.1.3: Selected vegetation survey results, shrubs (‘+’ indicates presence)............... 53
Table 2.7.1.4: Selected vegetation survey results, herbaceous plants ( '+ ’ indicates
presence).............................................................................................................................................54
Table 3.4.3.1: Seedling level comparison o f frequency and % abundance o f ECM root tips
with standard error o f the mean (unshared letters within rows denote significance).
ANOVA used for comparison o f mean values with a post-hoc Tukey HSD test across sites.
ECM morphotypes are arranged in decreasing frequency rank. The P value represents the
significance level (a=0.05, n=20)..................................................................... :........................... 90
Table 3.4.4.1: Comparison o f four diversity indices by using pooled ECM root totals from
each tree (Gini-Simpson, Shannon, Shannon Evenness, and Margalef) for each site (n=20).
100
Table 3.4.4.2: Comparison o f four diversity indices using individual tree data for each site
(Gini-Simpson, Shannon, Shannon Evenness, and Margalef). Means were compared by
ANOVA with a post-hoc Tukey HSD test. Unshared letters denote significance (n=20). 101
Table 3.4.6.1: Comparison o f DNA identity and morphotyping identification. DNA
sequences were compared with sequences from BLAST.........................................................106
Table 3.5.4.1: Kuzkwa (calcareous) soil horizon chemical data showing Iron, Aluminum,
and Silicon concentrations by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*...............................................

124

Table 3.5.4.2: Pinchi Hill (calcareous) soil horizon chemical data showing Iron,
Aluminum, and Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*.................................................................................................. 127

Table 3.5.4.3: Spencer's Ridge (glacial) soil horizon chemical data showing Iron,
Aluminum, and Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*.................................................................................................. 130
Table 3.5.4.4: Tezzeron (glacial) soil horizon chemical data showing Iron, Aluminum, and
Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid ammonium oxalate
extractions*......................................................................................................................................133
Table 3.5.4.5: Murray Ridge-East (ultramafic) soil horizon chemical data showing Iron,
Aluminum, and Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*..................................................................................................136
Table 3.5.4.6: Pinchi Mountain (ultramafic) soil horizon major element oxides and
chemical data showing Iron, Aluminum, and Silicon by sodium citrate-dithionite, sodium
pyrophosphate, and acid ammonium oxalate extractions.*.................................................... 139
Table 3.5.4.7: Pinchi Mountain (ultramafic) soil horizon minor elem ents...........................140
Table 3.5.4.8: Chemical and elemental data from soil hom ogenates....................................142
Table 3.5.4.9: Chemical and elemental data from soil homogenates (continued)*............ 143
Table 3.5.4.10: Detailed descriptions and possible identification o f all m orphotypes

144

List of Figures:
Figure 2.3.1.1: Study area location with respect to the boundaries o f the Sub-boreal Spruce
biogeoclimatic zone and the range o f Douglas-fir dominated stands......................................36
Figure 2.3.3.1: Locations o f Douglas-fir stands with respect to elevation within the study
area...................................................................................................................................................... 38
Figure 2.4.1.1: Soil sampling locations within the Fort St. James area.................................. 40
Figure 2.6.1.1: Soil nickel and chromium concentrations for each site.................................. 44
Figure 2.6.1.2: Mean soil nickel and chromium levels measured via four acid digestion and
ICP-AES............................................................................................................................................ 45
Figure 2.6.1.3: Soil cation exchange capacity by B aC h extraction for each site.................. 46
Figure 2.6.1.4: Mean cation exchange capacity by BaCU extraction o f each soil type with
standard error o f the mean.............................................................................................................. 46
Figure 2.6.1.5: Soil exchangeable calcium: magnesium ratio for each site............................47
Figure 2.6.1.6: Mean exchangeable calcium: magnesium ratio with standard error o f the
mean....................................................................................................................................................48
Figure 2.6.1.7: Soil pH (H 2 O) for each site..................................................................................49
Figure 2.6.1.8: Mean soil pH (H20 ) with standard error o f the mean..................................... 49
Figure 2.7.1.1: Stand density for all study sites. Groups that do not share letter designations
are significant.................................................................................................................................... 55
Figure 2.7.1.2: Mean basal area o f all trees per site (Douglas-fir, hybrid white spruce and
subalpine fir-including standing dead) showing standard deviation........................................ 56
Figure 2.7.2.1: Photo o f a standing fire-killed tree (Pinchi Hill) with younger regenerated
trees. Above are the two cores from the trees standing to the left and right which
established post-fire..........................................................................................................................57

Figure 3.4.1.1: Mean basal diameter o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p < 0.001, F= 58.05, n -5 9 4 )..........................................................................................................80
Figure 3.4.1.2: Mean seedling height o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p < 0.001, F=60.92, n=594)...........................................................................................................81
Figure 3.4.1.3: Mean total biomass o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p <0.001, F= 118.33, n=594).........................................................................................................82
Figure 3.4.1.4: Mean root:shoot ratio o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p <0.001, F= 14.22, n=594)...........................................................................................................83
Figure 3.4.1.5: Mean root and shoot weight as part o f total biomass o f greenhouse grown
Douglas-fir seedlings (n=594)........................................................................................................83
Figure 3.4.2.1: Mean foliar active iron levels from greenhouse grown Douglas-fir seedlings
with standard deviation (n=2).........................................................................................................84
Figure 3.4.2.2: Mean foliar concentrations o f nickel from greenhouse grown Douglas-fir
seedlings with standard deviation (n=2)....................................................................................... 85
Figure 3.4.2.3:Mean foliar concentrations o f chromium from greenhouse grown Douglasfir seedling with standard deviation (n=2)....................................................................................85
Figure 3.4.3.1: Dissecting microscope photos o f common and rare morphotypes. A:
Rhizopogon cf. villosulus, B: E-strain, C: Cenococcum geophilum, D: HebelomaAmphinema type, E: Russula/Lactarius-2, and F: Tuber sp...................................................... 87
Figure 3.4.3.2: Compound microscope images o f ECM hyphal and mantle structures. Top
left, A: Hebeloma-Amphinema like emanating hyphae, note the clamp connection, bottom
left C: Hebeloma-Amphinema sporocarp type. Top right, B: E-strain inner mantle showing

labyrinthic pattern, bottom right, Hebeloma-Amphinema sporocarp found fruiting in soil
from Murray Ridge-East, D: Rhizopogon cf. villosulus emanating hyphae, note the elbowtype bend on the pigmented hypha (arrow)..................................................................................88
Figure 3.4.3.3: Number o f ECM root tips o f the two major morphotypes, (n=22 039, both
types together represent 90.5% o f all root tips sampled)........................................................... 92
Figure 3.4.3.4: Number o f ECM root tips o f the 13 minor morphotypes (n=22 039, these
types together represent 9.5% o f all root tips sampled)............................................................. 93
Figure 3.4.4.1: Combined log-transformed rank-abundance curve. E-strain begins the curve
with the highest rank. Table 3.4.3.1 lists the morphotypes in their pooled rank order,
corresponding to the y-axis o f this figure.....................................................................................94
Figure 3.4.4.2: Site-level comparison rank-abundance curves o f ECM morphotypes o f each
site. Log-transformed abundance is plotted against the morphotype rank o f each site. A
point intersecting with the x-axis indicates absence o f the morphotype with the given rank.
............................................................................................................................................................ 95
Figure 3.4.4.3: Rank percent abundance o f morphotypes per site. Morphotypes are ranked
from lowest to highest abundance (proportion o f the community made up by each
morphotype per site). Non-mycorrhizal tips are also represented (n= 22 039)......................97
Figure 3.4.4.4: Number o f ECM morphotypes per site as a measure o f species richness...98
Figure 3.4.5.1: Two-way cluster analysis using a relative Sorensen index showing
presence-absence o f fungal species for comparison and using furthest neighbour clustering.
Sites are represented by two-letter codes, while morphotypes are represented by single­
letter codes. The designation “Un” represents uncolonized root tips (n= 22 039).............. 103
Figure 3.4.5.2: Two-way cluster analysis using a relative Sorensen index showing
relativized values for the number o f ECM colonized root tips for comparison and using
furthest neighbour clustering. Sites are represented by two-letter codes, while morphotypes
are represented by single-letter codes. The designation “Un” represents uncolonized root
tips (n=22 039)................................................................................................................................ 104
x

Figure 3.5.4.1: A: Seedling from Murray Ridge with lateral growth and a set apical bud. B:
Seedling from Pinchi Mountain with a swollen and bent stem. C: Small, unidentified
ascomycete, unconnected with any known morphotypes, found on two sites (Spencer's
Ridge and Pinchi Hill). D: Mature spider mite, with shed exoskeletons, webs, eggs and egg
cases.................................................................................................................................................. 113
Figure 3.5.4.1: Soil profile from Kuzkwa (calcareous) showing LF, Bmk, and Cca
horizons............................................................................................................................................ 125
Figure 3.5.4.2: Soil profile from Pinchi Hill (calcareous) showing LF, Ahk, Bmk and Ck
horizons............................................................................................................................................ 128
Figure 3.5.4.3: Soil profile from Spencer's Ridge (glacial) showing Ln, Fm, Bm, BC, and
C horizons...........................................................

131

Figure 3.5.4.4: Soil profile from Tezzeron (glacial) showing S/Ln, Fm, Ae, Bm, and Bt
horizons............................................................................................................................................ 134
Figure 3.5.4.5: Soil profile for Murray Ridge-East (ultramafic) showing S/Ln, Fm, Ahej,
Bm, and R horizons....................................................................................................................... 137
Figure 3.5.4.6: Soil profile for Pinchi Mountain (ultramafic) showing LF, Ah, B m l, Bm2,
and C horizons................................................................................................................................141

A cknow ledgem ents:
First I would like to thank my supervisor Dr. Hugues Massicotte. His boundless
enthusiasm for the mycorrhizal world and all things fungi first piqued my interest in
exploring ectomycorrhizas. Dr. Paul Sanborn has been invaluable in educating me literally
from the ground (or parent material) up. Because o f him, I have a new appreciation for the
ground I walk on. I am especially grateful to Linda Tackaberry for her ability to answer my
myriad questions and her patience in teaching me morphotyping technique. I thank Dr.
Keith Egger for his help with the molecular components o f my research. His wit and
advice on classic dramatic works helped brighten my non-school hours too.
I am grateful to the many volunteers on this study, whose knowledge and tireless
hands helped make my project possible. Doug Thompson and John Orlowsky o f the
UNBC EFL helped me keep my seedlings alive and often had answers to my most
outlandish questions. Joanne Vinnedge helped greatly in the identification o f areas o f
mature Douglas-fir in the Fort St. James area. I would also like to thank the many student
volunteers, especially Courtney Berdan for the help in processing all 714 o f my seedlings.
Thanks to the many UNBC faculty and staff that provided advice and a listening ear for
both thesis and non-thesis queries.
I am thankful to my friends both in town and across the country. Sara and Elizabeth
not only helped me through grad school growing pains, but also helped me plan a wedding
while finishing research. Laura and Carlyn were always ready with a cup o f tea as I
rambled on about my work. I am grateful to many o f my fellow grad students at UNBC,
especially Dan Williams and Serena Black. Both o f you provided a sounding board and
injected much needed humour into many a situation. I am also very grateful to Nicole
Sukdeo for help with woes o f many kinds, including methodological, writing and
statistical. Your friendship and humour helped me get around quite a few road blocks.
I thank my brothers Liam and Josiah and all my family for putting up with my
rather sudden shift into the world o f fungus and dirt. Most o f all, I am forever grateful to
my husband Neil who created all o f the maps in this thesis. Without you, the forestry
portions o f this work would be o f far lesser quality. Your keen eye for typos and flying
leaps o f logic helped to temper this thesis down from a collection o f ideas into a narrative.
Love always. Thanks also to the elder Mr. Thompson for his mature perspective on
ecology and writing style.
This research would not have been possible without financial support through an
NSERC Discovery Grant provided to Dr. Hugues Massicotte and a UNBC graduate
entrance and travel scholarship. Thanks to Spencer Reitenback for assistance with seed
selection. I am grateful to Dexter Hodder and the staff at the Cinnabar Resort in the John
Prince Research Forest for the use o f their facilities. The Canadian Botanical Association
and Canadian Society o f Soil Science also provided conference travel funds. UNBC
provided laboratory facilities. Additional analyses were conducted by the BC Ministry o f
Environment Analytical Chemistry Laboratory and ALS Geochemistry-Vancouver.

1. Introduction and literature review
Within the rhizosphere, the area o f soil directly influenced by plant roots, plants spread
their roots to acquire water and nutrients. Forest ecosystems depend on the connection
between above-ground inputs o f sunlight, precipitation, litter fall from trees and the belowground nutrient inputs from the soil. Mycorrhizal fungi form a mutualistic relationship by
colonizing root systems, spreading networks o f fungal hyphae throughout the soil (Smith
& Read, 2008). Ectomycorrhizal fungi, a sub-group o f mycorrhizal fungi, colonize a
majority o f forest tree species, including all conifers and most hardwoods found in British
Columbia.
Soils are not uniform providers o f nutrients and water to plants. Soil development
is impacted by bedrock lithology, glacial activity, age and climate. Weathering o f parent
material releases trace minerals bonded to insoluble complexes that are important to plants.
Weathering o f some parent materials also releases potentially toxic elements into bioavailable forms within the soil.
Within the soil, a broad consortium o f bacteria, unicellular eukaryotes,
invertebrates, and fungal organisms are housed, protected and supported. These organisms
respond and adapt to changes in the substrate. This collection o f interconnected organisms
is largely responsible for the nutrient and water cycling that supports the above-ground
ecosystem. The metabolic processes responsible for survival o f below-ground organisms
also bind and release materials vital to the proper functioning o f above-ground organisms.
The intersection between the immediate availability o f nutrients in any given soil and the
products o f soil organisms also shapes the above-ground plant community.

1

Mycorrhizal fungal communities, often described in terms o f functional guilds (see
below), have been linked to increased health and vigor o f plants, increased uptake o f water
and nutrients, drought resistance and translocation o f soil nutrients into forms that are
usually inaccessible to host plants (Parke e t a l , 1983; Li eta l., 1991; Gonsalves eta l.,
2007, 2009). A functional guild represents a grouping o f mycorrhizal fungi that share
similarities in purpose for a specific host in a particular area (Molina et a l, 1992;
Massicotte et a l, 1999). Stated simply, they act as if seeking a common goal (Perry et a l,
1989).
Fungi are the plant’s first direct contact with the soil surface and therefore are in
closest proximity to damaging compounds within the soil. In extreme soils, these toxic
conditions may derive from alkaline pH and heavy metals. Soil pH and nutrient uptake are
often intertwined as nutrients become bio-available as pH goes up or down. Studies have
documented a level o f resistance to cellular toxicity caused by certain types o f metals
including zinc, nickel, copper, and aluminum, when host root systems are colonized by
mycorrhizal fungi (Jones & Hutchinson, 1986, 1988; Wilkins, 1991; Jentschke & Godbold,
2000). Some studies have also shown mycorrhizal fungi communities have sensitivities to
pH (Fujimura & Egger, 2012; Walker et a l, 2014). By understanding some o f the
complexities o f the influences o f soil chemical characteristics on mycorrhizal fungi we can
increase our knowledge o f vast abiotic and biotic interactions that shape the soil system
and ecosystem.

1.1.

Mycorrhizal fungi
Mycorrhizal fungi form mutualistic relationships with plants through associations

with the fine roots. These fungi share a phylogenetic lineage with saprotrophic fungi

(decomposers that use dead plant and animal matter within the soil as a carbon source)
(Hibbett et al., 2000). Associations between roots and fungal hyphae were first described
by German scientist Albert Bernhard Frank in the late 1800s (Frank, 1885). Fungal hyphae
were discovered to penetrate, infect and envelop the roots o f many plants in both long term
and ephemeral unions. At the time o f discovery, however, it was unclear whether or not the
fungi were decomposing the plant.
Researchers in the early 20th century used improved microscopy to further
characterise three proposed mycorrhizal categories based on morphology: endotrophic,
ectotrophic, and ectendotrophic (Tourney & Korstian, 1947). Mycorrhizas are now broadly
divided into seven designations based on morphology and host species: arbuscular, ericoid,
arbutoid, orchid, monotropoid, ectomycorrhizas and ectendomycorrhizas (Peterson et al.,
2004; Peterson & Massicotte, 2004). O f this group, ectomycorrhizas (ECM) are the most
important to conifer tree species (Trappe, 1977), but are the least prevalent globally with
only 3% o f seed-bearing plants hosting ectomycorrhizal fungi (Moore et al., 2011).

1.2.

Arbuscular and orchid mycorrhizas: individual guilds
Arbuscular mycorrhizas are characterized by a diagnostic feature called an

arbuscule, made up o f a combination o f the root cell plasma membrane and tree-like
projections o f hyphae into the root cell. The arbuscule is the center for metabolite
exchange between the symbionts. These fungi, comprising the phylum Glomeromycota,
are common on crop species worldwide (Marschner & Dell, 1994; Kivlin et al., 2011).
While not phylogenetically diverse, these fungi are essential to the success o f many crop
species as they supply needed minerals to the host plants.
Orchid mycorrhizal fungi first partner with germinating orchid seeds, which have

no fat or nutrient reserves o f their own (Smith & Read, 2008). Fungal hyphae aid in
success after germination by penetrating the orchid embryo, forming intracellular coils,
and processing complex carbohydrates from the surrounding soil into simple sugars for the
developing plant. Later, the orchid fungi will invade the roots and perform the same
function (Rasmussen, 1995; Peterson & Massicotte, 2004). Both o f these types represent
individual functional guilds as they are usually restricted to their particular host (meaning
orchid mycorrhizal fungi would not be found with arbuscular mycorrhiza on the same
host).

1.3.

Ericoid, arbutoid, ectendo and monotropoid mycorrhizas: shared guilds
Ericoid mycorrhizas are fungal-root associations between ascomycetes, such as

Rhizoscyphus ericae (formerly Hymenoscyphus ericae) and plants within the order
Ericales, including various genera such as Vaccinium and Gaultheria (McKechnie, 2009).
Ericoid mycorrhizal fungi colonise the hair roots o f the Ericales hosts, forming a loose
mantle while also penetrating the epidermal root cells. Some mycorrhizal fungi are not
exclusively ectomycorrhizal in form, but may also form arbutoid mycorrhizas when grown
near plants in the Ericaceae family such as Pyrola, Arbutus, and Arctostaphylos
(Massicotte et al., 1993; Hagerman & Durall, 2004).
Ectendomycorrhizas are formed by fungi within the order Pezizales. Ascomycetes
in the genus Wilcoxina, commonly grouped under the name E-strain, are the predominant
ectendomycorrhizal fungi. These fungi can colonize a variety o f hosts to somewhat
different effect. For instance, Wilcoxina will form ectomycorrhizas with Pseudotsuga
menziesii (Massicotte et al., 1999) but will form ectendomycorrhizas with members o f the
Larix or Pinus genera (Yu et al., 2001).

Monotropa is a genus o f mycoheterotrophic achlorophyllous plants usually found
in conifer forests which also has a suite o f monotropoid mycorrhizal fungal associates.
Both arbutoid and monotropoid mycorrhizal fungi often belong to the same functional
guild as ectomycorrhizal fungi. All o f these fungi can potentially form networks between
their respective plants and make the trees and understory plants partners (Molina et al.,
1992). In these cases, mycorrhizal fungi act as a conduit, passing carbohydrates from
neighbouring trees to the plant (Simard et a l, 1997).

1.4.

Ectomycorrhizas

1.4.1. Structure and function
Ectomycorrhizas are a union between the external root surface o f plants and fungal
hyphae (Teste et al., 2009; Simard, 2009; O ’Brien et al., 2011). Colonization o f the fine
roots o f trees begins with the germination o f fungal spores within the soil or from direct
contact with fungal hyphae. Ectomycorrhizal fungal propagules are known to germinate in
response to the presence o f roots, and it is postulated that germination may be stimulated
by the secretion o f host root exudates (Molina et al., 1992). Fungal mycelia grow towards
and weave together to encase the root tip in a mantle. Hyphae from the mantle push
between the root epidermal and cortical cells to form the nutrient exchange structure
known as the Hartig net (Peterson et al., 2004).
Tuberculate species o f fungi have a different type o f mycorrhizal fungal
colonization shape: groups o f fungal colonized tips with mantles will have a peridium, or
rind, surrounding the root cluster. While research has not provided a clear function for
these larger ectomycorrhizal structures, some experiments on Douglas-fir have shown that

5

they still obtain soil nutrients from rhizomorphs, long, thick, parallel strands o f hyphae,
extending into the soil (Zak, 1971).
Extraradical mycelia are long emanating hyphae that extend from the mantle into
the surrounding soils. Mycelia increase the amount o f soil exploration possible for a tree,
allowing for greater uptake o f minerals and water from the soil (Trappe, 1977; Teste et a l,
2009). Rhizomorphs are used in the transport o f absorbed nutrients. Ectomycorrhizal fungi
have been shown to greatly increase the effective surface area o f colonized root systems,
allowing some levels o f drought resistance (Parke et a l, 1983).
Ectomycorrhizas differ from other types o f mycorrhizas in the lack o f live plant cell
penetration by the fungal hyphae. Dying root cells can be punctured by fungal hyphae, but
fungal hyphae remain on the surface o f the live plant root, outside o f the living cells (Yu et
a l, 2001). Once thought to be pathogenic (Tourney & Korstian, 1947), these fungi live in
symbiosis with plants in exchange for a carbon source from the plant (Jones & Hutchinson,
1986; Teste e t a l , 2009). A variety o f types o f angiosperms and gymnosperms are potential
hosts to ECM, but colonization is most common on perennial tree and shrub species
(Moore et al., 2011). Ectomycorrhizal fungi have been the topic o f much study because o f
the high timber value o f their hosts (Hunt, 1992).
Basidiomycetes form the majority o f ectomycorrhizal partnerships with many
forest plants, though some ascomycetes and rare Zygomycetes are also ectomycorrhizal. It
was estimated that as many as 6000 species o f fungi worldwide can form ectomycorrhizal
associations (Molina et a l , 1992). Recent DNA and molecular evidence has raised this
estimate to 20 000 potential species (Brundrett, 2009).
Host species influences the shape and structure o f the Hartig net. Gymnosperms

6

usually have Hartig nets that penetrate between the root epidermal and cortical cells. In
contrast, angiosperms form epidermal Hartig nets which do not extend into the cortical
cells (Moore et al., 2011). Hartig net formation depends on the stage o f growth o f the root.
Often the Hartig net is less developed at the growing apical end o f the root tip (Massicotte
et al., 1989). Mantle thickness is also influenced by the growth o f the root, with uneven
growth o f mantles on older lateral roots compared to younger laterals (Massicotte et al.
1986).

1.4.2. Ecosystem impact and importance
Mycorrhizal fungi differ in the types o f preferred hosts. Some are restricted to only
one host (narrow host range), some can form partnerships with one family o f plants
(intermediate host range), and others can form partnerships with almost no restrictions
(broad host range). ECM fungi are typically grouped between the intermediate and narrow
host ranges, with some exceptions (Molina et al., 1992). Structure o f colonized plant roots
will vary depending on the species o f plant and the species o f fungi. The host plant can
also determine the number o f ECM species that can form mutualistic associations.
Most host trees will often have multiple species colonizing their root systems at one
time (Massicotte eta l., 1999; Robertson, 2003; Branco & Ree, 2010). Single roots can also
be co-infected by different species trying to establish a symbiosis with the host (Bruns,
1995). In the case o f Douglas-fir, only a small number o f the fungal partners are restricted
to the species (e.g. Rhizopogon cf. villosulus and Suillus caerulescens) while the others are
capable o f forming mycorrhizas with other tree and shrub species (e.g. Cenococcum
geophilum Fr., Russula spp., and Tuber spp.) thus expanding the potential functional guild
(Molina et a l, 1992). Access to broad host range fungi also provides a more secure source

o f inoculum as other plants can retain the fungi in the event o f a disturbance that damages
the networks connected to the original hosts (e.g. regeneration in a clear cut with all
Douglas-fir removed).

1.4.3. Development o f modem methods o f ECM identification
Ectomycorrhizal species have typically been described by morphotyping root tips.
This process developed due to the lack o f known fruiting bodies o f a majority of
ectomycorrhizal species (Smith & Read, 2008). Morphotyping involves visual analysis,
complemented by the use o f light microscopy, to identify the particular species colonizing
a root system. Colour and rhizomorph presence are used as diagnostic features, but fungal
mantle structure is often the best recognition factor (Ingleby et al., 1990). A gerer (1987)
and Ingleby et al. (1990) pioneered the in-depth identification o f fungi through
morphological means. Improved methods o f compound and electron microscopy aided
greatly in the characterization o f mycorrhizas (Massicotte et al., 1986, 1992). Agerer later
compiled a 5 volume work consolidating all photographic and descriptive data on
mycorrhizas from around the world (Agerer, 1987-2008).
Prior to advances in DNA technology, ectomycorrhizal fungi would often be
cultured on agar-based medium in the lab in order to isolate specific genets for population
studies (Dahlberg & Stenlid, 1994). Once pure cultures o f ECM fungal mycelium were
obtained, they could be compared based on somatic compatibility (Baar et al., 1994). In
other words, mycelium that could interact positively were deemed to be related or o f the
same species. This process was very time consuming and incomplete as many groups o f
ectomycorrhizal fungi do not culture in a laboratory setting and some different species do
not display incompatibility with each other (Smith & Read, 2008).

DNA methods have improved the identification o f many fungi. Extensive work has
been done with Terminal Restriction Fragment Length Polymorphisms (T-RFLP) in the
1990s and early 2000s (Erland eta l., 1994, 1999; Allen et al., 2003; Moser et al., 2005).
This process uses bacterial enzymes to cut polymerase chain reaction (PCR) amplified
sequences o f interest, usually the ITS DNA band (Schoch et a l, 2012), and measure the
resulting fragments on the basis o f size using gel electrophoresis. DNA fragments o f the
same size will assort together and are therefore indistinguishable from each other during
gel visualization. Different ECM species can produce DNA sequences o f the same length
which limits the resolution o f this technique (Gonsalves et al., 2009).
Amplification o f portions o f the ribosomal DNA fragment ITS through PCR and
the advent o f Sanger sequencing systems allowed for greater differentiation between
fungal species (Bruns et al., 1998; Horton et a l, 1999; Branco & Ree, 2010). Instead o f
basing separate identifications on fragment size, the order o f base pairs could be recorded
using dye-tagged nucleotides and compared to fungal sequences housed at GenBank ® or
the purely fungal database UNITE using BLAST (Basic Local Alignment Search Tool)
(Koljalg et al., 2005). This approach depends on the accurate vouchering o f fungal
specimens and maintenance o f sequence databases.
Recently, next-generation sequencing (NGS) has become a point o f great interest in
the study o f ECM fungal DNA. The speed o f the process is advantageous over previous
sequencing methods. Massively parallel (‘454’) pyrosequencing, one o f the three main
platforms o f NGS, can generate a large number o f sequences up to 500 base pairs in length
(Henrik Nilsson eta l., 2011). Instead o f using dye-terminators and linear processing, NGS
runs sequencing reactions in parallel, greatly reducing the time required. NGS uses and

9

generates a very large volume o f data which must be handled properly. However, it is
important to note that NGS will still rely on proper curation o f database information and is
vulnerable to database errors, lack o f information about recorded sequences, or simply
information overload (Taylor & Harris, 2012). Given the possibilities, this method will
most likely continue to be the focus o f much research in the next decade.

1.5.

Ecological influence of soil parent material

1.5.1. Pedogenesis in central British Columbia
Pedogenesis refers to the natural processes that lead to the formation o f a soil
(Schaetzl & Anderson, 2005). These processes are influenced by climate and time from last
disturbance. Pedogenesis is directly linked to the weathering o f the soil parent material,
when biotic and abiotic factors break down the original physical and chemical structure of
the parent material. Moist, warmer sites increase the rate o f production o f secondary
minerals from parent material while cooler, drier sites retard weathering (Brady & Weil,
2008). Organic matter is also cycled faster or slower from deposition to decomposition
dependent on climate (Schaetzl & Anderson, 2005). The blending o f the organic and
inorganic inputs creates the unique signature o f the soil (Brady & Weil, 2008).
Most Canadian soils are comparatively young, sharing a recent history o f glacial
disturbance. Central British Columbia was under a thick layer o f ice during the Late
Wisconsinan glaciation approximately 20 000 years ago. Ice flowing eastward from the
Coast Mountains generated large volumes o f glacial till, glaciofluvial and glaciolacustrine
deposits (Plouffe, 2001). Retreat o f the glaciers led to ice-free conditions in central British
Columbia by 10 000 years ago (Clague, 1981). Colder continental climate and moderately

10

dry conditions coupled with recent glaciation led to the development o f high proportions o f
Luvisolic and Brunisolic soils in central British Columbia (Clayton et al., 1977; Meidinger
eta l., 1991).
Soils vary over extremely small spatial scales (Cheng et al., 2011). A pedon is the
smallest unit that can be designated a soil. Pedons are usually approximately a square
metre, though this area may increase up to several meters to obtain proper soil definition,
and 1 or more meters deep (Brady & Weil, 2008). This variation extends into layers within
the vertical structure o f soils referred to as horizons (Soil Classification Working Group,
1998).
Horizons are visually distinct bands o f different chemical and physical properties.
Organic horizons, formed from litter deposition in forests are usually referred to as L, F, or
H layers dependent on the degree o f decomposition. Mineral soil horizons are classified as
A, the uppermost horizon which is affected by leaching and organic matter deposition, B,
the second horizon, enriched with clays and other weathering products, and C, the lowest
horizon, usually unaffected by most pedogenic processes (Clayton et a l, 1977).
Secondary, lowercase letters are used to designate specifics o f each horizon. Luvisols will
often have a Bt layer (with high amounts o f illuvial clay), while Brunisols may have a Bm
(very little pedogenesis has occurred) (Soil Classification Working Group, 1998). These
minute variations within pedons necessitate on-site sampling through soil pits to properly
classify the conditions found in a particular area.

1.5.2. Nutrient mobility within soils
Weathering o f parent material makes many minerals and nutrients more
bioavailable. However, this mobility within the soil profile can also release more toxic

elements (Kierczak et a l, 2007; Cheng et a l, 2011). Conversely, atmospheric inputs of
material, or newly released minerals, can be bonded to other compounds within the soil,
which renders them insoluble or not readily bio-available (Schaetzl & Anderson, 2005;
Brady e t a l , 2005).
Nitrogen, phosphorus and potassium are all essential to the survival o f plants.
Phosphorus, for example, is often difficult to access on soils with calcareous parent
material (Kishchuk, 2000). High pH within the soil causes the formation o f orthophosphate
ions which in turn forms complexes with carbonates to create insoluble minerals (Thorne
& Seatz, 1955). Both serpentine and ultramafic derived soils may be high in heavy metals
which are phytotoxic. Soils derived from serpentinized bedrock may have readily available
nickel due to the ease o f weathering serpentine and olivine. Nickel is a metal that is
beneficial in trace amounts, but toxic to plants at high levels (Rencz & Shifts, 1980;
Kierczak et a l, 2007). Drainage, or lack thereof, also impacts translocation o f nutrients
and minerals throughout the soil horizons (Schaetzl & Anderson, 2005).
Biotic factors also play a role in the movement o f nutrients. Bulk soil, or nonrhizosphere soil, is usually under the influence o f water drainage patterns and parent
material. The rhizosphere features complex interactions between plant roots, mycorrhizal
fungi, soil bacteria and the soil itself. Saprotrophic fungi decompose much o f the organic
material deposited on top o f the upper forest floor horizons (Rayner & Boddy, 1988).
Mycorrhizal fungi can transport these newly accessible nutrients to their host tree
while also increasing nutrient accessibility themselves. Extracellular enzymes released by
the fungi into the soil will mobilize complex molecules containing nitrogen and
phosphorus within the soil organic matter (Courty et a l, 2010). It is also hypothesized that

12

some species o f ECM can prevent uptake o f toxic metals and minerals, though specific
mechanisms and evidence are not clear (Wilkins, 1991; Azcon & Barea, 1992). It is
theorized that the fungi bind the metals into the mantle tissue, stopping the host plant from
absorbing the metal compounds (Moore et al., 2011).

1.6.

Douglas-fir in Central British Columbia

1.6.1. Habitat and range
Douglas-fir (Pseudotsuga menziesii) is a long-lived conifer found in western North
America from Mexico to central British Columbia (Silen, 1978). It can live for upwards of
500 years (Hermann & Lavender, 1990), and the Interior Douglas-fir subspecies
(Pseudotsuga menziesii var. glauca (Beissn.) Franco) has been documented at 625 years o f
age in the Chilcotin region o f British Columbia (N. Thompson, pers. comm. 2015).
Mature Douglas-fir is easily recognizable by its thickly grooved reddish bark,
spreading crown, scaled cones and single spaced needles. Thick cork-like bark makes the
mature tree resistant to ground fires, allowing for greater survival compared to other tree
and shrub species within the same habitat (Hermann & Lavender, 1990). Seeds disperse
from the cones through wind and may be transported great distances. Trees can form
almost pure stands through natural regeneration, or can be a component o f mixed stands
usually dependant on soil and climate. Douglas-fir prefers well-drained soils and is not
successful in compacted or wet areas (Hermann & Lavender, 1990).
Douglas-fir can grow on slopes and valley sides that represent too dry an
environment for competing species like hybrid white spruce (Picea glauca var.
engelmannii (Parry) Boivin) and subalpine fir (Abies lasiocarpa (Hook) Nutt.). (Hermann

& Lavender, 1990; Delong, 1999). It is often found with understory plants such as
Arctostaphylos uva-ursi (L.) Spreng. which may host the some of the same species o f
ectomycorrhizal fungi as Douglas-fir (Hagerman et a l, 2001; Hagerman & Durall, 2004).
Douglas-fir seedlings are sensitive to frost damage and are said to have a preference for
sunny, southwest-facing slopes in cooler areas (Delong, 1999; Griesbauer & Green, 2010).
It is unclear whether soil, competition, or climate impacts the establishment o f Douglas-fir
more.

1.6.2. ECM associates
A wide variety o f fungi that span coastal, montane and interior plateau ecosystems
are available to Douglas-fir as fungal associates. Douglas-fir’s long lifespan has led to the
hypothesis that the trees act as mycorrhizal refugia, providing inoculum for trees and
shrubs after disturbance (Wiensczyk & Gamiet, 2002; Simard, 2009). Approximately 205
to 2000 different species o f fungi have been estimated to form mycorrhizal partnerships
with Douglas-fir (Trappe, 1977; M olina eta l., 1992).
Some o f the most common genera o f epigeous, or above-ground fruiting bodies,
genera include: Cortinarius spp., Suillus spp., and Russula spp (Arora, 1986; Smith et al.,
2002). Below-ground, or hypogeous, fruiting mycorrhizal fungal partners include
Rhizopogon spp and Tuber spp (Zak, 1971; Hunt, 1992; Massicotte et al., 1994). Other
species that do not display known fruiting bodies o f any kind, such as Cenococcum spp.,
are also colonizers o f Douglas-fir roots (Jones et al., 2010).

1.7.

Current research into ECM on extreme parent material
Some species o f ECM are hypothesized to provide a measure o f resistance to
14

impacts o f soil metals (Wilkinson & Dickinson, 1995). An understanding o f the belowground fungal network o f ECM communities grown on extreme soils can help shed light
on forest success in regions influenced by strong chemical signatures o f parent material.
This in turn aids in the preservation and management o f these areas in both forest
production and habitat conservation. For instance, if certain species o f ectomycorrhizal
fungi improve growth on areas with harsh parent material, planting seedlings that are pre­
inoculated with those species could improve seeding performance. Continued research into
ECM fungi that survive and succeed on extreme substrate helps to illuminate a small facet
o f the world o f below-ground soil interactions.

1.7.1. Mycorrhizal fungi o f serpentine and ultramafic-derived soils
Serpentine soil, a broad term that is often used to describe soil with both ultramafic
and serpentinized parent materials, has been a research topic for many decades as it relates
to ectomycorrhizal fungi. John Maas and Daniel Stuntz (1969) did pioneering research to
characterize the presence o f epigeous fungi growing on serpentine soils o f the Washington
Cascade Mountains. While few sporocarps were found on serpentine, a greater proportion
o f the fungi found on serpentine were ectomycorrhizal (Russula sp., Amanita sp. and
Suillus sp.), compared with the non-serpentine sites, which may indicate that both plants
and ectomycorrhizal fungi need each other to survive on serpentine soils. Since that time,
much research has been done to describe and quantify the species richness (the number of
species found) and diversity (the variety o f species) o f ECM fungi on serpentine areas
around the world.
A study by Moser et al. (2005) on Quercus garryana Dougl. (Fagaceae) (Garry
oak) in Oregon determined, by field sampling ECM root tips, that fungal communities did

not significantly differ between serpentine and non-serpentine sites. However, this research
did find that 43% o f the 74 morphotypes in the study were unique to serpentine soils.
Many o f these unique morphotypes were rare and present as a single root tip, which were
therefore not included in statistical analysis by the researchers. This study used
morphological techniques coupled with the analysis o f restriction fragment length
polymorphisms (RFLP) (Moser et al., 2005). Gladish et al. (2010) studied conifer (Pinus
ponderosa Douglas ex C.Lawson and Pinus Jeffreyi Balf.) ECM communities o f Oregon
serpentine soils by field sampling six sites (3 paired serpentine and non-serpentine). In this
study, both ectomycorrhizas and hypogeous sporocarps associated with conifers were
compared using DNA sequencing, instead o f RFLP to identify fungi. Less hypogeous fungi
diversity was found on serpentine soils which the researchers concluded may be more
related to host populations than soil conditions. When comparing mycorrhizal fungi on
serpentine and non-serpentine areas, the researchers concluded that limitations in the host
species dispersal were driving fungal diversity, as opposed to differences in the underlying
soil (Gladish et al., 2010).
Gonsalves et al. (2007) focused on Cenococcum geophilum isolates from a
serpentine and non-serpentine site in Portugal using amplified fragment length
polymorphisms (AFLP). AFLP uses a process similar to RFLP, but instead the data are
usually treated as presence-absence. The serpentine isolates were not affected by nickel
addition to growth media compared to non-serpentine isolates which may indicate
resistance to nickel toxicity. However, all isolates were genetically distinct from one
another and both serpentine and non-serpentine isolates were equal in levels o f genetic
diversity (Gon 9 alves et a l , 2007). This is in contrast to a previous study conducted in

16

Maryland, USA which used similar techniques to show that serpentine Cenococcum
geophilum isolates were more similar to each other than to isolates from non-serpentine
soils (Panaccione et al., 2001).
Branco and Ree (2010) conducted several studies on serpentine ECM in oak forests
in Portugal. These oaks were located on serpentine soils and harboured ECM fungal
communities that had little overlap with non-serpentine communities, with only 15% of
sampled species shared between sites (Inocybe sp., Tricholoma sp, Cenococcum sp. and
others). Though there was low overlap between the fungi sampled, many species were
documented only once and therefore, both serpentine and non-serpentine sites were not
significantly different (Branco & Ree, 2010). Branco also conducted another study using
reciprocal transplantation o f seedlings into both serpentine and non-serpentine soils to
compare the ECM grown on Quercus ilex spp. ballota (holm oak). Serpentine soils
produced a higher fungal richness than non-serpentine soils, but were less rich and diverse
than the previous study. Fungi were also shared between both soils, which leads Branco to
conclude that fungi have a tolerance to the extremes within the soil (Branco, 2010).
Recently in Deer Isle, Maine, Davoodian etal. (2012) compared the arbuscular
mycorrhizal communities o f the host Hypericum perforatum L. (St. John’s wort) between
soils derived from serpentine and granite parent materials. In this case, number o f species
was not considered; instead, colonization was compared in proportion to the entire root
system. Percent colonization did not differ between serpentine and granite sites at any o f
the sampling times. Colonization did differ for both sites during stages o f flowering, with
the highest level found post-flower (Davoodian et al., 2012).
Research on serpentine soils to date is summarized in a review by Southworth et al.

17

(2014). They conclude that the majority o f studies conducted indicate that ECM fungal
communities o f serpentine soils do not differ from those on non-serpentine soils. Some
species, including Cenococcum geophilum, may be locally adapted to serpentine sites and
hypogeous fungi are less prevalent on serpentine soils (Southworth et al., 2014). However,
some studies o f ECM do indicate that the harshness o f serpentine soils may increase
biodiversity or richness instead o f hindering it (Branco & Ree, 2010; Branco, 2010).
However, this review and these studies also demonstrate the variability often found when
conducting mycorrhizal research and the confounding problem o f rare species.

1.7.2. Mycorrhizal fungi o f calcareous soils
Arbuscular mycorrhizal (AM) fungi o f calcareous soils have had much attention
from an agricultural perspective. Studies have shown that cereal and legume crops grown
in calcareous soil benefit from root inoculation with AM fungi (Kothari et al., 1991; Li et
al., 1991). Low levels o f accessible phosphorus in calcareous soils can be compensated for
by inoculation with AM fungi (Chen et al., 2003; Feng et al., 2003). Commercial varieties
of AM inoculum are now widely available and little attention is given to the biodiversity o f
the root systems o f these crop species.
Ectomycorrhizal research involving calcareous soils is much rarer. Much o f the
work focuses on the survival o f plant species based on the presence or absence o f
mycorrhizal associates. A majority o f these studies pre-date the now common molecular
DNA techniques used in parallel with morphotyping for identifications. Few o f the studies
in question describe the ECM community in great detail. An exception is a study
conducted on eight Salix spp. (S. reticulata L. (reticulate willow), S. herbacea L. (least
willow), S. myrsinites L. (myrtle willow), S. glauca L. (northern willow), S. phylicifolia
18

(L.) sm. (tea-leaved willow), S. lanata L. (woolly willow), S. hastata L. (halbert-leaved
willow), S. nigricans Sm. (dark leaved willow)) grown in Norwegian calcareous and noncalcareous soils described prolific Cenococcum geophilum and 6 species o f ECM including
the genus Tuber and Laccaria through morphotyping. This study was an exploration o f
mycorrhizal conditions o f Salix spp. at both boreal and alpine ecosystems (Dhillion, 1994).
Presence o f ECM fungi has been connected to the survival o f plants grown on
calcareous soils or analogues. A study by Le Tacon (1978) on Pinus nigra J.F. Arnold
(black pine) and Picea excelsa Link (Picea abies (L.) H. Karst-Norway spruce) compared
uncolonized and seedlings already colonized by ECM fungi by planting them in sand
substrate laced with calcium carbonate (CaCOs). Picea excelsa was able to tolerate the
increased levels o f calcium without ECM, but Pinus nigra required colonization to survive.
While this substrate was not a calcareous soil, the author used the addition o f CaCCh to
mirror naturally occurring levels o f calcium within the soil (Le Tacon, 1978). Another
species o f pine, Pinus halepensis Miller (Aleppo pine), was also shown to require ECM
fungal partners to survive on calcareous soils. Seedlings planted in sterile calcareous soil
did not develop ectomycorrhizas and were stunted and stressed compared to seedlings
planted in unsterile soil which were successful in partnering with ECM fungi (Piou, 1979).
In both cases, survival was directly tied to the presence o f mycorrhizal fungi.
Laypeyrie and Chilvers (1985) conducted a study involving both ecto and
endomycorrhizal fungi comparing the success o f sterile and non-sterile calcareous and
acidic soils amended with potting mix and additional CaC 0 3 . Eucalyptus dumosa (White
Mallee) grown in sterile acidic soil displayed no signs o f stress, whereas the calcareous
grown trees displayed very slow and inhibited growth on the sterile substrate. However,

19

when the sterile substrate was inoculated with unsterile soil, no difference in growth was
observed between the acidic and calcareous soils, leading the researchers to conclude that
ECM fungi were necessary for reducing the stress caused by calcareous soils (Lapeyrie &
Chilvers, 1985). In another study, Cenococcum graniforme (=geophilum) sclerotia were
used to inoculate sterile calcareous soil and successfully prevent plant death and chlorosis
o f leaves in Helianthemum chamaecistus (-nummularium or Common rock-rose)
(Kianmehr, 1978). These two studies point to the potential for ECM fungi to protect the
host plant from the damages associated with calcareous soils. While specific mechanisms
are not documented in these studies, it is likely that limiting nutrients, such as phosphorus,
are made more accessible to the host plants through the ECM fungi (Lapeyrie, 1990).
While serpentine soils have been extensively studied worldwide as extreme,
edaphic habitats, no research has been done on ECM diversity in soils derived from the
Stuart Lake Belt ultramafic rocks. Calcareous rocks and deposits within the Fort St. James
region have been documented by Plouffe (2000) and information on calcareous soils and
ecosystem impacts throughout the south o f British Columbia and west Alberta has been
gathered by Kishchuk (2000) but likewise, no research on ECM populations has been
conducted. Little is known o f the impacts o f the physical, biological and geochemical
signature o f soils in the Fort St. area on ECM diversity within the rhizosphere.

20

1.8.

Objectives of study
As a prelude for my biology work, the first objective is to provide an initial

assessment o f soils found on the diverse lithologies in the Fort St. James area, including
ultramafic and calcareous bedrock. These soils are distinct from other ultramafic and
calcareous areas due to the relatively recent glaciation and to my knowledge, no other
study has made such a comparison. While Douglas-fir is o f high economic importance in
central British Columbia, little research has been done on the stand conditions o f the
ultramafic and calcareous grown stands in the Fort St. James area. This study will provide
a description o f the Douglas-fir forest attributes and associated vegetation grown on the
strongly contrasting soils derived from ultramafic, calcareous, and glacial parent material.
Both calcareous and serpentine soils have been observed to have severe health
impacts on plants on unglaciated areas worldwide (Lapeyrie, 1990; Gladish et a l, 2010),
but no research has been done on the plant health impacts o f ultramafic, calcareous and
glacial-derived soils in the Fort St. James area. My study will use a greenhouse bioassay to
preliminarily assess the health and growth o f Douglas-fir grown in these extreme soils.
Research on the ECM communities worldwide has shown that ECM fungi are usually
equally diverse on and off serpentine soil (Southworth et a l, 2014). However, no study has
explored the ECM fungal communities o f Douglas-fir on ultramafic-derived soils in central
British Columbia, or compared those communities to calcareous and glacial derived fungal
assemblages. My study will use morphological identification coupled with DNA
sequencing to categorize and quantify the unknown ECM fungal communities o f Douglasfir.

21

1.9.

References

A gerer R. (Ed.) 1987-2008. Colour Atlas o f Ectomycorrhizae. 1 s t - 14th ed. EinhomVerlag, Schwabisch Gmiind
Allen TR , M illar T, Berch SM , Berbee M L. 2003. Culturing and direct DNA extraction
find different fungi from the same ericoid mycorrhizal roots. New Phytologist 160: 255—
272.
A ro ra D. 1986. M ushrooms Demystified; A Comprehensive Guide to the Fleshy Fungi.
Berkeley, CA: Ten Speed Press.
Azcon R, B area JM . 1992. The effect o f vesicular-arbuscular mycorrhizae in decreasing
Ca acquisition by alfalfa plants in calcareous soils. Biology and Fertility o f Soils 1 3 :1 5 5 159.
B a a r J , O zinga W A, K u y p er TW . 1994. Spatial distribution o f Laccaria bicolor genets
reflected by sporocarps after removal o f litter and humus layers in a Pinus sylvestris forest.
Mycological Research 98: 726-728.
B rady KU, K ru ck eb erg AR, B radshaw H D J. 2005. Evolutionary ecology o f plant
adaptation to serpentine soils. Annual Review o f Ecology, Evolution, and Systematics 36:
243-266.
B rady NC, W eil RR. 2008. The Nature and Properties o f Soils. 14th Ed. Prentice Hall.
B ranco S. 2010. Serpentine soils promote ectomycorrhizal fungal diversity. M olecular
Ecology 19: 5566-5576.
B ranco S, Ree RH . 2010. Serpentine soils do not limit mycorrhizal fungal diversity. PLoS
ONE 5: el 1757.
B ru n d re tt M C. 2009. Mycorrhizal associations and other means o f nutrition o f vascular
plants: Understanding the global diversity o f host plants by resolving conflicting
information and developing reliable means o f diagnosis. Plant and Soil 320: 37-77.
B runs T. 1995. Thoughts on the processes that maintain local species diversity o f
ectomycorrhizal fungi. Plant and Soil 170: 63-73.
B runs TD, Szaro TM , G ardes M , Cullings KW , Pan J J , T aylor DL, H o rto n TR ,
K retze r A, G arbelotto M , Li Y. 1998. A sequence database for the identification o f
ectomycorrhizal basidiomycetes by phylogenetic analysis. Molecular Ecology 7: 257-272.

22

Chen BD, Li XL, Tao HQ, Christie P, Wong MH. 2003. The role o f arbuscular
mycorrhiza in zinc uptake by red clover growing in a calcareous soil spiked with various
quantities o f zinc. Chemosphere 50: 839-846.
Cheng CH, Jien SH, Iizuka Y, Tsai H, Chang YH, Hseu ZY. 2011. Pedogenic
chromium and nickel partitioning in serpentine soils along a toposequence. Soil Science
Society o f America Journal 75: 659-668.
Clague JJ. 1981. Late Quaternary geology and geochronology o f British Columbia, Part 2:
summary and discussion o f radiocarbon-dated Quaternary history. Geological Survey o f
Canada, Paper 80-35.
Clayton JS, Ehrlich WA, Cann DB, Day JH, Marshall IB. 1977. Soils o f Canada.
Agriculture Canada Publ. 1544. 2 vols.
Courty PE, Buee M, Diedhiou AG, Frey-Klett P, Le Tacon F, Rineau F, Turpault
MP, Uroz S, Garbaye J. 2010. The role o f ectomycorrhizal communities in forest
ecosystem processes: New perspectives and emerging concepts. Soil Biology and
Biochemistry 42: 679-698.
Dahlberg A, Stenlid JAN. 1994. Size, distribution and biomass o f genets in populations of
Suillus bovinus (L.: Fr.) Roussel revealed by somatic incompatibility. New Phytologist 128:
225-234.
Davoodian N, Bosworth J, Rajakaruna N. 2012. Mycorrhizal colonization o f Hypericum
perforatum L . (Hypericaceae) from serpentine and granite outcrops on the Deer isles,
Maine. Northeastern Naturalist 19: 517-526.
Delong C. 1999. Ecology o f Douglas-fir at its Northern Limits. In: Lousier JD, Kessler
WB, eds. Ecology and management o f interior Douglas-fir {Pseudotsuga menziesii var.
glauca) at the northern extreme o f its range. Proceedings o f a workshop held October 7-9,
1996 in Fort St. James, British Columbia. Faculty o f Natural Resources and Environmental
Studies, University o f Northern British Columbia, Prince George, B.C., 27-28.
Dhillion SS. 1994. Ectomycorrhizae, arbuscular mycorrhizae and Rhizoctonia sp. o f alpine
and boreal Salix spp. Arctic and Alpine Research 26: 304—307.
Erland S, Henrion B, Martin F, Glover LA, Alexander IJ. 1994. Identification o f the
ectomycorrhizal basidiomycete Tylospora fibrillosa Donk by RFLP analysis o f the PCRamplified ITS and IGS regions o f ribosomal DNA. N ew Phytologist 126: 525-532.
Erland S, Jonsson T, Mahmood S, Finlay RD. 1999. Below-ground ectomycorrhizal
community structure in two Picea abies forests in southern Sweden. Scandinavian Journal
o f Forest Research 14: 209—217.

23

Feng G, Song YC, Li XL, Christie P. 2003. Contribution o f arbuscular mycorrhizal fungi
to utilization o f organic sources o f phosphorus by red clover in a calcareous soil. Applied
Soil Ecology 22: 139-148.
Frank AB. 1885. Ueber die auf Wurzelsymbiose beruhende Emahrung gewisser Baume
durch unterirdische Pilze. Ber. Deut, Bot. Gesell 3: 128-145.
Fujimura KE, Egger KN. 2012. Host plant and environment influence community
assembly o f High Arctic root-associated fungal communities. Fungal Ecology 5: 409-418.
Gladish S, Frank J , Southworth D. 2010. The serpentine syndrome below ground:
ectomycorrhizas and hypogeous fungi associated with conifers. Canadian Journal o f
Forest Research 40: 1671—1679.
Gonsalves SC, Martins-Lou^ao MA, Freitas H. 2009. Evidence o f adaptive tolerance to
nickel in isolates o f Cenococcum geophilum from serpentine soils. Mycorrhiza 19: 221—
230.
Gonsalves SC, Portugal A, Gonsalves MT, Vieira R, Martins-Lou?ao MA, Freitas H.
2007. Genetic diversity and differential in vitro responses to N i in Cenococcum geophilum
isolates from serpentine soils in Portugal. Mycorrhiza 17: 677-686.
Griesbauer HP, Green DS. 2010. Assessing the climatic sensitivity o f Douglas-fir at its
northern range margins in British Columbia, Canada. Trees 24: 375-389.
Hagerman S, Durall D. 2004. Ectomycorrhizal colonization o f greenhouse-grown
Douglas-fir (.Pseudotsuga menziesii) seedlings by inoculum associated with the roots o f
refuge plants sampled from a Douglas-fir forest in the southern interior o f British
Columbia. Canadian Journal o f Botany 82: 742-751.
Hagerman SM, Sakakibara SM, Durall DM. 2001. The potential for woody understory
plants to provide refuge for ectomycorrhizal inoculum at an interior Douglas-fir forest after
clear-cut logging. Canadian Journal o f Forest Research 31:711-721.
Henrik Nilsson R, Tedersoo L, Lindahl BD, Kjoiler R, Carlsen T, Quince C,
Abarenkov K, Pennanen T, Stenlid J, Bruns T, Larsson K-H, Koljalg U, Kauserud
H. 2011. Towards standardization o f the description and publication o f next-generation
sequencing datasets o f fungal communities. New Phytologist 191: 314-318.
Hermann R, Lavender D. 1990. Pseudotsuga menziesii (Mirb.) Franco. Silvics o f North
America Vol 1. United States Department o f Agriculture, 527-540.
Hibbett DS, Gilbert LB, Donoghue MJ. 2000. Evolutionary instability o f
ectomycorrhizal symbioses in basidiomycetes. Nature 407: 506-508.

24

Horton TR, Bruns TD, Parker VT. 1999. Ectomycorrhizal fungi associated with
Arctostaphylos contribute to Pseudotsuga menziesii establishment. Canadian Journal o f
Botany 77: 93-102.
Hunt GA. 1992. Effect o f mycorrhizal fu n g i on quality o f nursery stock and plantation
performance in the southern interior o f British Columbia. FRDA Report 185, Copublished
by the BC Ministry o f Forests.
Ingleby K, Mason PA, Last FT, Fleming L V. 1990. Identification o f ectomycorrhizas.
London: HMSO.
Jentschke G, Godbold DL. 2000. Metal toxicity and ectomycorrhizas. Physiologia
Plantarum 109: 107-116.
Jones MD, Hutchinson TC. 1986. The effect o f mycorrhizal infection on the response o f
Betula papyrifera to nickel and copper. New Phytologist 102: 429-442.
Jones MD, Hutchinson TC. 1988. Nickel toxicity in mycorrhizal birch seedlings infected
with Lactarius rufus or Scleroderma flavidum II. Uptake o f nickel, calcium, magnesium
phosphorus and iron. N ew Phytologist 108: 461—470.
Jones MD, Twieg BD, Ward V, Barker J, Durall DM, Simard SW. 2010. Functional
complementarity o f Douglas-fir ectomycorrhizas for extracellular enzyme activity after
wildfire or clearcut logging. Functional Ecology 24: 1139-1151.
Kianmehr H. 1978. The response o f Helianthemum chamaecistus Mill, to mycorrhizal
infection in two different types o f soil. Plant and Soil 50: 719-722.
Kierczak J, Neel C, Bril H, Puziewicz J. 2007. Effect o f mineralogy and pedoclimatic
variations on Ni and Cr distribution in serpentine soils under temperate climate. Geoderma
142: 165-177.
Kishchuk BE. 2000. Calcareous soils, their properties and potential limitations to conifer
growth in southeastern British Columbia and Western Alberta: a literature review.
Resources Canada, Canadian Forest Service, Northern Forestry Centre, Edmonton,
Alberta. Information Report NOR-X-370.
Kivlin SN, Hawkes C V., Treseder KK. 2011. Global diversity and distribution of
arbuscular mycorrhizal fungi. Soil Biology and Biochemistry 43: 2294-2303.
Koljalg U, Larsson K-H, Abarenkov K, Nilsson RH, Alexander IJ, Eberhardt U,
Erland S, Hoiland K, Kjoller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo
L, Vralstad T, Ursing BM. 2005. UNITE: a database providing web-based methods for
the molecular identification o f ectomycorrhizal fungi. New Phytologist 166: 1063-8.

25

K o th ari SK, M arsch n er H, R om held V. 1991. Contribution o f the VA mycorrhizal
hyphae in acquisition o f phosphorus and zinc by maize grown in a calcareous soil. Plant
and Soil 131: 177-185.
L apeyrie F. 1990. The role o f ectomycorrhizal fungi in calcareous soil tolerance by
“symbiocalcicole” woody plants. Annales des Sciences Forestieres 47: 579-589.
L apeyrie F, C hilvers G. 1985. An endomycorrhiza-ectomycorrhiza succession associated
with enhanced growth o f Eucalyptus dumosa seedlings planted in a calcareous soil. New
Phytologist 100: 93-104.
Le Tacon F. 1978. La presence de calcaire dans le sol. Influence sur le comportement de
l’Epicea commun {Picea excelsa Link.) et du Pin noir d'Autriche {Pinus Nigra nigricans
Host.). Annales des Sciences forestieres 35: 165-174.
Li XL, G eorge E, M arsch n er H. 1991. Extension o f the phosphorus depletion zone in
VA-mycorrhizal white clover in a calcareous soil. Plant and Soil 136: 41^18.
M aas JL , S tuntz DE. 1969. Mycoecology on Serpentine Soil. Mycologia 61: 1106-1116.
M arsch n er H , Dell B. 1994. Nutrient uptake in mycorrhizai symbiosis. Plant and Soil
159:89-102.
M assicotte HB, M elville LH , Li CY, P eterson R L . 1992. Structural aspects o f Douglasfir [Pseudotsuga menziesii (Mirb.) Franco] tuberculate ectomycorrhizae. Trees 6: 137-146.
M assicotte HB, Melville LH , M olina R, P eterson R L. 1993. Structure and
histochemistry o f mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two
basidiomycetes, Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae).
Mycorrhiza 3: 1—11.
M assicotte HB, M olina R, Luom a DL, Sm ith JE . 1994. Biology o f the ectomycorrhizal
genus Rhizopogon: II. Patterns o f host-fimgus specificity following spore inoculation o f
diverse hosts grown in monoculture and dual culture. New Phytologist 126: 677-690.
M assicotte HB, M olina R, T ack ab erry LE , Sm ith JE , A m aran th u s M P. 1999.
Diversity and host specificity o f ectomycorrhizal fungi retrieved from three adjacent forest
sites by five host species. Canadian Journal o f Botany 77: 1053-1076.
M assicotte HB, Peterson R L, A ckerley CA, Piche Y. 1986. Structure and ontogeny o f
Alnus crispa-Alpova diplophloeus ectomycorrhizae. Canadian Journal o f Botany 64: 177—
192.

26

Massicotte HB, Peterson RL, Melville LH. 1989. Hartig net structure o f ectomycorrhizae
synthesized between Laccaria bicolor (Tricholomataceae) and two hosts: Betula
alleghaniensis (Betulaceae) and Pinus resinosa (Pinaceae). American Journal o f Botany
76: 1654-1667.
McKechnie 1 .2009. Propagation methods and the effectiveness o f fungal inoculation on
Vaccinium species native to central British Columbia. M aster’s Thesis, University o f
Northern BC.
Meidinger D, Pojar J, Harper W. 1991. Sub-boreal spruce zone. Ecosystems o f British
Columbia. Special Report Series 6: 209-221.
Molina, R., H. Massicotte, and J.M. Trappe. 1992. Specificity phenomena in
mycorrhizal symbioses: community-ecological consequences and practical implications,
357-423. In: M.F. Allen (ed.), Mycorrhizal Functioning: An Integrative Plant-fungal
Process. Chapman and Hall, NY.
M oore D, Robson GD, Trinci APJ. 2011. 21st Century Guidebook to Fungi. Cambridge,
UK: Cambridge University Press.
Moser AM, Petersen CA, D ’Allura JA, Southworth D. 2005. Comparison o f
ectomycorrhizas o f Quercus garryana (Fagaceae) on serpentine and nonserpentine soils in
southwestern Oregon. American Journal o f Botany 92: 224-230.
O ’Brien MJ, Gomola CE, Horton TR. 2011. The effect o f forest soil and community
composition on ectomycorrhizal colonization and seedling growth. Plant and Soil 341:
321-331.
Panaccione DG, Sheets NL, Miller SP, Cumming JR. 2001. Diversity o f Cenococcum
geophilum isolates from serpentine and non-serpentine soils. Mycologia 93: 645-652.
Parke EL, Linderman RG, Black CH. 1983. The role o f ectomycorrhizas in drought
tolerance o f Douglas-fir seedlings. New Phytologist 95: 83-95.
Perry DA, Amaranthus MP, Borchers JG, Borchers SL, Brainerd RB. 1989.
Bootstrapping in ecosystems. BioScience 39: 230-237.
Peterson RL, Massicotte HB. 2004. Exploring structural definitions o f mycorrhizas, with
emphasis on nutrient-exchange interfaces. Canadian Journal o f Botany 82: 1074-1088.
Peterson RL, Massicotte HB, Melville LH. 2004. Mycorrhizas: Anatomy and Cell
Biology. NRC Research Press Ottawa, Canada.
Piou D. 1979. Importance de la mycorhization dans la resistance au calcaire de diverses
especes forestieres. Revue Forestiere Frangaise 31:116—125.

27

Plouffe A. 2000. Quaternary geology o f the Fort Fraser and M anson River map areas,
central British Columbia. Geological Survey o f Canada, Bulletin 554.
Plouffe A. 2001. The glacial transport and physical partitioning o f mercury and gold in till:
implications for mineral exploration with examples from central British Columbia,
Canada. Geological Society, London, Special Publications 185: 287-299.
Rasmussen HN. 1995. Terrestrial orchids fro m seed to mycotrophic plant. Cambridge,
UK: Cambridge University Press.
Rayner AD, Boddy L. 1988. Fungal decomposition o f wood. Its biology and ecology.
John Wiley & Sons Ltd.
Rencz A, Shilts W. 1980. Nickel in soils and vegetation o f glaciated terrains. Ottawa:
Geological Survey o f Canada.
Robertson SJ. 2003. Morphological and M olecular Assessment o f Ectomycorrhizal
Communities Associating with Black Spruce (Picea mariana (Mill.) Bsp) in Wetland and
Upland Forests in Central British Columbia. M aster’s Thesis, University o f Northern BC.
Schaetzl RJ, Anderson S. 2005. Soils: Genesis and Geomorphology. Cambridge
University Press.
Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W.
2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA
barcode marker for Fungi. Proceedings o f the National Academy o f Sciences o f the United
States o f America 109: 6241-6.
Silen RR. 1978. Genetics o f Douglas-fir. US Department o f Agriculture. Research Paper,
WO-35.
Simard SW, Perry DA, Jones MD, Myrold DD, Durall DM, Molina R. 1997. Net
transfer o f carbon between ectomycorrhizal tree species in the field. Nature 388: 579-582.
Simard SW. 2009. The foundational role o f mycorrhizal networks in self-organization o f
interior Douglas-fir forests. Forest Ecology and Management 258: S95-S107.
Smith JE, Molina R, Huso MM, Luoma DL, McKay D, Castellano MA, Lebel T,
Valachovic Y. 2002. Species richness, abundance, and composition o f hypogeous and
epigeous ectomycorrhizal fungal sporocarps in young, rotation-age, and old-growth stands
o f Douglas-fir (Pseudotsuga menziesii) in the Cascade Range o f Oregon, U.S.A. Canadian
Journal o f Botany 80: 186-204.
Smith SE, Read DJ. 2008. Mycorrhizal Symbiosis. Academic Press.

28

Soil Classification W orking G roup. 1998. The Canadian System o f Soil Classification.
Agriculture and Agri-Food Canada Publication 1646.
S outhw orth D, T ackaberry LE, M assicotte HB. 2014. Mycorrhizal ecology on
serpentine soils. Plant Ecology & Diversity 7: 445-455.
T aylor H R , H a rris W E. 2012. An emergent science on the brink o f irrelevance: a review
o f the past 8 years o f DNA barcoding. Molecular Ecology Resources 12: 377-88.
Teste FP, S im ard SW , D urall DM , G uy RD, Jones M D, S choonm aker A L. 2009.
Access to mycorrhizal networks and roots o f trees: importance for seedling survival and
resource transfer. Ecology 90: 2808-2822.
T horn e D, Seatz L. 1955. Acid, alkaline, alkali, and saline soils. Pages 219-252 in F.E.
Bear, ed. Chemistry o f the soil. Reinhold Publishing Commision. New York, NY.
American Chemical Society monograph 126.
Toum ey JW , K orstian C F. 1947. Foundations o f silviculture upon an ecological basis.
New York, NY: John Wiley & Sons, Inc.
T rap p e JM . 1977. Selection o f fungi for ectomycorrhizal inoculation in nurseries. Annual
Review o f Phytopathology 15: 203-222.
W alk er JK M , Phillips LA, Jones M D. 2014. Ectomycorrhizal fungal hyphae
communities vary more along a pH and nitrogen gradient than between decayed wood and
mineral soil microsites. Botany 92: 453-463.
W iensczyk A, G am iet S. 2002. Ectomycorrhizae and forestry in British Columbia: A
summary o f current research and conservation strategies. Journal o f Ecosystems and
Management 2: 1-20.
W ilkins DA. 1991. The influence o f sheathing (ecto-)mycorrhizas o f trees on the uptake
and toxicity o f metals. Agriculture, Ecosystems & Environment 35: 245-260.
W ilkinson DM, Dickinson NM. 1995. Metal resistance in trees: the role o f mycorrhizae.
Oikos 72: 298-300.
Yu T, E gger K, P eterson L. 2001. Ectendomycorrhizal associations-characteristics and
functions .M ycorrhiza 11: 167-177.
Z a k B. 1971. Characterization and classification o f mycorrhizae o f Douglas fir. II.
Pseudotsuga menziesii+ Rhizopogon vinicolor. Canadian Journal o f Botany 49: 1079—
1084.

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2. Fort St. James: Soils and forest ecosystems related to diverse lithologies

2.1.

Description o f bedrock origins

2.1.1. Ultramafic bedrock, serpentine rocks and soils
British Columbia has several distinct ultramafic bedrock deposits formed by ocean
crusts, or ophiolite complexes, accreting against the North American Craton (Bulmer,
1992; Plouffe, 2000). Accreted fragments o f ocean crust, known as terranes, form a
patchwork o f distinct bedrock types in central British Columbia. The Trembleur ultramafic
rocks o f the Cache Creek terrane are the underlying parent material o f the ultramafic soils
located in Fort St. James. The Trembleur ultramafic rocks are made up o f a combination of
ultramafic and serpentinized rocks including harzburgite and peridotite, and serpentinite
(Plouffe, 2000). These rocks were formed during the Carboniferous period, approximately
360 to 300 million years ago, and the Lower Jurassic period, approximately 200 to 175
million years ago (Plouffe, 2000; Cohen et al., 2013).
Ultramafic rocks are igneous, formed from molten parts o f the earth’s mantle.
These rocks are low in silica and usually dark in colour due to the high levels o f iron and
magnesium. Heavy metals such as nickel and chromium can be enriched within ultramafic
deposits. Serpentinization is a metamorphic process that occurs when ultramafic deposits
are exposed to water at temperatures less than 400°C. The original rocks are oxidized and
hydrolyzed by the water, weakening the structure o f the rock (Alexander et al., 2006). Both
serpentine and ultramafic parent material contribute high levels o f iron, magnesium,
chromium and nickel to the soils developed on the deposits (Bulmer & Lavkulich, 1994).

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2.1.2. Above and below-ground effects o f ultramafic and serpentine bedrock
Serpentine soils are one o f the most widely recognized examples o f strong
chemical signatures within soil conditions (Harrison & Rajakaruna, 2011). True serpentine
soils reflect the chemical character o f the serpentine rocks and parent material. While both
serpentine and ultramafic parent materials are often grouped together, ultramafic rocks are
more stable than serpentinized rocks and will often have a slightly different chemical
signature within the soils horizons (Bulmer & Lavkulich, 1994). Serpentine and ultramafic
soils worldwide often present a very visible change in ecosystem type at the location where
the bedrock transitions from the harsher serpentine and ultramafic parent material to one
with a weaker chemical signature (Kruckeberg, 2004; Alexander et al., 2006).
High levels o f magnesium lead to a low Ca:Mg ratio within the soil producing
strain on plants grown in serpentine areas (Brady et al., 2005; Fitzsimons & Miller, 2010;
Armbruster, 2014). Nickel levels within all soil horizons on serpentine soils can potentially
be phytotoxic, interfering with cellular metabolism and growth o f plants (Miller &
Cumming, 2000; Panaccione et al., 2001). Plants usually attempt to reduce accumulation
o f nickel by exclusion in the rooting zone but this too reduces vigor (MesjaszPrzybyfowicz et a l, 2007). ECM fungi may play a role in the prevention o f absorption o f
nickel and other metals (Brown & Wilkins, 1985; Wilkins, 1991; Wilkinson & Dickinson,
1995). Nutrient stress leading to chlorosis is also a factor o f ultramafic and serpentine
ecosystems due to relatively alkaline pH, low phosphorus and potassium availability
(Alves et a l, 2011). The fragility o f serpentinized rocks frequently leads to soil instability,
mass wasting and water loss, which can damage or remove some plants completely
(Cleaves et al., 1974; Schreier, 1989).

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Plant endemism on serpentine soils has been well documented in California, Cuba,
Italy and New Caledonia (Kruckeberg, 2004; Brady et a l, 2005; D ’Amico & Previtali,
2012). What little research has been done on British Columbia serpentine and ultramafic
ecosystems has not shown the same level o f above-ground ecosystem change, often
described as barrens, found elsewhere (Alexander et a l, 2006). There is some evidence
that northern and western maidenhair fern, Adiantum pedatum L. and Adiantum aleuticum
L. respectively, show preference for serpentine soils (Paris, 1991); however, this is still
anecdotal in British Columbia. The “serpentine syndrome” does not seem to produce the
same level o f plant endemism in British Columbia as described in areas less recently
glaciated.

Fungi also display sensitivity to changes in soil chemistry. Soil pH and phosphorus
levels have been found to influence fungal community structure. In the same study,
dolomitic soils differed in community structure from granitic sites (Fujimura & Egger,
2012). Nickel has been shown to inhibit germination o f some types o f fungi in vitro,
though isolates from ultramafic soils were generally more tolerant o f heavy metals (Amir
& Pineau, 1998). These factors have led to the hypothesis that the below-ground
environment is as taxing for mycorrhizal fungi as it is to plants. Several studies have been
conducted on the ECM communities o f serpentine soils outside Canada, but few have
found statistically quantifiable changes in diversity between serpentine and non-serpentine
sites (Southworth et a l, 2014). Specific mechanisms o f ECM fungal success on serpentine
soils have also not yet been documented.

32

2.1.3. Calcareous bedrock and soils
Calcareous bedrock in British Columbia is high in primary carbonate minerals and
is found in patches throughout the province (Kishchuk, 2000). Dolostone or marine
sedimentary limestone deposits make up the bedrock found underneath the majority o f
calcareous soils found in British Columbia. The sedimentary deposits in the Fort St. James
area date from the Carboniferous and lower Jurassic periods (Plouffe, 2000). Calcareous
soils on grassland ecosystems outside o f British Columbia have been studied extensively
due to their potential value as farm or ranch land (Azcon-Aguilar et al., 1986; Kothari et
al., 1991; Azcon & Barea, 1992) but little research has been done on the recently glaciated
calcareous soils o f central British Columbia, especially those underneath Douglas-fir
forest.

2.1.4. Above and below-ground effects o f calcareous bedrock
High levels o f calcium and high pH within calcareous soils present a difficult
growth environment for many plants (Lapeyrie, 1990). Iron deficiency, shown by chlorosis
of leaves, is very common in many plants grown in calcareous soils (Loeppert et al., 1994).
Plants in this condition are limited in their ability to transport iron from the roots to the
leaves and the rest o f the plant. This may be a consequence o f the formation o f iron oxide
precipitates due to the alkaline conditions (Mengel and Geurtzen, 1986; Mengel, 1994).
Dissolved iron within the soil may also be present in suboptimum concentrations (Lindsay
& Schwab, 1982). Calcium coupled with high pH within calcareous soils will lead to the
formation o f H P O f2 ions and other insoluble bindings with phosphates, rendering
phosphorus inaccessible to plants (Lapeyrie, 1990; Azcon & Barea, 1992; Kishchuk,

33

2000). Other trace nutrients such as copper, manganese and zinc are less soluble in
calcareous soils due to the formation o f chemical complexes and the ionization caused by
the increase in pH (Thome & Seatz, 1955; Marschner & Marschner, 2012).
Mycorrhizal fungi are thought to ameliorate the effects o f calcareous soils on plants
leading to increased success (Piou, 1979; Li et al., 1991). Phosphorus uptake is greatly
increased by the inclusion o f ectomycorrhizal fungi (Lapeyrie & Chilvers, 1985). Iron
uptake may also be increased by both microbial and ectomycorrhizal mobilization
(Szaniszlo et al., 1981). However, the reasons for increased plant success with
ectomycorrhizal fungi on calcareous soils have not been fully explored. Individual species
or guilds o f ectomycorrhizal fungi that contribute to plant health in calcareous soils are
also not well characterized (Lapeyrie 1990).

2.2.

History o f glaciation

2.2.1. Glacial parent material and soils
The passage o f the Cordilleran ice sheet shaped the topography and water courses
o f the study area. Pressure from the ice sheet fragmented and ground down many o f the
bedrock surfaces and other consolidated deposits in the Fort St. James area (Plouffe, 2001).
These fragments were carried by the glacier and deposited as the ice sheet receded. Many
deposits are in the form o f eskers, the sandy remnants o f glacial river beds.
Glaciolacustrine deposits consist o f sorted and stratified, fine-grained sediments deposited
in glacial lakes. Unsorted, blended sediments, gravels, cobbles and boulders that were
directly deposited by the glacier are known as till. Material deposited from underneath the
glacial are known as basal till, while ablation till was left as the glacier melted (Brady &

34

Weil, 2008). In plateau lands with gentle topography, the chemical and mineralogical
composition o f glacial parent materials reflect a blending o f sediments and bedrock
fragments drawn from across a wide area.

2.3.

Sub-Boreal Spruce zone biogeoclimatic zone

2.3.1. Moisture and temperature regime
The Sub-Boreal Spruce biogeoclimatic zone (SBS) encompasses most o f northcentral British Columbia (Figure 2.3.1.1) and is typified by a continental climate with
moist, temperate summers with intermittent precipitation and long cold winters with heavy
snow. Summers can reach temperatures o f over 30 °C, but usually range from 20-25 °C.
Winters have average temperatures o f between -10 °C and -20 °C with lows o f -40 °C.
Precipitation data varies between long term and short term collecting sites, but often ranges
between 415 to 1650 mm yearly due to variety o f terrain. This precipitation is generally
split between snow and rain throughout the year (Meidinger et al., 1991).
Within the study area, precipitation averages approximately 550 mm per year. This
is divided into approximately 65% rain and 35% snow. Winters average -9.5 °C with lows
o f -49.5 °C. Summers average 15.4 °C with highs o f 36.7 °C. Mean annual temperature is
3.5 °C with 96 frost-free days (Fort St. James weather station, Environment Canada, 2014).

35

Legend
BEC Zone
*

SBS

VRI Forest Cover Type
■ I Douglas-fir
I 'r in c i G e o rg e

Map by Neil P. Thompson
Data courtesy o f BC Government
Open government license VI .0

^
Kilom eters

Figure 2.3.1.1: Study area location with respect to the boundaries o f the Sub-boreal Spruce
biogeoclimatic zone and the range o f Douglas-fir dominated stands.
2.3.2. Plant community structure
The SBS has large conifer populations o f hybrid white spruce (Picea glauca var.
engelmannii (Parry) Boivin) and subalpine fir {Abies lasiocarpa (Hook) Nutt.). These trees
are well adapted to snow loading and can withstand cold, snowy winters. Both are
opportunistic colonizers and have a wide range o f potential habitats across many different
soil types and drainages, but require more moisture than Douglas-fir (Bums & Honkala,
1990). The SBS forest type is typically found between elevations o f 500 to 1300 m in the
Fort St. James region (Hrinkevich & Lewis, 2011). Historically, lodgepole pine {Pinus
contorta Dougl. ex. Loud var. latifolia) has also had a large presence in the interior with

36

near monoculture stands on drier areas. However, the mountain pine beetle (Dendroctonus
ponderosae Hopkins) outbreak o f the late 1990s and 2000s has killed approximately 90%
o f all mature lodgepole pine (Safranyik & Wilson, 2007). The species often remains as a
component o f the understory, especially on drier sites.

Hardwood species are also present in the SBS. Black cottonwood (Populus
trichocarpa Torr. & Gray), balsam poplar (Populus balsamifera L.), trembling aspen
(Populus tremuloides Michx.) and paper birch (Betula papyrifera Marshall) are present in
mixedwood stands with many o f the conifers in the SBS. Underbrush species are varied
within the SBS depending on moisture and light regime. Species include kinnikinnick
(Arctostaphylos uva-ursi L. Spreng), blueberries and black huckleberries ( Vaccinium spp.),
Devil’s club (Oplopanax horridus (Sm.) Miq.) highbush cranberry {Viburnum edule
(Michx.) Raf.) and wild roses {Rosa spp.) (Meidinger et a l, 1991). Presence o f these
species is related to elevation and moisture levels at each site. For example, Devil’s club is
more common on lower, moister sites, while huckleberries are more common on higher,
drier sites.

2.3.3. Douglas-fir in the Fort St. James area
Interior Douglas-fir has the most extensive range of all conifer species west o f the
Rocky Mountains (Van Hooser et a l, 1991). Douglas-fir in the northern part o f its range
can grow at elevations ranging from 550 m to 2440 m (Hermann & Lavender, 1990). Most
stands o f Douglas-fir in the study area were found above 750 m (Figure 2.3.3.1). O f the
approximately 28 074 hectares o f calcareous parent material in the study area, 4812
hectares are Douglas-fir leading stands, and o f the 8998 hectares o f ultramafic parent

37

material, 3358 hectares are Douglas-fir leading stands. Douglas-fir naturally regenerates
better on areas with litter fall, compared to bare mineral soil (Ryker, 1975). Seedlings
range from shade tolerant to shade intolerant (Province o f BC, 1995), but generally grow
well under semi-open canopy conditions (Day, 1998). Douglas-fir has also been shown to
vary genetically both regionally (Hermann & Lavender, 1990) and over environmental
gradients as it adapts to changing conditions (Rehfeldt, 1991).

iV

Texeron- '

J

.

Parent Material
!§§§ Calcareous
m Ultramafie

\ f<

Stand Elevation (m)

^

439-750
1
751 - 1000
■ n 1001 • 1379
♦

Study Site

Ponce G e o rg e s

Vancouver

K ilo m etres

Map by Neil P. T h o m p s # n ^ ^ v &
Data courtesy o f BC GO^ernmen't
Open government license VI 0

Figure 2.3.3.1: Locations o f Douglas-fir stands with respect to elevation witnin the study
area.

38

2.4.

Methods o f soil collection

2.4.1. Site selection
Soils derived from three types o f parent material were selected for this study:
ultramafic, calcareous, and glacial. Six field sites, two for each soil type, were chosen.
Field sites were first determined for candidacy by using bedrock geology maps o f British
Columbia (Plouffe, 2000; Erdmer & Cui, 2009). Sites that showed ultramafic or calcareous
bedrock or glacial materials within less than a day o f travel from Prince George were
identified. Local knowledge from Ms. Joanne Vinnedge (Ministry o f Water, Land and Air
Protection) was used to isolate sites known to have mature Douglas-fir dominated stands,
mostly in the area o f Fort St. James. From the subset o f sites that met the criteria for soil
and tree population, sites that were further than 50 km away from each other were
eliminated in order to reduce potential climatic variability (Figure 2.4.1.1). Sites were each
visited in-person to determine Douglas-fir presence, accessibility and similarity o f site
aspect.

39

%v,

m .

Parent Material
M g ^ .
I
Calcareous

Fezzeron

k tu k w a

♦
at

Calcareous

L im estone, local dolostone,
m ostly u p p e r C arboniferous
or P erm ian, m in o r local T ria ssie
lim estone, m in o r c h e rt an d basa lt

/err

MMUltramafic

& £indn@f§$»tain

P yroxenite, harzburcite,
serpentiniic, Ihentolite,
d u n itc, talc, v$abbru

lltra n ia fic

V "
\

F u n hi Hill

/ ’ ■, i •

C alcareo u s

V 'tiirl
M u n a \J K id jm a s t

Lake

lltra n ia fic
z fW

Port St

Spencer's Ridge
Glacial
20
Vancouver

Created hv Kirsicn Ih i
and Neil J}«*tnps>n
«>t N oivw !jct.2« 1<*
Dala e,xu«r<y iif fiC O iivrrm rcra

OpMtCisvcfUuwRt License vl.f?

Figure 2.4.1.1: Soil sampling locations within the Fort St. James area.
2.4.2. Transect layout and soil collection
Composite soil samples were taken from each site to accurately represent the
average condition. Three transects 30 m in length were established parallel to the contour
spaced at a distance o f 5 m at mid-slope. Approximately 4 L o f the mineral soil were taken
to a depth o f 20 cm through both the A and B horizons at 6 m increments. All organic
horizons were scraped away prior to collection. Large debris and rocks were removed from
the soil in the field. All tools were sterilized with a 10% bleach solution prior to use on
each site to prevent cross-contamination. Approximately 60L o f soil was collected per site
and placed into sterilized 20L buckets for transport back to UNBC.

40

2.4.3. Soil pits and horizon description
Soil pits were dug at all 6 sites to classify the soil into orders. Each pit was dug to a
depth o f 1 m or until parent material was encountered. A majority o f sites allowed for a
lm pit, but some, like Murray-Ridge East had parent material within 75cm o f the surface
(see Appendix 1). The extent o f fine roots was noted throughout both the A and B mineral
horizons. Each soil was classified under the Canadian System o f Soil Classification with
field determination o f Order and Great Group. Samples from each horizon were sent to the
British Columbia Ministry o f Environment Laboratory for determination o f exchangeable
cations, pH, iron, aluminum, and silicon (by sodium citrate-dithionite, sodium
pyrophosphate and acid ammonium oxalate extractions) and particle analysis was
conducted by Maxxam Laboratories BC.
While organic horizons were measured and used in the soil classification o f each
site, organic material was not collected with the mineral soil for the greenhouse bioassay
(Chapter 3). ECM populations are known to have differential vertical distributions tied to
rooting depth, therefore sampling several o f the upper horizons was implemented increase
the chances o f a variable population (Pickles & Pither, 2014). Mineral horizons were also
chosen to better represent the chemical signature o f the soil as organic layers are less
influence by parent material.

2.4.4. Soils preparation and analysis
Soils were homogenized and sieved using a 5mm sieve to remove large
particulates. A sub-sample o f each soil was sent to ALS Geochemistry-Vancouver for whole
rock, or total elemental analysis. Major elements were extracted using lithium borate

41

fusion and measured by inductively coupled plasma atomic emission spectroscopy (ICPAES). Trace elements, including chromium, were extracted by the same protocol, but
instead inductively coupled plasma mass spectrometry (ICP-MS) was used for detection.
Nickel and any remaining metals in the soil were isolated by a four-acid digestion
(perchloric, nitric, hydrofluoric and hydrochloric acids) followed by ICP-AES. Samples
were also sent to the BC ministry o f environment lab to determine cation exchange
capacity (CEC) by the B aCh extraction method. Measurements o f pH were conducted at
UNBC by suspending lOg o f soil in water and measuring the pH with a pH meter. This
process was also repeated using a 0.01 M CaCh suspension as it gives a slightly lower pH
value (Schofield and Taylor 1955).

2.4.5. Statistical analysis o f soil data
As each soil sample sent to the lab was a composite, only one measurement was
taken. However, for the purposes o f comparing sites by parent material, data from sites
sharing the same parent material were pooled and compared using a one-way ANOVA
(StataCorp., 2011) to determine significant difference between sites (a=0.05). Pair-wise
mean comparisons were carried out using Tukey’s honestly significant difference (HSD)
post-hoc test (a=0.05).

2.5.

Methods o f vegetation and forest survey

2.5.1. Site characteristics
Sites were accessed during late September and early October 12-14th 2012 and
September 26-28th 2013. Understory vascular and non-vascular plants and mosses
overlaying the soil organic horizon were identified using Plants o f Northern British
42

Columbia (MacKinnon et al., 1992). The presence and identity o f fungal sporocarps were
also recorded whenever possible. Lichens were identified on both ground and trees. For all
non-tree species, presence and absence were the only measures taken.

2.5.2. Host stand characteristics
Douglas-fir age was determined through the use o f an increment borer on at least
10 trees per site. Each core was visually crossdated (Stokes & Smiley, 1968), ring width
was measured in the program WinDENDRO (Regent Instruments, 2012) and crossdating
was confirmed using the software COFECHA (Holmes, 1983).
Three circular fixed area plots o f both 1/40* hectare and 1/250* hectare were used
to count stems per hectare on each site. Trees greater than 15 cm in diameter at breast
height (DBH) were included in counts for the l/40th ha, while trees less than 15 cm DBH
were counted in the 1/250* ha. All trees within a plot were measured for DBH. Plot level
data were transformed to a per hectare basis (one tree on a 1/40* ha is equal to 40 trees per
hectare). These values were then summed for each plot and then averaged over the three
plots. Basal area was calculated by using the area o f a circle formula A — n r 2. Basal area
per tree values were summed and averaged across each site.

2.5.3. Statistical analysis o f forest inventory data
Stand data were compared statistically (basal area and trees per hectare), and
qualitatively (age classes) between each site (StataCorp., 2011). Mean basal area and mean
trees per hectare were compared using a one-way ANOVA to determine significant
difference between sites (a=0.05). Means were compared using Tukey’s HSD post-hoc test
(a=0.05).

43

2.6.

Soil analysis

2.6.1. Soil chemical and texture data
Soils derived from ultramafic and serpentinized rocks are expected to have elevated
levels o f some heavy metals in comparison with soils derived from other forms o f parent
material. Similarity was found in nickel and chromium concentrations between sites
derived from the same parent material (Figure 2.6.1.1). On average, soils developed on
ultramafic rocks in this study presented high levels o f nickel and chromium, 1383 mg/kg
and 3080 mg/kg respectively. These values were significantly higher than the glacial and
calcareous derived soils which were well below 200 mg/kg for both metals (Figure
2 . 6 . 1.2 ).

3500

■ Chromium
h Nickel

Kuzkwa Rd Pinchi Hill
Calcareous

Spencer's
Ridge

Tezzeron Murray Ridge Pinchi
- East | Mountain

Glacial
Soil Parent Material

Ultramafic

Figure 2.6.1.1: Soil nickel and chromium concentrations for each site.

44

3500 !

3000
2500 H

a Chromium
a Nickel

2000

'5k
s 1500
1000

500

Calcareous

Glacial
Soil Parent Material

Ultramafic

Figure 2.6.1.2: Mean soil nickel and chromium levels measured via four acid digestion and
ICP-AES.

Both calcareous and ultramafic derived soils shared a higher cation exchange
capacity (CEO) than glacial derived soils (Figure 2.6.1.3). These differences are not
statistically significant, due to the amount o f variation between individual sites (Figure
2.6.1.4). Both ultramafic and calcareous soils differed by over 10 cmol (+) kg '1 between
sites.

45

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's
Ridge

Tezzeron

Glacial
Soil Parent Material

Murray Ridge East

Pinchi
Mountain

Ultramafic

Figure 2.6.1.3: Soil cation exchange capacity by BaCl2 extraction for each site.

45 -j
40 35 -

Calcareous

Glacial
Soil Parent Material

Ultramafic

Figure 2.6.1.4: Mean cation exchange capacity by BaCl2 extraction o f each soil type with
standard error o f the mean.

46

Exchangeable calcium: magnesium (Ca:Mg) ratio was relatively low for ultramafic
derived soils and high for calcareous soils. Glacial parent material had Ca:Mg ratio
between that o f ultramafic and calcareous. However, given the very large variation
between the calcareous sites (Figure 2.6.1.4), these values do not differ significantly. This
variation is due to the difference between the Ca:Mg ratio between both calcareous sites
(Figure 2.6.1.5). It is important to note that the low Ca:Mg ratio for Pinchi hill does not
reflect a deficit o f calcium within the soil, but instead an unusually high level o f
magnesium (App. 1).

35 i
30
25
20

15 f
10

5
0

Kuzkwa Rd

.^I

Pinchi Hill

Calcareous

Spencer's
Ridge

Tezzeron

Glacial
Soil Parent Material

Murray Ridge East

Ultramafic

Figure 2.6.1.5: Soil exchangeable calcium: magnesium ratio for each site.

47

Pinchi
Mountain

Calcareous

Glacial

Ultramafic

Soil Parent Material
Figure 2.6.1.6: Mean exchangeable calcium: magnesium ratio with standard error o f the
mean.

Soil pH differed significantly for all soil types. While no soils in this study were
truly alkaline, calcareous and ultramafic derived soils had the highest pH with values o f
6.7 and 5.9 respectively. Glacial soil had the lowest pH with a mean value o f 5.2 (Figure
2.6.1.8). Sites that shared parent material had very similar pH values compared to each
other (Figure 2.6.1.7).

48

Kuzkwa Rd

Pinchi Hill

Spencer's Ridge

Tezzeron

Murray Ridge - Pinchi Mountain
East

Glacial

Calcareous

Ultramafic

Soil Parent Material
Figure 2.6.1.7: Soil pH (H 2 O) for each site.

Calcareous

Glacial
Soil Parent Material

Figure 2.6.1.8: Mean soil pH (H 2 O) with standard error o f the mean.

49

Ultramafic

Soil particle size was determined for each homogenized sample (Table 2.6.1.1).
Each soil had a slightly different texture, but all belong to the loamy designation (Soil
Classification Working Group, 1998).
Table 2.6.1.1: Soil texture data from each sample location.
Location

Parent Material

Texture

Kuzkwa Rd

Limestone-Calcareous

Silt Loam

Pinchi Hill

Limestone-Calcareous

Sandy Loam

Spencer's Ridge

Glaciofluvial-Glacial

Sandy Loam

Tezzeron

Till-Glacial

Loam

Murray Ridge - East

Ultramafic

Loam

Pinchi Mountain

Ultramafic

Clay Loam

50

2.7.

Forest overstory and understory data

2.7.1. Douglas-fir stand age, basal area, and understory data
Almost all sites sampled had one or more age classes represented. Pinchi Mountain
is the exception from this list (Table 2.7.1.1). This variety in ages suggests that there were
several periods o f disturbance, removing almost all mature trees within the stand, leaving
only a few survivors. In the case o f Murray Ridge, this disturbance history resulted in 4
separate cohorts o f trees, with some dating as far back as 1596.
Dates reported here do not include missing rings when pith was missed. This
underestimates the age o f trees when pith was missed by 5 to 15 years. Cohorts were
bounded by trees where pith was hit, allowing for accurate dating o f the tree’s
establishment. The designation o f age cohorts is such that trees with missing rings that
were dated a decade older than cohort boundaries were placed into the next cohort.
Table 2.7.1.1: Age cohorts for Douglas-fir on each study site.
Location

Cohort A

Cohort B

Cohort C

Cohort D

Cohort 0

Kuzkwa Rd

1893-1907

1814-1833

1778

-

-

Pinchi Hill

1898-1915

1798-1837

1730-1774

-

-

Tezzeron

1895-1918

1716

-

-

-

Murray Ridge - East

1851-1877

1836

1780-1792

1596-1650

-

Pinchi Mountain

1895-1909

-

-

-

-

Spencer's Ridge

1900-1910

1734

-

-

1947

51

Vegetation surveys o f all field sites showed similarity in the types o f overstory and
understory trees present. Douglas-fir, hybrid white spruce (Picea glauca var. engelmannii
(Parry) Boivin), trembling aspen (Populus tremuloides Michx.) and lodgepole pine (Pinus
contorta Dougl. ex. Loud var. latifolia) were found on all sites (Table 2.7.1.2). Understory
plants including highbush cranberry ( Viburnum edule (Michx.) Raf.), wild rose (Rosa spp.)
and a variety o f Vaccinium species (Table 2.7.1.3-4). All sites had dominant forest o f
conifers with some level o f understory represented. While ultramafic sites did not lack in
the number o f species found, these areas did appear more scrub-like, with smaller plants o f
the same species when compared to other sites. O f the fungal sporocarps noted, few were
ectomycorrhizal and are therefore not reported.
Table 2.7.1.2: Selected vegetation survey results, trees (‘+ ’ indicates presence).

Latin Name
Abies lasiocarpa
(Hooker) Nuttall
Acer glabrum Torr.
Alnus viridis subsp.
sinuata
Betula papyrifera
Marshall
Picea
engelmanniii *glauca
Pinus contorta subsp.
latifolia
Prunus pensylvanica
L.f.
Prunus virginiana L.
Pseudotsuga
menziesii var. glauca
(Beissn.) Franco

Common
Name
Subalpine
Fir
Douglas
Maple

Calcareous
Kuzkwa Pinchi Spencer's
Hill
Ridge
Rd
+

+

+

+

Paper Birch
Hybrid
White
Spruce
Lodgepole
Pine
Pin Cherry

Murray
Tezzeron
Ridge
+

+
+

Sitka Alder

Glacial

+

+

+

+

+

+

+

+

+

+

+

+

+

+
+

+

+

+

+

+

+

+

+

+

+

+

Chokecherry
Interior
Douglas-fir

Serpentine
Pinchi
Mountain

+

+

52

+

+

+

+

Table 2.7.1.3: Selected vegetation survey results, shrubs (‘+ ’ indicates presence).

Latin Name
Arctostaphylos
uva-ursi L.
Spreng
Cornus
canadensis L.
Fragaria
virginiana

Duchesne
Goodyera
oblongifolia
Raf.
Goodyera
repens (L.) R.
Br.
Pyrola
asarifolia
Michx.
Maianthemum
racemosum (L.)
Link

Common
Name

Kuzkwa
Rd

Calcareous
Pinchi Spencer's
Hill
Ridge

Kinnikinnick

+

Bunchberry

+

Strawberry

+

+

+

+

Western
Rattlesnake
Plantain
Dwarf
Rattlesnake
Plantain

Glacial
Tezzeron

Serpentine
Murray Pinchi
Ridge
Mountain
+

+
+

+

+

+

+

+

+

+

+

+

Bog
Wintergreen

+

+

+

+

False
Solomon's
Seal

+

+

53

+

+

Table 2.7.1.4: Selected vegetation survey results, herbaceous plants (‘+ ’ indicates
presence).

Latin Name
Spiraea betulifolia
L.
Ribes lacustre
(Pers.) Poir.
Rosa acicularis
Lindl.
Rubus parviflorus
Nutt.
Salix sp.
Shepherdia
canadensis (L.)
Nutt.
Vaccinium
caespitosum
Michx.
Vaccinium
membranaceum
Douglas ex Torr.
Viburnum edule
(Michx.) Raf.

Common
Name
Birch-leaved
Spirea
Prickly
Currant
Prickly Wild
Rose

Calcareous
Kuzkwa Pinchi Spencer's
Rd
Hill
Ridge
+

+

+
+

+

Glacial

Serpentine
Murray Pinchi
Tezzeron
Ridge
Mountain

+

+

+

+

+
+

Thimbleberry

+

Willow

+

+

+

Soopolallie

+

-f

*f

+

-f-

+

+

+

+

+

+

+
+

+

Dwarf
Bilberry

+

Black
Huckleberry

+

High Bush
Cranberry

-J-

+

54

+

4.

+

4-

4.

Stand density tended toward higher values for glacial derived sites compared to
calcareous and ultramafic derived sites. Both Murray Ridge and Pinchi Mountain have a
significantly lower number o f stems per hectare (all species) than the Tezzeron glacial site.
While calcareous and ultramafic sites did display lower stand density, the data are variable.
This variablity is best shown on the Tezzeron site which while having the largest count,
also had the greatest standard deviation (Figure 2.7.1.1).

7000 -i

6000 -

*

5000 -

o>
Um

Kuzkwa Rd

Pinchi Hill

Calcareous

Tezzeron

Spencer's
Ridge

Glacial
Soil Parent Material

Murray Ridge
- East

Pinchi
Mountain

Ultramafic

Figure 2.7.1.1: Stand density for all study sites. Groups that do not share letter designations
are significant.

55

Mean basal area (all species) differed between field sites, with higher values on
glacial sites compared to both ultramafic and calcareous derived sites. However, no
significant differences were found between sites. Standing dead basal area o f lodgepole
pine and Douglas-fir is also reported for Spencer’s Ridge as it had a large proportion o f
dead trees present. All other sites had small numbers o f dead trees present as wind-throw
(Figure 2.7.1.2).

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's
Spencer's
Ridge | Ridge Dead

Tezzeron

Glacial
Soil Parent Material

Murray Ridge
Pinchi
- East
Mountain
Ultramafic

Figure 2.7.1.2: Mean basal area o f all trees per site (Douglas-fir, hybrid white spruce and
subalpine fir-including standing dead) showing standard deviation.

56

2.7.2. Evidence o f fire
Figure 2.7.2.1 shows a photograph o f a standing dead, fire-killed stump on Pinchi
hill. The trees standing to the left and right o f the fire-killed stump were cored and
crossdated. Since both younger trees originated between 1898-1897, it can be inferred that
the fire removed most o f the overstory and younger, weaker trees prior to the sampled trees
establishment. Given the silvics o f Douglas-fir, this fire can be dated 5 to 15 years
previous, in the early 1890s or mid to late 1880s.

1897 -

‘

Figure 2.7.2.1: Photo o f a standing fire-killed tree (Pinchi Hill) with younger regenerated
trees. Above are the two cores from the trees standing to the left and right which
established post-fire.

57

2.8.

Discussion of field data

2.8.1. Soil properties

Sites with soil developed on parent material with distinctive chemical signatures
reflected those chemical signatures within the horizons sampled in this study. Ultramaficderived soils had the characteristic presence o f high levels o f nickel and chromium
(Figures 2.6.1.1 and 2.6.2.2). Both Murray Ridge and Pinchi Mountain also had the low
calcium to magnesium ratio expected o f ultramafic soils. Calcareous-derived soil in
contrast to ultramafic soil had very high calcium to magnesium ratio (Figures 2.6.1.5 and
2.6.1.6). Both calcareous and ultramafic soils had much higher pH than glacial-derived
sites (Figures 2.6.1.7 and 2.6.1.8).
These variations within the soil chemical conditions are known to contribute to
stress on the plants that grow on these soils (Kruckeberg, 1979; Mengel and Geurtzen,
1986; Pillon et al., 2010). In contrast to the “barrens” described in serpentine areas in other
countries, and the harshness o f calcareous areas, the sites sampled in this study had mature
forests comparable to each other and surrounding ecosystems found within central British
Columbia.

2.8.2. Forest conditions
Much o f the above-ground data on the established forest indicates that all sites have
experienced disturbance at one time or another. Logged stumps and presumed remains o f
old skid roads point to logging around the turn o f the 20th century. The Fort St. James area
has been influenced by European settlers for the past 200 years, leading to highgrading o f
stands (removal o f the largest, most desirable trees) which creates patchy stands o f trees
58

(MacGregor, 2002). However fire is likely the main historical cause o f the variety of age
classes found at the field sites that pre-date settlement. The characteristic variability o f fire
may have left patches o f forest unbumed. Larger, more robust trees may have been better
able to withstand the flames. These trees would in turn provide the seed source for the next
generation o f trees and retain ECM inoculum within their root systems.
The small scope o f this study can provide limited inference about the expected
differences between forest ecosystems that grow on extreme soils. However, given the
above data on trees per hectare and mean basal area per site, a more in-depth look at soil
origin may be warranted. While the data are extremely variable, on-site visual surveys of
vegetation and assessments o f tree volume show that with a larger data set, significant
differences between sites with a milder parent material (non-ultramafic and noncalcareous) may become apparent.
Knowledge o f the limitations o f sites depending on soil type could be helpful in
informing forestry practices on treatment o f specific sites. For example, a site that is
ultramafic or calcareous may not produce the same volume o f wood in the same timeframe
as glacial soils. If that is known to forest planners, harvesting and replanting schedules
may be modified to account for the expected changes. Ultramafic and calcareous soils may
also host unique communities o f below-ground organisms, including ECM fungi, which
may not be apparent to the above-ground observations.

2.8.3. The use o f sites o f the same parent material as replicates
While sites did segregate according to parent material, data were variable for
measures including cation exchange capacity (Figure 2.6.1.3), Ca:Mg ratio (Figure 2.6.1.5)
and soil texture (Table 2.6.1.1). Sites that shared parent material often had differences in
59

one or more o f these measures. For instance, Kuzkwa Rd and Pinchi Hill are a silt loam
and a sandy loam respectively. Pinchi Hill will therefore have a higher level o f drainage
and potentially less nutrients within the soil compared to Kuzkwa Rd (Schaetzl &
Anderson, 2005) even though they are both calcareous soils. Likewise both ultramafic sites
have different textures with Murray Ridge-East having a loam texture and Pinchi Mountain
having a clay loam texture. In this case, the clay in the Pinchi Mountain soil will cause
increased water holding (Brady & Weil, 2008) compared to Murray Ridge-East. The
change in nutrient balance and water holding capacity may impact the health and success
o f plants grown in those soils and may also impact the composition o f the below-ground
fungal communities. For this reason, while sites share parent material, they are not true
replicates o f one another and can be compared in general terms, but not pooled for
statistical comparisons.

2.9.

S um m ary
This study provides a first comparison o f the strongly contrasting chemical

differences found on the soils o f the Fort St. James area. No other study in this region links
the below-ground chemical and physical differences in soil to the above-ground conditions.
Ultramafic soils present potentially toxic levels o f chromium and nickel to Douglas-fir and
the ecosystems found in the Fort St. James area, but these plant communities do not show
the same level o f stress seen outside o f Canada. Mature trees are still capable o f growing
and establishing stands that appear to be only slightly less vigorous than comparable
glacial sites. Calcareous soils likewise have extremely high levels o f calcium within the
soil, but do not show depauperate landscapes compared with glacial areas.

60

The lack o f strain apparent on these ecosystems may be related to several
ameliorating factors. The geological youth o f these sites may mean that the soils have not
had time to develop the same level o f chemical signature compared to older areas in
Europe and the Southern United States. As the individual sites with Douglas-fir represent
drier areas, it is possible that the trees are more responsive to the favourable climate than to
the individual soil conditions. The combination o f drier climate, slope position and glacial
blending o f extreme parent materials and mixed sediments may have produced soil that is
balanced between chemical harshness and beneficial conditions (e.g. well drained and
enriched with organic material). It is also possible that the presence o f ECM fungal
partners and rhizosphere organisms may be acting as a first line o f defence preventing
uptake o f damaging levels o f calcium, nickel and chromium. In the following chapter,
these communities o f ECM fungi will be compared to ascertain if strongly contrasting soil
signatures produce equally contrasting populations o f ECM fungi.

61

2.10.

References

Alexander EB, Coleman RG, Keeler-W olfe T, Harrison SP. 2006. Serpentine
Geoecology o f Western North America: Geology, Soils, and Vegetation. New York, NY:
Oxford University Press.
Alves S, Trancoso MA, Gonsalves MDLS, Correia dos Santos MM. 2011. A nickel
availability study in serpentinised areas o f Portugal. Geoderma 164: 155-163.
Amir H, Pineau R. 1998. Effects o f metals on the germination and growth o f fungal
isolates from New Caledonian ultramafic soils. Soil Biology and Biochemistry 30: 20432054.
Armbruster WS. 2014. Multiple origins o f serpentine-soil endemism explained by
preexisting tolerance o f open habitats. Proceedings o f the National Academy o f Sciences o f
the United States o f America 111: 14968-9.
Azcon R, Barea JM. 1992. The effect o f vesicular-arbuscular mycorrhizae in decreasing
Ca acquisition by alfalfa plants in calcareous soils. Biology and Fertility o f Soils 13: 155—
159.
Azcon-Aguilar C, Gianinazzi-Pearson V, Fardeau JC, Gianinazzi S. 1986. Effect o f
vesicular-arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria on growth and
nutrition o f soybean in a neutral-calcareous soil amended with32P-45Ca-tricalcium
phosphate. Plant and Soil 96: 3-15.
Brady KU, Kruckeberg AR, Bradshaw HDJ. 2005. Evolutionary ecology o f plant
adaptation to serpentine soils. Annual Review o f Ecology, Evolution, and Systematics 36:
243-266.
Brady NC, Weil RR. 2008. The Nature and Properties o f Soils. 14th Ed. Prentice Hall.
Brown MT, Wilkins D A. 1985. Zinc tolerance o f mycorrhizal Betula. New Phytologist
99: 101-106.
Bulmer CE. 1992. Pedogenesis o f Soils Derived from Ultramafic Rocks and Tephra in
Southwestern British Columbia. PhD thesis. University o f British Columbia.
Bulmer CE, Lavkulich LM. 1994. Pedogenic and geochemical processes o f ultramafic
soils along a climatic gradient in southwestern British Columbia. Canadian Journal o f Soil
Science 1A: 165-177.
Burns RM, Honkala BH. 1990. Silvics o f North America: 1. Conifers; 2. Hardwoods.
Agriculture Handbook 654. U.S. Department o f Agriculture, Forest Service, Washington,
DC.
62

Cleaves E T , F ish er DW , B rick er OP. 1974. Chemical weathering o f serpentinite in the
eastern Piedmont o f Maryland. Geological Society o f America Bulletin 85: 437-444.
Cohen K M , Finney S, G ib b ard P L . 2013. International Chronostratigraphic Chart.
International Commission on Stratigraphy.
D ’Amico M E, Previtali F. 2012. Edaphic influences o f ophiolitic substrates on vegetation
in the Western Italian Alps. Plant and Soil 351: 73-95.
D ay JK . 1998. Selection Management o f Interior Douglas-fir for Mule Deer W inter
Range. M aster’s Thesis, University o f British Columbia.
E nviro n m en t C anada. 2014. Canadian Climate Normals (1981-2010). Fort St. James,
British Columbia, http://climate.weather.gc.ca/climate_normals/results_l 981_2010_
e. html?stnID=5 8 8&autofwd= 1
E rd m er, P, Cui, Y. 2009. Geological Map o f British Columbia; British Columbia
M inistry o f Energy, Mines and Petroleum Resources, Geoscience Map 2009-1, scale 1:1
500 000.
Fitzsim ons M S, M iller R M . 2010. Serpentine soil has little influence on the rootassociated microbial community composition o f the serpentine tolerant grass species
Avenula sulcata. Plant and Soil 330: 393—405.
F u jim u ra K E, Egger KN. 2012. Host plant and environment influence community
assembly o f High Arctic root-associated fungal communities. Fungal Ecology 5: 409—418.
H arriso n S, R a ja k a ru n a N. 2011. Serpentine: The Evolution and Ecology o f a M odel
System. University o f California Press.
H erm an n R, L av en d er D. 1990. Pseudotsuga menziesii (Mirb.) Franco. Silvics o f North
America Vol 1. United States Department o f Agriculture, 527-540.
Holmes RL. 1983. Computer-assisted quality control in tree-ring dating and measurement.
Tree-ring bulletin 43: 69-78.
H rinkevich K , Lewis K J. 2011. Northern range limit mountain pine beetle outbreak
dynamics in mixed sub-boreal pine forests o f British Columbia. Ecosphere 2: artl 16.
K ishchuk BE. 2000. Calcareous soils, their properties and potential limitations to conifer
growth in southeastern British Columbia and Western Alberta: a literature review.
Resources Canada, Canadian Forest Service, Northern Forestry Centre, Edmonton,
Alberta. Information Report NOR-X-370.

63

K o th ari SK, M arsch n er H, R om held V. 1991. Contribution o f the VA mycorrhizal
hyphae in acquisition o f phosphorus and zinc by maize grown in a calcareous soil. Plant
and Soil 131: 177-185.
K ruck eb erg A. 1979. Plants that grow on serpentine: A hard life. Davidsonia 10: 21-29.
K ru ck eb erg AR. 2004. Geology and Plant Life: The Effects o f Landforms and Rock Types
on Plants. University o f Washington Press.
L apeyrie F. 1990. The role o f ectomycorrhizal fungi in calcareous soil tolerance by
“symbiocalcicole” woody plants. Annales des Sciences Forestieres 47: 579-589.
L apeyrie F, C hilvers G. 1985. A n endomycorrhiza-ectomycorrhiza succession associated
with enhanced growth o f Eucalyptus dumosa seedlings planted in a calcareous soil. New
Phytologist 100: 93-104.
Li XL, G eorge E, M arsch n er H . 1991. Extension o f the phosphorus depletion zone in
VA-mycorrhizal white clover in a calcareous soil. Plant and Soil 136: 41-48.
Lindsay W , Schwab A. 1982. The chemistry o f iron in soils and its availability to plants.
Journal o f Plant Nutrition 5: 821-840.
L o ep p ert R H , W ei LC, O cum paugh W R. 1994. Soil factors influencing the mobilization
o f iron in calcareous soils, pages 343-360 in J. A. Manthey, D. E. Crowley, and D. G.
Luster, eds. Biochemistry o f metal micronutrients in the rhizosphere. Lewis publications,
Boca Raton, Florida.
M acG regor M. 2002. Developing reference conditions for ecological restoration o f forests
in sub-boreal British Columbia. M aster’s Thesis, University o f Northern BC.
M acK innon A, P o ja r J , C oupe R. 1992. Plants o f Northern British Columbia.
Vancouver: B. C. Ministry o f Forests and Lone Pine Publishing.
M arsch n er H , M arsch n er P. 2012. M arschner’s mineral nutrition o f higher plants.
Elsevier.
M eidinger D, P o ja r J , H a rp e r W . 1991. Sub-boreal spruce zone. Ecosystems o f British
Columbia. Special Report Series 6: 209-221.
M engel K. 1994. Iron availability in plant tissues-iron chlorosis on calcareous soils. Plant
and Soil 165: 275-283.
M engel an d G eurtzen G. 1986. Iron chlorosis on calcareous soils. Alkaline nutritional
condition as the cause for the chlorosis. Journal o f Plant Nutrition 9: 161-173.

64

M esjasz-Przybylow icz J , B arn ab as A, Przybylow icz W. 2007. Comparison o f cytology
and distribution o f nickel in roots o f Ni-hyperaccumulating and non-hyperaccumulating
genotypes o f Senecio coronatus. Plant and Soil 293: 61-78.
M iller SP, C um m ing JR . 2000. Effects o f serpentine soil factors on Virginia pine (Pinus
virginiana) seedlings. Tree Physiology 20: 1129-1135.
Panaccione DG, Sheets NL, M iller SP, C um m ing JR . 2001. Diversity o f Cenococcum
geophilum isolates from serpentine and non-serpentine soils. Mycologia 93: 645-652.
P aris CA. 1991 .Adiantum viridimontanum, a new maidenhair fern in eastern North
America. Rhodora 93: 105-122.
Pickles B J, P ith er J. 2014. Still scratching the surface: How much o f the “black box” o f
soil ectomycorrhizal communities remains in the dark? New Phytologist 201: 1101-1105.
Pillon Y, M unzinger J , A m ir H, L eb ru n M . 2010. Ultramafic soils and species sorting in
the flora o f New Caledonia. Journal o f Ecology 98: 1108-1116.
Piou D. 1979. Importance de la mycorhization dans la resistance au calcaire de diverses
especes forestieres. Revue Forestiere Frangaise 31: 116-125.
Plouffe A. 2000. Quaternary geology o f the Fort Fraser and M anson River map areas,
central British Columbia. Geological Survey o f Canada, Bulletin 554.
Plouffe, A. 2001. The glacial transport and physical partitioning o f mercury and gold in
till: implications for mineral exploration with examples from central British Columbia,
Canada. Geological Society, London, Special Publications, 185(1), 287-299.
Province of BC. 1995. Establishment to free growing guidebook: Cariboo Forest Region.
Victoria.
R egent Instrum ents. 2012. WinDendro.
R ehfeldt, J. 1991. The genetic resource o f Douglas-fir in the interior northwest. Interior
Douglas fir: the species and its management: symposium proceedings February 27 March
1-1990 Spokane Washington USA compiled and edited by David M Baugartner and James
E Lotan. WSU Dept. Nat. Res. Sci.
R yker, R.A. 1975. A survey o f factors affecting regeneration o f Rocky Mountain
Douglas-fir. USDA Forest Service Research Paper INT-174.
S afranyik L, W ilson B. 2007. The mountain pine beetle: a synthesis o f biology,
management and impacts on lodgepole pine. Canadian Forest Service.

65

Schaetzl R J, A nderson S. 2005. Soils: Genesis and Geomorphology. Cambridge
University Press.
S chreier H. 1989. Asbestos in the natural environment. Elsevier.
Schofield R K , T aylor AW . 1955. The Measurement o f Soil pH. Soil Science Society o f
America Journal 19: 164-167.
Soil Classification W orking G ro u p . 1998. The Canadian System o f Soil Classification.
Agriculture and Agri-Food Canada Publication 1646.
S tataC o rp . 2011. Stata Statistical Software: Release 12.
Stokes MA, Smiley T L . 1968. An introduction to tree-ring dating. University o f Chicago
Press.
Szaniszlo P J, Powell P E , Reid C PP, Cline G R. 1981. Production o f hydroxamate
siderophore iron chelators by ectomycorrhizal fungi. Mycologia 73: 1158-1174.
T h o rn e D, Seatz L. 1955. Acid, alkaline, alkali, and saline soils. Chemistry o f the soil.
Reinhold Publishing Commision.
V an-H ooser DD, W addell K L, Mills JR , Tym ico R P. 1991. The interior Douglas-fir
resource: current status and projections to the year 2040. Interior Douglas fir: the species
and its management: symposium proceedings February 27 March 1-1990 Spokane
Washington USA compiled and edited by David M Baugartner and James E Lotan WSU
Dept. Nat. Res. Sci.
W ilkins DA. 1991. The influence o f sheathing (ecto-)mycorrhizas o f trees on the uptake
and toxicity o f metals. Agriculture, Ecosystems & Environment 35: 245-260.
W ilkinson DM, Dickinson NM. 1995. Metal resistance in trees: the role o f mycorrhizae.
Oikos 72: 298-300.

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3. Characterization o f Pseudotsuga menziesii var. glauca ectomycorrhizal fungi
communities on ultramafic and calcareous soils in central British Columbia

3.1.

Douglas-fir fungal partners

3.1.1. Douglas-fir and ECM in the field
Douglas-fir is an important part o f western interior forest ecosystems from Mexico
to central British Columbia. It is also known to form ectomycorrhizas (ECM) with a
variety o f species o f fungi depending on the region (Smith eta l., 2002). In field studies,
Douglas-fir has been found in partnership with members o f the genera Rhizopogon,
Cenococcum, Russula, and others (Trappe, 1977; Molina eta l., 1992; Goodman &
Trofymow, 1998). ECM fungi are vital to the survival o f Douglas-fir and may help in the
perpetuation o f stands over time (Simard, 2009). ECM species may be segregated by depth
within the soil column and will differ over a variety o f soil conditions (Pickles & Pither,
2014; Walker eta l., 2014).
In field studies o f disturbed areas (e.g. logged areas, severe windthrow), a few
morphotypes (a grouping based on morphology) are usually dominant, with a variety of
ECM species that are present in low levels (Lang et al., 2011; Pickles & Pither, 2014).
Some o f the common symbionts o f Douglas-fir in disturbed areas include E-strain (usually
o f the genus Wilcoxina), MRA {Mycelium radicis atrovirens), and Cenococcum geophilum,
all o f which are broad host range fungi and capable o f colonizing other hosts (pine and
spruce) (Jones et al., 1998; Barker et al., 2013).
Areas that are not recently disturbed may produce a larger variety o f morphotypes
and species due to the retention o f a living mycorrhizal network (Smith & Read, 2008). A

67

study by Smith et al. (2002) o f ECM fungal sporocarps in younger and old growth
Douglas-fir stands in Oregon found that as stand age increased, so too did the likelihood of
finding unique or rare species. The authors postulated that this might be due to the age o f
the stand, producing conditions favorable to ECM fungi that fruit rarely. In mature stands
o f Douglas-fir, the age o f the root system may alter the composition o f the community
(Smith eta l., 2002).
This diversity within the community, however, does not usually aid in
differentiation between sites as rare morphotypes are not easily included in statistical
analysis (Robertson, 2003; Branco & Ree, 2010). In morphotyping studies, rare types are
often recorded and described, but not included in statistical analyses (Jonsson et al., 1999).
However, while these tips do not help distinguish between sites or treatments in a
quantitative fashion, they are informative from a community composition standpoint and
have value in both field and greenhouse studies.

3.1.2. Douglas-fir and ECM in greenhouse conditions
Douglas-fir is often grown in a nursery under greenhouse conditions to produce
seedlings for tree planting in forestry. Greenhouse Douglas-fir is often reticent to form
mycorrhizas and may be very slow to achieve colonization, taking over 10 months
(Kazantseva et al., 2010; Pickles et al., 2015). Species found in greenhouse studies mirror
those in the field, but rare species are not as common. Often the community will show
stronger levels o f colonization, but a slightly less diverse group o f species (Branco, 2010).
Species often include E-strain and Rhizopogon spp., however, greenhouse contaminants
from existing spore loads can also form ectomycorrhizas with Douglas-fir. For example,
members o f the Thelephora genus are often found in both the field and greenhouse settings
68

(Jones eta l., 1998; Barroetavena eta l., 2007).
High rates o f ECM colonization are helpful in ascertaining potential differences
between sites as it enables a greater statistical comparison o f ECM communities. A much
higher number o f root tips are generated; higher numbers in turn increase statistical power
(Magurran, 2004). While some species o f ECM may not be as prevalent, or as visible due
to absence o f pre-existing hosts (Jones et al., 1998), growing ECM and their hosts in a
nursery can provide higher resolution for the few types o f fungi that may be more
prevalent. ECM root tips generated from a greenhouse study also can be quickly processed
and analyzed using morphological and molecular techniques (see section 1.4.3) in a
controlled environment.

3.2.

Methods of greenhouse bioassay

3.2.1. Seed preparation
Seedlot provenances (CONIFEX 53613 and 30822) were selected from trees within
50 km o f the Fort St. James area (CONIFEX, Fort St. James). Parent trees from these
seedlots were located in areas overlain with deeper glacial deposits from the late
Wisconsinan Fraser Glaciation (Plouffe, 2000). Lack o f strong chemical signatures within
the soil minimized the risk o f planting with stock that has had selective environmental
pressures from soil chemical properties, such as elevated heavy metal concentrations.
All Douglas-fir seeds were surface sterilized to remove potential pathogens prior to
planting. Seeds were submerged in 3% H 2 O 2 with light agitation for 2 hours to ensure no
contaminants remained on the seed coat. Autoclaved deionized water was used to triple
rinse the seeds prior to soaking in fresh sterile deionized water for 24 hours. Seeds were

69

triple rinsed again with sterile water, dried with paper towels until seeds no longer adhered
to one another and stored in sterile jars in a 2°C refrigerator for 21 days to cold stratify
them to ensure germination (Kolotelo et al., 2001). Pre-soaked weight was recorded and
post-soaked weight was monitored weekly to ensure the seeds did not desiccate prior to
planting.

3.2.2. Seedling growth conditions
Following soil collection and transport to UNBC (see section 2.4.2), samples were
stored at 4°C to preserve the fungal propagules within the soil until use. Soils were sieved
through a 5mm mesh to remove pebbles and coarse woody debris. Homogenization o f the
soil was conducted with sterile equipment to ensure an even distribution o f fungal
inoculum within the soil sample. Homogenized soil was potted into 262mL Cone-tainer™
cells.
All cells were treated with AC Vertex™ fungicide prior to planting to remove any
greenhouse contaminants. Potting tools were also treated with AC Vertex™ fungicide
between site soils to prevent cross contamination. All tools and cells were well rinsed to
remove residual fungicide prior to planting. The bottom of each cell was filled with 9
autoclaved sterile clay balls to ensure proper drainage. Each soil was potted by hand into
160 cells, 80 cells for each Douglas-fir seedlot. Cell trays were shaken to ensure even
distribution o f soil and then tamped down with a wooden dowel to remove air pockets.
Each cell was planted with one seed and topped with a layer o f heat-treated and rinsed
forestry sand (coarse grit) to weigh seeds down during watering. In total, 960 cells were
filled and planted on December 5, 2012.
Seeds were germinated under sodium lamps in the University o f Northern BC
70

Enhanced Forestry Laboratory greenhouse-Pod B. Germination temperature was 25°C with
a 16 hours light (day) and 8 hours darkness (night) regime. Light began at 8:00 am and
finished at 10:00 pm to match the time o f sunrise. Trays were watered on a 1 to 2 day
schedule during germination and placed on a greenhouse grid bench to allow for complete
drainage. Water pressure was reduced by using a 1 gallon/minute mist-head hose to prevent
seeds from being dislodged from under the forestry sand. Germination was complete by
January 18, 2013.
Counts were taken o f the seedlings as germination progressed. Death o f new
seedlings and failure to germinate were also recorded. Temperature in the greenhouse pod
was reduced to 22°C to prevent stressing the seedlings. Watering was also reduced to twice
per week after germination to reduce the risk o f damping off. Reducing the amount of
water in the soil helps to prevent damping off as the microorganisms responsible for
damping off thrive in wet conditions (Hartley & Pierce, 1917).
Watering was changed to a 2 gallon/minute rain shower head six weeks after
planting. Seedling trays were rotated within the greenhouse pod to moderate any potential
lighting or temperature anomalies within the pod. Periodic counts o f seedlings still living
were conducted to assess survival rate until the time o f biomass sampling. Observation of
yellowing foliage in May 2013 after 5 months o f growth necessitated the application o f
water-soluble Tune-up fertilizer. This fertilizer has a 20-10-20 ratio o f nitrogen,
phosphorus and potassium (200 ppm, 100 ppm, 200 ppm respectively). Watering with
fertilizer was conducted once weekly for a period o f 2 months until foliage returned to
green.

71

3.2.3. Seedling growth and biomass
Seedlings (n=594) were destructively harvested for biomass sampling at 10 months
(40 weeks) o f age. Height and mean basal diameter were recorded to the nearest millimeter
and the nearest hundredth o f a millimeter respectively using a standard 30 cm ruler and
digital calipers. Root and shoot biomass were assessed by separating the roots and shoot o f
each seedling at the root collar and drying at 70°C for three days (Hagerman & Durall,
2004). Seedling roots and shoots were weighed separately on a Sartorius Micro scale to the
nearest milligram. Root and shoot weights were added together to produce total biomass
for each seedling.

3.2.4. Collection o f seedling foliar samples for chemical analysis
After seedlings were oven-dried, a sub-sample o f needles was harvested to quantify
potential uptake o f metals from the soil to the seedlings. Two grams o f needles were
required per sample, each made up o f composites from multiple trees grown on each soil
type. Microwave digestion was used to prepare samples for inductively coupled plasma
(ICP) spectrometry. Two samples were analyzed for each site (12 in total) at the BC
Ministry o f Environment Analytical Chemistry Laboratory.

3.2.5. Mycorrhizal colonization
Seedlings take weeks to months to become fully colonized by ectomycorrhizal
fungi (Erland & Finlay, 1992) and greenhouse grown Douglas-fir is especially reticent to
form ECM associations (Castellano & Molina, 1989; Kazantseva et al., 2010). Periodic
checks for fungal gnats were used as a non-invasive method o f checking for colonization.
The gnats feed on the fungi and only appear after the trees are fully colonized (H.

Massicotte, pers. comm. 2013). After 7 months o f growth, a few gnats were observed and 3
seedlings were randomly selected to assess the progress o f fungal colonization. These
seedlings were gently removed from the cell to not disturb any fungal tissue on the roots.
The roots were gently washed in cool tap water and fine roots were removed. Any lateral
root tips that showed any sign o f colonization were also removed.
A dissecting microscope was used to assess the presence or absence o f fungal tissue
on the roots. Squash mounts o f root tips were also used to assess the thickness o f any
fungal mantles or hyphae. Due to the very low number o f observed fungal tips (less than
10%), the seedlings were allowed to grow 3 months longer. More fungal gnats were
observed in the greenhouse at this time, and a single seedling was subsampled to check for
fungal colonization. A much higher level o f colonization was detected (approximately
80%) and both morphotyping and biomass sampling was initiated.

3.2.6. Morphology analysis
Morphotyping is the process o f identifying ectomycorrhizal fungal types using a set
o f microscopic characteristics. From 714 surviving seedlings, 120 were randomly
subsampled for morphotyping, 10 from each seedlot and soil type. Each seedling was
removed from the cell and gently shaken to remove the majority o f soil particles. Roots
were rinsed under cool free flowing tap water to dislodge the remaining soil. A stainless
steel dish was filled with tepid deionized water and the roots were soaked with gentle
agitation for 1-2 minutes at a time to ensure the roots were completely clean. The soaking
process was repeated until the water remained clear (Massicotte et al., 1994).
Cleaned roots were separated from the shoot at the root collar and floated in
deionized water in a stainless steel dish with a numbered centimetre grid in the bottom.
73

Grid patches were selected using a random number list that corresponded with the numbers
on the grid. Larger roots were clipped and the root mass was dispersed evenly over the
grid. Approximately 200 lateral and fine roots were cut from the root mass in 2 cm patches
and placed in a glass petri dish filled with deionized water. Remaining roots were stored in
the stainless steel tray in a 2°C refrigerator in case additional sampling was required,
whether due to an incorrect number o f tips or the discovery o f a rare morphotype. A
dissecting microscope was used to assess the visual traits with the tips floating in deionized
water in a petri dish. The entire root system o f a seedling was counted if the total number
o f roots was less than 200. During the course o f the experiment, 23039 root tips were
counted and morphotyped (Ingleby et al., 1990; Goodman et al., 1996; Agerer, 1987-2008).
Each root tip was counted as either uncolonized or colonized. Uncolonized root tips
either had mature root tissue and no evidence o f fungal hyphae, very young and fast
growing meristematic tissue, or a layer o f unorganized fungal hyphae too thin to measure
or visualize using a squash mount. Colonized tips were divided into morphotypes on the
basis o f visual and microscopic traits. Colour was assessed first as it is one o f the simpler
traits to categorize. Luster, or how the root tip reflects light, is also a diagnostic feature.
Many types o f mycorrhizal fungi have distinct emanating hyphae. The branching pattern of
the root tip, whether it is single, clustered, or coralline in shape is also diagnostic (see
Appendix 3 for full descriptions).
A compound microscope was used to view the cellular details o f several
representative root tips from each root system. Wet squash mounts were prepared using a
single root tip. Fungal mantles are made up o f many layers o f hyphae with particular
shapes that distinguish one from another. Mantles differ in cells interlocking with one

74

another, non-interlocking, have a net-like shape, or a stellate pattern. Fungal mantles or
hyphae may be studded with crystalline exudates o f many colours. Presence o f hyphal
clamps or anastomoses between hyphae also helps differentiate between morphotypes.
Presence or absence o f cystidia or hyphae, along with the hyphal and cell size, can help
separate morphotypes as well. Root tips were assigned a particular morphotype based on a
combination o f the above characteristics, using the Colour Atlas o f Ectomycorrhizae
(Agerer, 1987-2008) and Identification o f Ectomycorrhizas (Ingleby et al., 1990) labeled as
Types A-O, and counted.

3.2.7. Statistical analysis o f biomass and morphology data
Seedling biomass measurements (height, basal diameter, root:shoot ratio and total
biomass) were compared using statistical methods (StataCorp., 2011). A one-way ANOVA
was used to determine significant differences between sites (a=0.05). Pair-wise mean
comparisons were made using Tukey’s honestly significant difference (HSD) post-hoc test
(a=0.05). Seedlot was not included in the analysis as a preliminary data check produced no
differences in any data measures (biomass or morphology), between seedlots.
Morphotypes were counted and frequency (number o f seedlings with individual
morphotypes) and percent abundance (proportion o f the morphotype in the entire seedling
community) were calculated using the total sample and partitioned site values. From the
morphotype totals, rank abundance curves were generated using the log transformed
relative abundance (number o f root tips across entire study) o f each morphotype plotted
against morphotype rank as a comparison o f ECM communities between sites (Robertson,
2003; Kindt & Coe, 2005). Mean morphotype abundance values were calculated (averaged
across sites) and compared using a one-way ANOVA to determine the significant
75

difference between sites (a^O.OS). Pair-wise mean comparisons were made using Tukey’s
HSD post-hoc test (a=0.05).
Several diversity indices were used to compare differences between ECM
communities on different sites. These diversity indices are non-parametric and rely on
counts o f species and abundance data. For all indices, a high value is indicative o f higher
diversity or richness. Indices used were the Shannon, Shannon-Evenness, M argalef and the
Gini-Simpson. The Shannon index measures species richness and relative abundance,
while the Shannon-Evenness index is only a measure o f relative abundance. The M argalef
index is a measure o f species richness and the Gini-Simpson index measures relative
abundance, but is more sensitive to abundant types (Magurran, 1988, 2004). Diversity
index values were generated for each site using pooled morphology data. For comparison,
diversity index values were calculated using each seedling, then averaged across sites to
generate mean index values. Mean index values were then compared using a one-way
ANOVA with a post-hoc Tukey’s HSD test.
Sites were compared using morphology data to generate clusters using a two-way
cluster analysis in PC-ORD (McCune & Mefford, 2006). The amount o f root tips for a
particular morphotype was relativized using the maximum number o f root tips for that
type. Dendrograms were constructed using a farthest neighbour joining tree with both a
presence-absence matrix and a relative abundance matrix.

3.3.

Methods of molecular analysis

3.3.1. DNA extraction
Representative root tips o f each morphotype were collected for DNA extraction
76

during the morphotyping process. Only root tips with healthy, distinct mantles and little to
no evidence o f secondary fungal colonization were selected. Each tip, or cluster o f tips,
was removed from the root mass and dried on clean paper towel before being placed into
an autoclaved Eppendorf tube. The tubes were stored in a -20°C freezer until DNA
extraction could begin. Due to the poor quality o f the MRA or O type morphotype, no root
tips were deemed suitable for DNA extraction.
A pilot study was conducted using sporocarp tissue and DNeasy Plant Mini Kit
from Qiagen. This protocol involved lyophilizing the tissue using liquid nitrogen and a
mortar and pestle. Due to poor yields with the DNeasy Plant Mini Kit, the MO BIO
PowerSoil® DNA Isolation Kit was tested as a possible replacement. When the MO BIO
PowerSoil® DNA Isolation Kit was used on similar sporocarp tissue, the yields were
superior to the pilot study and this kit was adopted.
The low amount o f fungal tissue on root tips and the toughness o f fungal mantles
initially made it difficult to extract a useful amount o f DNA from the root tips. After
researching a variety o f methods, an additional heat step and an altered bead beating step
were added to the MO BIO PowerSoil® DNA Isolation Kit protocol (Koide, 2005). Prior
to bead beating (agitation with beads to lyse the cells), the reaction tubes were placed in
boiling water for 10 minutes to increase the activity o f the cell lysis buffer. Two 2mm
zirconium oxide beads were added to the garnet beads in each reaction tube to crush the
fungal cells as well as cut them. This increased the amount o f DNA released into solution.
Extracted DNA concentration was measured on a Thermo Scientific™ NanoDrop 1000.

3.3.2. DNA amplification
Fungal DNA extracts were used as the template for amplification through the
77

polymerase chain reaction (PCR). Initially a master mix protocol was implemented, using
aliquots o f Taq polymerase, dNTPs (ATP, GTP, TTP, CTP), MgCl2 , lOx buffer and
nuclease free water, plus ITS3 primer and NLB4 primer (Kennedy et al., 2015) for a 25pL
reaction with 1pL o f template. Reactions were run in a BioRad DNA Thermocycler for the
following times: Step 1: 95 °C for 5 minutes; Step 2: 94 °C for 30 seconds; Step 3: 55 °C
for 30 seconds; Step 4: 72 °C for 45 seconds; Step 5: repeat step 2-4 40 times; Step 6: 72
°C for 5 minutes; Step 7: hold at 4 °C.
Resulting amplifications were visualized on a 1% agarose gel, made from 0.4g
agarose, 40mL TBE buffer and 0.5 pL EtBr. Amplicons, or product DNA samples, from
the initial reaction were extremely poor, producing smears on the agarose gel. BSA (bovine
serum albumin) was added to the master mix, which helped improve yields (Kreader,
1996; Farell & Alexandre, 2012), however, the results were still highly variable.
Due to this variation, Fisher Scientific 5 PRIME™ MasterMix™ PCR Mix was
used instead o f lab-made master mix. Fisher Scientific 5 PRIME™ MasterMix™ PCR Mix
also produced less than adequate amplification and also necessitated the adding o f BSA.
All reactions were run as duplicates to increase the amount o f DNA amplicon produced. In
an effort to increase DNA yields, reaction times were also altered to: Step 1: 95 °C for 5
minutes; Step 2: 95 °C for 1 minute; Step 3: 52 °C for 1 minute; Step 4: 72 °C for 1
minute; Step 5: repeat step 2-4 29 times; Step 6: 72 °C for 5 minutes; Step 7: hold at 4 °C.
Samples that showed only one clear band when visualized on agarose were purified
using QIAquick PCR Purification Kit. Samples that showed two or more bands were set
aside for gel purification. A large 2% agarose gel was used to visualize the separate bands.
Each separate band was cut out o f the gel and purified by a QIAquick Gel Extraction Kit.

78

DNA concentration was determined by the use o f both a NanoDrop 1000 and a Qubit® 2.0
Fluorometer.

3.3.3. DNA sequencing
Purified samples were sent to the UNBC Genetics lab where they were sequenced
using an Applied Biosystems 3 130xL. A total o f 166 representative morphotype DNA
samples were sent for sequencing. Samples that were successfully sequenced were checked
using the CodonCode software (Pignone et al., 2006; CodonCodeCorperation, 2014) and
identified using The Basic Local Alignment Search Tool (BLAST). Samples with a lower
than 85% similarity match to GenBank sequences were discarded (Madden, 2002).

3.4.

Experimental results

3.4.1. Growth and biomass o f seedlings
Greenhouse grown Douglas-fir seedlings were measured for mean basal diameter
prior to destructive sampling. Murray Ridge and Pinchi Hill were the only sites that were
significantly different from all other groups (Figure 3.4.1.1). All measures o f mean basal
diameter were significantly different between sites that shared parent material.

79

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's Ridge

Tezzeron

Glacial

Murray Ridge East

Pinchi
Mountain

Ultramafic

Soil Parent M aterial

Figure 3.4.1.1: Mean basal diameter o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p < 0.001, F= 58.05, n=594).

Douglas-fir seedling height differed numerically between sites, with tallest
seedlings on glacial soil and shortest on calcareous soil (Figure 3.4.1.2). Pinchi Hill was
the only site that was significantly different from all others. Both ultramafic sites were not
significantly different from each other, as were both glacial sites. However, Tezzeron
(glacial) was also non-significant with Murray Ridge (ultramafic) and Kuzkwa
(calcareous) was non-significant to both ultramafic sites.

80

25

Kuzkwa Rd

Pinchi Hill

Spencer's Ridge

Calcareous

Tezzeron

Glacial

Murray Ridge East

Pinchi
Mountain

Ultramafic

Soil Parent M aterial

Figure 3.4.1.2: Mean seedling height o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p < 0.001, F=60.92, n=594).

Total biomass measurements were significantly different between groups that
shared parent material, meaning that each pair o f soil types (ultramafic, calcareous, and
glacial) did not have similar biomass measurements (Figure 3.4.1.3). Tezzeron and
Kuzkwa seedlings were not significantly different from each other, as were Spencer’s
Ridge and Pinchi Mountain. Only Murray Ridge and Pinchi Hill were significantly
different than all other seedlings. Pinchi Hill had the lowest amount o f total biomass with a
mean value o f 1.3 g. Spencer’s Ridge and Pinchi Mountain had the highest numeric values,
with 5.32 g and 4.85 g respectively.

81

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's
Ridge

Tezzeron

Glacial

Murray Ridge
- East

Pinchi
Mountain

Ultramafic

Soil Parent M aterial

Figure 3.4.1.3: Mean total biomass o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p <0.001, F= 118.33, n=594).

Root-shoot ratio was significantly different between soils sharing calcareous and
ultramafic parent material, but did not differ significantly between glacial soils. Pinchi Hill
and Murray Ridge were also non-significant compared to each other and to glacial soils.
Pinchi Mountain and Kuzkwa were both non-significant to each other, but significantly
different from the previous four sites (Figure 3.4.1.4). The evenness between root and
shoot values masks the large difference in total biomass between all sites (Figure 3.4.1.5)

82

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's Ridge

Murray Ridge East

Glacial

Pinchi
Mountain

Ultramafic

Soil Parent M aterial

Figure 3.4.1.4: Mean root:shoot ratio o f greenhouse grown Douglas-fir seedlings with
standard deviation. Groups that do not share letter designations are significantly different
(p <0.001, F= 14.22, n=594).
6 i

Si Shoot Weight

■ Root Weight

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's
Ridge

Tezzeron

Glacial

Murray Ridge East

Pinchi
Mountain

Ultramafic

Soil Parent M aterial

Figure 3.4.1.5: Mean root and shoot weight as part o f total biomass o f greenhouse grown
Douglas-fir seedlings (n=594).
83

3.4.2. Foliar metal content o f seedlings
Given the mass o f tissue required for foliar metals analysis, it was not possible to
generate enough replicate samples for an ANOVA. However, the data do manifest some
numerical trends. Foliar active iron for all sites ranged from 64.8 mg/Kg to 97.4 mg/Kg
with one exception. Pinchi Hill had the lowest level o f active iron, with 39.5 mg/Kg
(Figure 3.4.2.1).
Soils with both calcareous and glacial parent material produced seedlings with very
low levels o f nickel and chromium (<1.2 mg/Kg). Murray Ridge-East and Pinchi Mountain
seedlings had nickel levels o f 18.89 and 17.17 mg/Kg respectively (Figure 3.4.2.2). Needle
chromium levels were likewise high on ultramafic derived sites compared to glacial and
calcareous (Figure 3.4.2.3) with Murray Ridge levels at 1.2 mg/Kg and Pinchi Mountain
levels at 1.07 mg/Kg.

120.0

-

80.0 -

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's
Ridge

Tezzeron

Glacial

Murray Ridge
Pinchi
- East
Mountain
Ultramafic

Soil Parent M aterial

Figure 3.4.2.1: Mean foliar active iron levels from greenhouse grown Douglas-fir seedlings
with standard deviation (n=2).

84

25.00 -i

20.00 -

15.00 DA

a io.oo
5.00

*

-

-

Pinchi Hill

Spencer's
Ridge

Tezzeron

0.00

Kuzkwa Rd

Calcareous

Glacial

Murray Ridge
- East

Pinchi
Mountain

Ultramafic

Soil Parent M aterial

Figure 3A.2.2: Mean foliar concentrations o f nickel from greenhouse grown Douglas-fir
seedlings with standard deviation (n=2).

2.50

2.00
1.50

W)
I 100

a

0.50
0.00

Kuzkwa Rd

Pinchi Hill

Calcareous

Spencer's
Ridge

Tezzeron

Glacial

J

Murray Ridge
- East

Pinchi
Mountain

Ultramafic

Soil Parent M aterial

Figure 3.4.2.3:Mean foliar concentrations o f chromium from greenhouse grown Douglasfir seedling with standard deviation (n=2).

85

3.4.3. ECM abundance and frequency
Overall, 15 morphotypes were described based on a total o f 120 seedlings,
representing 22039 root tips. O f those 15 morphotypes (Appendix 3), 12 were
basidiomycetes and 3 were ascomycetes. Many morphotypes were identified to the genus
level including Cenococcum, Rhizopogon, and Tuber. Some o f the morphotypes were only
identifiable to families, such as Thelephoraceae. One morphotype, name Blonde because o f
a pale, thready mantle, was not identifiable to any taxonomic level. O f all root tips studied,
only 12% (n= 2641) were classified as non-mycorrhizal. Pinchi Hill (calcareous) had the
highest frequency o f seedlings with uncolonized tips (90%) while Kuzkwa (calcareous)
had the lowest frequency (50%).

Only three morphotypes (Cenococcum geophilum, Rhizopogon cf. villosulus, and
E-strain) were ubiquitous on all soil types and present on a large number o f seedlings. O f
the three, E-strain was the most prevalent, found on 94.2 % o f all seedlings morphotyped
(Table 3.4.3.1). Rhizopogon cf. villosulus was the next most prevalent (80%) and
Cenococcum geophilum was the third most prevalent (40%). All other morphotypes were
found only on 6 seedlings or less and are considered rare.

86

Some morphotypes were limited to a few seedlings, but present in fairly large
numbers, like the Hebeloma-Amphimena type (4 seedlings, 295 root tips). Others were
present in very small numbers, including Thelephoraceae/Tomentella-3 and 4 (1 seedling,
6 root tips, 1 seedling, 7 root tips). Both Thelephoraceae/Tomentella-3 and
Thelephoraceae/Tomentella-A were restricted to Pinchi Mountain. Russula/Lactarius-2 was
restricted to the Kuzkwa site. In contrast to ECM with more patchy distribution, Tuber was
restricted to only ultramafic sites (Table 3.4.3.1 and Figures 3.4.3.1 -F).

1 mm

Figure 3.4.3.1: Dissecting microscope photos o f common and rare morphotypes. A:
Rhizopogon cf. villosulus, B: E-strain, C: Cenococcum geophilum, D: HebelomaAmphinema type, E: Russula/Lactarius-2, and F: Tuber sp.

87

Figure 3.4.3.2: Compound microscope images o f ECM hyphal and mantle structures. Top
left, A: Hebeloma-Amphinema like emanating hyphae, note the clamp connection, bottom
left C: Hebeloma-Amphinema sporocarp type. Top right, B: E-strain inner mantle showing
labyrinthic pattern, bottom right, Hebeloma-Amphinema sporocarp found fruiting in soil
from Murray Ridge-East, D: Rhizopogon cf. villosulus emanating hyphae, note the elbowtype bend on the pigmented hypha (arrow).

88

In terms o f abundance o f mycorrhizal tips, only Rhizopogon cf. villosulus differed
significantly between sites (P<0.001, F=8.35) ranging from a high o f 31.5% on Kuzkwa
(calcareous) to a low o f 6.8% on Murray Ridge-East (ultramafic) (Table 3.4.3.1). E-strain
was consistently abundant for all sites, ranging from 52% on Tezzeron (glacial) to 74.4%
on Murray Ridge-East. While glacial and ultramafic derived soils were not significantly
different, the calcareous derived sites (Kuzkwa and Pinchi Hill) were significantly different
from one another. This pattern was repeated for the number o f uncolonized tips as well. All
other morphotypes had numerical, but non-significant, differences.

89

Table 3.4.3.1: Seedling level comparison o f frequency and % abundance o f ECM root tips with standard error o f the mean (unshared
letters within rows denote significance). ANOVA used for comparison o f mean values with a post-hoc Tukey HSD test across sites.
ECM morphotypes are arranged in decreasing frequency rank. The P value represents the significance level (a=0.05, n=20).
C a lc areo u s
S pencer's

T reatm en t
K uzkw a

E ffect

U ltram afic

Glacial

Ridge

P in c h i Hill

T ezzaron

M u rra y

P in ch i

R id g e E a s t

M o u n ta in

ECM M o rp h o ty p e

F

P

A bundance

F req

A bundance

Freq

A bundance

F req

A bundance

Freq

A bundance

F req

A bundance

U nco lo n ized

6.53

0

4.2 (1.7)a

50

33.9 (9.2)c

90

8.2 (2.9)ab

75

26.7 (5.6)bc

70

4.8 (1.4)a

55

11.7 (3.7)ab

75

E -strain

2.27

0.0518

53.8 (3.7)

100

54.1 (9.2)

75

59.7(5.7)

95

5 2.0(4.3 )

95

74.4 (6.0)

100

6 6 .4 (4 .1 )

100

8.35

0

F req

31.5 (4.0)b

95

6 .9 (3.8)a

15

2 3 .1 (3.5)b

100

19.8 (3.7)ab

85

6.8 (1.5)a

85

18.7 (2.7)ab

100

T h e le p h o r a c e a e /T o m e n te lla -I

0.9 (0.9)

5

2.4 (2.4)

5

4.8 (3.3)

20

O.O(O.O)

0

0.0 (0.0)

0

0 .0 ( 0 0 )

0

H e b e lo m a -A m p h in e m a like

0.2 (0.2)

5

0.0 (0.0)

0

3.2 (3.2)

5

O.O(O.O)

0

4.5 (3.5)

10

O.O(O.O)

0
55

R h iz o p o g o n cf. v illo s u lu s

C e n o c o c c u m g e o p h ilu m

3.3 (2.6)

45

0.3 (0.3)

5

1.0 (0.3)

70

0 .9 ( 0 8 )

35

0.4 (0.3)

30

1.0 (0.4)

Tuber sp .

0.0 (0.0)

0

0.0 (0.0)

0

0 0 ( 0 .0 )

0

O.O(O.O)

0

2.9 (2.8)

10

1.9 (1.9)

5

S u illu s c a e r u le s c e n s

0.3 (0.3)

5

2.4(1.8)

10

0 0 ( 0 .0 )

0

0.5 (0.5)

5

0.2 (0.1)

10

0 .0 ( 0 0 )

0

R h iz o p o g o n s u b c a e r u le s c e n s /

B londe

0.0 (0.0)

0

0.0 (0.0)

0

0 0 (0 .0 )

0

0.0 (0.0)

0

3.7 (3.5)

10

O.O(O.O)

0

R u ssu la / L a c ta r iu s -l

0.2 (0.2)

5

0.0 (0.0)

0

0 .0 (0 0 )

0

0.0 (0.0)

0

2.3 (2.3)

5

0.0 (0.0)

0

R u ssu la / L a c ta riu s -2

2.1 (1.4)

10

0.0 (0.0)

0

0 0 (0 .0 )

0

0.0 (0.0)

0

O.O(O.O)

0

0.0 (0.0)

0

MRA

0.0 (0.0)

0

0.0 (0.0)

0

3.3 (3.0)

10

0.0 (0.0)

0

0.0 (0.0)

0

O.O(O.O)

0
0

R u ssu la /L a c1 a riu s-3

1.8 (1.7)

10

0.0 (0.0)

0

0 0 (0 .0 )

0

0.0 (0.0)

0

0.0 (0.0)

0

0.0 (0.0)

T h e le p h o r a c e a e /T o m e n te lla -2

1.7(1.7)

5

0.0 (0.0)

0

0 0 (0 .0 )

0

0 .0 (0 .0 )

0

0.0 (0.0)

0

0.5 (0.5)

5

T h e le p h o r a c e a e /T o m e n te lla -4

0.0 (0.0)

0

0.0 (0.0)

0

0 0 (0 .0 )

0

0.0 (0.0)

0

0 0 ( 0 .0 )

0

1.2 (1.2)

5

T h e le p h o r a c e a e /T o m e n te lla -3

0.0 (0.0)

0

0.0 (0.0)

0

0 .0 (0 0 )

0

0.0 (0.0)

0

0 .0 ( 0 0 )

0

0.1 (0.1)

5

90

The two highest ranked morphotypes were also the most abundant, together
comprising 90.5% o f the root tips sampled. E-strain colonized tips represented 68.2% o f all
tips sampled. Rhizopogon cf. villosulus was the second most prevalent and made up 22.4%
o f all sampled tips (Figure 3.4.3.3). Combined, all other root tips only made up 9.5% o f the
number sampled. O f these, nine morphotypes account for less than 1% o f sampled root tips
due to their rarity. While many other morphotypes displayed locally higher numbers on one
soil compared to another, Cenococcum geophilum was the only morphotype within the
minority 9.5% found on all six sites (Figure 3.4.3.4).

91

4000

3500

B Rhizopogon cf. villosulus

Z 1500

BE-strain

Kuzkwa

Pinchi Hill

Calcareous

Spencer's Ridge
|

Tezzeron

Glacial
Soil Parent Material

Murray Ridge- Pinchi Mountain
East
|

Ultramafic

|

Figure 3.4.3.3: Number o f ECM root tips o f the two major morphotypes, (n=22 039, both types together represent 90.5% o f all root
tips sampled).
92

600

CM RA
■ Thelephoraceae/Tomentella-4
500

- Russula/Lactarius-3
P. Tuber sp.
400

? Russula/Lactarius-2

Vi

a

•■c

O
©

'■Blonde

'S 300

■ Russula/Lactarius-1

fa.

V

XI

s9

■ Thelephoraceae/Tomentella-3

z,

200

s. H ebelom a-Am phinem a like
K Thelephoraceae/Tomentella-2
100

Cenococcum geophilum
v Thelephoraceae/Tomentella-1
___ __________

Kuzkwa

Pinchi Hill

Spencer's Ridge

Tezzeron
|

Calcareous

1

Glacial

Murray RidgeEast

|

Pinchi
Mountain

w Rhizopogon subcaerulescens /Suillus
caerulescens

Ultramafic

Soil Parent M aterial

Figure 3.4.3.4: Number o f ECM root tips o f the 13 minor morphotypes (n=22 039, these types together represent 9.5% o f all root
tips sampled).
93

3.4.4. Diversity indices and rank abundance curves
Rank abundance curves show biodiversity o f the community and the relative role
that each morphotype plays (Magurran, 2004; Kindt & Coe, 2005). Based on the sampled
numbers, the rank abundance curve is plotted with an exponential curve (Figure 3.4.4.1).
The two highest points represent the most frequent and abundant ECM fungi, while the
two lowest points represent the least frequent and abundant colonizers. Relative abundance
o f each site is represented in Figure 3.4.4.2. For all sites the meeting o f the x-axis and the
curve represent the morphotypes that were not present on the site. The steepness o f the
curve indicates lower evenness of species, therefore, Kuzkwa has a more even distribution
o f species than Tezzeron.
4.5

0.5

0
0

2

4

6
8
10
Morphotype Abundance Rank

12

14

16

Figure 3.4.4.1: Combined log-transformed rank-abundance curve. E-strain begins the curve
with the highest rank. Table 3.4.3.1 lists the morphotypes in their pooled rank order,
corresponding to the y-axis o f this figure.

Kuzkwa
3.5
i Hill
Spencer's Ridge
S2.5
■*—Murray Ridge-East
a.
Pinchi Mountain
9 1.5

0.5

0

2

4

6

8

10

12

14

16

Morphotype Abundance Rank
Figure 3.4.4.2: Site-level comparison rank-abundance curves o f ECM morphotypes o f each
site. Log-transformed abundance is plotted against the morphotype rank o f each site. A
point intersecting with the x-axis indicates absence o f the morphotype with the given rank.

95

Both E-strain and Rhizopogon cf. villosulus had very high percent abundance
compared to the low abundance o f other ECM fungi. Pinchi Hill had the highest proportion
o f uncolonized tips (over 47 %, or 1422 root tips) with four trees completely uncolonized.
E-strain is the most evenly abundant morphotype on all sites. Many o f the other sites had
numeric, but not statistically significant variation in levels o f abundance. For example,
Rhizopogon subcaerulescens/Suillus caerulescens was only present on Kuzkwa, Tezzeron,
and Murray Ridge. O f these three occurrence o f Rhizopogon subcaerulescens/Suillus
caerulescens, Tezzeron and Murray Ridge had much higher percent abundances with
4.11% and 3.45% compared to only 0.2% for Kuzkwa (Figure 3.4.4.3).

96

% Abundance

Uncolonized Tips
E-strain
Rhizopogon cf. villosulus
Thelephoraceae/Tomentella-1

i Kuzkwa

Hebeloma-Amphinema like
i Pinchi Hill

Cenococcum geophilum
Tuber sp.

i Spencer's Ridge

Rhizopogon subcaerulescens/ Suillus caerulescens
• Tezzeron

Blonde
Russula/Lactarius-1

I Murray Ridge-East

Russula/Lactarius-2
MRA

sPinchi Mountain

Russula/Lactarius-3
Thelephoraceae/Tomentella-2
Thelephoraceae/Tomentella-4
Thelephoraceae/Tomentella-3
Figure 3.4.4.3: Rank percent abundance o f morphotypes per site. Morphotypes are ranked from low est to highest abundance
(proportion o f the community made up by each morphotype per site). Non-mycorrhizal tips are also represented (n= 22 039).

97

The number o f morphotypes found on seedlings from each site is an indicator o f
species richness. There is a wide range o f species richness across sites. For instance, the
glacial site Tezzeron had the lowest number o f ECM with only 4 morphotypes identified.
In contrast, Kuzkwa, a calcareous site, had the highest number o f morphotypes with 10
ECM types. Both ultramafic sites had more morphotypes than the glacial sites and Pinchi
Hill, and less than Kuzkwa (Figure 3.4.4.4).

i■

________________
___
MMbsa___

Kuzkwa

Pinchi Hill

Calcareous

Spencer's
Ridge

wmm______________ i
Tezzaron Murray RidgeEast

Glacial

Pinchi
Mountain

Ultramafic

Parent material

Figure 3.4.4.4: Number o f ECM morphotypes per site as a measure o f species richness.

98

Four diversity indices were compared to gauge the level o f diversity found on all
sites. Site level comparisons used the number o f morphotypes found on all seedlings o f a
particular sites and pooled the data. Site level indices are therefore not statistically
comparable. Seedling level comparisons use each individual seedling to generate an index
value. These values are then pooled on the basis o f site and analyzed using ANOVA
(Robertson, 2003).
ECM diversity was usually highest at the site level on the Kuzkwa site, in contrast
to the other calcareous site, Pinchi Hill, which usually had the lowest diversity. Mid-level
diversity was found on both glacial and ultramafic derived sites (Table 3.4.4.1). ECM
diversity compared at the seedling level was usually highest on the Kuzkwa site, in
contrast to the other calcareous site, Pinchi Hill, which usually had the lowest diversity
based on pooled values. Mid-level diversity was found on both glacial and ultramafic
derived sites. All diversity indices showed significance between sites (PO.OOl).
The M argalef index, which is a measure o f species richness, gives Spencer’s Ridge
as the site with the highest richness at the seedling level. Spencer’s Ridge had the highest
mean number o f mycorrhizal species per tree (3 species o f ECM). This is in contrast to the
site level species richness, as Kuzkwa had the higher number o f morphotypes (10 species)
compared to Spencer’s Ridge (6 species).

99

Mean Gini-Simpson index values showed a significant difference between both
calcareous sites (Table 3.4.4.2). The mean Shannon and Shannon evenness indices also
separated out Kuzkwa and Pinchi hill from each other. The M argalef index based on
pooled ECM morphotypes showed a much more even level o f diversity than the total site
data. Both types o f indices showed slightly higher levels o f diversity on the Kuzkwa sites
in comparison to all other. Pinchi hill also remained the site with the lowest diversity in
most cases.
Table 3.4.4.1: Comparison o f four diversity indices by using pooled ECM root totals from
each tree (Gini-Simpson, Shannon, Shannon Evenness, and Margalef) for each site (n=20).
Calcareous
Kuzkwa

Glacial

Ultramafic

Pinchi Spencer's
Murray Ridge- Pinchi
Hill
Ridge Tezzeron
East
Mountain

Gini-Simpson
Margalef

0.58
1.09

0.48
0.54

0.52
0.61

0.42
0.37

0.41
0.86

0.39
0.73

Shannon
Shannon Evenness

1.13
0.49

0.94
0.58

1.02
0.57

0.68
0.49

0.94
0.45

0.70
0.36

100

Table 3.4.4.2: Comparison o f four diversity indices using individual tree data for each site (Gini-Sim pson, Shannon, Shannon
Evenness, and Margalef). Means were compared by ANOVA with a post-hoc Tukey HSD test. Unshared letters denote significance
(n=20).
Glacial
Spencer's Ridge
Tezzaron

Ultramafc
Murray Ridge-East
Pinchi Mountain

Site

F

P

Calcareous
Pinchi Hill
Kuzkwa

Mean
Species
Richness

15.1

0

2.8 (0.2)ab

1.1 (0.2)

3.0 (0.1)b

2.2 (0.2)a

2.6 (0.3)ab

2.8 (0.1)ab

Mean Gini11.94
Simpson

0

0.476
(0.023)a

0.113 (0.046)c

0.376 (0.029)a

0.331 (0.042)ab

0.217 (0.042)bc

0.341 (0.033)ab

Mean
Shannon

12.65

0

0.759
(0.049)b

0.168 (0.068)c

0.598 (0.040)ab

0.505(0.060)a

0.388 (0.069)ac

0.558 (0.047)ab

Mean
Shannon
Evenness

11.54

0

0.779
(0.032)b

0.218 (0.088)c

0.554 (0.040)ab 0.598 (0.070)ab

0.360 (0.052)ac

0.583 (0.048)ab

Mean
Margalef

16.03

0

2.659
(0.209)ab

0.867 (0.170)

2.810 (0.073)b

2.404 (0.256)ab

2.559 (0.143)ab

101

2.014(0.167)a

3.4.5. Two-way cluster analysis
Morphology data was compared using the relative Sorensen index to test for
similarity between groups. Figure 3.4.5.1 shows clustering using presence-absence data
(black and white), while Figure 3.4.5.2 shows clustering with relative abundance data
(shading). Data were compiled into a dendrogram using a farthest neighbour clustering.
Sites appear do not cluster closely according to their parent material. Murray Ridge-east is
most distant from all other sites. Pinchi Mountain and Tezzeron appear to have the greatest
similarity to each other (Figure 3.4.5.1).
Rhizopogon cf. villosulus shares similarity between Tezzeron, Pinchi Mountain and
Spencer’s Ridge. Tuber sp. is shared only between ultramafic sites, and is present in higher
numbers on Murray Ridge-East. The two morphotypes with the highest abundance,
Rhizopogon cf. villosulus and E-strain, are similar in distribution and are clustered together
(Figure 3.4.5.2).

102

CK=K«zkwa
CP=Piiiclii Hill
LS=Spencei's Riclge
LT=Tezzeron
SM=Mmray Ridge-East
SP=Pmclu Mountain

A = Thelephoraceae/Tomentella-l
Rhizopogon subcaerulescens/
® ~ Suillus caemlescem
C = Cenococcum geophilum
D = Rhizopogon cf.villosulus
E = E-strain
F = Thelephoraceae/Tomeniella-2
G = Hebeloma-Amphinema like
H = Thelephbraceae/Tomenlella-3
t = Russula/Lactarius-J
J = Blonde
K= Russula/Laaarius-2
L ='Tuber sp.
M = Rmsula/Lactarius-3
N = ThelephordceaefTomentelld-4
O = MRA
Un = Uncolonized tips

CM

O

m

Matrix Coding
IPresence

o
o

Q Absence

Information Remaining (%)
0
L.

25

50

75

1
< O m O Q U J D u .s :5 C 5 _ -5 _ iT Z

100
i

- CK
■ C P

LS
LT
SP
SM

Figure 3.4.5.1: Two-way cluster analysis using a relative Sorensen index showing
presence-absence o f fungal species for comparison and using furthest neighbour clustering.
Sites are represented by two-letter codes, while morphotypes are represented by single­
letter codes. The designation “Un” represents uncolonized root tips (n= 22 039).

103

CK=Cnzkwa
CP=Pinchi Hill
LS=Spencer's Ridge
LT=Tezzeron
SM=Mtraay Ridge-East
SP=Piiielii Mountain

A= ThelephoraceaeJTomentella-1
Rhizopogon subcaerulescens /
® ~ Suillus caerulescens
C —Cenococcum geophilum
D = Rhizopogon cf villosulus
E = E-strain
F = Thelephoraceae/Tomeniella-2
G = Hebeloma-Amphinema like
H = Theiephoraceae/Tomentella-3
I = Russula/Lactarius-1
J = Blonde
K - Russula/Lactarius-2
L = Tuber sp.
M = Russula/Lactarm-3
N — Thelephoraceae/TomemeIla-4
0 = MRA
Un = Uncolonized tips

m

m

m

Matrix C oding

o
o J
T“

Information Remaining (%)
0

25
_ i_

50

75

c

< O c D O Q u J Z ) u - ic S O _ - ) _ ix z :

100

mmJ L m

Figure 3.4.5.2: Two-way cluster analysis using a relative Sorensen index showing
relativized values for the number o f ECM colonized root tips for comparison and using
furthest neighbour clustering. Sites are represented by two-letter codes, while morphotypes
are represented by single-letter codes. The designation “Un” represents uncolonized root
tips (n=22 039).

104

3.4.6. Confirmation o f identity using DNA
DNA extraction and amplification were used to further clarify the taxonomic
resolution o f the selected morphotypes for this study. Many o f the morphotypes were
identifiable to their genus level, including Rhizopogon, Suillus, and Tomentella. Species
level identification was achieved for both the Tuber type (confirmed at Tuber anniae) and
the Hebeloma-Amphinema type (Inocybe abjecta). Out o f 166 DNA samples sent for
sequencing, only 94 returned sequence data. The other 72 samples did not produce
readable data. O f the 94 returned sequences, only 69 had a strong enough signal and little
to no evidence o f a contaminating DNA sequence.
Out o f 14 morphotypes sent for sequencing, 8 produced usable sequence data. All
other morphotypes tested did not sequence well enough to produce a readable DNA trace
(Table 3.4.6.1). The morphotype Rhizopogon subcaerulescens/Suillus caerulescens
encompassed both Suillus caerulescens and Suillus lakei. The Rhizopogon cf. villosulus
type also encompassed Rhizopogon subclavitisporus, Rhizopogon pedicellus, and
Rhizopogon villosulus. These two morphotypes represent species-complexes and were
common to all sites on which the original morphotype was found. Overall, a total o f 18
ECM species were determined in this study using combined morphological and DNA
methods.

105

Table 3.4.6.1: Comparison o f DNA identity and morphotyping identification. DNA
sequences were compared with sequences from BLAST.
M orphotype
Label

Possible Identity

DNA Confirmed Identify

Tomentella sp,
Suillus caerulescens/Suillus lakei

Type C

Thelephoraceae/Tomentella-1
Rhizopogon
subcaerulescens/Suillus
caerulescens
Cenococcum geophilum

TypeD

Rhizopogon cf. villosulus

Rhizopogon subclavitisporus, Rhizopogon
pedicellus, Rhizopogon villosulus

TypeE
Type F
Type G

E-strain
Thelephoraceae/Tomentella-2
Hebeloma-Amphinema like

I n o c y b e a b j e c ta

Type H
Type I
Type J

Thelephoraceae/Tomentella-3
Russula/Lactarius-1
Blonde
Russula/Lactarius-2
Tuber sp.
Russula/Lactarius-3

Poor Signal
Poor Signal
Poor Signal
Poor Signal
Tuber anniae
Poor Signal

Thelephoraceae/Tomentella-4
Mycelium radicis atrovirens
(MRA)_________________

Tomentella sp.

Type A
Type B

TypeK
Type L
Type M
TypeN
Type 0

3.5.

Cenococcum geophilum

Wilcoxina sp.
Poor signal

n/a

Discussion

3.5.1. Impacts on seedlings grown on ultramafic, calcareous, and glacial soils
Seedlings displayed varied biomass responses to the different soil types. For all
biomass measures taken, trees grown in the same soil type often differed between sites.
This further supported the choice to not pool data on the basis o f soil type. Soils are
inherently variable and therefore, other properties such as texture may be more important
than chemical composition. For example, trees grown on Pinchi Hill (calcareous) displayed
very slow and stunted growth compared to their calcareous Kuzkwa counterparts. Pinchi
Hill seedlings had the smallest height and the smallest root mass (Figure 3.4.1.5). The root
106

systems o f the seedlings were also extremely fragile and largely uncolonized (Figure
3.4.4.3). Trees grown on ultramafic derived soils did not display signs o f growth stress
(often described as serpentine syndrome) comparable to trees grown on Pinchi Hill.
Foliar metal assessment may shed some light on the changes in growth o f the
Pinchi Hill trees. Active iron levels within the needles o f the Pinchi Hill seedlings were
extremely low when compared with all other seedlings (Figure 3.4.2.1). Iron deficiency is
often linked with leaf chlorosis and plant death (Loeppert et al., 1994; Kishchuk, 2000).
While pH values were not extremely high on Pinchi Hill, the measure o f % CaC 0 3
equivalent was much higher than all other soils (see Appendix 1). It is possible that the
specific balance o f carbonate mineralization with the Pinchi Hill soils rendered iron largely
inaccessible to the seedlings, resulting in low levels o f iron availability.
The lack o f ECM colonization may also be a factor in the lack o f success for the
Pinchi Hill seedlings. Without the fungi, iron may not have been properly mobilized to the
plants, resulting in further stress. Kuzkwa seedlings, also grown in calcareous soils,
displayed no signs o f undue stress. However, Kuzkwa also hosted the largest complement
o f mycorrhizal species. These trees also had very few uncolonized tips compared to the
Pinchi Hill trees (186 tips uncolonized compared to 1422 uncolonized tips).
It is impossible to identify the specific reasons for the relative lack o f colonization
on the Pinchi Hill seedlings, however, it is possible that some combination o f factors
within the soil severely retarded the seedlings’ ability to photosynthesize. In this case,
because the seedlings are poor providers o f sugars for mycorrhizal fungi, there is little
incentive for the fungi to colonize the root system. It has been shown in pinyon pine (Pinus
edulis Engelm.J that seedlings under attack by defoliator insects have reduced levels o f

107

mycorrhizal colonization due to the lower levels o f photosynthates produced. When foliage
returned to normal, the mycorrhizal communities likewise recovered (Del Vecchio et a l,
1993; Gehring & Whitham, 2003). The inability o f the seedlings to thrive may create a
negative feedback loop where ECM fungi will not form partnerships due to the poor health
o f the plant, which remains poor due to the lack o f nutrients supplied by the fungi.
Nickel and chromium levels within the foliage o f seedlings grown on ultramafic
soil were quite high (Figure 3.4.2.2-3). Nickel levels were much higher than chromium,
data which corresponds to established research on serpentine soils (Gough et a l, 1989).
Though not statistically assessed in this study, ultramafic seedlings were observed to
display a loss o f epinastic, or dominant leader, control (Mattheck, 1990). Seedlings
displayed bushy, bonsai-like foliage and often had extremely long lateral branches and
short leaders with set apical buds (Figure 3.5.4.1). It is possible that this morphology
resulted from the apical buds receiving more o f the heavy metals along with nutrients and
sugars usually supplied to the apical leader to encourage growth. This may have impeded
metabolic actively o f the leaders, halting growth. Due to the lack o f tissue on leaders that
had set bud, no analysis could be performed to assess potential differences in the heavy
metal levels within different tree tissues.

3.5.2. Diversity o f morphotypes and community structure
The three common species Rhizopogon cf. villosulus, Cenococcum geophilum, and
E-strain all correspond to dominant ECM species found in many other studies on Douglasfir (Parke et al., 1983; Hunt, 1992; Jones et al., 1998; Massicotte et a l, 1999; Hagerman &
Durall, 2004). Some species within the Rhizopogon genus are known to be common
mycorrhizal associates o f Douglas-flr and do not form associations with other potential
108

host species (Zak, 1971; Massicotte et al., 1994). E-strain and Cenococcum geophilum are
both generalist species that form mycorrhizas with many species o f trees (Yu et al., 2001;
Bourne et al., 2014).
My research produced ectomycorrhizal distributions for Douglas-fir that are
comparable to other areas sampled within central and eastern British Columbia. Pickles et
al. (2015) found Douglas-fir grown in soils from around the province produced ECM roots
dominated by species o f Rhizopogon and Pyronemataceae (E-strain). A species o f Tuber
was also found in the Fort St. James area. The soils in their study were separated by a
much larger geographic area, but in each case, the distribution o f fungi matched the pattern
found in our own study: dominance o f one or two types with a few rare species.
Previous research indicates that mycorrhiza may benefit from mixed stands with
succession o f one species to another, divided into early-stage and late-stage (Jones et al.,
1998; Massicotte et al., 1999). Early-stage ECM fungi are readily able to colonize host
roots from soil inoculum in areas under recent disturbance where refuge plants are few.
Late-stage fungi cannot form associations directly from spores, but instead require a pre­
existing living host to support a living hyphal network that will colonize new seedlings
root systems (Jones et a l, 1998). Members o f the genus Russula, for example, belong to
the group o f late-stage colonizers (Jones et al., 1998) and may have appeared less
prevalent in this study because o f the use o f monoculture pots.
The three ubiquitous ECM morphotypes may represent early-stage colonizers, ones
that are best adapted to colonize seedlings quickly. This adaptation explains the high
numbers and widespread nature o f these morphotypes. Out o f the other observed
morphotypes, MRA and Thelephora spp. are also part o f the early-stage colonizers, and do

109

not require refuge trees to support an establishing ECM association. Since soil collection
inherently disturbs the soil, this study may over represent the proportion o f the early-stage
fungi found on all soils.
Recent glaciation may also play a role in the low numbers o f ECM fungi retrieved
from these soils. Areas now populated by Douglas-fir were dominated by pine, spruce and
fir immediately following the last glaciation (Hansen, 1955). The recent advance of
Douglas-fir may mean that the ECM species that colonize its roots have not yet had
enough time for speciation to occur. What ECM species that are currently present may
instead be descendants o f fungi with a broad host range that were capable o f transitioning
from being spruce and fir symbionts to Douglas-fir partners.
A common problem faced by mycologists studying ECM communities is the patchy
distribution and the appearance o f rare species (Branco, 2010; Molina et a l, 2011; Barker
et a l, 2013). ECM species may be present on only one root tip, indicating that the species
is present, but providing a very small contribution to a species diversity index or an
ordination. Ultramafic soils in this study produced the rare type, Tuber anniae, but not in
numbers that could be usefully compared across sites. To ecologists, it is perhaps more
useful to document rare species o f ectomycorrhizas and treat the data as purely
observational and focus on the wider distributed species.

3.5.3. DNA sequence enhanced identification
Out o f 14 morphotypes isolated for DNA sequencing, only 8 were deemed to have
sufficiently clear sequences to confirm identification (n=69 sequences). O f those types
visually grouped together, only Rhizopogon subcaerulescens/Suillus caerulescens and
Rhizopogon cf. villosulus were found to represent 2 and 3 different species respectively.
110

Two o f the three Rhizopogon species identified (R . subclavitisporus and R.villosulus) have
been grouped previously in Rhizopogon subgen. Villosuli sect. Vinicolores (Grubisha et al.,
2002) which confirms our previous identification. The remainder o f morphotypes tested
corresponded to a single type o f DNA sequence. This identification process, while vital to
separating species complexes, continues to have complications.
Contamination was a consistent problem in the DNA samples. Growth media in
studies such as this is by necessity non-sterile. ECM fungi themselves are opportunistic
and hyphae from multiple species were often observed on one root tip at the same time.
While root tips were harvested from deionized water after first being thoroughly rinsed,
removal o f all secondary hyphae is impossible. Root tips that appeared clean under a
dissecting scope often would show other hyphae when observed under the compound
microscope. This leads to multiple signals within one DNA sample, and obscures the
desired sequence. Multiple fungal samples are also difficult to differentiate and may not
produce viable data (Avis et al., 2010). Some fungal DNA does not sequence well, as was
the case with a small unknown ascomycete found on both Spencer’s Ridge (glacial) and
Pinchi Hill (calcareous) soil (Figure 3.5.4.1).

A secondary concern is the vast number o f DNA sequences available online.
Higher resolution curation o f DNA sequences is imperative for proper identification o f
collected sequences. For example, all types o f Thelephora identified by DNA sequences in
this study did not have a listed species name with the online sequence. Others, such as
many o f the E-strain samples either corresponded to only the genus Wilcoxina or the order
Pezizales. Morphotyping remains a vital tool in species identification. If vouchered
specimens o f ECM fungi were carefully sequences and those sequences curated,
111

identification o f species o f interest would likely become simpler, increasing the resolution
o f ECM community studies.

3.5.4. Complications and limits to greenhouse studies
Greenhouse growing conditions were selected in order to optimize growth o f
Douglas-fir, based on previous greenhouse studies (Massicotte eta l., 1999; Hagerman &
Durall, 2004). Field studies can be difficult as the age o f mycorrhizal roots is often difficult
to access. The number o f root tips recovered is often much lower than what is obtained in a
greenhouse study, produced a more diverse group o f fungi, but with far less resolution
(Branco & Ree, 2010; Branco, 2010). While colonized roots may be found, the evidence
for succession o f ECM on the same root tip (Massicotte et al., 1999) leads to the problem
o f competing DNA signals during extraction procedures. However, even with relatively
controlled conditions, things occasionally go awry. In the case o f this experiment, two
large stressors could have potentially impacted the health o f the seedlings: growth
conditions and pest presence.
Lighting and temperature were adjusted to encourage the seedlings to germinate
and then were reduced to encourage steady growth. However, due to several power failures
at the Enhanced Forestry Lab (EFL) at UNBC, temperature and lighting conditions were
reset back to the 16:8 hours o f light and darkness and 25°C o f the germination conditions.
This change may have been a contributing factor in the chlorotic tissue that was observed,
leading to preventative application o f fertilizer to prevent seedling mortality. An infestation
o f defoliating spider mites may have impacted the health o f the remaining seedlings
(Figure 3.5.4.1). Stressed and dying trees do not have the same mycorrhizal communities
as healthy trees (Del Vecchio et al., 1993) therefore, morphotyping had to be done quickly.
112

Even with the expedient harvest and characterisation o f the root systems, it is possible that
the stress on the trees masked the manifestation o f some o f the more rare potential ECM
species.

Figure 3.5.4.1: A: Seedling from Murray Ridge with lateral growth and a set apical bud. B:
Seedling from Pinchi Mountain with a swollen and bent stem. C: Small, unidentified
ascomycete, unconnected with any known morphotypes, found on two sites (Spencer's
Ridge and Pinchi Hill). D: Mature spider mite, with shed exoskeletons, webs, eggs and egg
cases.

113

3.6.

Summary
Based on morphology and DNA analysis, 3 morphotypes are present on all sites,

with 12 others present in patchy distributions across a variety o f sites. O f these
morphotypes, E-strain was present in the highest abundance. Tuber anniae was restricted
to the ultramafic sites, but this may be an artifact o f distribution o f spores, as opposed to an
indication o f an endemic species. Both ultramafic sites had higher species richness than the
glacial sites, while one o f the calcareous sites (Kuzkwa) had the greatest number o f
morphotypes present and the highest species diversity. Extreme parent material did not
usually produce low levels o f ECM diversity with one exception (Pinchi Hill-calcareous).
Douglas-fir seedling biomass varied across all soil types. No one parent material
produced consistently larger trees than another. Pinchi Hill (calcareous) seedlings were the
smallest, averaging only 1.3 g in weight, showing fragile roots and low levels o f ECM
colonization. However, even with the variations in biomass, root:shoot ratios remained
fairly consistent. Douglas-fir seedlings may also show some level o f tolerance to nickel
within the soil. Seedlings grown on ultramafic soil had elevated levels o f foliar nickel
compared with all other seedlings and still had comparable, or larger, total biomass than
their glacial and calcareous counterparts. Calcareous parent material in one case (Pinchi
Hill) caused perceived detrimental changes in biomass, but parent material in general did
not largely affect seedling biomass.

114

3.7.

References

Agerer R. (Ed.) 1987-2008. Colour Atlas ofEctomycorrhizae. 1 s t - N th ed. EinhomVerlag, Schwabisch Gmund.
Avis P, Branco S, Tang Y, Mueller GM. 2010. Pooled samples bias fungal community
descriptions. Molecular Ecology Resources 10: 135-141.
Barker JS, Simard SW, Jones MD, Durall DM. 2013. Ectomycorrhizal fungal
community assembly on regenerating Douglas-fir after wildfire and clearcut harvesting.
Oecologia 172: 1179-1189.
Barroetavena C, Cazares E, Rajchenberg M. 2007. Ectomycorrhizal fungi associated
with ponderosa pine and Douglas-fir: a comparison o f species richness in native western
North American forests and Patagonian plantations from Argentina. Mycorrhiza 17: 355—
73.
Bourne EC, Mina D, Gonsalves SC, Loureiro J, Freitas H, Muller LAH. 2014. Large
and variable genome size unrelated to serpentine adaptation but supportive o f cryptic
sexuality in Cenococcum geophilum. Mycorrhiza 24: 13-20.
Branco S. 2010. Serpentine soils promote ectomycorrhizal fungal diversity. M olecular
Ecology 19: 5566-5576.
Branco S, Ree RH. 2010. Serpentine soils do not limit mycorrhizal fungal diversity. PLoS
ONE 5: e l 1757.
Castellano M, Molina R. 1989. Mycorrhizae. The Container Tree Nursery Manual, Vol.
5.
CodonCodeCorperation. 2014. CodonCode Aligner.
Del Vecchio TA, Gehring CA, Cobb NS, Whitham TG. 1993. Negative Effects o f Scale
Insect Herbivory on the Ectomycorrhizae o f Juvenile Pinyon Pine. Ecology 74: 22972302.
Erland S, Finlay R. 1992. Effects o f temperature and incubation time on the ability o f
three ectomycorrhizal fungi to colonize Pinus sylvestris roots. M ycological Research 96:
270-272.
Farell EM, Alexandre G. 2012. Bovine serum albumin further enhances the effects o f
organic solvents on increased yield o f polymerase chain reaction o f GC-rich templates.
BM C research notes 5: 257.

115

Gehring C, Whitham T. 1991. Herbivore-driven mycorrhizal mutualism in insectsusceptible pinyonpine. Nature 353: 556-557.
Goodman DM, Durall DM, Trofymow JA, Berch SM. 1996. A manual o f concise
descriptions o f North American ectomycorrhizae including microscopic and molecular
characterization. Natural Resources Canada, Canadian Forest Service, Pacific Forestry
Centre, Victoria, BC, British Columbia Ministry o f Forests.
Goodman DM, Trofymow JA. 1998. Distribution o f ectomycorrhizas in micro-habitats in
mature and old-growth stands o f Douglas-fir on southeastern Vancouver Island. Soil
Biology and Biochemistry 30: 2127-2138.
Gough L, Meadows G, Jackson L, Dudka S. 1989. Biogeochemistry o f a highly
serpentinized, chromite-rich ultramafic area, Tehama County, California. U. S. G. S.
Bulletin 1901., U. S. Dept, o f the Interior, Washington, D. C.
Grubisha LC, Trappe JM, Molina R, Spatafora JW. 2002. Biology o f the
ectomycorrhizal genus Rhizopogon. VI. Re-examination o f infrageneric relationships
inferred from phylogenetic analyses o f ITS sequences. Mycologia 94: 607-619.
Hagerman S, Durall D. 2004. Ectomycorrhizal colonization o f greenhouse-grown
Douglas-fir (Pseudotsuga menziesii) seedlings by inoculum associated with the roots o f
refuge plants sampled from a Douglas-fir forest in the southern interior o f British
Columbia. Canadian Journal o f Botany 82: 742-751.
Hansen H. 1955. Postglacial forests in south-central and central British Columbia.
American Journal o f Science 253: 640-658.
Hartley C, Pierce R. 1917. The control o f damping-off o f coniferous seedlings. US
Department o f Agriculture Bui. 453.
Hunt GA. 1992. Effect o f mycorrhizal fu n g i on quality o f nursery stock and plantation
performance in the southern interior o f British Columbia. FRDA Report 185, Copublished
by the BC Ministry o f Forests.
Ingleby K, Mason PA, Last FT, Fleming L V. 1990. Identification o f ectomycorrhizas.
London: HMSO.
Jones M, Durall D, Harniman S, Classen D, Simard S. 1998. Ectomycorrhizal diversity
o f paper birch and Douglas-fir seedlings grown in single-species and mixed plots in the
ICH zone o f southern British Columbia. Ministry o f Forests Research Program. Extension
Note 19.
Jonsson T, Kokalj S, Finlay R, Erland S. 1999. Ectomycorrhizal community structure in
a limed spruce forest. M ycological Research: 501-508.

116

K azantseva O, B ingham M , S im ard SW, B erch SM. 2010. Effects o f growth medium,
nutrients, water, and aeration on mycorrhization and biomass allocation o f greenhousegrown interior Douglas-fir seedlings. Mycorrhiza 20: 51-66.
K ennedy NM, R obertson SJ, G reen DS, Scholefield SR, A rocena JM , T ack ab erry LE,
M assicotte HB, E gger KN. 2015. Site properties have a stronger influence than fire
severity on ectomycorrhizal fungi and associated N-cycling bacteria in regenerating postbeetle-killed lodgepole pine forests. Folia Microbiologica 60: 1-12.
K in d t R, Coe R. 2005. Tree diversity analysis: a manual and software fo r common
statistical methods fo r ecological and biodiversity studies. World Agroforestry Centre.
K ishchuk BE. 2000. Calcareous soils, their properties and potential limitations to conifer
growth in southeastern British Columbia and Western Alberta: a literature review.
Resources Canada, Canadian Forest Service, Northern Forestry Centre, Edmonton,
Alberta. Information Report NOR-X-370.
K oide R T. 2005. Nucleic acid isolation from ecological sam ples-fungal associations,
mycorrhizae. Methods in enzymology 395: 58-72.
Kolotelo D, V an Steenis E, P eterson M , B ennett R, T ro tte r D, D ennis J . 2001. Seed
handling guidebook. British Columbia Ministry o f Forests, Tree Seed Centre.
K read er CA. 1996. R elief o f amplification inhibition in PCR with bovine serum albumin
or T4 gene 32 protein. Applied and Environmental Microbiology 62: 1102—1106.
L ang C, Seven J, Polle A. 2011. Host preferences and differential contributions o f
deciduous tree species shape mycorrhizal species richness in a mixed Central European
forest. Mycorrhiza 21: 297-308.
L o ep p ert R H , W ei L C , O cum paugh W R. 1994. Soil factors influencing the mobilization
o f iron in calcareous soils, pages 343-360 in J. A. Manthey, D. E. Crowley, and D. G.
Luster, eds. Biochemistry o f metal micronutrients in the rhizosphere. Lewis publications,
Boca Raton, Florida.
M adden T. 2002. The BLAST Sequence Analysis Tool. The NCBI Handbook. Bethesda:
National Center for Biotechnology Information.
M ag u rran AE. 1988. Ecological Diversity and its Measurement. Princeton, NJ: Princeton
University Press.
M ag u rran AE. 2004. M easuring Biological Diversity. Oxford, UK: Blackwell Science.

117

Massicotte HB, Molina RL, Luoma DL, Smith JE. 1994. Biology o f the
ectomycorrhizal genus Rhizopogon: II. Patterns o f host-fungus specificity following spore
inoculation o f diverse hosts grown in monoculture and dual culture. New Phytologist 126:
677-690.
Massicotte HB, Molina R, Tackaberry LE, Smith JE, Amaranthus MP. 1999.
Diversity and host specificity o f ectomycorrhizal fungi retrieved from three adjacent forest
sites by five host species. Canadian Journal o f Botany 77: 1053-1076.
Mattheck C. 1990. W hy they grow, how they grow: The mechanics o f trees.
Arboricultural Journal: The International Journal o f Urban Forestry 14: 1-17.
McCune B, Mefford M. 2006. PC-ORD: multivariate analysis o f ecological data.
Version 5.32. MjM Software. Gleneden Beach.
Molina R, Horton TR, Trappe JM, Marcot BG. 2011. Addressing uncertainty: How to
conserve and manage rare or little-known fungi. Fungal Ecology 4: 134—146.
Molina, R., H. Massicotte, and J.M. Trappe. 1992. Specificity phenomena in
mycorrhizal symbioses: community-ecological consequences and practical implications,
357-423. In: M.F. Allen (ed.), Mycorrhizal Functioning: An Integrative Plant-fungal
Process. Chapman and Hall, NY.
Parke EL, Linderman RG, Black CH. 1983. The Role o f Ectomycorrhizas in Drought
Tolerance o f Douglas-fir Seedlings. N ew Phytologist 95: 83—95.
Pickles BJ, Gorzelak MA, Green DS, Egger KN, Massicotte HB. 2015. Host and habitat
filtering in seedling root-associated fungal communities : taxonomic and functional
diversity are altered in “ novel ” soils. Mycorrhiza. Article in press.
Pickles BJ, Pither J. 2014. Still scratching the surface: How much o f the “black box” o f
soil ectomycorrhizal communities remains in the dark? N ew Phytologist 201: 1101-1105.
Pignone M, Greth KM, Cooper J, Emerson D, Tang J. 2006. Identification o f
mycobacteria by matrix-assisted laser desorption ionization-time-of-flight mass
spectrometry. Journal o f Clinical Microbiology 44: 1963-1970.
Plouffe A. 2000. Quaternary geology o f the Fort Fraser and Manson River map areas,
central British Columbia. Geological Survey o f Canada, Bulletin 554.
Robertson SJ. 2003. Morphological and Molecular Assessment o f Ectomycorrhizal
Communities Associating with Black Spruce (Picea mariana (Mill.) Bsp) in W etland and
Upland Forests in Central British Columbia. M aster’s Thesis, University o f Northern
British Columbia.

118

S im ard SW. 2009. The foundational role o f mycorrhizal networks in self-organization o f
interior Douglas-fir forests. Forest Ecology and Management 258: S95-S107.
Sm ith JE , M olina R, H uso M M , Luom a D L, M cK ay D, C astellano M A, Lebel T,
V alachovic Y. 2002. Species richness, abundance, and composition o f hypogeous and
epigeous ectomycorrhizal fungal sporocarps in young, rotation-age, and old-growth stands
o f Douglas-fir (Pseudotsuga menziesii) in the Cascade Range o f Oregon, U.S.A. Canadian
Journal o f Botany 80: 186-204.
Sm ith SE, R ead D J. 2008. Mycorrhizal Symbiosis. Academic Press.
T rap p e J. 1977. Selection o f fungi for ectomycorrhizal inoculation in nurseries. Annual
Review o f Phytopathology 15: 203-222.
W alk er JK M , Phillips LA, Jones MD. 2014. Ectomycorrhizal fungal hyphae
communities vary more along a pH and nitrogen gradient than between decayed wood and
mineral soil microsites. Botany 92: 453-463.
Y u T, E gger K, P eterson L. 2001. Ectendomycorrhizal associations-characteristics and
functions. Mycorrhiza 11: 167-177.
Z ak B. 1971. Characterization and classification o f mycorrhizae o f Douglas fir. II.
Pseudotsuga menziesii+ Rhizopogon vinicolor. Canadian Journal o f Botany 49: 1079—
1084.

119

4. Conclusions
Interior Douglas-fir was used as a host to compare ECM diversity on
ultramafic, calcareous and glacial-derived soils. ECM communities developed on
greenhouse-grown seedlings were assessed using a combination o f morphological
identification and DNA sequencing. Ultramafic and calcareous derived soils did not
produce fungal communities that were depauperate compared to other sites.
Preliminary field assessments o f the Douglas-fir forest conditions also did not show
large differences between soil parent materials. Both Murray-Ridge East and Pinchi
Mountain (ultramafic) produced seedlings that were o f similar vigour and had more
morphotypes compared to the glacial sites.
Kuzkwa (calcareous) also had healthy seedlings and the highest number o f
morphotypes. Excluding Pinchi Hill (calcareous), which was discussed above, both
glacial sites had the lowest number o f morphotypes and did not display stressed
seedlings. The presence o f more fungal species on the ultramafic sites and Kuzkwa
may indicate that the trees require more fungal partners to survive on the harsher soil.
The poorer qualities o f the site may also reduce the ability o f a few morphotypes to
entirely dominate, leaving room for less prolific but more tolerant ECM species.
Seedlings in this study shared common ECM fungi found in greenhouse studies o f
other parts o f British Columbia, but did not have the same diversity o f ECM as field
studies on Douglas-fir have shown throughout North America.
Biomass o f seedlings differed across sites o f the same soil type with no clear
pattern. While Douglas-fir appeared largely resistant to the chemical signatures within

120

the soil, Pinchi Hill was the only site to produce severely stunted and stressed trees,
while also showing the least amount o f ECM colonization. Based on these differences,
knowledge o f underlying bedrock and resultant soils will most likely be o f some
benefit to afforestation efforts, but therefore necessitates on the ground assessment o f
habitat. Given the uneven history o f disturbance on all o f the sites, it is unlikely that
any site faced complete removal o f all Douglas-fir and understory plants, and by
extension, the removal o f live ECM networks, in the last 100 years.
Given the extreme stress observed in the greenhouse-grown Pinchi Hill
seedlings, it is possible that these sites with extreme soils may have difficulty with
seedling recruitment after complete removal o f all trees, whether by logging or by fire.
Without mature trees or understory plants to house living mycorrhizal networks, new
seedlings under the duress o f high calcium levels may not be capable o f initiating
mycorrhizal relationships with the early-stage ECM species found in this study. Mature
Douglas-fir are already often left as seed sources when logging in central BC, but
special attention should be given to the distribution and protection o f reserve trees and
advance regeneration on soils derived from calcareous or ultramafic bedrocks (see
Figure 2.3.3.1 for Douglas-fir stands in question).
Seedlings grown on ultramafic soils were also shown to have uptake o f nickel
into their foliage. While plants grown on serpentine soils have been known to transport
nickel and chromium (to a lesser extent), it had not been demonstrated to happen in
Douglas-fir in central British Columbia before now. With increasing interest in the
conversion o f logged timber to bio-fuel, trees containing heavy metals may be a
hindrance and possibly a health hazard due to the release o f heavy metals into biomass
121

furnaces and the atmosphere. Awareness o f the bedrock underlying areas earmarked for
harvest may improve safety and functionality o f these new energy solutions.
Elucidation o f the below-ground networks o f ECM remains difficult to
quantify. In future, studies similar to this one would be augmented with both a field and
lab component. Next generation DNA sequencing (NGS) could be implemented to
quickly characterise the ECM component o f soils around seedlings grown in the field,
compared with soils from greenhouse seedlings. However, it must be stressed that the
use o f NGS will amplify all DNA within the soil, and not differentiate between spores,
inoculum, and active ECM. For this reason, morphotyping will remain a critical tool
for characterising the fungal communities involved directly on root systems.
Repeated sampling over the course o f several years may also improve the
assessment o f the ECM communities as different species may manifest over time. All
DNA samples generated should be vouchered in a university herbarium if possible,
photographed, and then sequenced. These sequences should only be added to those on
GenBank if the ECM identity is clear. Morphotype descriptions, coupled with the host
and growing conditions should also be made available. While DNA technology
continues to improve, the information gathered from a holistic study will ultimately
provide better resolution to questions o f fungal diversity.

122

Appendices

Appendix 1: Soil profile descriptions and analytical data
Site: Kuzkwa (BC 12-17)
Latitude: 54° 42’ 54.3” N
Aspect: 225°

Longitude: 124° 38’ 49.4” W Elevation: 806 mm

Slope: 70%

Slope position: midslope

Vegetation: Mature Douglas-fir stand with sparse understory
Parent material:

Limestone colluvium

Soil classification:

Orthic Eutric Brunisol

Comments: most coarse fragments in Bmk and Cca have continuous mammillated
pendants on their lower surfaces
Horizon
(Sample)
LF
(BC12-17-01)

Depth
(cm)
2-0

Bmk
(BC 12-17-02)

0-25

Cca
(BC 12-17-03)

25-70+

Description
Very dark brown (10YR 2/2 m); fresh and semi-decomposed
Douglas-fir needle litter; abrupt, wavy boundary; 0.5-3.0 cm
thick; slightly acid (pH 6.31).
Dark yellowish brown (10YR 3/4 m); silt loam; weak, fine
subangular blocky; very friable; plentiful, very fine, fine, and
medium, oblique roots; 60-70% angular gravel and cobbles;
weakly effervescent; gradual, wavy boundary; 20-35 cm
thick; mildly alkaline (pH 7.38).
Brown (10YR 5/3 m); loam; single grain; loose; few, very
fine, fine, and medium, oblique roots; 80% angular gravel
and cobbles; strongly effervescent; mildly alkaline (pH 7.45).

123

Table 3.5.4.1: Kuzkwa (calcareous) soil horizon chemical data showing Iron, Aluminum,
and Silicon concentrations by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*
__________________________________________________ %_________________________________
Silt
Sam ple No. Horizon Depth (cm) Sand
Clay Org-C Tot-N Tot-S C aC 0 3-eq
LF

38.79

BC12-17-01
BC12-17-02

Bmk

2-0
0-25

35.9

BC12-17-03

Cca

25-70+

41.8

50.3
47.2

Sam ple

Horizon

Depth (cm)

Al3*

E xchangeable (cmol (+) 1 kg)
Mg
Ca2*
K*
Mn2*
Fe3*

BC12-17-01
BC12-17-02
BC12-17-03

LF
Bmk
Cca

2-0
0-25
25-70+

0.031
0.003
0.001

70.75
17.71
16.44

< 0.001
<0.001

0.97
0.13
0.07

Sam ple

Horizon

Depth (cm)

Alp

Al0

Fep

BC12-17-01
BC12-17-02

LF
Bmk

0.041

0.243

BC12-17-03

Cca

2-0
0-25
25-70+

0.032

0.239

13.8
11.0

0.007

1.99
1.34

0.1075
0.0082

4.35

0.056

0.0042

8.17

0.41

0.244
0.001
< 0.001

Fe0

Fed

Sip

0.072

0.328

0.044

0.315

1.372
1.049

0.081
0.094

pH

6.62

1.360
0.087

2.62
0.49

pH
(H20 )
7.71
7.87

Na*

Sum

0.054
0.020

74.68
18.36

0.021

16.95

%

Alp + Fep Fep/Fed

0.112
0.076

0.24
0.30

*Org-C=organic carbon, Tot-N= total nitrogen, Tot-S=total sulfur. Subscripts for Al, Fe,
and Si extractions: p = pyrophosphate, o=oxalate, d=dithionite.

124

Figure 3.5.4.1: Soil profile from Kuzkwa (calcareous) showing LF, Bmk, and Cca
horizons.

125

Site:

Pinchi Hill (BC10-12)

Latitude: 54° 34’ 41.5” N
Aspect: 225 0

Longitude: 124° 29’ 7.6” W Elevation: 804 m

Slope: 45%

Slope position: midslope

Vegetation: Open Douglas-fir forest, with shrubby understory
Parent material:

Limestone / dolomite colluvium.

Soil classification:

Orthic Melanie Brunisol

Horizon
(Sample)
LF

Ahk
(BC10-12-01)

Depth
(cm)
2-0
0-15

Description
Douglas-fir needle litter and partially decomposed organic
matter; abrupt, wavy boundary; 2 cm thick.
Very dark grayish brown (10YR 3/2 m); sandy loam; weak,
fine and medium granular; very friable; abundant, very fine,
fine, and medium, oblique roots; 50% angular gravel; weakly
effervescent; gradual, wavy boundary; 12-20 cm thick;
m ild ly a lk a lin e (p H 7 .4 7 ).

Bmk
(BC10-12-02)

15-40

Brown (10YR 4/3 d); sandy loam; weak, fine and medium
granular; very friable; abundant, very fine, fine, and medium,
oblique roots; 70-80% angular gravels; weakly effervescent;
gradual, wavy boundary; 20-30 cm thick; mildly alkaline (pH
7.65).
Ck
40-65+ Brown (10YR 5/3 d); sandy loam*; single grain; very friable;
plentiful, very fine, fine, and medium, oblique roots; 90%
(BC10-12-03)
angular gravels and cobbles; moderately effervescent; mildly
alkaline (pH 7.61).
*field texture estimate only; insufficient sample available for analysis.

126

Table 3.5.4.2: Pinchi Hill (calcareous) soil horizon chemical data showing Iron,
Aluminum, and Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*
_________________________________________________ %
Clay
Silt
Sample No. Horizon Depth (cm) Sand

Org-C Tot-N

Tot-S

CaC03-eq

pH
(H20 )

BC10-12-01

Ahk

0-15

69.3

25.8

5.0

8.31

0.413

0.0493

20.37

7.65

BC10-12-02

Bmk

15-40

64.2

27.4

8.4

3.22

0.217

0.0335

20.34

7.80

BC10-12-03

Ck

40-65+

n.d

n.d.

n.d.

3.95

0.187

0.0241

18.96

7.72

Al3*

Ca2*

Exchangeable (cmol (+) / kg)
K*
Mg
Fe
Mn2*

Na*

Sum

Sample

Horizon Depth (cm)

BC10-12-01
BC10-12-02

Ahk
Bmk

0-15
15-40

0.011
0.002

46.989
23.504

<0.001
<0.001

0.183
0.063

11.537
6.026

0.001
< 0.001

0.040
0.080

58.762
29.676

BC10-12-03

Ck

40-65+

0.008

24.177

<0.001

0.072

8.037

< 0.001

0.041

32.335

Alp

Al0

Fep

Fe»

Fed

Sip

pH

%

Sample

Horizon Depth (cm)

Alp + Fep Fep/Fed

BC10-12-01

Ahk

0-15

0.254

0.388

0.241

0.426

0.792

0.037

0.495

0.54

BC10-12-02

Bmk

15-40

0.175

0.260

0.094

0.168

0.544

0.036

0.269

0.31

BC10-12-03

Ck

40-65+

0.138

0.211

0.082

0.162

0.542

0.031

0.220

0.30

*Org-C=organic carbon, Tot-N= total nitrogen, Tot-S=total sulfur. Subscripts for Al, Fe,
and Si extractions: p = pyrophosphate, o=oxalate, d—dithionite.

127

Figure 3.5.4.2: Soil profile from Pinchi Hill (calcareous) showing LF, Ahk, Bmk and Ck
horizons.

128

Site: Spencer’s Ridge (BC12-19)
Latitude: 54° 21’ 28.7” N
Aspect: n/a

Longitude: 124° 20’ 9.7” W Elevation: 821m

Slope: level

Slope position: crest

Vegetation: Mature Douglas-fir forest, with some paper birch
Parent material:

Gravelly sandy glaciofluvial ridge (esker)

Soil classification:

Orthic Dystric Brunisol

Comments: Discontinuous, broken Aej horizon < 1 cm thick.
Horizon
(Sample)
Ln
(BC 12-19-01)

Depth
(cm)
4-3

Fm
(BC 12-19-02)

3-0

Bm
(BC 12-19-03)

0-40

BC
(BC 12-19-04)

40-60

C
(BC 12-19-05)

60-85+

Description
Fresh Douglas-fir and paper birch leaf litter, minor
feathermoss cover; abrupt, smooth boundary; 1 cm thick;
extremely acid (pH 4.10).
Very dark brown (7.5 YR 2.5/2 m); semi-decomposed
organic matter, with pockets o f brown rotted wood up to 5
cm thick; abundant mycelia; compact matted; abundant, very
fine and fine, horizontal and oblique roots; abrupt, wavy
boundary; 2-5 cm thick; strongly acid (pH 5.53).
Brown (7.5YR 4/4 m); loamy sand; weak, fine and medium
subangular blocky; very friable; abundant, very fine, fine,
and medium, oblique, and few, coarse, oblique roots; 50%
rounded gravel and cobbles; gradual, wavy boundary; 35-45
cm thick; very strongly acid (pH 4.91).
Yellowish brown (10YR 5/4 m); loamy sand; single grain;
very friable; plentiful, very fine, fine, and medium, oblique,
and few coarse, oblique roots; 60-70% rounded gravel and
cobbles; gradual, wavy boundary; very strongly acid (pH
4.57).
Light yellowish gray (2.5Y 6/2 m); sand; single grain; loose;
few, fine, medium, and coarse, oblique roots; 60-70%
rounded gravel and cobbles; strongly acid (pH 5.10).

129

Table 3.5.4.3: Spencer's Ridge (glacial) soil horizon chemical data showing Iron,
Aluminum, and Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*
pH
(H20 )

pH
(CaCI2)

0.0746
0.1500
0.0048

4.28
5.86
5.57

0.0057
0.0038

5.29
5.85

4.10
5.53
4.91
4.57
5.10

%

Sam ple No. Horizon Depth (cm) Sand
BC12-19-01
BC12-19-02
BC12-19-03
BC12-19-04
BC12-19-05

Sample
BC12-19-01
BC12-19-02
BC12-19-03
BC12-19-04
BC12-19-05

Ln
Fm
Bm
BC
C

4-3
3-0
0-40
40-60
60-85+

Horizon Depth (cm)
Ln
Fm
Bm
BC
C

4-3
3-0
0-40
40-60
60-85+

82.2

Silt

Clay

Org-C Tot-N Tot-S

3.8

52.90
47.18
0.83

0.711
1.572
0.044

3.5
1.6

0.44
0.40

0.024
0.021

C aC 0 3-eq

78.0
93.7

13.9
18.5
4.8

AT

E xchangeable cmol (+) / kg
K*
Mg2* Mn2*
Ca2*
FeJ*

Na*

Sum

6.42
2.77
0.18
0.18
0.17

0.079
0.064
0.019
0.033
0.024

30.09
93.77
4.13
4.05

0.168
0.020
0.283
0.398
0.062

14.88
83.28
3.22
2.87
3.21

0.008
0.004
0.008
0.009
0.002

5.51
6.20
0.40
0.54

3.025
1.434
0.024
0.022

0.53

0.015

Alp

Al„

Fep

Fe0

Fed

Sio

0.144
0.050
0.027

0.311
0.158
0.100

0.200

0.636

0.068
0.042

0.338
0.244

1.213
0.922
0.713

0.057
0.047
0.041

4.01

%

Sample
BC12-19-01
BC12-19-02
BC12-19-03
BC12-19-04
BC12-19-05

Horizon Depth (cm)
Ln
Fm
Bm
BC
C

4-3
3-0
0-40
40-60
60-85+

Alp + Fep Fe„/Fed

0 .3 4 4
0 .1 1 9
0 .0 7 0

0 .5 2
0 .3 7
0 .3 4

*Org-C=organic carbon, Tot-N= total nitrogen, Tot-S=total sulfur. Subscripts for Al, Fe,
and Si extractions: p = pyrophosphate, o=oxalate, d=dithionite.

130

Figure 3.5.4.3: Soil profile from Spencer's Ridge (glacial) showing Ln, Fm, Bm, BC, and
C horizons.

131

Site:

Tezzeron (BC12-16)

Latitude: 54° 43'3.8" N
Aspect: n/a

Longitude: 124° 20'50.1" W Elevation: 868 m

Slope: level

Slope position: crest

Vegetation: Mature Douglas-fir forest
Parent material:

Fine gravelly morainal blanket over bedrock ridge.

Soil classification:

Brunisolic Gray Luvisol

Horizon
(Sample)
S/Ln
(BC12-16-01)
Fm
(BC 12-16-02)

Depth
(cm)
5-3

Ae
(BC 12-16-03)

0-25

Bm
(BC 12-16-04)

10-30

Bt
(BC 12-16-05)

30-75+

3-0

Description
Feathermoss and needle litter; abrupt, smooth boundary; 1-3
cm thick; extremely acid (pH 4.36).
Very dark brown (7.5YR 2.5/3 m); abundant, very fine, fine,
medium, and coarse oblique roots; abundant mycelia;
compact matted; abrupt wavy boundary; 2-4 cm thick; very
strongly acid (pH 4.94).
Brown (10YR 5/3 m); loam; weak, fine and medium
subangular blocky; very friable; abundant, very fine, fine,
and medium, oblique, and plentiful, coarse; horizontal roots;
30% angular gravel and cobbles; gradual, irregular boundary;
10-30 cm thick; extremely acid (pH 4.50).
Brown (7.5YR 4/4 m); loam; weak, fine subangular blocky;
loose; plentiful, very fine, fine, and medium, oblique roots;
70% angular gravel [predominantly reddish oxidized
sandstone]; gradual, broken boundary; 0-25 cm thick;
extremely acid (pH 4.52).
Brown (7.5YR 5/4 m); loam; strong, fine and medium
subangular blocky; firm; few, fine and medium, oblique
roots; abundant clay films; 50% angular gravel; medium acid
(pH 5.55).

132

Table 3.5.4.4: Tezzeron (glacial) soil horizon chemical data showing Iron, Aluminum, and
Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid ammonium oxalate
extractions*
%

S am p le No. H orizon

D epth (cm )

S and

Silt

C lay

O rg-C Tot-N

Tot-S

C a C 0 3-e q

pH
(H20 )

pH
(CaCI2)
4.36
4.94

BC 12-16-01

S/Ln

5-3

54.00

0.918

0.0886

4.54

BC12-16-02

Fm

3-0

1.628

0.1513

5.26

BC 12-16-03

Ae

0-25

44.0

46.5

9.6

50.45
1.42

B C 12-16-04

Bm

10-30

51.2

37.4

11.3

1.68

BC 12-16-05

Bt

30-75+

45.0

32.1

22.9

0.74

S am p le

H orizon

D epth (cm)

a i 3+

C al*

BC12-16-01
BC 12-16-02

S/Ln

5-3

0.088

20.75

Fm

3-0

0.045

BC 12-16-03

Ae

0-25

0.369

BC 12-16-04

Bm

10-30

0.379

4.85

0.010

0.21

B C 12-16-05

Bt

30-75+

0.010

13.46

< 0.001

0.28

S am p le

H orizon

D epth (cm )

Alp

Al0

FeP

Fe0

F ed

Si0

BC 12-16-01
BC12-16-02

S/Ln
Fm

5-3
3-0

BC 12-16-03
BC 12-16-04
BC 12-16-05

Ae
Bm
Bt

0-25
10-30
30-75+

0.067

0.121
0.146
0.169

0.112
0.149
0.100

0.408
0.475
0.295

1.901
3.592
4 .923

0.022
0.031
0,062

0.063

0.0095

5.11

4 .50

0.078
0.047

0.0142
0.0056

5.10
6.26

4.52
5.55

E x c h a n g e a b le (cm ol (+) / kg)
K+
Mg** Mn2t
Fe3+

Na+

Sum

0.005

6.21

7.52

2.308

0.095

36.97

63.38

0.006

4.41

3.95

0.022

0.17

7.33
0.74

2.901
0.032

0.129
0.024

78.21
5.30

1.06

0.072

0.020

6.60

3.44

0.015

0.046

17.24

%

0.080
0.101

Alp + Fep Fe0/F ed

0 .1 7 9
0 .2 2 9
0 .2 0 1

0 .2 1
0 .1 3
0 .0 6

*Org-C=organic carbon, Tot-N= total nitrogen, Tot-S=total sulfur. Subscripts for Al, Fe,
and Si extractions: p = pyrophosphate; o=oxalate, d=dithionite.

133

Figure 3.5.4.4: Soil profile from Tezzeron (glacial) showing S/Ln, Fm, Ae, Bm, and Bt
horizons.

134

Site: Murray Ridge (BC12-18)
Latitude: 54° 29’ 55.7” N
Aspect: 225°

Longitude: 124° T 19.2” W Elevation: 827 m

Slope: 50%

Slope position: upper

Vegetation: Open stand o f scattered, low productivity Douglas-fir, with mossy and lichendominated forest floor, 5-10% exposure o f rock outcrop.
Parent material:

Shallow colluvial veneer over hummocky ultramafic bedrock.

Soil classification:

Orthic Eutric Brunisol

Horizon
(Sample)
S/L
(not sampled)
Fm
(BC12-18-01)

Description

Depth
(cm)
2-1

Feathermoss and lichen, with scattering o f Douglas-fir needle
litter.
Black (10YR 2/1 m); semi-decomposed moss, lichen, and
1-0
needle litter; abundant mycelia; non-compact matted;
plentiful, very fine and fine horizontal roots; abrupt, wavy
boundary; 1-2 cm thick; strongly acid (pH 5.23).
Ahej
0-1
Brown (10YR 5/3 m); silt loam*; weak, fine subangular
blocky; friable; abundant, very fine, fine, and medium,
(BC 12-18-02)
oblique roots; clear, broken boundary; 0-2 cm thick; strongly
acid (pH 5.33).
Brown (10YR 4/3 m); loam; weak, fine subangular blocky;
Bm
1-30
friable; abundant, very fine, fine, and medium, oblique roots;
(BC12-18-03)
abrupt, irregular boundary; 20-40 cm thick; slightly acid (pH
6.17).
R
30+
Ultramafic bedrock.
*field texture estimate only; insufficient sample available for analysis.

135

Table 3.5.4.5: Murray Ridge-East (ultramafic) soil horizon chemical data showing Iron,
Aluminum, and Silicon by sodium citrate-dithionite, sodium pyrophosphate, and acid
ammonium oxalate extractions*
__________________________________________________ %__________________________________
pH
Silt
Clay Org-C Tot-N Tot-S C aC 0 3-eq (H20 )
Sam ple No. Horizon Depth (cm) Sand
BC12-18-01
BC12-18-02
BC12-18-03

Fm
Ahej
Bm

1-0
0-1
1-30

n.d.
35.3

n.d.
41.7

Sam ple

Horizon

Depth (cm)

Al3*

E xchangeable (cmol (+) / kg)
Ca2*
K*
Mg2* Mn2*
Fe3*

BC12-18-01
BC12-18-02

Fm
Ahej
Bm

0.019
0.019
0.002

41.43
12.96
2.54

0.008
0.013
< 0.001

1.55
0.75
0.12

Alp

Al0

Fep

0.075
0.034

0.153
0.172

0.398
0.174

BC12-18-03

1-0
0-1
1-30

n.d.
23.0

49.05
12.52
3.04

1.766
0.500

0.1334
0.0437

0.175

0.0215

44.54
26.12
20.64

0.687
0.638
0.010

Fe„

Fed

Si„

1.492
1.395

2.301
2.709

0.162
0.212

pH

5.65
5.64
6.69

Na*

Sum

0.095

88.33
40.54
23.33

0.035
0.020

%

Sam ple
BC12-18-01
BC12-18-02
BC12-18-03

Horizon Depth (cm)
Fm
Ahej
Bm

1-0
0-1
1-30

Alp + Fep Fe„/Fed
0 .4 7 2
0 .2 0 8

0 .6 5
0 .5 2

*Org-C=organic carbon, Tot-N= total nitrogen, Tot-S=total sulfur. Subscripts for Al, Fe,
and Si extractions: p = pyrophosphate, o=oxalate, d-dithionite.

136

Figure 3.5.4.5: Soil profile for Murray Ridge-East (ultramafic) showing S/Ln, Fm, Ahej,
Bm, and R horizons

137

Site:

Pinchi Mountain (B C 11 -06)

Latitude: 54° 38.973’ N
Aspect:

180°

Longitude: 124° 29.293’ W Elevation: 961 m

Slope: 40%

Slope position: midslope

Vegetation: Open Douglas-fir forest, with juniper, spiraea, grass understory
Parent material:

Colluvial veneer over bedrock.

Soil classification:

Orthic Eutric Brunisol

Horizon
(Sample)
LF
(BC 11-06-01)

Depth
(cm)
3-0

Ah
(BC 11-06-02)

0-10

Bml
(BC 11-06-03)

10-28

Bm2
(BC 11-06-04)

28-53

C
(BC11-06-05)

53-70

R

70+

Description
Shrub, tree, and herbaceous leaf litter, and semi-decomposed
organic matter; non-compact matted; plentiful, very fine,
fine, and medium, oblique and horizontal roots; abrupt, wavy
boundary; 2-4 cm thick; medium acid (pH 5.68).
Very dark brown (7.5YR 2.5/2 m); clay loam; weak, coarse
granular; friable; plentiful, very fine, fine, and medium,
oblique, and few, coarse, horizontal roots; 20% angular and
subangular gravel; gradual, wavy boundary; 8-12 cm thick;
slightly acid (pH 6.25).
Brown (7.5YR 4/3 m); sandy clay loam; weak, fine and
medium subangular blocky; friable; plentiful, very fine, fine,
and medium, oblique roots; 20-30% angular and subangular
gravel and cobbles; clear, wavy boundary; 15-20 cm thick;
neutral (pH 6.58).
Brown (10YR 4/3 m); sandy loam; single grain; friable; few,
very fine, fine, and medium, oblique roots; 60-70% angular
gravel; abrupt, wavy boundary; 15-30 cm thick; neutral (pH
6.95).
Very dark gray (2.5Y 3/1 m); sandy loam; massive; friable;
few, very fine, fine, and medium, oblique roots; 50-60%
angular and subangular gravel; abrupt, wavy boundary;
neutral (pH 6.77).
Fractured bedrock.

138

Table 3.5.4.6: Pinchi Mountain (ultramafic) soil horizon major element oxides and
chemical data showing Iron, Aluminum, and Silicon by sodium citrate-dithionite, sodium
pyrophosphate, and acid ammonium oxalate extractions.*
Sam ple No. Horizon

BC11-06-01
BC11-06-02
BC11-06-03
BC11-06-04
BC11-06-05

LF
Ah
Bm1
Bm2
C

D epth (cm) S an d

Silt

%__________________________________
pH
Clay Org-C Tot-N Tot-S C aC 0 3-eq (H20 )

3-0
0-10
10-28
28-53
53-70

44.5
53.1
67.0
58.1

28.5
24.3
22.8
35.0

27.0
22.6
10.2
7.0

C az*

Fe

46.49
5.17
2.34
0.45
0.66

1.450 0.1244
0.245 0.0246
0.126 0.0124
0.031 0.0050
0.040 0.0031

cm ol (+) / kg
K*
Mgz*

5.85
6.57
6.83
7.30
7.35

Mnz*

S am ple

Horizon

D epth (cm)

Al3*

BC11-06-01
BC11-06-02
BC11-06-03
BC11-06-04
BC11-06-05

LF
Ah
Bm1
Bm2
C

3-0
0-10
10-28
28-53
53-70

0.008
0.005
< 0.001
< 0.001
< 0.001

54.78
13.31
5.86
1.89
5.19

0.010
<0.001
< 0.001
<0.001
<0.001

Sam ple

H orizon

D epth (cm)

Alp

Al0

Fep

BC11-06-01
BC11-06-02
BC11-06-03
BC11-06-04
BC11-06-05

LF
Ah
Bm1
Bm2
C

3-0
0-10
10-28
28-53
53-70

0.070
0.078
0.006
0.006

0.171
0.131
0.108
0.453

0.530 1.838 4.239 0.236
0.418 1.283 3.370 0.222
0.049 0.618 1.499 0.150
0.067 1.471 2.027 0.238

1.89 35.39 0.261
0.41 24.34 0.050
0.17 17.93 0.016
0.08 8.80 0.007
0.08 43.56 0.006

pH
(CaCI2)

Na*

S um

0.098
< 0.001
< 0.001
< 0.001
< 0.001

92.43
38.10
23.97
10.74
48.80

5.68
6.25
6.58
6.95
6.77

%
Fe0

Fep

SiQ

Alp+ F e p Fep/Fed

0.600
0.496
0.055
0.073

0.43
0.38
0.41
0.73

Major Element Oxides:

%
S a m p le No.

H orizon

D ep th (cm )

S i0 2

a i 2o 3

F e 20 3

C aO

MgO

Na20

K20

C r20 3

BC11-06-01
BC11-06-02
BC11-06-03
BC11-06-04
BC11-06-05

LF
Ah
Bm1
Bm2
C

3-0
0-10
10-28
28-53
53-70

39.9
40.6
47.8
42.0

3.14
2.60
4.14
4.88

12.75
11.75
8.37
9.78

0.72
0.37
0.68
0.45

19.25
25.10
25.10
22.10

0.38
0.25
0.70
0.06

0.30
0.20
0.43
0.12

0.57
0.47
0.32
0.30

S a m p le No.

H orizon

D ep th (cm )

T i0 2

MnO

P 2O s

S rO

B aO

LOI

T otal

BC11-06-01
BC11-06-02
BC11-06-03
BC11-06-04
BC11-06-05

LF
Ah
Bm1
Bm2
C

3-0
0-10
10-28
28-53
53-70

0.32
0.25
0.35
0.74

0.31
0.16
0.14
0.31

0.30
0.12
0.05
0.09

0.01
0.01
0.01
<0.01

0.05
0.02
0.03
0.02

22.30
18.40
12.75
18.70

100.30
100.30
100.87
99.55

%

*Org-C=organic carbon, Tot-N= total nitrogen, Tot-S=total sulfur, LOI=loss on ignition.
Subscripts for Al, Fe, and Si extractions: p = pyrophosphate, o=oxalate, d=dithionite.

139

Table 3.5.4.7: Pinchi Mountain (ultramafic) soil horizon minor elements
Minor Elements:

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ E£HL
Sample No. Horizon Depth (cm)
LF

BC11-06-01
BC 11-06-02

Ah

3-0
0-10

BC 11-06-03
BC 11-06-04

Bm1
Bm2

10-28
28-53

BC11-06-05

C

53-70

Ag

Ce

Co

Cr

Cs

Cu

Dy

Er
0.50
0.42

492

13.0

182.0

5740

1.06

29

0.84

1

168.5
320

9.5
14.8

149.5
109.0

4570
3240

0.68
0.82

26
27

0.72
1.27

1

153

18.8

123.5

3040

0.64

49

1.67

0.79
0.91

Eu

Ga

Gd

Hf

Ho

La

Lu

Mo

Nb

0-10

0.28

4.8

1.02

1.2

0.16

6.4

0.06

<2

4.9

10-28
28-53

0.26
0.37
0.75

3.6
5.5
8.2

0.97
1.50

1.1
1.6

0.14
0.26

0.06
0.11

<2
<2

2.31

1.8

0.32

4.8
7.6
9.4

0.10

<2

5.0
7.6

Sample No. Horizon Depth (cm)

Nd

Ni

Pb

Pr

Rb

Sm

Sn

Sr

Ta

BC 11-06-01
BC11-06-02

LF
Ah

3-0
0-10

6.2

2310

<5

1.46

15.4

1.16

<1

56.9

0.3

BC11-06-03
BC 11-06-04

Bm1
Bm2
C

5.5
8.1
12.0

2870
2140
2100

<5
<5
<5

1.19
1.80
2.50

10.1
16.2
5.1

1.09
1.62
2.42

<1

BC 11-06-05

10-28
28-53
53-70

1

36.2
59.6
19.1

0.3
0.3
0.5

Tb

Th

Tl

Tm

U

V

W

Y

Yb

Zn

Zr

0.14
0.13
0.21
0.29

0.97
0.76
1.35
1.18

<0.5
<0.5
<0.5

0.06
0.05
0.10
0.12

0.31
0.27
0.58

245
191
167

2

4.7
4.1
7.6

0.44
0.40
0.71

206
105
67

0.35

162

9.0

0.74

91

44
41
64
71

Sample No. Horizon Depth (cm)
BC11-06-01

LF

BC11-06-02

Ah
Bm1

BC11-06-03
BC11-06-04
BC11-06-05

Bm2
C

3-0

53-70

Sample No. Horizon Depth (cm)
BC 11-06-01
BC 11-06-02
BC 11-06-03
BC 11-06-04
BC 11-06-05

LF
Ah
Bm1
Bm2
C

4
1

Ba

3-0
0-10
10-28
28-53
53-70

<0.5

140

<1

1
1
1

3.9

Figure 3.5.4.6: Soil profile for Pinchi Mountain (ultramafic) showing LF, Ah, B m l, Bm2,
and C horizons

141

Appendix 2: Elemental analyses o f soil composites used in greenhouse experiment
Table 3.5.4.8: Chemical and elemental data from soil homogenates
Si02
A1203 Fe203 CaO
Na20 K20
Cr203 Ti02
MgO
Location
%
%
%
%
%
%
%
%
%
Kidcwa Rd
51.2
4.84
6.17
3.76
1.24
0.53
9.17
1
0.02
Pinchi Hill
66.6
11.9
1.64
1.1
0.02
5.05
2.91
1.58
0.68
Spencer's Ridge
68.6
1.38
0.91
2.62
0.03
0.74
11.35
4.35
1.49
Tezzeron
4.04
2.41
1.25
1.24
0.02
0.53
69.8
8.67
1.1
0.37
48.4
15.95
1.01
0.62
0.43
Murray Ridge - East
5.38
8.87
0.91
53.4
0.71
0.45
0.66
Pinchi Mountain
11
1.08
8.19
0.96
6.09
!
1
1
!
t
(
I
1
SrO
MnO
P205
BaO
C
S
Ba
Ce
Cr
Location
%
%
%
ppm
%
%
%
ppm
ppm
Kudcwa Rd
0.17
0.02
7.37
170
0.69
0.11
0.01
920
31.3
0.2
Pinchi Hill
0.41
0.04
0.11
2.28
27.1
160
0.01
932
210
0.14
2.62
1285
25.7
Spencer's Ridge
0.24
0.03
0.16
0.01
3.63
160
Tezzeron
0.08
0.07
0.02
610
31.6
0.19
0.01
4.72
3180
0.2
0.05
0.02
457
18.6
Murray Ridge - East
0.19
0.01
4.47
21.6
2980
Pinchi Mountain
0.25
0.21
0.01
0.07
0.02
528
Dy
ppm
3.73
2.6
2.11
3.3
1.68
1.7

La
ppm
19.3
13
12.4
16.4
8.5
9.6

Location
Kidcwa Rd
Pinchi Hill
Spencer's Ridge
Tezzeron
Murray Ridge - East
Pinchi Mountain

Nb
ppm
7.9
7.6
7.4
8
4.5
7.8

Nd
ppm
17.3
11.9
11.4
16
8
9.6

Pr
ppm
4.34
3.04
2.96
4.01
2.07
2.42

Rb
ppm
39.3
45.7
54.6
34.7
21.3
25.4

Sm
PPm
3.57
2.42
2.12
3.36
1.7
2

Sn
ppm
1
<1
1
1
<1
1

Sr
ppm
115
307
231
111
95.9
92.8

Ta
ppm
0.5
0.5
0.5
0.5
0.3
0.5

Location
Kidcwa Rd
Pinchi Hill
Spencer's Ridge
Tezzeron
Murray Ridge - East
Pinchi Mountain

Tb
ppm
0.66
0.41
0.36
0.58
0.28
0.33

Th
PPm
3.94
3.44
2.83
2.81
1.83
1.9

T1
ppm
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5

Tm
ppm
0.38
0.26
0.23
0.33
0.15
0.15

U
PPm
1.32
1.52
1.29
1.84
0.72
0.71

V
PPm
93
105
131
82
21
20

W
ppm
1
1
1
1
1
3

Y
ppm
31.1
14.8
13.3
22.3
9.4
9.7

Yb
PPm
2.27
1.83
1.48
2.06
1
0.97

.

1

!

142

Gd
PPm
4.37
2.45
2.18
3.79
1.72
2.04

Ho
ppm
0.8
0.56
0.46
0.71
0.35
0.34

Lu
ppm
0.36
0.28
0.24
0.33
0.16
0.15

!

Eu
ppm
1.03
0.76
0.6
0.86
0.45
0.65

Hf
PPm
2.7
4
3.4
2.7
1.8
1.9

Location
Kidcwa Rd
Pinchi Hill
Spencer's Ridge
Tezzeron
Murray Ridge - East
Pinchi Mountain

1

Er
ppm
2.65
1.69
1.5
2.15
1.01
1.07

Ga
ppm
11.8
14.2
13.7
10.8
6.8
8.1

Cs
ppm
2.29
2.22
3.41
1.87
1.05
1.97

1

i

Table 3.5.4.9: Chemical and elemental data from soil homogenates (continued)*
Zr
ppm
118
157
140
107
71
81

As
ppm
2.6
5.1
13.4
5.4
1.4
1.4

Bi
ppm
0.13
0.11
0.09
0.09
0.06
0.06

Hg
ppm
0.084
0.027
0.11
0.059
0.053
0.108

Sb
ppm
1.41
0.37
6.8
0.7
0.19
0.37

Se
ppm
0.6
0.3
0.3
0.5
0.3
0.4

Te
ppm
0.05
0.03
0.03
0.03
0.03
0.03

LOI
%
22.2
7.61
8.17
10.7
17.25
17.85

Total
%
101.12
99.85
100.21
100.11
99.64
100.93

Ag
Location
ppm
Kuzkwa Rd
<0.5
Pinchi Hill
<0.5
<0.5
Spencer's Ridge
Tezzeron
<0.5
Murray Ridge - East. <0.5
Pinchi Mountain
<0.5
*LOI= loss on ignition.

Cd
ppm
5.4
0.7
<0.5
0.7
<0.5
<0.5

Co
ppm
9
11
9
8
101
126

Cu
ppm
24
17
25
20
14
31

Mo
ppm
<1
<1
<1
1
<1
<1

Ni
ppm
53
22
25
44
1250
1515

Pb
ppm
9
10
9
9
5
4

Sc
ppm
10
10
10
10
11
15

Zn
ppm
385
270
138
150
121
146

Location
Kuzkwa Rd
Pinchi Hill
Spencer's Ridge
Tezzeron
Murray Ridge - East
Pinchi Mountain

143

Appendix 3: Morphotype descriptions
Table 3.5.4.10: Detailed descriptions and possible identification o f all morphotypes
Morphotype
Label

Type A

Type B

TypeC

Type D

Composite Description

Possible Identity

ECM Dark brown, monopodial with a
reflective luster, straight and
unbranched tips. Mantle has an
interlocking to non-interlocking
irregular synenchyma, 2-3 pm wide
with pigment blotches and a deep
golden brown color.
ECM Unbranched, straight,
monopodial to cluster- tuberculate,
matte bright white surface with matte
grey-purple rind. Cottony texture.
Mantle structure obscured by crystals.
Emanating hyphae hyaline and
branched 2-4 pm wide with garnet
colored crystal ornamentation and thick
cell walls. Highly differentiated
rhizomorphs, hyaline in color, + 100pm
wide with garnet crystalline deposits.
ECM Monopodial, very black with a
stringy texture. Matte luster with
copious emanating hyphae. Stellate
patterned net synenchyma. Hyphal
cells approximately 3 pm wide. Some
sclerotia visible.
ECM Monopodial to pinnate, silverywhite with a thick black/brown rind.
Texture varies from reflective to matte
and cottony. Mantle structure obscured
by thick silver-white crystals. Many
dark grey-brown branching
rhizomorphs and emanating brown
hyphae. Hyphae 2-3 pm thick with
slight globules, thick cell walls, and
knee-like bends. Rhizomorphs +90 pm
thick with some gray-silver crystals,
slight globular deposits, thick cell
walls, and knee-like bends.

144

Thelephoraceae/

Tommtella-1

Rhizopogon
subcaerulescens/Suillus
caerulescens

Cenococcum geophilum

Rhizopogon cf. villosulus

TypeE

Type F

TypeG

TypeH

Type I

Type J

ECM Reddish brown single
monopodial with a smooth texture and
reflective luster. Net prosenchyma with
hyphae approximately 6*7 pm with
some +13 pm wide.
ECM Monopodial, pale yellow-brown.
Smooth texture with a matte luster.
Interlocking to non-interlocking
irregular synenchyma with no pigment
blotches. One emanating hyphae,
unbranched, septate hyaline, clamps,
3 pm in width.
ECM Monopodial, pale yellow brown
with a very cottony and wooly texture
and a matte appearance. Mantle is a
very loose felt prosenchyma with 23pm cells wide. Emanating hyphae
branching with some anastomoses.
Pale yellow in color to hyaline, 1-3 pm
in width. Septate with clamps at almost
every septum. Differentiated
rhizomoiph with both septa and
clamps. Pale golden yellow in color.
ECM Monopodial, very dark brown
with a shiny luster. Interlocking to non­
interlocking synenchyma. Some pale
brown emanating hyphae with septa
and clamps, approximately 4 pm wide.
ECM Monopodial, pale gold-brown to
darker brown. Very smooth texture
with a matte luster. Interlocking to
non-interlocking irregular synenchyma
with cells 8-9 pm thick. Laticifers
visible. Some emanating hyphae,
unbranched, septate hyaline, 4pm in
width.
ECM Blonde, smooth, reflective
branched tip. Felt prosenchyma with
long, thin hyphae approximately 1-2
pm wide. Hartig net visible.
Unbranched emanating hyphae, hyaline
in color, 1 pm wide.

145

E-strain

Thelephoraceae/
Tomentella-2

Hebeloma-Amphinema like

Thelephoraceae/
Tomentella-2

Russula/
Lactarius-1

Blonde

TypeK

Type L

TypeM

Type N

Type O

ECM Pale-golden brown, smooth
texture with a reflective luster.
Branched tips with a monopodial
pinnate structure. Golden brown
interlocking irregular synenchyma,
cells 5-7 pm thick. Some laticifers
visible,
ECM Pale golden brown, smooth
texture with a reflective luster.
Straight, unbranched to monopodial
pinnate, some tips very long. Fine
cystidia on the tip.
ECM Monopodial, very pale goldbrown. Very smooth texture with a
reflective luster. Mantle with
interlocking to non-interlocking
irregular synenchyma, cells pale
yellow under microscope 3-4 pm in
width.
ECM Dark brown, monopodial with a
reflective luster, straight and
unbranched tips. Mantle has an
interlocking to non-interlocking
irregular synenchyma, 2-3 pm wide
with a visible Hartig net.
ECM Monopodial, dark brown with a
smooth texture and matte luster. Net
prosenchyma with some swollen cells.
Mantle hyphae 2-3 pm in width.
Branched emanating hyphae dark
brown-grey in color, finely varicose
with anastomoses._________________

146

Russula/
Lactarius-2

Tuber

Russula/
Lactarius-3

Thelephoraceae/
Tomentella-4

Mycelium radicis
atrovirens (MRA)