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ORIGINAL PAPER
Diversity and species distribution of ectomycorrhizal fungi
along productivity gradients of a southern boreal forest
J. M. Kranabetter &D. M. Durall &W. H. MacKenzie
Received: 26 May 2008 / Accepted: 9 October 2008 / Published online: 22 October 2008
#Springer-Verlag 2008
Abstract Coniferous forests with diverse ectomycorrhizal
fungus (EMF) communities are associated with nutrient-
poor, acidic soils but there is some debate whether EMF
can be equally adapted to more productive, nitrogen-rich
sites. We compared EMF species distribution and diversity
along a replicated productivity gradient in a southern boreal
forest of British Columbia (Canada). Roots from subalpine
fir (Abies lasiocarpa) saplings of the understory were
sampled and EMF species were identified by morphotypes
supplemented with ITS rDNA analysis. There were
significant changes in the distribution and abundance of
74 EMF species along the productivity gradient, with as
little as 24% community similarity among contrasting sites.
Species richness per plot increased asymptotically with
foliar nitrogen concentrations of subalpine fir, demonstrat-
ing that many EMF species were well suited to soils with
high rates of nitrogen mineralization. EMF species abun-
dance in relation to site productivity included parabolic,
negative linear, and positive exponential curves. Both
multi-site and more narrowly distributed EMF were
documented, and a diverse mix of mantle exploration types
was present across the entire productivity gradient. The
results demonstrate strong associations of EMF fungal
species with edaphic characteristics, especially nitrogen
availability, and a specialization in EMF communities that
may contribute to the successful exploitation of such
contrasting extremes in soil fertility by a single tree host.
Keywords Ectomycorrhizal species richness .Nitrogen .
Boreal forests .Diversity–productivity relationships .
Ectomycorrhizal exploration type
Introduction
Ectomycorrhizal fungi (EMF) are the key mediating agent
between soils and many tree species, and research into the
diverse communities EMF may form continues to expand
upon abiotic–biotic relationships fundamental to forest
ecology. These investigations include the association of
particular EMF assemblages with edaphic and climatic
factors (Gehring et al. 2006), the role of EMF species and
fungal networks in forest nutrition and productivity (Paul et
al. 2007; Selosse et al. 2006), and the dynamics of EMF
communities in primary or secondary forest succession
(Nara 2006; Twieg et al. 2007). Ultimately, the insights into
EMF community ecology gained from these lines of inquiry
should provide a better understanding of forest soil
ecosystems and tree species autecology (especially survival,
nutrition, and productivity), and enable a more thorough
evaluation of forest ecosystem response to stressors such as
forest harvesting, atmospheric pollution, invasive species,
and climate change.
Mycorrhiza (2009) 19:99–111
DOI 10.1007/s00572-008-0208-z
J. M. Kranabetter (*)
British Columbia Ministry of Forests and Range,
4300 North Road,
Victoria, BC, Canada V8Z 5J3
e-mail: Marty.Kranabetter@gov.bc.ca
D. M. Durall
University of British Columbia—Okanagan,
3333 University Way,
Kelowna, BC, Canada V1V 1V7
e-mail: Daniel.Durall@ubc.ca
W. H. MacKenzie
British Columbia Ministry of Forests and Range,
Bag 6000,
Smithers, BC, Canada V0J 2N0
e-mail: Will.MacKenzie@gov.bc.ca
One fundamental aspect of EMF ecology is the
relationship between soil nitrogen (N) supply and EMF
species distribution and diversity. It is increasingly apparent
that plant nutrition in cold, less productive forests is
dependent on organic N to a large degree (Lipson and
Näsholm 2001), and that many EMF of boreal and
subalpine forests can facilitate organic N availability
and uptake (Chalot and Brun 1998; Read and Perez-
Moreno 2003). In addition, a number of experimental
studies with N fertilizer or of atmospheric N deposition
have demonstrated large shifts in EMF species distribution
with increased inorganic N availability (Peter et al. 2001;
Lilleskov et al. 2002; Avis et al. 2003) and often losses in
‘specialist’or stress-tolerant EMF species (Wallenda and
Kottke 1998; Taylor et al. 2000). These results suggest, at
least for conifer species, that a primary niche of EMF is
nutrient-poor, acidic organic soils with negligible rates of N
mineralization (Read et al. 2004). For these reasons, we
might expect EMF diversity in coniferous forests to decline
with increasing soil N availability (Parrent et al. 2006;
Taniguchi et al. 2007), to the extent even of non-
mycorrhizal root proliferation (Berch et al. 2006), and
shifts in forest dynamics to favor arbuscular mycorrhizal
plant and tree species (Nilsson et al. 2005).
Alternatively, many conifer species establish across quite
wide gradients in soil moisture or nutrient regimes, and
investigations of more pristine habitat have revealed an
array of EMF species able to thrive on N-rich sites
(Toljander et al. 2006). Rather than changes in simply
species richness, the effect of soil fertility might be revealed
through shifts in the distribution of genera such as
Cortinarius and Tricholoma (Trudell and Edmonds 2004),
in the functional attributes suggested by mantle character-
istics (Nilsson and Wallander 2003), or in the abundance of
mushroom fruiting (Kårén and Nylund 1997; Jonsson et al.
2000). Few studies have thoroughly examined EMF
communities across naturally contrasting soils or habitat
types, but it is apparent that both widely tolerant, generalist
species and more niche-specialized species can be expected
within mature forest landscapes (Nantel and Neumann
1992; Gehring et al. 1998; Kernaghan and Harper 2001;
Toljander et al. 2006; Robertson et al. 2006). Soil N
availability can vary temporally during cycles of forest
disturbance as well, although the duration of this effect and
influence on EMF communities appears to be subtle
(Kranabetter et al. 2005; Yamashita et al. 2007; B. Twieg,
unpublished).
Detailed study of EMF species distribution across well-
defined and replicated natural edaphic gradients would help
clarify the significance of soil fertility to EMF communi-
ties. One such gradient was described for upland plant
associations of southern boreal forests in British Columbia
(Canada), where stand productivity and foliar N concen-
trations were positively correlated to dissolved organic N
mass and N mineralization rates of the soil profile
(Kranabetter et al. 2007). In addition, key differences in
soil biota were suggested by forest floor morphology
(Green et al. 1993), which shifted from purportedly
fungal-dominated, matted mor humus forms on poorer sites
to faunal-dominated, aggregated moder humus forms on
richer sites. These contrasting sites under a uniform
macroclimate provided an ideal setting for isolating edaphic
influences on late-seral EMF communities, and we were
able to minimize the possible effects of host diversity
(DeBellis et al. 2006) and tree size by sampling a single
understory species, subalpine fir (Abies lasiocarpa [Hook.]
Nutt.), which had naturally regenerated throughout these
old-growth forests.
In this study, we report on the relationships between
natural gradients in soil productivity and the EMF
communities of A. lasiocarpa, including diversity esti-
mates, species distribution, and hyphal exploration types
(Agerer 2001). We compare our findings with vascular
plant diversity–productivity relationships to discuss com-
monalities in aboveground and belowground community
ecology, and discuss some of the possible broader implica-
tions of diverse, site-specific EMF communities in boreal
landscapes.
Materials and methods
Site descriptions
The southern boreal forest of British Columbia is designat-
ed as the Sub-Boreal Spruce Zone (SBS), and is located in
the montane landscape of the central interior, within the
closed forest portion of the Cordilleran boreal region (Pojar
1996). The SBS has a continental climate characterized by
severe, snowy winters and short, warm, moist summers.
Upland coniferous forests are comprised of lodgepole pine
(Pl) (Pinus contorta Dougl. ex Loud), hybrid white spruce
(Sx) (Picea glauca×Picea englemanii [Moench] Voss), and
subalpine fir (Bl). Soils are free of permafrost and are
predominantly deep blankets of glacial tills with coarse
fragments of mixed lithology.
The study sites were located in the moist cold (mc)
subzone of the SBS near Smithers, British Columbia,
Canada (54°49′N 127°10′W; elevation 522 m). Four site
series (represented by climax plant communities
corresponding to soil moisture and nutrient regime; Pojar
et al. 1987) were sampled to provide a wide range in upland
edaphic conditions: (02) xeric and poor Pl–Cladonia; (01)
mesic and medium Sx–Huckleberry; (06) subhygric and
rich Sx–Oak fern; and (09) subhygric and very rich Sx–
Devil’s club (Banner et al. 1993). Site series are hereafter
100 Mycorrhiza (2009) 19:99–111
referred to by their nutrient regime and plant association
name.
Experimental design
Five blocks were located along a 25-km portion of the
McDonnell Forest Service Road (54°40′to 47′N and 127°16′
to 36′W) at approximately 900 m elevation. Mean annual air
temperature of these sites is estimated, based on ClimateBC
extrapolation (Spittlehouse 2006) at 2.3°C, with mean annual
precipitation of 987 mm (477 mm as snow). One replicate of
each plant association was located per block, generally
within a radius <1 km (four plant associations×five blocks=
20 plots). We were unable to find a suitable Sx–Devil’sclub
plot at the fourth block, therefore the study was limited to 19
plots. Each plot was 50 m×30 m (0.15 ha) in size. Further
descriptions of stand, soil, and vegetation characteristics of
the study plots are listed in Kranabetter et al. (2007)and
Kranabetter and Simard (2008). Some key site properties
published previously are summarized in Tables 1and 2and
briefly described below.
Site properties
All plots had mixed, late-seral coniferous forests (~180 years)
but with differences in relative canopy composition across the
gradient; lodgepole pine was the dominant species on
nutrient-poor, xeric sites, and was less abundant than
subalpine fir or hybrid spruce on moister and richer sites
(Table 1). Trees of the canopy had ceased height growth (i.e.,
reached an asymptote) decades earlier, and we used the
height of three co-dominant trees of each species per plot as
a measure of site potential (in some of the poor-Cladonia
stands only lodgepole pine comprised the overstory). Site
index was determined for one co-dominant tree per species
per plot using the British Columbia Ministry of Forests Site
Tool (Version 3.2B). A fixed area subplot of 0.01 ha was
located near the center of each plot to determine stand basal
area.
As described in Kranabetter et al. (2007), the mass per
hectare (forest floor and mineral soil) of dissolved organic
N, NH
4+
, and NO
3
−
were determined from a 5-week in situ
incubation initiated in early June, 2006. Forest floor F and
H horizons were sampled as intact cores, avoiding pure
decayed wood, and mineral soils were sampled down to
20 cm with an auger. Mineral soils were sealed in a
polyethylene bag within the sample hole, and forest floors
were placed on top of this sample in a separate bag.
Dissolved organic N (DON) and inorganic N (DIN) was
determined colorimetrically using a modified persulfate
solution, and forest floor and mineral soil N concentration
data was converted to mass per hectare using depth and
coarse fragment content values from each plot.
Foliar N concentrations (N
%
) of understory subalpine fir
were determined in mid-September of 2006. The sapling
cohort established naturally under the canopy and had been
suppressed for some decades on all plots. Current year
foliage was clipped from 15 subalpine fir saplings and
bulked together to form three subsamples per plot. Foliar
samples were oven dried (70°C for 24 h), ground with a
Wiley mill, and analyzed for N by dry combustion.
Soil moisture was measured gravimetrically every
3 weeks throughout the summer of 2006 and converted to
content (kg ha
−1
) for the soil profile using the same depth
and coarse fragment content values as N determinations.
Forest floor pH was measured in water, and total organic
phosphorus (P) was determined indirectly with a dry ash
and sulfuric acid extraction and an UV–visible spectropho-
tometer (Varian Inc., Palo Alto, USA).
Ectomycorrhizal fungus assessment
Roots for EMF assessment were sampled June 13–15, 2007
from the understory subalpine fir saplings. Understory
saplings are ideal as they limit root sampling to one tree
species, and typically host EMF communities comparable
to the larger overstory trees (Jonsson et al. 1999; Richard et
al. 2005). Soil was removed from around the base of the
Table 1 Stand characteristics and understory A. lasiocarpa foliar N concentrations by plant association (means with SE in brackets)
Plant association
a
Stand height (m) Site index
(m @
50 years)
Foliar N
%
(g kg
−1
)
Basal area
(m
2
ha
−1
)
Relative cover (%)
nPl Ba Sx
P—Cladonia 5 21a* (1.4) 12a (1.3) 9.7a (0.12) 33a (3) 80a (3) 15a (4) 5 (2)
M—Huckleberry 5 28b (0.5) 15b (1.4) 11.5b (0.17) 54b (4) 44b (5) 49b (5) 7 (1)
R—Oak fern 5 32c (0.4) 19c (0.3) 12.6c (0.14) 75c (7) 13c (5) 65b (9) 22 (8)
VR—Devil’s club 4 36d (0.7) 24d (1.0) 13.6d (0.17) 119d (6) 19c (2) 67b (3) 15 (1)
*Means within columns separated by letters are significantly different (p< 0.05)
a
Soil nutrient regimes Ppoor, Mmedium, Rrich, VR very rich
Mycorrhiza (2009) 19:99–111 101
sapling to reveal the larger, radiating structural roots (5–
10 mm in diameter). Three of these roots were clipped and
gently excavated from the surrounding soil as completely as
possible. Roots were positioned primarily above or along
the humus–mineral soil interface and occasionally through
buried wood, so feeder roots were extracted from all
substrate types to some degree. Five healthy, widely spaced
saplings (minimum 10 m apart) were selected per plot, for a
total of 95 (5×19 plots) saplings in the study. The root
systems were wrapped in moss to keep the root tips fresh,
placed into a plastic bag, and returned to the laboratory.
Sixty root systems were refrigerated and examined imme-
diately, while the remaining 35 root systems were frozen
until the fall before completing the ectomycorrhizal
assessment.
The three root segments from each sapling were washed
gently in warm water to remove most of the soil and
organic debris. Once all surface debris was removed, the
clean roots were cut into approximately 2.5-cm-long
sections and placed in a glass pan filled with water.
Sections were continuously mixed and individual segments
randomly selected to determine the number of root tips
colonized by each EMF morphotype. Successive root
sections were examined until 200 root tips had been
classified from each of the saplings. EMF colonization
rates were virtually 100%, and in rare cases a root segment
was discarded and replaced if the mantle was too young and
undeveloped to identify so that a complete census of 200
colonized root tips could be made. The total number of fine
roots assessed for the study was 19,000 (95 saplings× 200
root tips per sapling).
Each root tip was examined stereoscopically (10× to
40× magnification) for features such as color, shape,
size, and texture of the root tip as well as emanating
elements, if present. The root tips were examined with a
compound microscope at 1,000× magnification for
characteristics of the mantle layers and emanating
elements such as mantle type, ornamentation, cell
contents, clamp frequency, and lengths and widths of
hyphal cells. Slides were prepared using either mantle
squashes or mantle peels if fungal layers of the mantle
were exceptionally thick. When necessary, the root tips
were stained with either 0.1% (w/v) aqueous toluidine
blue O, 10% (w/v) KOH, or Meltzer’s reagent to
emphasize the mantle features. We named the morphotype
if it matched descriptions of species published by the
British Columbia Ectomycorrhizal Research Network
(2007). In addition, we characterized the hyphal explora-
tion type of each morphotype based on Agerer (2001):
‘contact’types had smooth mantles and no rhizomorphs;
‘short’types had emanating hyphae with no rhizomorphs;
‘fringe’types had long emanating hyphae with diffuse
rhizomorphs; ‘mat’types had short emanating hyphae with
cottony rhizomorphs; ‘smooth’types had few or no
emanating hyphae and undifferentiated rhizomorphs; and
‘long’types had smooth mantles and highly differentiated
rhizomorphs.
Molecular techniques
DNA information was used to clarify the taxonomy of
distinct but unknown EMF morphotypes and to distinguish
between highly similar morphotypes. This latter objective
was especially important for Cortinarius, as most of these
species share a similar morphology (bent to tortuous root
tips with thick, white emanating hyphae [4–5μmin
diameter with large clamps] and few other notable features).
Five to ten root tips were collected from 96 fungal
colonies of interest (a cluster of root tips colonized by the
same EMF morphotype on an individual sapling) and
frozen for subsequent DNA extraction and PCR amplifica-
tion of the fungal ITS region of nuclear rDNA. Samples of
one to three tips were placed into a fast prep extraction tube
containing AP1 solution of the DNeasy 96 Plant Kit
(Qiagen, Mississauga, Canada). The tips were pulverized
with a ceramic bead in a FastPrep (FP120) high-speed
shaker (Thermosavant, Holbrook, USA). After centrifuging
briefly, the supernatant was transferred into wells of a 96-
Table 2 Soil nitrogen indices (dissolved organic N, ammonium, and nitrate after a 5-week in situ incubation) and selected properties by plant
association (means with SE in brackets)
Plant association
a
DON
(kg ha
−1
)
NH
4+
(kg ha
−1
)
NO
3
−
(kg ha
−1
)
Mineral soil
pH (H
2
O)
Forest floor
pH (H
2
O)
Soil moisture
(kg m
−2
)
Organic P
(kg ha
−1
)
n
P—Cladonia 5 16.7a (2.7) 0.9a (0.2) 0a 4.8 (0.06) 4.0a (0.05) 13.4a (1.2) 137a (8)
M—Huckleberry 5 27.1b (1.6) 3.2b (1.0) 0a 4.6 (0.05) 4.1a (0.07) 18.7a (1.5) 179ab (25)
R—Oak fern 5 33.1b (1.4) 7.5c (1.0) 0.2b (0.1) 5.2 (0.20) 4.7b (0.15) 29.3b (2.1) 246b (24)
VR—Devil’s club 4 32.0b (3.3) 9.2c (3.6) 5.5c (3.3) 5.3 (0.09) 4.8b (0.14) 27.6b (2.0) 446c (39)
*Means within columns separated by letters are significantly different (p< 0.05)
a
Soil nutrient regimes Ppoor, Mmedium, Rrich, VR very rich
102 Mycorrhiza (2009) 19:99–111
well plate supplied by the DNeasy Plant Kit. As per the
instructions of the DNeasy Plant Kit, 130 μl of the AP2
buffer was added to each well and shaken for 15 s, then
stored at −20°C for 10 min followed by centrifuging at
4,000 rpm for 10 min. Six hundred microliters of the AP3/E
solution was added to 400 μl supernatant and the resultant
solution was shaken vigorously for 15 s, centrifuged to
3,000 rpm, and then immediately stopped. One milliliter of
each sample was added and vacuumed from each well of a
new DNeasy plate. Four hundred microliters of the AW
buffer was added and vacuumed from each well after which
this step was repeated, and the plate was dried at 40°C. The
DNeasy plate containing the DNA was eluted into elution
tubes by adding 100 μl of the AE buffer, waiting 1 min and
then vacuuming, after which this step was repeated. The
DNeasy plate was centrifuged for 3 min at 2,000 rpm to
remove final amounts of DNA. The resultant genomic
DNA was stored at −20°C. Primer pairs used in PCR
amplifications were either ITS1F-ITS4B or NSI1-NLC2.
Samples were cycle sequenced using the Big Dye Termi-
nator Kit (Applied Biosystems, Foster City USA) and the
primer set ITS1f and ITS4. Sequencing was performed on a
3,130×1 capillary sequencer (Applied Biosystems). For-
ward and reverse sequences were aligned and manually
corrected in Sequencher 4.2 (GeneCodes, Ann Arbor, MI,
USA). Sequences were BLAST searched (Altschul et al.
1997) against the GenBank database to suggest taxonomic
affinities of the samples.
Data analysis
EMF species diversity was described in three ways,
following Newmaster et al. (2003): species richness per
sapling, species richness by plot (alpha diversity, α), and
cumulative species richness by plant association (gamma
diversity, γ). Shannon’s diversity index for the EMF
community of each plot (five saplings combined) was
determined using PC-ORD 5.0 (McCune and Grace 2002).
The study was organized in a randomized incomplete
block design. Species richness and hyphal exploration
type abundance was tested among plant associations
using Proc Mixed in SAS (SAS Inc 2004)withblock
and block interactions set as random factors. Residuals
from the analyses were examined for normal distributions
and found to meet the assumptions of equal variance.
Significant differences between least square means of each
plant association were tested using pairwise ttests at a
significance level of 0.05. The general linear model
procedure in SAS using Type 1 Sums of Squares was
used to test linear and curvilinear correlations between
plot means of dependent and independent variables (n=
19). No significant effect of block or block× treatment
interactions was found in any of the correlations. We
chose a significance pvalue of 0.010 for correlations of
EMF species abundance because of the inherently high
variation in species occurrence and scale of sampling.
A comparison of EMF fungal communities among plots
was undertaken by non-metric multi-dimensional scaling
(NMS), using the relative Sorenson measure for species
abundance. Computations were undertaken with PC-ORD
5.0 software, using the random starting configurations
(McCune and Grace 2002). The ordination of axes was
tested against plot soil measures using Pearson and Kendall
correlations and the ordination graph rotated to the variable
with the strongest correlation. Separation of EMF commu-
nities by plant association was tested in pairwise compar-
isons using the multi-response permutation procedure
(MRPP) with the Sorenson (Bray–Curtis) distance measure
(presence/absence) (McCune and Grace 2002). EMF
community similarity based on species abundance (% root
colonized) was determined by percentage similarity (PS)
(Pielou 1984).
Results
Initial morphotyping distinguished 63 EMF taxa, and 75%
of the distinct but unknown morphotypes were identified to
the closest aligned species through ITS rDNA analysis
(Appendix). We were able to separate the Cortinarius
colonies into 24 species using ITS rDNA as well; a small
number of inconclusive results were designated as Corti-
narius sp. A decision was made to lump together a few
infrequent but similar morphotypes (likely from the
Thelephoraceae family) when we were unable to confirm
unique species identification or if consistent separation of
morphotypes was not possible. With these adjustments, the
total number of taxa used in the statistical analysis was 74
species. This included, in part, a dark septate fungus
(MRA), four species of Piloderma, seven of Tomentella
and Pseudotomentella, eight of Russula,27ofCortinarius,
two of Lactarius, one of Tricholoma, and a variety of
unknown fungi (Appendix;apartiallistofthemore
common EMF taxa is presented by plant association in
Tab le 2). We lacked the resources to sequence every
morphotype on every sapling in this survey, and so certain
fungi treated as a single species, such as Cenoccocum
geophilum, may represent species complexes (Jany et al.
2002).
The number of EMF species per sapling ranged from one
to 14, and average richness per sapling was significantly
lower (p=0.006) on poor-Cladonia sites compared to the
other plant associations (Table 3). A similar trend was
found in αdiversity of the plots, with approximately 20
species on average for the medium to very rich plant
associations (Table 3). The extent of EMF αdiversity
Mycorrhiza (2009) 19:99–111 103
increased asymptotically with soil fertility in regression
analysis, as demonstrated by the positive curvilinear correla-
tion with foliar N
%
of the saplings (Fig. 1). Removing one
outlier contributed by a rich-Oak fern site improved the
precision of the equation (r
2
from 0.59 to 0.71) but had little
effect on the significance or shape of the curve. Shannon’s
diversity index averaged 2.36 overall (Table 3), and we were
unable to detect significant differences among plant associ-
ations (p=0.153). Plant association (γ) diversity peaked at 41
species on rich-Oak fern and very rich-Devil’sclubsites
(Table 3), equal to an approximately 20% increase over
poor-Cladonia and medium-Huckleberry sites.
The EMF communities showed a progressive separa-
tion by plant association in the NMS analysis that
followed the productivity rankings (Fig. 2; the proportion
of variance along axes 1 and 2 were 0.577 and 0.226,
respectively, for a cumulative r
2
of 0.803). Pearson and
Kendall correlations were most significant between axis 1
and soil N indices, including inorganic N mass (r
2
=0.803)
and DIN:DON ratio (r
2
=0.758). Axis 2 was best defined
by the geochemistry variables of exchangeable K (r
2
=
0.391) and mineral soil pH (r
2
=0.200). Asymptotic stand
height was also a significant correlate for axis 1 (r
2
=
0.715), although all site potential indices covary strongly
Table 3 Diversity measures (means with SE in brackets) and abundance (mean % root colonization) for the more frequent ectomycorrhizal fungi
grouped by plant association (% frequency by sapling; 25 in total for poor, medium and rich plant associations, 20 for very rich)
Poor—Cladonia
(n=5)
Medium—Huckleberry
(n=5)
Rich—Oak fern
(n=5)
Very Rich—Devil’s club
(n=4)
Richness per sapling 6.0a* (0.3) 7.2b (0.4) 7.7b (0.4) 7.5b (0.4)
αdiversity (per plot) 15.6a (0.8) 19.6b (1.0) 20.2b (1.2) 20.8b (0.9)
Shannon’s index (per plot) 2.18 (0.15) 2.36 (0.08) 2.43 (0.08) 2.50 (0.03)
γdiversity (all replicates) 33 34 41 41
Percent root colonization (% frequency)
Cenococcum geophilum 9.6 (80) 16.6 (92) 13.7 (100) 9.1 (65)
MRA 25.6 (72) 20.9 (88) 9.6 (56) 1.0 (15)
Unknown fungus VI 10.1 (44) 4.0 (36) 4.6 (36) 1.8 (20)
Unknown fungus VIII 8.7 (56) 3.3 (36) 3.4 (28) 3.8 (40)
Amphinema byssoides 0.2 (4) 2.2 (16) 4.7 (32) 13.4 (55)
Laccaria laccata 1.8 (8) 2.8 (20) 5.4 (52) 9.9 (65)
Piloderma fallax 5.6 (60) 10.6 (68) 6.4 (60) 1.5 (25)
Piloderma I 2.0 (36) 5.0 (40) 11.5 (60) 9.5 (75)
Piloderma II 0 0 1.7 (12) 3.6 (15)
Piloderma III 1.2 (16) 0.7 (8) 0 0
Cortinarius cf. semisanguineus 1.5 (32) 2.4 (40) 0.6 (8) 0
Cortinarius neofurvolaesus 2.2 (8) 0 0 0
Cortinarius cinnamomeus 0 1.2 (24) 0.9 (20) 0
Cortinarius III 0 0.3 (4) 0 3.6 (5)
Cortinarius hemictrichus 0 0.7 (16) 1.1 (24) 2.2 (20)
Inocybe lanuginosa-like 0 0.5 (4) 0.5 (12) 1.1 (10)
Inocybe I 0 0 0.3 (4) 1.5 (10)
Leccinum aurantiacum 1.1 (12) 0 0 0
Russula decolorans 4.7 (8) 1.6 (8) 0 0
Russula III 2.2 (4) 0 0 0
Russula bicolor 0 0.4 (12) 2.2 (36) 2.0 (20)
Russula I 0 0 1.2 (16) 3.0 (10)
Russula II 0 0 0 3.7 (15)
Thaxterogaster cf. pinguis 0 5.3 (20) 3.8 (24) 1.8 (15)
Sarcodon sp. 7.9 (24) 0 0 0
Tomentella cf. stuposa 0 0 0 8.1 (55)
Cortinarius XII 0 0.7 (4) 3.0 (16) 1.7 (5)
Unknown fungus II 0.2 (4) 2.8 (24) 1.7 (12) 0
Unknown fungus V 0 4.0 (28) 5.8 (24) 0.4 (5)
Unknown fungus VI 0.9 (8) 3.4 (24) 2.7 (16) 0
*Means within columns (diversity measures only) separated by letters are significantly different (p< 0.05)
104 Mycorrhiza (2009) 19:99–111
with soil N indices and foliar N
%
(Tables 1and 2;
Kranabetter and Simard 2008).
Significant differences in EMF community composi-
tion (presence/absence) were detected in MRPP compar-
isons of species assemblages between poor, medium, and
very rich sites (Table 4). PS analysis also revealed an
increasing dissimilarity in EMF fungal distribution and
abundance with soil fertility, equal to a 24% overlap in
EMF communities between the extreme contrasts in plant
associations (Table 4). An intermediate degree of shared
EMF species was found between medium-Huckleberry
and rich-Oak fern sites (Fig. 2,Table4), likely reflecting
the consistency in forest floor N supply between these two
plant associations (N mineralization potential of 624 and
656mgkg
−1
, respectively; Kranabetter et al. 2007). Very
few EMF species were evenly distributed across plant
associations, and some of the more common EMF species
had significant trends (p<0.010) in abundance in relation
to soil fertility. This was demonstrated for six EMF
species, and included parabolic, negative linear, and
positive exponential curves in correlations with foliar N
%
(Fig. 3).
There were few generalizations that could be drawn on
the distribution of EMF genera. Inocybe and Tomentella
species tended to favor richer soils, but other speciose
genera such as Cortinarius and Russula had individual
species better adapted to either end of the productivity
spectrum (Table 3). The distribution of EMF by exploration
type was quite consistent among plant associations,
averaging seven contact, 11 short-distance, 14 medium-
fringe, two medium-mat, and three medium-smooth species
per plot. The abundance of three exploration types changed
significantly with plant association (Fig. 4); short explora-
tion fungi declined on the rich-Oak fern and very rich-
Devil’s club sites (p=0.028), as medium-fringe and
medium-smooth fungi increased (p=0.033 and p=0.037,
respectively).
Discussion
The significant and consistent changes in distribution
and abundance of 74 EMF species demonstrated a high
degree of community specialization along these grada-
tions in soil fertility. It is difficult, however, to isolate
the exact causes of EMF species distribution because of
the number of covarying site properties which may
influence EMF communities, including N and P avail-
ability, soil moisture, and pH. Nitrogen is a useful focus
for analysis because its availability in boreal ecosystems
0
5
10
15
20
25
9111315
Foliar N (
g
k
g
-1)
Species richness
Poor
Medium
Rich
Very rich
Fig. 1 EMF species richness per plot (αdiversity) in correlation with
foliar N concentration of the A. lasiocarpa understory (n=18, one
rich-Oak fern plot not included). Richness= −41.8 + 9.14 (foliar N
%
)−
0.33(foliar N
%
)
2
;p<0.001; r
2
=0.71
Axis 1
Axis 2
Medium Huckleberry
Poor Cladonia
Rich Oakfern
Very rich Devil's club
Fig. 2 Non-metric multi-
dimensional scaling analysis of
EMF communities among the
19 plots (based on the abun-
dance of 74 species), rotated to
maximize correlation with inor-
ganic N mass on axis 1
Mycorrhiza (2009) 19:99–111 105
integrates underlying soil moisture and geochemical
drivers well, and correlates strongly with forest produc-
tivity, both as soil N indices and foliar N concentration
(Kranabetter and Simard 2008). Given the strong evi-
dence for direct effects of N on EMF physiology (e.g.,
Arnebrant 1994) it was most relevant to our objectives to
examine natural ranges in N availability, but we recognize
other ecosystem attributes could be influential on EMF
and deserve consideration. For example, an effort was
made to equalize the potential effect of neighbors (Hubert
and Gehring 2008) by choosing sites with mixed stands of
pine,fir,andspruce,although it was not possible to find
an equal distribution of the three conifer species across all
sites (Table 1). Perhaps then the EMF community
parameters would differ under pure Abies lasiocarpa
forests to some degree, but we would argue a high degree
of EMF community specialization with soil properties
would still exist. The differences in overstory tree size and
rates of C fixation were controlled by sampling a
0
5
10
15
20
25
9 11 13 15 9 11 13 15
9 11 13 15 9 11 13 15
9 11 13 15 9 11 13 15
Foliar N (g kg-1)
Percent colonization
Cenococcum
geophilum
0
5
10
15
20
25
30
Foliar N (g kg-1)
Percent colonization
Piloderma
fallax
0
5
10
15
20
Foliar N (g kg-1)
Percent colonization
Unknown fungi VI
0
10
20
30
40
50
60
Foliar N (g kg-1)
Percent colonization
Dark septate
fungi 'MRA'
0
5
10
15
20
25
Foliar N (
g
k
g
-1)
Percent colonization
Amphinema
byssoides
0
5
10
15
20
Foliar N (
g
k
g
-1)
Percent colonization
Laccaria
laccata
Fig. 3 Abundance of six EMF
species in correlation with foliar
N concentration of A. lasiocarpa
C. geophilum=−245+ 45.2
(Foliar N
%
)−1.9 (Foliar N
%
)
2
;
p=0.010, r
2
=0.43; P. fallax=
−191+ 35.4 (Foliar N
%
)−1.6
(Foliar N
%
)
2
;p=0.090, r
2
=0.26;
MRA=84−5.8 (Foliar N
%
); p=
0.004, r
2
=0.40; unknown fungi
VI= 27.6 −1.9 (Foliar N
%
);
p=0.002, r
2
=0.45; A.
byssoides=0.28 + 0.000014e
(Foli-
ar N%)
;p=0.001, r
2
=0.59; L.
laccata= 2.1 + 0.0000083e
(Foliar
N%)
;p=0.003, r
2
=0.50
Table 4 Matrix of ectomycorrhizal fungal community similarity
between plant associations by MRPP (pvalues based on presence/
absence of species) and, in brackets, percentage community similarity
(based on mean % root colonization by species)
M—Huckleberry 0.003 (56)
R—Oak fern 0.005 (42) 0.340 (66)
VR—Devil’s
club
0.004 (24) 0.007 (35) 0.126 (52)
Poor—
Cladonia
Medium—
Huckleberry
Rich—Oak
fern
106 Mycorrhiza (2009) 19:99–111
comparable cohort of suppressed advanced regeneration
(~1.5 m in height) on all plots; in any case, it is uncertain
how significant photosynthesis rates might be since there
is little evidence for differences in EMF communities
between illuminated overstory and shaded understory trees
(Jonsson et al. 1999; Richard et al. 2005).
TheincreaseinEMFαdiversity with foliar N
%
demonstrated that many of these EMF species were well
suited to soils with high rates of N mineralization, at least
within the context of these cool, moderately productive
boreal landscapes. A hump-backed or unimodal distribu-
tion of plant diversity with soil fertility is often proposed
(and widely debated) by ecologists, where relatively few
plant species are successful on both the most stressful and
competitive sites (Mittlebach et al. 2001). Some parallels
can be drawn to this EMF community since there was a
reduction in the number of species on the driest, N-poor
soils, but no corresponding reduction on the most
productive sites. Species such as A. byssoides,L. laccata,
and T. stuposa were gaining in dominance, but the rates of
N mineralization and nitrificationonveryrich-Devil’s
club sites were perhaps never high enough to allow more
complete competitive success. For this reason, we suspect
the peak in EMF diversity coincided with the more
heterogeneous supply of all three N forms (amino acids,
NH
4+
,andNO
3
−
) associated with rich and very rich soils
(Kranabetter et al. 2007), and we are unaware of any
(ultra-rich) ecosystems supplied entirely by inorganic N in
these boreal landscapes. In addition, productive ecosys-
tems have a component of poor microsites, such as buried
wood, that would contribute to niche diversity and species
richness (Buée et al. 2007;Iwański and Radawska 2007).
Positive productivity–species richness relationships such
as these are not entirely uncommon among plant or animal
taxa,especiallywhencomparedwithinacommunitytype
or over a limited productivity range (Mittlebach et al.
2001).
The increase in EMF diversity and medium-distance
exploration types with soil fertility were largely at odds
with results reported from N fertilization or N deposition
studies (Lilleskov et al. 2002; Nilsson and Wallander
2003). Toljander et al. (2006) noted a similar discrepancy,
and suggested that the range of N concentrations among
natural soils is of a much smaller magnitude than those
experimentally applied, resulting in more dramatic effects
of N fertilizer on EMF communities. For example, the
anthropogenic fertility gradient for Picea glauca (Lilleskov
et al. 2002) had foliar N concentrations of 13.9 g kg
−1
under the lowest N inputs, which would actually be
comparable to our richest sites (13.6 g kg
−1
). Certain
tree species, especially of Pinus, may be better adapted to
poor soils and organic N forms and consequently respond
differently than Abies to inorganic N availability (Berch et
al. 2006;Parrentetal.2006; Taniguchi et al. 2007).
Another consideration is that EMF evolved with niches
that occur naturally in forests such as the high soil
moisture and inorganic N availability found together
on rich-Oak fern and very rich-Devil’s club sites
(Kranabetter and Simard 2008). Perhaps then high
amounts of N deposition on mesic sites would be
unsuitable for eutrophic EMF species that cannot tolerate
soil droughtiness or higher acidity. It is very likely that
various soil properties (nitrogen, moisture, pH, etc.) must
be aligned to create suitable habitat (Trudell and Edmonds
2004), and any perturbations to forest ecosystems resulting
in habitats with no natural analogue could be detrimental
to EMF.
Among these diverse communities were EMF species
which varied in abundance but were present at least to
some degree on all site types (e.g., C. geophilum,
Piloderma I, unknown fungus VIII). This wide habitat
distribution (‘multi-site’) could be a significant contribu-
tion to the resiliency of these forest ecosystems as it would
allow quick responses to any positive or negative changes
in soil resource availability (resiliency defined as the
capacity to absorb disturbances without undergoing
change to a fundamentally different state; Drever et al.
2006). An example is the flush of inorganic N commonly
occurring after forest disturbances, and it is likely of some
importance that many of these multi-site EMF species are
also multi-seral and multi-host fungi, able to persist and
thrive in regenerating stands with many tree species
(Kranabetter 2004). The capacity of EMF to buffer
disturbances, in a resilience context, might also include
severe drought events (Swaty et al. 2004)ormoregradual
but significant climatic trends (e.g., Pacific Decadal
0
20
40
60
80
100
Poor Med. Rich V. Rich
Percent colonization
long
smooth
mat
fringe
short
contact
Fig. 4 Hyphal exploration types of EMF as a mean percent of root
colonization grouped by plant association (n=5 for poor, medium, and
rich plant associations; 4 for very rich)
Mycorrhiza (2009) 19:99–111 107
Oscillation) that could affect soil processes and nutrient
availability. Along with generalist fungi, there were also
EMF species more limited in distribution (e.g., R.
decolorans, unknown fungi VI, T. stuposa), that may be
well adapted to specific edaphic niches and contribute to
increased utilization of soil resources. Ectomycorrhizal
fungus communities may have a degree of functional
similarity, as with many soil biota, but certainly a mix of
species attributes (multi-site and site-specific species,
multi-seral and late-seral species, multi-host and host-
specific species) should insure resiliency and sustain
productivity in a stressful, dynamic, and unpredictable
forest environment (Perry and Amaranthus 1997).
It is perhaps not surprising there were only minor
trends in the distribution of fungal exploration types with
soil fertility in comparison to N fertilizer treatments
given the relatively subtle shift in N amounts and forms.
The consistent mix of mantle types could reflect high
functional diversity in response to the heterogeneity of
microsites and resources found throughout the fertility
gradient (Baier et al. 2006). Presumably EMF would
contribute significantly to microbial biomass across the
entire productivity gradient, with some effect of rooting
density, while shifts in ericoid and arbuscular fungi would
correspond to the distribution of understory plants (Nilsson
et al. 2005). The visual perception of EMF abundance, as a
characteristic of humus forms (Green et al. 1993), is more
likely a reflection of shifts in EMF communities on sites
such as these because conspicuous mat-forming fungi,
especially bright yellow P. fallax, declined as dark-colored
Tomentella spp. gained in abundance on the richest sites.
Categorizing more diverse fungal genera such as Corti-
narius or Russula into habitat types would be an
oversimplification as these species occupied all manner
of niches, similar to the patterns in genera distribution
with forest succession (Kranabetter et al. 2005;Twieget
al. 2007). Tracking the full complement of EMF species
through root sampling would be exceedingly difficult
(Taylor 2002), however, and sporocarp surveys may help
define the distribution of the more infrequent fungi.
Presumably EMF communities in older forests such as
these have little change in dominant species composition
over time (Izzo et al. 2005), but possible seasonal effects
might also be of interest in studies of soil abiotic–biotic
relationships (Koide et al. 2007).
Other than poor-Cladonia sites, the difference in α
diversity or Shannon’s diversity index among plant
associations was quite insignificant compared to the more
profound shifts in EMF species distribution and commu-
nity composition. For this reason, we would suggest
diversity parameters are not always the most relevant
variable compared to the identity and abundance of the
EMF species themselves in evaluating forest processes
(Wallenda et al. 2000; Dahlberg 2001). Likewise, it is
possible that controlled studies with ad hoc EMF species
assemblages could draw incongruous conclusions if ill-
suited EMF species were selected for the experimental soil
conditions. For example, greenhouse studies of tree
nutrition and N forms do not always account for EMF
species composition (e.g., Bennett and Prescott 2004),
which is understandable given the inability to recreate
such specialized and complex EMF communities, but this
simplification could affect the outcome of these experi-
ments. Plant ecologists are acutely aware of hidden
treatment effects in experimental manipulation of plant
communities (Huston 1997), and we would caution that
similar confounding effects of EMF species need to be
considered in testing of tree–soil interactions.
In conclusion, the results suggest that EMF species
distribution across landscapes, like many vascular and
non-vascular forest plants, is largely defined by adapta-
tion and competition for niches related to stress
tolerance (e.g., drought, soil acidity) and resource
availability (especially organic N, NH
4+
,andNO
3
−
)in
soils (Dickie et al. 2002; Koide et al. 2005). The
significance of such extensive EMF βdiversity with a
single tree host, especially in contrast to the much greater
aboveground diversity of plants with arbuscular fungi
(Allen et al. 1995), is worth further consideration. A
reasonable conjecture, from both this and similar results
(Gehring et al. 1998; Toljander et al. 2006; Robertson et
al. 2006), is that the wide ecological amplitude of
relatively few tree species across vast boreal and temperate
landscapes would depend to some degree on partnerships
with well-adapted EMF fungal assemblages. Consequent-
ly, the simplification of EMF communities through
anthropogenic activities might hamper the survival of
conifers on stressful sites, impede their ability to compete
with arbuscular plants on productive sites, or reduce the
stability of forests in dynamic and unpredictable environ-
ments. These hypotheses are not easily validated, but
present some of the possible long-term risks to consider in
the evaluation of stressors (intensive forestry, atmospheric
pollution, invasive species, and climate change) on forest
ecosystems.
Acknowledgements We thank Michaela Byrne and Jenna Benson
of the UBC Okanagan molecular lab for undertaking the ITS
analysis. Marcel Lavigne and Bill Borrett assisted in the plot layout
and field sampling, and Rick Trowbridge of Boreal Research and
Development Ltd. was consulted on soil and plant association
classification. Clive Dawson and Dave Dunn of the B.C. Ministry
of Forests Analytical Laboratory undertook the soil N and foliar
chemical analysis. Peter Ott and Wendy Bergerud of the B.C.
Ministry of Forests were consulted on the statistical analysis. Funds
for the research project were provided by the Forest Investment
Account of British Columbia.
108 Mycorrhiza (2009) 19:99–111
Appendix
Table 5 List of morphotypes with successful ITS rDNA analysis
Provisional name Closest GenBank match % Match Comment
Mycelium radicis atrovirens (MRA) DQ481971 638/648 (98%) Uncultured ectomycorrhiza
Cortinarius cf. anomalus EU525957 575/577 (99%)
Cortinarius boulderensis DQ499466 630/636 (99%)
Cortinarius calopus FJ039571 492/494 (99%)
Cortinarius canabarba FJ039562 545/549 (99%)
Cortinarius clandestinus FJ039583 587/587 (100%)
Cortinarius firmus AF389163 544/544 (100%)
Cortinarius hemitrichus AY669680 498/502 (99%)
Cortinarius malicoria DQ481917 715/721 (99%)
Cortinarius neofurvolaesus DQ140002 478/479 (99%)
Cortinarius saturnius FJ039551 664/673 (98%)
Cortinarius cf. semisanguineus DQ481909 580/595 (97%) Morphotype matched except color was light pink
Cortinarius triformis FJ039573 536/540 (99%)
Cortinarius vibratilis EU821696 658/663 (99%)
Cortinarius I AY669687 342/348 (98%) Cortinarius umbilicatus
Cortinarius II AJ889975 370/377 (98%) Cortinarius praestigosus
Cortinarius III FJ039675 249/257 (96%) Cortinarius paragaudis
Cortinarius IV DQ102683 428/446 (95%) Cortinarius cf. saniosus
Cortinarius V EF218763 511/516 (99%) Uncultured (Cortinarius)
Cortinarius VI AJ438981 578/599 (96%) Cortinarius obtusus
Cortinarius VII DQ481963 549/552 (99%) Uncultured (Cortinarius)
Cortinarius VIII AF325590 487/504 (96%) Cortinarius brunneus
Cortinarius IX DQ481959 635/648 (97%) Uncultured cf. Dermocybe
Cortinarius X EF218758 444/446 (99%) Uncultured (Cortinarius)
Cortinarius XI EU693242 509/520 (97%) Cortinarius testaceofolius
Cortinarius XII EF077497 321/328 (97%) Uncultured (Cortinarius)
Unknown fungi I FJ152525 371/392 (94%) Uncultured (Helotiales)
Unknown fungi II DQ481700 438/439 (99%) Uncultured ectomycorrhiza
Unknown fungi III DQ481971 486/488 (99%) Uncultured ectomycorrhiza
Unknown fungi IV AY825525 684/699 (97%) Uncultured Thelephoraceae
Unknown fungi V EU057086 583/590 (98%) Uncultured Thelephoraceae
Unknown fungi VI AY822734 614/623 (98%) Uncultured ectomycorrhiza
Unknown fungi VII AY702742 271/279 (97%) Uncultured ectomycorrhiza
Unknown fungi VIII AY394895 619/670 (92%) Uncultured ectomycorrhiza
Inocybe I DQ093854 396/413 (95%) Inocybe geophylla
Lactarius rufus EF685089 498/498 (100%)
Piloderma fallax DQ658864 406/406 (100%)
Piloderma I‘Green globs’EU057111 388/420 (92%) Uncultured Piloderma
Piloderma II ‘Glass shards’DQ474735 521/538 (96%) Uncultured Piloderma
Piloderma III ‘Peaches’DQ377372 504/544 (92%) Uncultured Piloderma
Russula I AY061685 421/435 (96%) Russula laricina
Russula II EF433961 713/725 (98%) Uncultured Russula
Russula III AB211253 432/442 (97%) Uncultured Russula
Tomentella cf. stuposa AF272902 439/440 (99%) Tomentella stuposa
Tomentella I AJ534911 625/649 (96%) Tomentella sp. O53
Tomentella II DQ974777 491/517 (94%) Tomentella lateritia
Tomentella III TSU83470 612/617 (99%) Thelephoraceae ‘Taylor #6’
Pseudotomentella humicola AM490945 555/559 (99%)
Pseudotomentella sp. AJ893352 611/617 (99%) Uncultured Pseudotomentella
Thaxterogaster cf. pinguis DQ328112 357/364 (98%) Thaxterogaster pinguis
Sarcodon sp. AF103896 649/672 (96%) Sarcodon squamosus
Tricholoma sp. AY656987 424/425 (99%) Uncultured Tricholoma
Note: additional species recognized through morphotype characters included Cenococcum geophilum,Amphinema byssoides,Cortinarius
cinnamomeus,Laccaria laccata,Leccinum aurantiacum,Lactarius kaufmanii,Rozites caperata,Russula aeruginea,Russula bicolor, and Russula
decolorans (British Columbia Ectomycorrhizal Research Network 2007). A further 11 taxa were characterized as morphotypically distinct but
were unsuccessful in DNA sequencing. Species identity was assumed when the match with GenBank was 98% or better at >450 base pairs,
otherwise the closest matching species name was noted under comments
Mycorrhiza (2009) 19:99–111 109
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