The effects of ectomycorrhizal fungal networks on seedling establishment are contingent on species and severity of overstorey mortality

Abstract

For tree seedlings in boreal forests, ectomycorrhizal (EM) fungal networks may promote, while root competition may impede establishment. Thus, disruption to EM fungal networks may decrease seedling establishment owing to the loss of positive interactions among neighbors. Widespread tree mortality can disrupt EM networks, but it is not clear whether seedling establishment will be limited by the loss of positive interactions or increased by the loss of negative interactions with surrounding roots. Depending upon the relative influence of these mechanisms, widespread tree mortality may have complicated consequences on seedling establishment, and in turn, the composition of future forests. To discern between these possible outcomes and the drivers of seedling establishment, we determined the relative importance of EM fungal networks, root presence, and the bulk soil on the establishment of lodgepole pine and white spruce seedlings along a gradient of beetle-induced tree mortality. We manipulated seedling contact with EM fungal networks and roots through the use of mesh-fabric cylinders installed in soils of lodgepole pine forests experiencing a range of overstorey tree mortality caused by mountain pine beetle. Lodgepole pine seedling survival was higher with access to EM fungal networks in undisturbed pine forests in comparison with that in beetle-killed stands. That is, overstorey tree mortality shifted fungal networks from being a benefit to a cost on seedling survival. In contrast, overstorey tree mortality did not change the relative strength of EM fungal networks, root presence and the bulk soil on survival and biomass of white spruce seedlings. Furthermore, the relative influence of EM fungal networks, root presence, and bulk soils on foliar N and P concentrations was highly contingent on seedling species and overstorey tree mortality. Our results highlight that following large-scale insect outbreak, soil-mediated processes can enable differential population growth of two common conifer species, which may result in species replacement in the future.

Full text

ORIGINAL ARTICLE
The effects of ectomycorrhizal fungal networks on seedling
establishment are contingent on species and severity
of overstorey mortality
Gregory J. Pec
1,2
&Suzanne W. Simard
3
&James F. Cahill Jr
1
&Justine Karst
1,4
Received: 11 October 2019 / Accepted: 12 February 2020
#Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
For tree seedlings in boreal forests, ectomycorrhizal (EM) fungal networks may promote, while root competition may impede
establishment. Thus, disruption to EM fungal networks may decrease seedling establishment owing to the loss of positive
interactions among neighbors. Widespread tree mortality can disrupt EM networks, but it is not clear whether seedling estab-
lishment will be limited by the loss of positive interactions or increased by the loss of negative interactions with surrounding
roots. Depending upon the relative influence of these mechanisms, widespread tree mortality may have complicated conse-
quences on seedling establishment, and in turn, the composition of future forests. To discern between these possible outcomes
and the drivers of seedling establishment, we determined the relative importance of EM fungal networks, root presence, and the
bulk soil on the establishment of lodgepole pine and white spruce seedlings along a gradient of beetle-induced tree mortality. We
manipulated seedling contact with EM fungal networks and roots through the use of mesh-fabric cylinders installed in soils of
lodgepole pine forests experiencing a range of overstorey tree mortality caused by mountain pine beetle. Lodgepole pine seedling
survival was higher with access to EM fungal networks in undisturbed pine forests in comparison with that in beetle-killed stands.
That is, overstorey tree mortality shifted fungal networks from being a benefit to a cost on seedling survival. In contrast,
overstorey tree mortality did not change the relative strength of EM fungal networks, root presence and the bulk soil on survival
and biomass of white spruce seedlings. Furthermore, the relative influence of EM fungal networks, root presence, and bulk soils
on foliar N and P concentrations was highly contingent on seedling species and overstorey tree mortality. Our results highlight
that following large-scale insect outbreak, soil-mediated processes can enable differential population growth of two common
conifer species, which may result in species replacement in the future.
Keywords Tree mortality .Mountain pine beetle .Pinus contorta .Picea glauca .Insect outbreaks .Ectomycorrhizal fungi .
Forest disturbance
Introduction
Seedling establishment can be a key process influencing the
structure and function of forest ecosystems (Oliver and Larson
1990). Many factors influence seedling establishment, such as
light and soil resource availability, can be mediated by shoot
and root competition (Pickett and White 1985;Coomesand
Grubb 2000). In addition to competition, there is increasing
evidence that access to mycorrhizal fungal networks can in-
fluence seedling growth and survival for many species
(Horton et al. 1999; Leake et al. 2004; Simard and Durall
2004;Nara2006; Selosse et al. 2006;McGuire2007;
Johnson et al. 2017). Mycorrhizal networks, which are com-
posed of fungal hyphae that connect roots of the same indi-
vidual, different individuals and even different host species,
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00572-020-00940-4) contains supplementary
material, which is available to authorized users.
*Gregory J. Pec
pecg@unk.edu
1
Department of Biological Sciences, University of Alberta, B717a,
Biological Sciences Building, Edmonton, Alberta T6G 2E9, Canada
2
Department of Biology, University of Nebraska at Kearney,
Kearney, NE 68849, USA
3
Department of Forest and Conservation Sciences, University of
British Columbia, Forest Sciences Centre #3601-2424 Main Hall,
Vancouver, British Columbia V6T 1Z4, Canada
4
Department of Renewable Resources, University of Alberta, 442
Earth Sciences Building, Edmonton, Alberta T6G 2E3, Canada
Mycorrhiza
https://doi.org/10.1007/s00572-020-00940-4

serve as sources of fungal inoculum for seedlings and can
function as channels for C, nutrient, and water movement
among plants (Leake et al. 2004; Simard and Durall 2004;
Simard et al. 2012). For example, ectomycorrhizal (EM) fun-
gal networks may facilitate establishment of tree seedlings
where resources or fungal propagules are deficient in soils
(McGuire 2007; Teste and Simard 2008), mitigate effects of
overstorey tree competition on seedling growth (Simard et al.
1997; Booth and Hoeksema 2010), and facilitate natural re-
generation of seedlings in association with the transfer of C
and N from mature trees (Teste et al. 2009). However, contrary
to these findings, other studies have reported that the benefits
provided by EM fungal networks are dwarfed in comparison
to the negative growth effects of shading and competition for
soil resources (Kranabetter 2005; Brearley et al. 2016).
Studies disentangling roots and hyphal effects on seedling
establishment are rare, yet critical to a broader understanding
of seedling establishment in natural forests.
Seedling establishment is an outcome of the net effects of
positive and negative ecological processes acting simulta-
neously. The local ecological context can vary the strength
of each process independently, with potentially complex con-
sequences for net effects on seedling establishment. For in-
stance, the abiotic environment can shift the balance of com-
petition and facilitation in plant communities (Callaway 1997;
Brooker et al. 2008). Similarly, the relative strength of biotic
processes may alter the net effects on seedling establishment.
In particular, the widespread mortality of dominant trees
supporting EM fungal networks may have a pronounced ef-
fect on positive and negative interactions underlying seedling
establishment. Host tree mortality may cause EM networks to
degrade as fungi lose their main source of carbon following
tree death (Treu et al. 2014; Karst et al. 2014). However, the
death of dominant trees may also mean the death of a sizeable
fraction of roots (Cigan et al. 2015), depending on succession-
al trajectories. Thus, following widespread tree mortality, we
may expect seedling establishment to decrease owing to the
loss of positive interactions maintained through EM fungal
networks, or increase owing to the loss of negative interac-
tions with surrounding roots. Understanding the net effects of
these belowground processes is critical for predicting the com-
position of the succeeding forest.
Landscape-level tree mortality has increased in the past
decades (van Mantgem et al. 2009;Pengetal.2011;
Williams et al. 2013), with insect outbreaks predicted to in-
crease in severity, frequency, and scale as a result of climate
change (Weed et al. 2013). In particular, mountain pine beetle
(Dendroctonus ponderosae), an insect native to temperate co-
nifer forests, has expanded east of the Rocky Mountains into
novel pine habitats (Cullingham et al. 2011; Cigan et al.
2015). Our previous work in this region has shown that the
widespread mortality of pines has altered nutrient cycling
(Cigan et al. 2015), forest overstorey and understorey
structure (Pec et al. 2015), and the abundance of aboveground
EM fungal fruiting bodies and the belowground EM fungal
composition (Treu et al. 2014; Pec et al. 2017). Toward
disentangling net belowground effects, we investigated the
relative importance of EM fungal networks, root presence,
and the bulk soil on seedling establishment (growth, nutrition,
and survival) in beetle-killed stands. Specifically, we exam-
ined how dominant, conspecific lodgepole pine (Pinus
contorta) and subordinate, heterospecific white spruce
(Picea glauca) seedling establishment responded to a gradient
of tree mortality caused by a mountain pine beetle outbreak.
Methods
Field site description
We located eleven forest stands within a 625-km
2
region in the
Lower Foothills natural subregion southwest of Grande
Prairie, Alberta, Canada (54° 39′N, 118° 59′W; 950–
1150 m above sea level) (see Treu et al. (2014) for specific
stand locations) in 2011. The regional climate consists of long
cold winters and short cool summers with a mean annual
precipitation of 445 mm and mean annual temperature of
2.2 °C (Environment Canada 2011). These stands bordered
provincial permanent sampling plots and had experienced
mountain pine beetle activity since 2009. Stands were domi-
nated (≥80% basal area) by even-aged (120 ± 0.4 SE years
old) lodgepole pine (Pinus contorta Dougl. ex. Loud.), and
across stands, a gradient of beetle-induced tree mortality was
captured (0 to 82% lodgepole pine basal area killed) (Cigan
et al. 2015). Across these eleven forest stands, Abies balsamea
(L.) Mill, Betula papyrifera Marshall, Picea glauca (Moench)
Voss, Picea mariana Mill. Britton, Sterns, & Pogenb., and
Populus tremuloides Michx. were interspersed in the
subcanopy and mixed with diverse understory vegetation
(Pec et al. 2015). White spruce (Picea glauca) was the most
abundant tree species in the subcanopy (49% cover) (Cigan
et al. 2015). Successful mountain pine beetle attack can pro-
mote growth of preexisting shade-tolerant conifer species
(Nigh et al. 2008) and further, as lodgepole pine and white
spruce have different associations with species of EM fungi,
we reasoned they may show differential responses along the
tree mortality gradient.
Experimental design
We tested the relative importance of EM fungal networks, root
presence, and the bulk soil on the performance (survival,
growth and nutrition) of seedlings along a gradient of beetle-
induced tree mortality. In May 2011, we established a 900-m
2
(30 m × 30 m) plot within each of the eleven stands. In
June 2011, we recorded all trees within each plot and noted
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species, diameter at breast height and health status. Attack by
mountain pine beetle was confirmed by the presence of pitch
tubes, boring dust, exit holes, and subcortical galleries. Basal
area by tree species was calculated for each plot, and tree
mortality was calculated as beetle-killed P. contorta basal area
over total basal area expressed as a percentage. In August
2011, ten evenly distributed focal trees of lodgepole pine (>
20 cm diameter at breast height) were identified within each
plot and six locations, i.e., “subplots,”were located within
three meters from each focal tree in a random cardinal or
intercardinal direction.
The experiment consisted of three mesh treatments: (1)
access to an EM fungal network (+EM network−roots), (2)
access to an EM fungal network and roots (+EM network+
roots), and (3) contact with neither EM fungal networks nor
roots (−EM network−roots), namely seedlings have access to
the bulk soil only (i.e., soil that is outside of the rhizosphere,
not penetrated by plant roots but with potential to contain an
abundance of EM propagules). To vary the extent of fungal
contact with seedling roots, mesh of different pore sizes was
used. The “+EM network−roots”and the “−EM network
−roots”treatments were created by placing 44 μmand
0.5 μm mesh bags (Plastok®Meshes and Filtration, Ltd.,
Birkenhead, UK), respectively, of 15 cm diameter into holes
dug in the soil to a depth of 35 cm. Both mesh sizes prevent
roots from passing while allowing for diffusion of solutes,
whereas the 0.5 μm mesh also prevents hyphal passage of
EM fungi (Teste et al. 2006). We applied the three mesh treat-
ments to the two-tree species (lodgepole pine, white spruce).
Each of the six treatments was randomly assigned to one of
the six subplots. Each mesh bag was refilled with the previ-
ously removed field soil, keeping horizontal soil layers intact.
The “+EM network+roots”treatment entailed refilling holes
with previously dug field soil, but not installing a mesh bag. In
total, there were 660 experimental units (eleven stands × ten
focal trees × three treatments × 2 species).
To determine whether mesh treatments introduced a con-
founding factor of altered water and nutrient flow, we mea-
sured soil moisture levels within and directly next to each of
the mesh bags monthly during the growing season using a
Theta Probe soil moisture sensor (Delta-T Devices,
Cambridge, UK). We found no differences in soil moisture
content levels within and directly next to a mesh bag (P=
0.735) as well as among mesh treatments (P=0.904),which
is similar to previous studies on soil water movement across
mesh (Teste, Karst, Jones, Simard, & Durall, 2006; Teste et al.
2009).
Five months later, in October 2011, twenty lodgepole pine
seeds or white spruce seeds were sown into soil of each
subplot. Seeds were provided by Smoky Lake Forest
Nursery,Alberta(lodgepolepineseedlotnumber:NWB1
64-8-6-1981; white spruce seedlot number: NES3 60-20-5
1983) and sourced from the same origin as where the study
area was located. Bags were left in the field over the winter to
allow for EM fungal networks to form. In May 2012, we
found no successful germination and immediately reseeded
each subplot with an additional 20 seeds. During the first
growing season (2012), an open-topped cylindrical mesh
(6 mm) cage was used to protect seedlings from herbivory.
A 0.5 m buffer zone was created around each subplot and all
vegetation within the buffer zone was periodically clipped
throughout the growing season to limit growth of neighbor-
ing plants.
Survival, growth, and nutrition of field seedlings
Survival of 2-year-old germinants was assessed in May 2013
and was calculated as the percentage of live seedlings out of
the total number of seeds that germinated per subplot and
averaged across each plot. Seedlings were randomly thinned
to two seedlings per subplot in May 2013. All seedlings were
destructively sampled in August 2013. The first seedling was
harvested for biomass (shoots and roots). To determine shoot
biomass, stems were cut at the soil surface, oven dried at 70 °C
for 48 h and weighed. To determine root biomass, roots of
seedlings were carefully removed from mesh bags with soil
intact, placed in plastic bags, transported, and stored at −
20 °C until further processing. For seedlings established in
treatments without a mesh bag, roots were carefully removed
with the same volume of soil as that contained in mesh bags
(15 cm diameter, 35 cm deep), which encompassed the entire
root system. Roots were extracted from thawed soil by care-
fully washing under running tap water, oven dried at 70 °C for
48 h, and weighed. The second seedling was harvested to
measure foliar concentration of N, P, as well as mycorrhizal
fungal community composition on seedling root tips (see be-
low). Shoots and roots were harvested as described for bio-
mass determination. Needles were first ground and homoge-
nized to a fine powder using a Brinkmann ball grinder (Retsch
Type MM 220; Retsch GmbH, Haan, Germany). Foliar N was
analyzed by the Dumas Combustion Method (Nelson and
Sommers 1996) using a Costech 4010 Elemental Analyzer
System (Costech Analytical Technologies Inc., Valencia,
CA, USA). Foliar P was analyzed by nitric acid digestion
(Halloran and Cade-Menun 2007) and determined spectro-
photometrically on a SmartChem®wet chemistry discrete
analyzer (Westco Scientific Instruments, Inc., Brookfield,
CT, USA). Foliar analysis of N and P was performed at the
University of Alberta Natural Resources Analytical
Laboratory.
Sampling and identification of ectomycorrhizal fungi
on seedlings
To determine whether fungi colonizing seedling roots differed
by mesh treatment and along the tree mortality gradient, we
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identified fungi using Sanger sequencing. Roots of seedlings
were carefully washed under tap water and cut into 1-cm frag-
ments. Samples were morphotyped using both dissecting and
compound microscopes based on color, tip shape, branching
pattern, and texture (Goodman et al. 1998). DNA was extract-
ed from a single root tip for each morphotype on each seed-
ling. Amplification of the internal transcribed spacer (ITS)
region of fungal nuclear rDNA was performed in 16 μlreac-
tions using primers NSI1 and NLB4 (Martin and Rygiewicz
2005), and cycle sequencing was performed in 10 μlreactions
following methods outlined in Karst et al. (2015). Sanger se-
quencing reactions were cleaned using EtOH precipitation and
run on an ABI 3730 prism genetic analyzer (Applied
Biosystems, Foster City, CA, USA).
Initial sequence processing, quality filtering, sequence
clustering, and taxonomic identities of sequences were proc-
essed using the QIIME pipeline v.1.8 (Caporaso et al. 2010).
In brief, sequences were manually formatted to .fasta and .qual
files using Geneious v10 (Biomatters Ltd., Auckland, New
Zealand). Sequences were edited using the
add_qiime_labels.py to modify the sample ID for all .fasta
sequences. Files were preprocessed using the
convert_fastaqual_fastq.py and paired-end sequences were
merged using join_paired_ends.py with a minimum overlap
of 10 bp. Joined sequences were processed using
split_libraries_fastq.py with a minimum Phred quality score
of 25. Merged sequences were clustered into OTUs using
pick_open_reference_otus.py (Rideout et al. 2014) using a
97% similarity threshold and with the
suppress_taxonomy_assignment flag. Any resulting singleton
OTUs were included as these were representative of fungal
species based on previous morphotyping of root tips.
Taxonomic affiliations were assigned by searching represen-
tative sequences from each OTU against GenBank and
UNITE+INSD databases using the BLAST option in
assign_taxonomy.py. Sequences of all EM fungal OTUs were
submitted in the GenBank database under accession numbers
(KX498030-KX498065) (Table S1).
Statistical analysis
Prior to analyses, all data were pooled within each stand to
obtain plot-level estimates of seedling survival, growth and
nutrition and ectomycorrhizal fungi found on the roots of
field seedlings (data points for plot level estimates; one seed-
ling per experimental unit × ten focal trees × three treatments
per species = 30). We used a generalized linear model with a
binomial distribution and a logit link function to test the main
effect of seedling species (white spruce or lodgepole pine),
extent of overstorey tree mortality, and their interaction on
seedling survival. We only used seedling survival data from
the “+EM network+roots”treatment as seedlings in this
treatment experienced in situ conditions. That is, seedlings
establishing in beetle-killed stands could be in contact with
both EM fungal networks and roots under natural field
conditions.
Further, we used linear models to determine the relative
importance of EM fungal networks, root presence and bulk
soil on seedling survival, growth and nutrition. Prior to this
analysis, a quantitative index of the effect size in each re-
sponse (i.e., survival, biomass, foliar N and P) was calculated
by taking the natural log of individual response ratios.
Response ratios for survival, biomass, foliar N and foliar P
were calculated as follows: (1) EM fungal networks (+EM
network−roots/−EM network−roots), (2) root presence
(+EM network+roots/+EM network−roots), and (3) bulk soil
(+EM network+roots/−EM network−roots). The first re-
sponse ratio tests for the effects of EM fungal networks by
holding root presence constant, the second tests for the effects
of root presence by holding EM fungal network status con-
stant, and the third tests for the effects of the bulk soil by
comparing seedling responses when EM fungal networks
and roots are present versus absent. Model assumptions were
checked with diagnostic plots of the residuals.
To test for differences among EM fungal communities col-
onizing seedlings in the three mesh treatments across the gra-
dient of beetle-induced tree mortality, partial redundancy anal-
ysis (RDA) was performed in the vegan package (Oksanen
et al. 2013) with permutations set to 9999. Indicator species
analysis was performed to identify EM fungi strongly
responding to seedling species differences within each of the
mesh treatments and across the gradient of tree mortality using
the multipatt function in the R package indispecies (Cáceres
and Legendre 2009). All statistical analyses were performed
using R v3.3.2 (R Development Core Team 2018).
Results
Seedling survival following beetle-induced tree
mortality of the overstorey
Seedling survival differed between species (F
1,9
=47.11;
P< 0.0001), with white spruce seedling survival (79% ± 6
SE) six-fold higher than that of lodgepole pine (12% ± 4
SE). Lodgepole pine seedling survival decreased with
beetle-induced tree mortality (F
1,9
=18.12; P= 0.002), while
white spruce seedling survival was invariant across the same
gradient (F
1,9
=2.77; P= 0.13) (Fig. 1).
Effects of EM fungal networks, presence of roots
and bulk soil on seedling performance
Seedling responses to EM fungal networks, root presence, and
the bulk soil were highly variable, depending upon seedling
species, extent of overstorey tree mortality, and the response
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variable (Table 1). Overstorey tree mortality caused increas-
ingly negative effects of access to the EM fungal network on
lodgepole pine survival (Fig. 2a). In contrast, there was no
effect of access to EM fungal networks on white spruce sur-
vival, regardless of the extent of overstorey tree mortality (Fig.
2b). Root presence between seedlings and neighbors reduced
survival of lodgepole pine but had no effects on white spruce
(Fig. 2c, d). Relative to seedlings in the presence of root and
EM fungal networks, the survival response to bulk soil varied
between species. Bulk soil increased lodgepole pine seedling
survival, while no effect of bulk soil on white spruce seedling
survival was found, independent of overstorey tree mortality
(Fig. 2e, f).
In contrast to the highly contingent effects of treatments on
seedling survival, treatment effects on seedling biomass were
negligible. We found no effect of access to EM fungal
Table 1 Summary of linear models used to test the separate and
combined effects of ectomycorrhizal fungal networks, roots and bulk
soil on the survival, growth, and nutrition of lodgepole pine and white
spruce seedlings across a gradient of beetle-induced tree mortality.
*P<0.05, **P< 0.01, ***P<0.001
Lodgepole pine White spruce
Response Factors Main effect Overstorey tree mortality Interaction Main effect Overstorey tree mortality Interaction
t
2,27
t
1,27
t
2,27
t
2,27
t
1,27
t
2,27
Survival EM network −0.46 x −3.47** 0.78 −0.10
Root presence −4.92*** −1.32 −0.83 −1.33
Bulk soils 3.79** 2.29* 0.05 1.38
Combined 0.01 −1.49
Biomass EM network 0.58 −0.30 1.36 −0.60
Root presence −1.73 1.35 −1.14 −0.04
Bulk soils −0.70 0.60 −0.77 −0.85
Combined 0.86 −0.75
Foliar N EM network 2.63* −0.02 0.58 −0.15
Root presence −4.10*** −2.06* −1.34 −0.23
Bulk soils −2.65* −2.19* −0.77 −0.46
Combined 1.93 −0.48
Foliar P EM network 1.76 1.48 −1.48 −1.08
Root presence −2.87* −2.79* −1.31 −2.84*
Bulk soils −1.03 −0.01 −1.70 −1.90
Combined −0.03 −2.87**
Fig. 1 Survival of lodgepole pine
and white spruce seedlings across
a gradient of overstorey tree
mortality (% lodgepole pine basal
area killed) caused by mountain
pine beetle
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networks or root presence on seedling biomass for both spe-
cies (lodgepole pine and white spruce), regardless of
overstorey tree mortality (Table 1). However, with increasing
overstorey tree mortality, access to EM fungal networks in-
creased foliar N concentrations of lodgepole pine, whereas no
effect was observed for white spruce (Table 1;Fig.3a, b). In
the presence of neighboring roots and in bulk soil alone, foliar
N concentrations of lodgepole pine decreased across the
overstorey tree mortality gradient. In contrast, we found no
effect on foliar N concentrations from the presence of neigh-
boring roots and bulk soil across the overstorey tree mortality
gradient for white spruce (Table 1;Fig.3c–f). Access to EM
fungal networks and bulk soil alone had little effect on foliar P
concentrations of lodgepole pine and white spruce, which was
constant across the overstorey tree mortality gradient(Table 1;
Fig. S1a–bande–f). However, with increasing overstorey tree
mortality, the presence of roots decreased foliar P
concentrations of both lodgepole pine and white spruce
(Table 1; Fig. S1c–d).
Fungal community colonizing seedlings
A total of 31 EM fungal taxa were found on lodgepole pine
seedlings and 30 on white spruce seedlings, with 26 taxa
shared between the two species (Fig. 4). Overall, EM fungal
community composition differed between lodgepole pine and
white spruce seedlings (Table S2;Fig.S2). Many fungal taxa
were abundant on both lodgepole pine and white spruce seed-
lings; however, Laccaria bicolor,Thelephoraceae 1,
Thelephora terrestris,Tomentellopsis submollis, and Tuber
pacificum were found exclusively on lodgepole pine
(Fig. 4). Overstorey tree mortality led to shifts in the commu-
nity composition of EM fungi for both pine and spruce seed-
lings (Table S2;Fig.S2) and an overall increase in EM fungal
Fig. 2 Relative importance of ectomycorrhizal (EM) fungal networks,
roots and bulk soil for the survival of lodgepole pine (left panels) and
white spruce (right panels) seedlings across a beetle-induced tree
mortality gradient. Isolated effects of EM fungal networks in (a) and
(b), root presence in (c) and (d), and bulk soil in (e) and (f). Colored
dots and lines (solid—main effect; dashed—interaction) represent the
following effects on seedling survival: gray (EM fungal networks),
black (root presence), and white (bulk soil). The strength and direction
of each factor was determined through linear models using a quantitative
index of the effect size in each response by calculating the natural log of
individual response ratios (rr). Individual response ratios of biomass were
calculated as follows: EM networks (+EM network−roots/−EM network
−roots), root competition (+EM network+roots/+EM network−roots),
and bulk soil (+EM network+roots/−EM network−roots); see text for
descriptions of categories
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richness on seedling root tips (Fig. S3). There were a total of
nine indicator fungal taxa across the overstorey tree mortality
gradient (Table S3). Pine seedlings in undisturbed forests har-
bored substantially fewer species of EM fungi than seedlings
in forests with overstorey tree mortality, and included species
such as Peziza sp. and Piloderma sp. By contrast the number
of EM fungi species colonizing roots of pine and spruce seed-
lings in forests with high tree mortality was greater compared
with undisturbed forests and were dominated by fungal spe-
cies such as Laccaria bicolor and Russula bicolor (Table S3).
Mesh treatments did not alter community composition of EM
fungi (Table S2).
Discussion
Our results demonstrate that soil-mediated processes can en-
able differential population growth of two common conifer
species. Specifically, we found that the potential of pine seed-
lings to form EM fungal networks declines with overstorey
tree mortality. As the effects of root presence did not change,
this finding indicates that pine seedling survival along the
overstorey tree mortality gradient likely decreased due to a
loss of positive interactions maintained through EM networks.
Contrary to pine, white spruce had high overall seedling sur-
vival, with EM networks having a negligible effect regardless
of the degree of overstorey tree mortality. In white spruce, the
relative influence of EM fungal networks, the presence of
roots and the bulk soils was maintained regardless of
overstorey conditions. That the response of seedlings to EM
networks differs between white spruce and lodgepole pine
may favor the recovery of the former and the demise of the
other in beetle-killed stands. In this region of mountain pine
beetle outbreak, white spruce may replace lodgepole pine
allowing forests to persist on these landscapes, however with
a change in tree composition.
White spruce and lodgepole pine seedlings are widely
distributed tree species within boreal forests of North
America (Lotan and Perry 1983). Following stand-
replacing disturbance (e.g., fire), early seral, shade-
intolerant lodgepole pine has much faster juvenile growth
rate and establishment compared to the more shade tolerant
Fig. 3 Relative importance of ectomycorrhizal (EM) fungal networks,
roots and bulk soil for foliar N concentrations of lodgepole pine (left
panels) and white spruce (right panels) seedlings across a beetle-
induced tree mortality gradient. Isolated effects of EM fungal networks
in (a) and (b), root presence in (c) and (d), and bulk soil in (e) and (f).
Colored dots and lines (solid—main effect; dashed—interaction)
represent the following effects on seedling survival: gray (EM fungal
networks), black (root presence), and white (bulk soil). The strength
and direction of each factor was determined through linear models
using a quantitative index of the effect size in each response by
calculating the natural log of individual response ratios (rr). Individual
response ratios of biomass were calculated as follows: EM networks
(+EM network−roots/−EM network−roots), root competition (+EM
network+roots/+EM network−roots), and bulk soil (+EM network+
roots/−EM network-roots); see text for descriptions of categories
Mycorrhiza

white spruce. As canopy closure ensues through time, white
spruce is able to increase in growth in the understory due to
more favorable environmental conditions (Despain 2001;
Gärtner et al. 2011). However, insect-induced tree mortality
has little physical effect on understory vegetation and soils,
as these are intact following disturbance (Burton 2008). In
our study, greater survival of white spruce across the tree
mortality gradient may be due more to favorable seedbeds,
less-deteriorated mineral soils, and thicker organic matter
layers (Simard et al. 1998; Purdy et al. 2002; Paudel et al.
2015) and less due to EM network connectivity (Kranabetter
2005). In contrast, the decline in lodgepole pine seedling
survival across the tree mortality gradient may alternatively
be due, in part, to increased residual vegetative productivity
with increasing resource availability in the understory post-
disturbance (Despain 2001), similar light levels present
across the tree mortality gradient (< 5 years post-
disturbance) due to standing snags (Pec et al. 2015), as well
as pine having a greater mortality rate than white spruce at
low growth rates (Kobe and Coates 1997).
Furthermore, that EM networks and root presence had
varying effects on foliar N and P of the two tree species in
our field experiment was surprising for several reasons. First,
N is assumed to be more limiting than P in boreal forest soils
(Perry et al. 2008; Högberg et al. 2017). We found that supply
rates of soil NO
3
−
increased with extent of tree mortality and
were elevated for several years following beetle attack in the
same stands of the current field experiment (Cigan et al.
2015). The pulse of soil N with litter deposition and root
mortality may have shifted limitation from N to P in soils of
these stands. Second, though both root presence and EM net-
works affected foliar N and P, they acted in differing ways for
pine. In undisturbed forests, the presence of roots had neutral
effects on foliar N and P concentrations of lodgepole pine and
white spruce but became more pronounced in their negative
effects with extent of tree mortality. We suggest that a possible
mechanism underlying foliar N and P concentrations may be
an increase in root presence from both herbaceous and woody
perennials in the understory in stands with high tree mortality,
as in these beetle-killed stands, understory diversity and pro-
ductivity were nearly double as compared to undisturbed for-
ests (Pec et al. 2015).
Overall, applying physical barriers allowed us to test the
relative importance of EM fungal networks, root competition
and the bulk soil on the establishment of lodgepole pine and
white spruce seedlings along a gradient of beetle-induced tree
mortality. Physical barriers are designed to exclude in-growth
of EM fungi from the surrounding soil, and in consequence,
limit the formation of EM fungal networks. As there is no
current method in the field to allow root in-growth without
allowing fungal in-growth, resulting treatments cannot direct-
ly test the effects of roots independent of EM fungal networks.
Owing to this type of experimental manipulation, the benefits
of EM fungal networks tested in the field are confounded with
the absence of roots. While there may be no experimental
method to circumvent this issue, response ratios were used
Fig. 4 Frequency of occurrence
of ectomycorrhizal fungal taxa
found on lodgepole pine and
white spruce seedlings
established in undisturbed and
beetle-killed stands within the
Lower Foothills natural subregion
southwest of Grande Prairie,
Alberta, Canada
Mycorrhiza

to indirectly estimate the separate effects of EM fungal net-
works, root presence and the bulk soil on seedling establish-
ment. Conceptually similar approaches have been used on
observational data to parse out the spatial component of com-
munity structure (Borcard et al. 1992), decompose beta diver-
sity into its various components (Legendre and De Cáceres
2013), and explore the nature of species interactions (Grace
2008).
Shifts in fungal communities with overstorey tree
mortality
In agreement with our previous research, the composition of
EM fungal communities shifted with beetle-induced tree
mortality. We have demonstrated that, when characterized
by sporocarps (Treu et al. 2014), mixed DNA from soils
(Pec et al. 2017), or EM root tips of seedlings (current study),
the death of dominant, mature lodgepole pines changes EM
fungal communities. Shifts in fungal communities with tree
mortality have also been reported across a range of insect
outbreaks including spruce bark beetle (Ips typographus)in
Norway spruce (Picea abies) forests of Central Europe
(Stursova et al. 2014) and insect defoliation by geometrid
moths in mountain birch (Betula pubescens ssp.
czerepanovii) forest of northern Finland (Saravesi et al.
2015). Thus, as a consequence of tree mortality and a subse-
quent loss in carbon flow from hosts, compositional shifts in
EM fungal communities are likely to occur following large-
scale, intense outbreaks. However, Stursova et al. (2014) and
Saravesi et al. (2015) reported that richness and abundance
of EM fungi declined by 70–80% following insect outbreak,
and though our previous results align with these findings
(Treu et al. 2014; Pec et al. 2017), the current study demon-
strates that EM fungal richness on root tips of seedlings in-
creased with overstorey tree mortality (F= 29.01,
P< 0.0001) (Fig. S3). Within our study site, we suggest that
communities represented by different types of fungal tissues
(sporocarps, mycelium, and ectomycorrhizas) may be sorted
by different environmental filters. Along with overstorey
trees, the understory plant community provides the structure
of these forests, shaping above- and belowground microhab-
itat conditions. For example, in temperate mountain forests
of northern China (Chen et al. 2018), sporocarp composition
was shown to differ among various microhabitats, suggest-
ing that microhabitat variability favors occurrence of differ-
ent macrofungal species. Whereas, following widespread
drought-induced forest die-off, including Eucalyptus
marginata and Corymbia calophylla forest in the Northern
Jarrah Forest of southwestern Australia, revealed that overall
fungal richness in the rhizosphere were similar between trees
in drought-affected plots compared to unaffected plots
(Hopkins et al. 2018).
Forest recovery following insect outbreaks
In our study, we demonstrate that a balance of often opposing
belowground processes underpin seedling establishment.
Moreover, for some tree species, the ecological context can
shift the balance of these opposing processes. This contingen-
cy may partly underlie differences in pine seedling establish-
ment across regions of forests experiencing various severities
of mountain pine beetle outbreaks. For instance, in beetle-
killed pine forests that do not regenerate naturally via canopy
disturbance such as wildfire, seedling establishment is rela-
tively low (Harvey et al. 2014). However, new post-beetle
recruitment in pine forests of central British Columbia has
been shown to clearly increase with the loss of the overall
canopy basal area in naturally regenerating stands (Astrup
et al. 2008) and pine seedlings in beetle-killed pine-dominated
subalpine forests of Colorado were found to be established in
50% of naturally regenerating stands (Collins et al. 2011).
Though the complexity in belowground interactions under-
mines our ability to generalize across forest ecosystems, it
may also buffer forests against disturbance. For example, the
importance of residual trees, such as white spruce, as refugia
for EM fungi has also been shown to be vital to the survival
and growth of seedlings (Kranabetter 2000; Smith and Read
2008). Residual trees, either conspecific or heterospecific,
growing in the subcanopy of forest stands can potentially
serve as surrogate hubs for networking fungi to establishing
seedlings (Simard 2009;Beileretal.2010,2015). For exam-
ple, EM networks of residual trees have been shown to facil-
itate regeneration of Pseudotsuga menziesii seedlings under
drought and root competition in interior dry forests of
British Columbia (Bingham and Simard 2012). In our study,
residual trees in the subcanopy of beetle-killed stands may act
as legacy trees for EM fungi following disturbance, providing
a robust network for the establishment, survival, and growth
of white spruce seedlings.
Conclusions
Through physically manipulating EM fungal networks in the
field and indirectly parsing the separate effects of EM fungal
networks, root presence and bulk soils on seedling perfor-
mance, we show that the potential to form EM fungal net-
works declines combined with an increase in non-EM plant
competition leads to shifts in pine and spruce seedling survival
and nutrition. Importantly, we show that processes occurring
belowground are often opposing and are of different combi-
nations across the landscape. This complexity of interactions
may not permit generalizations on seedling establishment
across forest ecosystems but may make ecosystems and land-
scapes resilient to disturbance. As disturbances, such as insect
outbreaks, continue to intensify in forested systems of western
Mycorrhiza

North America (Weed et al. 2013), complex belowground
interactions may favor the recovery of individual tree species
over others, thereby facilitating the persistence of forests, al-
beit differing in composition.
Acknowledgments We thank members of the Cahill Lab for providing
helpful comments during the development of this manuscript. We also
thank P.W. Cigan, M. Devine, M. Randall, and A. Sywenky with field
assistance, F. Najari with sample processing, and C. Narang with molec-
ular assistance.
Authors’contributions All authors conceived the ideas and designed
methodology; GJP and JK collected the data; GJP analyzed the data;
GJP and JK led the writing of the manuscript. All authors contributed
critically to the drafts and gave final approval for publication.
Funding information This work was funded by a Natural Sciences and
Engineering Research Council of Canada Strategic Grant (NSERC)
awarded to J. Cooke, N. Erbilgin, S.W. Simard, and J.F. Cahill, Jr. and
NSERC Discovery Grants awarded to N. Erbilgin, S.W. Simard, and J.F.
Cahill, Jr.
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