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Importance of Soil, Stand, and Mycorrhizal Fungi in Abies balsamea

Establishment in the Boreal Forest

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DOI: 10.3390/f11080815

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Article
Importance of Soil, Stand, and Mycorrhizal Fungi in
Abies balsamea Establishment in the Boreal Forest
Mélissande Nagati 1,2 , Mélanie Roy 2, * , Annie DesRochers 1 , Yves Bergeron 1
and Monique Gardes 2
1 Chaire Industrielle UQAM-UQAT en Aménagement Forestier Durable, Institut de Recherche sur les Forêts,
Université du Québec en Abitibi-Témiscamingue, 445 Boul. de l’Université,
Rouyn-Noranda, QC J9X 4E5, Canada; melissande.nagati@UQAT.ca (M.N.);
Annie.Desrochers@uqat.ca (A.D.); Yves.Bergeron@uqat.ca (Y.B.)
2 CNRS_IRD Laboratoire Evolution et Diversité Biologique, Université Paul Sabatier, UMR5174, 118 route de
Narbonne, 31330 Toulouse, France; monique.gardes@univ-tlse3.fr
* Correspondence: melanie.roy@univ-tlse3.fr




Received: 3 June 2020; Accepted: 26 July 2020; Published: 28 July 2020


Abstract: Research highlights: To understand differences in the establishment of balsam fir regeneration
observed in the boreal forest, we examined how soil layer and microorganisms explained differences
in growth and mycorrhization in three different stand types. Our experiment revealed positive and
negative effects on growth of seedlings, and highlights the importance of biotic interactions in balsam
fir establishment. Background and Objectives: In a context of climate change, understanding tree
migration can be examined through changes in tree regeneration. At the ecotone between mixed
and conifer boreal forest, regeneration of balsam fir northward is of particular interest because it
thrives better under aspen-dominated stands as compared to adjacent spruce-dominated stands.
As the understorey differs between these stands, with more Ericaceae under spruce and different
ectomycorrhizal fungal communities in organic and mineral horizons, we hypothesized that biotic
factors could explain differences in balsam fir establishment. Materials and Methods: Using a growth
chamber experiment, we tested if differences in soil layers and modification of soil fungal communities
would affect germination, mycorrhization, and growth of balsam fir seedlings in three different
stand vegetation. We compared 12 treatments and followed 120 seedlings over three growth seasons.
Results: We found similar survival in soils from aspen- and spruce-dominated stands, and a greater
biomass on organic layers. In addition to this, a greater mycorrhization rate was found in aspen
soils but improved germination in spruce soils. The presence of Ericaceae in spruce soils was
associated with lower mycorrhization but did not affect other traits. Sterilization and therefore
microorganisms affected mainly the number of ectomycorrhizae and the investment in root biomass.
Finally, mycorrhization and biomass were correlated, but independent from N nutrition measured
in needles. Conclusions: Our results highlighted the positive effects of organic soil layers and of
mycorrhization on biomass, and showed that mycorrhization was increased under aspen as compared
to other stand types. Our experiment also revealed positive effects of spruce soil on fir germination
and showed that fir was able to grow and survive in all conditions. Our study suggests that fir
establishment is affected by belowground multi-species interactions, and therefore highlights that
biotic interactions shall be taken into account to understand and predict future tree migrations in the
boreal forest.

Keywords: ectomycorrhizae; trembling aspen; black spruce; Ericaceae; seedbed

Forests 2020, 11, 815; doi:10.3390/f11080815 www.mdpi.com/journal/forests

Forests 2020, 11, 815 2 of 16

1. Introduction
In a context of climate change, species migration and changes in tree species distribution are
expected to occur. Migrations to the North or in elevation have already been observed for birds,
butterflies, and grasses [1]. Tree responses to increasing temperatures seem more heterogeneous
and Zhu et al. [2] concluded that only northern tree species would migrate north, these representing
about 20% of tree species in North America. Tree migration models may include more than climatic
parameters [3], and take into account local processes and heterogeneity [4]. Indeed, tree establishment
into a new area depends not only on latitude or climatic conditions, but also on abiotic and biotic
conditions, as well as on the available space [5]; all these factors delineate the realized niche of a tree
species. New models try to integrate the dependence toward symbionts [6] or competition with other
tree species [7], but may miss local heterogeneity of biotic interactions that could buffer the effect of
stressors on population dynamics and maintenance of biotic interactions. Understanding mechanisms
controlling tree regeneration would then greatly improve migration models and give guidelines for
assisted migration programs [3].
The boreal forest represents a key ecosystem to study tree migration today, as fast changes
are already observed [8] and threaten the largest forest biome on Earth [9]. In Quebec (Canada),
boreal forests are mainly dominated by black spruce (Picea mariana (Miller) B.S.P.; [10]) and
feather-mosses. Local changes in tree dominance are increasingly observed, such as patches of trembling
aspen (Populus tremuloides Michaux), possibly favored by climate change and forest management
practices [11]. In this heterogeneous landscape, tree species are extending their distribution northward,
such as balsam fir (Abies balsamea (L.) Miller; [12]). Interestingly, balsam fir establishes and grows
better under trembling aspen- than under black spruce-dominated stands of the boreal forest [13],
suggesting a positive interaction between aspen and fir. Both stands occur in similar climatic
and edaphic conditions but host different understorey vegetation [14], soil fungal communities,
and more specifically saprotrophic and symbiotic fungi communities [15]. Investigations of these
micro-organisms communities have also revealed strong differences within stands between organic
and mineral layers [15], which could increase local heterogeneity and complexify interactions around
balsam fir seedlings.
Among interactions, ectomycorrhizal symbioses might be of peculiar importance for tree
establishment. These interactions between Asco and Basidiomycetes and tree roots are particularly
rich and diverse in north temperate and boreal forests [16] and provide nutritional benefits to most
tree species in these biomes [17], including balsam fir, trembling aspen, and black spruce [18]. In a
previous study, Nagati et al. [19] observed ectomycorrhizae on balsam fir seedlings growing under
trembling aspen or black spruce, showing that associations were possible for this migrating species.
The colonization by ectomycorrhizal fungi, estimated by ramification indices and molecular markers,
was greater under trembling aspen than under black spruce, especially when black spruce stands
contained ericaceous shrubs [19]. Overall, this study showed that facilitation of fir under trembling
aspen was most likely mediated by ectomycorrhizal fungi. On the other hand, ericaceous shrubs are
known for their negative effects on coniferous regeneration [20–23] in conifer-ericaceous communities
in boreal and temperate forests. Indeed, poor tree recruitment and slow seedling growth is often
observed in nutrient-stressed environments dominated by ericaceous plants [20]. In a first study [19],
we found that the presence of Ericaceae affected mycorrhizal communities on the surrounding fir roots,
which may lead to a decrease of mycorrhization as observed in other ecosystems [22–24]. In addition
to this, different fungal species with ectomycorrhizal or ericoid status can coexist in this particular
habitat, and potentially compete in plant root systems [23], leading to differences in plant nutrient
uptake or functioning.
To further disentangle these complex interactions and test the relative importance of soil microbial
communities, soil layers and dominant vegetation (aspen or spruce) on balsam fir establishment,
we launched an ex-situ experiment in a growth chamber. We did not isolate ectomycorrhizal fungi, but rather
tested the effect of fungi occurrence by comparing sterile and non-sterile soils. Controlled conditions
Forests 2020, 11, 815 3 of 16

are particularly useful as ecological factors shape species traits and the outcome of biotic interactions,
and experimental tests have already allowed to show the link between mycorrhizae and facilitation in other
biomes (e.g., [25]). Moreover, our objective was also to monitor the early-stage steps of tree establishment,
that are germination, seedling survival, and growth, under different conditions. We hypothesized that
ectomycorrhizal fungi would play a role on seedling growth but not on seed germination, and
Forests 2020, 11, x FOR PEER REVIEW
tested that
3 of 17
germination and survival was dependent mostly on the seedbed [26] and therefore soil layer (organic or
mineral)monitor
and soil theorigin
early-stage
(fromsteps
standsof tree establishment,
dominated eitherthat are germination,
by aspen or spruce,seedling
with orsurvival,
withoutand ericaceous
growth, under different conditions. We hypothesized that ectomycorrhizal fungi would
shrubs). In line with our field observations [19], we expected that mycorrhization, growth and survival play a role
on seedling growth but not on seed germination, and tested that germination and survival was
would be greater in non-sterile soils and organic layers, and greater on soils collected from aspen-dominated
dependent mostly on the seedbed [26] and therefore soil layer (organic or mineral) and soil origin
stands than
(fromfromstandsspruce-dominated
dominated either by stands,
aspen particularly
or spruce, withwith Ericaceae.
or without Finally,shrubs).
ericaceous we compared our results
In line with
with in our
situfield
interactions
observations drawn
[19], from sapling
we expected growth
that and mycorrhization,
mycorrhization, to sketch
growth and survival a synthesis
would be greaterof direct
and indirect interactions
in non-sterile leading
soils and organictolayers,
balsam andfirgreater
establishment in the boreal
on soils collected forest, and conclude
from aspen-dominated stands on the
than of
importance fromsoilspruce-dominated
fungi and mycorrhiza stands, for
particularly
balsam fir with Ericaceae. Finally, we compared our results
colonization.
with in situ interactions drawn from sapling growth and mycorrhization, to sketch a synthesis of
direct and
2. Materials andindirect
Methods interactions leading to balsam fir establishment in the boreal forest, and conclude
on the importance of soil fungi and mycorrhiza for balsam fir colonization.
2.1. Soil Origin and Sterilization
2. Materials and Methods
Soils were sampled in August 2016 in three adjacent stands distant 20–100 m, either dominated
2.1. Soil aspen
by trembling Origin and
(TA)Sterilization
or black spruce (BS), the latter with or without the presence of ericaceous
shrubs (Ledum groenlandicum;
Soils were sampled in BSE
August(BS2016 in three
with adjacentstand).
Ericaceae) stands distant
Stands 20–100
werem,previously
either dominated
described in
by trembling aspen (TA) or black spruce (BS), the latter with or without the
Nagati et al. [15] (site 4, see Table S1 for soil characteristics). For each soil origin (stand) presence of ericaceous
we collected
shrubs (Ledum groenlandicum; BSE (BS with Ericaceae) stand). Stands were previously described in
10 L of organic soil and 10 L of mineral soil independently. As texture of mineral soil was heavy clay
Nagati et al. [15] (site 4, see Table S1 for soil characteristics). For each soil origin (stand) we collected
the transition between organic and mineral horizons was clear in the field, at circa 20–30 cm depth and
10 L of organic soil and 10 L of mineral soil independently. As texture of mineral soil was heavy clay
allowedthe totransition
separatebetween
the twoorganic
horizons (Figurehorizons
and mineral 1a,b). Towas test the
clear ineffect of biotic
the field, at circafactors
20–30 cm aside
depthfrom the
dominant andvegetation, we sterilized
allowed to separate the two our wet (Figure
horizons soils by1a,b).
microwaving themoffor
To test the effect 5 min
biotic at maximum
factors aside from power
the dominant
(1000 watts), vegetation,
three times: wetime,
A first sterilized
threeour wet later,
hours soils by microwaving
and them for 5 min atby
24 h later (recommended maximum
M. Bidartondo
based on power
[27]).(1000 watts),
This three times:
sterilization A first was
method time, supposed
three hours tolater, and 24 hall
decrease later (recommended
microbial bybut
life [27] M. we did
Bidartondo based on [27]). This sterilization method was supposed to decrease all microbial life [27]
not test the sterilization on plate. We therefore had 12 different soil treatments (Figure 1c), allowing to
but we did not test the sterilization on plate. We therefore had 12 different soil treatments (Figure 1c),
test for allowing
soil layerto (organic
test for soilvs. mineral),
layer (organic and sterilization
vs. mineral), (sterile, non-sterile)
and sterilization effects,
(sterile, non-sterile) and comparing
effects, and
stand type (BS, BSE, and TA).
comparing stand type (BS, BSE, and TA).

Figure 1.Figure 1. Soilfrom

Soil cores coresblack
from spruce
black spruce (BS)
(BS) (a) and(a)trembling
and trembling
aspen aspen
(TA)(TA) (b) stands
(b) stands showing
showing thethe
difference
difference between organic and mineral soil layers, and (c) experimental scheme showing the 12
between organic and mineral soil layers, and (c) experimental scheme showing the 12 different treatments
different treatments and three growth seasons : TAO (Trembling aspen organic layer), TAM
and three growth aspen
(Trembling seasons: TAO
mineral (Trembling
layer), aspen
BSO (Black organic
spruce layer),
organic layer),TAM (Trembling
BSM (Black spruce aspen
mineralmineral
layer), layer),
BSO (Black
BSEOspruce organic
(Black spruce layer),
and BSMorganic
Ericaceae, (Blacklayer),
spruce mineral
BSEM (Blacklayer),
spruceBSEO (Black spruce
and Ericaceae, mineraland Ericaceae,
layer),
organicNS layer), BSEM
(non sterile (Black spruce
treatment, and treatment)).
and S (sterile Ericaceae, mineral layer), NS (non sterile treatment, and S
(sterile treatment)).
Forests 2020, 11, 815 4 of 16

2.2. Germination Process

Balsam fir seeds were provided by the Centre des semences forestières de Berthier, Quebec Ministry
of Forests, Wildlife, and Parks. Seeds were stratified for germination by immersing bags containing
circa 2000 seeds in running cold fresh water for 24 h (tap was let opened to ensure a water flow of
0.5 L/min), air dried for 48 h before putting them at 3 ◦ C in a polyethylene bag for 28 days and mixed
each week to ensure oxygenation. For each treatment, we put 10 seeds per pot and replicated it 10 times
(100 seeds per treatment). Seeds were planted at 0.5 cm depth for two months in a growth chamber
(Conviron model CG-108, Winnipeg, MB, Canada). The following conditions were set for germination
period: 16h photoperiod with a photosynthetically active radiation at pot level of 450 µmol m−2 s−1 ,
day/night temperature 20/10 ◦ C and relative humidity 80%. After two months, we counted the number
of germinated seeds per pot and calculated the germination rate for each pot and treatment (Table S2).
One randomly selected seedling per pot was kept for the next part of the experiment. The selected
seedlings were grown under these conditions for one supplementary month corresponding to the first
growth phase.

2.3. Growing Period

In addition to the first growth phase, two additional growing seasons were simulated,
each preceded by a dormancy period. First, a three-month period corresponded to the first growth
season. Two additional growth seasons were carried out, and each growing season lasted 3 months
under the same conditions as for the germination phase. Each dormancy phase lasted 2 months
with a 4 h photoperiod with photosynthetically active radiation at pot level of 450 µmol m−2 s−1 ,
temperature at 4 ◦ C and humidity at 80%. At the beginning of the second growth phase, soils were
fertilized with 20/20/20 (N/P/K) fertilizer at a 2 g/L concentration. Each pot received around 0.5 L of
fertilized water.

2.4. Seedlings Mycorrhizal Status, Biomass, Anatomy, and Physiology

At the end of the experiment, we counted the number of ectomycorrhizal (EM) and non-EM
apices under a magnifying glass for each seedling. The total number of EM apices and the percent
of EM apices were reported (Table S2). Height and root collar diameter were measured before stems
(with needles) and roots were separated and separately weighed fresh. Stem and roots were then
oven-dried at 40 ◦ C for 48 h for dry weight measurements. Needles were finely ground with a ball
mill (Pulverisette 0, Fritsch, Idar-Oberstein, Germany). Nitrogen and carbon contents in needles were
determined by combustion elemental analysis using a CNS-2000 Elemental Analyzer (Leco Instruments
Ltd., St-Josephs, MI, USA). Element concentrations were calculated as the sum of the absolute amounts,
divided by the total dry weight of the sample. Isotope analyses were performed with a PDZ Europa
ANCA-GSL elemental analyzer, interfaced with a PDZ Europa 20–20 isotope-ratio mass spectrometer
(Sercon Ltd., Crewe, Cheshire, UK) at the Stable Isotope Facility of University of California Davis
(Davis, CA, USA). The δ13 C was determined as:

δ13 C = ((Rsample − Rstandard )/Rstandard ) × 1000 (1)

where Rsample and Rstandard are, respectively, the 13 C/12 C ratios of the needle sample and the international
standard V-PDB (Vienna PeeDee Belemnite). The δ15 N followed the same formula, and Rsample and
Rstandard were respectively the 15 N/14 N ratios of the needle sample and the atmospheric N2 . This analysis
was possible only for the larger seedlings with at least 1 mg of dried needles (i.e., from 3 to 10 individuals
per treatment, see Table S2).

2.5. Statistical Analyses

Statistical analyses were done with R software (version 3.5.2, [28]). To take our experimental design
into account, we analyzed all our data with general linear mixed effect models (GLMM) and linear mixed
Forests 2020, 11, 815 5 of 16

effect models (LMM). These models are robust to assumptions on data distribution [29], and assume
that our treatment are not completely independent. As soils were collected independently in each stand,
the layer was considered as a fixed effect per se, and so was the sterilization nested in the soil layer.
The stand was not replicated, as only one site was studied and could not be included as a fixed effect.
However, to differentiate the variability due to stands from the variability within treatment (and between
the ten pseudoreplicates), we included a random effect in models, where treatment was nested into the
stand. We did not nest the presence of Ericaceae within spruce stands, as soils were collected independently.
The final formula of our model was: response variable ~Layer + Layer/Steril + (1|Stand/Treatment).
To choose the family of models to use for each variable, we first plotted the distribution of our
data through histograms and tested the normality with a Shapiro-Wilcoxon test. Height, Root collar
diameter, total, shoot and root dry weight, δ13 C, δ15 N, and N quantity followed a Gaussian distribution
(p-value > 0.05 for all Shapiro-Wilcoxon tests) and a LMM was used. For count variables (survival,
germination, number of mycorrhizal apices), we plotted the variance and means for each treatment.
If variance and means were correlated, we used a Poisson law in the GLMM. If no correlation
was detected, we used a different law, notable a Poisson negative binomial law for the GLMM on
germination. For survival, i.e., the presence or absence of seedling after three growth seasons, we used
a binomial law. For the percent of mycorrhizal apices, percent of C and N, and root/shoot ratio, we used
a quasi-binomial law, recommended for percent analysis. Finally for GLMM, to check if we used the
right law, we used the TestDispersion test (DHARMA package, [30]) and checked that the p-value was
non-significant, and therefore that residuals were neither over or under-dispersed.
For LMM, an ANOVA was performed on the model to detect the effect of stand, soil layer within
stand and sterilization within soil layer. For factors showing significantly different estimates in models,
differences between pairs of treatments were compared and represented based on contrasts between
marginal means of the models (using emmeans package, [31]).
To better describe differences between stand, we finally extracted the random effect from our
models (ranef function in nlme package, [32]). No statistical test was performed, as we could
not differentiate our stand effect form a site effect, but the difference of variance between stand
and treatments in LMM and GLMM allowed to identify traits more variable between stands than
within treatments.
Finally, to examine the correlations between variables, we performed pairwise Spearman
correlations, and corrected our p-value using a Bonferroni correction for the number of comparisons.

3. Results

3.1. Germination
Germination was successful for all treatment (Figure 2, Table S2). As no clear correlation was
detected between germination variance and mean, we analyzed germination with a GLMM following
a Poisson negative binomial distribution. No significant overdispersion was detected on residuals of
this model (p-value = 0.448). Only seedlings growing under TA showed a slightly lower germination
rate (Figure 2a), while soil layer and sterilization did not significantly influence germination (Table S3).

3.2. Survival
Over the 120 seedlings that remained for the growth experiment, 16 did not survive after 3 growing
seasons. The number of seedlings at the end of the experiment varied between 6 and 10 per treatment
(over 10 seedlings; Figure 2b; Table S2). For these binomial data, we analyzed the effect of stand,
layer and sterilization through a GLMM, assuming a binomial distribution of the data. The GLMM did
not detect any significant effect of soil layer and sterilization (Table S3) and the variance linked with
stand was null in our model. These results reject the idea that survival after three growing seasons
would be affected by stand, soil layer, or sterilization.
Forests 2020, 11, 815 6 of 16
Forests 2020, 11, x FOR PEER REVIEW 6 of 17

a BS BSE TA b BS BSE TA
6 10.0

# of germinated seeds
7.5
4

Survival
5.0

2
2.5

0 0.0

MNS
MS
ONS
OS

MNS
MS
ONS
OS

MNS
MS
ONS
OS
MNS

MNS

MNS
ONS

ONS

ONS
MS

MS

MS
OS

OS

OS
Figure 2. Number
Figure 2. Number (#) of germinated
(#) of germinated seeds
seeds perpot
per pot and
andperpertreatment
treatmentover over
10 seeds
10 (a) and Survival
seeds (a) and Survival
after threeafter three growth seasons (b). BS: Black spruce stand, BSE: BS with Ericaceae, TA: Trembling Aspen
growth seasons (b). BS: Black spruce stand, BSE: BS with Ericaceae, TA: Trembling Aspen
stand, M: Mineral, O: Organic, S, Sterile (blue), NS: non sterile (red). For the Survival (b), the
stand, M: Mineral, O: Organic, S, Sterile (blue), NS: non sterile (red). For the Survival (b), the proportion
proportion of dead seedlings is represented in black, while live seedlings at the end of the experiment
of dead seedlings is represented in black, while live seedlings at the end of the experiment are in grey.
are in grey.

3.3. Mycorrhization
3.2. Survival
Over the 120
Ectomycorrhizal seedlings
apices werethat remainedon
observed for seedlings
the growth experiment, 16 did not
in all treatments survive
after the after
three3 growing
growing seasons. The number of seedlings at the end of the experiment varied between 6 and 10 per
seasons. Seedlings growing on sterile soils also developed EM apices after three growth seasons but in
treatment (over 10 seedlings; Figure 2b; Table S2). For these binomial data, we analyzed the effect of
lower proportion, with
stand, layer and1.8 to 7.2 times
sterilization more
through ectomycorrhizae
a GLMM, on non-sterile
assuming a binomial treatments
distribution asThe
of the data. compared to
sterile treatments
GLMM did (mean = 4.32).
not detect any significant effect of soil layer and sterilization (Table S3) and the variance
Theselinked
numberwith stand
and was null in of
percent ourectomycorrhizal
model. These results reject thewere
apices idea that survival after
analyzed withthree growingassuming,
GLMM
seasons would be affected by stand, soil layer, or sterilization.
respectively, a Poisson and a quasibinomial distribution, based on the relationship between variance
and means.3.3.No overdispersion was detected for these models (p-value > 0.05). The number and percent
Mycorrhization
of EM apices was lower under
Ectomycorrhizal BSE
apices werethan underonBS
observed and TA,
seedlings and
in all the higher
treatments after variance between stands
the three growing
than within treatment
seasons. supported
Seedlings growing on this difference
sterile (Table 1, EM
soils also developed Figure
apices 3a,b). Across
after three stands,
growth the
seasons butnumber of
EM apices in waslower proportion,
always higherwith 1.8 to 7.2than
on organic timesonmore ectomycorrhizae
mineral soil (Table 1, on Figure
non-sterile treatments
3c) and always as higher on
compared to sterile treatments (mean = 4.32).
non-sterile soils as compared with sterile soils (Table 1, Figure 3c). All these differences were supported
These number and percent of ectomycorrhizal apices were analyzed with GLMM assuming,
by pairwise comparisons
respectively, of and
a Poisson marginal meansdistribution,
a quasibinomial estimates based
(Figure
on the3c). These differences
relationship between soil
between variance
layers were andnot observed
means. on the percent
No overdispersion was of mycorrhizal
detected for these apices
models (Table
(p-value1).
> 0.05). The number and
Forests 2020, 11, x FOR PEER REVIEW 7 of 17
percent of EM apices was lower under BSE than under BS and TA, and the higher variance between
stands than within treatment supported this difference (Table 1, Figure 3a,b). Across stands, the
a b c
number ofBSEM apices
BSE TA
was always BS
higher on BSE
organic TA
than on mineral soil (Table 1, Figure 3c) and
0.8
5 higher on non-sterile soils as compared with sterile soils (Table 1, Figure 3c). All these
always
EMM for # of mycorrhizal apices

4
differences were supported by pairwise comparisons of marginal means estimates (Figure 3c). These
differences
4 between soil layers were not observed on the percent of mycorrhizal apices (Table 1).
0.6
Percent of EM apices
log(# EM Apices)

3
3
0.4

2 2

0.2
1
1

0 0.0

M O M O M O M O M O M O NS M SM NS O SO

Figure 3. Number (#) of ectomycorrhizal (EM) apices (a, log transformed), Percent of ectomycorrhizal
Figure 3. Number (#) of ectomycorrhizal (EM) apices (a, log transformed), Percent of ectomycorrhizal
apices (b) and comparison of estimated marginal means (EMM, c) between stands, layers and
apices (b) and comparison of estimated marginal means (EMM, c) between stands, layers and
sterilization treatments.
sterilization BS: Black
treatments. spruce
BS: Black sprucestand, BSE:BS
stand, BSE: BSwith
with Ericaceae,
Ericaceae, TA: Trembling
TA: Trembling Aspen stand,
Aspen stand,
NS: non NS:
sterile, S: sterile, M: Mineral, O: Organic. In (a,b), blue dots refer to sterile treatments,
non sterile, S: sterile, M: Mineral, O: Organic. In (a) and (b), blue dots refer to sterile treatments, and
red dots and
to non-sterile
red dots to treatments. In (c), blue
non-sterile treatments. bars
In (c), indicate
blue confidence
bars indicate intervals
confidence of estimated
intervals of estimatedmarginal
marginalrefer
means, arrows means,
to arrows refer to comparisons
comparisons among marginal
among marginal means. means.
If twoIfarrows
two arrows overlap,
overlap, Tukeypost-hoc
Tukey
post-hoc tests are non-significant and p-value for pairwise comparisons are > 0.05.
tests are non-significant and p-value for pairwise comparisons are > 0.05.
Table 1. General linear mixed effect models (GLMM) and linear mixed effect models (LMM) estimates
for the number (#) and percent (%) of ectomycorrhizal (EM) apices. Std: standard error, p: p-value. *:
p-value < 0.01, **: p-value < 0.001, ***: p-value < 0.0001. Model formula: ~Layer + Layer/Steril +
(1|Stand/Treatment).

#EM Apices (GLMM) % of EM Apices (LMM)

Factor Estimate Std. Error Significance Estimate Std. Error Significance
Forests 2020, 11, 815 7 of 16

Table 1. General linear mixed effect models (GLMM) and linear mixed effect models (LMM) estimates
for the number (#) and percent (%) of ectomycorrhizal (EM) apices. Std: standard error, p: p-value.
*: p-value < 0.01, **: p-value < 0.001, ***: p-value < 0.0001. Model formula: ~Layer + Layer/Steril +
(1|Stand/Treatment).
#EM Apices (GLMM) % of EM Apices (LMM)
Factor Estimate Std. Error Significance Estimate Std. Error Significance
Fixed effects Intercept (Mineral) 2.7393 0.3879 *** 0.27064 0.05809 **
Organic Layer 1.0532 0.3122 *** 0.09448 0.05049
Layer M: Sterile −1.4386 0.3310 *** −0.18923 0.05116 **
Layer O: Sterile −1.2573 0.3138 *** −0.30239 0.04971 ***
Variance Std. Error Variance Std. Error
Random effects Treatment:Stand 0.1406 0.375 0.002488 0.04988
Stand 0.3025 0.550 0.006203 0.07876
Residuals 0.011555 0.10750

3.4. Growth and Biomass

For height, total, shoot and root dry weight and root collar diameter, we tested the effect of soil
layer and sterilization through LMM, considering the Gaussian distribution of the data. Across stand
types, soil layer explained most differences of anatomy and biomass (Table 2 and Table S3 for model
estimates, and Table S4 for ANOVA). Indeed biomass (total, shoot and root dry weight), height and
root collar diameter were always significantly greater for seedlings growing on organic than on mineral
layers (Table 2, Table S3, Figure 4). ANOVA on LMM also supported an effect of sterilization only on
root dry weight (Table S4, Figure 4d). The variance attributed to stands within the random effects was
null or lower than the variance attributed to treatments, suggesting a lack of effect of the stand on these
variables (Table 2 and Table S3).

Table 2. LMM estimates for height, root collar (RC) diameter, total dry weight (DW). Std: standard
error, p: p-value. *: p-value < 0.01, **: p-value < 0.001, ***: p-value < 0.0001. LMM formula: ~Layer +
Layer/Steril + (1|Stand/Treatment).
Height RC diameter Total DW
Factor Estimate Std. Error Sig. Estimate Std. Error Sig. Estimate Std. Error Sig.
Fixed effects Intercept (Mineral) 56.374 6.089 *** 1.4646 0.1350 *** 0.29900 0.09784 **
Organic Layer 31.051 8.378 *** 0.5410 0.1869 * 0.35967 0.13836 *
Layer M: Sterile −8.146 8.701 −0.1534 0.1918 −0.07233 0.13836
Layer O: Sterile 16.795 8.067 * 0.2673 0.1818 0.33967 0.13836 *
Variance Std. Error Variance Std. Error Variance Std. Error
Treatment: Stand 0 0.00 0.01639 0.128 0.01401 0.1183
Random effects Stand 0 0.00 0.00000 0.000 0.00000 0.0000
Residuals 927 30.45 0.31469 0.561 0.14709 0.3835
Forests 2020, 11, x FOR PEER REVIEW 8 of 17

a b c 1.00
d 0.4
EMM for root collar diameter (mm)

100
EMM for total DW (g)
EMM for Height (cm)

EMM for root DW (g)

0.75 0.3
2.0

80

0.50 0.2

60 1.5

0.25 0.1

40
M O M O M O NS M S M NS O S O

Figure 4. Comparison of estimated

Figure 4. Comparison marginal
of estimated means
marginal (EMM)
means (EMM) showing
showing significant
significant differences ofdifferences
height of height
(a), root collar (a), root collar(b),
diameter diameter
total(b),dry
total weight
dry weight(c,
(c, DW),
DW), and root root
and DW (d)DW of seedlings
(d) ofper soil layer per soil layer
seedlings
(Mineral: M, Organic: O) and sterilization treatment (S: sterile, NS: non sterile). Blue bars indicate
(Mineral: M, Organic: O) and sterilization treatment (S: sterile, NS: non sterile). Blue bars indicate
confidence intervals of estimated marginal means, arrows refer to comparisons among marginal
confidence intervals of estimated
means. If two marginal
arrows overlap, Tukeymeans,
post-hoc arrows refer to comparisons
tests are non-significant among
and p-value for pairwisemarginal means.
comparisons are > 0.05.
If two arrows overlap, Tukey post-hoc tests are non-significant and p-value for pairwise comparisons
are > 0.05. Table 2. LMM estimates for height, root collar (RC) diameter, total dry weight (DW). Std: standard
error, p: p-value. *: p-value < 0.01, **: p-value < 0.001, ***: p-value < 0.0001. LMM formula: ~Layer +
Layer/Steril + (1|Stand/Treatment).

Height RC diameter Total DW

Factor Estimate Std. Error Sig. Estimate Std. Error Sig. Estimate Std. Error Sig.
Fixed effects Intercept (Mineral) 56.374 6.089 *** 1.4646 0.1350 *** 0.29900 0.09784 **
Organic Layer 31.051 8.378 *** 0.5410 0.1869 * 0.35967 0.13836 *
Layer M: Sterile −8.146 8.701 −0.1534 0.1918 −0.07233 0.13836
Layer O: Sterile 16.795 8.067 * 0.2673 0.1818 0.33967 0.13836 *
Variance Std. Error Variance Std. Error Variance Std. Error
Forests 2020, 11, 815 8 of 16

3.5. Anatomy and Physiology

All measurements of root/shoot ratio, percent of C and N content were analyzed with GLMM
assuming a quasibinomial distribution of the variables. Measurements of δ13 C and δ15 N followed
a Gaussian distribution and were analyzed with a LMM. No overdispersion was detected for these
models (p-value > 0.05).
Among these variables, only the root/shoot ratio was significantly different between soil layers
(Table S4), with more investment in roots than in shoots in the mineral layer (Table S3, Figure 5a).
Moreover, the root/shoot ratio was less variable between stands than within treatments (Table S3),
suggesting that seedlings growing on soil from TA stand would develop more roots than shoots,
as compared with soils from other stands.
Forests 2020, 11, x FOR PEER REVIEW 9 of 17

a 0.7
b BS BSE TA c BS BSE TA

15
30

0.6
EMM for RS ratio

10
20
N (%)

d15N
0.5
5

10
0.4
0

0
NS M SM NS O SO M O M O M O M O M O M O

Figure 5. Comparison of estimated marginal means (EMM) showing significant differences of Root/shoot
Figure 5. Comparison of estimated marginal means (EMM) showing significant differences of
ratio between soil layers and sterilization treatments (a), and distribution of percent of N (b) and
Root/shoot ratio between soil layers and sterilization treatments (a), and distribution of percent of N
15
δ N (c). BS: Black spruce
(b) and δ15N (c).stand,
BS: BlackBSE:
spruceBS with
stand, BSE: Ericaceae, TA:TA:
BS with Ericaceae, Trembling Aspen
Trembling Aspen stand,stand,
NS: nonNS: non sterile,
sterile, S: sterile, M: Mineral, O: Organic. In (a), blue bars indicate confidence intervals of estimated
S: sterile, M: Mineral, O: Organic. In (a), blue bars indicate confidence intervals of estimated marginal
marginal means, arrows refer to comparisons among marginal means. If two arrows overlap, Tukey
means, arrows post-hoc
refer totests comparisons among
are non-significant marginal
and p-value means.
for pairwise If two
comparisons are >arrows
0.05. In (b)overlap,
and (c), blueTukey post-hoc
dots refer to sterile
tests are non-significant p-value and
and treatments, forred dots to non-sterile
pairwise treatments.are > 0.05. In (b,c), blue dots refer to
comparisons
sterile treatments, and red
3.6. The Effects dots toShrubs
of Ericaceous non-sterile treatments.
We expected that the presence of Ericaceae would result in a negative effect on seedling growth
Variations of percenttoofBSC,
as compared δ13
soil. C, N and
According δ15 N in
to different needles
models, basedwere
on thenot explained
comparison byvariability
between layer and sterilization
within stand15
(Table S3). Measures of δ N and percent of N were more variable within stand than stands
and between treatments, only few traits were different between BS and BSE between treatments,
(Table 3), and the main difference was observed on mycorrhization, as seedlings growing in BSE soil 15
but showed thathad seedlings growing on soils from TA stand had a lower N and higher δ N than those
less mycorrhizal apices (in number and percent) than those growing in BS soil (Figure 3a,b, Table
growing on soil3).from BSobserved
We also standthat (Figure
seedling5b,c).
growing under BSE developed less roots than shoots, as compared
to seedlings growing under other stands.
3.6. The Effects of Ericaceous Shrubs
Table 3. Random effects attributed to stands extracted from different GLMM and LMM (full model
estimates reported in Tables 1 and 2, Table S3). In grey, the variance attributed to stands was larger
We expected that the attributed
to variance presence of Ericaceae would result in a negative effect on seedling growth
to treatment.
as compared to BS soil. Germination
Stand According to # EMdifferent
Apex models,
%EM Apex based
d15N on the comparison
N (µg) %N between variability
R/S Ratio
within stand and BS between0.1314 treatments, only few
0.1526 traits −2.3987
−0.0124 were different
5.1111 between
1.7067 BS and BSE stands
−0.0178
BSE 0.0142 −0.6867 −0.0829 1.0662 −0.5412 −0.4002 −0.0081
(Table 3), and the main
TA difference
−0.1382 was0.5500observed0.0953
on mycorrhization,
1.3319 −4.5698 as−1.3064
seedlings growing in BSE soil
0.02600
had less mycorrhizal apices (in number and percent) than those growing in BS soil (Figure 3a,b, Table 3).
We also observed that seedling growing under BSE developed less roots than shoots, as compared to
seedlings growing under other stands.

Table 3. Random effects attributed to stands extracted from different GLMM and LMM (full model
estimates reported in Tables 1 and 2, Table S3). In grey, the variance attributed to stands was larger to
variance attributed to treatment.
Stand Germination # EM Apex %EM Apex d15N N (µg) %N R/S Ratio
BS 0.1314 0.1526 −0.0124 −2.3987 5.1111 1.7067 −0.0178
BSE 0.0142 −0.6867 −0.0829 1.0662 −0.5412 −0.4002 −0.0081
TA −0.1382 0.5500 0.0953 1.3319 −4.5698 −1.3064 0.02600
 Forests 2020, 11, 815 9 of 16
Forests 2020, 11, x FOR PEER REVIEW 10 of 17

3.7.Correlations
3.7. Correlationsbetween
betweenVariables
Variables
In order
In ordertotohave a global
have overview
a global of seedlings
overview response,response,
of seedlings and notably
andof notably
the effect of
of mycorhization,
the effect of
we finally investigated
mycorhization, the correlations
we finally investigatedbetween variables. between
the correlations The use of Bonferroni
variables. Thecorrections decreased
use of Bonferroni
the numberdecreased
corrections of significant correlations,
the number and we observed
of significant different
correlations, and wegroups of correlations
observed (Figureof
different groups 6).
The number(Figure
correlations of EM 6).
apices
The was significantly
number and was
of EM apices positively correlated
significantly and with the percent
positively of EM
correlated apices,
with the
height and
percent of EMtotalapices,
dry weight of seedlings
height and total (Figure
dry weight of seedlingsδ15(Figure
6). Interestingly, N, N content and N concentration
6). Interestingly, δ15N, N
were correlated
content and N (or anti-correlated)
concentration with
were each other,(or
correlated butanti-correlated)
never significantly correlated
with with mycorrhizal
each other, but never
indices (Figure
significantly 6). These
correlated withresults show that
mycorrhizal growth
indices was6).
(Figure globally responding
These results showtothatmycorrhization,
growth was
throughresponding
globally a positive interaction, but not through
to mycorrhization, throughNa nutrition.
C positive interaction, but not through N nutrition.

C 1
t
igh
He

Height 0.11 1
otD
Ro

lDW

RootDW 0.12 0.83 1

ta
To

tDW

TotalDW 0.13 0.9 0.96 1

oo
Sh

ShootDW 0.14 0.91 0.91 0.99 1

5N
d1

yc

0.03 − 0.06 − 0.07 − 0.08 − 0.08 1

.M

d15N
ex
Ap

c
my
nt.

Apex.Myc 0.11 0.42 0.35 0.4 0.41 0.1 1

rce
Pe

.
rog

Percent.myc − 0.03 0.08 − 0.01 0.02 0.04 0.15 0.8 1

mic
N.

N.microg. 0.13 − 0.12 − 0.25 − 0.19 − 0.16 − 0.34 − 0.08 − 0.06 1

N

ot
o
Sh

N 0.09 − 0.06 − 0.2 − 0.13 − 0.1 − 0.34 − 0.04 − 0.02 0.96 1

ot.
Ro

Root.Shoot 0.1 − 0.37 − 0.03 − 0.25 − 0.34 − 0.07 − 0.2 − 0.13 − 0.13 − 0.19 1
3C
d1

d13C 0.21 0.11 0.21 0.15 0.13 0 0 − 0.08 − 0.08 − 0.09 0.23 1

−1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1

Figure 6. Significant correlations between variables, after Bonferroni corrections for multiple
Figure 6. Significant correlations between variables, after Bonferroni corrections for multiple
comparisons. N. microg: N content (µg).
comparisons. N. microg: N content (μg).
4. Discussion
4. Discussion
Our experiment on the germination, growth and survival of balsam fir on boreal soils has revealed
Our experiment
complex interactions on the germination,
between growth and
soil layers, dominant survivaland
vegetation, of balsam fir on boreal
microorganisms. soils has
Soil layer was
revealed complex interactions between soil layers, dominant vegetation, and
the main structuring factor affecting seedling biomass and anatomy, and soil sterilization only affected microorganisms. Soil
layer was the main structuring
the mycorrhization factor affecting
and the root/shoot seedling biomass
ratio. Differences between and anatomy,
stand and soil
were rather sterilization
observed than
only affected the mycorrhization and the root/shoot ratio. Differences between
tested, but revealed differences of mycorrhization, germination, and root/shoot ratio. Interestingly, stand were rather
observed
results werethandifferent
tested, but
onrevealed differences
germination, survival,of mycorrhization,
and growth, allowing germination, and root/shoot
to disentangle ratio.
interactions
Interestingly,
between soil, results
fungi andwere different
stand along onthegermination, survival,
first steps of balsam firand growth, allowing
establishment into the to disentangle
boreal forest.
interactions
Following the experiment of McLaren and Janke [26] in Michigan, boreal forest soilsinto
between soil, fungi and stand along the first steps of balsam fir establishment canthebe
boreal forest.
favorable to seed germination, especially the organic layer. In our experiment, seed germination was
Following
similar betweenthe experiment
organic horizonsof (14
McLaren
to 28%)and andJanke [26]soil
mineral in (9
Michigan,
to 22%). boreal forest soilsrate
Our germination canwasbe
favorable to seed germination, especially the organic layer. In our experiment,
relatively high, confirming that boreal soils are favorable to balsam fir establishment and that this seed germination was
similar
speciesbetween organic
is relatively horizonsto(14
well adapted to 28%)
these and mineral
soil conditions. soil (9 to 22%).
Interestingly, soils Our
fromgermination
spruce stands rate
(BSwas
and
relatively high, confirming that boreal soils are favorable to balsam fir establishment
BSE) were more favorable to fir germination that those from TA stands (Table 3), while we expected and that this
species is relatively
the opposite. well adapted
It is possible that thetopresence
these soil ofconditions.
mosses andInterestingly, soils from spp.
particularly Pleurozium spruce
in stands (BS
the organic
and
layerBSE)
of BSwere
standsmore favorable
represents to fir
a good germination
seedbed thatfir
for balsam those
seedsfrom TA stands
contrary (Table 3),litter
to the broadleaf while we
[26,33].
expected the opposite. It is possible that the presence of mosses and particularly
However, beside this initial difference in germination rate, final survival after three growth seasons Pleurozium spp. in
the
wasorganic
similarlayer of BStreatments.
between stands represents a good seedbed for balsam fir seeds contrary to the broadleaf
litter [26,33]. However, beside this initial difference in germination rate, final survival after three
growth seasons was similar between treatments.
Forests 2020, 11, 815 10 of 16

While germination was variable but successful in all soil types, highlighting the ability of fir
seedlings to grow on contrasted substrates, we observed more variability in seedling growth, in terms
of biomass, anatomy and physiology. Based on field observations [19], we expected growth to be
better in TA soils than in BS soils. The responses were more complex, and for example only root/shoot
ratio differed between stands, with seedlings growing on soil from TA stand producing more roots
than shoots as compared to other stands. As the effect of stand relied only on differences within
a site, more sites shall be sampled to confirm both the lack of effect of stands on growth, and the
peculiar difference of root/shoot ratio. Considering that previous studies including more sites detected
consistent differences between soils of closely located stands [14,15,34], we expect to observe similar
results with soils from other local sites. Across stands, most differences were observed between soil
layers, and indeed seedlings growing in organic soils were taller, and developed more dry weight
than seedlings growing in mineral soils. This result was consistent across soils from different stands,
showing the importance of soil characteristics and organic layer on fir seedling growth. Interestingly,
for other trees of the boreal forests, such as black spruce and trembling aspen, their germination
and establishment are on the contrary favored by bare mineral soil, a situation encountered after
severe fire [35–40]. Black spruce are also able to grow on thick layer of organic soil [35], a situation
encountered later along post-fire succession in the boreal fire [41]. The response of fir to organic layer is
therefore more typical of a late colonizer [41], and indeed the forest at our site resulted from a post-fire
recolonization dated around 1916 [42].
Beside the contrasted soil layers and differences between stands, seedlings managed to form
ectomycorrhizae, but developed up to 7.2 times more ectomycorrhizae in non-sterile compared to sterile
soils, after three growth seasons. The difference in mycorrhization between sterile and non-sterile soils
confirms that our sterilization targeted micro-organisms, but the growth chamber did not exclude a
possible post-colonization by airborne spores (a frequent phenomenon at least in greenhouses, [43]).
We assume that these spores would come from local forests around the lab (campus of Amos),
located 100 km apart from our field site. Beside the colonization of sterile soils, our results showed
that mycorrhization was greater in TA soils, and significantly higher in organic layers (in number,
not in percent). Moreover, the mycorrhization was correlated with seedlings biomass and height
(whatever the treatment, Figure 6). The difference in mycorrhization observed between stand types
shall be confirmed on other sites of course, but these results are consistent with our hypothesis and the
results obtained on root ramification indices of naturally-growing and older saplings [19]. Interestingly,
soil analyses did not detect differences of abundance and diversity of ectomycorrhizal fungi on the
very same site and other neighboring sites [15], suggesting that young fir encounter not more EM
fungi under TA, but more compatible mycorrhizal fungi. In this experiment, we did not sequence the
mycorrhizal community in soils or on roots, but such data might be useful to identify fungi possibly
linked with the better in-situ growth of young balsam fir in boreal forest soils dominated by trembling
aspen, as responses to mycorrhization are also sensitive to fungi identity (e.g., [44,45]).
Although we expected N nutrition to be better in TA than in BS soils, seedlings physiology did
not reveal such a trend. Indeed, N concentration was more variable within treatments than between
stand, soil layer or sterilization treatments. Moreover, N concentration in needles was uncorrelated
with mycorrhizal parameters, while in general, mycorrhizal symbioses are expected to provide N and
P benefits to their partners [17]. We shall confirm this trend with a larger sampling, but the lack of
correlation between N parameters, and even growth, also invite to consider the different N forms
(ammonium and nitrate) accessible to young balsam fir. The greater mycorrhization and slightly
higher δ15 N of seedlings growing on soils from TA stand suggests that balsam fir rather relies on
mycorrhizae for N acquisition in these conditions, perhaps also linked with increased P availability in
soils under aspen [46]. Indeed, δ15 N variation may be related to the use of different N sources [47]
and notably from ectomycorrhizal fungi. For example Mayor et al. [48] revealed that higher δ15 N
of black spruce needles reflected a greater dependency on ectomycorrhizal fungi. On the contrary,
the lower mycorrhization and slightly lower δ15 N in black spruce soil suggests that balsam fir rather
Forests 2020, 11, 815 11 of 16

Forests
relied on2020, 11, xNFOR
other PEER REVIEW
sources in these treatments, notably ammonium, directly acquired from soil. 12 In
of pots
17

containing soil from BS stand, we indeed noticed the development of Pleurozium species, mosses able
containing soil from BS stand, we indeed noticed the development of Pleurozium species, mosses able
to fix N [49]. These mosses could have released ammonium in BS treatments. Interestingly, in Northern
to fix N [49]. These mosses could have released ammonium in BS treatments. Interestingly, in
Quebec, black spruce δ15 N values15are generally low [50], suggesting a minor dependence toward
Northern Quebec, black spruce δ N values are generally low [50], suggesting a minor dependence
mycorrhizal fungi. More
toward mycorrhizal generally,
fungi. conifers conifers
More generally, and early-succession speciesspecies
and early-succession are known for their
are known for greater
their
preference for ammonium
greater preference than for nitrate
for ammonium than for[51,52]. A [51,52].
nitrate more precise
A more analysis
preciseon balsamon
analysis firbalsam
preferences
fir
would be complementary,
preferences but our data but
would be complementary, already suggest
our data thesuggest
already ability the
of young
ability balsam
of youngfirbalsam
to acquire
fir to N,
whatever
acquire their mycorrhization
N, whatever rate and possibly
their mycorrhization rate and from different
possibly from sources.
different sources.
OurOur results
resultsdiffered
differedfrom previous
from previousfield observations
field observationswhere
wherewe we
hadhaddetected
detecteda negative effect
a negative of the
effect
presence of ericaceous shrubs on growth of fir seedlings, possibly mediated
of the presence of ericaceous shrubs on growth of fir seedlings, possibly mediated by mycorrhizal by mycorrhizal fungi [19].
This growth
fungi [19].chamber
This growth study did notstudy
chamber confirm didany notnegative
confirm effect from microorganisms
any negative present in soils
effect from microorganisms
from ericaceous
present in soils shrubs,
from except that mycorrhization
ericaceous shrubs, except that was lower in these soils.
mycorrhization was Interestingly,
lower in these soils from
soils.
BSEInterestingly,
did not decrease soils needle
from BSE did not decreasecontrary
N concentrations, needle N toconcentrations,
the field [19]. We contrary to thethat
also found field [19]. We
germination,
also found
survival and that
growth germination, survival and
were all relatively highgrowth were
in black all relatively
spruce soils inhigh
our in black spruce
experiment as soils in our to
compared
experiment
field observations.as compared to field observations.
Several explanations could beSeveral explanations
formulated to explain could
thesebedifferences
formulatedbetween
to explain field
these differences between field and growth chamber conditions (Figure
and growth chamber conditions (Figure 7). Among them, root competition for nutrient acquisition 7). Among them, root
competition
with BS roots [53] for and
nutrient
water acquisition with BS roots
logging constrain in BS[53] and [54]
stands water logging
have both constrain in BS standsin[54]
been demonstrated these
have both been demonstrated in these very same forest
very same forest stands. The presence of a common mycorrhizal network between young stands. The presence of a common
and older
mycorrhizal network between young and older balsam fir or TA in TA stands could also explain
balsam fir or TA in TA stands could also explain these differences.
these differences.

Observed pattern on growth :

Factors
independent TA > BS > BSE
from stand
type
Variable d13C, C
related to other

- mycorrhization
factors

+ mycorhization + ammonium
effect on N in close to
needles? Ericaceae
+ germination
organic layer
biomass of
+ effect on

Organic

+ Less competition with TA roots

mineral layer
- Growth on

Mineral

+ Other N
alone

source in situ
?

Increasing number of mycorrhizal root tips

Figure
Figure 7. 7. Syntheticsummary
Synthetic summaryofofpositive
positive and
and negative effect
effect of
ofsoil
soillayer,
layer,stand,
stand,trees,
trees,and
andfungi based
fungi based
on on ex-situ
ex-situ andand in-situ
in-situ observations.
observations.
Forests 2020, 11, 815 12 of 16

Except for mycorrhization, we did not detect such a positive effect of trembling aspen soils on
balsam fir germination and growth in the growth chamber. We had expected a more direct effect of
trembling aspen soils or other co-occurring species, since balsam fir thrived better under aspen and
fewer adult trees occurred on our black spruce adjacent sites [19]. As mycorrhizae are essential for
balsam fir growth, more direct interactions with neighboring tree roots suggest that balsam fir could
depend on a common mycorrhizal network, linking them to trembling aspen roots and organic C
(e.g., [55–58]). To go further with this hypothesis, a field experiment would be required to exclude
mycorrhizal networks by regularly cutting soils around seedlings or setting a fine mesh around the
young seedlings [58]. Moreover, such experiment would allow testing for root competition, from both
ericaceous shrubs and black spruce trees. Such an experiment, handled in a Pinus radiata forest,
revealed that canopy tree roots reduced growth of seedlings but that common mycorrhizal networks
counteracted this competition [25]. Tracing of marked N injected to adult balsam fir trees has also
revealed a possible transfer of nitrogen to balsam fir seedlings [59]. Such transfers could occur on
our study sites, as balsam fir adults were scattered but more abundant under trembling aspen than
under black spruce [19]. Bent et al. [60] also suggested common mycorrhizal network involving
trembling aspen in boreal forests of Alaska, but did not observe any balsam fir seedlings in these forests.
Setting an experiment, excluding root competition and common mycorrhizal networks, and tracing N
from adult aspen and balsam fir would definitely allow us to understand how boreal forests favor
balsam fir establishment today.

5. Conclusions
Our results show that young fir were able to germinate and survive in all conditions, and that
their growth were mainly favored by the organic layer. Aspen stands appear more favorable
to mycorrhization of balsam fir compared to other stands, and across stands, mycorrhization,
seedlings height, and biomass were positively correlated. Therefore, the increase in aspen due
to climate change, natural or anthropogenic disturbances [61,62] offers an opportunity to increase
balsam fir abundance in the black spruce dominated forests to the north. For future predictions in a
context of climate change, our results highlight the dependency of balsam fir toward biotic interactions,
involving mycorrhizal fungi, but also interactions with trees. These interactions are essential for
species migration [63,64], and taking them into account could definitely improve our models of tree
distribution as illustrated by several recent studies, notably on the boreal forest [65,66]. The case of
balsam fir is not isolated, and for example Acer saccharum establishment in boreal forest also depends
on soil characteristics and is limited by the access to mycorrhizal fungi [67]. Our study shows that
setting controlled experiments can greatly improve our understanding of tree adaptability to new
conditions, migration, and possible climate change effect on boreal forest.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4907/11/8/815/s1.

Table S1: Soil characteristics measured in situ from [15]. Table S2: Means (and standard deviation) of seedlings
measurements during germination and the three growth seasons. Diameter: root collar diameter, R/S: root/shoot
ratio and DW: dry weight. Germination refers to the number of germinated seedlings over 100 (10*10 replicates,
survival to the number of saplings at the end of the three growth season (over 10) and EM to ectomycorrhizal
apices. For physiological measurements, only seedlings producing >1 mg of needles were used (n: number of
individuals used for these measures). Table S3: Estimates of LMM and GLMM on Germination, survival rate,
shoot and root dry weigh (DW), d13C, percent of C, d15N, percent of N, N content (µg) and Root/Shoot (RS) ratio.
Table S4: Statistics from ANOVA on seedling height, root collar diameter, δ13 C, δ15 N, total C and N content,
total dry weight, shoot and root dry weight.
Author Contributions: Conceptualization, M.N., M.G., M.R., A.D. and Y.B.; methodology, M.N., M.G., A.D. and
M.R.; statistical analysis, M.N. and M.R.; writing—original draft preparation, M.N. and M.R.; writing—review
and editing, M.N., M.R., M.G., A.D. and Y.B.; visualization, M.N. and M.R.; supervision, M.G., A.D., Y.B. and M.R.;
project administration, Y.B., A.D. and M.R.; funding acquisition, Y.B. and M.R. All authors have read and agreed
to the published version of the manuscript.
Forests 2020, 11, 815 13 of 16

Funding: This work was financed by a Mitacs acceleration fellowship to MN in collaboration with Norbord Inc.
and from The UQAT-UQAM NSERC industrial chair in sustainable forest management to Y.B. We benefitted
from the support of an “Investissement d’Avenir” grants also managed by Agence Nationale de la Recherche
(CEBA, ref. ANR-10-LABX-25-01; ANAEE-France: ANR-11-INBS-0001, TULIP: ANR-10-LABX-0041), and from a
Champlain grant from the CFQCU.
Acknowledgments: Authors sincerely thank Lyne Blackburn for field assistance, Aurélie Suzanne for laboratory
assistance and Martin Bidartondo for giving helpful advice for soil sterilization. We deeply thank Philippe
Marchand for his help on statistics.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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