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Tree species effects on nutrient cycling and soil biota: A feedback
mechanism favouring species coexistence
Cristina Aponte
a,b,
⇑
, Luis V. García
a
, Teodoro Marañón
a
a
Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNASE) CSIC, Av de la Reina Mercedes, 10 41012 Sevilla, Spain
b
Department of Forest and Ecosystem Science, Melbourne School of Land and Environment, The University of Melbourne, 500 Yarra Boulevard, Richmond, Victoria 3121, Australia
article info
Article history:
Available online 12 June 2013
Keywords:
Feedback processes
Microbial biomass
Mycorrhizal fungi
Nutrient cycling
Plant–soil interactions
Quercus
abstract
We synthesise a series of independent but integrated studies on the functioning of a mixed Mediterra-
nean oak forest to demonstrate the tree–soil interactions underpinning a positive feedback process that
sustains the coexistence of two oak species. The studies focused on the foliar functional traits, plant
regeneration patterns, biogeochemical cycles, soil microbial biomass and ectomycorrhizal (ECM) fungal
diversity associated with the co-dominant evergreen Quercus suber and deciduous Quercus canariensis
in a Mediterranean forest in southern Spain.
Foliar attributes differed between oak species, with Q. canariensis having higher nutrient content
and lower carbon to nutrient ratios and leaf mass per area than Q. suber. These attributes reflected
their distinct resource use strategies and adaptation to high and low resource-availability environ-
ments, respectively. Leaf-fall nutrient concentrations were higher in Q. canariensis than in Q. suber
and were correlated with concentrations in the fresh leaves. Leaf-fall nutrient concentrations influ-
enced nutrient return, leaf-fall decay rate and the proportion of nutrients released from decomposing
leaf-fall, all of which were higher for Q. canariensis than for Q. suber. This generated a differential net
nutrient input into the soil that led to increased soil nutrient concentrations under the canopy of Q.
canariensis as compared to Q. suber. The fraction of slowly decomposing leaf-fall that builds up soil
organic matter was higher for Q. canariensis, further raising the nutrient and moisture retention of
its soils. Differences between species in soil properties disappeared with increasing soil depth, which
was consistent with the hypothesised leaf-fall-mediated effect. Tree-species-generated changes in soil
properties had further impacts on soil organisms. Soil microbial biomass (Cmic) and nutrients (Nmic,
Pmic) were higher under Q. canariensis than under Q. suber and were positively related to soil mois-
ture content and substrate availability (particularly soil N). The composition of the ECM fungal com-
munity shifted between the two oaks in response to changes in the soil properties, particularly soil Ca
and pH. Lower ECM phylogenetic diversity and higher abundance of mycorrhizal species with sapro-
phytic abilities were related to the greater soil fertility under Q. canariensis. Overall, the two oak spe-
cies generated soil conditions that aligned with their resource-use strategies and would enhance their
own competitive capabilities, potentially creating a positive feedback. The two Quercus created soil
spatial heterogeneity that could enable their coexistence through spatial niche partitioning. This study
demonstrates the critical role of aboveground-belowground interactions underpinning forest commu-
nity composition.
Ó2013 Elsevier B.V. All rights reserved.
1. Introduction
Plant species coexistence has always intrigued ecologists,
particularly in relation to environmental variability (Grime,
1979; Tilman, 1988). Recently a call has been made to move from
describing patterns to understanding the mechanisms driving
coexistence (Agrawal et al., 2007). As a result there has been a
rapid increase in the number of studies suggesting that above-
ground and belowground processes, and particularly plant–soil
feedbacks, are among the main mechanisms underpinning
species abundance, coexistence and succession (Kardol et al.,
2006; Kulmatiski et al., 2008; Miki et al., 2010; van der Putten
et al., 2013).
Plant–soil feedbacks occur whenever a plant causes species-
specific changes to soil biotic or abiotic properties that in turn
affect the establishment, growth or reproduction of their own
0378-1127/$ - see front matter Ó2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.foreco.2013.05.035
⇑
Corresponding author. Current address: Department of Forest and Ecosystem
Science, Melbourne School of Land and Environment, The University of Melbourne,
500 Yarra Boulevard, Richmond, Victoria 3121, Australia. Tel.: +61 3 9035 6862.
E-mail addresses: caponte@unimelb.edu.au (C. Aponte), ventura@cica.es (L.V.
García), teodoro@irnase.csic.es (T. Marañón).
Forest Ecology and Management 309 (2013) 36–46
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species (Bever, 1994; Ehrenfeld et al., 2005). Both positive and
negative feedbacks can promote coexistence: negative feedbacks
diminish the fitness differences between species via equalising
mechanisms (sensu (Barot, 2004) leading to dynamic coexistence
whereas positive feedbacks generate multiple steady states and
promote coexistence via space and/or time partitioning (Pacala
and Levin, 1997; Barot, 2004). In both cases the underlying
mechanisms rely on the ability of the species to generate environ-
mental conditions that alter competitive interactions and facilitate
or prevent other species establishment.
Many studies have shown that trees are ecosystem engineers
able to generate species-specific effects on soil properties and soil
communities that could potentially lead to a feedback effect
(Gómez-Aparicio and Canham, 2008; Vesterdal et al., 2008;
Mitchell et al., 2012; Vesterdal et al., 2012; Prescott and Grayston,
2013 and references therein). However, few of them have inves-
tigated the processes underpinning those effects and their conse-
quences for ecosystem properties (Reich et al., 2005; Mitchell
et al., 2007; Ayres et al., 2009). Furthermore, these studies are of-
ten focused on a particular aspect of tree–soil interactions. For in-
stance, studies have separately addressed tree species effect on
light availability, soil chemical properties, decomposer commu-
nity or the effects of soils on species distributions (Canham
et al., 1994; Van Breemen et al., 1997; Hobbie et al., 2006; Turk
et al., 2008). To our knowledge only a few studies have presented
a holistic vision of the multiple concomitant tree–soil interaction
processes occurring at a single site despite its importance to eco-
system functioning (Ayres et al., 2009). Thus a major effort is
needed to integrate the current knowledge on the multiple func-
tional processes and ecological mechanisms that underpin ecosys-
tems composition and dynamics.
To address this knowledge gap we bring together a series of
independent but integrated studies on the functioning of a
mixed Mediterranean forest ecosystem. The studies investigated
the effects of the coexisting evergreen Quercus suber and the
winter deciduous Q. canariensis on different ecosystem proper-
ties. In particular they characterised the foliar traits of both
oak species (Domínguez et al., 2012) and addressed species ef-
fects on nutrient cycling (Aponte et al., 2011), litter decomposi-
tion (Aponte et al., 2012), and soil biota (Aponte et al., 2010a,b).
The aim of this synthesis is to review the results of these studies
and to discuss whether these interactions could sustain a feed-
back mechanism driving the coexistence of the two Quercus
species.
Our overarching hypothesis is that the two oak species,
through differences in their leaf-fall nutrient concentration, gen-
erate species-specific changes in the soil abiotic properties that
further affect the soil biota and that could ultimately increase
their own fitness. To that end we sequentially examined the fol-
lowing hypotheses: (1) Q. canariensis has higher leaf nutrient con-
tent and different morphological traits than Q. suber, which reflect
their different ecological strategies; (2) The attributes of the fresh
leaves are inherited by the leaf-fall, resulting in Q. canariensis
having higher leaf-fall quality and nutrient return than Q. suber;
(3) Higher leaf-fall quality leads to higher decomposition rate
and nutrient release into soil; (4) The higher nutrient return
and release from Q. canariensis leaf-fall increases its soil fertility
levels as compared to Q. suber; (5) Species-induced changes in
soil nutrient content affect the size and properties of the soil
microbial biomass and alter the species community composition
of the ectomycorrhizal fungal community; (6) Tree species gener-
ate a soil environment where their competitive abilities are en-
hanced, thus increasing their fitness and leading to a positive
feedback. At a stand scale, this creates a mosaic of soil conditions
that allows for a spatial niche separation and sustains their
coexistence.
2. Materials and methods
2.1. Study area
The studies were conducted in a mixed oak forest located in
southern Spain, near the Strait of Gibraltar. This area of about
1000 km
2
holds high ecological value. The rough relief and acidic
nutrient-poor soils, which made the area unsuitable for cultivation,
its frontier location, which limited deforestation and settlement
during medieval times, and the rise of the value of the cork har-
vested from the Q. suber have contributed to the ecological mainte-
nance of this area now protected as ‘‘Los Alcornocales’’ (meaning
the cork oak woodlands) Natural Park (Marañón and Ojeda, 1998).
The forest grows on Oligo–Miocene sandstone bedrock that is
interspersed with layers of marl sediments. The area has sub-hu-
mid Mediterranean climate, the annual mean temperature is
16.5 °C and the annual rainfall ranges from 701 to 1331 mm
(Anonymous, 2005). Two oak species coexist in the area distributed
along a topographic gradient: the evergreen Quercus suber
dominates on the nutrient-poor soils on the ridges whereas the
deciduous Q. canariensis dominates at the valley bottoms. Both
species co-dominate in mixed stands on the midslope (Urbieta
et al., 2008).
The studies were conducted in two 1-ha mixed forest stands lo-
cated on the midslope of two forest sites (30 km apart) named San
Carlos del Tiradero (36°9
0
46
00
N, 5°35
0
39
00
W) and La Sauceda
(36°31
0
54
00
N, 5°34
0
29
00
W). The stand in Tiradero (335–360 m a.s.l)
had a higher density of trees (768 stems ha
1
) and a close canopy
(LAI 2.26 m
2
m
2
) compared to La Sauceda (530–560 m a.s.l; 219
stems ha
1
; LAI 1.84 m
2
m
2
). Soils in Tiradero had similar carbon
content (3.13% vs. 3.27%), C/N ratios (15.6 vs. 16.1) and pH (4.0 vs.
4.8) but higher sand content (58% vs. 47%) than those in La Sauceda
(mean values over the first 50 cm). At each 1-ha stand ten individ-
uals of the evergreen Q. suber and ten individuals of the deciduous
Q. canariensis were selected. The selected trees in the mixed forest
stands had their nearest hetero-specific neighbour within approx-
imately 4–10 m.
2.2. Methods
This study draw upon several datasets gathered over more than
five years of studies in the above-mentioned forest stands (Tirader-
o and La Sauceda). Some of these datasets had been separately ana-
lysed to address specific questions on plant functional traits, litter
decomposition or soil heterogeneity among others and the results
have been previously published (Aponte et al., 2010a,b, 2011,
2012; Domínguez et al., 2012). Other datasets had remained
unpublished. Here we reviewed the results of these studies and
reanalysed the combined datasets to obtain an integrated view of
the ecosystem functioning.
Leaf traits, including four morphological and 19 chemical traits
were analysed in 17 woody plant species (including both oak spe-
cies) from the forest community in La Sauceda. Leaf mass per area
(LMA, g m
2
) and leaf dry matter content (LDMC, g g
1
) were mea-
sured following methods in Cornelissen et al. (2003). Leaf carbon
concentration was determined in an elemental analyser (CHNS
Eurovector EA-3000). Nitrogen was analysed by Kjeldahl digestion
(Jones and Case, 1990). Leaf macronutrients (Ca, K, Mg, P, and S)
and micronutrients (Cu, Co, Fe, Mn, Ni and Zn) concentrations were
determined by acid digestion followed by ICP-OES analysis.
Isotopic analyses of C (d
13
C) and N (d
15
N) in leaf samples were
performed using a continuous-flow elemental analyser – isotopic-
ratio mass spectrometer (EA Thermo 1112-IRMS Thermo Delta V
Advantage). For more details on leaf trait analysis see Domínguez
et al. (2012).
C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46 37

Tree species nutrient return and effects on soil fertility were
examined for the 40 selected oak trees. Fresh leaves, leaf-fall, litter,
topsoil (0–25 cm depth) and subsoil (25–50 cm depth), were
sampled in November 2006. Leaf-fall, litter and soil were sampled
beneath the canopy of each selected oak. Accumulated annual
leaf-fall was collected in four traps under each tree. Litter was con-
sidered dead plant material relatively undecomposed standing on
the ground and it was harvested within two 30 30 cm quadrats.
Soil cores were extracted with a cylindrical auger after removing
the litter layer. Soil pH was determined in a 1:2.5 soil:CaCl
0.01 M solution. Soil carbon was estimated using a total organic
carbon analyser (TOCVesh), available soil P was estimated using
the Bray–Kurtz method and soil NH
þ
4
was estimated by extraction
with KCl (2 M) and steam distillation. Total concentration of
several nutrients (Ca, K, Mg, P, S, Mn, Cu and Zn) in plant tissues
and soils was determined using wet oxidation with HNO
3
(for
plants) or HCl + HNO
3
(for soils) under pressure in a microwave
digester followed by ICP-OES analysis. Plant and soil N was
determined by Kjeldahl digestion. Further details on the methods
can be found in Aponte et al. (2011).
Leaf-fall decay and nutrient release were examined using a lit-
terbag decomposition experiment. Litterbags containing freshly se-
nesced leaves from Q. suber or Q. canariensis were incubated
beneath the canopies of the selected oak trees, beneath shrubby
cover and in open areas. Litterbags were harvested every 6 months
for 2 years. Upon harvest, leaf litter was removed from the bags,
dried and weighed for mass loss. Samples were ground and ana-
lysed for C, N, Ca, K, Mg, P, S, Mn, Cu and Zn to assess changes in
nutrient content over time. Biomass loss (in this case carbon loss)
was fitted with an asymptotic model, Mt = m + (1 m)

e
kt
, where
Mt was the proportion of remaining mass at time t,mwas the frac-
tion of the initial mass with a decomposition rate of zero (that is,
the asymptote) and kwas the decomposition rate of the remaining
fraction (1 m). The asymptotic model implied that there was a
limit value (m) for mass loss. This value corresponded to a very sta-
ble fraction of the litter that decomposed extremely slow over the
time span of the experiment (Berg et al., 2003). See further meth-
odological details in Aponte et al. (2012).
Soil microbial C, N and P content were estimated on soil sam-
ples extracted at two depths (0–8 cm and 8–16 cm after removing
the litter layer) beneath the selected oak trees. Soil samples were
taken in spring (May–June), summer (September) and autumn
(December) 2007, and spring (May) 2008. Microbial C, N and P
were estimated using a chloroform fumigation-extraction proce-
dure (Brookes et al., 1982, 1985; Vance et al., 1987). Two soil subs-
amples were extracted using 0.5 M K
2
SO
4
or 0.025 N HCl + 0.03 N
NH
4
F for subsequent determination of microbial C and N or micro-
bial P, respectively. Other two soil subsamples were fumigated
with chloroform for 24 h in a vacuum desiccator, followed by the
same extraction procedure as the unfumigated samples. Carbon
and N in fumigated and unfumigated soil extracts were determined
using a Total Dissolved Organic Carbon and Nitrogen Analyzer
(TOC-Vesh). Microbial C and N were estimated as the difference
in K
2
SO
4
-extractable dissolved organic carbon or nitrogen between
fumigated and unfumigated soils using as extractability correction
factors: K
C
= 0.45 for C and K
N
= 0.40 for N (Jonasson et al., 1996).
Available P in NH
4
F soil extracts was measured using the Bray–
Kurtz method (Bray and Kurtz, 1945). Microbial P was estimated
as the difference in available P between fumigated and unfumigat-
ed soil using a correction factor K
P
= 0.40 (Brookes et al., 1982). For
more methodological details see Aponte et al. (2010b).
The community composition of the ectomycorrhizal fungi asso-
ciated with the roots of the selected oak trees was identified using
PCR-based molecular method. Superficial roots (15 cm depth)
approximately equal in length (20 cm) were taken from each se-
lected tree, close to the litter and soil sampling points, in Novem-
ber 2007. From each tree 20 mycorrhizal root tips were randomly
picked. Mycorrhizal DNA was extracted using the Wizard Genomic
DNA Purification Kit (Promega, Charbonnieres, France) and the
internal transcribed spacer regions I and II and the nuclear 5.8S
rRNA gene were amplified using the primer sets ITS-1F/ITS-4B
(Gardes and Bruns, 1993) or ITS-1F/ITS-4 (White et al., 1990).
The sequencing of the final amplification products was done by
MilleGen (Labège, France). Ectomycorrhizal species (‘‘Operational
taxonomic units’’ sensu (Blaxter et al., 2005) were determined by
BLAST searches against GenBank and the UNITE database. See
methodological details in Aponte et al. (2010a).
2.3. Data analysis
A range of multivariate ordination techniques, namely Principal
Component Analysis (PCA), Canonical Correspondence Analysis
(CCA) and path analysis, were used to better understand the mul-
tivariate patterns present in the data. Principal Component Analy-
sis was applied to single tables to explore the variability within
datasets. Canonical Correspondence Analysis was used to analyse
the relationship between the ECM community composition and
environmental conditions. Path analysis with d-sep tests was used
to evaluate alternative causal relationships among the properties
of the ecosystem components (Shipley, 2000). Differences between
Quercus species in the univariate or multivariate space were eval-
uated using Analysis of Variance (ANOVA) or Mann–Witney non-
parametric test for small sample sizes.
3. Results and discussion
3.1. Leaf traits
The traits of the fresh leaves of Q. canariensis and Q. suber, to-
gether with other 15 woody species of the plant community in
La Sauceda, were studied by Domínguez et al. (2012) using a Prin-
cipal Component Analysis (Fig. 1a). The first PCA axis accounted for
26% of the variability of the traits attributes and it was negatively
related to leaf nutrient concentration and positively related to leaf
mass per area (LMA), leaf dry matter content (LDMC), carbon con-
centration, dC
13
and carbon to nitrogen ratio. We analysed the dif-
ferences between the two Quercus in the multivariate space
defined by the PCA and observed that the two species had signifi-
cantly different scores along the first axis (Mann–Whitney U test,
n= 10 individuals, P< 0.009): the evergreen Q. suber grouped with
other sclerophyllous species at the positive end of the first axis,
whereas Q. canariensis was on the negative side of the same axis
(Fig. 1a). Similar results were obtained when we analysed the attri-
butes of the fresh leaves sampled from the 40 oak trees (20 Q. suber
and 20 Q. canariensis) in the two forest sites (La Sauceda and Tira-
dero; Fig. 1b); the first PCA axis accounted for 36% of the variability
of the dataset and clearly separated the two Quercus species
(P< 0.001). Both analyses indicated that Q. suber had a higher
LMA, LDMC, C, C:N and dC
13
and lower nutrient concentrations
than Q. canariensis.
Species leaf trait values reflect their functional strategy to man-
age resources such as water, light and nutrients (Poorter et al.,
2009; Pérez-Ramos et al., 2012). Two main opposite strategies
can be distinguished from the global range of traits variation that
defines the leaf economics spectrum (Wright et al., 2004): a conser-
vative resource-use strategy and resource-acquisition strategy. The
first one is defined by slow rates of resource acquisition and min-
imum resource loss and it is characteristic of species adapted to re-
source-limited environment, whereas the opposite is true for the
second one (Aerts, 1995). The differences in the foliar attributes
of the studied oaks align each species with one of the divergent
38 C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46

ecological strategies. That is, Q. suber could be considered to have a
conservative-resource strategy because of its higher values of LMA,
higher density tissues, higher efficiency in the use of water (high
C
13
(Farquhar et al., 1989)) and higher carbon-to-nutrient ratios.
In contrast Q. canariensis, which exhibited opposite attributes,
would be ascribed to the resource-acquisition strategy (Wright
et al., 2005; Villar et al., 2006). The divergence in their strategies
is consistent with their distinct decomposability (lower for Q. sub-
er), which would lead to slower nutrient loss during decomposition
(Gallardo and Merino, 1993; Aponte et al., 2012).
Species resource-use strategies are the result of their adaptive
evolution to environmental conditions (Reich et al., 2003), which
suggests that the two Quercus would be adapted to environments
with distinct resource (water, light and nutrients) availability. This
was sustained by Urbieta et al. (2008) who examined the domi-
nance of both oak species along a topographic gradient in a 284-
ha mixed forest stand within Los Alcornocales National Park. They
observed that the abundance of Q. suber increased with altitude
and distance from the valley bottom (i.e., decreasing water and
nutrient availability) whereas the opposite was observed for Q.
canariensis. Higher water availability also increased seedling per-
formance and survival rate of Q. canariensis but did not affect Q.
suber in greenhouse and field experiments (Quero et al., 2006;
Pérez-Ramos, 2007). Studies on the regeneration of both oak spe-
cies demonstrated that in low-light environments (i.e., dense plant
cover, high litter depth and nutrient availability) Q. canariensis had
higher survival and growth rate than Q. suber. However in high-
light, nutrient-poor environments the evergreen Q. suber seedlings
outgrew the deciduous species (García et al., 2006; Gómez-Apari-
cio et al., 2008b; Pérez-Ramos et al., 2010). The differential re-
sponse of Q. canariensis and Q. suber to resource availability
suggests that their coexistence might be mediated through niche
partitioning.
Species leaf attributes are not only a response to environmental
conditions but also determine species effect on the ecosystem
properties (Diaz et al., 2004; Ayres et al., 2009). For instance, the
chemical and morphological attributes of fresh leaves and se-
nesced leaves (leaf-fall) influence soil nutrient availability through
its effects on biogeochemical processes (e.g., nutrient throughfall,
decomposition) (Facelli and Pickett, 1991; Prescott, 2002; Hobbie
et al., 2006). Therefore the distinct foliar attributes of the studies
species would likely generate contrasting effects on the ecosystem
properties.
3.2. Leaf-fall and nutrient return
The nutrient concentration of the fresh leaves and leaf-fall of
the 40 oak trees in the two forest sites was highly correlated (Apo-
nte et al., 2011), resulting in Q. canariensis having higher leaf litter
quality than Q. suber (Fig. 2). This was consistent with results from
the leaf-fall decomposition study by Aponte et al. (2012), who re-
ported higher concentrations of N, Ca, Mg, P and S (P< 0.001) in the
leaf-fall of Q. canariensis as compared to Q. suber. Differences be-
tween species were particularly high for macronutrients such as
Ca (51% higher in Q. canariensis leaf-fall), P (28%) and Mg (26%).
These results indicate that fresh leaf attributes, and thus differ-
ences between species, were inherited by the leaf-fall. However,
the relationship between fresh leave and leaf-fall mineral content
cannot be generalised since leaf-fall nutrient content might be
influenced by nutrient resorption during the senescing process
(Aerts, 1996). Nutrient resorption minimises nutrient losses and
therefore high resorption efficiency would be expected from spe-
cies exhibiting a conservative use of resources. Conversely, in our
study Q. suber and Q. canariensis did not differ in proportional
resorption of N (39% Q. canariensis vs. 36% Q. suber) or P (39.7% Q.
canariensis vs. 41% Q. suber). Other nutrients measured (Ca, Mg, S,
Mn, Cu, Fe and Zn) were not resorbed but instead accumulated in
the leaf-fall. Aerts (1996) also observed small (47% vs. 54%) or no
differences in the resorption of N and P between evergreen and
deciduous trees and concluded that the lower nutrient concentra-
tion in evergreen leaf-fall contributed more to nutrient conserva-
tion than did nutrient resorption.
Annual leaf-fall production of Q. canariensis and Q. suber was
similar in amount (0.30 and 0.29 kg/m
2
respectively). Comparable
leaf-fall production values were found for the winter deciduous Q.
pyrenaica (0.237 kg/m
2
) in the centre of Spain (Salamanca), and the
evergreens Q. lanuginose (0.246 kg/m
2
) and Q. ilex (0.243 kg/m
2
)in
the south of France (Montpellier) (Rapp et al., 1999), suggesting
that, at least for this genus, leaf-fall productivity is not necessarily
related to foliar habit. Both leaf-fall nutrient content and leaf-fall
quantity determine tree species nutrient return and their impact
on ecosystem properties (Facelli and Pickett, 1991; Washburn
and Arthur, 2003). Leaf-fall quantity could be more influential than
quality in terms of net nutrient return to soil if the different masses
of leaf-fall overrode the differences in nutrient concentrations
(Chabot, 1982; Cuevas and Lugo, 1998). However this was not
the case for Q. suber and Q. canariensis, which had comparable
LMA
LDMC
N
C
C:N
P
N:P
B
Ca
Fe
K
Mg
S
d15N
d13C
Co
Cu
Mn
Ni
Zn
-1.0 -0.5 0.0 0.5 1.0
Factor 1 : 26.2%
-1.0
-0.5
0.0
0.5
1.0
Factor 2 : 15.5%
Q. canariensis
Q. suber
Other species
LMA
LDMC
C
N
C:N
CA
CU
K
MG
MN
P
S
ZN
-1.0 -0.5 0.0 0.5 1.0
Factor 1 : 35.75%
-1.0
-0.5
0.0
0.5
1.0
Factor 2 : 25.11%
Q. canariensis
Q. suber
(a) (b)
Fig. 1. (a) PCA ordination of leaf traits of the woody plant species sampled at La Sauceda, including Q. canariensis (filled symbol) and Q. suber (hollow symbol). Species scores
represent the centroid of 5 individuals of the same species except for the Quercus (modified from Domínguez et al. (2012). (b) PCA ordination of 20 individuals of Q. canariensis
and Q. suber sampled in La Sauceda and Tiradero. Abbreviations are LMA: leaf mass per area, LDMC: leaf dry matter content, d15 N: isotope N
15
, d13C: isotope C
13
and symbols
of each element indicating their concentration in fresh leaves.
C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46 39

leaf-fall production. Therefore the nutrient concentration of the
leaf-fall created the distinct nutrient return of the two species.
3.3. Leaf-fall decomposition and nutrient release
Leaf-fall decomposition of Q. suber and Q. canariensis were mea-
sured in a two-year litterbag experiment (Aponte et al. 2012). Dur-
ing the early stages of decomposition leaf-fall of Q. canariensis had
higher chemical quality, particularly the higher concentrations of N
(lower C:N) and Ca, and decayed faster than that of Q. suber. Leaf-
fall quality largely controls leaf litter decomposition and release of
nutrients into soil and thus could potentially explain species ef-
fects on soil fertility (Norris et al., 2012). High leaf-fall quality
has been related to high nutrient content and low carbon to nutri-
ent ratios but also to low non-structural and recalcitrant carbohy-
drate concentrations (e.g. lignin, tannins and phenolic) and low leaf
mass per area and foliar toughness (Gallardo and Merino, 1993;
Pérez-Harguindeguy et al., 2000; Aerts et al., 2003; Hättenschwiler
and Jørgensen, 2010). Most of these properties differed between Q.
suber and Q. canariensis in keeping with their distinct decay rates
(Gallardo and Merino, 1993).
In contrast to the pattern observed during early decomposition,
the limit value of decomposition, i.e. the fraction of leaf litter mass
that remains stable at late stages of decomposition and builds up
soil organic matter, was higher for Q. canariensis than for Q. suber
(40% vs. 31%, p< 0.0001; Aponte et al. 2012). This was consistent
with the larger concentrations of soil organic matter measured un-
der the canopy of Q. canariensis than under Q. suber (Aponte et al.,
2010b, 2011). Differences in species limit value were related to
their distinct N, Ca and Mn content. Nitrogen and Ca, which en-
hanced early decay rates, hindered late stage decomposition, thus
exerting counteracting effects over time. In high-N substrates mi-
crobes are not N-limited and have higher substrate use efficiency.
This results in a faster initial decomposition but also in a greater
accumulation of microbial products and residues over the long
term. These microbial products, when bonded with metal polyva-
lent cation such as Ca, are the precursors of stable SOM formation
(Davey et al., 2007; Cotrufo et al., 2013). Manganese was the only
nutrient which concentration was higher in Q. suber than in Q.
canariensis leaf-fall and it emerged as the most important driver
of carbon loss during late decomposition. The effect of Mn was re-
lated to its role as a cofactor in a lignin degrading enzyme (Eriksson
et al., 1990; Davey et al., 2007).
Aponte et al. (2012) also observed that the rate and proportion
of nutrients loss from decomposing leaf-fall was higher for Q.
canariensis than for Q. suber. For example, after 6 months Q. canari-
ensis leaf-fall had lost 49% and 17% of its P and N content respec-
tively. In contrast Q. suber had lost 29% of its P content and none
of its N. The species differences in their relative nutrient loss dur-
ing decomposition added to the differences in species nutrient re-
turn. As a result, Q. canariensis released a higher net amount of
nutrients into the soil than Q. suber (Fig. 3). For instance, after
two years Q. canariensis would have released 12 kg ha
1
of N (31%
of the initial input), 8.6 kg ha
1
of Ca (19%) and 2.1 kg ha
1
of P
(67%) whereas Q. suber would have released 2.1 kg ha
1
of N
(8%), 3.1 kg ha
1
of Ca (11%) and 0.9 kg ha
1
of P (50%). Nutrient re-
turn from Q. canariensis could have been slightly higher since, due
to its marcescent habit, a fraction of the soluble nutrients in its
leaf-fall could have been leached over the winter before our sam-
pling (Ibrahima et al., 1995). Nevertheless this would further in-
crease the differences in nutrient release between oak species.
Leaf-fall decomposition is not only influenced by its quality it
can also be affected by the soil biota and environmental conditions
(i.e., moisture, UV radiation and temperature (Hobbie, 1996; Aus-
tin and Vivanco, 2006; Negrete-Yankelevich et al., 2008). Aponte
et al. (2012) evaluated the relative importance of substrate (leaf-
fall) quality vs. tree-generated environmental conditions on the
decomposition of Q. suber and Q. canariensis leaf-fall by incubating
litterbags beneath the canopies of both species. Leaf-fall quality ex-
plained a greater percentage of the variation of early and late decay
parameters (35.2% and 19.6% respectively) than topsoil environ-
mental conditions (4.4% and 4.5%). Nevertheless, the higher mois-
ture content and higher N and P concentration of the soils beneath
Q. canariensis positively influenced leaf-fall decay of both species.
These results are in accordance with other studies that suggest that
long term tree–soil interactions can shape topsoil properties and
0.34 0.38 0.42 0.46 0.50
Log concentration of leaf fall
0.20
0.24
0.28
0.32
0.36
0.40
Q. canariensi
s
Q. suber
N
0.0 0.1 0.2 0.3 0.4 0.5
0.20
0.25
0.30
0.35
0.40
0.45
0.50 Ca
Log concentration of fresh leaves
0.02 0.03 0.04 0.05
0.00
0.01
0.02
0.03
0.04
0.05 P
r=0.54***r=0.70***r=0.59***
Fig. 2. Correlation between the chemical composition of the fresh leaves and leaf-fall for the evergreen Q. suber and the deciduous Q. canariensis. Dots represent the average
value of the element concentration for 20 evergreen and 20 deciduous trees.
06121824
Nutrient released (kg ha-1)
-5
0
5
10
15 NCa P
06121824
-5
0
5
10
06121824
0.0
0.5
1.0
1.5
2.0
2.5
Fig. 3. Differences in nutrient loss from litter during decomposition of leaf-fall of the deciduous Q. canariensis (filled symbol) and the evergreen Q. suber (hollow symbol).
40 C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46

organisms thus allowing for a potential indirect effect of trees spe-
cies on leaf-fall decay via changes in soil environment (Mitchell
et al., 2007; Vivanco and Austin, 2008; Freschet et al., 2012). For in-
stance, Reich et al. (2005) and Hobbie et al.(2006) showed that tree
species affected leaf-fall decomposition through variation in leaf-
fall quality, soil temperature and earthworm community. Chad-
wick et al. (1998) observed that leaf-fall decay rate was influenced
by the nutrient content of the layer of litter on which leaf-fall was
incubated. Recently Vesterdal et al. (2012) correlated the leaf-fall
quality (N, Ca and Mg) and microclimatic conditions generated
by five deciduous tree species with forest floor C turnover rates.
Overall, the distinct nutrient return and decay patterns of both
Quercus species, controlled by their leaf-fall quality, resulted in a
differential nutrient input into the soils that could in turn alter soil
nutrient availability. In addition, the higher limit value of decom-
position of Q. canariensis lead to higher levels of SOM and thus
higher retention of nutrients and moisture, further reinforcing
the ability of Q. canariensis to change soil conditions.
3.4. Soil nutrient content
We analysed the chemical and textural characteristics of the
subsoil (25–50 cm) sampled beneath the 40 Q. canariensis and Q.
suber trees within each forest site as a proxy of the original soil
conditions. No differences were found between the two species
(Supplementary Fig. S2), suggesting that the parent material, i.e.
the original soil conditions, within stands was homogeneous. In
contrast, topsoil (0–25 cm) carbon and nutrient concentration
and pH were significantly (P< 0.05) higher under Q. canariensis
than under Q. suber, consistent with the differences in nutrient re-
turn and decomposition dynamics. The multivariate analysis of the
chemical composition of the leaf-fall and topsoil of the two species
(Fig. 4) indicated that nutrient concentration in the topsoil was
strongly related to that measured in the leaf-fall, as evidenced by
the correlation of all variables along the main axis. This meant that
the foliage attributes (in this case chemical composition) of each
tree were mirrored in the topsoil, which was consistent with a
leaf-fall-mediated tree species effect on soil properties.
We conducted a more detailed study on the chemical composi-
tion along the soil profile beneath Q. canariensis and Q. suber in La
Sauceda (Fig. 5). Samples were taken every 10 cm along the first 0–
60 cm of soil. In accordance to the previous results, differences in
soil nutrient concentration between species were larger in the
uppermost soil layers but they gradually disappeared with soil
depth.
One of the key issues when examining tree species induced soil
changes is the confounding effect of soil variability prior to species
establishment. If experiments are conducted on an initially homo-
geneous substrate, then any changes in soil variables between spe-
cies can be fully attributed to species effects. Otherwise differences
in the soils under different species may not conclusively confirm
the species ability to modify soil conditions, but could be the result
of the initial species distribution governed by the differences in
species soil and nutrient requirements. As a result most studies
investigate tree species influence on soil conditions using experi-
mental plantations with monocultures in common garden designs
on homogeneous substrates (Menyailo et al., 2002; Hagen-Thorn
et al., 2004; Oostra et al., 2006; Vesterdal et al., 2008). Descriptive
studies from mixed forests overcome this limitation by assessing
the homogeneity of the deeper soil layers, as a surrogate of the ini-
tial substrate conditions (Boettcher and Kalisz, 1990; Finzi et al.,
1998a; Finzi et al., 1998b). In mature (50–100 years old), stands
species influence can be found in the deeper mineral soils layer
(Nordén, 1994). However changes in soil chemistry due to differ-
ences in leaf-fall quality are much more distinct in the upper most
layers, as was the case in the studied forest (Hagen-Thorn et al.,
2004).
Our study focused on the effects via leaf-fall properties but
other concurrent mechanisms might also induce changes in the
soil conditions, such as differences in interception of atmospheric
deposition, canopy interactions, leaching and root exudates as well
as alterations to microclimate (Augusto et al., 2002; Berger et al.,
LF.N
LF.Ca
LF.Mg
LF.Mn
LF.S
TOP.pH
TOP.K
TOP.P
TOP.S
TOP.Zn
Factor 1: 43.96%
Factor 2: 17.36%
Q. canariensis
Q. suber
P <0.001
TOP.Mg
TOP.Mn
TOP.Ca
LF.Zn
LF.K
TOP.N
LF.P
Fig. 4. PCA ordination of the chemical composition of the leaf-fall (LF) and topsoil
(TOP) of 20 Q. canariensis (filled symbols) and 20 Q. suber (hollow symbols) sampled
in La Sauceda and Tiradero. Differences (ANOVA) between Q. canariensis and Q.
suber scores along the factor1 axis are indicated.
Soil N (g kg -1)
012345
Soil depth (cm)
0
10
20
30
40
50
60
Soil Ca (mg kg-1 )
0 102030405060
0
10
20
30
40
50
60
Soil P (mg kg -1 )
1234
0
10
20
30
40
50
60
Q. canariensi
s
Q. suber
Fig. 5. Variation in N, Ca and P concentrations along the soil vertical profile beneath the canopy of the deciduous Q. canariensis (filled symbol) and the perennial Q. suber
(hollow symbols).
C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46 41

2009). However, the strong relationship between the chemical
composition of the leaf-fall and the soil beneath the canopy of each
tree, and the finding that the differences between oak species de-
clined with depth in the soil profile suggest that the changes in soil
chemistry were largely due to leaf-fall properties.
3.5. Soil microbial biomass
Soil microbial biomass (Cmic) and microbial nutrients (Nmic,
Pmic) were higher under Q. canariensis than under Q. suber (18%;
24%; 9% respectively), as reported by Aponte et al. (2010b).To
determine whether this effect was mediated by tree species influ-
ence on soil properties we analysed the covariation of microbial
nutrients and soil properties (Fig. 6). Two main significant axes
accounting for 34% and 18% of the variance emerged from the ordi-
nation analysis as determined by the broken stick method. The
variables loading on these two axes revealed that microbial C, N
and P variability was strongly related to the abiotic soil properties.
The analysis of bivariate relationships indicated that among all soil
parameters, soil total N was the best predictor of Cmic (R
2
= 0.84),
Nmic (R
2
= 0.87) and Pmic (R
2
= 0.69).
Several studies have reported differences in microbial C, N and P
in soils from under different tree species (Malchair and Carnol,
2009; Smolander and Kitunen, 2011; Huang et al., 2013). In most
cases the mechanisms underlying those effects remain unclear,
while others found that microbial biomass was positively related
to the availability of limiting resources such as water, organic mat-
ter and nutrients (Billore et al., 1995; Nielsen et al., 2009; Lucas-
Borja et al., 2012). Therefore the increased levels of soil microbial
C, N and P under Q. canariensis could be explained by the higher
nutrient concentrations (particularly total N), soil organic matter
content and soil water-holding capacity of its soils as compared
to Q. suber. Furthermore, Aponte et al. (2010b) observed a positive
correlation between microbial and available inorganic N and P
(r= 0.44 and r= 0.37 respectively; p< 0.001). These relationships
suggest that tree species, through their influence on soil microor-
ganisms, can affect nutrient mineralisation and availability further
reinforcing their effect on soil fertility (Smolander and Kitunen,
2011; Huang et al., 2013).
The differences observed by Aponte et al. (2010b) in the micro-
bial pools between Quercus species were only significant in the
uppermost soil layer (0–8 cm) whereas they diluted with soil
depth (8–16 cm). The pattern of differences in microbial nutrients
(being greatest in the upper soil and disappearing along the soil
profile) mirrored that found for soil nutrient concentrations
(Fig. 5). Furthermore, these layers (0–8 cm and 8–16 cm) would
roughly correspond to the organic F and H layers, as the average
depth of the organic soil in these sites was 20 cm. The F layer often
shows the largest differences in microbial communities composi-
tion and activity among tree species as opposed to the H layer
and the mineral soil, which are less influenced by tree species
and thus show less detectable differences (Grayston and Prescott,
2005; Ushio et al., 2010). Root litter and root exudates could also
influence microbial communities through input of labile C and
nutrients (Billore et al., 1995; Brimecombe et al., 2000). However
the correlation between soil and microbial nutrients and the dilu-
tion of differences between species along the vertical soil profile
suggest that species indirectly affected soil microbial biomass
through leaf-fall-mediated changes in soil abiotic properties.
Both Nmic and Pmic showed a strong seasonal variability, with
differences between species being significant in spring but not in
summer (Aponte et al., 2010b). This was attributed to changes in
soil water content, which varied almost twofold from spring
(21%) to summer (12%). That is, drought limited microbial activity
during summer, equalising the levels of Nmic and Pmic between
species. However high soil water availability in spring increased
the accessibility of nutrients (Nielsen et al., 2009), thus allowing
for a differential microbial growth beneath the two Quercus. Higher
microbial activity in the wet than in the dry season had been pre-
viously found in the same forest (Quilchano and Marañón, 2002).
The effect of changes in water availability could further interact
with seasonal differences in substrate availability associated to
species phenology (Rinnan et al., 2008). In the studied forest, the
evergreen Q. suber showed a clear seasonal pattern, shedding most
of its annual leaf-fall during early summer as a strategy to reduce
evapo-transpiration and withstand summer drought (Supplemen-
tary Fig. S1). In contrast the winter deciduous Q. canariensis had
marcescent habit and shed most (60%) of its leaf-fall throughout
the winter and spring (Navarro et al., 2005). Therefore, Q. canarien-
sis provided more and higher quality substrate at the peak time of
microbial activity, explaining why the differences observed in the
soil microbial properties between the two oaks were significant
only in spring.
3.6. Ectomycorrhizal community composition
The ECM community on the roots of Q. canariensis and Q. suber
was examined to evaluate to which extent host species and host-
generated soil conditions influenced the symbiotic community
(Aponte et al. 2010a). The ECM community composition of the
two oaks was largely dissimilar with only 13 of the 69 identified
species (18%) occurring in both Quercus species. Thelephoraceae
species dominated the roots of Q. canariensis (38.9% of the identi-
fied mycorrhizae) whereas species from Russulaceae family domi-
nated the roots of Q. suber (46.6%). The taxonomic distinctness
(Warwick and Clarke, 1995) and the phylogenetic structure of
the community also shifted between oak species (P< 0.001): Q.
canariensis harboured a segregated phylogeny (lower taxonomic
distinctness) with a high abundance of the resupinate tomentelloid
species and a lack of epigeous taxa. In contrast, Q. suber ECM com-
munity showed a high taxonomic distinctness (i.e., lower phyloge-
netic relatedness among species) and a higher abundance of
epigeous species.
The shift in ECM species composition was related to changes in
litter and topsoil properties (Aponte et al. 2010a). In particular, Ca
concentration emerged as the best predictor of the ECM commu-
nity composition (P< 0.001; 8% of the overall ECM species
Moisture.Sp
SOM.Sp
Pi.Sp
Pmic.Sp
NH4.Sp
Cmic.Sp
Nmic.Sp
Moisture.Su
SOM.Su
Pi.Su
Pmic.Su
NH4.Su
Cmic.Su
Nmic.Su
Factor 1: 34%
Factor 2: 18%
Q. canariensis
Q. suber
P <0.03
Fig. 6. PCA ordination of the properties of soil and soil microbial biomass in the
upper 0–8 cm measured in spring (Sp) and summer (Su) under Q. canariensis (filled
symbols) and Q. suber (hollow symbols). Differences (ANOVA) between Q. canari-
ensis and Q. suber scores along the factor1 axis are indicated.
42 C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46

variance). Calcium concentrations were strongly related to soil pH
suggesting that calcium-induced changes in soil acidity could also
be driving the shift observed in the fungal communities. Based on
their observations of distinct Ca contents of the leaf-fall, litter
and topsoil of Q. canariensis and Q. suber they conducted a path
analysis to evaluate whether the changes in the ECM composition
could be attributed to the leaf-fall mediated changes on the litter
and topsoil Ca concentrations (Supplementary Fig. S3). Several
alternative models were tested but only those which included
the indirect effects of host species on litter and topsoil properties
via leaf-fall Ca were significant as opposed to those which only
included the direct effects of soil or host species. These results
suggested that Q. canariensis and Q. suber influenced the ECM
community composition by altering litter and topsoil acidity and
Ca concentration.
Other studies have observed shifts in the composition of the
ECM fungal communities, such as changes in species richness
and dominance from epigeous to resupinate and from Basidiomy-
cetes to Ascomycetes, related to variations in soil nutrient avail-
ability (Avis et al., 2008; Buée et al., 2011; Kluber et al., 2012).
Under high nutrient availability tree dependence on ECM symbio-
sis for nutrient uptake decreases and so might the transference of
carbohydrates to the symbionts. This would favour the presence of
tomentelloid species, which have certain saprophytic capacity and
are able to obtain part of their carbon through litter and soil organ-
ic matter decomposition (Kõljalg et al., 2000; Pena et al., 2013).
Thus the soil conditions generated by Q. canariensis imposed an
environmental filter selecting for a cluster of closely related ‘toler-
ant’ species. On the other hand, the higher taxonomic distinctness
observed in the nutrient-poor soils under Q. suber suggests a func-
tional diversification of the ECM community driven by limiting re-
sources and competitive interactions. Soil acidity has also been
shown to affect species performances (e.g., production of fruit
bodies, mycelial growth and enzymatic capabilities) and thus influ-
ence their competitive abilities leading to changes in the commu-
nity composition (Agerer et al., 1998; Rosling et al., 2004; Courty
et al., 2005). Nonetheless, these changes were treated as abiotic
host-independent influence. Morris et al. (2008) conducted a sim-
ilar study to Aponte et al. (2010a) and also found differences in the
abundance and diversity of epigeous ECM species between the
roots of coexisting evergreen and deciduous oaks and related those
differences with changes in host species and soil nutrient content.
However, in contrast to Aponte et al. (2010), they did not attempt
to demonstrate the soil-mediated indirect effect of host tree spe-
cies on ECM fungal assemblages.
Tree species effect on ECM fungi could further lead to changes
in the microbial community activity and composition as their pro-
duction of exudates can further affect other soil microorganisms
(Högberg and Högberg, 2002; Jones et al., 2004; Frey-Klett et al.,
2005). Some studies have related changes in the microbial commu-
nity composition (PLFA and TRFLP) with variation in litter and soil
pH and Ca (Ayres et al., 2009; Thoms et al., 2010). Whether these
changes are mediated by shifts in the ECM community composi-
tion remains unclear. The recent increase in the number of studies
exploring the indirect effects of plant species on soil communities
highlights the important role that these interactions have in the
ecosystem functioning (Thoms et al., 2010; Sagova-Mareckova
et al., 2011; Lucas-Borja et al., 2012; Mitchell et al., 2012; Vesterdal
et al., 2012).
3.7. Feedback effects and species coexistence
The mechanisms sustaining evergreen and deciduous species
coexistence are still unclear (Givnish, 2002). Most studies suggest
that species coexistence is maintained by differences in their
regeneration niche, demographic characteristics, susceptibility to
soil pathogens or responses to gap disturbance regime (Tang and
Ohsawa, 2002; Taylor et al., 2006; Gómez-Aparicio et al., 2012).
In a recent analysis of the mechanisms promoting species coexis-
tence Barot (2004) suggested that species-induced spatial hetero-
geneity of resources (‘endogenous heterogeneity’) could sustain
species coexistence through self-generated niche differentiation.
For example, if the species-specific changes in ecosystem proper-
ties generated a positive feedback by leading to soil conditions in
which the species are more competitive, then the endogenous
environmental heterogeneity would promote stable species coex-
istence through space partitioning (Pacala and Levin, 1997; Brandt
et al., 2013). Our results indicated that coexisting deciduous Q.
canariensis and evergreen Q. suber, through their capacity to modify
the soil properties and communities beneath their canopies, cre-
ated a mosaic of soil conditions, i.e. endogenous environmental
heterogeneity. However, only if the species’ self-generated soil
conditions increased their own fitness in a positive feedback would
this heterogeneity promote coexistence. Aponte et al. (2011) tested
the feasibility of this positive feedback effect using a path analysis
that fitted several alternative causal models to the empirical data
collected on the field. In particular they analysed the causal rela-
tionships between the oak species and the chemical composition
of the fresh leaves, leaf-fall, topsoil and subsoil (Supplementary
Fig. S4). The main hypothesis underlying the models tested were
(1) oak species affect soil conditions via nutrient return, and in turn
this affects species distribution and generates a positive feedback
effect; (2) species modify topsoil conditions via nutrient return
but species distribution is only affected by subsoil properties, thus
there are no feedback effects; and (3) soil affects species distribu-
tion, but trees have no effect on soil conditions. Only the model
based on the feedback hypothesis matched field data. Also, obser-
vational and experimental works in the study area have shown
that the studied Quercus species differ in their regeneration niches,
as mentioned above (Section 3.1). The probability of successful
recruitment, growth rate and abundance of seedlings and saplings
of both oaks was positively related to the presence of conspecific
adults and negatively related to the presence of the other species
(Maltez-Mouro et al., 2005; Pérez-Ramos et al., 2010). In addition,
the emergence and recruitment of Q. canariensis increased with soil
fertility (Maltez-Mouro et al., 2009; Pérez-Ramos and Marañón,
2012). Furthermore, the soil conditions generated by each species
aligns with their life-history and nutritional strategies and reflect,
at a local scale, the different environments where each species
dominate (Gómez-Aparicio et al., 2008b; Urbieta et al., 2008;
Pérez-Ramos et al., 2010). All of the above suggest that each oak
species generates a space where it is the best competitor, leading
to a potential positive feedback effect that would underpin species
coexistence (Catovsky and Bazzaz, 2000; Barot, 2004; Brandt et al.,
2013).
The importance of the role of plant–soil feedbacks as drivers of
plant community composition and species coexistence is increas-
ingly being recognised (Gómez-Aparicio et al., 2008a; Kulmatiski
et al., 2008; Kardol et al., 2013). For instance, Brandt et al. (2013)
observed that soil heterogeneity generated by plant–soil feedbacks
had species-specific effects on germination and establishment,
with consequences for recruitment dynamics. Interestingly, most
reported plant–soil feedback effects are negative, often mediated
by soil pathogens and root herbivores (Bever, 2003; Bonanomi
et al., 2005; Kardol et al., 2006; Kulmatiski and Kardol, 2008).
Gómez-Aparicio et al. (2012) analysed the spatial patterns of soil
pathogens in Q. canariensis–Q. suber mixed forests and found no
evidence of plant–soil feedback effects via soil pathogens. Further-
more, as stated in a recent review on plant–soil feedbacks (van der
Putten et al., 2013), most negative feedbacks results emerge from
simulations, monoculture experiments under controlled indoor
conditions or field studies in agricultural systems. Feedback
C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46 43

studies in natural forest systems are still scarce and essential to
understand plant population dynamics and functioning of forest
ecosystems.
Although this empirical evidence sustains our hypothesis
regarding the capacity of oak species to modify ecosystem proper-
ties, our conclusions on the positive feedback processes and coex-
istence mechanisms are tentative. Reciprocal field-based
transplant experiments where species are planted next to con-
and hetero-specific individuals are pathways for future investiga-
tions into the tree–soil feedbacks in these mixed oak forest. Glass-
house experimental approaches such addition of soil inocula in
sterilized soils and soil conditioning by ‘own’ vs. ‘foreign’ plant spe-
cies could further help teasing apart the influence of the biotic and
abiotic soil conditioning on the feedback processes (Brinkman
et al., 2010; Brandt et al., 2013).
4. Conclusions
We have reviewed the existing knowledge on multiple and con-
current tree–soil interactions in a mixed forest of deciduous Q.
canariensis and evergreen Q. suber. In this forest, oak species leaf-
fall quality (particularly nutrient content) determined nutrient re-
turn, leaf-fall decomposition and nutrient release into soil, leading
to different levels of soil fertility. In turn oak species generated
changes in soil nutrient concentrations, particularly N and Ca, fur-
ther affected the size and composition of the soil microbial com-
munity. Through this integration we have gained a
comprehensive understanding of the mechanisms underlying oak
species effect on soil abiotic properties and soil communities. In
addition, we have presented evidence supporting the hypothesis
that tree-species-induced changes in soil conditions create a posi-
tive feedback which favours tree species coexistence though niche
partitioning. Understanding the mechanisms sustaining long-term
species coexistence in mixed communities is critical to foresee
changes in the structure and composition of plant communities.
Our results reinforce the suggestion that plant–soil feedbacks
influence species abundance, persistence and succession and
thereby underpin species coexistence (Bonanomi et al., 2005;
Brandt et al., 2013; van der Putten et al., 2013).
Acknowledgements
We are most grateful to Cindy Prescott, Lars Vesterdal and two
anonymous reviewers for their very constructive comments. We
thank the Consejería de Medio Ambiente (Andalusian Government)
and the then Director and staff of Los Alcornocales Natural Park, for
the facilities and support to carry out our field work. We are grate-
ful to Eduardo Gutiérrez, Ana Pozuelos, María Navarro, Marga San-
taella, Daniel Caballos, Carlos Ros, Manuel del Pozo, Paula Delgado-
Cuzmar, Susana Hito, Ramón Redondo, Sophie Manzi and Juliet
Roche for field and/or lab assistance. Our deepest gratitude for
Ignacio M. Pérez-Ramos, Maria T. Dominguez and Monique Gardes
for their help and support throughout this research. This study was
supported by a FPI-MEC grant and a postdoctoral-MEC grant (EX-
2010-0148) to CA, by the Spanish MEC projects DINAMED
(CGL2005-5830-C03-01 and -02), INTERBOS (CGL2008-4503-C03-
01 and -02) and DIVERBOS (CGL2011-30285-C02); the Andalusian
CICE projects GESBOME (P06-RNM-1890) and ANASINQUE
(PE2010-RNM-5782); the Subprograma de Técnicos de Apoyo MIC-
INN (PTA2009-1782-I) and European FEDER funds.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foreco.2013.05.035.
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46 C. Aponte et al. / Forest Ecology and Management 309 (2013) 36–46

SUPPLEMENTARY MATERIAL 1
Supplementary Fig S1. Leaf fall temporal patterns in the studied forest for the deciduous Q. 2
canariensis (filled symbol) and the evergreen Q. suber (hollow symbols). 3
4
5
6
7
Feb Apr Jun Aug Oct Dec
Percentage of annual leaf fall
0
10
20
30
40
50 Q. canariensis
Q. suber

Supplementary Fig S2. Chemical and textural properties of the subsoil (25- 50cm) sampled 8 under Q. canariensis and Q.suber in the two study sites (La Sauceda and Tiradero). 9 Differences between species (ANOVA) are indicated. 10 11
12

13 Supplementary Fig S3. Alternative models used in the path analysis to determine the causal 14
relationships between host species (OAK), calcium concentration in the fresh leaves (LV), 15
leaf fall (LF), litter (LI), topsoil (TOP), subsoil (SUB), and the ECM community composition 16
(expressed as the 1st correspondence analysis axis). Model 1: Direct effect of soil intrinsic 17
properties; Model 2: Direct effect of host genetic specificity; Model 3: Indirect effect of host 18
species via leaf fall and litter quality; Model 4: Direct and indirect effects of host species; 19
Model 5: Direct effect of soil intrinsic properties and indirect effect of host species; Model 6: 20
Direct effect of soil intrinsic properties and direct and indirect effects of host species. The 21
result of the path analysis (d-sep test) for each model are presented in brackets. A model is 22
considered significantly feasible when the underlying relationships cannot be falsified (i.e. P 23
> 0.05). Modified from Aponte et al.. {Aponte, 2010 #460}. 24
25
26

Supplementary Fig S4. Alternative models used in the path analysis to determine the causal 27
relationships between oak species (Q. canariensis/Q. suber), nutrient concentration in the 28
fresh leaves, leaf fall, topsoil (0-25 cm) and subsoil (25-50 cm) (expressed as the 1st axis of 29
the PCA on the concentrations of N, Ca, K, Mg, S, P, Mn, Zn and Cu). The result of the path 30
analysis (d-sep test) for each model are presented in brackets. A model is considered 31
significantly feasible when the underlying relationships cannot be falsified (i.e. P > 0.05). 32
Modified from Aponte et al. {, 2011 #882} 33
34
35
36
37
38