Journal of Vegetation Science && (2016)

Grassland root functional parameters vary according to

a community-level resource acquisitionconservation
trade-off
Florian Fort, Pablo Cruz, Eric Lecloux, Leandro Bittencourt de Oliveira, Ciprian Stroia,
Jean-Pierre Theau & Claire Jouany

Keywords
Deep root; Nitrogen; Phosphorus; Root
acquisitionconservation trade-off; Root
functional trait; Shallow root; Soil depth;
Traitenvironment relations
Abbreviations
CFI = comparative fit index; RLD = root length
density; RMSEA = root mean square error of
approximation; RTD = root tissue density;
SRL = specific root length; SEM = structural
equation model.
Nomenclature
Euro+Med PlantBase
Received 29 July 2015
Accepted 19 February 2016
Co-ordinating Editor: Francisco Pugnaire

Fort, F. (corresponding author,

fort.florian@gmail.com)1,
Cruz, P. (Pablo.cruz@toulouse.inra.fr)2,
Lecloux, E. (eric.lecloux@toulouse.inra.fr)2,
Bittencourt de Oliveira, L.
(leandroliveira86@hotmail.com)3,
Stroia, C. (cipistroia2001@yahoo.com)4,
Theau, J.P. (jean-pierre.theau@
toulouse.inra.fr)2,
Jouany, C. (claire.jouany@toulouse.inra.fr)2
1

Montpellier SupAgro, UMR 5175 CEFE,

de Montpellier Universit
e Paul
Universite

ry EPHE, 1919 route de Mende, F-34293
Vale
Montpellier Cedex 5, France;
2
INRA, UMR1248 AGIR, INRA Universit
e de
Toulouse INPT, F-31326 Castanet-Tolosan,
France;
3
Animal Sciences Department, Ecological
Natural Grassland Research Group,
Universidade Federal de Santa Maria, Santa
Maria 97150-900, Brazil;
4
Department of Biology and Plant Protection,
Banat University of Agricultural Sciences and
Veterinary Medicine King Michael I of
Romania Timisoara, Calea Aradului 119,
300645 Timisoara, Romania

Abstract
Questions: The fundamental trade-off between fine root trait attributes related
to resource acquisition and conservation is well documented at species and
community levels. However, relations remain unclear between this trade-off
and community adaptation to environmental factors. As a result we ask: (1)
how do fertility changes influence community position along the root
acquisitionconservation trade-off; (2) how does root position along the soil
profile influence its functional parameter; and (3) do fertility and soil depth
have an interactive effect on community root parameters?
Location: Native multi-species temperate grassland in the French Massif
Central.

Methods: We assessed botanical composition and measured community root

functional parameters (at plot and soil layer levels) in 16 plots differentiated by
the amounts of N and P fertilizers applied over 16 yr and a soil depth gradient.
Structural equation models were used to analyse relations among environmental factors, botanical composition and root functional parameters.

Results: Botanical composition and plot-level root functional parameters vary

according to fertility and soil depth. Communities from low fertility plots display
a resource conservation strategy, i.e. high root tissue density, low specific root
length (SRL) and low root length density (RLD), while communities from high
fertility plots display a resource acquisition strategy, i.e. opposite parameter values. This demonstrates the importance of root resource management strategies
in community adaptation to resource acquisition. Roots display different parameter values with soil depth. Roots in the surface horizon have small diameters
and high SRL and RLD, suggesting intensive soil exploration and a high nutrient
acquisition capacity. Roots in deep horizons have large diameters and low SRL,
suggesting high water transport capacity per root length unit. This pattern is
affected by plot fertility, i.e. communities from high fertility plots show higher
root strategy differentiation with depth than communities from low fertility
plots. We hypothesize that this root specialization along the soil profile enables
species to be competitive for nutrient uptake in shallow soil layers and water
uptake and transport in deep soil layers.

Conclusion: Our study provides evidence that fertility and soil depth modified
root functional parameters, in agreement with predictions from the economics
spectrum theory.

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Root adaptation to fertility change

F. Fort et al.

Introduction
Functional and botanical plant community composition
results from the interaction of abiotic and biotic filters that
select species individuals able to colonize a habitat and
maintain a population (Lortie et al. 2004). Species ability
to colonize a habitat results from the interaction between
habitat characteristics and species functional trait attributes, e.g. species position on the resource economics spectra.
These spectra relate to the fundamental trade-off between
resource acquisition strategies, which maximize plant
growth rate and competitiveness in low-stress, nutrientrich environments, and resource conservation strategies,
which maximize plants ability to tolerate stressful nutrient-poor environments (Chapin et al. 1993). This trade-off
has been well documented in studies that mainly focus on
the above-ground compartment (Westoby & Wright
2006). Rarely is the below-ground compartment considered; it is responsible for nutrient and water uptake and
likely plays a major role in mitigating water and nutrition
stress effects on plant growth (Fort et al. 2015) and plant
interactions (Mommer et al. 2011; Fort et al. 2014).
Recent studies indicate the existence of a community-level
root economics spectrum related to community adaptation to
resource availability gradients (Craine et al. 2001; P

erezRamos et al. 2012; Prieto et al. 2015).
Resource availability gradients can be modified by
human activities. For example, grassland functioning and
composition in agricultural systems is impacted through
cutting for forage production and frequent fertilization
(mainly N and P fertilization) to ensure the quantity and
quality of forage. It was demonstrated that such practices
change both specific and functional composition (Louault
et al. 2005). Species found in N-fertilized and disturbed
(by cutting) grasslands tend to display above- and belowground acquisitive strategies characterized by low root
tissue density (RTD); Craine et al. 2001). Leuschner et al.
(2013) showed that grassland species improve resource
acquisition capacities by increasing specific root length
(SRL) and decreasing root dry matter content in response
to fertilization.
However, these results were obtained at the species
level, and little is known about root system adaptation to
agricultural practices at the community level. Prieto et al.
(2015) demonstrated that the functional trade-off between
resource acquisition and resource conservation strategies is
maintained over a large range of communities (from grasslands to forests). This trade-off is related to community
adaptation to environmental gradients. Communities with
functional parameter (functional traits measured at the
community level) values associated with an acquisition
strategy (thin roots with high SRL and low dry matter content) were found in environments highly disturbed by

agricultural practices and poor in nutrients. Conversely,

communities with functional parameter values associated
with a conservation strategy (coarse roots with low SRL
and high dry matter content) were located in undisturbed
and nutrient-rich environments. This contradicts previous
results that indicate a decrease in RTD or root dry matter
content with an increase in nutrient availability (Craine
et al. 2001; P

erez-Ramos et al. 2012). This could be related
to the difficulty in distinguishing which part of root parameter variation is due to the effect of nutrient availability
and/or disturbance modification along the gradient studied
(Prieto et al. 2015). Despite contradictory results, these
studies demonstrate the importance of root functional
parameters as an indicator of plant adaptation to environmental changes, especially changes in nutrient availability.
Soil depth also affects community functional parameter
position along a resource acquisitionconservation tradeoff. Roots from surface soil layers within the same soil profile display a more acquisitive strategy than roots from deep
soil layers (Prieto et al. 2015). This can be explained by differences in resource partitioning along the soil profile, i.e.
larger nutrient stocks in surface soil layers than deep soil
layers, and larger water stocks in deep soil layers than surface soil layers. As a result, roots from surface soil layers
should acquire most of the plants nutrients. Under this
condition, thin roots with high SRL and root length density
(RLD) must have an advantage because they allow plants
to compete when nutrients are highly available (Mommer
et al. 2011; Fort et al. 2014). Conversely, deep soil layers
that have lower nutrient availability but higher water storage ability should contain roots that preferentially acquire
water and transport it from the deep soil to the shoot. It has
been demonstrated that large roots have higher conductance capacity than thin roots (Hern

andez et al. 2010); as a
result large diameter roots in deep soil layers should give
plants a competitive advantage by increasing their water
export capacity. We hypothesized that root parameter variations along the soil profile result from root specialization
to acquire different resources (nutrients or water).
Despite their importance for community structure,
below-ground mechanisms that promote plant community adaptation to fertility changes, i.e. root functional
parameter response to fertility changes at the community
level, are poorly understood. It would be interesting to test
whether changes in community root functional parameters are due to changes in species composition or are simply
within-species trait variations in response to these
gradients.
We tested the two following hypotheses. First, contrasting long-term fertilization regimes that highly modify soil
nutrient availability and consequently also modify grassland botanical and functional composition by favouring
acquisitive species in fertilized plots and conservative ones

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Root adaptation to fertility change

F. Fort et al.

in unfertilized plots. Second, for a given soil layer, root

functional parameters change according to the interaction
between nutrient availability and root position along the
soil profile, i.e. fertilized plots have more differentiated
root systems, with acquisitive thin roots in surface soil layers to ensure competitiveness for nutrient acquisition, and
coarse roots in deep soil layers to ensure water transport
from deep horizons. Unfertilized plots would have less differentiated root functional parameters along the profile
due to the establishment of conservative strategies in both
deep and surface soil layers.
Our objectives were to determine how environmental
factors, i.e. fertility (N and P) and soil depth, influence
grassland botanical composition and root functional
parameters. We studied botanical composition and root
functional parameters (depth of 95% root biomass, root
diameter, root biomass, RLD, RTD and SRL) at the plot
level and their variations along the soil profile for 16 grassland communities differentiated by 16 yr of contrasting N
and P fertilization regimes.

Methods
Study site and experimental design
The study site was located in the French Massif Central, in
a native multi-species agricultural grassland (44 N, 2 E,
607 m a.s.l.). The mean annual air temperature is 11.8 C,
with mean annual rainfall of 960 mm. The experiment,
started in 1998, aimed to study long-term effects of N and
P fertilization on plant community and soil fertility dynamics (Stroia et al. 2007). A factorial design with two levels of
N and P was applied, resulting in four treatments: N0P0,
N0P+, N+P0 and N+P+, where N0 and P0 correspond to no
N and no P fertilization, respectively; N+ corresponds to N
fertilization of 160 kg
N
ha 1 yr 1; and P+ corresponds to
P fertilization of 50 kg
P
ha 1
yr 1. Each treatment was
replicated four times in plots 5-m long and 3-m wide. The
plots were arranged randomly in four blocks, with one
replicate of each treatment per block, to consider a deep
soil gradient along the field. Vegetation on all plots was cut
three times per year during the 16 experimental years.
Root and soil sampling
During the first week of early spring in March 2014, seven
soil cores were removed from the centre of each plot
(diameter: 5 cm, length: until maximum soil depth). Each
soil core was divided into five layers (05, 510, 1020,
2040, 40maximum soil depth, in cm). Three soil cores
were dedicated to root analysis. Each layer of these soil
cores was frozen separately at 18 C to preserve it until
roots were washed. The other four soil cores were dedicated to soil parameter analysis. They were mixed by layer

and air-dried, sieved (2 mm) and stored at ambient

temperature before analysis (Appendix S1).
Soil parameter measurement
Soil depth was measured during the sampling. For each of
the seven soil cores, we measured the rock source (micaschist) depth. Total soil organic N concentration and plant
available P were determined at the soil layer level using
the Kjeldahl method (NF ISO 11261) and the Olsen
method (Olsen et al. 1954), respectively. Plot organic N
and P concentrations were calculated as the mean of layer
concentrations multiplied by the soil mass (soil volume 9
soil density) within each soil layer.
Root parameter measurements and calculation
Each soil sample was defrosted, and soil particles attached
to the roots were washed off. When roots were totally free
of soil and organic matter fragments, the coarse roots
within each layer were separated from fine roots (<2-mm
diameter). Fine roots were stained for at least 12 h with
methylene blue (1%) to improve contrast during scanning.
Roots were spread in water in a clear acrylic tray and then
scanned at 400 dots per inch. WinrhizoTM Pro 2007 software (Regent Instrument, Sainte-Foy, Quebec, CA) was
used to measure the total root length of each root segment
with a constant diameter. This method estimates the total
length, surface area and volume of each root sample. After
scanning, each root sample was drained, dried at least 72 h
at 60 C and weighed.
Mean root diameter was calculated as the mean of the
median root diameter of each class size weighted by the
root length of each class. SRL was calculated by dividing
the sample root length by its dry mass. Root tissue density
was the ratio of the sample root dry mass to its volume.
RLD was calculated by dividing the total root length of
each sample by the soil volume of this sample. These
parameters were calculated for each soil layer of each soil
core dedicated to root parameter measurement. Afterward,
they were calculated (1) at the soil layer level for a plot, i.e.
mean parameter values of the same soil layer for the three
soil cores in the plot; and (2) at the soil core level, i.e. mean
of the soil layer level parameter, weighted according to
root biomass, for the three soil cores in a plot. The plot
parameter values were calculated as the mean of these soil
core parameter values. Total root biomass was calculated
for each soil core as the sum of each root sample dry mass.
To analyse root distribution along the profile, we used the
depth of 95% of the total root biomass, calculated using a
linear regression of cumulative fine root length with depth
(log). For other parameters, the plot total root biomass and
depth of 95% of the root biomass were the mean of the

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Root adaptation to fertility change

F. Fort et al.

three soil cores total biomass and depth of 95% of the root
biomass, respectively.
Above-ground measurements
Species composition and abundance were assessed just
before soil sampling within a 15-cm radius around the centre of the soil cores dedicated to root measurement (three
per plot). Within each circle, the most abundant species
were identified and ranked using the visual de Vries
method (De Vries & De Boer 1959). This method is used to
assess grassland species composition (Lavorel et al. 2008).
Above-ground peak biomass was determined (Jun 2014)
in three 0.19-m quadrats placed in the centre of each plot.
The harvested biomass was dried (48 h at 60 C), weighed
and ground. Next, the dry biomass was ground, and N concentration was determined using a CHN elemental analyser. P concentration was measured with the malachite
green method (van Veldhoven & Mannaerts 1987). These
concentrations were used to calculate the N and P nutrient
indices (NNI and PNI, respectively; Jouany et al. 2004;
Gastal et al. 2015).
Data analysis
ANOVAs were used to test (1) the influence of treatments
on soil fertility and above-ground biomass production, and
(2) the independence of soil depth and fertilization treatment; we used the Spearman correlation test to test
whether soil N and P concentrations were related to soil
depth. Principal component analysis (PCA) was performed
using species abundance in the 16 plots to determine
whether plot botanical composition differed according to
environmental factors (soil depth, N and P concentration)
and, if so, which species were responsible for the differences. Grassland botanical composition before the start of
the experiment was projected along the PCA components,
but was not used to construct them. The correlation
between PCA components, N and P nutrient indices and
above-ground biomass at the peak of vegetation was
assessed with the Spearman correlation test. To analyse
the influence of soil depth on root parameters, we used a
mixed linear model with depth as an explanatory factor
and plot identity as an error factor in case parameters
among plot layers were not independent.
For both plot and soil layer analysis, structural equation model (SEM) analyses were performed with R 3.2.0
software (R Foundation for Statistical Computing, Vienna,
AT) using the sem function from the lavaan package (Rosseel 2012). For plot-level analysis, all the direct and indirect paths from environmental factors (N, P availability
and soil depth) to root parameters (diameter, depth of
95% root length, root biomass, RLD, RTD and SRL) calcu-

lated at the plot level (mean of plot three soil core root
parameters, n = 16) were included in the initial models.
The indirect path extends from environmental factors to
root parameters through botanical composition. PCA of
the first three components allowed estimation of the
portion of root parameter change caused by change in
botanical composition. Model reliability was assessed with
a chi-square test. A significant chi-square statistic indicated
that the model did not fit the data. After the first run of the
plot-level model all non-significant paths were removed
from the model and only significant paths were kept for
the analysis and presented in this work. For the soil layer
level SEM analysis, we kept the significant path from environmental factors to PCA components because path values
were the same as those of the plot-level analysis. In the
SEM, we included root functional parameter values calculated per layer (n = 16 for each soil layer except the deepest, for which n = 15) to analyse the SEM results that
included a soil layer effect (group effect). Two other indices
of fit, (1) comparative fit index (CFI) and (2) root mean
square error of approximation (RMSEA), which are often
used in SEM, are reported to assess the closeness of fit.
Good models have RMSEA values <0.05, P-values > 0.05
and CFI values > 0.95 (Vile et al. 2006). To ensure the
robustness of P-value estimation we used a Bollen-Stine
bootstrap procedure (Rosseel 2012). The standard P-value
was validated by computing the proportion of test statistics
from the bootstrap samples that exceed the value of the
test statistic from the original sample. The number of generated samples was 1000. Once the models were validated,
path coefficient estimates were used to study direct and
indirect effects among variables.

Results
Environmental factors
The soil depth in all plots ranged from 0.41 to 0.76 m, and
varied irrespective of the fertilization treatment (ANOVA,
P = 0.97) and N and P concentrations (Spearman correlation test, P = 0.32 and 0.38, respectively). The combination of N and P fertilization treatments highly
differentiated the fertility of the 16 plots (Table 1). Mean
total N concentration over the soil profile ranged from
0.87  0.3 g
kg 1 without N and P fertilization to
1.04  0.3 g
kg 1 with N and P fertilization. This divergence in N stock resulted in twice as much above-ground
biomass production in the N+P+ treatment as in the N0P0
treatment (Table 1). Mean plant available P concentration
over the soil profile strongly varied among treatments,
from 0.010  0.001 mg
kg 1 for plots of the N+P0 to
0.046  0.003 mg
kg 1 for plots of the N0P+. This indicated an interactive effect of N and P fertilization on the
plant available concentration of soil P, which had a strong

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Root adaptation to fertility change

F. Fort et al.

Table 1. : Soil nutrient (N and P) concentrations, biomass production, N nutrient index (NNI) and P nutrient index (PNI) means  SE (n = 4) for the four
fertilization treatments (n = 16).
Treatments
Nitrogen

Phosphorus

N Soil Concentration
(g
kg 1)

N0
N0
N+
N+

P0
P+
P0
P+

0.87
0.93
1.03
1.04






0.3 a
0.2 a
0.2 b
0.3 b

P Soil Concentration
(Olsen P, mg
kg 1)
0.016
0.046
0.010
0.036






0.001 a
0.003 c
0.001 a
0.004 b

Biomass Production
(t
ha 1)
2.3
2.9
4.4
4.6






0.3 a
0.5 a
0.5 b
0.4 b

NNI (%)

53.9
55.8
70.5
64.6






PNI (%)

3.7 a
2.2 a
3.5 b
3.7 ab

123.1
144.1
98.7
131.8






6.1 b
6.0 c
4.1 a
6.0 bc

N0: unfertilized by N; N+: fertilized by N; P0: unfertilized by P; P+: fertilized by P. Letters indicate significant differences within columns (ANOVA and Tukey
post-hoc test, a = 0.05).

effect on PNI. Despite the significant differences in PNI

between N+P0 and N+P+ treatments, above-ground biomass production measured at the peak of vegetation in
2014 was not significantly affected by P fertilization
(Table 1). NNI was also affected by the N and P fertilization
interaction. NNI values for all plots were far below the
threshold of 100, indicating sub-optimal levels of N nutrition (Gastal et al. 2015).
Botanical composition-based PCA
Thirteen species typical of southwestern French grasslands
were found in the area around root sampling points, and
large differences existed among botanical compositions of
the plots (Fig. 1). The PCA revealed three major independent sets of correlations among species abundance, which
explained 59% of the total variability. The first set of correlations (Component 1) explained 26% of the total PCA

variation. It represents a gradient of plots from those dominated by Achillea millefolium to those dominated by Lolium
perenne, Plantago lanceolata, Poa trivialis and Trifolium repens
(Fig. 1). The second set of correlations (Component 2)
explained 18% of the total PCA variation. It represents a
gradient of plots from those with a high abundance of
Festuca rubra and Holcus lanatus to those dominated by
Trisetum flavescens (Appendix S2). Component 3 explained
15% of the total PCA variation. Plot coordinates in these
components were positively related to Dactylis glomerata
and negatively to Taraxacum officinale abundance (Fig. 1).
Component 1 plot coordinates were positively correlated with plot PNI (r = 0.53, P = 0.03, Spearman correlation test) but not with their NNI or above-ground biomass
production. Component 2 was not correlated with NNI or
above-ground biomass production (Spearman correlation
test P = 0.50 and 0.83, respectively). Component 3 plot
coordinates were positively correlated with plot NNI
(r = 0.59, P = 0.02, Spearman correlation test) and aboveground biomass production (r = 0.79, P < 0.001, Spearman correlation test) but not with their PNI. Moreover,
plots receiving N fertilization had higher coordinates on
Component 3 than other plots (Fig. 1).
Plot-level SEM model

Fig. 1. Coordinates of the plots along botanical composition PCA

Components 1 and 3 (black axes) and plot-level species abundance
loadings for these components (grey axes). Labels and grey arrows
represent species with a significant contribution to PCA Components 1
and 3. Botanical composition in 1998, before the experiment (grey circle),
is included for comparison. Species: Achmil: Achilea millefolium; Dacglo:
Dactylis glomerata; Lolper: Lolium perenne; Plalan: Plantago lanceolata;
Poatri: Poa trivialis; Trirep: Trifolium repens; Taroff: Taraxacum officinalis.

After removing all non-significant paths in the SEM

model, we assessed the relation between environmental
factors, botanical composition (PCA components) and
plot-level functional parameters, which provided a good fit
to the data (chi-square = 21.39, df = 27 and P = 0.77,
CFI = 1.0, RMSEA < 10 4 and P = 0.81). The model
showed significant paths between plot coordinates along
the PCA component based on botanical composition, plot
fertility (N and P) and soil depth (Fig. 2).
Relations between environmental factors and botanical
composition
The SEM indicated significant paths from environmental
factors and species abundance based on PCA components,

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Root adaptation to fertility change

F. Fort et al.

Fig. 2. SEM for assessing the relation among environmental factors (N: total soil N concentration; P: soil P concentration; Soil depth), community botanical
composition (PCA components) and below-ground functional parameters. Root diameter and biomass are not represented because no significant paths
linked them to PCA components. A significant path occurred from soil depth to root diameter ( 0.39), and two significant paths occurred from soil depth
and P concentration to root biomass (0.72 and 0.14, respectively). R values are given for dependent variables except for Diameter (R: 0.53) and root
biomass (R: 0.38). Goodness-of-fit statistics are chi-square = 21.39, df = 27 and P = 0.77, CFI = 1.0, RMSEA < 10 4 and P = 0.81. RLD: root length
density; Root depth: depth of 95% root biomass; RTD: root tissue density; SRL: specific root length.

i.e. community botanical composition changed according

to differences in environmental factors. Significant paths
occurred from soil depth (negative) and P concentration
(positive) to plot coordinates along Component 1 (Fig. 2).
Conversely, Component 2 was connected to soil N concentration but not to soil depth or P concentration (Fig. 2).
The two significant paths from soil depth and soil N concentration to Component 3 coordinates appeared to be the
strongest paths relating environmental factors to a component. These indicate that soil depth and N concentration
had negative and positive significant effects, respectively,
on Component 3 plot coordinates (Fig. 2).
Effect of environmental factors and botanical
composition on functional parameters
Plot-level functional parameters were influenced by environmental factors and botanical composition (Fig. 2). Soil
depth had the strongest effect on these traits among all
environmental factors (Fig. 2). Plot-level mean root diameter decreased with soil depth, while root biomass, RTD
and depth of 95% root mass increased with soil depth
(Fig. 3a,b,c,e). SRL and RLD were not significantly affected
by soil depth (Fig. 3d,f) or soil N and P concentration
(Table 2). A positive and significant path occurred from
soil N concentration to the depth of 95% root mass
(Fig. 2). Total soil N concentration also had a negative

effect on plot-level RTD, while the available soil P concentration had a negative effect on root biomass (Fig. 2).
Significant paths also occurred from PCA components to
functional parameters, except for root diameter and root
biomass, which were related only to environmental factors
(P concentration and soil depth). Four significant paths
occurred from Component 1 to root parameters: two positive, to RLD and SRL, and two negative, to depth of 95%
root mass and RTD (Fig. 2). As a result, plots dominated by
L. perenne, P. lanceolata, P. trivialis and T. repens had higher
RLD, SRL and lower RTD and root depth than plots dominated by A. millefolium (Fig. 2). Two paths were significant
from Component 2, to depth of 95% root mass (negative)
and to SRL (positive; Fig. 2), demonstrating that plots with
high abundance of T. flavescens tended to have higher SRL
and lower depth of 95% root mass than those with high
abundance of F. rubra and H. lanatus (Fig. 2). Three significant paths occurred from Component 3 to RTD, RLD and
SRL. The first was negative while the others were positive
(Fig. 2), highlighting that plots in which D. glomerata was
well represented had higher RTD and lower RLD and SRL
than plots dominated by T. officinale (Fig. 2).
Soil layer SEM models
All functional parameters varied along the soil profiles
(Appendix S3). Root biomass and root length density

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Root adaptation to fertility change

F. Fort et al.

top soil layers (05-cm depth). The path from these components to RLD were positive and significant for the top
soil layer (05-cm depth) and negative for the deep soil
layers, the path to the root biomass similarly varied, but
only for Component 1 (Table 2).
Component 2 displayed less interaction between soil layers and path sign or significance. All paths were negative
from this component to root diameter and root biomass.
For Component 1, paths to RLD were negative for deep soil
layers and non-significant for surface soil layers. Paths from
Component 2 to SRL were positive and significant for
intermediate soil layers (520-cm depth; Table 2). Unlike
the two other components, the path from Component 2 to
RTD was positive and significant for deep soil layers.

Discussion
We demonstrated that 16 yr of contrasting N and P fertilization regimes led to a strong differentiation of grassland
community biomass production. This change is the result
of changes in soil fertility and functional community composition. Different functional parameter values are noticeable at plot and soil layer levels, highlighting an overall
adaptation of community root parameters to fertility
changes and contrasting parameter response to soil layer
depth.

Fig. 3. Variation in plot-level fine root functional parameters along the

gradient of soil depth. Graphs show linear regressions between soil depth
and plot-level functional parameters. Only significant model regressions
(R2) are shown (** P < 0.01, *** P < 0.001, n = 16).

decreased from surface soil layers to deep soil layers. RTD

and SRL also decreased along the soil profile, while the
mean root diameter increased with depth (Appendix S3).
The SEM models that assessed relations between environmental factors, PCA components and plot-level functional
parameters analysed with a soil layer effect (group effect)
provided a good fit to the data (chi-square = 30.39, df = 35
and P = 0.69, CFI = 1.0, RMSEA < 10 4 and P = 0.74).
The significance and sign of paths from PCA components to root parameters changed with soil layer depth.
Components 1 and 3 tended to have the same relations
with functional parameters, but the strength and significance of the paths were higher for Component 1. These
component paths to root diameter were negative or close
to zero for surface soil layers and positive for the deep soil
layers (Table 2). Conversely, for the SRL, they were positive for surface soil layers and negative for deep soil layers
(Table 2). These results are schematically represented on
Fig. 4. The path from Components 1 and 3 to RTD was
negative for all soil layers and was more significant for the

Root functional parameter response to environmental

factors at the community level
As previously reported regarding this experimental site
(Stroia et al. 2007), N fertilization was the only fertilization
treatment that induced a change in biomass production. N
fertilization also induced a modification of environmental
filters that affected species community composition, i.e.
communities with the highest N fertility were dominated
by T. officinale, a species considered a good indicator of high
N availability and soil organic matter content (Grime et al.
1988; Ellenberg et al. 1991). Even though P was non-limiting for biomass production, the PCA showed that P availability was, together with soil depth, one of the main
factors driving community composition. Communities
with higher P availability were dominated by L. perenne,
P. trivialis, T. repens and, to a lesser extent, P. lanceolata.
These species are typical in nutrient-rich grasslands (Grime
et al. 1988; Ellenberg et al. 1991).
Environmental factors directly and indirectly affected
root functional parameters and explained differences
among plots. Community adaptation to high N and/or P
fertility led to changes in species abundance and species
replacement, which induced, at the plot level, an increase
in RLD and SRL and a decrease in RTD. High SRL and RLD
are related to an efficient soil exploration capacity (Hodge

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Root adaptation to fertility change

F. Fort et al.

Table 2. Path coefficient (standardized partial regression coefficient) estimates and significance from biotic factors (PCA based on species abundance components) to root functional parameters measured at the soil layer level. Goodness-of-fit statistics of the SEM analysed with a group effect (soil layer depth)
are chi-square = 30.39, df = 35 and P = 0.69, CFI = 1.0, RMSEA < 10 4 and P = 0.74. Boldface indicates P < 0.05. Italics indicate P > 0.05 and <0.10.
Component and Depth

Diameter
Estimate

Component 1
05 cm
510 cm
1020 cm
2040 cm
40-max depth, cm
Component 2
05 cm
510 cm
1020 cm
2040 cm
40-max depth, cm
Component 3
05 cm
510 cm
1020 cm
2040 cm
40-max depth, cm

Root biomass
P

Estimate

Root Tissue Density

Specific Root Length

Estimate

Estimate

Estimate

0.64
0.04
0.37
0.35
0.50

0.001
0.80
0.02
0.08
0.01

0.30
0.32
0.69
0.60
0.08

0.10
0.07
<0.001
<0.001
0.72

0.94
0.14
0.58
0.61
0.33

<0.001
0.44
<0.01
<0.001
0.04

0.33
0.37
0.51
0.25
0.30

0.04
<0.01
<0.001
0.02
0.14

0.61
0.33
0.08
0.07
0.38

<0.01
0.03
0.69
0.78
0.06

0.29
0.57
0.46
0.28
0.04

0.12
<0.001
<0.01
0.15
0.85

0.56
0.37
0.20
0.53
0.64

<0.01
0.03
0.19
<0.001
<0.01

0.22
0.30
0.14
0.65
0.92

0.13
0.09
0.46
<0.001
<0.001

0.24
0.13
0.26
0.31
0.26

0.14
0.26
0.03
<0.01
0.19

0.07
0.62
0.43
0.24
0.07

0.74
<0.001
0.03
0.33
0.73

0.09
0.22
0.15
0.41
0.49

0.66
0.15
0.34
0.04
0.01

0.09
0.04
0.16
0.05
0.20

0.62
0.80
0.31
0.74
0.38

0.30
0.17
0.32
0.15
0.30

0.04
0.36
0.10
0.15
0.07

0.36
0.04
0.32
0.18
0.11

0.03
0.76
0.01
0.11
0.59

0.39
0.27
0.36
0.30
0.64

0.06
0.08
0.08
0.25
<0.01

Fig. 4. Diagram of the relation between fertility and community root

functional strategies in surface (05 cm) and deep (>40 cm) soil layers.
Solid lines show that the same trends are observed for N and P fertility. For
surface root diameter dashed line show that only P fertility affect surface
root diameter, while N fertility had no effect (dotted line). Diameter: root
diameter; RLD: root length density; SRL: specific root length.

2006), higher nutrient uptake (Mommer et al. 2011; Bardgett et al. 2014) and higher competitiveness for high nutrient availability (Craine 2006; Mommer et al. 2011; Fort

Root Length Density

et al. 2014), but also induced short root life spans and
higher resource turnover (McCormack et al. 2012). We
hypothesized that these changes in root functional parameters were due to environmental filters (Lortie et al. 2004)
selecting the most competitive species under conditions of
high nutrient availability. In these conditions, even though
nutrients were abundant and highly available, plants
should invest much more root biomass than was theoretically expected to acquire such nutrients (Gersani et al.
2001; Maina et al. 2002), and should produce high RLD to
ensure that they acquire enough nutrients or water to
remain competitive. Conversely, communities adapted to
low fertility generally displayed conservative resource management strategies, with high RTD and low RLD and SRL,
associated with long root life spans and higher resource
conservation (Withington et al. 2006; McCormack et al.
2012). We hypothesized that highly competitive acquisitive
species were excluded or weakened by the low nutrient
availability and replaced by conservative species, which are
more adapted to manage limiting growth conditions. Our
results indicated the existence, at the community level, of a
resource acquisitionconservation trade-off resulting from
communities adaptation to gradients of fertility.
Interactive effect of fertility and root position on root
parameters along the soil profile
We found that root position along the soil profile had a significant effect on their functional parameters. Roots from
surface soil layers exhibited more acquisitive strategies

Journal of Vegetation Science

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Root adaptation to fertility change

F. Fort et al.

than roots from deep soil. This indicated that roots from
surface soil layers were able to efficiently forage the soil to
acquire nutrients (Craine 2006; Mommer et al. 2011;
Bardgett et al. 2014). They should be less efficient than
coarse roots in transporting nutrients and water (Hern

andez et al. 2010). These results agree with resource partitioning along the soil profile (nutrients and water in
surface and in deep soil layers, respectively) and with previous observations (Prieto et al. 2015). RTD decreased with
soil depth, which contradicted what is expected based on
root diameter and SRL variations. This could be due to
roots adapting to different soil conditions, i.e. deep roots
were less subject to soil dehydration than surface roots and
consequently did not need a particular defence via cell wall
reinforcement or secondary compound accumulation.
They were more often subject to anoxic conditions and
might need to develop aerenchyma to maintain good
tissue oxygenation (Hodge et al. 2009). Consequently,
root position along the soil profile appeared to be one of
the main drivers of root functional parameter values.
The differences on community functional strategies for
roots from the surface or the deep soil layer increased with
fertility (Fig. 4). Communities from fertile plots had roots
with more acquisitive strategies in surface soil layers than
communities from less fertile plots, showing a potential
increase in competitive ability for resource acquisition with
increased nutrient availability. Conversely, communities
adapted to high fertility displayed thicker roots in deep soil
layers (Fig. 4) than communities adapted to low fertility.
The root diameter increase and SRL decrease could be
related to increased water transport efficiency (Hern

andez
et al. 2010) along the soil profile, to satisfy the substantial
water demand of the above-ground compartment due to
vigorous and fast biomass production. The relation
between RTD and the community degree of adaptation to
fertility did not change with soil depth, which demonstrated a community-level trade-off between high RTD
and adaptation to fertility, independent of soil layer depth.
Soil depth effect on plot-level root parameters
Soil depth appeared to be a major factor affecting root
parameter values at the plot level. The depth of 95% of the
root biomass and total root biomass increased with soil
depth. These relations were expected because increased
soil depth allows roots to grow deeper and provides a larger
soil volume to explore. However, the plot-level root diameter decreased and RTD increased with soil depth. As
described previously, small diameter and high RTD represent the surface soil layer parameter syndrome of roots.
This could be explained by deep soils containing a larger
water stock than shallow soils (P
erez-Ramos et al. 2012).
Larger water stocks can sustain more biomass production

by communities, but this increased the requirement of the

above-ground biomass for nutrients. We hypothesize that
in deep soil, high community nutrient requirement caused
plants to develop a large proportion of their roots with efficient nutrient acquisition strategies, i.e. with surface soil
layer parameter syndrome (small diameter and high RTD).

Conclusion
Our results demonstrate that grassland adaptation to longterm fertility changes undergoes important root functional
parameter modifications at the community level, in agreement with predictions from the economics spectrum theory.
Communities from high-fertility plots display botanical
composition changes that induce the establishment of
acquisitive strategies in surface soil layers and conservative
strategies in deep soil layers. We hypothesize that this root
specialization enables species to be competitive in terms of
both nutrient and water uptake.

Acknowledgements
This work was supported by the R

egion Midi Pyr

en

ees project Legumip (13050966). We thank Jean Foucras and the
staff of the Chambre dAgriculture de lAveyron (CA 12)
for their support in conducting portions of the fieldwork.
We thank H

elose Bessi
ere for participation in field and lab
work. We would also like to thank Mr. Estival for the use
of his field.

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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Diagram of the sampling procedure
for a plot (a replicate for a fertilization treatment).
Appendix S2. Species abundance loadings on the
first three components of principal component analyses.
Appendix S3. Variation in soil layer-level fine root
functional parameters along soil layer depth.

Journal of Vegetation Science

Doi: 10.1111/jvs.12405 2016 International Association for Vegetation Science