Plant, Cell and Environment (2009) 32, 1346–1356
doi: 10.1111/j.1365-3040.2009.02002.x
Bulk leaf d 18O and d 13C reflect the intensity of intraspecific
competition for water in a semi-arid tussock grassland
pce_2002
1346..1356
DAVID A. RAMÍREZ1, JOSÉ I. QUEREJETA2 & JUAN BELLOT3
1
Departamento de Ciencias Ambientales, Universidad de Castilla-La Mancha, Ap. 45071, Toledo, Spain, 2Departamento de
Conservación de Suelos y Aguas, Centro de Edafología y Biología Aplicada del Segura-Consejo Superior de Investigaciones
Científicas (CEBAS-CSIC), Campus Universitario de Espinardo E-30100, Murcia, Spain and 3Departamento de Ecología,
Universidad de Alicante, Ap. 99-E- 03080, Alicante, Spain
ABSTRACT
We investigated the extent to which plant water and nutrient status are affected by intraspecific competition intensity
and microsite quality in a monodominant tussock grassland.
Leaf gas exchange and stable isotope measurements were
used to assess the water relations of Stipa tenacissima tussocks growing along a gradient of plant cover and soil depth
in a semi-arid catchment of Southeast Spain. Stomatal conductance and photosynthetic rate decreased with increasing
intensity of competition during the wet growing season,
leading to foliar d 18O and d 13C enrichment. A high potential for runoff interception by upslope neighbours exerted
strong detrimental effects on the water and phosphorus
status of downslope S. tenacissima tussocks. Foliar d 15N
values became more enriched with increasing soil depth.
Multiple stepwise regression showed that competition
potential and/or rhizosphere soil depth accounted for large
proportions of variance in foliar d 13C, d 18O and d 15N among
target tussocks (57, 37 and 64%, respectively). The results
presented here highlight the key role that spatial redistribution of resources (water and nutrients) by runoff plays in
semi-arid ecosystems. It is concluded that combined measurement of d 13C, d 18O and nutrient concentrations in bulk
leaf tissue can provide insight into the intensity of competitive interactions occurring in natural plant communities.
Key-words: Stipa tenacissima; d 13C; d 15N; d 18O; drought
stress; plant competition; runoff redistribution.
INTRODUCTION
Arid and semi-arid steppes dominated by the perennial
tussock grass Stipa tenacissima L. (esparto or alpha grass)
constitute one of the major vegetation types in the drier
parts of the western Mediterranean region, where they
occupy around 2.8 million hectares (Le Houérou 2001).
S. tenacissima is a drought-adapted species that shows
intermittent, opportunistic growth when soil moisture is
adequate (Pugnaire et al. 1996). This species has a waterspender strategy and responds rapidly to rainfall pulses by
Correspondence: J. I. Querejeta. Fax: +34 968 39 62 13; e-mail:
querejeta@cebas.csic.es
1346
sharply increasing transpiration, photosynthesis and new
leaf production and growth (Pugnaire & Haase 1996; Pugnaire et al. 1996). However, S. tenacissima is highly responsive to water shortage: below a certain soil moisture
potential threshold, leaves fold and roll in, stomata become
concealed in deep grooves and gas exchange is sharply
restricted (Pugnaire et al. 1996; Haase et al. 1999; Ramírez
et al. 2008a). Further, the proportion of photosynthetically
active green leaf biomass in the canopy of S. tenacissima
tussocks is heavily dependent on plant water status and
decreases sharply in drought-stressed individuals due to
xanthophyll and chlorophyll loss and reversible leaf senescence (Haase et al. 1999; Balaguer et al. 2002).
Tussock spatial patterns play a key role in runoff generation and infiltration, nutrient cycling and overall
ecosystem functioning in S. tenacissima grasslands (Puigdefábregas et al. 1999; Maestre & Cortina 2006). Semi-arid
ecosystems often show a two-phase mosaic structure of
high and low plant-cover areas with distinctly different
attributes (Aguiar & Sala 1999). Bare and vegetated
patches behave as sources and sinks of runoff water,
sediments and nutrients, leading to significant resource
redistribution and high spatial connectivity in semi-arid
landscapes (Ludwig et al. 2005; Bautista et al. 2007).
Source-sink resource dynamics may be particularly important in S. tenacissima grasslands growing on slopes with
shallow soils of low water-storage capacity and fertility.
When growing on sloping terrain, S. tenacissima tussocks
are usually arranged in rows perpendicular to the slope to
intercept runoff fluxes (Puigdefábregas 2005). Interception
and deposition of sediments (organic debris and soil particles) in the upslope side of tussocks eventually leads to
the formation of small contour terraces (hereafter called
terracettes), which have higher infiltration rate, water
holding capacity, nutrient content and microbial activity
than the surrounding bare areas (Bochet, Poesen & Rubio
2000; Goberna et al. 2007). Cerdá (1997) suggested that
runoff from neighbouring bare areas may sustain the survival and growth of S. tenacissima tussocks in unfavourable sites where rainfall alone is insufficient.
The stable isotope composition of bulk leaf material can
provide much insight into the major abiotic or biotic factors
affecting plant water status in semi-arid ecosystems (e.g.
© 2009 Blackwell Publishing Ltd
Intraspecific competition for water in a semi-arid tussock grassland 1347
Leffler & Caldwell 2005; Querejeta et al. 2006, 2007, 2008).
The carbon isotope composition of plant tissues (d 13C) provides a useful index for assessing intrinsic water use efficiency, that is the ratio of photosynthetic carbon fixation
to stomatal conductance (Farquhar, Ehleringer & Hubick
1989a; Dawson et al. 2002). The oxygen isotope composition
of plant organic material (d 18O) reflects the isotope composition of soil water taken up by the plant, evaporative
and diffusional effects in transpiring leaves, and isotopic
exchange between oxygen atoms in organic molecules and
plant water (Barbour 2007). Recent studies have suggested
that leaf d 18O could serve as a time-integrated measure of
plant stomatal conductance when other sources of variation
(mainly source water d 18O) are minimized (Barbour & Farquhar 2000; Barbour et al. 2000; Barbour 2007; Farquhar,
Cernusak & Barnes 2007). Further, the simultaneous measurement of d 13C and d 18O in leaf material can help separate the independent effects of carbon fixation and stomatal
conductance on d 13C, because d 18O shares dependence on
stomatal conductance with d 13C, but is unaffected by photosynthetic rate (Scheidegger et al. 2000; Grams et al. 2007;
Sullivan & Welker 2007). The nitrogen isotope composition
of leaf material (d 15N) reflects the net effect of a wide range
of processes, including the isotopic signature of the soil N
sources used by the plant, mycorrhizal associations, temporal and spatial variation in N availability or changes in plant
demand (Högberg 1997; Dawson et al. 2002).
Monodominant S. tenacissima grasslands typically show a
regular spacing pattern in the distribution of tussocks
(Maestre et al. 2005), which suggests that intense intraspecific competition for soil resources (water and/or nutrients)
may occur in these systems (Ramírez et al. 2008c; Ramírez
& Bellot 2009). In the present study, we measured leaf gas
exchange, leaf isotopic composition and foliar nitrogen and
phosphorus concentration to assess the water and nutrient
status of tussocks growing along a natural gradient of soil
depth and S. tenacissima cover in a semi-arid catchment in
Southeast Spain. Our primary goal was to evaluate whether
bulk leaf d 13C and d 18O can provide a proxy measure of the
intensity of interference/competition for water experienced
by individual tussocks in monodominant S. tenacissima
grasslands (Ehleringer 1993). We predicted that stomatal
conductance and photosynthetic rate in S. tenacissima tussocks would decrease with increasing potential for runoff
interception (PRI) by upslope conspecific neighbours
and/or increasing density of neighbours, and that this would
result in enhanced water-use efficiency and more enriched
(positive) bulk leaf d 13C and d 18O values.
MATERIALS AND METHODS
Study area
The experimental area was located in a sub-catchment
(19 ha) of ‘El Ventós’ range watershed (38°28′N, 0°37′W)
near the town of Agost (Alicante province) in Southeast
Spain. Elevation at the study sites ranges from 400 to 700 m
above sea level, and slope ranges from 37 to 70%, with
predominantly SE and SW aspects. Soils are lithosols and
calcareous regosols (FAO-UNESCO 1988) of silt loam
texture. The experimental area has a semi-arid Mediterranean climate, with hot dry summers and high spatiotemporal variability of precipitation. The mean annual
rainfall is 296.6 mm, and the average annual temperature is
17.4 °C, with average monthly temperatures ranging from
11.7 °C (January) to 26.3 °C (August). Mean vegetation
cover in the catchment is 42.9 ⫾ 2.1%. Alpha grass (Stipa
tenacissima L.) is by far the dominant species, accounting
for 53% of the total plant cover. Other abundant grass
and shrub species in the experimental area are Brachypodium retusum Pers., Globularia alypum L. and Quercus
coccifera L.
Experimental design
Based on the results of previous studies (Ramírez et al.
2008c; Ramírez & Bellot 2009), we selected three distinct
sectors within the experimental catchment that span a
natural gradient of soil depth and S. tenacissima cover
(Table 1). The three selected catchment sectors are 167–
470 m apart from each other. In each of the three catchment
sectors, ten tussocks of S. tenacissima were randomly
selected for sampling (10 tussocks per sector ¥ 3 catchment
sectors, n = 30). Following the criteria detailed by Ramírez
et al. (2007a,b), only sexually mature plants (tussock external diameter > 1.2 m) were included in the study. Within
each catchment sector, sampled tussocks were at least 2 m
apart from each other.
Microsite characteristics
Mean soil depth within the rhizosphere of each target S.
tenacissima tussock (a circular area of 0.5 m radius around
the plant) was measured by driving a steel stake (0.5 m
long) into the soil. We considered eight 0.5-m-long axes (N,
NW, NE, E, SE, S, SW and W) originating at the centre of
each tussock, and measured soil depth at 0.1 m intervals
along them (40 points per tussock). Rock outcrop cover in
the rhizosphere of each tussock (%) was estimated as the
number of sampling points located on rock outcrops (soil
depth = 0) divided by the total number of sampling points
(40).The spatial pattern of soil depth within the rhizosphere
of target tussocks was evaluated using the ‘aggregation
index’ (Ia) of the Spatial Analysis by Distance Index
method (SADIE; Perry 1995).We performed 26 678 permutations using the SADIEShell Software 1.22. Soil depth
values were ranked into six classes: (1) 0 cm; (2) 0–5 cm; (3)
5–10 cm; (4) 10–15 cm; (5) 15–20 cm; and (6) >20 cm. The
spatial pattern of soil depth in the rhizosphere of target
tussocks was considered clumped, random or regular when
Ia values were >1, ~1 or <1, respectively.
Plant morphological parameters
We measured the mean external diameter of the target
tussocks, as this parameter has been used as an indicator of
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
1348 D. A. Ramírez et al.
Altitude (m a.s.l.)
Alpha grass cover (%)
Soil depth (m)
Rock outcrop cover (%)
PRI (%)
HIC (m-1)
Ia (unitless)
LAIgreen (m2 m-2)
d 13C (‰)
d 15N (‰)
d 18O (‰)
N (%)
P (%)
Sector I
Sector II
Sector III
415
41.4
0.17 ⫾ 0.04
2.61 ⫾ 0.05
0.63 ⫾ 0.006b
4.54 ⫾ 0.03c
1.43 ⫾ 0.004a
0.79 ⫾ 0.002a
-24.09 ⫾ 0.18b
-2.49 ⫾ 0.20c
30.56 ⫾ 0.37b
0.66 ⫾ 0.01a
0.056 ⫾ 5.10-4a
640
8.9
0.14 ⫾ 0.04
0.82 ⫾ 0.02
0.13 ⫾ 0.001a
0.27 ⫾ 0.004a
1.84 ⫾ 0.006ab
1.30 ⫾ 0.006b
-24.96 ⫾ 0.25a
-3.37 ⫾ 0.23b
29.28 ⫾ 0.26a
0.71 ⫾ 0.01b
0.062 ⫾ 7.10-4b
675
18.1
0.08 ⫾ 0.06
29.6 ⫾ 0.2
0.54 ⫾ 0.004b
1.22 ⫾ 0.009b
2.23 ⫾ 0.004b
0.66 ⫾ 0.003a
-23.40 ⫾ 0.27c
-4.46 ⫾ 0.11a
30.58 ⫾ 0.43b
0.68 ⫾ 0.01a
0.061 ⫾ 7.10-4b
Table 1. Site and S. tenacissima stand
characteristics, green leaf area index of
tussocks (LAIgreen) and leaf isotopic
composition and foliar nitrogen and
phosphorus concentration in three distinct
sectors of the experimental catchment
Mean values ⫾ 1 standard error are shown (n = 30; 10 tussocks per sector). Different letters
indicate significant differences among sectors according to the U-Mann–Whitney test
(P < 0.05).
HCI, Hegyi’s competition index; Ia, soil patchiness in the rhizosphere; PRI, potential for
runoff interception by upslope conspecific neighbours.
tussock age and biomass in S. tenacissima (Ramírez et al.
2007a, 2008a). We also calculated the green leaf area of 30
tillers (one tiller sampled per target tussock) using a calibration function developed by Ramírez et al. (2006):
y = 0.982 x + 0.063
(1)
(n = 100, r2 = 0.94, P < 0.05), where x = leaf area, calculated
as the product of leaf length multiplied by leaf width;
y = scanned leaf area. In order to estimate leaf-specific
weight (g m-2), the 30 sampled tillers were dried and
weighed. We fitted the following allometric equation:
y = 20.42 10 −4 x − 6.25 10 −4
(2)
(n = 30, r2 = 0.98, P < 0.05) where: x = green leaf biomass (g)
and y = green leaf area (m2). The green leaf area index
(LAIgreen) of each target tussock was calculated using a
destructive method. In June 2007, a metal parallelepiped
frame (0.01 m2 and 0.3 m height) was randomly inserted
into the tussock canopy (five times per tussock, n = 5), and
all the green leaves within the frame were sampled, dried
(60 °C, 72 h) and weighed. Equation 2 was used to calculate
the green leaf area within the frame, and LAIgreen (m2 m-2)
was estimated by dividing the green leaf area by the parallelepiped area (0.01 m2).
Leaf nutrient concentrations and
isotopic composition
Leaf sampling was conducted at the end of the leaf growth
season in late spring (13 June 2007). For each target tussock,
we chose three stems located at the growing front in the
downslope side of the tussock, and sampled three to four
basal segments of young and mature leaves per stem (following the leaf age classification of Haase et al. 1999). Leaf
samples were dried (80 °C, 48 h) and ground using a ball
mill. The finely ground leaf material was subjected to
digestion with HNO3 : HClO4 (2:1, v : v) before measurement of foliar phosphorus concentration by atomic absorption spectrometry (Perkin Elmer ICP 5500, Norwalk, CT,
USA). Foliar nitrogen concentration was determined with
an automated 1500 Carlo Erba elemental analyser.
All stable isotope analyses for this study were conducted
at the Stable Isotope Facility of the University of
California-Davis. Leaf d 13C and d 15N were measured on a
continuous-flow isotope ratio mass spectrometry (Europa
Scientific Hydra 20/20, Cheshire, UK), interfaced with a
C/N elemental analyser. The standards were Pee Dee
Belemnite for d 13C and atmospheric N2 for d 15N. Leaf material was analysed for oxygen isotope composition using a
Heckatech HT Oxygen Analyser interfaced to a PDZ
Europa 20–20 isotope ratio mass spectrometer (Sercon
Ltd., Cheshire, UK), following the method described in
Kornexl et al. (1999). Leaf samples were converted by
pyrolysis in a glassy carbon reactor at 1400 °C to CO and
H2O, and oxygen was analysed as CO. The oxygen isotope
signature is expressed in d 18O, relative to the internationally
accepted standard (Vienna Standard Mean Oceanic Water,
VSMOW). The working standard for d 18O analysis of bulk
leaf tissue was microcrystalline cellulose at 30.5‰ VSMOW.
Three replicate samples of bulk leaf tissue per target
tussock were analysed for d 13C, d 15N and d 18O (30 target
tussocks ¥ 3 replicates per tussock, n = 90 samples for each
isotope). Average precision of the isotopic analyses was
⫾0.27‰ for d 13C and d 18O, and ⫾0.23‰ for d 15N (see also
error bars in Fig. 4).
Intraspecific competition
Ephemeral stream channels provide the only hydrological
connection among distinct catchment sectors in El Ventós
range watershed. All target tussocks were far removed from
any ephemeral stream channels, so runoff inputs from other
catchment sectors, or competition for runoff water and
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
Intraspecific competition for water in a semi-arid tussock grassland 1349
nutrients among tussocks in different sectors, can be ruled
out. Within each catchment sector, the PRI by upslope conspecific neighbours was estimated for each target tussock, as
the proportion of its external diameter that was overlapped
by the projection of the external diameter of upslope tussocks located within a distance of 2 m (in %). Belowground
competition intensity was calculated for each target tussock
using a modification of Hegyi’s index (Daniels 1976). This
index considered the number, size and proximity of conspecific neighbours within a circular zone of influence (2 m
radius) around each target tussock. The Hegyi’s competition index (HCI) was calculated using the following
equation:
N
( Di2 Dj−2 )
i =1
DISTij
HCI i = ∑
maximum daily values of PPFD. Micrometeorological
conditions in the LI-COR chamber during gas exchange
measurements were fixed (air flow rate = 500 mmol s-1,
PPFD = 1800 mmol m-2 s-1, relative humidity = 30%, reference CO2 concentration = 370 ppm, leaf temperature =
30 °C). Fixed values of relative humidity, temperature and
PPFD were chosen according to mean values for the month
of June in the study area (‘Ventós 2’ meteorological station:
38°27′N, 0°37′W). Intrinsic water use efficiency (WUEintrinsic)
was calculated as the ratio of photosynthetic rate to stomatal
conductance (A/g) for each target tussock.
Statistical analyses
(3)
where HCIi is the competition index for target tussocks
(m-1), D is tussock external ⭋ (m), DISTij is the distance
between target tussock i and competitor j (m), and N is the
number of conspecific competitors.
Leaf gas exchange measurements
Preliminary leaf gas exchange measurements were conducted on four representative S. tenacissima tussocks in
each of the three catchment sectors described in the experimental design section (4 tussocks per sector ¥ 3 catchment
sectors, n = 12). Measurements were conducted during both
the wet growing season (21–23 January 2004) and the
peak dry season (10–12 August 2004). January 2004 was
preceded by a relatively rainy autumn (145.3 mm rainfall
during October–December 2003), and volumetric mean soil
water content in the experimental area was 21.0 ⫾ 0.8% at
the time. Soil water content during the peak dry season was
much lower (5.8 ⫾ 0.3%). Measurements were conducted
on S. tenacissima tussocks with an external diameter >1.2 m,
and followed the procedures described in detail in Ramírez
& Bellot (2009). Relative humidity of the air (%) was
similar in all the sectors of the experimental catchment
when this parameter was measured in January 2004
(55.9 ⫾ 0.1 in sector I; 55.7 ⫾ 0.1 in sector II; 57.0 ⫾ 0.1 in
sector III). Photosynthetic photon flux density (PPFD) was
also very similar in all the catchment sectors at time of gas
exchange measurements (for further details, see Ramírez
et al. 2008c).
Immediately before leaf sampling for isotopic analyses,
leaf gas exchange measurements were conducted (on 11–12
June 2007) on the exact same 30 target tussocks, using a
portable, open photosynthesis system (LI-6400, LI-COR
Biosciences, Lincoln, NE, USA). For each tussock, measurements were conducted on six bundles of leaves (three to four
leaf sections per bundle), four located in the tussock periphery (N,S,E andW aspect),and two more located in the centre
of the tussock. Each leaf bundle was placed into the LI-COR
chamber, and photosynthetic rate and stomatal conductance
were measured between 11:00 and 13:00. This time of the
day is characterized by low variability of PPFD, and
Correlation (using the Spearman index) and regression
(linear and non-linear) analyses were performed to evaluate the relationships among measured variables. Variables
were year-1 transformed when required to achieve homogeneity of variances and normality of the residuals. Forward
multiple stepwise regression was used to determine the best
combination of predictor variables for bulk leaf d 13C, d 18O
and d 15N. We used the SPSS software for Windows 14.0
(SPSS, Chicago, IL, USA) for all statistical analyses.
RESULTS
Microsite quality
Across sectors, soil depth in the rhizosphere of target S.
tenacissima tussocks ranged from 2 to 23 cm, whereas rock
outcrop cover ranged from 0 to 60%. Mean soil depth was
greatest in sector I, whereas sector III had the thinnest soil
(Table 1). Mean rock outcrop cover was much greater in
sector III than in the other sectors.Across sectors, soil depth
and rock outcrop cover were strongly negatively correlated
with each other (rSpearman = -0.82, P < 0.05). Spatial aggregation of soil depth values in the rhizosphere of target tussocks (soil patchiness: Ia, dimensionless) ranged from 0.8
to 3.2. Microsites with thin soil over bedrock in sector
III showed the highest Ia values, due to the clumped spatial
distribution of rock outcrops and soil pockets in such
locations.
Intraspecific competition/interference
There was a large variability in the potential for intraspecific competition experienced by target S. tenacissima tussocks in the experimental catchment. Across sectors, HCI
values for target tussocks ranged between 0 and 24.8 m-1,
whereas PRI by upslope neighbours ranged between 0 and
90%. HCI and PRI were positively correlated with each
other (rSpearman = 0.67, P < 0.05), as both parameters were
influenced by conspecific neighbour size and distance from
the target plant. Sector I (high S. tenacissima cover) showed
the greatest values of HCI and PRI, whereas sector II (low
S. tenacissima cover) showed the lowest values for both
parameters (Table 1).
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
1350 D. A. Ramírez et al.
Table 2. Results of the multiple stepwise regression analyses for leaf d 13C, d 18O, d 15N and LAIgreen in target Stipa tenacissima tussocks
(n = 30)
Response
variable
Selected
predictor variables
d 13C
Soil depth (x1)
PRI (x2)
PRI (x1)
HCI (x2)
Soil depth (x1)
Ia (x2)
HCI (x1)
PRI (x2)
d 18O
d 15N
1/LAIgreen
r2
F(2,27) anova
bx1
bx2
Equation
0.57
16.32**
-0.59**
-0.32*
y = -23.43-9.25x1 + 1.30x2
0.37
5.86**
0.48*
-0.32 n.s.
y = 29.19 + 2.02x1 + 0.03x2
0.64
22.98**
0.48**
-0.46**
y = -2.94 + 7.57x1 - 0.78x2
0.66
24.2**
0.50**
0.48**
y = 0.77 + 0.05x1 + 0.99x2
The predictor variables considered in the analyses were: soil depth in the tussock rhizosphere, potential for runoff interception by upslope
conspecific neighbours (PRI, in %), soil patchiness in the rhizosphere (Ia, unitless) and Hegyi’s competition index (HCI, m-1). The significance
of the F test and the standardized coefficients for each predictor variable (b) are shown (**significant at P < 0.01, *significant at P < 0.05).
anova, analysis of variance.
Tussock size and LAI green
The external diameter of target S. tenacissima tussocks
ranged between 1.2 and 2.4 m. Tussock external diameter
was positively correlated with soil depth (rSpearman = 0.45,
P < 0.05). Across sectors, the LAIgreen of target tussocks
ranged between 0.14 and 2.95 m2 m-2, indicating large interplant variability in the proportion of green versus dead or
senescent leaves. Mean LAIgreen was highest in sector II and
lowest in sector III (Table 1). LAIgreen was positively correlated with soil depth (rSpearman = 0.42, P < 0.05) and negatively
correlated with rock outcrop cover (rSpearman = -0.52,
P < 0.05). LAIgreen was also negatively related to both HCI
(rSpearman = -0.48, P < 0.05) and PRI by upslope neighbours
(rSpearman = -0.56, P < 0.05). Forward stepwise multiple
regression showed that HCI and PRI together accounted for
66% of the variability in LAIgreen across sectors (Table 2).
Plant nutrient status
Tussocks in sector II showed the highest mean foliar concentrations of nitrogen and phosphorus, whereas tussocks
in sector I showed the lowest (Table 1). Across sectors,
foliar nitrogen concentration in target tussocks ranged
between 0.49 and 0.87%, and tended to decrease as HCI
increased (rSpearman = -0.37, P < 0.05). Foliar phosphorus
concentration ranged between 0.051 and 0.071%, and
tended to decrease as the PRI by upslope neighbours
increased (Fig. 1). Foliar phosphorus concentration was
negatively related to HCI (rSpearman = -0.51, P < 0.05) as well.
during the wet growing season increased with decreasing
stomatal conductance (Fig. 3b), and was greatest in the
sector with the densest cover of S. tenacissima tussocks
(sector I; Fig. 2).
Stomatal conductance and photosynthetic rate in S. tenacissima decreased markedly during the peak dry season
(August 2004), but the decline was much sharper in areas
with thinner soil (94% decrease in stomatal conductance
with respect to wet season levels in sector III) than in areas
with deeper soil (sector I, 66% decrease in stomatal conductance) (Fig. 2). Intrinsic water-use efficiency increased
in sector II but remained unchanged in sector I during the
peak dry season. Interestingly, intrinsic water-use efficiency
actually decreased during the peak dry season in sector III,
indicating photosynthetic impairment due to severe plant
water stress at this location (see Ramírez & Bellot 2009 for
further details).
Leaf gas exchange measurements conducted on the exact
same target tussocks as leaf isotopic measurements in
y = 0.06 – 0.008x
r2 = 0.3**
Leaf gas exchange measurements
Preliminary measurements conducted during the wet
growing season in January 2004 showed that mean stomatal
conductance was highest in the area with the sparsest cover
of S. tenacissima (sector II), and lowest in the area with the
densest cover (sector I; Fig. 2). Mean photosynthetic rate
was also lower in sector I than in the other sectors during
the wet growing season. Intrinsic water use efficiency (A/g)
Figure 1. Foliar phosphorus concentration in target Stipa
tenacissima tussocks decreased with increasing potential for
runoff interception by upslope conspecific neighbours
(**significant at P < 0.01). Samples collected in June 2007
(n = 30).
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
Intraspecific competition for water in a semi-arid tussock grassland 1351
water-use efficiency and water stress among target tussocks.
Mean bulk leaf d 13C was highest in sector III and lowest in
sector II (Table 1). Leaf d 13C was strongly positively correlated with PRI, and was negatively correlated with soil
depth and LAIgreen (Fig. 4b–d). Forward stepwise multiple
regression revealed that soil depth in the rhizosphere and
PRI by upslope neighbours were the strongest predictors of
foliar d 13C, together accounting for 57% of the variance
across sectors (Table 2).
Bulk leaf d 18O ranged between 27.6 and 33.5‰ across
sectors, suggesting large inter-tussock differences in timeintegrated stomatal conductance. Mean bulk leaf d 18O was
significantly less enriched in sector II than in the other
sectors (Table 1). Foliar d 18O was positively correlated with
foliar d 13C and PRI (Fig. 4a,f), and was negatively related to
LAIgreen (Fig. 4e). Stepwise multiple regression showed that
PRI by upslope neighbours and HCI were the best predictors of bulk leaf d 18O, as they explained 37% of the variance
across sectors (Table 2).
Foliar d 15N ranged between -5.2 and -1.48‰, and was
strongly positively correlated with soil depth in the
y = – 0.004x + 0.14
r2 = 0.08 n.s.
Figure 2. Leaf gas exchange parameters in target Stipa
tenacissima tussocks at three different sampling periods (A = net
photosynthesis, g = stomatal conductance, A/g = intrinsic water
use efficiency) For each sampling period, columns with different
letters indicate significant differences among sectors according to
the U-Mann–Whitney test (P < 0.05). n = 12 in January and
August 2004; n = 30 in June 2007.
June 2007 showed that stomatal conductance and photosynthetic rate in S. tenacissima were higher in sector I than in
the other sectors during the early dry season (Fig. 2). Both
photosynthetic rate and stomatal conductance were
strongly positively correlated with soil depth at this time
(rSpearman = 0.56 and 0.58, respectively: P < 0.05). Intrinsic
water-use efficiency was lower in sector I than in the other
sectors during the early dry season.
Plant isotopic composition
Across sectors, bulk leaf d 13C ranged between -26.6
and -21.9‰, thus indicating large differences in intrinsic
y = – 0.49x + 0.16
r2 = 0.68**
Figure 3. (a) Intrinsic water use efficiency (WUEintrinsic) in
target Stipa tenacissima tussocks was not significantly affected by
changes in photosynthetic rate (A). (b) However, WUEintrinsic
increased sharply with decreasing stomatal conductance (g).
Measurements conducted during the wet growing season in
January 2004 (n = 12). **significant at P < 0.01; n.s., not significant
at P > 0.05.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
1352 D. A. Ramírez et al.
y = –0.04 – 0.004x + 0.001x2
r2 = 0.42**
y = –42.61 + 0.86x – 0.008x2
r2 = 0.29**
y = –11.42x – 22.82
r2 = 0.47**
y = 2.46 x – 25.09
r2 = 0.45**
y = 29.8x–0.05
r2 = 0.44**
y = 29.20 + 2.28x
r2 = 0.30**
y = 11.17 – 4.80x
r2 = 0.48**
Figure 4. Main relationships among measured variables in target Stipa tenacissima tussocks in June 2007 (n = 30). (a) Leaf d13C versus
leaf d18O, (b) leaf d13C versus green leaf area index of tussocks (LAIgreen), (c) leaf d13C versus soil depth in the rhizosphere of tussocks,
(d) leaf d13C versus potential for runoff interception by upslope conspecific neighbours (PRI), (e) leaf d18O versus LAIgreen, (f) leaf d18O
versus PRI, (g) leaf d15N versus soil depth in the rhizosphere of tussocks. The fitted function shown in (b) was obtained after inverse
(year-1) transformation of green leaf area index (LAIgreen, m2 m-2) and d 13C data to achieve homogeneity of variances and normality of
the residuals year-1 (**significant at P < 0.01).
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
Intraspecific competition for water in a semi-arid tussock grassland 1353
rhizosphere of target tussocks (Fig. 4g). Mean bulk leaf
d 15N was highest in sector I and lowest in sector III
(Table 1). Leaf d 15N was negatively correlated with soil
patchiness – Ia (rSpearman = -0.61, P < 0.05) and rock outcrop
cover (rSpearman = -0.67, P < 0.05). Forward stepwise multiple
regression indicated that the best predictors of bulk leaf
d 15N were soil depth and Ia, which together explained 64%
of the variance across sectors (Table 2).
DISCUSSION
Interpretation of bulk leaf d 18O and d 13C data
An increase in bulk leaf d 18O would be consistent with
reduced stomatal conductance in S. tenacissima tussocks
(Barbour 2007; Farquhar et al. 2007). However, potential
differences among catchment sectors in the d 18O of soil
water available to plants could have also influenced the d 18O
of leaf tissue (Barbour 2007). Differences among catchment
sectors in soil depth or soil evaporation rate may lead to
differences in the isotopic composition of source water available to plants in the different sectors. Evaporative enrichment of surface soil water can cause the formation of
gradients of soil water d 18O with depth (Leffler & Caldwell
2005; Barbour 2007), so that tussocks growing on deeper soil
might have access to isotopically depleted water that is not
available to tussocks growing on shallower soil. Therefore,
the evidence provided by bulk leaf d 18O data alone is not by
itself unambiguous and needs other supporting data.
We found good agreement between stomatal conductance data collected during the wet growing season (when
leaf growth occurs) and bulk leaf d 18O data across catchment sectors. Mean bulk leaf d 18O (Table 1) was least
enriched in sector II (highest mean stomatal conductance
in January 2004, Fig. 2). Tussocks in sector I (lowest mean
stomatal conductance) and sector III (intermediate mean
stomatal conductance) showed significantly more enriched
mean bulk leaf d 18O values (Table 1). Interestingly,
bulk leaf d 18O in S. tenacissima was not correlated
with mean soil depth in the rhizosphere of target tussocks
(rSpearman = 0.03, P = 0.86), which suggests that differences in
d 18O among tussocks may not have been exclusively (or
even primarily) caused by differences in source water
d 18O. Mean bulk leaf d 18O was the same in catchment
sectors I (mean soil depth 0.17 m) and III (mean soil depth
0.08 m), even though mean soil depth was more than twice
greater in the former than in the latter sector (Table 1). By
contrast, tussocks in catchment sector II (mean soil depth
0.14 m) had significantly less enriched bulk leaf d 18O.
Taken together, these results suggest that variability in
bulk leaf d 18O across the experimental catchment primarily reflects differences in stomatal conductance (Farquhar
et al. 2007), rather than (or in addition to) differences in
source water d 18O.
Foliar d 18O and d 13C were strongly positively correlated
with each other across catchment sectors (Fig. 4a), suggesting that water-use efficiency increased with decreasing
stomatal conductance (Barbour, Walcroft & Farquhar
2002; Keitel et al. 2003). Leaf gas exchange measurements
conducted during the wet growing season in 2004 also
showed that intrinsic water-use efficiency in S. tenacissima
is tightly controlled by stomatal regulation of transpiration
(Fig. 3b; see also Ramírez et al. 2008c). Enriched foliar
d 13C was therefore interpreted as evidence of enhanced
water stress and stomatal limitation to photosynthesis,
according to the conceptual models developed by Farquhar et al. (1989b), Sternberg, Mulkey & Wright (1989),
Scheidegger et al. (2000) and Grams et al. (2007). In agreement with leaf gas exchange measurements conducted
during the wet growing season (January 2004; Fig. 2), tussocks in sector II showed less enriched leaf d 13C (and,
therefore, lower water use efficiency) than those in the
other sectors, due to milder water stress and less severe
stomatal constraints on photosynthesis.
Photosynthetic rate, water use efficiency and d 13C
usually increase in response to improved plant nutrient
status (Farquhar et al. 1989b; Dawson et al. 2002), which
can sometimes confound the effects of soil water availability on d 13C (e.g. Maestre & Cortina 2006). Analysis of
foliar nutrient concentration can therefore aid the interpretation of bulk leaf d 13C data. Interestingly, we found no
significant correlation between leaf d 13C and foliar N or P
concentration across sectors. Stomatal response to soil
moisture availability (rather than plant nutrient status)
appears to be the key mechanism controlling the leaf d 13C
and water-use efficiency of S. tenacissima tussocks in this
semi-arid ecosystem.
Leaf gas exchange measurements taken during the early
dry season in June 2007 did not show the anticipated variation among tussocks across the competition gradient.
Further, neither bulk leaf d 18O nor d 13C were correlated
with stomatal conductance, photosynthetic rate or intrinsic
water-use efficiency when both gas exchange and leaf isotopic composition were measured in June 2007. This suggests that the stable isotope composition of bulk foliar
tissue in S. tenacissima may be largely determined by plant
water status and physiological activity during the wet
growing season, when leaf structural carbohydrates are
formed. New leaf production and growth in S. tenacissima
are primarily controlled by soil moisture availability, and
occurs exclusively during the wetter months of the year
(October–May; Pugnaire & Haase 1996; Pugnaire et al.
1996). Plant water status and physiological activity during
the dry season appear to have comparatively little influence
on bulk leaf d 18O and d 13C in S. tenacissima.
LAIgreen was strongly negatively correlated with foliar
d 18O and d 13C across sectors (Fig. 4b,e), indicating that the
proportion of green leaf biomass in the canopy of tussocks
tended to decrease with decreasing stomatal conductance
and increasing plant water stress. When the soil dries out,
stomatal conductance declines rapidly, carbon assimilation
rate falls near or below the photosynthetic compensation
point, leaf growth stops, leaf pigment concentrations
decrease and reversible leaf senescence sets in (Haase et al.
1999; Balaguer et al. 2002), all of which tend to reduce the
LAIgreen of S. tenacissima tussocks.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
1354 D. A. Ramírez et al.
Effects of intraspecific competition on plant
water and nutrient status
In agreement with leaf gas exchange measurements conducted during the wet growing season (January 2004), tussocks in sectors I (high competition intensity, low stomatal
conductance) and III (intermediate competition intensity,
intermediate stomatal conductance) showed more enriched
leaf d 18O than those in sector II (low competition intensity,
high stomatal conductance). Plant water stress in S. tenacissima appeared to increase sharply with increasing PRI by
upslope neighbours (Fig. 4d,e). Stepwise regression showed
that PRI was the best predictor of leaf d 18O across sectors
(Table 2), indicating that time-integrated transpiration in
target tussocks decreased with increasing interference by
upslope neighbours. Runoff interception by neighbours
also exerted a strong detrimental effect on the phosphorus
status of downslope tussocks in this P-deficient ecosystem
(foliar N : P ratios = 11.1–11.8; Fig. 1). Overall, the data
indicate that ‘trapping’ of runoff water, sediment and
nutrient fluxes by upslope neighbours is an important
mechanism of conspecific interference in monodominant
S. tenacissima grasslands. Our results are in agreement
with – and extend those of – Puigdefábregas et al. (1999),
who found that experimental exclusion of runoff inputs
using artificial barriers caused a significant reduction in the
growth of S. tenacissima tussocks.
Across sectors, the LAIgreen of tussocks decreased linearly with HCI, indicating that belowground competition
from conspecific neighbours affected S. tenacissima performance negatively (Fowler 1986; Casper & Jackson 1997;
Casper, Schenk & Jackson 2003). Foliar N and P concentrations were also negatively correlated with HCI across
sectors, which strongly suggests that the root systems of
adjacent tussocks may overlap and compete for soil
resources in bare interspaces (Ramírez, Domingo & Bellot
2008b; Ramírez & Bellot 2009). Tussocks in sector II
(lowest competition intensity) showed the highest foliar N
and P concentrations, whereas those in sector I (highest
competition intensity) showed the lowest values. Interestingly, foliar N concentration in S. tenacissima tussocks
was unaffected by PRI by upslope neighbours, but was
strongly influenced by direct belowground competition
from adjacent neighbours (HCI).
Effects of microsite quality on plant water and
nutrient status
Bulk leaf d 13C was strongly negatively correlated with soil
depth, suggesting that greater moisture storage capacity in
microsites with deeper soil allowed for improved plant
water status in this semi-arid catchment (Ehleringer &
Cooper 1988). Although alpha grass is considered a
shallow-rooted species (rooting depth <0.5 m; Puigdefábregas & Sánchez 1996), d 13C data suggest that tussocks
growing in shallow soil pockets over bedrock (sector III)
experienced more severe water stress than those growing
on deeper soil (sectors I and II). Both root lateral spread
and rooting depth are severely constrained in tussocks
growing in thin soil pockets over bedrock in sector III
(Ramírez et al. 2008b). Ramírez & Bellot (2009) found that
S. tenacissima individuals in rock outcrop areas showed
intense photoinhibition during prolonged rainless periods,
which is an indication of severe water stress in this species
(Pugnaire et al. 1996).
In contrast to leaf d 13C, bulk leaf d 18O was not negatively
correlated with soil depth. This puzzling result can be
largely explained by the highly enriched d 18O values of
tussocks in catchment sector I (deepest mean soil depth of
all sectors, see Table 1). Intense intraspecific competition
for water appeared to overwhelm any positive effects of
greater soil depth on leaf d 18O in this catchment sector.
When tussocks in sector I were excluded from the statistical
analysis, bulk leaf d 18O was actually found to be negatively
correlated with soil depth in this semi-arid ecosystem
(rSpearman = -0.53, P < 0.05, data not shown).
Across sectors, bulk leaf d 15N increased with soil depth
in the rhizosphere of tussocks, which suggests that plants
growing on thicker soil may have had access to isotopically enriched sources of nitrogen at depth that were
unavailable to those growing on shallow soil patches
(Nadelhoffer et al. 1996; Högberg 1997). A consistent,
widespread pattern of increasing abundance of the heavy
isotope of N (15N) with depth has been observed in the
soil profile of many forest and rangeland ecosystems
(Nadelhoffer & Fry 1988 and references therein). We
found that tussocks in sector III (shallowest soil) had the
most depleted mean foliar d 15N, whereas those in sector I
(deepest soil) had the most enriched mean foliar d 15N
values (Table 1). Heavy reliance on N from contour terracettes may have contributed to the depleted foliar d 15N
of tussocks growing on thin soil over bedrock in sector III.
The sediment and organic debris that accumulate in
contour terracettes are expected to be considerably less
enriched in 15N than ‘regular’ mineral soil, as they originate from laminar erosion of upslope topsoil layers
(including the litter layer) which are depleted in 15N relative to deeper soil horizons (Nadelhoffer & Fry 1988).
Greater isotopic fractionation as a consequence of slower
growth and lower N demand may have also contributed to
low d 15N values in tussocks growing in poor-quality microsites in sector III (McKee et al. 2002).
In conclusion, the combined measurement of leaf gas
exchange and bulk leaf d 13C and d 18O yielded insight into
the major biotic and abiotic factors controlling plant water
status in a semi-arid grassland. To our knowledge, this is
the first study reporting a significant response of bulk leaf
d 18O to the intensity of competition for water experienced
by individual plants in a natural community. In agreement
with previous studies, we found that a high PRI by
upslope conspecifics tended to exacerbate water and nutrient stress in downslope S. tenacissima tussocks. The results
presented here highlight the key roles that resource
(water and nutrients) redistribution by runoff and
intraspecific competition play in the functioning of semiarid plant communities.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
Intraspecific competition for water in a semi-arid tussock grassland 1355
ACKNOWLEDGMENTS
This study was supported by the Spanish Comisión Interministerial de Ciencia y Tecnología (Grant number
CGL2004-03627). J.I.Q. acknowledges financial support
from the ‘Ramón y Cajal’ Program of the Spanish Ministerio de Educación y Ciencia and the European Social Fund.
We thank José Abad for his assistance with field work. The
authors are grateful to Prof. G.D. Farquhar, V. Resco and
two anonymous reviewers for helpful comments on earlier
versions of this paper.
REFERENCES
Aguiar M.R. & Sala O.E. (1999) Patch structure, dynamics and
implications for the functioning of arid ecosystems. Trends in
Ecology & Evolution 14, 273–277.
Balaguer L., Pugnaire F.I., Martínez-Ferri E., Armas C., Valladares
F. & Manrique E. (2002) Ecophysiological significance of
chlorophyll loss and reduced photochemical efficiency under
extreme aridity in Stipa tenacissima L. Plant and Soil 240, 343–
352.
Barbour M.M. (2007) Stable oxygen isotope composition of plant
tissue: a review. Functional Plant Biology 34, 83–94.
Barbour M.M. & Farquhar G.D. (2000) Relative humidity- and
ABA-induced variation in carbon and oxygen isotope ratios of
cotton leaves. Plant, Cell & Environment 23, 473–485.
Barbour M.M., Fischer R.A., Sayre K.D. & Farquhar G.D. (2000)
Oxygen isotope ratio of leaf and grain material correlates with
stomatal conductance and grain yield in irrigated wheat. Australian Journal of Plant Physiology 27, 625–637.
Barbour M.M., Walcroft A.S. & Farquhar G.D. (2002) Seasonal
variation in d 13C and d 18O of cellulose from growth rings of
Pinus radiata. Plant, Cell & Environment 25, 1483–1499.
Bautista S., Mayor A.G., Bourakhouadar J. & Bellot J. (2007) Plant
spatial pattern predicts hillslope runoff and erosion in a semiarid
Mediterranean landscape. Ecosystems 10, 987–998.
Bochet E., Poesen J. & Rubio J.L. (2000) Mound development as
an interaction of individual plants with soil, water erosion and
sedimentation processes on slopes. Earth Surface Processes and
Landforms 25, 847–867.
Casper B.B. & Jackson R.B. (1997) Plant competition underground. Annual Review Ecology and Systematics 28, 545–570.
Casper B.B., Schenk H.J. & Jackson R.B. (2003) Defining a plant’s
belowground zone of influence. Ecology 84, 2313–2321.
Cerdá A. (1997) The effect of patchy distribution of Stipa tenacissima L. on runoff and erosion. Journal of Arid Environments 36,
37–51.
Daniels R.F. (1976) Simple competition indices and their correlation with annual loblolly pine tree growth. Forest Science 22,
454–456.
Dawson T.E., Mambelli S., Plamboeck A.H., Templer P.H. & Tu
K.P. (2002) Stable isotopes in plant ecology. Annual Review of
Ecology and Systematics 33, 507–559.
Ehleringer J.R. (1993) Variation in leaf carbon isotope discrimination in Encelia farinosa: implications for growth, competition
and drought survival. Oecologia 95, 340–346.
Ehleringer J.R. & Cooper T.A. (1988) Correlations between
carbon isotope ratio and microhabitat in desert plants. Oecologia
76, 562–566.
FAO-UNESCO (1988) Soil Map of the World. Revised Legend.
World Soil Resources, Report 60, Rome, Italy.
Farquhar G.D., Ehleringer J.R. & Hubick K.T. (1989a) Carbon
isotope discrimination and photosynthesis. Annual Reviews in
Plant Physiology and Plant Molecular Biology 40, 503–537.
Farquhar G.D., Hubick K.T., Condon A.G. & Richards R.A.
(1989b) Carbon isotope fractionation and plant water use efficiency. In Stable Isotopes in Ecological Research (eds P.W.
Rundel, J.R. Ehleringer & K.A. Nagy) pp. 21–46. SpringerVerlag, New York, NY, USA.
Farquhar G.D., Cernusak L.A. & Barnes B. (2007) Heavy water
fractionation during transpiration. Plant Physiology 143, 11–
18.
Fowler N. (1986) The role of competition in plant communities in
arid and semiarid regions. Annual Review of Ecology and Systematics 17, 89–110.
Goberna M., Pascual J.A., García C. & Sánchez J. (2007) Do plant
clumps constitute microbial hotspots in semiarid Mediterranean
patchy landscapes? Soil Biology & Biochemistry 39, 1047–1054.
Grams T.E.E., Kozovitz A.R., Häberle K.-H., Matyssek R. &
Dawson T.E. (2007) Combining d 13C and d 18O analyses to
unravel competition, CO2 and O3 effects on the physiological
performance of different-aged trees. Plant, Cell & Environment
30, 1023–1034.
Haase P., Pugnaire F.I., Clark S.C. & Incoll L.D. (1999) Environmental control of canopy dynamics and photosynthetic rate in
the evergreen tussock grass Stipa tenacissima. Plant Ecology
145, 327–339.
Högberg P. (1997) 15N natural abundance in soil-plant systems. New
Phytologist 137, 179–203.
Keitel C., Adams M.A., Holst T., Matzarakis A., Mayer H., Rennenberg H. & Gessler A. (2003) Carbon and oxygen isotope composition of organic compounds in the phloem sap provides a shortterm measure for stomatal conductance of European beech
(Fagus sylvatica L.). Plant, Cell & Environment 26, 1157–1168.
Kornexl B.E., Gehre M., Höfling R. & Werner R.A. (1999) On line
d 18O measurement of organic and inorganic substances. Rapid
Communications in Mass Spectrometry 13, 1685–1693.
Le Houérou H.N. (2001) Biogeography of the arid steppeland
north of the Sahara. Journal of Arid Environments 48, 103–128.
Leffler A.J. & Caldwell M.M. (2005) Shifts in depth of water extraction and photosynthetic capacity inferred from stable isotope
proxies across an ecotone of Juniperus osteosperma (Utah
juniper) and Artemisia tridentata (big sagebrush). Journal of
Ecology 93, 783–793.
Ludwig J.A., Wilcox B.P., Breshears D.D., Tongway D.J. & Imeson
A.D. (2005) Vegetation patches and runoff-erosion as interacting ecohydrological processes in semiarid landscapes. Ecology
86, 288–297.
McKee K.L., Feller I.C., Popp M. & Wanek W. (2002) Mangrove
isotopic (d 15N and d 13C) fractionation across a nitrogen versus
phosphorus limitation gradient. Ecology 83, 1065–1075.
Maestre F.T. & Cortina J. (2006) Ecosystem structure and soilsurface conditions drive the variability in the foliar d 13C and
d 15N of Stipa tenacissima in semiarid Mediterranean steppes.
Ecological Research 21, 44–53.
Maestre F.T., Rodríguez F., Bautista S., Cortina J. & Bellot J. (2005)
Spatial associations and patterns of perennial vegetation in a
semi-arid steppe: a multivariate geostatistics approach. Plant
Ecology 179, 133–147.
Nadelhoffer K.J. & Fry B. (1988) Controls on natural Nitrogen-15
and Carbon-13 abundances in forest soil organic matter. Soil
Science Society of America Journal 52, 1633–1640.
Nadelhoffer K.J., Shaver G., Fry B., Giblin A., Johnson L. &
McKane R. (1996) 15N natural abundances and N use by tundra
plants. Oecologia 107, 386–394.
Perry J. (1995) Spatial analysis by distance index. Journal of Animal
Ecology 64, 303–314.
Pugnaire F.I. & Haase P. (1996) Comparative physiology and
growth of two perennial tussock grass species in a semiarid environment. Annals of Botany 77, 81–86.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356
1356 D. A. Ramírez et al.
Pugnaire F.I., Haase P., Incoll L.D. & Clark S.C. (1996) Response of
the tussock grass Stipa tenacissima to watering in a semi-arid
environment. Functional Ecology 10, 265–274.
Puigdefábregas J. (2005) The role of vegetation patterns in structuring runoff and sediment fluxes in drylands. Earth Surface
Processes and Landforms 30, 133–147.
Puigdefábregas J. & Sánchez G. (1996) Geomorphological
implications of vegetation patchiness on semi-arid slopes. In
Advances in Hillslopes Processes, vol. 2 (eds M.G. Anderson &
S.M. Brooks) pp. 1027–1060. John Wiley and Sons Ltd., New
York, NY, USA.
Puigdefábregas J., Sole A., Guitierrez L., del Barrio G. & Boer M.
(1999) Scales and processes of water and sediment redistribution
in drylands: results from the Rambla Honda field site in Southeast Spain. Earth-Science Reviews 48, 39–70.
Querejeta J.I., Allen M.F., Caravaca F. & Roldán A. (2006) Differential modulation of host plant d 13C and d 18O by native and
nonnative arbuscular mycorrhizal fungi in a semiarid environment. New Phytologist 169, 379–387.
Querejeta J.I., Allen M.F., Alguacil M.M. & Roldán A. (2007) Plant
isotopic composition provides insight into mechanisms underlying growth stimulation by AM fungi in a semiarid environment.
Functional Plant Biology 34, 683–691.
Querejeta J.I., Barberá G.G., Granados A. & Castillo V.M. (2008)
Afforestation method affects the isotopic composition of planted
Pinus halepensis in a semiarid region of Spain. Forest Ecology &
Management 254, 56–64.
Ramírez D.A. & Bellot J. (2009) Linking population density and
habitat structure to ecophysiological responses in semiarid
Spanish steppes. Plant Ecology 200, 191–204.
Ramírez D.A., Valladares F., Blasco A. & Bellot J. (2006) Assessing
transpiration in the tussock grass Stipa tenacissima L.: the crucial
role of the interplay between morphology and physiology. Acta
Oecologica 30, 386–398.
Ramírez D.A., Bellot J., Domingo F. & Blasco A. (2007a) Stand
transpiration of Stipa tenacissima grassland by sequential scaling
and multi-source evapotranspiration modelling. Journal of
Hydrology 342, 124–133.
Ramírez D.A., Bellot J., Domingo F. & Blasco A. (2007b) Can
water responses in Stipa tenacissima L. during the summer
season be promoted by non–rainfall water gains in soil? Plant
and Soil 291, 67–79.
Ramírez D.A., Valladares F., Blasco A. & Bellot J. (2008a) Effects
of tussock size and soil water content on whole plant gas
exchange in Stipa tenacissima L.: extrapolating from the leaf
versus modelling crown architecture. Environmental and Experimental Botany 62, 376–388.
Ramírez D.A., Domingo F. & Bellot J. (2008b) Water interactions
between bare soil and vegetation in semiarid Mediterranean
steppes: some new evidences. In Soil Ecology Research Developments (ed. T.-X. Liu) pp. 7–17. Nova Science Publishers, Inc.,
New York, NY, USA.
Ramírez D.A., Valladares F., Domingo F. & Bellot J. (2008c) Seasonal water-use efficiency and chlorophyll fluorescence response
in alpha grass (Stipa tenacissima L.) is affected by tussock size.
Photosynthetica 46, 222–231.
Scheidegger Y., Saurer M., Bahn M. & Siegwolf R. (2000) Linking
stable oxygen and carbon isotopes with stomatal conductance
and photosynthetic capacity: a conceptual model. Oecologia 125,
350–357.
Sternberg L.S.L., Mulkey S.S. & Wright S.J. (1989) Oxygen isotope
ratio stratification in a tropical moist forest. Oecologia 81, 51–
56.
Sullivan P.F. & Welker J.M. (2007) Variation in leaf physiology of
Salix arctica within and across ecosystems in the High Arctic: test
of a dual delta C-13 and delta O-18 conceptual model. Oecologia
151, 372–386.
Received 16 January 2009; received in revised form 23 April 2009;
accepted for publication 30 April 2009
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1346–1356