Journal of Ecology 2017, 105, 163–175 doi: 10.1111/1365-2745.

12662

Precipitation, not air temperature, drives functional

responses of trees in semi-arid ecosystems
Charlotte Grossiord1,*, Sanna Sevanto1, Henry D. Adams2, Adam D. Collins1,
Lee T. Dickman1, Natalie McBranch1, Sean T. Michaletz1, Elizabeth A. Stockton1,
Miguel Vigil1 and Nate G. McDowell1
1
Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; and
2
Department of Plant Biology, Ecology, and Evolution, Oklahoma State University, Stillwater, OK 74078-3013, USA

Summary
1. Model scenarios of climate change predict that warming and drought will occur simultaneously
in the future in many regions. The capacity of woody species to modify their physiology and mor-
phology in response to environmental conditions is widely recognized, but little is known about the
responses of trees to reduced precipitation and increased temperature acting simultaneously.
2. In a semi-arid woodland, we assessed the responses in physiological (needle emergence, maxi-
mum photosynthesis, stomatal conductance, water use efficiency (WUE) and shoot elongation) and
morphological (needle length and thickness, and leaf mass per area (LMA)) foliar traits of pi~non
pine (Pinus edulis) in response to three years of a 45% reduction in precipitation, a 4.8 °C increase
in air temperature and their simultaneous effects.
3. A strong change in physiological and morphological traits in response to reduced precipitation
was observed. Precipitation reduction delayed needle emergence, decreased photosynthesis and
stomatal conductance, increased WUE, decreased shoot elongation and induced shorter needles with
a higher LMA. Trees subjected to simultaneous reductions in precipitation and warming demon-
strated a similar response. However, atmospheric warming did not induce a response in any of the
measured traits.
4. Physiological and morphological traits of trees in this semi-arid climate were more responsive to
changes in soil moisture than air temperature. Long-term exposure to seasonal drought stress in arid
sites may have resulted in strong plastic responses to this first stressor. However, atmospheric warm-
ing probably was not experienced as a stress for trees in this warm and dry climate. Overall, our
results indicate that in semi-arid ecosystems where tree functioning is already highly limited by soil
water availability, atmospheric warming as anticipated with climate change may have less impact on
foliar trait responses than previously thought.
Key-words: climate change, ecophysiology, foliar traits, growth, needle structure, phenology,
photosynthesis, Pinus edulis Engelm, plasticity, water use efficiency

capacity of woody species to tune their traits to changing

Introduction
Climate change will increase global temperatures and the fre-
quency and the severity of drought events in many regions
(Pachauri et al. 2014). Higher temperatures and altered pre-
cipitation regimes will affect the exchange of energy, carbon,
water and nutrients between plants and the environment. To
persist and thrive under projected climate change, plants will
need to undergo rapid adjustments in their physiology and
morphology (Nicotra et al. 2010). However, although the
*Correspondence author: E-mail: cgrossiord@lanl.gov
© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society
environmental conditions is widely recognized, climate–vege-
tation models rarely consider changes in species traits, limit-
ing our current ability to make long-term predictions (Reed,
Schindler & Waples 2011; Schwartz 2012; Smith & Dukes
2013). It thus becomes essential to quantify plastic responses
in tree physiology and morphology in their natural conditions
under projected warming and drought intensification to enable
improved model assumptions.
Plastic changes may include developmental, morphological
and physiological adjustments of an individual to changes in
the environment. Under drier climatic conditions, plants often
undergo adjustments that are directed towards a water saving

164 C. Grossiord et al.
strategy. Manipulative experiments controlling incoming pre-
cipitation have been established in various climates world-
wide (e.g. Nepstad et al. 2007; Limousin et al. 2010; Pangle
et al. 2012), providing consistent evidence on trait responses
to precipitation reduction. Among the primary responses to
drought, changes in short-term dynamic processes occur that
limit excessive water loss and damage to the hydraulic system
(Ogaya & Pe~nuelas 2003; Leuzinger et al. 2005; Brando
et al. 2008; Limousin et al. 2015). For instance, trees must
manage the trade-off between optimum carbon gain and water
loss through transpiration which usually results in an increase
in water use efficiency (WUE), with a concomitant decrease
in net photosynthesis and stomatal conductance (Farquhar,
Ehleringer & Hubick 1989; Keitel et al. 2003; Limousin
et al. 2015). Observations of delayed leaf emergence leading
to shorter growing seasons have also been reported for trees
subjected to precipitation reduction (Ogaya & Pe~nuelas 2004;
Pe~nuelas et al. 2004). These changes in physiology during
drought stress are usually followed by modifications in mor-
phological traits such as the growth of new branches and
leaves. Indeed, morphological traits such as needle structure
can also influence responses to water stress (Jaindl, Eddleman
& Doescher 1995) as they impact gas exchange and drought
resistance through indirect effects on light absorption and
control of water loss (Abrams, Kubiske & Mostoller 1994;
Niinemets 2001; Woodruff et al. 2009). In combination with
needle morphological traits, reduction in shoot elongation is
also a reliable indicator of tree response to drought (Girard
et al. 2011) and may even exhibit greater changes than tree
ring widths in conifer species (Vennetier et al. 2013).
Although less studied, atmospheric warming may also
induce short- and long-term adjustments in traits of woody
plants. Most evidences of warming effects on tree species trait
responses arise from observations in natural conditions.
Manipulative experiments of atmospheric temperature are still
rare for trees and are usually conducted in controlled condi-
tions or on tree seedlings (e.g. Tjoelker, Reich & Oleksyn
1999; Morin et al. 2010; Fisichelli et al. 2014; Rich et al.
2015) limiting our current understanding on woody species
responses to warming. Overall, it seems that rising tempera-
tures caused by climate change could alter ecosystem func-
tions if foliar temperature exceeds an optimum temperature
for net carbon gain. In addition, warming may alter plant car-
bon acquisition and water use through its direct effect on
vapour pressure deficit (VPD) (McDowell et al. 2008; Allen,
Breshears & McDowell 2015; Williams et al. 2013). Indeed,
increasing VPD can enhance the physical process of diffusion
for water loss while at the same time inducing stomatal clo-
sure to an increasingly desiccating atmosphere (Sandford &
Jarvis 1986). However, evidence in forest ecosystems, mostly
arising from natural mesic environments, demonstrates that
locally elevated temperatures enhance rather than reduce bio-
chemical and physiological processes. For instance, long-term
studies have found that atmospheric warming over the last
century induced an extension of the growing season through
earlier bud burst (Linderholm 2006; Miller-Rushing &
Primack 2008; Primack, Higuchi & Miller-Rushing 2009) and
© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175
13652745, 2017, 1, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.12662 by Cochrane Chile, Wiley Online Library on [05/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
consequently enhanced secondary and primary growth (Saxe
et al. 2001; Boisvenue & Running 2006). Photosynthesis is
also known to increase under atmospheric warming, provided
that optimum temperatures are not exceeded (Larcher 2003).
However, the few experimental studies conducted in xeric
ecosystems contradict previous findings from mesic sites. In
seasonally dry climates, atmospheric warming was shown to
delay phenological development (Adams et al. 2015) and
reduce growth (Peterson & Peterson 2001; Williams et al.
2013) in forests. Furthermore, the response to temperature rise
seems to vary between functional groups, with no or even
negative effects observed on evergreen growth rates compared
to broadleaved species (reviewed in Way & Oren 2010). This
contrasting response suggests that warming effects on physiol-
ogy and morphology may depend on water availability and
species functional groups.
Overall, physiological and morphological changes in
response to drought and warming have been individually
studied in various ecosystems, but little is known about the
response of trees to the two stresses acting at once (Adams
et al. 2015; Garcia-Forner et al. 2016). Moreover, almost no
literature exists on the response of foliar traits to long-term
simultaneous warming and precipitation reduction of mature
trees in natural forest ecosystems. Scenarios of climate change
predict that environmental stresses such as drought and warm-
ing will most likely occur simultaneously in many regions
(Pachauri et al. 2014). Atmospheric warming may be favour-
able to forests at high elevation or high latitudes where the
growing season is currently limited by low temperatures or
snow cover (Peterson & Peterson 2001). However, warming
may not necessarily promote enhanced ecosystem functioning
if moisture is not adequate or if temperatures exceed the opti-
mum temperatures for physiological processes. Moreover,
warming is expected to advance snowmelt, increase evapo-
transpiration and could amplify seasonal drought stress in
environments that are already water-limited (Duan et al.
2013; Will et al. 2013; Williams et al. 2013). As a conse-
quence, dry summers in semi-arid and arid ecosystems may
become even drier with projected climate change.
Here, we tested whether a 45% reduction in precipitation, a
4.8 °C increase in air temperature and both combined affected
physiological and morphological traits of mature pi~ non pine
(Pinus edulis) trees growing in a semi-arid ecosystem. A
widely distributed tree species in semi-arid regions of North
America, pi~ non pine, demonstrates a high potential for plastic
responses (Van de Water, Leavitt & Betancourt 2002; Cole
et al. 2008; Gottfried, Shaw & Ford 2008). Pi~ non pine toler-
ates high temperatures (Spaulding, Robinson & Paillet 1984)
and demonstrates relatively high resistance to water stress
(Linton, Sperry & Williams 1998; Garcia-Forner et al. 2016).
Here, we tested foliar traits that represent tree physiology
(needle emergence, maximum photosynthesis, stomatal con-
ductance, WUE and shoot elongation) and morphology (nee-
dle length and thickness, and leaf mass per area (LMA)). We
hypothesized that exposure to reduced precipitation will
induce a more conservative physiology (delayed needle emer-
gence, lower photosynthesis and stomatal conductance, higher

WUE and lower shoot elongation) and a shift towards water
saving morphological trait values (shorter and thicker needles
with a higher LMA). We also hypothesized that trees sub-
jected to atmospheric warming will have a similar response to
trees subjected to precipitation reduction due to a higher
evaporative demand indirectly reducing soil water availability
and thus inducing a warming-mediated drought condition in
this semi-arid climate. Finally, we hypothesized that trees
exposed to a simultaneous reduction in precipitation and
atmospheric warming will develop a similar but more pro-
nounced physiological and morphological response than trees
exposed to single stressors due to the exacerbation of drought
stress.
Materials and methods
STUDY SITE
The study was conducted at the Los Alamos Survival–Mortality
(SUMO) experiment located in Los Alamos County, New Mexico
(35.49° N, 106.18° W, 2175 m a.s.l). The site is characterized by
Hackroy clay loam soils derived from volcanic tuff with a soil depth
ranging from 40 to 80 cm (Soil Survey Staff, Natural Resources Con-
servation Service, United States Department of Agriculture, 2016,
http://websoilsurvey.nrcs.usda.gov). The vegetation is dominated by
pi~
non pine (Pinus edulis Engelm.) and juniper (Juniperus mono-
sperma (Engelm.) Sarg.). Grasses, cacti and other tree species such as
Gambel oak (Quercus gambelli Nutt.) can be found in intercanopy
spaces, but they do not contribute substantially to total stand biomass.
The climate is semi-arid, with a mean annual temperature of 9.2 °C
(1987–2011 mean) and a mean annual precipitation of 415 mm
(1987–2012 mean), half of which falls between July and September
during the North American Monsoon season (http://environweb.lanl.
gov/weathermachine, Los Alamos National Laboratory, 2016).
In June 2012, a manipulative experiment was established at the
site, consisting of open-top chambers that increase air temperature by
4.8
0.3 °C and a precipitation exclusion structure consisting of
clear polymer troughs that reduces precipitation by 45% (i.e. percent-
age cover of soil surface) (Fig. 1). An increase in atmospheric
(a)
Fig. 1. Experimental design implemented at
the Los Alamos Survival–Mortality (SUMO)
site. Five treatments were assigned to the
selected trees: ambient when heating and rain
exclusion were not applied (A) (a), open-top (c)
chambers where temperatures were either
regulated to match ambient temperatures
(CC) or maintained at 5 °C above ambient
temperatures (H) (b), drought for trees within
the rain exclusion structure where 45% of
incoming precipitation was withheld (D) (c)
and heat and drought when both treatments
were applied simultaneously (HD) (d). [Colour
figure can be viewed at wileyonlinelibrary.com]
© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175
13652745, 2017, 1, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.12662 by Cochrane Chile, Wiley Online Library on [05/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Tree responses to warming and drought 165
temperatures of 4.8 °C and a decrease of 45% in precipitation were
selected at our experimental site to match projected values of climate
change models for 2100 under a business-as-usual scenario (Pachauri
et al. 2014). In total, 64 trees (>10 cm diameter at breast height) were
selected for the experiment (32 juniper and 32 pi~non pine trees). We
selected trees between 2 and 5 m tall and ensured that each size class
was equally represented in each treatment. For the purpose of this
study, we only considered pi~non pine because of the significant diffi-
culty and error associated with chronological foliage assessments in
juniper. Mean tree age was 56
5 years. Tree characteristics are
given in Table 1.
The selected trees were assigned to one of five treatments: ‘ambi-
ent’ for trees that were not subjected to temperature or precipitation
manipulations (A), ‘chamber control’ for trees that were situated
inside chambers with temperatures regulated to match ambient tem-
peratures (CC), ‘heat’ for trees inside chambers where the tempera-
tures were maintained at 4.8 °C above ambient temperatures (H),
‘drought’ for trees located within the precipitation exclusion struc-
ture (D) and ‘heat and drought’ for trees where both treatments
were applied simultaneously (HD) (Fig. 1). Chambers footprints ran-
ged from 6 to 20 m2 and contained between one and two trees
located at a minimum distance of 1.5 m from the chamber boundary
(Table 1). Although some roots of trees inside the chambers may
have extended beyond the chamber boundary, we expect the major-
ity of roots to be growing inside the chambers. The selected trees
in the drought treatment were situated at a minimum of 10 m from
the border of the precipitation exclusion structure (equivalent to two
times the height of the tallest tree in the drought treatment) to
ensure that the majority of roots were subjected to the drought treat-
ment. A minimum of six trees was selected for each treatment.
However, additional trees were added to D treatment (Table 1) as
they were situated within the drought exclusion structure and fitted
our selection criteria. To avoid any effect related to light intercep-
tion, three trees (one in the CC treatment and two in the HD treat-
ment) were discarded from the final analyses because they occupied
a suppressed position in the stand.
Climatic conditions were measured continuously and recorded
with two weather stations located on site. Atmospheric temperature
and relative humidity were measured in all chambers (Fig. 2,
Table S1 in Supporting Information) and used for controlling the
industrial-scale air conditioning units that controlled chamber
(b)
(d)
 13652745, 2017, 1, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.12662 by Cochrane Chile, Wiley Online Library on [05/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
166 C. Grossiord et al.

Table 1. Characteristics of selected trees. Number of trees sampled (N), number of chambers (N chambers), mean diameter at breast height
(d.b.h., m) measured at the beginning of the study, mean tree height (m), mean distant-dependent competition index (CI, unitless) and mean
crown radius (CR, m) with standard errors of the mean per treatment

Treatment N N chambers Mean d.b.h. (m) Mean tree height (m) Mean CR (m) Mean CI

Ambient 6 – 0.12
0.04 3.1
1.2 2.0
0.3 3.2
1.1

Chamber control 5 5 0.12
0.07 2.6
1.2 1.9
0.6 3.9
2.7
Heat 6 4 0.13
0.06 3.0
1.0 1.9
0.4 2.9
1.1
Drought 8 – 0.13
0.06 3.5
1.1 1.8
0.3 2.6
0.6
Heat and drought 4 4 0.15
0.05 2.9
1.1 1.7
0.4 2.1
0.5

(a)
35
SOIL WATER CHARACTERISTICS
Temperature (°C)

25
In order to characterize the soil drought intensity in the different treat-
15 ments for the 2012–2014 period, we estimated the daily relative
extractable water in the soil over the whole root zone (REW, unit-
5 less). REW represents the ratio between available soil water and max-
–5 imum extractable water. This value varies between 1, that is field
capacity, and 0, that is permanent wilting point. We used the forest
–15 water balance model BILJOU (Biljou – water balance model, 2016,
(b) A D H HD https://appgeodb.nancy.inra.fr/biljou/) to estimate REW at a daily
10
80 time-scale by using measurements of daily climatic conditions at the
Precipitation (mm)

8 site (rainfall, global radiation, wind speed, and air temperature and
PET (mm)

60 humidity; Granier et al. 1999). This model calculates the different

6
water fluxes at a daily time-scale in the ecosystem: tree transpiration,
40
4 understorey evapotranspiration, rainfall interception and drainage,
which are all dependent on leaf area index (LAI, m2 m2) and evapo-
2 20
rative demand (i.e. the potential evapotranspiration (PET), mm). The
0 0 model required multiple site-specific inputs for parameterization. We
(c) measured LAI with a LAI-2000 Plant Canopy Analyzer (PCA; LI-
1.0 COR, Lincoln, NE, USA) on June 2015. Measurements were taken at
–1.0
dawn at 12 locations within the site but outside the rain exclusion
0.8
structure and averaged to plot LAI (1.5
3 m2 m2). As climatic
ΨPD (MPa)

–2.0
REW

0.6 input data for the simulations, air temperature and humidity inside the
chambers were used to simulate REW in the CC, H and HD treat-
0.4
–3.0 ments and 45% of incoming precipitation was withheld for simula-
0.2 tions of REW in the D and HD treatments (Fig. 2). Soil
characteristics used as input data in the model include maximum
0.0 –4.0 extractable water and bulk density. Maximum extractable water in this
2

4
2

4
01

01

01

01

01

01
01

01

01

01

soil type was assumed to be 120 mm (Soil Survey Staff, Natural

n2

y2

p2

p2

n2

y2

c2
n2

y2

p2

Resources Conservation Service, United States Department of Agri-

Ma
Ma

De
Ma
Ja

Se

Se

Ja
Ja

Se

culture, 2016, http://websoilsurvey.nrcs.usda.gov). Bulk density was

Fig. 2. Climatic and soil water conditions during the 2012–2014 per- measured at the site in July 2015 and was equal to 1.4 g cm3. Simu-
iod for trees in all treatments (A, D, H and HD). Mean daily atmo- lations with the BILJOU model were performed online.
spheric temperature (°C) (a), mean daily potential evapotranspiration Additionally, we measured predawn leaf water potential (ΨPD,
(PET, mm) and daily sum of precipitation (mm) (b), relative extracta- MPa) monthly on all selected trees from June to August, a period
ble water (REW, unitless) and mean predawn leaf water potential when water potential was related to needle emergence and growth at
(WPD ), MPa, filled circles) (c), with standard errors of the mean. The
the site (Adams et al. 2015) from 2012 to 2014. Two branch samples
bold black arrow indicates the installation of the rain exclusion struc-
from each tree were taken before sunrise, placed in plastic bags and
ture and of the open-top chambers in June 2012. [Colour figure can
be viewed at wileyonlinelibrary.com] stored in a refrigerator until they were measured at the study site
within one hour of collection with a Scholander-type pressure cham-
ber (PMS Instruments, Albany, OR, USA). Mean ΨPD for each date
temperature. Increasing air temperature in the heat and heat-drought was determined as the mean of the two branch samples for each tree.
treatments induced an increase in VPD between 0.93 and 0.06 kPa Mean growing season ΨPD per tree (WPD ) was calculated as the mean
(average of 0.35 kPa) (Fig. S1). We measured soil water content of ΨPD over the June–August period for each year.
from 0 to 50 cm in depth periodically with a Diviner 2000 at 58 A strong relationship was found between REW and mean soil
locations in the site (Sentek Sensor Technologies, Stepney, water content over the 0–50 cm soil profile (Fig. S2a;
Australia) starting in April 2013. A more detailed description of y = 23.95x + 6.02; R2 = 0.68; P < 0.001) and REW and ΨPD
the design is provided by Adams et al. (2015) and Garcia-Forner (Fig. S2b; y = 0.47 ln(x)  1.09; R2 = 0.60, P < 0.001) demonstrat-
et al. (2016). ing that simulations of REW from the model reflected the actual soil

© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175

water content and drought intensity experienced by the trees at our
site.
PHYSIOLOGICAL TRAITS
Needle emergence
In each treatment, a subset of four trees was selected for repeated
measurement of needle emergence (NE) timing. On each of these
trees, we followed needle emergence on two or three branches per
tree, for a total of 11 branches per treatment (Adams et al. 2015). For
each branch, measurements were conducted on the primary axis of
growth following the method of Girard et al. (2011, 2012). Observa-
tions started in March and were repeated periodically through the
growing season of 2013 and 2014 until needle emergence occurred.
Maximum photosynthesis and stomatal conductance
We measured maximum photosynthesis (Amax, lmol m2 s1) and
stomatal conductance (gs, mol m2 s1) monthly on one current-year
or previous-year (i.e. depending on whether new needles had
emerged) sun-exposed shoot on the Southern Hemisphere of the
selected trees during the growing season (i.e. from June to August)
over the 2012–2014 period. The measurements were carried out on
the same day as predawn leaf water potential measurements. Amax and
gs were measured using a Li-Cor LI-6400 infrared gas exchange ana-
lyzer system. The measurements took place in the morning when the
highest stomatal conductance could be expected. Measurements were
conducted with the following settings with the 2 9 3 LED chamber:
380 ppm of reference CO2 concentration, 1500 lmol m2 s1 light-
saturating photosynthetic photon flux density, block temperature at 20
or 25 °C to match ambient air temperature and relative humidity on
full scrub. At the start of the experiment, CO2 concentration was mea-
sured each time before starting gas exchange measurements. CO2 con-
centration always fluctuated between 360 and 400 ppm at our site
and thus the average was chosen for the measurements (i.e.
380 ppm). To keep it consistent throughout the years, all measure-
ments were done with this fixed value. However, the impact of this
difference in our results is likely to be very small and does not influ-
ence the relative comparisons between treatments. Gas exchange mea-
surements were conducted with relative humidity on full scrub
because relative humidity was very low (4–15%) during the measure-
ments. As a consequence, the ambient VPD values were very close to
conditions in the chamber (Fig. S3). Leaf temperature was measured
using the energy balance. Measurements were recorded after steady
state gas exchange rates had been maintained for at least 2 min.
Projected leaf area of the measured foliage was determined using a
Li-Cor LI-3100C area meter and was used to correct Amax and gs.
Water use efficiency and shoot elongation
In June 2015, we sampled three branches in sun-exposed positions near
the top of the crowns of all selected trees. Current-year bud break had
not yet occurred at the time of the sampling. Before the sampling, we
ensured that selected branches included needles from the last three years
(i.e. 2012, 2013 and 2014). The length of the shoot (i.e. shoot elonga-
tion, mm) was measured for each year.
Needles were dried at 65 °C for 48 h and dry samples were finely
ground for each year and tree. About 1 mg of the powdered material
was placed into tin capsules for analysis of carbon isotope composition
(d13C, &). d13C in C3 plants constitutes an integrated record of the
© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175
13652745, 2017, 1, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.12662 by Cochrane Chile, Wiley Online Library on [05/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Tree responses to warming and drought 167
intercellular to atmospheric CO2 concentration ratio during the period
in which the carbon was fixed (Farquhar, Ehleringer & Hubick 1989).
All analyses were conducted on an elemental analyzer (Costech Inc.,
Valencia, CA, USA) interfaced to a Thermo Delta V stable isotope ratio
mass spectrometer (Thermo Scientific, Bremen, Germany) at Los Ala-
mos National Laboratory. d13C values are reported in the standard delta
notation relative to the Vienna Pee Dee Belemnite standard. Precision
of d13C measurements was 0.05& (n = 19). Carbon isotope discrimina-
tion (D13C, &) was calculated as (O’Leary 1981):
d13 Cair  d13 Cplant
M13 C ¼ ; eqn 1
1 þ d13 Cplant
where d13Cair (&) corresponds to the carbon isotope composition of
the air and d13Cplant (&) to the carbon isotope composition of the
plant sample. D13C (&) is related to the ratio of intercellular to atmo-
spheric CO2 concentration (Ci/Ca) through the following equation:
Ci
M13 C ¼ a þ ðb  aÞð Þ; eqn 2
Ca
where a (4.4&) is the fractionation caused by diffusion of CO2 from
the atmosphere into the intercellular space of leaves and b (27&) is
the fractionation caused by isotopic discrimination of RuBP carboxy-
lase against 13CO2 (Farquhar, O’leary & Berry 1982). As leaf conduc-
tance to water vapour is 1.6 times the conductance to CO2, D13C can
be converted to intrinsic WUE (lmol CO2 mol1 H2O) as:
h
M  ai C
a
WUE ¼ 1  ; eqn 3
b  a 1:6
where Ca is the CO2 concentration in the atmosphere. d13Cair and Ca
were obtained from the NOAA ESRL website (NOAA, 2016, http://
www.esrl.noaa.gov) (Dlugokencky et al. 2015; White, Vaughn &
Michel 2015). A close link exists between leaf gas exchange and car-
bon incorporation in conifer needles over seasonal and interannual
time-scales (Keitel et al. 2003; Klein et al. 2005). It is important to
note that needle cohorts formed in 2012, 2013 and 2014 were sam-
pled in 2015, so our data may reflect age-related changes in d13C.
However, we expect very little effect of age on needle d13C, as lim-
ited temporal variation is usually observed (Fessenden & Ehleringer
2003; Keitel et al. 2003). Indeed, the highest variation in d13C usu-
ally occurs during needle elongation while the carbon assimilated dur-
ing the following growing seasons was shown to have little influence
on d13C of needle tissues (Fessenden & Ehleringer 2003; Klein et al.
2005). To test for potential age effects at our site, we sampled cur-
rent-year sun-exposed needles on a subsample of trees (n = 10) in
July 2013 and measured d13C using the same method as described
above. We found a mean difference of 0.27& (
0.36&) for d13C
between needle sampled in 2013 and needles formed in 2013 but
sampled in 2015 suggesting little age effect on our measurements.
MORPHOLOGICAL TRAITS
Before drying needle samples for d13C measurements, we measured
needle length (mm) and needle thickness (mm) on a subsample of
five needles per branch and per year. The needles of all three
branches per tree were then pooled together for each year and mea-
sured for leaf area by using a scanner (HP Scanjet 5590; Hewlett-
Packard, Palo Alto, CA, USA) and the image processing software IM-
AGEJ. Needles were then dried at 65 °C for 48 h and their dry mass
was determined with a precision scale (MX5; Mettler Toledo, Colum-
bus, OH, USA) before being ground for d13C measurements. Leaf
mass per area (LMA, g m2; needle dry mass/fresh leaf area) was
then calculated. As previous studies suggested, some morphological

168 C. Grossiord et al.
adaptations such as needle length or leaf area are permanent once
needle development is complete (e.g. Sprugel, Brooks & Hinckley
1996; Vennetier et al. 2013). For instance, a study on Pinus sp.
showed that several foliar morphological traits are mostly affected by
precipitation during the growing season of its development while nee-
dle age by itself has little impact (Oleksyn et al. 1997). However,
some age-related trends in increasing needle width and leaf mass area
have also been reported in conifers and could influence our retrospec-
tive analysis of needle traits. Although care must be taken during our
interpretations, it must be noted that these evidences often arise from
trees with important differences in age classes (Day, Greenwood &
White 2001) and that this influence was shown to be far less impor-
tant than influence of external climatic conditions during needle for-
mation and/or position in the canopy (Day, Greenwood & White
2001). To insure little age effect on our measurements, we measured
LMA on the subsample of trees sampled in July 2013 for d13C and N
%. We found a mean difference in LMA of 19 g m2 (
10.5 g m2)
between needle sampled in 2013 and needles formed in 2013 but
sampled in 2015 suggesting little age effect on structural foliar traits.
STATISTICAL ANALYSES
All analyses were performed using the software R (3.2.1, R Core
Team 2015).
Mean growing season temperature (T, °C) and relative extractable
water (REW, unitless) were calculated as the mean daily temperature
and daily REW, respectively, from June to August (i.e. during the
measurement period) for each year. For T we used for each treatment
either ambient temperature (A and D treatments) or air temperature
inside the chambers (H and HD treatments).
The size and amount of space between interacting trees within an
immediate neighbourhood can have a strong effect on the growth
strategy of a given tree. To take this confounding effect into account
in our analyses, we calculated the distant-dependent size-ratio Hegyi
competition index (CI) (Hegyi 1974) (Table 1) within a 5 m radius of
each tree. This index assumes that the relative d.b.h. and distance
between the subject tree and the competitor trees reflects their
competitive interaction. CI was calculated as:


D
X Dji
CI ¼ ; eqn 4
DISTij
where Di (m) is the d.b.h. of the subject tree i, Dj (m) is the d.b.h.
of the competitor tree j, and DISTij (m) is the distance between the
subject tree i and the competitor tree j.
Response of the measured traits (needle emergence, Amax, gs,
WUE, shoot elongation, needle length, needle thickness and LMA) to
precipitation reduction, atmospheric warming and the combination of
the two treatments was determined through linear mixed effects, ran-
dom-intercepts models. For Amax and gs, the analyses were performed
on individual values measured monthly. The year, CI, heating (yes or
no), precipitation reduction (yes or no) and their interaction were used
as explanatory variables in the fixed part of the models. The individ-
ual trees nested in the chambers were input as random effects. The
model selection procedure started with all variables and by removing
progressively the variables with the lowest explanatory power until
the minimal model with the lowest Akaike information criterion was
obtained that still included the drought:heating interaction. Similar
mixed models were used to assess the relationships between the mea-
sured traits and T, REW and WPD . Similar models were used to deter-
mine differences in climatic conditions (mean daily air temperature,
relative humidity and REW) between treatments where the day, the
© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175
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year and the treatment were used as explanatory variables. Post hoc
analysis was performed with Tukey’s HSD post hoc test. R-squared
(r2) was obtained for linear mixed effects models following Naka-
gawa & Schielzeth (2013) and adapted by Jon Lefcheck (Sample
(Ecology) 2016, http://jonlefcheck.net/2013/03/13/r2-for-linear-mixed-
effects-models/). An alpha critical value of a = 0.05 was used to
determine statistical significance. Statistical analyses were performed
using the package nlme and the function lme.
No significant differences in climatic parameters were found
between ambient conditions and control chambers (P = 0.760 for
mean air temperature; P = 0.997 for VPD and P = 0.999 for REW).
In addition, we found no differences between traits in ambient and
CC conditions; we thus pooled measurements together for statistical
analyses.
We calculated an index of phenotypic plasticity in response to the
treatments ranging from zero to one for each trait and each year as
the positive difference between the means obtained in the ambient
treatment and a given treatment (H, D and HD) divided by the maxi-
mum mean (Valladares et al. 2000; Martınez-Ferri et al. 2001). A
given trait was only considered to be plastic in response to a given
treatment when the treatment effect was significant (P < 0.05).
Results
SOIL WATER CHARACTERISTICS
All three years were characterized by contrasting climatic con-
ditions (Fig. 2). Precipitation during the growing season was
119, 107 and 240 mm for 2012, 2013 and 2014, respectively.
Atmospheric temperatures during the growing season were sim-
ilar among years (22, 21 and 20.5 °C for 2012, 2013 and 2014,
respectively). In 2012, soil water content had reached very low
levels before the installation of the rain exclusion structure and
the chambers at the end of May (Fig. 2).
Drought (D) significantly decreased REW (P < 0.001) while
heating (H) had no effect (P = 0.273). The combination of HD
also significantly decreased REW (P < 0.001). Overall, differ-
ences in REW started to occur among treatments by the begin-
ning of July 2012 with slightly lower REW appearing in the D
and HD treatments as compared to the other treatments (Fig. 2).
The combination of HD induced marginally lower REW values
throughout the experiment as compared to the D treatment
because of slightly higher PET values (P = 0.042, Fig. 2).
Predawn leaf water potential varied among years
(P < 0.001) and treatments (P < 0.001). ΨPD decreased in
2013 as compared to 2012 with drier conditions due to
lower precipitation and increased with milder conditions in
2014 in all treatments (Fig. 2). ΨPD did not vary among
treatments in 2012. In 2013, D and HD significantly
decreased ΨPD (Fig. 2). In 2014, ΨPD was similar in the H,
D and HD treatments but slightly less negative in the A
treatment (Fig. 2).
PHYSIOLOGICAL TRAITS
Needle emergence
Needle emergence varied significantly among years
(P < 0.001) and in response to precipitation reduction
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Tree responses to warming and drought 169

(P = 0.026) but not to warming (P = 0.522) (Table S2). Post
hoc analyses revealed that needle emergence was significantly
delayed in the D and HD treatments treatment in 2013 but
not during other years (Fig. 3).
Maximum photosynthesis and stomatal
conductance
Amax and gs varied significantly among years (P < 0.001) and
in response to precipitation reduction (P = 0.004 and
P < 0.001, respectively) but not in response to warming
(P = 0.178 and P = 0.223, respectively) (Tables S3 and S4).
Post hoc analyses revealed that gs was significantly lower in
the D and HD treatments in 2012 and 2014 (Fig. 3). In 2013
and 2014, Amax was significantly lower in the D and HD
treatment as compared to the A treatment (Fig. 3). Significant
differences between treatments were found during August for
all three years and in July 2014 for Amax and June 2014 for
gs (Fig. S4).
Water use efficiency
WUE varied significantly among year (P < 0.001), and pre-
cipitation reduction significantly changed WUE but to a vary-
ing extent based on the years (P < 0.0001 for the
year:drought interaction) (Table S5). Warming did not modify
WUE (P = 0.705). Post hoc analyses revealed significantly
higher WUE in 2014 in the D and HD treatments than in
ambient trees (Fig. 3). In 2013, higher WUE was found in
the HD treatment only (Fig. 3).
Shoot elongation
Shoot elongation varied significantly among years
(P = 0.012), and precipitation reduction significantly changed
shoot elongation depending on the year (P = 0.006 for the
year:drought interaction) (Table S6). Warming did not modify
shoot elongation (P = 0.448). Post hoc analyses revealed that
in 2014 shoot elongation was significantly lower in the D and
HD treatments as compared to the A treatment (Fig. 3).

280 (a) *** A (e)

***

Shoot elongation (mm)

D
Needle emergence

21
240 H ***
***
HD
(DOY)

14
200

160 7

120 0
(b) (f)
*
*** ***
Amax (μmol m–2 s–1)

Needle length (mm)

7 *** 40

5 *
* 30

3 20

1 10
(c) *** (g)
*** 0.8
Needle thickness (mm)

0.08
gs (mol m–2 s–1)

*
*
0.7

0.04
0.6

Fig. 3. Mean (a) timing of needle 0.00 0.5

emergence, (b) maximum photosynthesis
(Amax), (c) stomatal conductance (gs), (d) (d) (h) ***
* 500
*
WUE (μmol mol–1)

water use efficiency (WUE), (e) shoot 145

elongation, (f) needle length, (g) needle *** ***
*** ***
LMA (g m–2)

thickness and (h) leaf mass per area (LMA) 400

130
for each treatment and for each year, with
standard errors of the mean. Asterisks denote
significant differences between treatments (D, 115 300
H and HD) and control (A) for a given year
(*P < 0.05, ***P < 0.0001) using post hoc
analyses. [Colour figure can be viewed at 100 200
wileyonlinelibrary.com] 2012 2013 2014 2012 2013 2014

© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175
 13652745, 2017, 1, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.12662 by Cochrane Chile, Wiley Online Library on [05/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
170 C. Grossiord et al.

MORPHOLOGICAL TRAITS needle length and thickness decreased, and LMA increased as
WPD and REW decreased (Figs 4 and S5).
Needle length and thickness Needle emergence, Amax, gs, WUE, shoot elongation, nee-
dle length, needle thickness and LMA were all significantly
Needle length varied significantly among years (P < 0.001),
influenced by seasonal predawn leaf water potential (WPD )
and precipitation reduction significantly influenced needle
(P = 0.004, P < 0.001, P < 0.001, P < 0.001, P = 0.002,
length depending on the year (P < 0.001 for the year:drought
P < 0.001, P = 0.004, P < 0.001, respectively) and seasonal
interaction) (Table S7). Warming did not influence needle
relative extractable water (REW) (P < 0.001, P =0 .002,
length (P = 0.4536). Post hoc analyses revealed that in 2014,
P < 0.001, P = 0.004, P = 0.070, P < 0.001, P < 0.001,
needle length was significantly lower in the D and HD treat-
P < 0.001, P = 0.021, respectively), but neither was influ-
ment as compared to the A treatment (Fig. 3). Needle thick-
enced by mean growing season temperature (T), or only mar-
ness showed significant variation among years (P < 0.001)
ginally (P = 0.062, P = 0.378, P = 0.458, P = 0.685,
but not in response to warming (P = 0.062) and precipitation
P = 0.087, P = 0.205, P = 0.624, P = 0.622, respectively).
reduction (P = 0.444) (Table S8, Fig. 3).

Leaf mass per area Discussion

LMA varied strongly throughout the 2012–2014 period Overall, pi~non pine exhibited significant changes in most
(P < 0.001) and the range of variation was significantly physiological traits (i.e. needle emergence, WUE, Amax and
influenced by precipitation reduction (P < 0.001) but not by gs) after only one year of treatment (Fig. 3). Contrary to
warming (P = 0.1759) (Table S9). Post hoc analyses physiological traits, morphological traits mainly responded
revealed that in 2013 and 2014, LMA was significantly after the second year of treatment (i.e. needle length, LMA
higher in the D and HD treatment as compared to ambient and shoot elongation) (Fig. 3). Furthermore, phenotypic plas-
trees (Fig. 3). ticity indexes revealed that the mean plasticity of morphologi-
cal traits was usually lower than the one of physiological
traits (Table S10). This result is in agreement with previous
Relationships between traits and climatic parameters
studies, suggesting that physiological and biochemical
Overall, we found that needle emergence occurred later, Amax responses tend to be larger and more rapid than morphologi-
and gs decreased, WUE increased, shoot elongation decreased, cal ones (Drew & Saker 1978; Fransen, Blijjenberg & De
Shoot elongation (mm) Needle length (mm) Needle thickness (mm)

300 30
y = –66.7x + 76.9 y = 3.4 x + 14.9
Needle emergence

R2 = 0.27
H R2 = 0.15
P < 0.001 A P = 0.020
250 20
(DOY)

D
HD
200 10

(a) (e)
150 0
y = 3.1x + 9.7 y = 9.8 x + 40.6
AMax (μmol m–2 s–1)

R2 = 0.26 R2 = 0.45
8 P < 0.001 P < 0.001
45

4 25

(b) (f)
0 5 Fig. 4. Relationships between growing
y = 0.03 x + 0.09 y = 0.1 x + 0.9 season (a) needle emergence, (b) maximum
R2 = 0.20 R2 = 0.35 0.9 photosynthesis (Amax), (c) stomatal
gs (μmol mol–1)

0.08 P < 0.001 P < 0.001

conductance (gs), (d) water use efficiency
(WUE), (e) shoot elongation, (f) needle
0.04 0.7 length, (g) needle thickness, and (h) leaf
mass per area (LMA), and mean growing
(c) (g) season predawn leaf water potential (WPD ).
0.00 0.4 For Amax and gs, growing season data
y = –10.8 x + 102.1 y = –78.1 x + 149.9 represent the mean of the three measurements
WUE (μmol mol–1)

R2 = 0.27 R2 = 0.20
140 P < 0.001 P = 0.005 440 that were conducted each year (i.e. from June
LMA (g m–2)

to August). Very similar relationships were

340 found for Amax and gs when using the
120 individual measurements conducted during
240
the growing season (Fig. S9). For each
(d) (h) measured trait, the equation, the R2 and P
100 140
–4 –3 –2 –1 0 –4 –3 –2 –1 0 value is given in the upper left corner.
–
ΨPD (MPa) –
ΨPD (MPa)
[Colour figure can be viewed at
wileyonlinelibrary.com]

© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175

Kroon 1999; Valladares et al. 2000; Hodge 2004). Indeed, a
greater benefit for the tree is achieved under adverse condi-
tions when the rate of change in a given trait is as fast as pos-
sible. However, once extreme conditions are removed,
physiological traits are more likely to rapidly return to previ-
ous levels than morphological ones that are accompanied by
supplementary construction or maintenance cost (Metlen,
Aschehoug & Callaway 2009).
Effects of precipitation reduction on physiology and
morphology
After being exposed to the treatments for only one year, sig-
nificant differences in traits could be observed for trees
exposed to precipitation reduction. When plants are faced with
a drier environment, foliar traits usually change consistent with
a water conserving strategy, although whether this response is
adaptive or simply symptomatic to water stress is difficult to
determine. When subjected to a precipitation reduction of 45%
at our experimental site, physiological and morphological traits
of the trees responded in a manner consistent with previous
observations: needle emergence occurred later, Amax and gs
decreased, WUE increased, shoot elongation decreased, needle
length decreased, and LMA increased, relative to ambient trees
(Fig. 3). These results confirm previous studies conducted
both in natural and controlled conditions on coniferous species
suggesting that phenology, foliar gas exchange and primary
growth processes can be particularly useful traits for identify-
ing plastic responses to drought (Van de Water, Leavitt &
Betancourt 2002; Cole et al. 2008; Girard et al. 2011; Limou-
sin et al. 2012; Vennetier et al. 2013). The observed delayed
phenological development of approximately 28 days in 2013
under reduced precipitation is also consistent with previous
studies on Mediterranean tree species subjected to natural or
experimental drought stress (Ogaya & Pe~nuelas 2004; Pe~ nue-
las et al. 2004; Limousin et al. 2012). As previously reported
by Limousin et al. (2013, 2015) under controlled precipitation
reduction, higher WUE combined with lower Amax and gs
demonstrates that trees in exerted strong stomatal regulation
for a longer period during the growing season to reduce water
loss (Figs 2 and 3). Shorter needles and higher LMA (Fig. 3)
also highlight the important changes in morphology of pi~ non
pine in the face of drought stress through an overall decrease
in water use by the reduction of transpiring leaf surface. This
large capacity for changes in both physiology and morphology
in response to drought may allow pi~non pine to adjust to tem-
porally variable soil moisture and reflects its large distribution
range in semi-arid climates.
Interestingly, we found that most trait values did not
change between the extremely dry conditions experienced in
2013 and the relative wet conditions of 2014 under reduced
precipitation (Fig. 3, Table S11), although water availability
was less restrictive in 2014 as compared to 2013 (Fig. 2).
Such a response is well known in ring width studies where
radial growth can be severely diminished for years to decades
after a major drought event, even in the case of non-restric-
tive climate conditions (e.g. Orwig & Abrams 1997). The
© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175
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Tree responses to warming and drought 171
absence of an immediate recovery has been previously attrib-
uted to high demand for post-drought repairs such as recovery
of hydraulic failure in the tree sapwood (e.g. Breda et al.
2006). Individuals may thus have not yet recovered from the
extremely dry conditions experienced in 2013 and were still
investing newly acquired carbon to repair functions during
the 2014 growing season. Furthermore, although REW was
the highest in 2014 compared to the two previous years, trees
in exposed to reduced precipitation still experienced restrictive
soil moisture conditions during the summer of 2014 (Fig. 2)
that could have prevented full recovery.
Although the immediate response of physiological and mor-
phological parameters observed in 2013 for pi~ non pine may be
considered advantageous to withstand sudden environmental
changes (Sultan 2000), long recovery or low resilience to cli-
matic stresses could eventually impair this species’ competitive
interactions and survival. Shorter and thicker needles could first
be advantageous in a drier habitat (Givnish 1984; Fonseca et al.
2000; Wright, Westoby & Reich 2002); however, reductions in
size and thickening of needles are usually accompanied by
lower photosynthetic capacity (Wright et al. 2005), which can
become detrimental in the long term when climatic conditions
become more favourable. Although we observed a strong
reduction in photosynthesis and a concomitant increase in leaf
mass area in trees exposed to precipitation reduction, our
results, however, do not suggest that changes in photosynthesis
were solely driven by changes in foliar morphology (Fig. S6).
It must be noted that not all traits responded rapidly or
strongly to precipitation reduction in the initial three years of
treatment exposure. Needle thickness for instance was mostly
unchanged and showed no plastic response (Fig. 3 and
Table S10). Although needle thickness is in general sensitive
to climate, rapid changes in trait values during or after a
drought event are not usually observed, particularly compared
to other morphological traits such as needle length or surface
(Vennetier et al. 2013). Indeed, needle thickness is mainly
dependent on twig diameter and health (Vennetier et al.
2013) and thus might not be a reliable indicator of tree
responses to drought.
Effects of warming on physiology and morphology
While precipitation reduction had a strong impact on the
physiological and morphological traits of trees, warming had
relatively modest effects (Fig. 3) and the reasons for this are
likely twofold. First, our heat treatment may have been too
small to impact photosynthetic physiology in this species. Our
site is characterized by warm summers where mean daily air
temperatures can reach above 25 °C during the growing sea-
son (Fig. 2). Foliar temperatures higher than 25 °C can cause
reversible or irreversible injuries that can reduce or prevent
photosynthesis (Berry & Bjorkman 1980; Levitt 1980, Nilsen
& Orcutt 1996). Indeed, rates of net photosynthesis generally
increase with leaf temperature to a maximum value at some
optimal temperature and then decrease with leaf temperature
above this optimum. The temperature sensitivity of photosyn-
thesis can be characterized by the thermal breadth (T90),

172 C. Grossiord et al.
which is the leaf temperature range across which net photo-
synthesis equals 90% of its maximum value (Nicotra et al.
2008; Michaletz et al. 2015). An increase of 4.8 °C in air
temperature would result in average leaf temperatures of
about 29 °C which is well within the T90 for pi~non pine at
this site (15.92
1.13 °C; Fig. S7) even if leaf temperatures
rates are generally variable in time. Furthermore, the increase
in atmospheric temperature induced by our experimental
warming during the 2012–2014 period did not induce temper-
ature outside of the long-term (1987–2015) daily temperature
range during the growing season (36.6 to 3.2 °C).
Secondly, as our site is already characterized by high water
limitation under ambient conditions, drought stress may have a
decisive influence over temperature effects, including increas-
ing VPD. Indeed, although we found strong negative relation-
ships between Amax and gs, and VPD in each treatment
(Fig. S8), these relationships were non-significant once Amax
and gs were corrected for REW (Fig. S8), suggesting that soil
moisture was the main limiting factor for gas exchange at our
site. Our results could therefore suggest that warming may not
promote strong responses in ecosystems where plants are
already restricted by other constraints such as water or nutri-
ents, and/or where they do not grow near the high temperature
threshold above which photosynthesis and growth usually
decline dramatically. Previous studies in mesic and/or cold cli-
mates generally show that warming promotes earlier bud
break, higher photosynthesis and growth (e.g. Chmielewski &
R€otzer 2001; Linderholm 2006; Miller-Rushing & Primack
2008; Primack, Higuchi & Miller-Rushing 2009). However, it
must be noted that at the same experimental site, Adams et al.
(2015) found that shoot elongation was slightly reduced under
experimental warming. The discrepancy between the two stud-
ies is likely related to method differences (i.e. branches from
the top of the canopy vs. mid-level branches and yearly vs.
seasonal measurements). Nevertheless, our results indicate that
it may be too early to draw general conclusions on the effects
of warming on forest ecosystems; instead, our results suggest
that the consequences of atmospheric warming must be evalu-
ated based on the limitations of each specific site.
Effects of simultaneous warming and drought on
physiology and morphology
It has been commonly assumed that concomitant atmospheric
warming and precipitation reduction in dry regions would
increase soil and vegetation water stress as a consequence of
larger evapotranspiration compared to precipitation reduction
alone. Surprisingly, we found no significant drought:heat
interaction for any of the measured traits (Tables S2–S9). Fur-
thermore, our results showed no differences in foliar trait
responses and phenotypic plasticity between the D and HD
treatments (Fig. 3, Table S10), supporting that water
availability has a more decisive impact on physiology and
morphology than heat at our site. Similarly, simulations from
the soil water balance model showed that an increase of atmo-
spheric temperatures by 4.8 °C does not induce a large
increase in evapotranspiration (Fig. 2) and thus does not
© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175
13652745, 2017, 1, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.12662 by Cochrane Chile, Wiley Online Library on [05/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
generate larger reduction in REW in the HD treatment above
that of the D treatment (Fig. 2). Using the same experimental
site, Garcia-Forner et al. (2016) also found no evidence for
exacerbated stress under simultaneous precipitation reduction
and atmospheric warming.
Consistently, for most traits, we found that WPD and REW
explained responses across all treatments (Fig. 4), while TMean
did not. Water availability is one of the most limiting factors
for tree functioning and survival in the south-west (Hidalgo
et al. 2001; Adams & Kolb 2005). At our site, atmospheric
warming was superimposed on ongoing drought stress for the
trees. Plants growing in arid or semi-arid climates are known
to develop dense and thick leaves with a high LMA, which
can be beneficial for moisture conservation and maintenance
of physiological favourable foliar temperatures (Leigh et al.
2012; Michaletz et al. 2015), and could also explain an inher-
ent low response to atmospheric warming. Furthermore, long-
term exposure to drought could have resulted in strong
response to this first stressor that altered the response to
superimposed warming. Previous studies showed that physio-
logical and morphological responses may indeed depend on
which stress occurs first and on the degree to which the plant
has acclimated to this first stress factor (e.g. Valladares &
Pearcy 2002). An increase in atmospheric temperatures of
4.8 °C and a decrease of 45% in precipitation were selected
at our experimental site to match projected values of climate
change models for 2100 under a business-as-usual scenario
(Pachauri et al. 2014). Our results suggest that these projec-
tions might largely affect the functioning of trees in semi-arid
regions in response to water availability change but not to
temperature change. However, whether this response is char-
acteristic of all tree species growing in dry environments and
can thus be generalized to other species growing in arid and
semi-arid climates requires validation.
Conclusion
Our study demonstrated that pi~ non pine is able to modify its
foliar traits in response to drier conditions, which could affect
its functioning and survival under projected changes in pre-
cipitation regimes. On the contrary, no response in foliar traits
was found under experimental atmospheric warming. Conse-
quently, counter to our expectations, simultaneous exposure
to precipitation reduction and atmospheric warming did not
exacerbate tree response. As pi~ non pine is inherently adapted
to water stress, drought-adapted foliar traits may be more
responsive to changes in soil water availability and could fur-
ther provide a better dampening of temperature variations.
Furthermore, atmospheric warming may not have been experi-
enced as a limiting factor for trees at our site as the tempera-
ture increase during the growing season fell well within the
thermal breadth of photosynthesis of this species.
Plastic trait responses have often been invoked in studies
focusing on the impact of climate change on plant and com-
munities (Maron et al. 2004). The role of these responses in
resistance to climate change and eventually as a buffer against
mortality has not yet been evaluated. However, rapid

physiological and morphological changes in response to
warming and drought are particularly relevant parameters to
climate change models and mechanistic plant physiology
models. This work also highlighted the need to study changes
in foliar traits in response to climate change in contrasting cli-
mates. Milder and cooler environments where light, tempera-
ture or nutrients rather than water are the main limiting
factors for tree growth might present a differential response to
reduced precipitation and atmospheric warming. Long-term
experiments where precipitation and atmospheric temperature
are manipulated and combined are particularly useful for
anticipating the impacts of climate change on vegetation, par-
ticularly as the effect of the treatments may not be constant
over time. Several systems have been established where
drought or heat stress effects are tested on tree functioning,
but to our knowledge no other experiments considered the
combination of multiple stressors in natural and mature condi-
tions. New experiments, in different climates, combining
multiple abiotic stresses are urgently needed to help bring
some light on tree functioning and on the longer-term resis-
tance and survival to climate change of forest ecosystems.
Acknowledgements
The Los Alamos Survival–Mortality Experiment (SUMO) is funded by the US
Department of Energy, Office of Science, Biological and Environmental
Research. We thank Heath Powers for his technical help during the building of
the site. We also thank Michel Vennetier, Samuel Briggs and Nuria Garcia-For-
ner for assistance with the measurement protocol and data collection of needle
emergence. We thank George Perkins for isotopic analyses. HDA and STM
were supported by a Director’s Fellowship from the Los Alamos National
Laboratory.
Data accessibility
All data used in this manuscript are present in the manuscript and its Support-
ing Information.
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(gs).
Received 28 June 2016; accepted 31 August 2016
Handling Editor: Laura Huenneke
Table S5. Summary of the linear mixed model of water use efficiency
(WUE).
Supporting Information
Table S6. Summary of the linear mixed model of shoot elongation.
Additional Supporting Information may be found in the online
version of this article: Table S7. Summary of the linear mixed model of needle length.

Figure S1. Differences between mean daily vapor pressure deficit
(DVPD, kPa) in the chamber control treatment (CC-A), heat treatment
(H-A) and heat and drought treatment (HD-A), and ambient condi-
tions for the 2012–2014 period.
Figure S2. Relationship between (a) soil water content (%) and daily
relative extractable water (REW, unitless), and (b) daily predawn leaf
water potential (ΨPD, MPa) and REW, measured monthly between
June and August during the 2012–2014 period.
Figure S3. Relationship between ambient vapor pressure deficit
(Ambient VPD, kPa) and vapor pressure deficit measured with the
Li-Cor inside the chamber (Li-Cor VPD, kPa) for all measurements
over the 2012–2014 period.
Figure S4. Mean maximum photosynthesis (Amax) and stomatal con-
ductance (gs) for each treatment and each measurement date, with
standard errors of the mean.
Table S8. Summary of the linear mixed model of needle thickness.
Table S9. Summary of the linear mixed model of leaf mass area (LMA).
Table S10. Phenotypic plasticity indexes for timing of needle emergence
(DOY), water use efficiency (WUE, lmol mol1), maximum photosyn-
thesis (Amax, lmol m2 s1), stomatal conductance (gs, lmol mol1),
shoot elongation (mm), needle thickness (mm), needle length (mm) and
leaf mass per area (LMA, g m2) in response to each treatment and for
each year.
Table S11. Mean timing of needle emergence (DOY), water use effi-
ciency (WUE, lmol mol1), maximum photosynthesis (Amax, lmol
m2 s1), stomatal conductance (gs, lmol mol1), shoot elongation
(mm), needle thickness (mm), needle length (mm) and leaf mass per area
(LMA, g m2) for each treatment and for each year, with standard errors
of the mean.

© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 105, 163–175