Research

Stomatal opening ratio mediates trait coordinating network

adaptation to environmental gradients
Jiangbo Xie , Zhongyuan Wang and Yan Li
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China

Summary
Author for correspondence:
A trait coordination network is constructed through intercorrelations of functional traits,
Yan Li which reflect trait-based adaptive strategies. However, little is known about how these net-
Email: liyan2016@zafu.edu.cn
works change across spatial scales, and what drivers and mechanisms mediate this change.

This study bridges that gap by integrating functional traits related to plant carbon gain and
Received: 25 October 2021 water economy into the coordination network of Siberian elm (Ulmus pumila), a eurybiont
Accepted: 22 April 2022
that survives along a 3800 km environmental gradient from humid forest to arid desert.

Our results demonstrated that both stomatal density and stomatal size reached a physiolog-
New Phytologist (2022) 235: 907–922 ical threshold at which adjustments in these traits were not sufficient to cope with the
doi: 10.1111/nph.18189 increased environmental stress. Network analysis further revealed that the mechanism for
overcoming this threshold, the stomatal opening ratio, gratio, was represented by the highest
Key words: covariation, eurybiont, guard
values for centrality across different spatial scales, and therefore mediated the changes in the
cells, maximum theoretical stomatal trait coordination network along environmental gradients. The mediating roles manifested as
conductance (gsmax), Siberian elm (Ulmus creating the highest maximum theoretical stomatal conductance (gsmax) but lowest possible
pumila), stomatal, water availability. gratio for pathogen defense in humid regions, while maintaining the gratio ‘sweet spot’ (c. 20%
in this region) for highest water use efficiency in semihumid regions, and having the lowest
gsmax and highest gratio for gas exchange and leaf cooling in arid regions.

These results suggested that the stomatal traits related to control of stomatal movement
play fundamental roles in balancing gas exchange, leaf cooling, embolism resistance and
pathogen defense. These insights will allow more accurate model parameterization for differ-
ent regions, and therefore better predictions of species’ responses to global change.

the trait coordination networks change across spatial scales and

Introduction
Coordination networks of key traits related to plant carbon and
water economy and their variation along environmental gradients
reflect trait-based adaptive strategies and their interactions with
climatic, edaphic or topographic drivers (Wright et al., 2004; Liu
et al., 2010; Poorter et al., 2013; Rosas et al., 2019). Quantifying
these patterns at multiple spatial scales is essential to understand-
ing the ecology and evolution of diversity in plant form and func-
tion (Messier et al., 2010, 2017; Poorter et al., 2013; Osnas
et al., 2018) and to identifying the dominant drivers and strate-
gies behind species distributions and diversity in plant form and
function (Liu et al., 2017). This, in turn, helps in understanding
and predicting the consequences of climatic changes on ecosys-
tems (Du et al., 2021). There have been significant advances in
our understanding of coordination networks for functional traits
related to carbon and water economy at different biological levels,
such as stomatal (Franks et al., 2009; McElwain et al., 2016;
Lawson & Matthews, 2020; Haworth et al., 2021), leaf (Wright
et al., 2004; Rosas et al., 2019) and whole plant (Poorter et al.,
2013; Messier et al., 2017) levels, but little is known about how
what drivers and mechanisms mediate this change.
A trait coordination network is constructed by intercorrelating
functional traits (Poorter et al., 2013; Messier et al., 2017), and
therefore changes in the network depend on variations in plant
functional traits that are brought about by evolutionary and envi-
ronmental drivers operating at different spatial scales. Previous
interspecitic analyses have revealed that higher variation in plant
traits relating to carbon or nutrient economy appear at finer scales
(i.e. local sites and plant communities) rather than coarser scales
(i.e. region) (Ackerly & Cornwell, 2007; Liu et al., 2010).
Indeed, a global synthesis has shown that higher interspecific vari-
ations in leaf economic traits are found within sites (Wright
et al., 2004). However, the above conclusions based on inter-
specitic analyses may collapse at the species level (Rosas et al.,
2019). An experimental study of Metrosideros polymorpha (which
survives over a broad range of annual precipitation, from < 400
to > 10 000 mm yr–1) in Hawai‘i showed that traits related to
drought tolerance (i.e. stem vulnerability to embolism) were
inflexible across sites; however, several other traits related to water
supply and gas exchange (i.e. photosynthetic capacity and

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hydraulic conductivity) differed among sites (Cornwell et al., mechanisms for stomatal control under different environmental
2007). More critically, different spatial scales (i.e. regions or sites) conditions (Haworth et al., 2021), and relevant experimental
may be accompanied by different environmental factors, and this studies have produced a rich library of literature on stomatal con-
further complicates the issue. Thus, the contributions of different trol that has fostered numerous hypothetical correlations with
spatial scales to trait variance depend on the species and traits photosynthesis (light-saturated CO2 assimilation rate (Asat), oper-
considered. To realize the adaptive value of trait combinations in ational stomatal conductance (gop), water use efficiency (WUE),
different environments, it is best to elucidate intraspecific trait Table 1), stomatal anatomy (stomatal size (Ss), stomatal density
covariance structures or carry out direct intraspecific analyses (Sd), stomatal area fraction (Sf)), stomatal function (anatomical
(Rosas et al., 2019). maximum stomatal conductance (gsmax), stomatal opening ratio
The ability of eurybionts to grow under a wide range of envi- (gratio)), leaf economics (leaf mass per unit leaf area (LMA), light-
ronmental conditions has long attracted the attention of scien- saturated CO2 assimilation rate per unit leaf mass (Amass)) and
tists, because eurybionts minimize phylogenetic signals (Lens water relation (leaf water potential at the turgor loss point (π tlp),
et al., 2011) and maximize the detection of intraspecific trait Dv, Huber value (Hv)).
variability (Messier et al., 2017). More critically, eurybionts can Previous studies have demonstrated that, in dry conditions,
survive along continuous environmental gradients, and therefore about half of plants show opportunistic and rapid growth during
may provide evidence for the physiological threshold of trait- short periods of relative high water availability with high
based adaptive strategies. Previous studies have revealed a operational stomatal conductance, gop, and therefore a high
parabolic response in stomatal density, Sd, which comprises an light-saturated CO2 assimilation rate, Asat (Hetherington &
increase in Sd under moderate drought but a reduction under sev- Woodward, 2003; Cornwell et al., 2007; Liu et al., 2017).
ere drought (Fraser et al., 2008; Xu & Zhou, 2008). These Generally, gop is determined by stomatal anatomy (Ss, Sd and Sf),
results imply that the plants may have reached a physiological stomatal behavior (gratio = gop/gsmax) and the water supply capac-
threshold (Fraser et al., 2008) at which increasing the Sd is not ity of the veins (Dv) (McElwain et al., 2016; Henry et al., 2019;
sufficient to cope with the increased environmental stress, and Kostaki et al., 2020; Lawson & Matthews, 2020). Therefore,
plasticity in some other traits is needed to compensate for it there are at least three nonexclusive ways to increase Asat and gop.
(Forsman, 2015; Xie et al., 2015a; Wang et al., 2022). If there is
only one trait (i.e. Sd) showing a physiological threshold along Table 1 Definitions of traits studied and units used.
the environment gradient, it should regulate a change in the trait
coordination network. Therefore, the physiological threshold Units
represents a turning point in plant behavior (i.e. a change in the Symbol Definition employed
trait coordination network) driven by environmental change, and Photosynthesis Asat Light-saturated CO2 μmol m−2 s−1
thus has great significance in predicting the consequences of cli- assimilation rate per unit leaf
matic changes on ecosystems. area
Stomata are turgor-driven valves on the leaf epidermis that gop Maximized operational mol m−2 s−1
stomatal conductance
perform at least four key functional roles: regulation of water loss
WUE Water-use efficiency, the ratio g CO2 kg–1
in exchange for CO2 uptake; evaporative cooling and the mainte- of CO2 uptake for Asat by the H2O
nance of optimal leaf temperature for metabolic processes; (3) water lost through the
protection against catastrophic embolisms (McElwain et al., stomata
2016); and pathogen defense (Muir, 2020; Ye et al., 2020). The Stomatal Ss Stomatal size μm2
anatomy Sd Number of stomatal pores per mm−2
stomata influence physiological/ecological processes across scales
unit leaf area
(Yamauchi et al., 2019), from carbon gain and water loss in Sf Stomatal area fraction, the –
leaves to global carbon and water cycling (Hetherington & average fraction of the leaf
Woodward, 2003; Wolf et al., 2016; Henry et al., 2019; West- epidermis that is allocated to
brook & Mcadam, 2020; Haworth et al., 2021). Recent evidence stomata
Stomatal gsmax Anatomical maximum mol m−2 s−1
has established their central role in physiology, evolution and
function stomatal conductance
global ecology (Hetherington & Woodward, 2003; Wolf et al., gratio Stomatal opening ratio during –
2016; Lawson & Matthews, 2020; Matthews et al., 2020). Thus, gas exchange at light-
potential traits that mediate change in the trait coordination net- saturated conditions
work may be related to stomatal traits with a physiological Leaf economic LMA Leaf mass per area g m−2
Amass Light-saturated CO2 μmol g−1 s−1
threshold along the environmental gradient. Photosynthesis and
assimilation rate per unit leaf
transpiration are mediated by stomatal conductance, which is mass, Amass = Asat/LMA
determined by short-term stomatal physiological responses (the Water relation π tlp Leaf water potential at turgor MPa
regulation of stomatal pore aperture), long-term anatomy adap- loss point
tion (the density, size and total epidermal area allocated to the Hv Huber values calculated as mm−2 cm−2
sapwood cross-sectional area
stomata) and hydraulic acclimation (hydraulic supply according
relative to leaf area
to leaf vein density, Dv) (Lawson & Matthews, 2020). These Dv Vein density mm mm−2
traits operate in a coordinated manner to produce distinct

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First, both Asat and gop are positively related to gsmax, which is cal-
culated from Ss and Sd (Franks & Beerling, 2009b; Dow et al.,
2014; McElwain et al., 2016). Larger stomata have been demon-
strated to acquire nonoptimal behavior (Franks & Beerling,
2009b; Franks et al., 2009). Thus, smaller, denser stomata (de-
creased Ss and increased Sd) result in higher gsmax, and therefore
higher Asat and gop (Franks et al., 2009). However, denser stom-
ata need more air spaces and higher hydraulic supply from veins,
all of which are at the expense of the photosynthesizing cells
(Fiorin et al., 2016; Lawson & Matthews, 2020). Furthermore, a
recent study along the north–south transect of eastern China
(NSTEC) has revealed that gop is independent of, and even
opposes, gsmax across communities regionally (Liu et al., 2017).
Moreover, previous studies have shown that Asat is not always
related to Sd, as increased Asat can cope with no change in Sd, sug-
gesting that the link between Asat and stomatal anatomy is com-
plex (Lawson & Matthews, 2020). The second way to enhance
Asat is to manipulate gop close to gsmax (increasing gratio), which is
equally important in the plasticity of stomatal anatomy (Henry et
al., 2019; Lawson & Matthews, 2020). However, increasing gop
is at the expensive of WUE, although this has facilitated greater
CO2 diffusion and therefore higher Asat (Lawson & Matthews,
2020). Furthermore, the scaling relationship between gop and
gsmax of plants along the environmental gradients remains poorly
understood, and knowledge is even more limited regarding its
adaptability to the varing environment. Finally, the way to
enhance Asat and gop is to increase Dv, as the water supply via
high-density veins to the stomata enhances photosynthetic rates
(Lawson & Matthews, 2020). Thus, the vein density hypothesis
predicts positive relatinships between gop, gsmax and Dv (Brodribb
& Feild, 2010; McElwain et al., 2016; Westbrook & Mcadam,
2020; Wilson et al., 2020; Du et al., 2021). However, increased
Dv has a cost linked with it due to the space that would otherwise
be occupied by photosynthesizing cells. Thus, results in support
of this hypothesis are mixed (Lawson & Matthews, 2020). These
conflicting findings come from studies of different species in dif-
ferent envrionments, implying that trait-based adaptive strategies
are species-specific. Equally, however, this may reflect the weight-
ing of the functional roles (balancing gas exchange, leaf cooling,
embolism resistance and pathogen defense) of stomata under dif-
ferent environmental stresses. Under field conditions, plants are
routinely subjected simultaneously to a number of different envi-
ronmental factors, and therefore trade-offs among the different
stomatal functions produce distinct mechanisms for stomatal
control (Zandalinas et al., 2018; Guerrieri et al., 2019). How-
ever, how stomatal control adapts to the combination of different
environmental factors remains unclear.
Stomatal opening to maintain photosynthesis is partly com-
pensated for by other efficient morphological adjustments. For
example, higher LMA (Rosas et al., 2019) and/or higher Hv (the
plants that grew under the no precipitation treatment had a
higher defoliation rate; Xu & Li, 2006; Xu et al., 2007) in low
precipitation habitats maintain Asat and prevent water loss
through the leaf surface. However, to date, the aforementioned
traits (photosynthesis, stomatal anatomy, stomatal function, leaf
economics and water supply) have not been studied
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simultaneously within a species across a wide range of environ-
mental conditions. The present study bridges this gap by inte-
grating these traits into a coordination network for Siberian elm
(Ulmus pumila L.), a eurybiont that survives along continuous
environmental gradients.
Siberian elm is naturally distributed throughout China, Mon-
golia, Korea and Russia and covers a broad range of environmental
gradients (Go et al., 2016). It is a deciduous tree with a broad,
upright crown. Its narrow leaves have single teeth along the
margin, and its winged seeds are hairless. The abundant, wind-
dispersed seeds allow this plant to spread rapidly. The trees are
very drought- and cold-tolerant, which allows this species to grow
in areas that others cannot. Thus, in China, Siberian elm has been
used widely in rehabilitating arid and semiarid areas to combat
desertification (Go et al., 2016). The trees have also been intro-
duced into North America, where they have been planted
throughout the Midwest and Great Plains as windbreaks and for
lumber. They form dense thickets that close open areas and dis-
place native vegetation, thereby reducing forage for wild animals
and livestock. The trees self-sow freely and sometimes escape from
cultivation. In fact, they have been listed as ‘invasive’ in some areas
such as the American Midwest. Many studies (Shi et al., 2004;
Jiang et al., 2006; Song et al., 2006; Li et al., 2007; Dulamsuren
et al., 2009; Tanaka-Oda et al., 2010; Wesche et al., 2011) have
revealed huge variations in the morphological and physiological
characteristics of Siberian elm between arid regions and mesic
areas. Although the striking ability of Siberian elm to establish at
the very wet and dry ends of an enormous precipitation gradient,
little is known about how the coordination network of functional
traits and its variation across and within spatial scales allow for the
species’ success in such varied conditions.
To address these critical issues, we studied the variability in a
set of photosynthetic, stomatal anatomy, stomatal function, leaf
economic and water supply traits along a water availability gradi-
ent in Siberian elm from the southeast to northwest transect of
China (SENWTC), focusing on the following questions:
(1) How much trait variation was observed and how was it strati-
fied among spatial scales? We hypothesized that differences
among regions (e.g., humid, semihumid and arid) explain most
trait variability in Siberian elm along environmental gradients.
(2) What traits exhibit physiological thresholds along environ-
mental gradients? We hypothesized that at least one trait (i.e. Sd)
related to stomata would exhibit a physiological threshold.
(3) How does the trait coordination network change on transect
and region scales? What drives the change and what trait or
mechanism mediates it? We hypothesized that the coordination
network would change on different spatial scales and the stomatal
traits with physiological thresholds would be central to mediating
changes in coordination network along environmental gradients.
Materials and Methods
Study area and field sampling
A survey along the SENWTC was conducted in 2019. Siberian
elm was found to be a eurybiont in this transect, and systematic
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sampling of the trees was conducted in 2020. From July to
September 2020, we studied 10 Siberian elm populations in situ
at 10 sites along the SENWTC, a length of 3800 km (Fig. 1a).
We carried a mobile laboratory fully equipped to study plant
water relations and anatomy (Fig. 1a,b). The 10 sites belonged to
either the Chinese Ecosystem Research Network (CERN) or Chi-
nese Forest Ecosystem Research Network (CFERN); they were
far from cities and thus free from significant human interference.
The SENWTC passes through arid (from Xinjiang to Hexi Cor-
ridor, China), semiarid/humid (Loess Plateau, China) and humid
(southeast mountainous region, China) regions and is perpendic-
ular to the isohyet line along the precipitation gradient, which
facilitates systematic comparisons of the zonal vegetation. The
vegetation passes from temperate desert to subtropical forest. The
10 sites (four sites in arid, three in semiarid/humid and three in
humid regions) spanned a longitudinal range from 87°460 E to
119°280 E and latitude range from 30°110 N to 45°300 N (Fig. 1a).
Mean annual precipitation (MAP) ranged from 54 to 1390 mm,
mean daily photosynthetically active radiation (PAR) from 24.59
to 37.70 mol m−2 d−1 in the growing season (from May to
September), aridity index (AI = mean annual potential evapo-
transpiration (PET)/MAP) from 12.62 to 0.59 and mean annual
temperature (MAT) from 6 to 15.3°C (Table 2; Supporting
Information Fig. S1). Climate data, including MAT, MAP and
AI, were collected from the China Meteorological Background
Data Set (Xu & Zhang, 2017) using ARCGIS v.10.2 (ESRI, Red-
lands, CA, USA). For each site, the coordinates (including lati-
tude, longitude and altitude) were recorded using a geographical
positioning system (Thales, USA).
Photosynthetic light response curves
At each site, 15 dominant trees were selected in a 100 × 100 m
plot. Before measurement, we labeled three healthy branches
(length > 30 cm) of similar diameter (5.19  0.08 mm) in the
middle of the south-facing crown for each tree using a mobile
scaffold (purchased from Taobao, Alibaba; maximum scaffold
height 15 m). We then selected two fully developed sun-exposed
leaves (i.e. alternate leaves, similar in size) on each labeled branch
to measure the light curves. Thus, there were six replicates (two
leaves × three branches) for each tree. Photosynthetic light
response curves were measured with a LI-6800 portable photo-
synthesis system (Li-Cor Inc., Lincoln, NE, USA). In-chamber
photosynthetic photon flux density was controlled using a 30 ×
30 mm leaf chamber with a light source (6800-02). The maxi-
mum photon flux density was 1800 μmol photons m−2 s−1 to
ensure light saturation and was dropped in steps during measure-
ment (i.e. 1800, 1500, 1200, 1000, 800, 600, 400, 300, 200,

(a) (b)

Fig. 1 Geographic distribution of the sampling sites of Ulmus pumila along the precipitation gradient in China (a) and the scheme for coordination of leaf
photosynthetic, gas exchange, stomatal and water relationship traits (b). The transect from arid (Hexi Corridor) to semiarid and semihumid (Loess Plateau)
to humid (southeast mountainous region) is perpendicular to the isohyet line along the precipitation gradient. This transect also avoids the Mongolian
Plateau, China, and systematically compares the difference in zonal vegetation under the precipitation gradient. TMS, Tianmushan Forest Ecosystem
Observation and Research Station, Hangzhou, Zhejiang, China; JGS, Jigongshan National Forest Ecosystem Observation and Research Station, Xinyang,
Henan, China; BTM, Baotianman Forest Ecosystem Observation and Research Station, Nanyang, Henan, China; JY, Jiyuan Forest Ecological Station, Jiyuan,
Henan, China; AS, Ansai Field Experiment Station of the Chinese Academy of Sciences, Yan‘an, Shanxi, China; GY, Guyuan Ecological Station of the
Chinese Academy of Sciences, Guyuan, Ningxia, China; SPT, Shapotou Desert Research and Experiment Station of the Chinese Academy of Sciences,
Zhongwei, Ningxia, China; MQ, Minqin National Studies Station for Desert Steppe Ecosystem, Wuwei, Gansu, China; GZ, Guazhou Anxi Extra-arid Desert
Nature Reserve, Jiuquan, Gansu, China; FK, Fukang Station of Desert Ecology of the Chinese Academy of Sciences, Changji, Xinjiang, China. Abbreviations
for the traits studied are given in Table 1.

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Table 2 The 10 study sites and their environmental features.

Region Site Lon. (°E) Lat. (°N) Elevation (m asl) MAT (°C) MAP (mm yr−1) AI PAR (mol m−2 d−1) VPD (kPa)

Humid TMS 119.44 30.34 1142.00 15.30 1390.00 0.59 37.94 0.64
JGS 114.09 31.87 260.00 15.20 1118.00 0.99 40.29 0.74
BTM 111.93 33.52 1426.50 14.00 823.30 1.02 40.10 0.72
Semihumid JY 112.50 35.05 384.75 9.60 614.50 1.27 42.35 1.08
AS 109.32 36.86 1218.00 7.90 534.00 2.23 45.41 1.04
GY 106.40 35.97 1831.00 6.80 495.70 1.85 43.60 0.70
Arid SPT 104.99 37.46 1247.00 6.60 186.00 4.90 49.98 1.38
MQ 103.14 38.66 1376.00 7.90 113.20 9.10 52.08 1.79
GZ 95.78 40.50 1139.00 7.60 54.40 12.62 56.05 2.43
FK 87.93 44.29 465.00 6.00 158.80 6.50 51.66 1.99

Lon., longitude; Lat., latitude; asl, above sea level; MAT, mean annual temperature; MAP, mean annual precipitation; AI, aridity index (AI = PET/MAP);
PAR, mean daily photosynthetically active radiation in the growing season (from May to September); VPD, vapor pressure deficit in the growing season
(from May to September). See details in Supporting Information Fig. S1. Abbreviations for sites are the same as in Fig. 1.

150, 100, 75, 50, 25 and 0 μmol m−2 s−1). The leaf tempera-
ture, CO2 concentration, humidity and flow rate were set to
25°C, 400 μmol mol−1, 60% and 500 µmol s−1, respectively.
Measurements were carried out from 08:30 to 11:30 h.
Following the photosynthetic measurements, the leaf in the
chamber was marked using an oily pen and scanned for opera-
tional stomatal conductance (gop) and stomatal anatomy (see fol-
lowing section). The scanned image was processed and the surface
area of the leaf calculated using IMAGEJ (1.8.0v for PC, W. Ras-
band, National Institute of Health, Bethesda, MD, USA). The
photosynthetic rate was then converted to a leaf-specific value. We
used PHOTOSYNTHESIS software (1.1v for PC; LI-COR Inc.) to fit
the light response curves for each set of measurements and obtain
the Asat value. Intrinsic instantaneous WUE was calculated as Asat/
Esat (transpiration rate at the light-saturated point). The average
Asat and WUE of each tree were then calculated as the average of
six replicates (two leaves × three branches).
Maximized operational stomatal conductance (gop)
The value of gop is affected by dynamic environmental signals, and
there are many protocols used to measure it. In this study we did
not follow a ‘variance protocol’ to capture mean gop (Drake et al.,
2013; McElwain et al., 2016). Instead, our purpose was to capture
the utilization of the anatomical potential in terms of gas exchange
under optimal conditions, which include saturating light intensity,
and optimal soil, water and nutrient conditions (Drake et al.,
2013). Thus, the gop used here refers to maximized stomatal con-
ductance under optimal conditions, which was measured over a
7 d period at each site, following the definitions provided by
Drake et al. (2013); Dow et al. (2014) and McElwain et al.
(2016). The marked leaves used for the photosynthetic measure-
ments were also used to measure gop in the field, with two measure-
ments per leaf taken over the course of the day, one during
midmorning (08:30–11:30 h) and one during early afternoon
(13:00–15:00 h), respectively. Maximum gop was measured using
an LI-6800 photosynthesis system at 400 μmol mol−1 CO2 and
saturating light intensity (obtained from preliminary light curves).
Leaf temperature and vapor pressure deficit (VPD) were set to
25°C and 1.0 kPa, respectively (McElwain et al., 2016). The leaf
was kept under optimal conditions for at least 20 min, until the
stomatal conductance reached a plateau. The gop of each leaf at
each measurement was then calculated as the average of three
recordings when stomata were fully open. The maximum gop for
each leaf was taken as the highest value for of the 7 d of measure-
ments. The maximum gop for each tree was then calculated as the
average of six leaves (two leaves × three branches). To ensure our
gop measured in 2020 produced a robust maximum gop in each
site, the entire photosynthetic light response measurement was
repeated in July–September 2021 (for details see Figs S2, S3;
Table S1 in Notes S1).
The branches (length > 30 cm) used in the gop measurement
were then removed from the trees and recut immediately under
water, while simultaneously keeping the incision under water
(simulating optimal water supply) to avoid embolism. (Two
selected small terminal twigs that did not contain the marked
leaves were also excised under water, and these were used to mea-
sure leaf water potential at the turgor loss point, π tlp; see follow-
ing section.) After the leaves were excised under water, they were
divided into three groups: the marked leaves on each branch were
used to measure stomatal anatomy and vein density (group 1);
the selected terminal twigs (including leaves) from each branch
were used to measure π tlp (group 2); and all remaining leaves
were used to measure LMA (group 3).
The leaves in group 3 were scanned and their areas measured
using IMAGEJ. Then, the leaves were oven-dried at 60°C and
weighed. LMA was calculated as follows:
LMA ¼ leaf dry mass=area
The cross-sectional sapwood area was measured as the total
xylem-filled area on images of dyed (safranine-methyl blue) 15
μm thick sections using IMAGEJ. The images were taken under a
Leica DM3000 microscope (DM3000; Leica, Wetzlar, Ger-
many). The Hv is the ratio of the cross-sectional sapwood area to
leaf area (area of group1 + group2 + group3). The average LMA
and Hv of each tree were then calculated as the average of three
replicates (three branches), respectively. Then, Amass = Asat/LMA
(Wright et al., 2004).

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Stomatal anatomy and anatomical maximum stomatal
conductance
The leaves used for the gop measurement were immediately taken
for stomatal anatomical measurements (Fig. 1b) using the nail-
polish imprint method (Russo et al., 2010; Zhao et al., 2016).
Clear and transparent nail polish was applied to the middle of
the abaxial leaf surface (avoiding the major veins; stomata on the
adaxial leaf surface were rare), and it was left to dry naturally (<
10 min). Then the nail polish was peeled off the leaf and
mounted ont a glass slide. At this point, the marked leaves used
for photosynthetic measurement were collected for scanning, and
the surface area was calculated using IMAGEJ. The stomatal prints
were photographed under a Leica DM3000 microscope at a mag-
nification of 200× to measure Sd and 400× to measure the stom-
atal size and pore size.
The number of stomata (Ns) was recorded in each image, and
Sd = Ns/0.3072, where 0.3072 was the measured image area
(mm2). Sd was the mean value from more than 25 digital image
fields from each leaf (five images per leaf).
Stomatal length (SL), stomatal width (SW), maximum stomatal
pore area (amax) and guard cell width (w) were averaged from >
20 open stomata per leaf (400× magnification). Then, Ss was cal-
culated as π/4 × SL × SW and Sf was calculated as Sd × Ss × 10–4
(Liu et al., 2017). The average value for the stomatal anatomical
traits for each tree was then calculated as the average of six repli-
cates (two leaves × three branches).
The maximum stomatal conductance of water vapor, gwmax,
can be calculated using the following formula, which assumes an
elliptical stomatal shape for kidney-shaped guard cells:
d
 S d  a max
g wmax ¼ v
pffiffiffiffiffiffiffiffiffiffiffiffiffiffi Eqn 1
w þ π2 a max =π
where d and v are both constants (d = 0.0000249 m2 s−1, which
is the diffusivity of water vapor in air at 25°C;
v = 0.0245 m3 mol−1, which is the molar volume of air), Sd is
the stomatal density (m−2), amax is the maximum stomatal pore
area (m2) and l is the stomatal pore depth (m; considered to be
equivalent to the width (w) of an inflated, fully turgid guard cell)
(Franks & Beerling, 2009a,b; Boer et al., 2016). Here, gsmax was
considered to be the maximum stomatal conductance to CO2,
gsmax = gwmax/1.6 (Farquhar & Sharkey, 1982; Franks et al.,
2009). Thus, the stomatal opening ratio, gratio, was equal to gop/
gsmax:
g ratio ¼ g op =g smax
Vein density
Following the stomatal anatomical measurements, the marked
leaves were used to measure Dv. The leaf samples were placed in
5% sodium hydroxide aqueous solution for 3 d, followed by
sodium hypochlorite solution for 12 h. The leaves were then
soaked in deionized water for 30 min, dyed with 1% toluidine
blue, mounted on glass slides (Zhao et al., 2016) and
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photographed using a Leica DM3000 microscope at 100× mag-
nification. The length of the veins was measured manually using
IMAGEJ. Dv was then measured as the mean value of more than
five digital image fields on each leaf. The average value of Dv for
each tree was then calculated as the average of six replicates (two
leaves × three branches).
Leaf water potential at turgor loss point (π tlp)
The value of π tlp was measured using a pressure chamber
(1505D-EXP; PMS Instrument Co., Albany, NY, USA), follow-
ing the protocol described by Leuschner et al. (2019). The peti-
oles of Siberian elm were too short to insert into the pressure
chamber; therefore, we measured small terminal twigs (which
were collected after the gop measurement). The average π tlp of
each tree was then calculated as the average of six replicates (two
selected twigs × three branches).
Statistical analysis
Trait variability was assessed via the coefficient of variation. The
spatial distribution of variability for each trait studied was tested
using nested ANOVA. A four-level nested ANOVA was designed
as follows: the first level was among-region (different climatic
regions); the next level was among-site (among-site within-
region); the third level was within-site among all trees (the trees
at each site); and the final level was within-tree (the replications
within each tree). The commonly measured traits (Asat, gop,
WUE, Ss, Sd, LMA, π tlp, Dv and Hv) were tested by a four-level
nested ANOVA. The calculated traits (Amass, gsmax, gratio and Sf,
which were calculated as a combination of mean measured traits
at the tree level) were tested by a three-level nested ANOVA
(among-region, among-site and within-site). All variables were
checked for normality and loge-transformed whenever required
to ensure normality. To further explore the overall variability in
all measured traits, we performed a principal component analysis
(PCA).
Before exploring the environment–trait relationships, a PCA
was performed to summarize the environmental variables (all
variables were nondimensionalized). Most studied environmental
variables were correlated with one another (collinear; Fig. S4).
Thus, the environment–trait relationships were first examined by
a stepwise generalized linear model (GLM) that started with the
‘saturated’ model for each trait, including all seven environmental
variables as fixed explanatory variables (without interactions; all
variables in the GLM were dimensionless), and iteratively
removed the least significant explanatory variable until a best
model with the lowest Akaike information criterion (AIC) was
obtained. The best model corrected the collinearity among envi-
ronmental variables and simultaneously obtained a reasonable
amount of explanatory power that affected trait variability. Based
on the stepwise GLM, the environment–trait relationships and
intercorrelation of functional traits were further examined using a
modified Mantel’s test. Standardized Mantel’s statistics calculate
the Pearson’s correlation coefficient between two correlation
matrices. Here, in the modified Mantel’s test, we used the
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coefficients between environmental variables and traits within the (a)

GLM instead of Pearson’s correlation coefficients. The parabolic
relationships between environmental variables and traits were
then also tested. Because some traits appeared physiological
thresholds along the transect (although the above linear relation-
ships between these traits and environmental variables were not
significant) and thus showed parabolic relationships with some
environmental variables.
Trait coordination networks at different spatial scales were
described by statistically significant correlations among traits and
illustrated using the IGRAPH package. Traits in the network were
represented as vertices and their correlation as the edges linking (b)
them. Network centrality was characterized by two indicators: D,
the number of its adjacent edges (the degree of a vertex); and Dw,
the sum of the edge weights of the adjacent edges for each vertex.
Data were loge-transformed to improve the linearity of the rela-
tionships. All analyses were performed in R (R 3.5.3; R Core
Team, 2019).

Results

Climatic regional differentiation of the studied traits

The original data for the studied traits are presented in Fig. S5.
The gratio, gop and Asat were the most variable traits, whereas π tlp,
Dv, gsmax and Sd showed the least variation (Fig. 2a). Ten out of
the 13 measured traits (π tlp, Asat, gratio, gop, Ss, Sd, Sf, gsmax, Dv
and Amass) exhibited substantial variability among-region and
among-site within-region (sites nested into region), but a much
higher variability occurred among-region than among-site
within-region. Only three measured traits (LMA, WUE and Hv)
showed higher variability among-site within-region than among-
region (especially Hv, for which the contribution of climatic
region was close to 0).
To explore the overall variability in the measured traits, we
performed a PCA, which revealed that regional climate (humid,
semihumid or arid) explained 53% of the variance (PC 1 in Fig.
3a). In addition, the first axis (PC 1) was mainly described by the
10 traits that showed higher variability among-region than
among-site within-region (see Fig. 2b). Furthermore, the second
axis (PC 2) was mainly related to LMA, WUE and Hv, which
contributed another 15% of the total variation. These results
were consistent with the nested ANOVA (Fig. 2b) and suggested
that the functional traits were stratified by regional climatic
parameters such as light, rainfall and temperature. We called this
phenomenon climatic regional differentiation. Given this cli-
matic regional differentiation, we further characterized the mea-
sured traits among the three regions. Leaf water potential at the
turgor loss point (π tlp), Asat, gop, gratio, Dv and LMA at arid sites
were higher than at humid sites, whereas gsmax, Sd, Ss and Sf at
arid sites were lower than at humid sites. No difference in the Hv
was found among the three regions. In particular, there were
physiological thresholds in WUE, Sd (Fig. 3h,i) and Ss (although
Ss was not statistically well supported in Fig. 3j, and the follow-
ing analysis (Fig. 4c) confirmed this point).
Fig. 2 (a) Coefficient of variation of the studied traits in Ulmus pumila;
(b) variance components of the studied traits based on nested ANOVAs
(except for the TMS site). Four-level nested ANOVAs for measured traits
(black x-axis tick labels): the first level was among-region (different cli-
matic regions); the next level was among-site (among-site within-region);
the third level was within-site among-trees (the trees at each site); and the
final level was within-tree (the replications within each tree). Three-level
(among-region, among-site and within-site) ANOVAs for calculated traits
(red x-axis tick labels). Traits are ordered (left to right) from higher to lower
percentage variation among-region in (b). Abbreviations for the traits
studied are given in Table 1.
Environmental driver of trait variance and intercorrelation
of functional traits
Hv and LMA were decoupled from the studied environmental
variables. Apart from Hv, the other measured traits were signifi-
cantly related to at least one but not more than three environ-
mental variables (Fig. 4a; Table 3). Among them, Asat, gop and
LMA were solely associated with MAP. Ss, Sd and Sf showed
physiological thresholds along the environmental gradient
(Fig. 4b–d). Furthermore, MAP was significantly associated with
most studied traits (Fig. 4a; Table 3; there were parabolic rela-
tionship between MAP and Sd and Sf, but no linear relationship
among them, Fig. 4b–d).

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(c) (d) (e)

(a)

(f) (g) (h)

(i) (j) (k)

(b)
(l) (m) (n)

Fig. 3 Climatic regional differentiation of the studied traits in Ulmus pumila along the transect (except for the TMS site). (a) Principal component analysis
(PCA) performed on the studied traits shows that the samples are significantly separated by climatic regions (i.e. humid, semihumid or arid). Boxplots of the
first PCA illustrate differences between climatic regions (b). The first two axes accounted for 68% of the variance. The confidence ellipses are drawn at the
90% level. (c–n) Boxplots of each trait at climatic regions. Horizontal lines within boxes indicate the median for each trait based on measurements (circles)
within a region. Boxes indicate the upper (75%) and lower (25%) quartiles. Whiskers represent the ranges of the minimum and maximum values. Circles
outside whiskers are statistical outlier values. Different letters indicate a significant difference between means at P < 0.05 (one-way ANOVA, followed by
multiple comparisons of treatments by means of a Tukey test). Abbreviations for studied traits are given in Table 1.

The intercorrelations between the functional traits are illus-
trated in the upper triangle in Fig. 4a and r1–r10. The Hv
appeared to have no significant relationships with the other mea-
sured traits, and the same held true for WUE and LMA. For
these three traits, the within-site variation was greater than the
among-region variation, according to the ANOVA (Fig. 2b).
The turgor loss point, π tlp (the π tlp values used were converted
from negatives to positives), was higher in elm occupying arid
sites, exhibiting significant positive relationships with Asat, gop
and gratio and significant negative relationships with gsmax, Ss and
Sf. The Asat value was significantly positively correlated with gop,
gratio and Dv but significantly negatively correlated with gsmax and
Sf. There were significantly positive correlations between gop and
gratio, π tlp and Dv (gratio was positively correlated with Dv and π tlp
but significantly negatively correlated with gsmax). Furthermore,
gratio was significantly negatively correlated with gsmax, whereas
gsmax was significantly positively correlated with Sf but signifi-
cantly negatively correlated with Dv. Finally, Sf was significantly
negatively related to Dv and significantly positively related to Ss
rather than Sd.
A higher Asat value was achieved through greater gop, which
was enhanced by higher gratio and Dv (gratio was positively corre-
lated with Dv) values but not gsmax. Counterintuitively, both Asat
and gop were significantly negatively related to gsmax. No signifi-
cant negative relationship appeared between Amass and LMA.
Trait coordination network changes at different spatial
scales
Trait coordination differed on the transect scale and region scale
(Fig. 5). On the transect scale, gratio exhibited the highest value of
centrality. Surprisingly, Asat and WUE were unrelated across the
regions (Fig. 5a), although a consistent, negative relationship
appeared in each region (Fig. 5b–d). At the regional scale
(Fig. 5b–d), gratio also showed the highest value of centrality in
semihumid and arid regions but not in the humid region (Ss
showed the highest centrality). The results were almost the same
as in Fig. S7.
At the site scale (Fig. S8), gratio showed the highest value of
centrality at eight (TMS and BTM in humid, JY, AS and GY in

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(a)

(b) (c) (d)

(e) (f) (g)

Fig. 4 Environmental drivers of trait variance of Ulmus pumila along the transect (except for the TMS site). (a) Edge width (r-size) corresponds to the coef-
ficients between each trait and environmental variables within the best model (obtained from the GLM; see details in Table 3; the intercorrelations of envi-
ronmental factors and traits based on bivariate analyses are illustrated in Supporting Information Fig. S6). The edge color denotes the statistical significance
(P-value; only significant correlations are shown (P ≤ 0.05)) between each trait and environmental variables within the best model. The parabolic relation-
ships between environmental variables and traits are described in detail below the panel (b–e). The solid black curve represents the significant parabolic
relationship between the environmental variable and trait across the sites. The broken gray line (f–g) represents no significant relationship between the
environmental variable and trait; however, when the TMS site was excluded from the analysis, the relationship was significant (solid red line). Pairwise com-
parisons of traits (in upper triangle of (a)) are shown, with a color gradient denoting Pearson’s correlation coefficient. The numbered squares (r1–r10) in the
upper triangle are described in detail in the left panel. Abbreviations for the traits and environmental variables are given in Tables 1, 2, respectively.

semihumid, SPT, MQ and GZ in arid) of the 10 sites. The high-
est centrality at sites JGS and FK was shown by WUE and LMA,
respectively.
Because gratio was a combination of gop and gsmax, which inde-
pendently showed responses, the following trait network analysis
omitted gratio and included gop and gsmax to study the robustness
of the inferred centrality of gratio. The results showed that gop or
gsmax did not show the highest centrality at the transect scale,
within humid and arid sites even when gratio was omitted (Fig.
S9). Thus, the highest centrality of gratio was not achieved by gop
or gsmax alone.
Discussion
We found that most of the studied traits (except Hv) of
Siberian elm along environmental gradients (i.e. MAP) were
clearly stratified by climatic region, which means that traits
varied primarily among-region. Most studied traits responded

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Table 3 Results of the generalized linear models examining the relationships between traits of Ulmus pumila and environmental variables.

π tlp Asat Amass gop gsmax gratio WUE Sd Ss Sf† Dv† LMA Hv

MAP −0.55** −0.95** −1.15* −0.90** −1.86* ni 1.51* ni −1.84* −1.06 −1.66* −1.27* ni
PAR 0.80* ni ni ni −2.35 1.76** −1.17 ni −2.18** −4.46* 3.86* −1.36 4.32
AI ni −0.42 −0.59 ni ni ni 1.66 ni ni −0.23 ni ni ni
VPD −0.46 ni ni −0.28 1.23 −1.27** ni 0.97 ni 2.53* −2.51* 1.01 −2.95
Lon −0.09 −0.36 ni −0.33 −2.82* ni 5.60** −2.25 ni −0.90* 0.68 ni ni
Lat ni ni ni ni −3.30* 0.37 6.27** −2.83* ni −1.04 ni ni ni
RH ni ni ni ni 0.83 ni ni 0.69 ni −0.55* 1.77* ni 1.32

ni, not included in the best model.

Abbreviations for traits and environmental variables are the same as in Tables 1, 2, respectively.
The model’s standardized coefficients are shown. Significant correlations (*, P < 0.05; **, P < 0.01) are shown in bold type.
†
Data except for the TMS site.

(a) (b)

(c) (d)

Fig. 5 Trait correlation networks of Ulmus pumila at the transect scale (n = 9 sites except for the TMS site) (a) and for each climatic region separately
(b–d). gsat and gsmax were excluded from the network, because of the inherent relationship (gratio = gsat/gsmax) between gratio and gsat and gsmax. Amass
(Amass = Asat/LMA) was also excluded from the network. The full coordination networks including these three traits are illustrated in Supporting
Information Fig. S7. The coordination network at each site is illustrated in Fig. S8. Figs 2, 3 show that the samples are significantly separated by climatic
regions (i.e. arid, semihumid and humid), and thus the samples within a region are considered to be homogeneous. Therefore, the network within each
region (b–d) is based on resampling of the data within the region. Namely, the intercorrelation of the functional traits is calculated by using the data within
each 90%-level confidence ellipse (see Fig. 3a). Solid and dashed edges show positive and negative correlations, respectively. The correlation strength is
represented by edge thickness. Only significant correlations are shown (P ≤ 0.05). Traits identified by red circles show the highest centrality value in terms
of both degree (the number of edges of a node) and weighted degree (the sum of all the significant coefficients of correlation of a node). All traits were
loge-transformed before analysis. Abbreviations for the traits studied are given in Table 1.

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to water availability, with increasing π tlp, Asat, gop, gratio, Dv
and LMA and decreasing gsmax and Sf with MAP. However,
two traits (Sd and Ss) demonstrated physiological thresholds
along the environmental gradient. Although the trait correla-
tion network was scale-dependent, the gratio had the highest
values of centrality across different spatial scales, suggesting
that stomatal traits related to control of stomatal movement
mediate changes in the trait coordination network along
environmental gradients.
Climatic regional differentiation of the studied traits
Our results showed that traits differed substantially in their vari-
ability along the same environmental gradient (i.e. MAP; Fig. 4a;
Table 3), with a four- to five-fold magnitude difference in the
coefficient of variation between the most variable (gratio, gop and
Asat) and least variable traits (gsmax and Sd; Fig. 2). The high vari-
ability of gratio, gop and Asat was consistent with previous studies
within species. McElwain et al. (2016) revealed that there is large
variability in gratio (the scaling relationship between gop and gsmax)
within some tropical crops. Moreover, Cornwell et al. (2007)
showed that large variation in photosynthetic capacity exists in
M. polymorpha across a broad range of precipitation conditions.
The high variability in gas exchange capacity has significant eco-
logical implications, as discussed further below. By contrast,
species with narrow habitats often showed high variation in Hv
and low variation in LMA, WUE and Asat. For example, the pho-
tosynthetic capacity and leaf-specific apparent hydraulic conduc-
tance of three xeric shrubs (Tamarix ramosissima, Haloxylon
ammodendron and Reaumuria soongorica) were stable under
drought and after rain (Xu & Li, 2006; Xu et al., 2007). The
observed stability in Asat was associated with their defoliation
under drought (higher defoliation rate, and thereby higher Hv
values under drought). These xeric shrubs even evolved succulent
leaves that allowed them to reduce water loss (Xie et al., 2015b),
and therefore always kept higher LMA than that of eurybionts.
The large variance in most studied traits explained by among-
region differences demonstrated that all traits (except Hv) were
stratified by bioclimatic zones (i.e. humid, semiarid/humid and
arid; Figs 2, 3), which is of particular relevance when studying
mechanisms of species distribution. Bioclimatic zones were classi-
fied by moisture regime (i.e. MAP) and temperature regime (i.e.
MAT), and therefore, not surprisingly, variability was mostly dis-
tributed among-region, reflecting the combination of tempera-
ture and moisture regimes (i.e. AI) as determinants for the
distribution of plants. PAR was another factor of influence at the
region level. Furthermore, other edaphic or topographic variables
are also important, mainly because they modify macroclimates
into mesoclimates and microclimates. Three traits (LMA, WUE
and Hv) showed higher variability among-site within-region than
among-region, especially Hv (the contribution of region climate
was close to 0; Fig. 2b), which suggested that these traits were in
fact adaptations to local edaphic or topographic features. An
alternative interpretation is that Siberian elm regulates water sup-
ply capacity per unit of demand (Hv) to compensate for the other
inflexible traits among-sites within-region. This result agrees with
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a previous study, which demonstrated that intraspecific adjust-
ments along water availability gradients relied primarily on
changes in Hv (Rosas et al., 2019). These results suggested that
the contributions of the different spatial scales to trait variance
depend on the species and the measured trait. To understand the
functional variability of a plant in different environments, the
best way is to elucidate intraspecific trait covariance structures or
use direct intraspecific analysis.
Environmental variables affect trait variance and
intercorrelation
The trait–environment relationships were very tight, suggesting
that the studied environmental variables associated trait variance.
Most traits were linearly (π tlp, Asat, Amass, gop, gsmax, WUE, Dv
and LMA, Fig. 4a) or parabolically (Sd, Ss and Sf, Fig. 4b–d)
related to MAP. Thus, MAP showed the strongest association
with trait variation (Fig. 4a). PAR ranked second and was signifi-
cantly associated with stomatal traits (Sf, Ss and gratio), Dv and π tlp
(Fig. 4a). VPD ranked third and was strongly associated with Dv,
Sf and gratio. These results indicated that MAP, PAR and VPD
significantly affected leaf functions related to water supply and
water consumption along the transect.
With decreasing precipitation, Asat, Amass, gop, gratio, π tlp,
LMA and Dv increased, whereas gsmax decreased (Fig. 3).
Moreover, the PAR–trait relationships were opposite to the
MAP–trait relationships (PAR showed an opposite trend to
MAP; Fig. S4). The Asat and gop values were significantly posi-
tively related to, and both increased with, decreasing precipita-
tion (Fig. 3d,e), which indicated that the Siberian elm in arid
conditions displays opportunistic and rapid growth during
short periods of water availability, which is consistent with pre-
vious studies (Hetherington & Woodward, 2003; Cornwell et
al., 2007; Liu et al., 2017). This opportunistic nature under
arid conditions was explained by a recent model (Lu et al.,
2020) that simulated stomatal behavior under stochastic rain-
fall in a competitive environment (water was ‘stolen’ by other
plants, similar to deficiency in soil water availability). The
model proposed that plants maximized carbon gain and there-
fore stomata remained open to allow gas exchange and xylem
was partly damaged (because of embolism) until the direct car-
bon cost of xylem recovery exceeded the potential future profit
(xylem recovery no longer increased plant net carbon gain in
this scenario). Dang et al. (2017) examined the capacity for
embolism recovery in six drought-resistant tree species on the
Loess Plateau and showed that Siberian elm ranked second.
Therefore, the opportunistic characteristic of Siberian elm in
arid conditions allows it to store carbon so that it can recover
the damaged xylem using stored energy. Xeric trees and shrubs
tend to maintain Asat under drought conditions by increaseing
nonstructural carbohydrates in the roots (stored carbon), which
increases water capture (Xu & Li, 2006; Xu et al., 2007, 2016)
and even facilitates plant resprouting following drought (Zhang
et al., 2016).
Stomatal conductance is determined by stomatal anatomy and
behavior (Lawson & Matthews, 2020). However, gsmax was
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significantly decreased (excluding at the TMS site) with decreas-
ing precipitation, whereas gratio strongly increased (Fig. 3f,g).
This resulted in Asat and gop being strongly positively related to
gratio but significantly negatively related to gsmax, which implied
that the higher gop and Asat in the arid region was regulated by
gratio rather than by gsmax (Fig. 4). The gratio in the arid region
was two- to four-fold larger than that in the humid region and
even reached 60% of gsmax for some plants in the Guazhou
extraarid desert (GZ site, Fig. S5E), which also suggested that
Siberian elm in arid regions has an innately higher Asat, which
takes greater advantage of the anatomical potential in terms of
gas exchange than individuals in humid regions with lower Asat,
which aligns with a previous study (McElwain et al., 2016). At
the same time, caution should be taken when considering these
results, because the in-chamber conditions (temperature and
VPD were set to 25°C and 1.0 kPa) were far less stressful that
the real growth conditions in arid. These in-chamber conditions
may lead plants adapted to dry conditions to open their stomata
more than the stomata of plants adapted to more humid condi-
tions. However, as discussed above, the opportunistic characteris-
tic of Siberian elm in arid conditions allows its xylem to be partly
damaged in order to allow stomata to remain open for carbon
gain, which suggests that the elm could increase gratio with
increasing temperature (or VPD) within a certain temperature
(or VPD) range. To reveal the opportunistic characteristics under
arid conditions, the best way is to infer gs by using δ18O instead
of in-chamber measurements (Lin et al., 2016, 2019, 2021).
In arid regions, the aggressive stomatal behavior of Siberian
elm suggests that the stomata operated near the limit of gsmax and
therefore is vulnerable in the context of climate variation. Hence,
the aggressive stomatal behavior must have been compensated for
by other conservative traits to ensure the survival of Siberian elm.
The key drought tolerance trait, π tlp, showed a significant rela-
tionship with precipitation, with higher π tlp (more negative) asso-
ciated with drier conditions (Fig. 3a), allowing the maintenance
of guard cell turgor pressure and stomatal opening under drier
conditions (Rosas et al., 2019; Deans et al., 2020). LMA
increased with decreasing precipitation (Fig. 3m), which also
enhanced plant resistance to drought (Rosas et al., 2019;
Haworth et al., 2021).
Hydraulic capacity and supply also influence stomatal conduc-
tance. The hydraulic supply trait, Dv, increased with decreasing
precipitation and increasing PAR (Fig. 3l), which aligned with
previous studies showing that higher gop was associated with
higher hydraulic supply capacity (Brodribb & Feild, 2010;
McElwain et al., 2016; Lawson & Matthews, 2020). Further-
more, higher PAR resulted in higher temperatures on the leaf sur-
face under drier conditions, which led to a higher water supply
via a higher Dv to stomata, enabling greater gop for leaf cooling
(Boyce et al., 2009; Brodribb & Feild, 2010; McElwain et al.,
2016; Lawson & Matthews, 2020) and ultimately enhancing the
Asat. These did not, however, prevent a decrease in gsmax under
drier conditions (Fig. 3f). The trend of increasing Dv (Fig. 3l),
decreasing gsmax (Fig. 3f) with decreasing precipitation and there-
fore a strong negative correlation between Dv and gsmax (Fig. 4a)
was unexpected in the current study and contrasted with the
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results of a previous study that showed a strong positive correla-
tion between Dv and gsmax (McElwain et al., 2016). By contrast,
a strong positive relationship between Dv and gratio (Fig. 4a)
linked the higher Dv with higher gratio in arid regions (Fig. 3g,l),
suggesting that supplying more water in an arid region main-
tained guard cell turgor pressure and ultimately enhanced gratio
rather than gsmax. As discussed above, the stomata operate near
the limit of gsmax in arid regions, and therefore the margin of dif-
ference between gsmax and gop also decreased with increasing Dv.
These results agree with the vein density hypothesis (Brodribb &
Feild, 2010; McElwain et al., 2016). However, they might also
reflect the different weighting of the functional roles (gas
exchange, leaf cooling, embolism resistance and pathogen
defense) of stomata under field conditions that are routinely sub-
jected to a number of different environmental stresses and there-
fore produce distinct mechanisms for stomatal control. Our data
showed that the Siberian elm in arid regions with high PAR
developed moderate Sd and Ss compared with those in humid
and semiarid/humid regions (Fig. 3i,j), consistent with a previous
study proposing that the requirement for leaf cooling in arid
regions (hot climates with high PAR) may select for species with
moderate densities of midsized stomata (McElwain et al., 2016).
This stomatal anatomical pattern was not conducive to increasing
gsmax in arid conditions, and therefore enhancing gratio through
high Dv was the alternative path to increasing gop. Our results
revealed a strong negative relationship (Fig. 4a, r6 and r8)
between adaptation for stomatal opening (denoted by gratio and
gop) and anatomical adaptation of maximum stomatal conduc-
tance (donated by gsmax and Sf), in agreement with a recent study
(Liu et al., 2017). Liu et al. (2017) also demorestrated that gop is
independent of, and even opposes, gsmax across communities
regionally along the NSTEC.
At least three traits (WUE, Sd and Ss; Sf was calculated using Sd
and Ss) showed a parabolic response to MAP and PAR, with WUE
and Ss reaching a maximum at intermediate levels of MAP and
PAR, while simultaneously Sd reached a minimum (Figs 3, 4b–e).
A recent field experiment on the same species (Siberian elm) along
a similar environmental gradient (from humid, through semiarid,
to arid) also reported the same results (table 2 and fig. 5 in Go et
al., 2016): Ss showed a maximum under semiarid and was reduced
under humid (or river side) and arid (or sand dune) conditions,
while simultaneously, Sd reached a minimum under semiarid and
increased under semiarid and was reduced under humid (or river
side) and arid (or sand dune) conditions. Simultaneously, Go et al.
(2016) also reported that Sf (calculated by multiplying Ss by Sd) of
Siberian elm was highest in humid and lowest in arid conditions,
which aligned with our findings for Sf (Fig. 2k). Several reports
have shown a parabolic relationship between stomatal anatomy and
environmental variables. For example, Liu et al. (2017) demon-
strated a parabolic relationship between climate factors (i.e. MAT,
MAP) and community-weighted means of Sd, Sf and Ss along the
NSTEC. In particular, Ss also reached a maximum at an intermedi-
ate level of MAP (see fig. 6e in Liu et al., 2017). In grasses, an
increase in Sd was observed under moderate drought (Sd reached a
maximum), but a decrease occurred under severe drought (Fraser
et al., 2008; Xu & Zhou, 2008).
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The largest stomata enhanced the distance for gas molecule
diffusion (Franks & Beerling, 2009b), and thereby compared
with the smaller, denser stomata, the larger, less dense stomata
resulted in a lower gsmax (Fig. 3), which was accompanied by an
improvement in WUE (Franks & Beerling, 2009b; Franks et al.,
2009) in semiarid/humid regions. For the same reason, a decrease
in WUE in drier and wetter conditions was brought about by
smaller stomata (Fig. 3). The highest WUE in semiarid/humid
environments was also associated with the ‘sweet spot’ of gratio (c.
20% in this region; Fig. 3g). Maintenance of stomatal function
in this sweet spot indicates faster responses to environmental sig-
nals (Franks et al., 2012; Dow et al., 2014; Mcelwain et al.,
2016) and facilitates greater CO2 uptake, while simultaneously
avoiding unnecessary water loss, which improves intrinsic WUE
(Lawson & Matthews, 2020). An alternative interpretation of
lower WUE in humid regions is that WUE is not selected in a
wet environment, whereas stomatal functions related to pathogen
defense are selected. A related extreme example appeared in
Siberian elm at the TMS site: the smallest stomata (Fig. S5H)
and lower gratio and Sf (Fig. S5E,I) were unfavorable to photosyn-
thesis and evaporative cooling; however, they were key to
pathogen defense (Muir, 2020; Ye et al., 2020). Stomata control
gas exchange, but also provide a channel for many foliar
pathogens to enter plant tissues (Ye et al., 2020); therefore,
trade-offs between pathogen defense and other stomatal functions
(gas exchange, leaf cooling and embolism resistance) always exist.
The weighting of pathogen defense tends to be greater in humid
regions due to the hot climate and high humidity, which may be
fundamental to why Siberian elm in humid regions has devel-
oped a low gratio. Nevertheless, further research on pathogen
defense at the stomatal level and the trade-offs between pathogen
defense and other stomatal functions is required.
Trait coordination network changes at different spatial
scales
To the best of our knowledge, our study is the first to test the
covariation within traits related to photosynthesis (Asat, gop,
WUE), stomatal anatomy (Ss, Sd, Sf), stomatal function (gsmax,
gratio), leaf economics (LMA, Amass) and water supply (π tlp, Dv,
Hv) simultaneously at different spatial levels. The trait correla-
tion network was region- and scale-dependent (Fig. 5). One of
the traits that received more attention was the leaf economic spec-
trum (LES), which highlights the trade-off between leaf longevity
and productivity. In arid regions (Fig. 5d), elm exhibited conser-
vative leaf economic strategies (i.e. higher LMA), possibly to sup-
port longer leaf life spans with higher Asat and Amass. This result
was opposite to the findings for the LES, which implied that an
increase in LMA coincided with a decrease in photosynthetic
capacity (Wright et al., 2004). The same applied at the transect
scale (Fig. 5a), where Amass and LMA were unrelated. By con-
trast, in humid and semihumid regions (Fig. 5b,c), we found evi-
dence for an LES with a strong negative correlation between
LMA and Amass. These results are consistent with previous find-
ings that the LES is not always conserved across scales, because
plants under field conditions are routinely subjected to several
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Research 919
different environmental stresses simultaneously and therefore
trade-offs between different traits produce distinct mechanisms,
which can effectively decouple traits at certain scales (Messier
et al., 2017; Rosas et al., 2019). Our results revealed a related
mechanism indicating that a higher LMA in arid regions was
largely achieved by higher Dv, which tended to supply more
water to maintain guard cell turgor pressure and ultimately
enhanced gratio, Asat and Amass, rather than enhancing leaf
longevity.
Despite the trait correlation network being region- and
scale-dependent, a general role (highest centrality) in trait coor-
dination networks holds for gratio (Figs 5, S7, S8; except for
the humid region in Fig. 5b). At the same time, caution is
needed when interpreting these results, because the measure-
ments are a relative snapshot of conditions that would occur
across the growing season at each site over a period of 7 d.
For example, Ss also appeared to be particularly important,
especially in the humid region where it showed the the highest
centrality (Fig. 5b). However, at site level, gratio also showed
the highest centrality at both TMS and JGS sites of the humid
region (Fig. S8a,b). In spite of the highest gsmax and Sf at the
BTM site in the humid region (Fig. S5d,i), Siberian elm also
maintained the lowest gratio (Fig. S5E) because of pathogen
defense. As discussed above, the trade-offs among stomatal
traits caused by pathogen defense require further study. gratio
participated in all stomatal functional roles. In particular, at
least two traits exhibited physiological thresholds along envi-
ronmental gradients; both Sd and Ss (Sf was calculated from Sd
and Ss; WUE also resulted from stomatal adjustment) reached
a physiological threshold, which implied that adjustment of
both traits was not sufficient to cope with the increase in envi-
ronmental stress. Therefore, the integrative traits, namely gratio
that combines Sd and Ss, must be changing to alleviate envi-
ronmental stress. Network analysis further demonstrated that
gratio offered the highest values of centrality across different
spatial scales, suggesting that stomatal traits related to control
of stomatal movement mediate the change in the trait coordi-
nation network along environmental gradients. Namely, the
interconnected traits elect an intermediate agency, gratio, to
adapt to environmental gradients. The mediating roles were
manifested as developing the highest gsmax but lowest gratio for
pathogen defense in humid regions, maintaining the ‘sweet
spot’ of gratio (c. 20% in this region) with the highest WUE in
semihumid regions and equipping the plant with the lowest
gsmax and highest gratio for gas exchange and leaf cooling in arid
regions. Siberian elm thus succeeds at the very wet and dry
ends of an enormous precipitation gradient. These results also
suggested a critical mechanism that mediates changes in trait
coordination networks along environmental gradients, namely
stomatal movement (i.e. gratio), which was independent of, and
even opposed, the stomatal pattern of development (i.e. gsmax).
Conclusion
Our study shows that Siberian elm, a eurybiont, is routinely sub-
jected to several different environmental stresses and harbors
New Phytologist (2022) 235: 907–922
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920 Research
suites of covariant traits to cope with them. The species highlights
the importance of the stomatal opening ratio in mediating the
trait coordination networks in response to environmental change.
We show that most studied traits vary primarily at the among-
region level, indicating that traits are clearly stratified by climatic
region. Second, the trait coordination networks of the eurybiont
along environmental gradients allowed us to determine the physi-
ological threshold of function traits and the mechanism to over-
come this threshold, which will allow more accurate model
parameterization and, therefore, better predictions of species’
responses to global change. Finally, we found that the stomatal
opening ratio provided the highest values of centrality across the
different spatial scales, and that stomatal traits related to control
of stomatal movement mediate changes in the trait coordination
network along environmental gradients, providing further evi-
dence that control of stomatal movement plays a fundamental
role in balancing gas exchange, leaf cooling, embolism resistance
and pathogen defense.
Acknowledgements
We thank Liu Xuecan, Chen Liang, Zhang Hui, Lu Shitong,
Chen Sen, Ding Xiaoran, Fang Jing, Pan Tiantian, Tang Luyao,
Zhang Bona and Ye Linfeng for the help with field sampling.
This research was funded by the National Natural Science Foun-
dation of China (nos. 41730638, 31770651 and 31901280).
The manuscript benefited greatly from the comments of two
anonymous reviewers and the editors. The authors declare no
competing interests.
Author contributions
YL and JX conceived the study; JX and ZW collected data, ana-
lyzed data and drafted the text; all authors edited the manuscript.
ORCID
Yan Li https://orcid.org/0000-0002-1503-0515
Zhongyuan Wang https://orcid.org/0000-0001-6018-4647
Jiangbo Xie https://orcid.org/0000-0003-1392-8396
Data availability
The data that support the findings of this study are openly available
in GitHub at https://github.com/allometry-dog/DataOfElm2020.
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Supporting Information Fig. S8 Trait correlation networks of Ulmus pumila within each
site.
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S9 Trait correlation networks (excepted Amass and gratio) of
Ulmus pumila at the transect scale (n = 9) and for each climatic
Fig. S1 Climate data.
region separately.
Fig. S2 The gop of Ulmus pumila measured in 2021.
Notes S1 The relationship between gop measured in 2020 and
2021.
Fig. S3 The relationship between stomatal conductance of Ulmus
pumila in 2020 and 2021.
Table S1 Operational stomatal conductance of Ulmus pumila
measured in 2020 and 2021.
Fig. S4 PCA summarizing environmental variables at the site
level.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any Supporting Information supplied by the
Fig. S5 Boxplot of each trait of Ulmus pumila at each site.
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
Fig. S6 The intercorrelations of environmental factors and traits
of Ulmus pumila based on bivariate analyses.

Fig. S7 Full trait correlation networks (including all traits) of

Ulmus pumila on the transect scale and for each climatic region
separately.

New Phytologist (2022) 235: 907–922 Ó 2022 The Authors

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