Linking Plant Leaf Nutrients/Stoichiometry to Water Use Efﬁciency On

Ecological Engineering 87 (2016) 124–131

Contents lists available at ScienceDirect

Ecological Engineering

jo urnal homepage: www.elsevier.com/locate/ecoleng

Linking plant leaf nutrients/stoichiometry to water use efﬁciency on

the Loess Plateau in China

a a a,b a,∗

Weiming Yan , Yangquanwei Zhong , Shuxia Zheng , Zhouping Shangguan

State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling, Shaanxi 712100, PR China

State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

r t a b

i s

c l e i n f o t r a c t

Article history: Nutrient and hydrological cycles are tightly coupled in ecosystems. However, little is known about the

Received 30 January 2015

relationship between leaf nutrient stoichiometry (nutrient mass ratios) and water use efﬁciency (WUE)

Received in revised form 22 October 2015

in ecosystems. To ﬁll this knowledge gap, we examined 132 plant samples distributed from the Qinling

Accepted 18 November 2015

Mountains to the north of the Loess Plateau of China and observed the relationship between leaf nutrient

stoichiometry and WUE in various ecosystems. Our ﬁndings suggest that a positive correlation exists

Keywords:

between the leaf nitrogen:phosphorus (N:P) ratio and WUE, and this relationship is sensitive to plant life

Leaf nutrients

forms and growth conditions. Additionally, potassium (K) was related to WUE in herbs and plants under

Stoichiometry

WUE P limitation. These results link plant nutrient stoichiometry to hydraulic processes in terrestrial plants

and provide useful information for ecologists studying nutrient and hydrological cycles in ecosystems.

Life form

1. Introduction required for the synthesis of proteins, which contain large amounts

of N and P (Cernusak et al., 2010). The N:P ratio (ratio of the N to

Carbon (C), nitrogen (N), phosphorus (P) and potassium (K) are P concentration) in terrestrial plant leaves can provide important

considered essential elements for plant growth and play a vital role information about potential nutrient limitation, which may affect

in plant functions (Marschner and Marschner, 2012). C provides primary productivity (Ågren, 2008; Cernusak et al., 2010). The bal-

the structural basis of the plant, constituting a relatively stable ance of N and P can inﬂuence the growth rate of plants, and the

50% of the dry mass; N is an important constituent of proteins and leaf N:P mass ratio has been widely used as an indicator of N or P

plays an essential role in all enzymatic activities; and P is involved deﬁcit. For example, based on studies conducted on European wet-

in energy transfer in cells. Additionally, P and N are important land plants, it has been suggested that an N:P ratio above a given

structural elements in nucleic acids (Ågren, 2008; Marschner and threshold (16 on a mass basis) indicates P limitation of biomass pro-

Marschner, 2012). These three elements (C, N and P) are strongly duction, whereas a ratio below a given threshold (c. 14 on a mass

coupled in terms of their biochemical functions. N and P, which basis) indicates N limitation (Koerselman and Meuleman, 1996;

are required for plant growth in relatively large quantities, are Aerts and Chapin, 1999; Tessier and Raynal, 2003; Güsewell, 2004;

classiﬁed as macronutrients and cannot be substituted with other Reich and Oleksyn, 2004; Cernusak et al., 2010). This parameter

elements in metabolic functions (Aerts and Chapin, 1999; Ågren, offers a powerful tool for ecological and physiological investiga-

2008). K is involved in the plant–water relationship (Babita et al., tions by providing a straightforward means of characterising the

2010) through plant osmotic control and improvement of stoma- relative availability of N vs. P (Cernusak et al., 2010). Relation-

tal function (Sangakkara et al., 2000; Babita et al., 2010; Laus et al., ships between N:P ratios and vegetation characteristics have also

2011; Rivas-Ubach et al., 2012). been used to describe functional differences between naturally N-

Plant growth requires photosynthetic products, and proteins are or P-limited plant communities and their responses to environ-

required for photosynthesis and growth. In addition, ribosomes are mental change or human management (Tessier and Raynal, 2003;

Güsewell, 2004; Reich and Oleksyn, 2004). K is also important, but

less information is available regarding the quantitative require-

∗ ments for this element (Reich and Oleksyn, 2004).

Correspondence to: Xinong Rd. 26, Institute of Soil and Water Conservation,

In many terrestrial ecosystems, soil water is variable and often

Yangling, Shaanxi, 712100, PR China. Tel.: +86 29 87019107; fax: +86 29 87012210.

the most important limiting soil resource. Therefore, plant water

E-mail addresses: [email protected] (W. Yan), [email protected]

(Y. Zhong), [email protected] (S. Zheng), [email protected] (Z. Shangguan). use efﬁciency (WUE) is of major importance for the survival,

http://dx.doi.org/10.1016/j.ecoleng.2015.11.034

W. Yan et al. / Ecological Engineering 87 (2016) 124–131 125

◦ ◦

productivity and ﬁtness of individual plants (Ponton et al., 2006) broadleaf forest located at 108 26 E and 33 26 N, all of the other

and is a measure of plant performance that has long been of inter- investigated stands, i.e., Yangling, Yongshou, Tongchuan, Fuxian,

est to agronomists, foresters and ecologists (Cernusak et al., 2007a; Ansai, Mizhi and Shenmu, are distributed from south to north on

Raven et al., 2009). In plant physiological ecology, leaf carbon-13 the Loess Plateau of China (Fig. 1). These stands are located at

␦13 ◦ ◦ ◦ ◦

( C) data are a useful index for assessing WUE when the leaf- 34 16 –38 47 N and 108 02 –110 21 E within the temperate zone,

to-air vapour pressure difference is known (Farquhar et al., 1989; and the vegetation type ranges from semi-humid forests to arid

Dawson et al., 2002), and these data have been widely used to eval- desert grasslands. The altitude, latitude and longitude were deter-

uate plant WUE under various environmental conditions and in mined using a global positioning system (GPS) at the sampling sites.

response to climatic variables (Hietz et al., 2005; Silva et al., 2009; The sampling sites were located far from human habitation (more

Nock et al., 2011; Penuelas˜ et al., 2011). Leaf ␦ C data not only than 1 km) to minimise the inﬂuence of human disturbance. The

precisely reﬂect water conditions but also reﬂect the physiologi- detailed geographical and climatic conditions of the eight sampling

cal status of the plant and have been proven to be the best means sites are summarised in Table 1.

of investigating WUE. As one of the most important physiological

characteristics involved in plant growth, WUE is considered to be an

2.2. Plant materials

objective index for evaluating water conditions (Cabrera–Bosquet

et al., 2007) and drought tolerance characteristics and is capable

A total of 132 plant samples (118 from the Loess Plateau and

of providing a theoretical basis for studying such characteristics

14 from the Qinling Mountains) belonging to 39 different species

in speciﬁc environments (Livingston et al., 1999; Condon et al.,

and 17 families, including 10 types of trees, 20 types of shrubs

2004). In addition, transpiration plays a role in modulating nutrient

and 10 types of herbaceous species, were collected at the eight

uptake by delivering nutrients to root surfaces through mass ﬂow

sites. Species from the 3 different plant life forms (herbs, shrubs

(Cernusak et al., 2011). However, little is known about the relation-

and trees) were selected based on the following criteria: the target

ship between plant WUE and nutrients in ecosystems. Therefore,

species should be the dominant species and relatively abundant

studying the relationship between WUE and nutrients across plant

at each site. Sun foliage samples were mainly collected from the

life forms and nutrient conditions would help us to better under-

upper canopies. Three to ﬁve healthy and fully expanded leaves

stand ecosystems.

from individual plants were randomly selected, and each sample

It has been reported that the leaf N:P ratio is positively correlated

included leaves from 4–5 individual plants of the same species.

with plant WUE in seedlings of tropical pioneer tree species, and it

has been suggested that the N:P ratio is expected to be correlated

with WUE according to the following argument (Cernusak et al., 2.3. C (carbon isotope discrimination) and calculation of

2007b): positive increases in plant C require N-rich proteins for WUE

plants to assimilate C and grow, as shown in the following equation

(Ågren, 2004): dC/dt = ˚CN NP (C, amount of carbon; t, time; CN, The leaf samples were ultrasonically washed with distilled

◦

rate factor; NP, amount of nitrogen in proteins used for growth). In water, air-dried, oven-dried at 70 C for at least 48 h to a con-

addition, P-rich ribosomes are required for protein synthesis. The stant weight, then ground to a ﬁne powder using a plant sample

amounts of N and P in plants exceed those required for growth and mill (Cyclotec sample Mill 1093; FOSS Tecator, Hoganas, Sweden),

␦

protein synthesis, but a balance between N and P is necessary for and ﬁnally sieved through a 1-mm mesh screen. C was ana-

normal plant functioning and could affect the normal growth of lysed using a MAT-251 mass spectrometer (Finnegan, San Jose, USA)

plants. Mass ﬂow, which partly depends on plant transpiration, is at the State Key Laboratory of Soil and Sustainable Agriculture,

the process by which P in the soil solution is transported to the Institute of Soil Science, Chinese Academy of Sciences. A 3–5 mg

surface of the roots, where it can subsequently be absorbed by the portion of each treated sample was placed in a vacuum quartz tube,

plant (Cernusak et al., 2007b; Craine et al., 2008; Cramer et al., 2008; mixed with an activator and desiccant, and then oxidised under

◦

Cernusak et al., 2010). Thus, C uptake is correlated with NP, and P an oxygen ﬂux at 850 C. The CO2 produced under these condi-

uptake is correlated with transpiration (T); consequently, the N:P tions was cryogenically puriﬁed using both a liquid N trap and a

ratio is correlated with the C:T ratio, which is equivalent to WUE dry ice–ethanol trap. Then, according to the PDB (belemnite from

(Cernusak et al., 2010). the Pee Dee Formation) standard, the C isotope of CO2 was analysed

To the best of our knowledge, no previous study has veriﬁed using a MAT-251 mass spectrometer with a precision of <0.02%. The

␦

the relationship between leaf nutrients, stoichiometry and WUE in resulting C value was determined using the following equation:

ecosystems. In the present study, the relationship between the leaf

␦13

= ×

nutrient stoichiometry and WUE of various plant life forms under C (‰) [ Rsample–Rstandard /Rstandard 1000] (1)

different nutrient conditions was analysed by examining 132 plant

13 12

samples (belonging to three different plant life forms) at eight geo- in which Rsample and Rstandard are the C/ C ratios in the samples

logical sites distributed from the Qinling Mountains to the north of and the controls, respectively (Farquhar et al., 1989). During CO2

the Loess Plateau of China. We hypothesised that there is a positive ﬁxation by leaves, C is related to the ratio between the CO2 in

relationship between the leaf nutrient stoichiometry and WUE in the leaf intercellular space and the atmospheric CO2 (Ci/Ca) by the

ecosystems. We tested the hypothesis that the leaf nutrients are following formula:

correlated with WUE across a diverse range of climates, plant life

C ‰ = a + (b − a) C /C (2)

forms and nutrient conditions. ( ) i a

where, a (4.4‰) represents the discrimination against CO2 dur-

2. Materials and methods ing the diffusion of CO2 through stomata, and b (27‰) represents

the discrimination associated with carboxylation (Farquhar and

2.1. Site description Richards, 1984; O’Leary, 1981).

The leaf conductance to water vapour (gH2O) is related to the

The study was conducted at eight geological sites in China. leaf conductance of CO2 (gCO2 ) as per the following equation:

With the exception of Ningshan County in the Qinling Moun-

gH O = 1.6 gCO (3)

tains, which belongs to the northern subtropical humid evergreen 2 2

126 W. Yan et al. / Ecological Engineering 87 (2016) 124–131

Fig. 1. Map of the study location in China, showing the sampling sites: Yangling, Yongshou, Tongchuan, Fuxian, Ansai, Mizhi and Shenmu distributed from south to north on

the Loess Plateau and Ningshan County in the southern part of the Qinling Mountains in China.

Table 1

Geographical and climatic conditions and number of distinct life forms at the eight study sites.

◦ ◦

Sampling site Geographical location Altitude(m) MAP(mm) MAT( C) ASH(h) AAT( C) Aridity index Trees Shrubs Herbs

◦ ◦

Ningshan 33 26 N,108 26 E 1614 1023 12.4 1668 3847 0.75 3 5 6

◦ ◦

Yangling 34 16 N,108 04 E 468 635 12.9 2163 4143 1.33 2 2 2

◦ ◦

Yongshou 34 49 N,108 02 E 1454 602 10.8 2166 3476 1.22 3 6 4

◦ ◦

Tongchuan 35 03 N,109 08 E 1324 555 12 2357 3413 1.16 5 11 5

◦ ◦

Fuxian 36 04 N108 32 E 1353 570 9.1 2492 3293 1.09 8 12 8

◦ ◦

Ansai 36 46 N109 15 E 1125 505 8.8 2397 3170 1.2 5 12 8

◦ ◦

Mizhi 37 51 N,110 10 E 1103 451 8.8 2731 3386 1.7 4 4 6

◦ ◦

Shenmu 38 47 N,110 21 E 1255 441 8.5 2876 3392 1.8 3 4 4

MAP: mean annual precipitation; MAT: mean annual temperature; ASH: annual sunshine hours; AAT: annual accumulated temperature.

Further, leaf net photosynthesis (A) is related to gH2O as Fick’s Unit, Foss Tecator, Hoganas, Sweden); the P concentration was

law: colourimetrically analysed using blue phosphor-molybdate (6505

UV spectrophotometer; Barloworld Scientiﬁc Ltd., Essex, UK); and

A gCO2 (Ca–Ci) (4)

the K concentration was quantiﬁed via ﬂame photometry (PE-5100

ZL atomic absorption spectrophotometer; Perkin Elmer, Waltham,

where, A is net photosynthesis, and gCO2 is leaf conductance of CO2

(Hietz et al., 2005; Penuelas˜ et al., 2011). USA) (Page, 1982). All chemical determinations were repeated

Given equations (2), (3) and (4), C can be converted to the three times with the same subsamples.

ratio A/gH2O (WUE) via the following equation (Hietz et al., 2005;

Osmond et al., 1980; Penuelas˜ et al., 2011):

2.5. Climate data

= +

C a (b–a) 1–1.6A/CagH2O (5)

The climate data for the study area, including the mean annual

which can be described as follows:

precipitation (MAP), mean annual temperature (MAT), annual sun-

13 shine hours (ASH), annual accumulated temperature (AAT) and

= = − −

WUE A/gH2O (b C)/1.6 (b a) (6)

aridity index, were provided by the Weather Bureau of Shaanxi

Province. Climate data for the eight sampling sites were fur-

2.4. Foliar chemical analysis

ther calculated using linear models and the latitude, longitude

◦ and altitude as variables, based on 20-year averaged observa-

The leaf samples were oven-dried at 105 C for 10 min, and then

◦ tion records obtained from the meteorological station in each

at 70 C to a constant mass. All leaves collected from the same

county.

species at a given site were mixed and subsequently ground into a

uniformly ﬁne powder using a plant sample mill, after which they

were sieved through a 1-mm mesh screen for chemical analysis. 2.6. Data analysis

To evaluate the N, P and K concentrations, the samples were ﬁrst

digested in a solution of H2SO4–HClO4, and the N concentration The Kolmogorov–Smirnov test was used to test for data nor-

was determined using a Kjeltec analyser (Kjeltec 2300 Analyser mality. The N, P and K mass ratios were used to represent the

W. Yan et al. / Ecological Engineering 87 (2016) 124–131 127

Table 2

stoichiometry. We calculated the Pearson correlation coefﬁcient

Correlation coefﬁcients between the leaf nutrient and climatic and geographical

to test the associations between the leaf nutrients (N, P and K)

variables.

and the climate data and WUE. Linear regression was performed

Leaf nutrient MAP MAT ASH Altitude Latitude

to analyse the relationships between the leaf nutrients and WUE.

All analyses were conducted using SPSS software (version 20.0, N −0.066 −0.019 0.064 −0.131 0.044

** ** **

USA). P 0.243 0.161 −0.232 0.118 −0.227

K 0.036 0.021 −0.062 −0.016 −0.056

* ** **

N:P −0.197 −0.164 0.239 −0.16 0.264

N:K −0.036 −0.011 0.081 −0.061 0.083

3. Results

K:P −0.121 −0.095 0.085 −0.083 0.103

MAP: mean annual precipitation; MAT: mean annual temperature; ASH: annual

3.1. Plant leaf nutrients, stoichiometry and WUE at the eight sites

sunshine hours.

Denote that the correlations are signiﬁcant at p = 0.05 (Pearson correlations)

The leaf N and K contents did not vary signiﬁcantly between Denote that the correlations are signiﬁcant at p = 0.01 (two-tailed Pearson cor-

relations).

the eight sites (Fig. 2), and both were highest at Mizhi. However,

there was a difference in the P concentration between sites, with

the highest and lowest values at Ningshan and Shenmu, respec- 3.2. The relationships of WUE with leaf nutrients and climatic

tively. The highest N:P ratio was measured at Shenmu, which was and geographical variables

18.98, whereas the lowest was 13.05, at Yongshou. The highest and

lowest N:K ratios, i.e., 2.32 and 1.52, were measured at Shenmu and The correlation coefﬁcients between the leaf nutrient with cli-

Fuxian, respectively, and with the exception of Shenmu, no signif- matic and geographical variables are presented in Table 2, and

icant differences were detected. The highest and lowest K:P ratios, the correlations between WUE and the nutrient or stoichiomet-

i.e., 12.36 and 7.76, were measured at Ansai and Yongshou, respec- ric ratios in all 132 plant samples are presented in Supplemental

tively, and with the exception of Ansai, no signiﬁcant differences Table 2. WUE was negatively correlated with the leaf P and K con-

were observed. tent but positively correlated with the leaf N:P and N:K ratios. We

WUE was signiﬁcantly lower at Ningshan than at the other sites; also observed that WUE was highly negatively correlated with MAP

the highest value was observed at Mizhi (Fig. 2). There were no sig- (p < 0.0001), MAT (p < 0.0001) and AAT (p < 0.0001), whereas it was

niﬁcant WUE differences between Yongshou, Tongchuan, Fuxian, signiﬁcantly positively correlated with ASH (p < 0.0001), the aridity

Ansai and Shenmu. index (p < 0.0001) and latitude (p < 0.0001). Additionally, WUE was

Fig. 2. Box-plots of the water use efﬁciency (WUE), leaf nutrient traits and stoichiometry of the eight sampling sites. Each box shows the interquartile ranges, maximum,

minimum, median and outliers (circles; 1.5–3 box lengths from the box edge). The lowercase letters above the vertical bars indicate signiﬁcant differences at p < 0.05 according

to the LSD test.

128 W. Yan et al. / Ecological Engineering 87 (2016) 124–131

Fig. 3. Relationships between leaf nutrients or stoichiometry and the water use efﬁciency (WUE) of the three plant life forms: (a) herbs; (b) shrubs; (c) trees.

Fig. 4. (a) The number of species under various types of nutrient limitation: N lim indicates N limitation; P lim indicates P limitation; and Co-lim indicates N and P limitation.

(b) Leaf N and P contents under the three types of nutrient limitation, in China and globally. The same lowercase letters below the vertical bars indicate non-signiﬁcant

differences at p < 0.05 according to the LSD test. (c–e) Relationship between leaf nutrients or stoichiometry and the water use efﬁciency (WUE) under three types of nutrient

limitation (c: N limitation; d: P limitation; e: Co-limitation).

signiﬁcantly negatively correlated with altitude (p < 0.0001, not P limitation exists when N:P > 16 and a co-limitation exists when

including Yangling; p = 0.0202, including Yangling) (Supplemental 14 < N:P < 16). The average N:P ratio across all plants was 15.23,

Table 1). and the high N:P ratios obtained in this study suggest that the

plants at these sites were more P- than N-limited. Thus, the plants

3.3. Relationship between leaf nutrient stoichiometry and WUE were divided into three groups according to their nutrient contents

across various life forms (Fig. 4a). The mean N:P ratios for the three groups were 10.82, 14.96

and 19.19, which corresponded to totals of 54, 28 and 50 plant

When the plant samples were divided into three life forms samples, respectively.

(herbs, shrubs and trees), a different result was obtained, as shown The average leaf N and P concentrations across all 132 species

−1

in Fig. 3. WUE exhibited a negative correlation with the leaf P and were 24.25 and 1.67 g kg , respectively; this N concentration is

K concentrations in the herbs, explaining 11.18% and 11.66% of the higher than that reported in other studies (Fig. 4b). The N con-

variation in P and K, respectively. In contrast, WUE was positively centrations measured under N limitation and co-limitation did

correlated with the N:P ratio in shrubs and trees, where it explained not differ but were signiﬁcantly lower than the N concentration

10.37% and 16.17% of the variation in the N:P ratio, respectively. measured under P limitation. Additionally, the P concentra-

tion measured under N limitation was signiﬁcantly higher than

3.4. Leaf nutrients and the relationship between leaf nutrient that measured under co-limitation and P limitation; no difference

stoichiometry and WUE under various nutrient conditions was observed in the P concentration between the co-limitation and

P limitation conditions.

The leaf N:P mass ratio has been used to detect conditions of In the plants growing under N-limited conditions (Fig. 4c), WUE

plant N or P limitation (i.e., an N limitation exists when N:P < 14, a was positively correlated with the leaf N:P ratio (p = 0.0015) and

W. Yan et al. / Ecological Engineering 87 (2016) 124–131 129

negatively correlated with the P concentration (p = 0.0262). When annual species. Light attenuation may be the main cause of the

the plants were limited by P (N:P > 16), WUE was not correlated lower WUE observed in the herbs, and plants in the upper canopy

with N, P or the N:P ratio (Fig. 4d), whereas it showed a negative exhibit higher ␦ C values than plants in the lower canopy (Brooks

correlation with the K concentration (p = 0.0256). When the plants et al., 1997). This phenomenon is most likely mediated through

were growing under co-limitation conditions (Fig. 4e), the rela- canopy sheltering effects, including light attenuation and increased

tionship between the N:P ratio and WUE was stronger, with WUE relative humidity (Dawson et al., 2002). Additionally, we observed

explaining 34.01% of the variation in the N:P ratio (p = 0.0011). that the nutrient contents of the herbs were higher than those of the

shrubs and trees (Supplemental Table 3). This result is consistent

with the ﬁndings of Han et al. (2005) and Güsewell and Koerselman

4. Discussion (2002), who indicated that short-lived, fast-growing species exhibit

higher N and P contents than long-lived, slow-growing species.

Many studies have been conducted to investigate the spatial Further, Wright et al. (2004) reported that shrubs and trees had

patterns of leaf nutrient traits and their relationships with the cli- a higher leaf mass per area and longer lifespan but lower N and P

mate at local, regional and global scales to develop a processing concentrations.

link between biogeographical models and biogeochemical models The leaf N:P ratios in the herbs, shrubs and trees were 14.88,

and to further understand the mechanisms underlying vegeta- 15.92 and 14.69, respectively (Supplemental Table 3). However, in

tion dynamics in response to global change (Hedin et al., 2003; contrast to the ﬁndings for the shrubs and trees, we did not observe

McGroddy et al., 2004; Reich and Oleksyn, 2004; Han et al., 2005; a correlation between the leaf-level WUE and N:P ratio in the herbs

Kerkhoff et al., 2005). Reich and Oleksyn (2004) reported that P (Fig. 3), indicating that the correlation between growth and WUE

and N decrease and the N:P ratio increases as latitude decreases was weak in the herbs despite the strong correlation found in the

(or MAT increases). Kerkhoff et al. (2005) demonstrated that leaf N shrubs and trees. Furthermore, we observed a negative correlation

and P were not related to latitude but that the N:P ratio decreased between WUE and leaf P in the herbs (Fig. 3a), which is consis-

markedly with increasing latitude. McGroddy et al. (2004) also tent with the ﬁndings of Raven et al. (2009), who reported that an

found that the leaf N:P ratio decreased signiﬁcantly with increasing increased P concentration sometimes reduced WUE. In addition,

latitude. Han et al. (2005) reported that leaf N and P were signiﬁ- the negative correlation between WUE and leaf K found in the herbs

cantly correlated with latitude and MAT, whereas the N:P ratios did may be caused by limited water resources. Water is an important

not show signiﬁcant changes. In this study, we observed that the limiting factor for plant growth in the study area, and plants suffer-

leaf N and K and N:K and K:P ratios were not related to MAP, MAT, ing from environmental stresses, such as drought, show a greater

ASH, altitude or latitude. However, the P concentration and the N:P internal requirement for K (Cakmak, 2005). Consequently, the herbs

ratio were both correlated with MAP, ASH and latitude (Table 2). may have accumulated more K to prevent a water deﬁciency. A

The plant ␦ C value, which can be inﬂuenced by both nutrients positive correlation between WUE and N:P ratio was observed in

(N and P) and environmental factors, including precipitation, tem- the shrubs and trees. This ﬁnding is consistent with the results

perature, humidity, light, atmospheric CO2 concentration and soil presented by Cernusak et al. (2007b) and Cernusak et al. (2010),

water content (Dawson et al., 2002), has been shown to be a useful indicating that the relationship between the N:P ratio and WUE

index for assessing plant WUE (Farquhar et al., 1989). The lowest may be general, which has important implications for ecosystem

WUE was recorded at Ningshan, compared with the highest values analysis because it links the plant N:P stoichiometry with plant

at Shenmu and Mizhi (Fig. 2). The various WUE values observed transpiration and thus integrates the nutrient and hydrological

among the different habitats may be related to the inherent phys- cycles.

iological variation in leaf gas exchange characteristics and varying The N:P ratio of vegetation is considered to be a reliable indicator

biological and environmental conditions across sites. For example, of nutrient limitation (Koerselman and Meuleman, 1996; Güsewell

air temperature and humidity can inﬂuence the atmospheric satu- and Koerselman, 2002; Tessier and Raynal, 2003) and has been

ration deﬁcit, which, in turn, can inﬂuence evapotranspiration and successfully applied to terrestrial plant species (Han et al., 2005;

ultimately lead to a difference in WUE (Ponton et al., 2006). Wang and Moore, 2014), bryophytes (Bragazza et al., 2004; Jirousekˇ

When using data from all species, we observed a positive corre- et al., 2011) and vascular plants (Güsewell and Koerselman, 2002;

lation between the leaf N:P ratio and WUE, which is consistent with Olde Venterink et al., 2003). When the plants were N limited, the

the work of Cernusak et al. (2007b) and Cernusak et al. (2010), who P content was signiﬁcantly higher, but the N content was lower.

observed a positive correlation between the N:P ratio and WUE. The lower N:P value observed under N limitation was mainly

In addition, we observed that WUE was negatively correlated with caused by a higher P content, but the higher N:P value observed

the P and K contents and positively correlated with the N:K ratio. under P limitation was mainly caused by a higher N content (Sup-

However, no correlation with leaf N was observed (Supplemental plemental Table 4). The N content measured in all plants was

Table 2), although many studies have reported that leaf-level WUE higher than the average value in China and worldwide, whereas

generally increases in response to increasing leaf N concentrations the P content was higher than the average measured in China

(Duursma and Marshall, 2006; Cernusak et al., 2007b; Raven et al., but lower than the global average (Fig. 4b) (Reich and Oleksyn,

2009). Our results are consistent with those reported by Cernusak 2004; Han et al., 2005). The relationship between leaf nutrients

et al. (2011), who found that WUE was more strongly positively and WUE changes under various nutrient limitation conditions.

correlated with the N:P ratio than with leaf N, which indicates that The WUE values were positively and negatively correlated with

the N:P ratio is a more likely modulator of WUE. the N:P ratio and P concentration, respectively, when the plants

Water and nutrient availability are the most important limiting were growing under N limitation conditions (Fig. 4c), indicating

factors for plant growth and have been observed to affect WUE that a higher N would increase WUE. This ﬁnding is consistent with

(Brooks et al., 1997). The leaf-level WUE of the shrubs and trees previous studies, which showed that WUE generally increases in

was signiﬁcantly higher than that of the herbs, demonstrating that response to increased N concentrations (Duursma and Marshall,

adaptive strategies to soil water in different life forms differ greatly. 2006; Cernusak et al., 2007b). In general, an increased P sup-

Speciﬁcally, the shrubs and trees performed better than the herbs ply can improve plant growth conditions when plants are limited

under the same soil moisture content, which is consistent with the by P. However, we did not observe a relationship between WUE

ﬁndings of Brooks et al. (1997) and Smedley et al. (1991), who indi- and leaf P. Additionally, the N content under P limitation condi-

cated that the WUE of perennial species was higher than that of tions was much higher than under N limitation conditions, and a

130 W. Yan et al. / Ecological Engineering 87 (2016) 124–131

supraoptimal N level has been associated with low transpiration Cakmak, I., 2005. The role of potassium in alleviating detrimental effects of abiotic

stresses in plants. J. Plant Nutr. Soil Sci. 168, 521–530.

(Wilkinson et al., 2007), which decreases the uptake of soil water

Cernusak, L.A., Aranda, J., Marshall, J.D., Winter, K., 2007a. Large variation in

and P by affecting the mass ﬂow of the soil solution (Cernusak

whole–plant water–use efﬁciency among tropical tree species. New Phytol. 173,

et al., 2007b; Craine et al., 2008; Cramer et al., 2008; Cernusak 294–305.

Cernusak, L.A., Winter, K., Aranda, J., Turner, B.L., Marshall, J.D., 2007b. Transpiration

et al., 2010). The plant K content is also related to environmental

efﬁciency of a tropical pioneer tree (Ficus insipida) in relation to soil fertility. J.

stresses, such as drought (Cakmak, 2005). We observed a negative

Exp. Bot. 58, 3549–3566.

relationship between K content and WUE (Fig. 4d), but the under- Cernusak, L.A., Winter, K., Turner, B.L., 2010. Leaf nitrogen to phosphorus ratios

of tropical trees: experimental assessment of physiological and environmental

lying mechanism requires further veriﬁcation. When plants grow

controls. New Phytol. 185, 770–779.

under co-limitation conditions (Fig. 4e), N and P should be in a bal-

Cernusak, L.A., Winter, K., Turner, B.L., 2011. Transpiration modulates phosphorus

anced supply, with an N:P ratio between 14 and 16 (Koerselman acquisition in tropical tree seedlings. Tree Physiol. 31, 878–885.

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WUE was strong, with WUE explaining 34.01% of the variation in the

Craine, J.M., Morrow, C., Stock, W.D., 2008. Nutrient concentration ratios and co-

N:P ratio. This result is consistent with the work of Cernusak et al.

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42% and 53%, respectively, of the variation in the N:P ratio, indicat-

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5. Concluding remarks

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Competing ﬁnancial interests: The authors declare no compet-

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Science Foundation of China (41390463) and the National Key Tech-

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nology R&D Programme (2015BAC01B03).

the stable carbon isotope composition, productivity and water use efﬁciency

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