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Ecological Engineering 87 (2016) 124–131

Contents lists available at ScienceDirect

Ecological Engineering

jo ur nal home p ag e: www.elsev ier .com/ locate /eco leng

inking plant leaf nutrients/stoichiometry to water use efficiency onhe Loess Plateau in China

eiming Yana, Yangquanwei Zhonga, Shuxia Zhenga,b, Zhouping Shangguana,∗

State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling, Shaanxi 712100, PR ChinaState Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

r t i c l e i n f o

rticle history:eceived 30 January 2015eceived in revised form 22 October 2015ccepted 18 November 2015

a b s t r a c t

Nutrient and hydrological cycles are tightly coupled in ecosystems. However, little is known about therelationship between leaf nutrient stoichiometry (nutrient mass ratios) and water use efficiency (WUE)in ecosystems. To fill this knowledge gap, we examined 132 plant samples distributed from the QinlingMountains to the north of the Loess Plateau of China and observed the relationship between leaf nutrient

eywords:eaf nutrientstoichiometryUE

ife formutrient limitation

stoichiometry and WUE in various ecosystems. Our findings suggest that a positive correlation existsbetween the leaf nitrogen:phosphorus (N:P) ratio and WUE, and this relationship is sensitive to plant lifeforms and growth conditions. Additionally, potassium (K) was related to WUE in herbs and plants underP limitation. These results link plant nutrient stoichiometry to hydraulic processes in terrestrial plantsand provide useful information for ecologists studying nutrient and hydrological cycles in ecosystems.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Carbon (C), nitrogen (N), phosphorus (P) and potassium (K) areonsidered essential elements for plant growth and play a vital rolen plant functions (Marschner and Marschner, 2012). C provideshe structural basis of the plant, constituting a relatively stable0% of the dry mass; N is an important constituent of proteins andlays an essential role in all enzymatic activities; and P is involved

n energy transfer in cells. Additionally, P and N are importanttructural elements in nucleic acids (Ågren, 2008; Marschner andarschner, 2012). These three elements (C, N and P) are strongly

oupled in terms of their biochemical functions. N and P, whichre required for plant growth in relatively large quantities, arelassified as macronutrients and cannot be substituted with otherlements in metabolic functions (Aerts and Chapin, 1999; Ågren,008). K is involved in the plant–water relationship (Babita et al.,010) through plant osmotic control and improvement of stoma-al function (Sangakkara et al., 2000; Babita et al., 2010; Laus et al.,

011; Rivas-Ubach et al., 2012).

Plant growth requires photosynthetic products, and proteins areequired for photosynthesis and growth. In addition, ribosomes are

∗ Correspondence to: Xinong Rd. 26, Institute of Soil and Water Conservation,angling, Shaanxi, 712100, PR China. Tel.: +86 29 87019107; fax: +86 29 87012210.

E-mail addresses: yanweiming0110@126.com (W. Yan), zyqwjay@gmail.comY. Zhong), zsx@ibcas.ac.cn (S. Zheng), shangguan@ms.iswc.ac.cn (Z. Shangguan).

ttp://dx.doi.org/10.1016/j.ecoleng.2015.11.034925-8574/© 2015 Elsevier B.V. All rights reserved.

required for the synthesis of proteins, which contain large amountsof N and P (Cernusak et al., 2010). The N:P ratio (ratio of the N toP concentration) in terrestrial plant leaves can provide importantinformation about potential nutrient limitation, which may affectprimary productivity (Ågren, 2008; Cernusak et al., 2010). The bal-ance of N and P can influence the growth rate of plants, and theleaf N:P mass ratio has been widely used as an indicator of N or Pdeficit. For example, based on studies conducted on European wet-land plants, it has been suggested that an N:P ratio above a giventhreshold (16 on a mass basis) indicates P limitation of biomass pro-duction, whereas a ratio below a given threshold (c. 14 on a massbasis) indicates N limitation (Koerselman and Meuleman, 1996;Aerts and Chapin, 1999; Tessier and Raynal, 2003; Güsewell, 2004;Reich and Oleksyn, 2004; Cernusak et al., 2010). This parameteroffers a powerful tool for ecological and physiological investiga-tions by providing a straightforward means of characterising therelative availability of N vs. P (Cernusak et al., 2010). Relation-ships between N:P ratios and vegetation characteristics have alsobeen used to describe functional differences between naturally N-or P-limited plant communities and their responses to environ-mental change or human management (Tessier and Raynal, 2003;Güsewell, 2004; Reich and Oleksyn, 2004). K is also important, butless information is available regarding the quantitative require-

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

In many terrestrial ecosystems, soil water is variable and oftenthe most important limiting soil resource. Therefore, plant wateruse efficiency (WUE) is of major importance for the survival,

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roductivity and fitness of individual plants (Ponton et al., 2006)nd is a measure of plant performance that has long been of inter-st to agronomists, foresters and ecologists (Cernusak et al., 2007a;aven et al., 2009). In plant physiological ecology, leaf carbon-13�13C) data are a useful index for assessing WUE when the leaf-o-air vapour pressure difference is known (Farquhar et al., 1989;awson et al., 2002), and these data have been widely used to eval-ate plant WUE under various environmental conditions and inesponse to climatic variables (Hietz et al., 2005; Silva et al., 2009;ock et al., 2011; Penuelas et al., 2011). Leaf �13C data not onlyrecisely reflect water conditions but also reflect the physiologi-al status of the plant and have been proven to be the best meansf investigating WUE. As one of the most important physiologicalharacteristics involved in plant growth, WUE is considered to be anbjective index for evaluating water conditions (Cabrera–Bosquett al., 2007) and drought tolerance characteristics and is capablef providing a theoretical basis for studying such characteristicsn specific environments (Livingston et al., 1999; Condon et al.,004). In addition, transpiration plays a role in modulating nutrientptake by delivering nutrients to root surfaces through mass flowCernusak et al., 2011). However, little is known about the relation-hip between plant WUE and nutrients in ecosystems. Therefore,tudying the relationship between WUE and nutrients across plantife forms and nutrient conditions would help us to better under-tand ecosystems.

It has been reported that the leaf N:P ratio is positively correlatedith plant WUE in seedlings of tropical pioneer tree species, and itas been suggested that the N:P ratio is expected to be correlatedith WUE according to the following argument (Cernusak et al.,

007b): positive increases in plant C require N-rich proteins forlants to assimilate C and grow, as shown in the following equationÅgren, 2004): dC/dt = ˚CN NP (C, amount of carbon; t, time; �CN,ate factor; NP, amount of nitrogen in proteins used for growth). Inddition, P-rich ribosomes are required for protein synthesis. Themounts of N and P in plants exceed those required for growth androtein synthesis, but a balance between N and P is necessary forormal plant functioning and could affect the normal growth oflants. Mass flow, which partly depends on plant transpiration, ishe process by which P in the soil solution is transported to theurface of the roots, where it can subsequently be absorbed by thelant (Cernusak et al., 2007b; Craine et al., 2008; Cramer et al., 2008;ernusak et al., 2010). Thus, C uptake is correlated with NP, and Pptake is correlated with transpiration (T); consequently, the N:Patio is correlated with the C:T ratio, which is equivalent to WUECernusak et al., 2010).

To the best of our knowledge, no previous study has verifiedhe relationship between leaf nutrients, stoichiometry and WUE incosystems. In the present study, the relationship between the leafutrient stoichiometry and WUE of various plant life forms underifferent nutrient conditions was analysed by examining 132 plantamples (belonging to three different plant life forms) at eight geo-ogical sites distributed from the Qinling Mountains to the north ofhe Loess Plateau of China. We hypothesised that there is a positiveelationship between the leaf nutrient stoichiometry and WUE incosystems. We tested the hypothesis that the leaf nutrients areorrelated with WUE across a diverse range of climates, plant lifeorms and nutrient conditions.

. Materials and methods

.1. Site description

The study was conducted at eight geological sites in China.ith the exception of Ningshan County in the Qinling Moun-

ains, which belongs to the northern subtropical humid evergreen

ering 87 (2016) 124–131 125

broadleaf forest located at 108◦26′E and 33◦26′N, all of the otherinvestigated stands, i.e., Yangling, Yongshou, Tongchuan, Fuxian,Ansai, Mizhi and Shenmu, are distributed from south to north onthe Loess Plateau of China (Fig. 1). These stands are located at34◦16′–38◦47′N and 108◦02′–110◦21′E within the temperate zone,and the vegetation type ranges from semi-humid forests to ariddesert grasslands. The altitude, latitude and longitude were deter-mined using a global positioning system (GPS) at the sampling sites.The sampling sites were located far from human habitation (morethan 1 km) to minimise the influence of human disturbance. Thedetailed geographical and climatic conditions of the eight samplingsites are summarised in Table 1.

2.2. Plant materials

A total of 132 plant samples (118 from the Loess Plateau and14 from the Qinling Mountains) belonging to 39 different speciesand 17 families, including 10 types of trees, 20 types of shrubsand 10 types of herbaceous species, were collected at the eightsites. Species from the 3 different plant life forms (herbs, shrubsand trees) were selected based on the following criteria: the targetspecies should be the dominant species and relatively abundantat each site. Sun foliage samples were mainly collected from theupper canopies. Three to five healthy and fully expanded leavesfrom individual plants were randomly selected, and each sampleincluded leaves from 4–5 individual plants of the same species.

2.3. �13C (carbon isotope discrimination) and calculation ofWUE

The leaf samples were ultrasonically washed with distilledwater, air-dried, oven-dried at 70 ◦C for at least 48 h to a con-stant weight, then ground to a fine powder using a plant samplemill (Cyclotec sample Mill 1093; FOSS Tecator, Hoganas, Sweden),and finally sieved through a 1-mm mesh screen. �13C was ana-lysed using a MAT-251 mass spectrometer (Finnegan, San Jose, USA)at the State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science, Chinese Academy of Sciences. A 3–5 mgportion of each treated sample was placed in a vacuum quartz tube,mixed with an activator and desiccant, and then oxidised underan oxygen flux at 850 ◦C. The CO2 produced under these condi-tions was cryogenically purified using both a liquid N trap and adry ice–ethanol trap. Then, according to the PDB (belemnite fromthe Pee Dee Formation) standard, the C isotope of CO2 was analysedusing a MAT-251 mass spectrometer with a precision of <0.02%. Theresulting �13C value was determined using the following equation:

�13C (‰) = [(

Rsample–Rstandard

)/Rstandard × 1000] (1)

in which Rsample and Rstandard are the 13C/12C ratios in the samplesand the controls, respectively (Farquhar et al., 1989). During CO2fixation by leaves, �13C is related to the ratio between the CO2 inthe leaf intercellular space and the atmospheric CO2 (Ci/Ca) by thefollowing formula:

�13C (‰) = a + (b − a)(

Ci/Ca

)(2)

where, a (4.4‰) represents the discrimination against 13CO2 dur-ing the diffusion of CO2 through stomata, and b (27‰) representsthe discrimination associated with carboxylation (Farquhar andRichards, 1984; O’Leary, 1981).

The leaf conductance to water vapour (gH2O) is related to theleaf conductance of CO2 (gCO2 ) as per the following equation:

gH2O = 1.6 gCO2 (3)

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

Table 1Geographical 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 6Yangling 34◦16′N,108◦04′E 468 635 12.9 2163 4143 1.33 2 2 2Yongshou 34◦49′N,108◦02′E 1454 602 10.8 2166 3476 1.22 3 6 4Tongchuan 35◦03′N,109◦08′E 1324 555 12 2357 3413 1.16 5 11 5Fuxian 36◦04′N108◦32′E 1353 570 9.1 2492 3293 1.09 8 12 8Ansai 36◦46′N109◦15′E 1125 505 8.8 2397 3170 1.2 5 12 8Mizhi 37◦51′N,110◦10′E 1103 451 8.8 2731 3386 1.7 4 4 6

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AP: mean annual precipitation; MAT: mean annual temperature; ASH: annual su

Further, leaf net photosynthesis (A) is related to gH2O as Fick’saw:

= gCO2 (Ca–Ci) (4)

here, A is net photosynthesis, and gCO2 is leaf conductance of CO2Hietz et al., 2005; Penuelas et al., 2011).

Given equations (2), (3) and (4), �13C can be converted to theatio A/gH2O (WUE) via the following equation (Hietz et al., 2005;smond et al., 1980; Penuelas et al., 2011):

13C = a + (b–a)(

1–1.6A/CagH2O)

(5)

which can be described as follows:

UE = A/gH2O = (b − �13C)/1.6 (b − a) (6)

.4. Foliar chemical analysis

The leaf samples were oven-dried at 105 ◦C for 10 min, and thent 70 ◦C to a constant mass. All leaves collected from the samepecies at a given site were mixed and subsequently ground into aniformly fine powder using a plant sample mill, after which they

ere sieved through a 1-mm mesh screen for chemical analysis.

o evaluate the N, P and K concentrations, the samples were firstigested in a solution of H2SO4–HClO4, and the N concentrationas determined using a Kjeltec analyser (Kjeltec 2300 Analyser

2876 3392 1.8 3 4 4

hours; AAT: annual accumulated temperature.

Unit, Foss Tecator, Hoganas, Sweden); the P concentration wascolourimetrically analysed using blue phosphor-molybdate (6505UV spectrophotometer; Barloworld Scientific Ltd., Essex, UK); andthe K concentration was quantified via flame photometry (PE-5100ZL atomic absorption spectrophotometer; Perkin Elmer, Waltham,USA) (Page, 1982). All chemical determinations were repeatedthree times with the same subsamples.

2.5. Climate data

The climate data for the study area, including the mean annualprecipitation (MAP), mean annual temperature (MAT), annual sun-shine hours (ASH), annual accumulated temperature (AAT) andaridity index, were provided by the Weather Bureau of ShaanxiProvince. Climate data for the eight sampling sites were fur-ther calculated using linear models and the latitude, longitudeand altitude as variables, based on 20-year averaged observa-tion records obtained from the meteorological station in eachcounty.

2.6. Data analysis

The Kolmogorov–Smirnov test was used to test for data nor-mality. The N, P and K mass ratios were used to represent the

ngineering 87 (2016) 124–131 127

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Table 2Correlation coefficients between the leaf nutrient and climatic and geographicalvariables.

Leaf nutrient MAP MAT ASH Altitude Latitude

N −0.066 −0.019 0.064 −0.131 0.044P 0.243** 0.161 −0.232** 0.118 −0.227**

K 0.036 0.021 −0.062 −0.016 −0.056N:P −0.197* −0.164 0.239** −0.16 0.264**

N:K −0.036 −0.011 0.081 −0.061 0.083K:P −0.121 −0.095 0.085 −0.083 0.103

MAP: mean annual precipitation; MAT: mean annual temperature; ASH: annualsunshine hours.

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W. Yan et al. / Ecological E

toichiometry. We calculated the Pearson correlation coefficiento test the associations between the leaf nutrients (N, P and K)nd the climate data and WUE. Linear regression was performedo analyse the relationships between the leaf nutrients and WUE.ll analyses were conducted using SPSS software (version 20.0,SA).

. Results

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

The leaf N and K contents did not vary significantly betweenhe eight sites (Fig. 2), and both were highest at Mizhi. However,here was a difference in the P concentration between sites, withhe highest and lowest values at Ningshan and Shenmu, respec-ively. The highest N:P ratio was measured at Shenmu, which was8.98, whereas the lowest was 13.05, at Yongshou. The highest and

owest N:K ratios, i.e., 2.32 and 1.52, were measured at Shenmu anduxian, respectively, and with the exception of Shenmu, no signif-cant differences were detected. The highest and lowest K:P ratios,.e., 12.36 and 7.76, were measured at Ansai and Yongshou, respec-ively, and with the exception of Ansai, no significant differencesere observed.

WUE was significantly lower at Ningshan than at the other sites;he highest value was observed at Mizhi (Fig. 2). There were no sig-ificant WUE differences between Yongshou, Tongchuan, Fuxian,nsai and Shenmu.

ig. 2. Box-plots of the water use efficiency (WUE), leaf nutrient traits and stoichiometrinimum, median and outliers (circles; 1.5–3 box lengths from the box edge). The lowerca

o the LSD test.

* Denote that the correlations are significant at p = 0.05 (Pearson correlations)** Denote that the correlations are significant at p = 0.01 (two-tailed Pearson cor-

relations).

3.2. The relationships of WUE with leaf nutrients and climaticand geographical variables

The correlation coefficients between the leaf nutrient with cli-matic and geographical variables are presented in Table 2, andthe correlations between WUE and the nutrient or stoichiomet-ric ratios in all 132 plant samples are presented in SupplementalTable 2. WUE was negatively correlated with the leaf P and K con-tent but positively correlated with the leaf N:P and N:K ratios. We

also observed that WUE was highly negatively correlated with MAP(p < 0.0001), MAT (p < 0.0001) and AAT (p < 0.0001), whereas it wassignificantly positively correlated with ASH (p < 0.0001), the aridityindex (p < 0.0001) and latitude (p < 0.0001). Additionally, WUE was

y of the eight sampling sites. Each box shows the interquartile ranges, maximum,se letters above the vertical bars indicate significant differences at p < 0.05 according

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-significantd rientsl

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ifferences at p < 0.05 according to the LSD test. (c–e) Relationship between leaf nutimitation (c: N limitation; d: P limitation; e: Co-limitation).

ignificantly negatively correlated with altitude (p < 0.0001, notncluding Yangling; p = 0.0202, including Yangling) (Supplementalable 1).

.3. Relationship between leaf nutrient stoichiometry and WUEcross various life forms

When the plant samples were divided into three life formsherbs, shrubs and trees), a different result was obtained, as shownn Fig. 3. WUE exhibited a negative correlation with the leaf P and

concentrations in the herbs, explaining 11.18% and 11.66% of theariation in P and K, respectively. In contrast, WUE was positivelyorrelated with the N:P ratio in shrubs and trees, where it explained0.37% and 16.17% of the variation in the N:P ratio, respectively.

.4. Leaf nutrients and the relationship between leaf nutrient

toichiometry and WUE under various nutrient conditions

The leaf N:P mass ratio has been used to detect conditions oflant N or P limitation (i.e., an N limitation exists when N:P < 14, a

or stoichiometry and the water use efficiency (WUE) under three types of nutrient

P limitation exists when N:P > 16 and a co-limitation exists when14 < N:P < 16). The average N:P ratio across all plants was 15.23,and the high N:P ratios obtained in this study suggest that theplants at these sites were more P- than N-limited. Thus, the plantswere divided into three groups according to their nutrient contents(Fig. 4a). The mean N:P ratios for the three groups were 10.82, 14.96and 19.19, which corresponded to totals of 54, 28 and 50 plantsamples, respectively.

The average leaf N and P concentrations across all 132 specieswere 24.25 and 1.67 g kg−1, respectively; this N concentration ishigher than that reported in other studies (Fig. 4b). The N con-centrations measured under N limitation and co-limitation didnot differ but were significantly lower than the N concentrationmeasured under P limitation. Additionally, the P concentra-tion measured under N limitation was significantly higher thanthat measured under co-limitation and P limitation; no difference

was observed in the P concentration between the co-limitation andP limitation conditions.

In the plants growing under N-limited conditions (Fig. 4c), WUEwas positively correlated with the leaf N:P ratio (p = 0.0015) and

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egatively correlated with the P concentration (p = 0.0262). Whenhe plants were limited by P (N:P > 16), WUE was not correlatedith N, P or the N:P ratio (Fig. 4d), whereas it showed a negative

orrelation with the K concentration (p = 0.0256). When the plantsere growing under co-limitation conditions (Fig. 4e), the rela-

ionship between the N:P ratio and WUE was stronger, with WUExplaining 34.01% of the variation in the N:P ratio (p = 0.0011).

. Discussion

Many studies have been conducted to investigate the spatialatterns of leaf nutrient traits and their relationships with the cli-ate at local, regional and global scales to develop a processing

ink between biogeographical models and biogeochemical modelsnd to further understand the mechanisms underlying vegeta-ion dynamics in response to global change (Hedin et al., 2003;

cGroddy et al., 2004; Reich and Oleksyn, 2004; Han et al., 2005;erkhoff et al., 2005). Reich and Oleksyn (2004) reported that Pnd N decrease and the N:P ratio increases as latitude decreasesor MAT increases). Kerkhoff et al. (2005) demonstrated that leaf Nnd P were not related to latitude but that the N:P ratio decreasedarkedly with increasing latitude. McGroddy et al. (2004) also

ound that the leaf N:P ratio decreased significantly with increasingatitude. Han et al. (2005) reported that leaf N and P were signifi-antly correlated with latitude and MAT, whereas the N:P ratios didot show significant changes. In this study, we observed that the

eaf N and K and N:K and K:P ratios were not related to MAP, MAT,SH, altitude or latitude. However, the P concentration and the N:Patio were both correlated with MAP, ASH and latitude (Table 2).

The plant �13C value, which can be influenced by both nutrientsN and P) and environmental factors, including precipitation, tem-erature, humidity, light, atmospheric CO2 concentration and soilater content (Dawson et al., 2002), has been shown to be a useful

ndex for assessing plant WUE (Farquhar et al., 1989). The lowestUE was recorded at Ningshan, compared with the highest values

t Shenmu and Mizhi (Fig. 2). The various WUE values observedmong the different habitats may be related to the inherent phys-ological variation in leaf gas exchange characteristics and varyingiological and environmental conditions across sites. For example,ir temperature and humidity can influence the atmospheric satu-ation deficit, which, in turn, can influence evapotranspiration andltimately lead to a difference in WUE (Ponton et al., 2006).

When using data from all species, we observed a positive corre-ation between the leaf N:P ratio and WUE, which is consistent withhe work of Cernusak et al. (2007b) and Cernusak et al. (2010), whobserved a positive correlation between the N:P ratio and WUE.n addition, we observed that WUE was negatively correlated withhe P and K contents and positively correlated with the N:K ratio.owever, no correlation with leaf N was observed (Supplementalable 2), although many studies have reported that leaf-level WUEenerally increases in response to increasing leaf N concentrationsDuursma and Marshall, 2006; Cernusak et al., 2007b; Raven et al.,009). Our results are consistent with those reported by Cernusakt al. (2011), who found that WUE was more strongly positivelyorrelated with the N:P ratio than with leaf N, which indicates thathe N:P ratio is a more likely modulator of WUE.

Water and nutrient availability are the most important limitingactors for plant growth and have been observed to affect WUEBrooks et al., 1997). The leaf-level WUE of the shrubs and treesas significantly higher than that of the herbs, demonstrating that

daptive strategies to soil water in different life forms differ greatly.

pecifically, the shrubs and trees performed better than the herbsnder the same soil moisture content, which is consistent with thendings of Brooks et al. (1997) and Smedley et al. (1991), who indi-ated that the WUE of perennial species was higher than that of

ering 87 (2016) 124–131 129

annual species. Light attenuation may be the main cause of thelower WUE observed in the herbs, and plants in the upper canopyexhibit higher �13C values than plants in the lower canopy (Brookset al., 1997). This phenomenon is most likely mediated throughcanopy sheltering effects, including light attenuation and increasedrelative humidity (Dawson et al., 2002). Additionally, we observedthat the nutrient contents of the herbs were higher than those of theshrubs and trees (Supplemental Table 3). This result is consistentwith the findings of Han et al. (2005) and Güsewell and Koerselman(2002), who indicated that short-lived, fast-growing species exhibithigher N and P contents than long-lived, slow-growing species.Further, Wright et al. (2004) reported that shrubs and trees hada higher leaf mass per area and longer lifespan but lower N and Pconcentrations.

The leaf N:P ratios in the herbs, shrubs and trees were 14.88,15.92 and 14.69, respectively (Supplemental Table 3). However, incontrast to the findings for the shrubs and trees, we did not observea correlation between the leaf-level WUE and N:P ratio in the herbs(Fig. 3), indicating that the correlation between growth and WUEwas weak in the herbs despite the strong correlation found in theshrubs and trees. Furthermore, we observed a negative correlationbetween WUE and leaf P in the herbs (Fig. 3a), which is consis-tent with the findings of Raven et al. (2009), who reported that anincreased P concentration sometimes reduced WUE. In addition,the negative correlation between WUE and leaf K found in the herbsmay be caused by limited water resources. Water is an importantlimiting factor for plant growth in the study area, and plants suffer-ing from environmental stresses, such as drought, show a greaterinternal requirement for K (Cakmak, 2005). Consequently, the herbsmay have accumulated more K to prevent a water deficiency. Apositive correlation between WUE and N:P ratio was observed inthe shrubs and trees. This finding is consistent with the resultspresented by Cernusak et al. (2007b) and Cernusak et al. (2010),indicating that the relationship between the N:P ratio and WUEmay be general, which has important implications for ecosystemanalysis because it links the plant N:P stoichiometry with planttranspiration and thus integrates the nutrient and hydrologicalcycles.

The N:P ratio of vegetation is considered to be a reliable indicatorof nutrient limitation (Koerselman and Meuleman, 1996; Güsewelland Koerselman, 2002; Tessier and Raynal, 2003) and has beensuccessfully applied to terrestrial plant species (Han et al., 2005;Wang and Moore, 2014), bryophytes (Bragazza et al., 2004; Jirouseket al., 2011) and vascular plants (Güsewell and Koerselman, 2002;Olde Venterink et al., 2003). When the plants were N limited, theP content was significantly higher, but the N content was lower.The lower N:P value observed under N limitation was mainlycaused by a higher P content, but the higher N:P value observedunder P limitation was mainly caused by a higher N content (Sup-plemental Table 4). The N content measured in all plants washigher than the average value in China and worldwide, whereasthe P content was higher than the average measured in Chinabut lower than the global average (Fig. 4b) (Reich and Oleksyn,2004; Han et al., 2005). The relationship between leaf nutrientsand WUE changes under various nutrient limitation conditions.The WUE values were positively and negatively correlated withthe N:P ratio and P concentration, respectively, when the plantswere growing under N limitation conditions (Fig. 4c), indicatingthat a higher N would increase WUE. This finding is consistent withprevious studies, which showed that WUE generally increases inresponse to increased N concentrations (Duursma and Marshall,2006; Cernusak et al., 2007b). In general, an increased P sup-

ply can improve plant growth conditions when plants are limitedby P. However, we did not observe a relationship between WUEand leaf P. Additionally, the N content under P limitation condi-tions was much higher than under N limitation conditions, and a

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C

30 W. Yan et al. / Ecological E

upraoptimal N level has been associated with low transpirationWilkinson et al., 2007), which decreases the uptake of soil waternd P by affecting the mass flow of the soil solution (Cernusakt al., 2007b; Craine et al., 2008; Cramer et al., 2008; Cernusakt al., 2010). The plant K content is also related to environmentaltresses, such as drought (Cakmak, 2005). We observed a negativeelationship between K content and WUE (Fig. 4d), but the under-ying mechanism requires further verification. When plants grownder co-limitation conditions (Fig. 4e), N and P should be in a bal-nced supply, with an N:P ratio between 14 and 16 (Koerselmannd Meuleman, 1996). The relationship between the N:P ratio andUE was strong, with WUE explaining 34.01% of the variation in the

:P ratio. This result is consistent with the work of Cernusak et al.2010) and Matimati et al. (2013), who found that WUE explained2% and 53%, respectively, of the variation in the N:P ratio, indicat-

ng that plant nutrient stoichiometry exhibits a strong relationshipith hydraulic processes in ecosystems.

. Concluding remarks

The results from this study provide strong support for theypothesis of a relationship between the leaf N:P ratio and WUE

n ecosystems. However, this relationship was stronger in the treesnd under conditions that included a balanced N and P supply. Ourndings link plant N:P stoichiometry with WUE and provide evi-ence of a link between stoichiometry and hydraulic processes inerrestrial plants, as well as useful information for ecologists study-ng nutrient and hydrological cycles.

dditional information

Competing financial interests: The authors declare no compet-ng financial interests.

cknowledgements

The study was financially supported by the National Naturalcience Foundation of China (41390463) and the National Key Tech-ology R&D Programme (2015BAC01B03).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.ecoleng.2015.11.34.

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