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ThecarbonandnitrogenisotopecompositionofAustraliangrassesinrelationtoclimate

ArticleinFunctionalEcology·June2009

DOI:10.1111/j.1365-2435.2009.01576.x

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The carbon and nitrogen isotope composition

of Australian grasses in relation to climate

Brett P. Murphy* and David M. J. S. Bowman

School of Plant Science, University of Tasmania, Hobart, Tasmania 7001, Australia

Summary

1. The carbon and nitrogen isotope composition of plants are known to be affected by environ-

mental factors, especially water availability. While d13C of C4 plants is generally assumed to be

much less responsive to environmental variables than that of C3 plants, it is unclear whether the

response of d15N differs between the two photosynthetic pathways.

2. Focussing on differences in the response of members of the C3 and C4 photosynthetic path-

ways to climate variables, we examined the foliar d13C and d15N of grasses collected from natural

vegetation in a wide range of climatic zones throughout Australia.

3. An index of water availability was clearly related to foliar d13C and d15N of both C3 and C4

grasses. There was a negative relationship between water availability and d13C of C3 grasses

(r2 = 0Æ21), similar to that documented extensively in other C3 plants. An opposite, positive

relationship was found between water availability and d13C in C4 grasses (r2 = 0Æ17), an effect

that has been reported only infrequently. Accounting for differences in d13C between the differ-

ent C4 types (NADP-ME, PCK, NAD-ME and Aristida) resulted in a substantial increase in the

fit of the model relating d13C to water availability (R2 = 0Æ48).4. There was a negative relationship between water availability and d15N (r2 = 0Æ40), that wassimilar in both C3 and C4 grasses, but temperature had no effect on d15N. This provides strong

support for the theory that water availability is the dominant factor determining soil and plant

d15N via its effect on the ‘openness’ of the nitrogen cycle.

5. We also found significant differences in d15N between the different C4 types, with the highest

values for PCK, followed by NADP-ME and NAD-ME, and the lowest values for Aristida. The

difference in d15N between PCK and Aristida was large, at 5Æ1&.

6. The importance of water availability as a predictor of d13C in both C3 and C4 grasses suggests

that variation in water availability should be considered when estimating C4 grass abundance

based on d13C measurements (e.g. in herbivore diets or as a contributor to biospheric carbon

sinks).

Key-words: C3, C4, photosynthetic pathway, Poaceae, rainfall, stable isotope, water availability

Introduction

Clear patterns can be seen in the stable carbon and nitrogen

isotope composition (expressed as d13C and d15N respec-

tively) of natural vegetation throughout the world, closely

reflecting climate and other environmental factors. Water

availability appears to be the most important factor, usually

showing a strong negative relationship with both d13C (e.g.

Stewart et al. 1995; Swap et al. 2004; Weiguo et al. 2005)

and d15N (e.g. Handley et al. 1999; Schuur & Matson 2001;

Swap et al. 2004). In the case of d13C however, the negative

relationship with water availability is usually only present

in plants using the C3 photosynthetic pathway. In C4 plants,

d13C is much less variable and relationships with climatic

factors are usually absent (Van Der Water et al. 2002; Swap

et al. 2004).

In C3 plants, d13C is primarily influenced by the ratio of

intercellular to ambient concentrations of CO2 (ci ⁄ ca) (Far-quhar et al. 1989a). Plants regulate ci ⁄ ca by opening and

closing their stomata, in response to a range of environmen-

tal factors, including light (Yakir & Israeli 1995), nutrients

(Raven & Farquhar 1990) and water availability (Winter

et al. 1982). A close relationship exists between ci ⁄ ca and

plant water use efficiency (WUE), which means that d13Ccan provide an estimate of the integrated long-term WUE

of a plant (Ehleringer 1989; Farquhar et al. 1989b). The

relationship between water availability and d13C of natural*Correspondence author. E-mail: brettpatrickmurphy@hotmail.

com

� 2009 The Authors. Journal compilation � 2009 British Ecological Society

Functional Ecology 2009, 23, 1040–1049 doi: 10.1111/j.1365-2435.2009.01576.x

vegetation arises in two ways. First, at an individual level,

plants respond to decreasing water availability by increasing

their WUE, and hence d13C (Farquhar et al. 1989b). Sec-

ondly, plants adapted to more arid environments tend to

have higher WUE, and hence d13C, than plants adapted to

more mesic environments, even when grown in the same

environment (Anderson et al. 1996).

In C4 plants, the effect of ci ⁄ ca on d13C is similar to that in

C3 plants, but greatly diminished, and confounded by post-

photosynthetic fractionation due to ‘leakiness’ of the bundle

sheath cells to CO2 (Farquhar 1983). Depending on the

degree of leakiness, the slope of the relationship between d13Cand water availability can range from positive (e.g. Weiguo

et al. 2005) to negative (e.g. Wang et al. 2005), but in general,

d13C in C4 plants tends to vary much less in response to envi-

ronmental factors than in C3 plants (Henderson et al. 1992).

Perhaps the largest source of variation in d13C in C4 plants is

biochemical subtype, with C4 plants generally divided into

three groups on the basis of biochemistry: NADP-ME,

NAD-ME and PCK, named after their respective C4 acid

decarboxylases. NADP-ME species tend to have the highest

d13C, followed by PCK and then NAD-ME species (Hatters-

ley 1982).

The causes of variation in d15N are much less clearly

understood than the causes of variation in d13C. Although

extensive fractionation may occur within plants (Evans

2001), foliar d15N tends to reflect soil d15N (Austin & Vito-

usek 1998; Handley et al. 1999), and a number of authors

have suggested that it is the ‘openness’ of the nitrogen cycle

that primarily influences soil d15N (Austin & Vitousek 1998,

Handley et al. 1999, Schuur & Matson 2001). In an open

nitrogen cycle, gains and losses of nitrogen are large relative

to the total nitrogen pool. This is typical of arid areas, where

water, rather than nitrogen, tends to be limiting. In more

mesic areas, nitrogen, rather than water, tends to be limiting,

such that it is efficiently recycled, with little leaving the nitro-

gen cycle.

Accurate prediction of foliar d13C and d15N has

numerous applications in ecological studies. Values of

d13C of plant and animal remains can be used to esti-

mate the proportion of plant biomass or diet that was

C4, but typical values of d13C for C3 and C4 plants must

be estimated (Vogel 1978; Witt et al. 1998; Cerling et al.

2006; Codron et al. 2007). Accurate estimates of d13C of

C3 and C4 vegetation are also required for use in global

carbon budgets that estimate the relative importance of

terrestrial and marine carbon sinks from d13C of atmo-

spheric CO2 (Lloyd & Farquhar 1994; Fung et al. 1997;

Suits et al. 2005). Values of d15N of animal remains can

be used to estimate trophic level, as animals tend to be

enriched by 1–5& with each increasing trophic level

(Hobson & Montevecchi 1991; Kwak & Zedler 1997).

However, variation in the d15N signature of plant mate-

rial at the base of the trophic structure must be

accounted for.

While most previous studies of variation in plant d13Cand d15N have focused on woody plants, grasses provide

an opportunity to directly compare the influence of the C3

and C4 photosynthetic pathways on the response of d13Cand d15N to environmental factors, within a single func-

tional group. This is not as readily achieved with woody

plants due to the limited occurrence of C4 photosynthesis

within this group. In this study, we examine the variation

in foliar d13C and d15N of grasses throughout the Austra-

lian continent, in relation to a range of environmental fac-

tors. Given the existing evidence for a close relationship

between water availability and both d13C and d15N in other

plants, we focus primarily on this factor, and evaluate three

hypotheses about isotopic variation in relation to water

availability:

1. there is strong a negative relationship between d13C and

water availability in C3 grasses;

2. there is no relationship between d13C and water availability

in C4 grasses; and

3. there is a negative relationship between d15N and water

availability, similar in both C3 and C4 grasses.

Materials and methods

S AM PL E C O L LE C T I ON A N D AN A L YS I S

Between March 2003 and November 2004, 408 grass specimens were

collected at 173 locations across Australia, encompassing a wide

range of climatic conditions, with locations accurately determined

using a global positioning system (Fig. 1). The vegetation at the col-

lection sites was relatively undisturbed; that is, the native woody vege-

tation had not been cleared. While many of the samples were

collected on land subject to pastoral grazing by sheep and cattle, dis-

turbance due to grazing was typically mild. We included exotic

grasses in our analysis, although these represent only a very small pro-

portion of specimens (Table 1).

Samples of live foliage were collected, pressed in newspaper and

air-dried. In about 25% of cases, no live foliage was available, so

senescent foliage was collected. A number of previous studies suggest

that both foliar d13C and d15N may change as leaves senesce, how-

ever reported trends are highly variable, ranging from negative (Cur-

rin et al. 1995; Cloern et al. 2002; Turney et al. 2002) to neutral

(Garten 1993; Currin et al. 1995; Kolb & Evans 2002) to positive

(Gebauer & Schulze 1991; Narholm 1991; Cloern et al. 2002) in vari-

ous taxa. While variation in d13C and d15N due to senescence is

clearly a potential source of error in this study, we consider it likely

that this would simply increase the overall variability in the data,

and not bias our results in any particular direction. Mature leaves

were selected from each sample, rinsed with deionized water, dried

and ground to a fine powder. Approximately 4 mg of each powdered

foliage sample was placed in tin capsules and analysed using an EA

1110 elemental analyser (CE Instruments, Rodano, Italy) coupled

with an IsoChrom mass spectrometer (Micromass, Manchester,

UK). d13C and d15N are expressed in per mil (&) units, relative

to the Vienna Pee Dee Belemnite and atmospheric N2 standards

respectively.

Specimens were classified as possessing either the C3 or C4 photo-

synthetic pathway (Table 1). If the specimen could be identified to

generic level, pathway classification was made on the basis of that

identification usingWatson&Dallwitz (1992 onwards). If a specimen

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Functional Ecology, 23, 1040–1049

Carbon and nitrogen isotopes in grasses 1041

could not be identified to genus, classification was made on the basis

of its foliar d13C. Those with d13C less than )19& were considered

C3, and those with d13C greater than )19& considered C4 (Cerling

et al. 1997). While the abundance of C4 grasses, in terms of both spe-

cies numbers and biomass, is sometimes assumed to be related to

water availability, with greater abundance of C4 grasses in arid areas

(Chazdon 1978), in Australia this is not the case. In Australia, C4

grasses are more closely associated with high growing season temper-

atures (Hattersley 1983;Murphy&Bowman 2007b).

Where possible, each C4 specimen was assigned to one of the three

C4 subtypes (NADP-ME, NAD-ME, or PCK), using the lists pro-

vided in Schulze et al. (1996a) and Watson & Dallwitz (1992

onwards). The subtypes are based on physiology and leaf anatomical

characteristics as defined by Prendergast et al. (1987). Following

Schulze et al. (1996a), we also included a fourth subtype, comprising

members of the genus Aristida. While this genus has typical NADP-

ME physiology, it is anatomically distinct from other NADP-ME

grasses, lacking a suberised lamella in its bundle sheath cells (Hatters-

ley 1992).

E N V I R O N M E N T A L D A T A

We examined variation in foliar d13C and d15N in relation to a num-

ber of climatic and environmental variables: a soil water availability

index (WAI), mean annual temperature (MAT), vegetation height

and canopy cover, chenopod abundance and proximity to coast. The

derivation of these variables, and the rationale behind their selection,

is provided below. These variables were selected because they were: (i)

the most likely to have large effects, based on the published literature

and (ii) were easily obtained, and relatively accurate, for each collec-

tion location.

Digital maps of mean annual areal actual evapotranspiration

(AET) and areal potential evapotranspiration (PET), covering Aus-

tralia at a resolution of 0Æ05�, were obtained from the Australian

Bureau ofMeteorology, Canberra. These maps had been constructed

following Morton (1983), and were used to estimate a soil WAI, cal-

culated as: 1 ) AET ⁄PET (Walker & Langridge 1997). MAT for

each collection location was estimated using the computer program

ANUClim 5.1 (Centre for Resource and Environmental Studies,

Australian National University, Canberra). As well as location co-

ordinates, ANUClim requires elevations, which were obtained from a

second resolution digital elevation model of Australia, obtained from

the Australian Spatial Data Directory (http://asdd.ga.gov.au/asdd/).

WAI ranged from 0Æ14 to 0Æ73, equivalent to a range in annual rainfallof 155–1605 mm, andMAT ranged from 6 to 26 �C.

Given that the presence of a tall, dense tree canopy can increase

d13C of understorey plants (Van Der Merwe & Medina 1989; Buch-

mann et al. 1997), we measured the average height of the surrounding

vegetation at each collection location using a clinometer, and esti-

mated canopy cover using a semi-quantitative index, ranging from 0

(no canopy) to 5 (overlapping crowns), based on crown separation

ratios (mean gap ⁄meanwidth:McDonald et al. 1998).

A useful indicator of site soil conditions in Australia is the presence

of chenopod (family Chenopodiaceae)-dominated vegetation, as it is

very closely associatedwith saline and sodic soils (Beadle 1981). These

Fig. 1. Locations of grass specimen collections within Australia. C3

and C4 specimens are indicated (grey and black circles respectively).

Contour lines refer to the water availability index (actual evapotrans-

piration ⁄ potential evapotranspiration).

Table 1. Number of specimens examined, broken down by

photosynthetic pathway and genus. Genera marked with an asterisk

are naturalized

Pathway Genus n

C3 Austrostipa 3

Elytrophorus 1

Hordeum* 1

Monachather 6

Thyridolepis 4

Vulpia* 2

Unidentified 41

Total 58

C4 Aristida 53

Astrebla 8

Bothriochloa 5

Brachyachne 5

Cymbopogon 4

Dactyloctenium 1

Dichanthium 1

Ectrosia 1

Enneapogon 25

Enteropogon 3

Eragrostis 7

Eriachne 3

Eulalia 1

Heteropogon 12

Hyparrhenia* 1

Iseilema 6

Melinis* 2

Mnesithea 8

Neurachne 2

Panicum 7

Perotis 2

Schizachyrium 5

Setaria 8

Sarga 7

Sporobolus 2

Thaumastochloa 2

Themeda 7

Tragus 2

Triodia 1

Uranthoecium 1

Urochloa* 2

Unidentified 156

Total 350

Total 408

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Functional Ecology, 23, 1040–1049

1042 B. P. Murphy & D. M. J. S. Bowman

soil types could be expected to affect plant d13C and d15N (Heaton

1987; Brugnoli & Lauteri 1991; Van Groenigen & Van Kessel 2002).

Each collection location was classified as either chenopod or non-

chenopod dominated. Chenopod sites were those where>50%of the

vegetation within a 1 km radius was described as ‘chenopod shrub-

land’, according to the National Vegetation Information System ver-

sion 1.0 (Department of Environment andHeritage, Australia, http://

www.deh.gov.au/erin/edd).

Given that previous studies have found elevated foliar d15N in close

proximity to the coast (Virginia & Delwiche 1982; Heaton 1987; but

see Vitousek et al. 1989 for absence of an effect), we calculated the

distance between each collection location and the nearest coastline

using a geographic information system. Collection locations were

classified as coastal if they were within 20 km of the coast. This dis-

tance was larger than that used in previous studies (8 km, Virginia &

Delwiche 1982; 2 km, Heaton 1987), simply because we had few col-

lection locations close to the coast.

S T A T I S T I C AL A N A L YS I S

We examined variation in d13C and d15N using least-squares linear

regression models. Water availability was thought to be the variable

of primary importance, so models containing only this variable were

constructed first. To examine the effect of additional environmental

variables, we added these to the models containing water availability.

All models were constructed in the computer program R (version

2.6.2) (Ihaka&Gentleman 1996).

Given that d13C is influenced by very different processes in C3 and

C4 plants, we analysed C3 and C4 specimens separately (Farquhar

1983; Farquhar et al. 1989a). In contrast, we could find no published

evidence that processes influencing d15N systematically vary between

C3 and C4 plants, and for this reason, we used a single analysis for

d15N. Visual inspection of foliar d13C of C4 grasses and foliar d15Nplotted againstWAI suggested a nonlinear relationship, sowe applied

an inverse transformation to WAI prior to any analysis, i.e. WAI)1.

The entire data set used in the analysis is provided in the Supporting

Information.

Results

G R A SS F O L I AR d 1 3 C

There were highly significant relationships between water

availability and foliar d13C for both C3 (P < 0Æ001,r2 = 0Æ21) and C4 grasses (P < 0Æ0001, r2 = 0Æ17). In the

case of C3 grasses, the relationship was negative, with a 2Æ8&difference between specimens from the wettest and driest sites

(Fig. 2a). In contrast, the relationship was positive for C4

grasses, with a 1Æ7& difference between specimens from the

wettest and driest sites.

Within C4 grasses, there were highly significant differences

in d13C between the four C4 types (P < 0Æ0001). While the

Aristida, NAD-ME and PCK types had similar d13C, theNADP-ME type had d13C about 1& higher than the other

types (Fig. 3a). Given that the NADP-ME type tends to be

associated with high rainfall areas (Hattersley 1992; Schulze

et al. 1996a), there is a possibility that the high d13C values

typical of this type may have been at least partly responsible

for the positive relationship between d13C and water

availability in C4 grasses. However, it is clear that the high

d13C values of the NADP-ME type were not entirely respon-

sible, because a significant positive relationship was present in

each of the C4 types, except PCK (NADP-ME P < 0Æ0001;NAD-MEP < 0Æ01;Aristida P < 0Æ01).There was little evidence that the relationship between d13C

and water availability varied between the four C4 types, as the

interaction between water availability and C4 type was not

significant (P = 0Æ82; Fig. 4). A simple linear model incorpo-

rating just water availability and C4 type was able to explain a

high proportion (48%) of the variation in foliar d13C of C4

grasses.

Neither vegetation height nor canopy cover had an effect

on d13C of C3 grasses (P = 0Æ98 and 0Æ22 respectively), but

both had a clear negative effect on d13C of C4 grasses

(P < 0Æ0001 and <0Æ01 respectively). For C4 grasses, d13Cdecreased by 1Æ4& between the shortest and tallest vegeta-

tion, and by 0Æ75& between the most open and closed vegeta-

tion. The presence of chenopod-dominated vegetation had no

clear effect on d13C of C4 grasses (P = 0Æ34).

–10 0·1 0·2 0·3 0·4 0·5 0·6 0·7 0·8

–5

0

5

10

15

20

Water availability index

–35

–30

–25

–20

–15

–10

Gra

ss fo

liage

δ13

C (

‰)

Gra

ss fo

liage

δ15

N (

‰)

δ13C

δ15N

C4 grasses

C3 grasses

P < 0·0001

r 2 = 0·17

WAI: P < 0·0001 WAI × pathway: P < 0·01

R 2 = 0·42

P < 0·001

r 2 = 0·21

(a)

(b)

Fig. 2. (a) d13C and (b) d15N of grass foliage collected throughout

Australia, plotted against water availability index (WAI). Filled cir-

cles represent C4 specimens; empty circles represent C3 specimens.

The lines represent the predictions of linear regressions of (a) d13Cand (b) d15N against WAI: the solid line is for C4 specimens; the

dashed line is for C3 specimens. For d13C, separate regressions wereused for C3 and C4 specimens, with r2 of 0Æ17 and 0Æ21 respectively.

For d15N, a multiple regression model was used:

d15N � WAI ·· photosynthetic pathway, for whichR2 was 0Æ42.

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Functional Ecology, 23, 1040–1049

Carbon and nitrogen isotopes in grasses 1043

G R A SS F O L I AR d 1 5 N

There was a highly significant negative relationship between

water availability and foliar d15N (P < 0Æ0001; r2 = 0Æ40),with a 9Æ5& difference between specimens from the wettest

and driest sites (Fig. 2b). Additionally, there was strong evi-

dence that the slope of the relationship between d15N and

water availability was steeper in C3 compared with C4 grasses

(P < 0Æ01). Over the entire range of water availability exam-

ined, foliar d15N of C3 grasses decreased by 4Æ4& more than

that of C4 grasses (Fig. 2b).

Within C4 grasses, there were highly significant differences

in d15N between the four C4 types (P < 0Æ0001). The most

conspicuous difference was that the PCK type had substan-

tially higher d15N than the other three types (Fig. 3b). d15Nwas lowest in Aristida and intermediate in the NADP-ME

and NAD-ME types. The magnitude of the effect was large,

with a 5Æ1& difference between the PCK and Aristida types

for a given level of water availability (Fig. 3b). There was lit-

tle evidence that the relationship between d15N and water

availability varied between the four C4 types, as the interac-

tion between water availability and C4 type was not signifi-

cant (P = 0Æ07).Of the other variables examined, only the effect of proxim-

ity to the coast was significant (P < 0Æ001). Contrary to

expectation, grasses from sites greater than 20 km from the

coast had foliar d15N about 2Æ1& higher than those from

coastal sites, opposite to the effect reported in the literature

(Virginia & Delwiche 1982, Heaton 1987). Inland sites gener-

ally have lower water availability than coastal sites, so it may

be that the observed effect is an artefact of the relationship

between water availability and foliar d15N.

Discussion

F O L I A R d 1 3 C

A negative relationship between water availability and foliar

d13C of C3 species has been extensively documented in a

–15

–14

–13

–12 δ13CG

rass

folia

ge δ

13C

(‰

)(a)

–2

0

2

4

6

AristidaNADP–ME PCK NAD–MEC4 type

δ15N

Gra

ss fo

liage

δ15

N (

‰)

(b)

Fig. 3. Modelled foliar (a) d13C and (b) d15N of grasses of the four C4

types examined, according to the linear regression model: d13C or

d15N � WAI)1 + C4 type. WAI was assumed to be 0Æ33, the mean

of the data set. R2 for was 0Æ48 and 0Æ46 for d13C and d15N respec-

tively. Standard errors are shown.

–18

–16

–14

–12

–10

Gra

ss fo

liage

δ13

C (

‰)

Gra

ss fo

liage

δ13

C (

‰)

Gra

ss fo

liage

δ13

C (

‰)

Gra

ss fo

liage

δ13

C (

‰)

NADP–ME

–18

–16

–14

–12

–10 PCK

–18

–16

–14

–12

–10 NAD–ME

–180·1 0·2 0·3 0·4 0·5 0·6

–16

–14

–12

–10

Water availability index

Aristida

(a)

(b)

(c)

(d)

Fig. 4. Foliar d13C of grasses of the four C4 types examined: (a)

NADP-ME, (b) PCK, (c) NAD-ME and (d) Aristida, plotted against

water availability index (WAI). The lines represent the predictions of

a least-squares linear regression model: d13C � WAI)1 + C4 type.

R2 for the regression was 0Æ48.

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Functional Ecology, 23, 1040–1049

1044 B. P. Murphy & D. M. J. S. Bowman

range of environments (Stewart et al. 1995; Korol et al.

1999; Van Der Water et al. 2002; Swap et al. 2004; Weiguo

et al. 2005). While most of these studies have focused on

woody plants, our results show that a similar pattern is pres-

ent in C3 grasses in Australia. Of greater significance, how-

ever, is the opposite, positive relationship we have clearly

demonstrated between water availability and foliar d13C of

C4 grasses. This relationship has been only infrequently

reported (Table 2), and certainly never on such a large geo-

graphical scale.

A positive relationship between water availability and

foliar d13C of C4 plants is clearly supported by physiological

theory. In C4 plants, the effect of ci ⁄ ca on d13C is confounded

by post-photosynthetic fractionation due to ‘leakiness’ of the

bundle sheath cells to CO2 (Farquhar 1983). The degree of

leakiness (F) affects the slope of the linear relationship

between ci ⁄ ca and d13C. When F is below about 0Æ35, d13C is

positively related to ci ⁄ ca, i.e. opposite to the relationship in

C3 plants, and F tends to remain below this level under a wide

range of environmental conditions and moderate levels of

stress (Henderson et al. 1992; Williams et al. 2001). For this

reason the relationship between water availability and d13C of

C4 species is usually positive, opposite to the direction of the

relationship in C3 species (Schulze et al. 1996a; Wang et al.

2005).

Within the C4 photosynthetic pathway, we found that the

NADP-ME type had d13C substantially higher than the other

three types. This is clearly consistent with the literature, with

numerous previous studies reporting high d13C values for

NADP-ME species (Hattersley 1982; Schulze et al. 1996a;

Cerling et al. 2003). Several previous studies have also

reported that the PCK type tends to have slightly higher d13Cthan the NAD-ME and Aristida types, although we did not

detect this difference. Importantly, a model of foliar d13C of

C4 grasses, incorporating both C4 type and water availability,

performed well (R2 = 0Æ48), substantially better than the bestmodel of foliar d13C of C3 grasses (r

2 = 0Æ21). Given that the

relative abundances of the four C4 types are closely related to

water availability (Hattersley 1992; Schulze et al. 1996a), it is

likely thatmean foliar d13C ofC4 grasses in a given geographi-

cal area could be reliably estimated, and such estimates are

commonly used in isotopic reconstructions of animal feeding

ecology and global carbon budgets, described under Implica-

tions, below.

Numerous authors have suggested that the relationship

between rainfall and foliar d13C of C3 plants is nonlinear

(Schulze et al. 1998; Korol et al. 1999; Miller et al. 2001; Van

Der Water et al. 2002; Weiguo et al. 2005). In these studies,

d13C increased rapidly at low levels of rainfall and then lev-

elled out at higher rainfall. From Australia, there have been

conflicting results. In eastern Australia, Stewart et al. (1995)

found a relationship between foliar d13C of woody species

and annual rainfall that was clearly linear. In contrast, over a

similar rainfall gradient in monsoonal northern Australia

both Schulze et al. (1998) and Miller et al. (2001) found that

the relationship between foliar d13C of Eucalyptus trees and

annual rainfall clearly levelled out at about 500–600 mm.

They suggested that this corresponded to the limit of a strong

monsoonal influence, and above this level, soils tend to be

uniformly saturated during the summer growing season when

foliar d13C is determined. The lack of C3 specimens from

intermediate levels of water availability in our study makes it

impossible to determine whether the relationship we observed

between d13C and water availability was also nonlinear.

Given that our specimens were collected from a range of areas

across the Australian continent, rather than from an unrepli-

cated water availability gradient, it is unlikely that a single

environmental feature, such as the limit of monsoon influence

Table 2. Summary of recently reported relationships between water availability (annual rainfall) and foliar d13C and d15N. Across each rainfall

gradient examined, the differences in d13C and ⁄ or d15N are specified. Differences in d13C and ⁄ or d15N were often visually estimated from figures

in the source literature, so are approximate only, rounded to the nearest 0Æ5&

Isotope Pathway Life forms

Rainfall

gradient (mm)

Isotopic

difference

(&) Location References

d13C C3 Grasses 220–640 )4Æ5 Chinese Loess Plateau Weiguo et al. (2005)

Trees, shrubs 350–1520 )4 NE Australia Stewart et al. (1995)

Trees, shrubs 230–970 )2Æ5 S Africa Swap et al. (2004)

Trees 220–1800 )2Æ5 N Australia Schulze et al. (1998)

Trees 630–1600 )2 SE Australia, New Zealand Korol et al. (1999)

Trees, shrubs 160–560 )3 to 0 SW United States Van Der Water et al. (2002)

Trees, shrubs, forbs, grasses 130–770 0 Patagonia Schulze et al. (1996b)

C4 Grasses 50–550 0 to 2 Namibia Schulze et al. (1996a)

Grasses 230–650 0 S Africa Swap et al. (2004)

Single shrub species 160–250 0 SW United States Van Der Water et al. (2002)

Single grass species 370–670 )2 Chinese Loess Plateau Weiguo et al. (2005)

d15N C3 Trees 220–1800 )5Æ5 N Australia Schulze et al. (1998), Austin &

Sala (1999)

Trees, shrubs 200–1300 )6Æ5 S Africa Swap et al. (2004)

Trees 500–5500 )4 Hawaii Austin & Vitousek (1998)

C3 + C4 All 20–2950 )9 Global Handley et al. (1999)

C4 Grasses 200–1300 )1 S Africa Swap et al. (2004)

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Functional Ecology, 23, 1040–1049

Carbon and nitrogen isotopes in grasses 1045

(e.g. Schulze et al. 1998; Miller et al. 2001), or a boundary

between major soil formations (e.g. Van Der Water et al.

2002) would result in a nonlinear relationship.

A number of studies have shown that d13C of plants grow-

ing beneath dense canopies tends to be lower than plants

growing in more open vegetation. This has been termed the

‘canopy effect’ and has been attributed to both the 13C deple-

tion of ambient CO2 during soil respiration (Van Der Merwe

& Medina 1989) and reduced light availability (Buchmann

et al. 1997).We found little evidence that vegetation height or

canopy cover affected foliar d13C of C3 grasses, but clearly

that of C4 grasses was affected, with a 0Æ7-1Æ3& decrease in

d13C between the shortest ⁄most open and tallest ⁄most closed

vegetation. This effect possibly reflects an increase in shade

with increasing vegetation height, demonstrated experimen-

tally by Buchmann et al. (1996). This explanation is more

likely than 13C depletion of ambient CO2 due to soil respira-

tion, which is typically associated with closed canopy vegeta-

tion that was not present in our data set. The effect is likely to

have been detectable in C4 but not C3 grasses because of their

much greater sample size (350 vs. 58).

F O L I A R d 1 5 N

The negative relationship between water availability and

foliar d15N that we have found throughout Australia is simi-

lar to that already recorded in northern Australia (Schulze

et al. 1998), on other continents (Heaton 1987; Schuur et al.

2001, Aranibar et al. 2004, Swap et al. 2004) and in an exten-

sive global synthesis (Handley et al. 1999). Many authors

now agree that the mechanism is the increased ‘openness’ of

nitrogen cycling in drier areas (Handley et al. 1999; Schuur

et al. 2001), a concept first developed by Austin & Vitousek

(1998). These authors suggested that where water is abun-

dant, nitrogen tends to be limiting, and is tightly recycled.

This limits the amount of nitrogen leaving the system through

fractionating pathways that would cause the system to

become enriched in 15N. In contrast, nitrogen is not recycled

so tightly in drier systems, because water is the limiting factor,

and nitrogen leaves the systemmore readily through fraction-

ating pathways.

The cause of the pattern in northern Australia has been the

subject of debate. Schulze et al. (1998) suggested that increas-

ing foliar d15N with increasing aridity may be due to higher

grazing pressure at arid sites, resulting in an acceleration of

nitrogen turnover and losses of 14N, although this hypothesis

has been disputed by Austin & Sala (1999). These authors

suggest that grazing intensity is in fact greater at wetter sites,

and that the pattern is more likely to be caused by the direct

effects of water availability on the ‘openness’ of the nitrogen

cycle, consistent with the hypothesis of Austin & Vitousek

(1998). Also working in northern Australia, Cook (2001)

added to the debate by demonstrating that grazing has only a

small effect on foliar d15N of grasses and trees, certainly not

sufficient to cause the large differences (�6&) observed

across the northern Australian rainfall gradient. Cook (2001)

also suggested that fire may play an important role, with fre-

quent fires, typical of the high rainfall end of the gradient,

possibly resulting in greater losses of 14N, and hence high

d15N. However, that the relationship between foliar d15N and

water availability holds across such a diverse range of climatic

and vegetation zones as examined in this study, clearly lends

support to the mechanism proposed by Austin & Vitousek

(1998) and Handley et al. (1999), that is, increased openness

of the nitrogen cycle in arid areas.

Recent work in the tropical forests of Hawaii by Houtton

et al. (2006) has provided additional insights into the mecha-

nism by which 14N is lost from relatively dry sites. Measuring

the isotopic composition of both inputs and hydrological out-

puts, these authors found that hydrological outputs from

drier sites were actually less enriched than inputs, suggesting

that 14N must be preferentially lost via another pathway, pre-

sumed to be gaseous loss via denitrification of NO3). While

nitrogen losses via denitrification were greater at wetter sites,

more complete consumption of NO3) was assumed to mini-

mize ecosystem enrichment with 15N. It remains unclear

whether the mechanism proposed by Houtton et al. (2006) is

also applicable outside tropical forest biomes, and particu-

larly whether denitrification remains an important pathway

of nitrogen loss in arid areas, where the globally maximum

values of ecosystem d15N tend to be found (Amundson et al.

2003).

Swap et al. (2004) concluded that the relationship between

plant d15N and rainfall in southern Africa was much stronger

and steeper in C3 than in C4 plants, but provided no

explanation for this phenomenon. Working in an arid

region of southern Africa, Aranibar et al. (2008) more

recently reported that d15Nwas higher in C3 than inC4 plants.

Unfortunately, from these two studies it is difficult to distin-

guish the direct effects of the photosynthetic pathway and

growth form on d15N, because C3 plants were almost always

non-grasses and C4 plants were almost always grasses. Our

finding that the response of d15N to increasing water avail-

ability is more negative in C3 grasses than in C4 grasses pro-

vides evidence that the effect previously reported by Swap

et al. (2004) and Aranibar et al. (2008) is directly related

to photosynthetic pathway, rather than growth form.

Discussions of the effects of photosynthetic pathway on plant

d15N are virtually absent from the published literature,

although there is abundant evidence of differences in nitrogen

metabolism between C3 and C4 plants, with substantially

higher nitrogen use efficiency in C4 plants, largely due to a

lower demand for Rubisco in leaves (Brown 1978; Schmidt

& Edwards 1981; Sage & Pearcy 1987; Makino et al. 2003).

Given these differences in nitrogen metabolism, and the large

fractionations that can occur within plants (Robinson 2001),

it is not unexpected that d15N may differ between the C3 and

C4 pathways. Similarly, differences in nitrogen metabolism

may also contribute to the differences we found in d15Nbetween C4 types, an effect that, to the best of our knowledge,

has not been previously reported. Work by Ghannoum et al.

(2005) suggests that there are clear differences in nitrogen

use efficiency and leaf nitrogen content between the NAD-

ME and NADP-ME types, although we found very little

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Functional Ecology, 23, 1040–1049

1046 B. P. Murphy & D. M. J. S. Bowman

difference in d15N between those types. The most notable

difference we found was between the PCK type and the other

three types, yet there is little in the published literature to sug-

gest that the PCK type differs substantially from the other

types in terms of nitrogenmetabolism. As such, we are unable

to provide an explanation for this effect at this stage, and it

clearly requires further investigation.

While it is well established that soil moisture is an impor-

tant determinant of soil and plant d15N (Austin et al. 1998,

Handley et al. 1999; Schuur et al. 2001), the effect of temper-

ature is poorly documented. Martinelli et al. (1999) were

among the first to note that tropical forests are enriched in15N relative to temperate forests, and Amundson et al. (2003)

analysed a global data set to conclude that soil and plant

d15N were more closely correlated with MAT than mean

annual rainfall. In contrast, we found no correlation between

MAT and foliar d15N. The lack of a relationship is probably

due to the limited temperature range over which samples were

collected in our study. In contrast, the study of Amundson

et al. (2003) utilized an extreme temperature range, from

tropical forests and deserts to high latitude boreal forest.

I M P L I C A T I O N S

Our findings also have implications for the use of d13C mea-

surements of plant and animal remains in ecological studies.

For example, in areas where most grass biomass is C4, d13Cmeasurements of herbivore remains are commonly used to

estimate the contribution of grass to the diet (Vogel 1978;

Witt et al. 1998; Cerling et al. 2006; Codron et al. 2007). Such

studies require accurate estimates of typical d13C values for

C3 and C4 plants, so-called ‘end-members’. In the majority of

cases, C3 and C4 end-members are each expressed as a single

mean, without accounting for effects such as water availabil-

ity (e.g. Witt et al. 1998; Sponheimer et al. 2003), while

recently, researchers have begun to account for such effects

and vary C3 end-members according to water availability

(e.g.Murphy et al. 2007a). However, in only several instances

have attempts been made to account for the effect of water

availability on C4 end-members (e.g. Cerling & Harris 1999;

Cerling et al. 2003). Our findings underscore the importance

of accounting for variation in foliar d13C of both C3 and C4

species in dietary studies, especially when attempting to make

precise estimates of parameters such as diet-tissue enrichment

factors, where errors of ±1& would be unacceptable (e.g.

Cerling &Harris 1999;Murphy et al. 2007a).

Models of 13C discrimination by terrestrial plants form an

important part of global carbon budgets. In these budgets,

changes in the isotopic concentration of atmospheric CO2 are

used to derive the location and magnitude of biospheric car-

bon sinks, particularly the relative contributions of the land

and oceans (Battle et al. 2000). However, the approach relies

on accurate estimates of 13C discrimination by terrestrial veg-

etation. While current models assume that discrimination by

C3 plants shows high spatial and temporal variability, espe-

cially in response to water availability, discrimination by C4

plants is assumed to be either constant, or vary little (Lloyd &

Farquhar 1994; Fung et al. 1997; Scholze et al. 2003; Suits

et al. 2005). We have shown that at a continental scale, 13C

discrimination by C4 plants is clearly influenced by water

availability. Indeed, the effect of water availability on d13C of

C4 grasses (1Æ7&) is of an order of magnitude similar to the

effect of water availability on d13C of C3 grasses (2Æ8&; this

study) and C3 plantsmore generally (2–4Æ5&; Table 2). Given

that the contribution of C4 grasses to terrestrial gross primary

production is high at about 23% globally (Still et al. 2003),

failure to account for the relationship between water avail-

ability and 13C discrimination by C4 grasses may represent an

important, yet easily corrected, source of error in current glo-

bal carbon budgets.

Acknowledgements

We thank Wendy Telfer, Lynda Prior and two anonymous referees for provid-

ing helpful comments on an early version of the manuscript, and Stephen

Clayton, Sue Wood and Hilary Stuart-Williams for assisting with the isotope

analysis. Grants from the Australian Research Council (DP0342788 and

DP0878177) supported this work.

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Handling Editor:Matthew Turnbull

Supporting information

Additional supporting information may be found in the online ver-

sion of this article.

Appendix S1.Grass foliar d13C and d15N data set used in the analysis.

Appendix S2. Regression equations relating grass d13C and d15N to

water availability.

Please note: Wiley-Blackwell is not responsible for the content or

functionality of any supporting information supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article

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