Comparison of ecosystem water-use efficiency amongDouglas-fir forest, aspen forest and grassland using eddycovariance and carbon isotope techniques

S T E P H A N E P O N T O N *1 , L A W R E N C E B . F L A N A G A N *, K A R R I N P. A L S T A D *,

B R U C E G . J O H N S O N *, K A I M O R G E N S T E R N w , N A T A S C H A K L J U N w 2 ,

T . A N D R E W B L A C K w , A L A N G . B A R R z*Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4,

wBiometeorology and Soil Physics Group, Faculty of Land and Food Systems, University of British Columbia, 135-2357 Main Mall,

Vancouver, BC, Canada V6T 1Z4, zClimate Research Branch, Meteorological Service of Canada, 11 Innovation Blvd., Saskatoon, SK,

Canada S7N 3H5

Abstract

Comparisons were made among Douglas-fir forest, aspen (broad leaf deciduous) forest

and wheatgrass (C3) grassland for ecosystem-level water-use efficiency (WUE). WUE was

defined as the ratio of photosynthetic CO2 assimilation rate and evapotranspiration (ET)

rate. The ET data measured by eddy covariance were screened so that they overwhel-

mingly represented transpiration. The three sites used in this comparison spanned a

range of vegetation (plant functional) types and environmental conditions within

western Canada. When compared in the relative order Douglas-fir (located on Vancouver

Island, BC), aspen (northern Saskatchewan), grassland (southern Alberta), the sites

demonstrated a progressive decline in precipitation and a general increase in maximum

air temperature and atmospheric saturation deficit (Dmax) during the mid-summer. The

average (� SD) WUE at the grassland site was 2.6� 0.7 mmol mol�1, which was much

lower than the average values observed for the two other sites (aspen: 5.4� 2.3, Douglas-

fir: 8.1� 2.4). The differences in WUE among sites were primarily because of variation in

ET. The highest maximum ET rates were approximately 5, 3.2 and 2.7 mm day�1 for the

grassland, aspen and Douglas-fir sites, respectively. There was a strong negative correla-

tion between WUE and Dmax for all sites. We also made seasonal measurements of the

carbon isotope ratio of ecosystem respired CO2 (dR) in order to test for the expected

correlation between shifts in environmental conditions and changes to the ecosystem-

integrated ratio of leaf intercellular to ambient CO2 concentration (ci/ca). There was a

consistent increase in dR values in the grassland, aspen forest and Douglas-fir forest

associated with a seasonal reduction in soil moisture. Comparisons were made between

WUE measured using eddy covariance with that calculated based on D and dR measure-

ments. There was excellent agreement between WUE values calculated using the two

techniques. Our dR measurements indicated that ci/ca values were quite similar among

the Douglas-fir, aspen and grassland sites, despite large variation in environmental

conditions among sites. This implied that the shorter-lived grass species had relatively

high ci/ca values for the D of their habitat. By contrast, the longer-lived Douglas-fir trees

were more conservative in water-use with lower ci/ca values relative to their habitat D.

This illustrates the interaction between biological and environmental characteristics

influencing ecosystem-level WUE. The strong correlation we observed between the two

independent measurements of WUE, indicates that the stable isotope composition of

respired CO2 is a useful ecosystem-scale tool to help study constraints to photosynthesis

and acclimation of ecosystems to environmental stress.

1Present address: UMR CIRAD-CNRS-ENGREF-INRA ECOFOG, Campus agronomique, BP709, F-97387 Kourou cedex, Guyane Francaise.

Correspondence: Lawrence B. Flanagan, fax 1 1 403 329 2082, e-mail: larry.flanagan@uleth.ca

2Present address: Institute for Atmospheric and Climate Science, ETH Zurich, Winterhurerstr. 190 CH-8057, Zurich, Switzerland.

Global Change Biology (2006) 12, 294–310, doi: 10.1111/j.1365-2486.2005.01103.x

294 r 2006 Blackwell Publishing Ltd

Keywords: boreal forest, conifer forest, eddy covariance, grassland, stable isotopes

Received 2 August 2004; revised version received 9 August 2005 and accepted 3 October 2005

Introduction

In many terrestrial ecosystems soil water is variable

and it can often be the most limiting soil resource

(Boyer, 1982). Therefore, water-use efficiency (WUE),

the ratio of carbon gain during CO2 assimilation (A,

mmol m�2 s�1) to water loss during transpiration (E,

mmol m�2 s�1), is of major importance to the survival,

productivity and fitness of individual plants (Osmond

et al., 1982). In addition, the boundaries between major

vegetation zones with different dominant plant func-

tional groups are often controlled by water availability

(Holdridge, 1947; Woodward, 1987; Hogg, 1994). We

expect plants in these contrasting ecosystems to have

different WUE because of inherent physiological varia-

tion in leaf gas exchange characteristics and because of

differences in environmental conditions among habitats

(Farquhar et al., 1989). Comparative studies of WUE are

important for helping to understand how future climate

change will affect the carbon and energy budgets of

ecosystems.

At the leaf level WUE can be calculated as

WUE ¼ A

E¼ ca 1� ci=cað Þ

1:6u; ð1Þ

where c represents the CO2 concentration (mmol mol�1),

and the subscript i refers to the leaf intercellular air

spaces and the subscript a refers to the ambient atmo-

sphere outside a leaf, u is the difference in water vapor

concentration (mmol mol�1) between the leaf intercel-

lular air spaces and the atmosphere. The value 1.6 is the

ratio of the stomatal conductance of water vapor to that

of CO2. Variation in leaf ci/ca and WUE among plant

species has been documented using carbon isotope

measurements (Farquhar et al., 1989). A relationship

between the carbon isotope composition of leaf tissue

and WUE occurs because both parameters are influ-

enced by leaf intercellular CO2 concentration, which in

turn depends on the ratio of photosynthetic rate to

stomatal conductance. Large differences in leaf ci/ca

and WUE occur among plant species with different

photosynthetic pathways (Osmond et al., 1982; Farqu-

har et al., 1989). In addition, plants with different life

histories or functional groups also show systematic

variation in leaf gas exchange characteristics including

leaf ci/ca when grown under similar environmental

conditions (Smedley et al., 1991; Ehleringer, 1993;

Brooks et al., 1997). For example, leaf ci/ca tends to be

higher in grasses than forbs, higher in annuals than

perennials, higher in broad-leaf deciduous trees than

conifer trees (Smedley et al., 1991; Brooks et al., 1997).

When scaling from the leaf to the canopy or ecosys-

tem there are additional complications that affect mea-

surements of WUE and their interpretation. Water lost

from the ecosystem can come from sources other than

transpiration, such as evaporation from soil or water on

leaf surfaces that was intercepted by canopy foliage

during precipitation events. Feedback processes can

also cause leaf level responses to vary from that ob-

served at the canopy scale. At the leaf level, reduction in

stomatal conductance usually results in higher WUE

because stomatal conductance limits transpirational

water loss more than CO2 assimilation (Farquhar &

Sharkey, 1982). At the ecosystem level, interactions

among stomatal conductance, aerodynamic conduc-

tance, entrainment of dry air in the planetary boundary

layer and changes in leaf temperature can compensate

so that evapotranspiration (ET) is increased despite

stomatal closure (Baldocchi et al., 2001). We expect that

canopy or ecosystem WUE should be negatively corre-

lated with variation in the atmospheric saturation def-

icit (D), despite the negative relationship between

stomatal conductance and D, because of the high eco-

system water vapor fluxes that occur when D is high.

At the ecosystem scale, carbon isotope discrimination

that occurs during net ecosystem carbon uptake should

reflect the photosynthesis-weighted average of discri-

mination in all plants in the ecosystem. In turn the

carbon isotope ratio of respired CO2 should reflect the

composition of the substrate used for respiration. Many

studies suggest that approximately 50–70% of ecosys-

tem photosynthesis is quickly released again as plant

and rhizosphere respiration (Amthor & Baldocchi, 2001;

Hogberg et al., 2001). In addition to plant and rhizo-

sphere components, total ecosystem respiration in-

cludes carbon dioxide released from organic matter

decomposition, but the carbon isotope ratio of soil litter

and humus decomposition is not likely to change on

time scales less than 1 year or much longer (Trumbore,

2000). We expect that measurements of the carbon

isotope composition of CO2 respired from the entire

ecosystem should represent a spatially integrated mea-

sure of whole ecosystem discrimination (Flanagan et al.,

1996; Buchmann et al., 1998; Flanagan & Ehleringer,

1998; Ekblad & Hogberg, 2001). The temporal compo-

nent of this integrated measurement depends on the

magnitude of leaf, stem and root respiration, as well as

that of respiration by rhizosphere organisms using

E C O S Y S T E M WA T E R - U S E E F F I C I E N C Y 295

r 2006 Blackwell Publishing Ltd, Global Change Biology, 12, 294–310

carbon exudates from plant roots. Shifts in the carbon

isotope composition of ecosystem respiration would

likely be dominated by fast-cycling carbon fixed from

the atmosphere a few days previous to sample collec-

tion (Ekblad & Hogberg, 2001; Bowling et al., 2002).

Consistent with these ideas, recent studies have demon-

strated the expected correlations between the carbon

isotope ratio of ecosystem respired CO2 and changes in

precipitation and soil moisture stress, vapor pressure

deficit and canopy conductance (Bowling et al., 2002;

Ometto et al., 2002; Fessenden & Ehleringer, 2003; Pataki

et al., 2003; McDowell et al., 2004b).

A potential complication for ecosystem studies of

the carbon isotope ratio of respired CO2 is isotopic

fractionation during plant metabolism. While labora-

tory studies have indicated that there is no isotopic

fractionation during leaf protoplast respiration (Lin &

Ehleringer, 1997), some recent studies have noted dif-

ferences between the carbon isotope composition of leaf

respired CO2 and the composition of the leaf organic

compounds thought to be the source of the respiratory

CO2 (Duranceau et al., 1999; Ghashghaie et al., 2001;

Tcherkez et al., 2003; Xu et al., 2004; Hymus et al., 2005).

This apparent fractionation can result from isotope

effects during the formation of glucose; carbon in posi-

tions 3 and 4 of the molecule tend to become enriched in13C, while those in the remaining four positions tend to

be depleted in 13C (Schmidt & Gleixner, 1998). During

glycolysis pyruvate is produced from glucose, and

subsequent decarboxylation of pyruvate releases CO2

with carbons originally from positions 3 and 4 in the

glucose molecule. As a result decarboxylation of pyr-

uvate produces CO2 relatively enriched in 13C, and the

acetyl coenzyme A (acetyl-CoA) molecule formed has

carbons that are relatively depleted in 13C. If the acetyl-

CoA produced from pyruvate is completely oxidized in

the Krebs cycle, then two molecules of CO2 depleted in13C are released and the mass balance of isotopes is

restored. However, if there are shifts in the proportion

of respired CO2 derived from decarboxylation of pyr-

uvate and that derived from the conversion of acetyl-

CoA in the Krebs cycle, it is possible to have up to 10%variation in the d13C of CO2 released in leaf respiration

(Tcherkez et al., 2003). At the moment the consequences

of apparent fractionation in leaf respiration for whole

plant and ecosystem-level studies is unclear (Pataki,

2005). It is possible that any partitioning and metabo-

lism of molecules with different 13C/12C ratios in the

shoot, could be counteracted by metabolic processes

occurring in the roots. Klumpp et al., (2005) showed

that shoot respired CO2 was enriched in 13C relative to

shoot biomass, while root respired CO2 was depleted in13C relative to root biomass, so that on a whole plant

basis there was no significant difference in the carbon

isotope composition of respired CO2 and whole plant

biomass.

The first objective of this paper, was to compare eddy

covariance measurements of ecosystem-scale WUE in

three contrasting ecosystems in western Canada, grass-

land in southern Alberta, a broad-leaf deciduous

(aspen) forest in central Saskatchewan and a coastal

evergreen conifer (Douglas-fir) forest on Vancouver

Island in British Columbia. We expected that ecosystem

WUE would vary in a pattern from low to high among

a sequential comparison of grassland, broad-leaf decid-

uous forest and coniferous forest because of inherent

differences in leaf ci/ca characteristics and large differ-

ences in D among these ecosystems. A second objective,

was to test whether measurements of the carbon isotope

composition (13C/12C) of ecosystem respired CO2

could be used as a proxy for short-term changes in

photosynthetic discrimination and associated shifts in

integrated canopy-level ci/ca as whole ecosystems

responded to changes in environmental conditions,

particularly seasonal reduction in soil moisture. If

measurements of the carbon isotope composition of

ecosystem respired CO2 can be used as a proxy for

canopy-level ci/ca, then such measurements could be

used along with air temperature and humidity mea-

surements to calculate ecosystem WUE for comparison

with independent ecosystem-scale WUE measurements

made by eddy covariance. If a strong correlation can

be developed between the two independent measure-

ments of WUE, then we will have increased confidence

that the stable isotope composition of respired CO2

provides an ecosystem-scale tool to help study con-

straints to photosynthesis and acclimation of an ecosys-

tem to environmental stress.

Materials and methods

Study sites

This study compared three sites, grassland in southern

Alberta, aspen forest in central Saskatchewan and Dou-

glas-fir forest on Vancouver Island in British Columbia.

The dominant vegetation in these three sites repre-

sented contrasting plant functional groups (C3 grass,

broad-leaf deciduous forest and evergreen coniferous

forest), and so we expected differences among sites in

leaf gas exchange characteristics that should influence

WUE. The aspen forest is located just north of a vegeta-

tion (climatic) transition zone with grassland and crop-

land ecosystems to the south and conifer-dominated

boreal forest to the north (Hogg, 1994). The comparison

between the grassland and aspen sites is relevant in the

context of climate change and shifts in vegetation

boundaries, because of recent observations of major

296 S . P O N T O N et al.

r 2006 Blackwell Publishing Ltd, Global Change Biology, 12, 294–310

aspen dieback in western Canada caused by severe

drought (Hogg et al., 2005). Douglas-fir is a major

forestry species that occurs in a range of habitats

including coastal forests and along the transition be-

tween grassland and montane vegetation on the eastern

slopes of the Rocky Mountains in southern Alberta. On

Vancouver Island, Douglas-fir is exposed to strong

reductions in late summer soil moisture, in a relatively

cool, humid environment. The comparison of the grass-

land and aspen sites with the coastal Douglas-fir site

was relevant for examining interactions among envir-

onmental conditions and leaf gas exchange character-

istics for their effects on ecosystem-level WUE. The two

forest sites are permanent flux stations in the Fluxnet-

Canada research network, whereas the grassland site is

part of the Ameriflux network and an associated site in

Fluxnet-Canada.

The aspen site, located in Prince Albert National

Park, Saskatchewan (53.631N, 106.201W, 601 m above

sea level), is an 80-year-old trembling aspen (Populus

tremuloides) forest with a thick understory of hazelnut

(Corylus cornuta). The canopy and understory heights

are 21 and 2 m, respectively. The base of the live canopy

for the aspen trees is at 15 m. The aspen stem density is

980 stems ha�1 (Griffis et al., 2003) and leaf area index

(LAI) was 2.0 and 2.1 m2 m�2 for the aspen and hazelnut

in 2003, respectively (Barr et al., 2002, 2004). The soil has

a silty-clay texture with a 5–10 cm deep surface layer of

organic material. The average annual precipitation is

424.3 mm (1971–2000 normals for the closest weather

station located in Prince Albert, 53.221N, 105.671W,

428 m above sea level) with 49% falling during summer

(June to August). The mean annual temperature is

0.9 1C and the average daily temperature during sum-

mer months (June–August) is 16.3 1C (Meteorological

Service of Canada, 2004). This site was established in

1993 as part of the Boreal Ecosystem-Atmosphere Study

(BOREAS, Sellers et al., 1995) and has been integrated

into the Boreal Ecosystem Research and Monitoring

Sites initiative (BERMS) since 1997.

The Douglas-fir site is located on Vancouver Island,

British Columbia (49.901N, 125.371W, 320 m above sea

level), 10 km southwest of the city of Campbell River.

The site was naturally regenerated after a forest fire in

1949. The stand is predominantly coastal Douglas-fir

(Pseudotsuga menziesii) with 17% western redcedar

(Thuja plicata) and 3% western hemlock (Tsuga hetero-

phylla). The canopy top and base are 33 and 15 m,

respectively. In 1998 the stem density was

1100 stems ha�1 and the overstory LAI (projected leaf

area) was 9.1 m2 m�2 (Morgenstern et al., 2004). The

sparse forest understory is dominated by Oregon grape

(Berberis nervosa), Salal (Gaultheria shallon) and Vanilla-

leaf deer foot (Achlys triphylla). The soil is a humo-ferric

podzol with a 1–10 cm thick litter/humus (LFH) organic

layer, a gravelly loamy sand layer and dense compacted

till at a depth of 1 m. The climate is characterized by

cool summers and mild winters (mean annual tempera-

ture is 8.6 1C according to 1971–2000 normals) resulting

in a long growing season. Average annual precipitation

is high with a value of 1451 mm but the summer period

is dry (only 150 mm from June to August; Meteorologi-

cal Service of Canada, 2004). Further details about the

site can be found in Drewitt et al. (2002).

The grassland site is a moist-mixed grassland situ-

ated 1.5 km west of the city limits of Lethbridge, Alberta

(49.431N, 112.561W, 951 m above sea level). The domi-

nant species are Agropyron dasystachyum and A. smithii.

This grassland has never been cultivated and has not

been grazed in over 20 years. The LAI varies greatly

from year to year and was 0.97 � 0.09 m2 m�2 (mean

SE, n 5 6) in 2003. The soil is a dark-brown chernozem

with a clay-loam to clay texture. This site has a moder-

ately cool and semiarid climate. The average annual

precipitation (1971–2000) is 401 mm with 30% falling in

May and June. The mean annual temperature is 5.7 1C

and the average daily temperature during the summer

months (June–August) is 17.1 1C (Meteorological Ser-

vice of Canada, 2004; Lethbridge station). Further de-

tails about the site can be found in Flanagan et al. (2002),

Wever et al. (2002) and Flanagan & Johnson (2005).

Flux and meteorological measurements

Fluxes of CO2, H2O and sensible heat were measured

with the eddy covariance technique at half-hourly inter-

vals. Air and soil temperatures, relative humidity, volu-

metric soil moisture content, precipitation and

incoming photosynthetically active photon flux density

(PPFD) were also measured. A complete description of

the equipment used and protocols for eddy covariance

data processing can be found in Flanagan et al. (2002),

Barr et al. (2002) and Morgenstern et al. (2004) for the

grassland, aspen and Douglas-fir sites, respectively (for

additional flux information see also Griffis et al. (2003)

for the aspen site, Humphreys et al. (2003) for the

Douglas-fir site, and Flanagan & Johnson (2005) for

the grassland site). Calculations of gross photosynthesis

were derived as the sum of daytime measurements of

net ecosystem CO2 exchange (NEE) and total ecosystem

respiration. Ecosystem respiration was estimated from

the relationship between nighttime NEE and soil tem-

perature (for the details see Barr et al., 2002; Flanagan

et al., 2002; Morgenstern et al., 2004). Soil moisture

content was presented on a relative scale (as available

soil water), defined as the ratio of actual extractable

water (difference between a given volumetric measure-

ment and the minimum volumetric soil water content)

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r 2006 Blackwell Publishing Ltd, Global Change Biology, 12, 294–310

to the maximum extractable water (difference between

maximum and minimum volumetric soil water con-

tents). These calculations were done separately for each

site based on the minimum and maximum soil water

content recorded during 2003.

Collection and analysis of atmospheric CO2 samples

Atmospheric CO2 samples were collected during night-

time sample periods on several dates during the 2003

growing season, with an average interval of 3 weeks

between sampling dates. Air samples were collected in

order to use a modified Keeling plot technique to

calculate the isotope composition of CO2 respired by

the ecosystem (Miller & Tans, 2003; Pataki et al., 2003).

The samples were collected in 200 mL glass flasks

equipped with high vacuum Teflon stopcocks. After

sample collection, the flasks were shipped to the Uni-

versity of Lethbridge where CO2 concentration was

determined (within 1–2 weeks after field collection)

according to the method developed by Bowling et al.

(2001). In this method a flask is connected to an evac-

uated inlet manifold and the sample gas is expanded

into the manifold and its associated variable volume

bellows. The bellows is then compressed to flush the

sample air through an infrared gas analyzer (IRGA LI-

6262, Li-Cor Inc., Lincoln, NE, USA). We slightly mod-

ified the inlet manifold system described by Bowling

et al. (2001) by including an additional section where a

portion (30 mL) of the sample gas could be isolated. The

air samples collected in this isolated 30 mL volume were

transferred via a helium carrier gas to a GasBench II

interface (ThermoFinnigan, Bremen, Germany) coupled

to a gas isotope ratio mass spectrometer (Delta Plus,

ThermoFinnigan) in order to measure the carbon iso-

tope ratio of the sample CO2. The carbon isotope ratios

were expressed as d13C:

d ¼Rsample

Rstd� 1

� �; ð2Þ

where R is the molar ratio of heavy to light isotope and

where the subscript std refers to the international

standard Pee Dee Belemnite (PDB). The d values are

conveniently expressed in parts per thousand or per mil

(%). The precision (standard deviation of 292 lab mea-

surements of a lab working standard) was 0.18 ppm for

[CO2] (as measured by the IRGA) and 0.14% for d13C.

Actual measurement precision for [CO2] for all samples

was likely slightly larger because of the broad range of

concentrations that were analyzed during our flask

sampling.

At the forest stations (aspen and Douglas-fir sites), an

automatic system was used to collect atmospheric CO2

samples (Schauer et al., 2003). The system included a

data logger (CR23X, Campbell Scientific, Logan, UT,

USA) and accessories that controlled the operation of a

multiposition valve (Valco Instruments Company Inc.,

Houston, TX, USA), solenoid valves on two manifolds,

a pump, and an infrared gas analyzer (LiCor LI-820, Li-

Cor Inc., Lincoln, NE, USA). The system also included a

set of 15 glass flasks. Air was sampled from three

different heights: (i) one meter above ground (level 1),

(ii) mid-way between the ground surface and the top of

the tree canopy (level 2), and (iii) at the top of the tree

canopy (level 3). Air was pulled down through tubing

(Synflex 1300, 0.625 cm OD, nonbuffering polyethylene

coating, Saint-Gobain, Mantua, OH, USA), through two

desiccant tubes (6200DP, Li-Cor Inc.) containing mag-

nesium perchlorate, and into a glass flask at a flow rate

of 2.5 L min�1 for a period of 5 min. An inverted funnel,

covered with mesh screen to prevent the entry of

insects, was placed over the tubing inlet to prevent

water entry into the tube. On a sampling date, the data

logger was programmed to collect a mid-afternoon

sample from the top of the canopy (level 3), then to

start sampling 2 h before dusk from levels 1 and 2.

Sampling was stopped 2 h after dawn, at the latest. At

night during each 15 min interval, a flask was flushed

with air from level 1 and the [CO2] was measured. After

collection of the first sample, each subsequent sample

from level 1 was compared to every other sample

previously collected from level 1 and kept only if its

[CO2] was different from the others. If not, the same

flask was flushed again 15 min later with new air. The

minimum [CO2] range for a modified Keeling plot

analysis was fixed to 50 ppm (Buchmann et al., 1998;

Pataki et al., 2003), corresponding to a minimum differ-

ence of 7.14 ppm between each of the samples at level 1.

Our sampling strategy was a tradeoff between the

maximization of the [CO2] range, in order to minimize

the uncertainty in the modified Keeling plot slope

calculation (Miller & Tans, 2003; Pataki et al., 2003),

and the minimization of the risk of failure in the

completion of a full set of flasks after two successive

nights. We considered that the risk of violation of the

basic assumption of the modified Keeling plot approach

(the system is composed of either (i) a single respiration

source with a constant d, or (ii) several sources that

might have different d but must have constant relative

contributions to the total respiratory flux) was too high

beyond a period of two successive nights. Once a

sample was validated, the Valco valve position was

advanced to the next flask and an air sample was

collected from level 2 and kept regardless of its [CO2].

This procedure was followed until all flasks had been

filled or the nighttime period ended (time of sunrise

plus 2 h). If all the flasks were not collected in the first

night, the automatic collection system would remain

298 S . P O N T O N et al.

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inactive during daylight hours and then would attempt

to collect the remaining flask samples on the subsequent

night. Automatic calibration of the LI-820 IRGA oc-

curred before the mid-afternoon sample, before the first

night sample and then every hour during nighttime

sampling. During all sampling dates at the aspen site,

flask samples were collected during one night of sam-

pling. At the Douglas-fir site, flask samples were col-

lected during one night of sampling for five of the eight

sampling dates, while two nights of sampling were

required on three sample dates.

In order to test for errors associated with the auto-

matic flask sampling system and our field collection

procedures, we filled 15 flasks with a working standard

gas from a high-pressure cylinder at a field site. The gas

(air) from the working standard cylinder was passed

through the entire sampling system including a flask for

4 min at a flow rate of 1 L min�1. The gas flow was then

shut off for 45 s before advancing the Valco valve to the

next flask. The flasks then remained in the automatic

system for various time intervals (24, 48 and 72 h) before

the stopcocks on the flasks were closed and they were

returned to the lab for analysis. The results of the

analyses of these flask samples indicated that there

were no significant systematic errors associated with

the sampling system or the collection procedures

(Table 1). The average [CO2] of flasks left with their

stopcocks open for only 24 h was slightly higher (by

1.1mmol mol�1) than that measured on flasks when the

stopcocks were left open for 48 or 72 h. However, the

measured [CO2] of the working standard tank was

407.1 � 0.18 mmol mol�1, so the flasks with the stop-

cocks left open for longer periods of time actually had

more accurate concentrations than the flasks with stop-

cocks left open for only 24 h. There were no significant

differences between the d13C values of flasks left open

for different periods of time (Table 1), and the measured

values on these flasks all agreed well with the value of

the working standard cylinder determined in lab ana-

lyses (�10.22 � 0.14%, n 5 292). We did note that there

was a slight decrease in the precision of our [CO2]

measurements on flasks left open in the field for longer

periods of time (indicated by a slight increase in the

standard deviation of replicate measurements). This

may have been associated with the slightly higher water

vapor concentration in the flasks when the stopcocks

were open for longer periods, although the water vapor

differences did not significantly affect the measured

d13C values (Table 1).

In the low stature grassland site, air samples were

manually collected from the headspace of four respira-

tion chambers (100 L volume), using procedures and

equipment previously described by Flanagan et al.

(1999). These chambers were made of a plexiglass top

that was clamped on collars installed in the ground the

year before. The collars sampled a ground area of

approximately 0.36 m2. An evacuated flask was con-

nected to the respiration chamber via a port equipped

with an Ultra-Torr connector. On sampling days, flask

samples of air were collected from each respiration

chamber after dusk. From each respiration chamber,

10 previously evacuated flasks (5–10 Pa) were filled

with air, with a 1–2 min interval between samples. We

attempted to obtain a CO2 range, across all ten flasks, of

at least 100 ppm. On average, air samples were collected

at intervals of approximately 10–15 ppm change in

chamber [CO2]. At the grassland site only three of four

chambers were sampled on July 14 (day of year (DOY)

195) because of a shortage of flasks.

The carbon isotope composition of CO2 respired by

the ecosystem (dR) was calculated from measurements

of [CO2] and carbon isotope composition on air samples

using the (modified Keeling plot) approach described

by Miller & Tans (2003):

dobscobs ¼ dRcobs � cbg dbg � dR

� �; ð3Þ

where c is the concentration of CO2 and d is the carbon

isotope composition of CO2, and the subscripts obs, bg

and R refer to observed, background and respired CO2

values, respectively. This equation describes a simple

linear function with a slope dR and an intercept

�cbg(dbg–dR). A geometric mean linear regression was

performed to estimate dR (Pataki et al., 2003). Uncer-

tainty in the slope was calculated as described by Miller

& Tans (2003). The analytical precision of the measure-

ments were used as a priori estimates of the errors in

Table 1 Comparison of measurements made on flasks filled

with dry air from a working standard gas cylinder

Hours with stopcocks open

24 48 72

[CO2]

(mmol mol�1)

408.9 � 0.2 407.8 � 1.1 407.7 � 2.3

d13C (%) �10.34 � 0.12 �10.44 � 0.09 �10.38 � 0.10

[H2O]

(mmol mol�1)

0.6 � 0.0 1.3 � 0.1 1.8 � 0.1

After flushing air through the automatic sample system and

flasks, the stopcocks were left open for different periods of

time while the flasks remained connected to the sample

system. After the stopcocks were closed, the flasks were

removed from the sample system and returned to the lab

for analysis. The working standard gas cylinder had the

following characteristics, based on the average ( � SD) of 292

replicate lab measurements: [CO2] 5 407.1 � 0.18 mmol mol�1,

d13C 5�10.22 � 0.14%. The values in the table represent the

mean � SD, n 5 5.

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cobs and dobs, (i.e. 0.18 ppm and 0.14% for [CO2] and

d13C, respectively). According to an error propagation

calculation, uncertainty associated with dobscobs was

calculated as

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffic � Ddð Þ2þ d � Dc

� �2q

where c and d corre-

spond to the mean value of [CO2] and d13C, respec-

tively, and Dd and Dc correspond to the errors reported

above. Using the routine ‘fitexy’ (Press et al., 1992),

data uncertainties were scaled until q 5 0.5 � 0.01, then

the corresponding uncertainty (SE) for the slope was

reported.

Any points where the absolute value of the residual

(orthogonal distance to the fitted line) was larger than

three standard deviations of all the absolute residuals

were considered as outliers and removed before the

final linear regression analysis (modified Keeling

plot) was done. This procedure was used for each

modified Keeling plot sample set independently. In

total, only 13 (i.e. 2.4%) flask samples were discarded

from the entire data set using this procedure. Only flask

samples collected during time periods when the light

intensity (PPFD) at the top of the canopy was below

100 mmol m�2 s�1 were used in the modified Keeling

plot analyses.

WUE calculations

For a comparison of environmental controls on ecosys-

tem-level WUE, we made calculations of WUE using

two approaches. First, ecosystem WUE was calculated

as the ratio of gross photosynthesis (GPP) to ecosystem

ET during the time of the day when D was at its

maximum. This daily average calculation made use

of the six half-hour time periods (i.e. 25% of the values)

between 8:30 and 20:30 hours when D was the highest.

In order to reduce the contribution of the evaporation

component of ET in these calculations (in contrast to

the transpiration component), days with recorded

precipitation and the day after a rain event greater

than 5 mm were excluded. In addition, we also

excluded any time periods with low water vapor

fluxes (o0.05 mmol m�2 s�1), inadequate turbulence

(u*o0.10 m s�1), or low PPFD (o25 mmol m�2 s�1) as

described in Wever et al. (2002). Seasonal patterns in

WUE were examined using a time-series graph during

the period of time when the deciduous leaf species were

near their peak photosynthetic activity (see below for

details).

The second approach made use of ci/ca calculations

determined from the carbon isotope ratio of ecosystem

respired CO2. The background for these calculations is

described below.

Farquhar et al. (1982, 1989) showed that the stable

isotope composition of leaf tissue (dp) was related

to ci/ca and the isotope composition of atmospheric

CO2 (da) by

dp ¼ da � a� b� að Þ ci

ca; ð4Þ

where a is the isotopic fractionation during photosyn-

thetic gas exchange caused by the slower diffusion of13CO2 in air (4.4%), b is the net fractionation associated

with RuBP carboxylase activity (27%), and ca and ci are

the atmospheric and intercellular CO2 concentrations,

respectively. The instantaneous WUE can be calculated

using Eqn (1) with input of ci/ca calculated from dp,

which was estimated from measurements of the carbon

isotope composition of CO2 respired by the ecosystem

(dR). In this study, the leaf intercellular spaces were

assumed to be at water vapor saturation and the leaf

temperature was assumed to be identical to that of air

temperature. As a consequence u was equal to the ratio

of D (kPa) to atmospheric pressure (kPa).

Combining Eqns (1) and (4) and using dp 5 dR, we

estimated the WUE of the ecosystem from dR measure-

ments using the following equation:

WUE ¼ A

E¼ ca � ca dR � da þ aÞ=ða� bð Þ

1:6uð5Þ

where ca was assumed to be 370 mmol mol�1 and

da 5�8%. In this case, we assume that all of the CO2

released by total ecosystem respiration (Reco) had the

carbon isotope signal set during recent photosynthetic

gas exchange in the ecosystem. In reality only the plant

leaf, stem, root and rhizosphere component of ecosys-

tem respiration should be influenced in the short-term

(days) by changes in carbon isotope discrimination

during photosynthetic gas exchange. We consider rhizo-

sphere respiration (respiration by rhizosphere organ-

isms using carbon exudates from plant roots) as a

component of autotrophic respiration (Ra) for this ana-

lysis. The heterotrophic component (Rh) of ecosystem

respiration (decomposition of surface litter and humus)

should have a carbon isotope composition that remains

relatively constant over the growing season, if not long-

er time scales. A mass balance equation can be written

for the autotrophic and heterotrophic components con-

tributing to total ecosystem respiration and their asso-

ciated carbon isotope compositions:

dRReco ¼ dRaRaþdRhRh; ð6Þ

where dRa is the carbon isotope composition of auto-

trophic respiration and dRh is the carbon isotope com-

position of heterotrophic respiration. The carbon

isotope composition of autotrophic respiration can be

calculated if the relative proportion of total ecosystem

respiration rate that is contributed by autotrophic

300 S . P O N T O N et al.

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respiration (f) is known, and dRh and dR are known:

dRa ¼dR�dRhð1� fÞ

f: ð7Þ

We used measurements of the carbon isotope composi-

tion of surface litter and the LFH component of the soil

profile in each study site to estimate the carbon isotope

composition of heterotrophic respiration. These mea-

surements were combined with modified Keeling plot

estimates of dR to estimate dRa. Lacking detailed infor-

mation on the relative proportion of total ecosystem

respiration rate that was contributed by autotrophic

respiration (f), we started with the initial simple as-

sumption that f was 0.5 (see below for further discus-

sion of this assumption). Ecosystem WUE was then also

calculated using Eqn (5), with our estimates of dRa in

place of dR.

In our calculations of ecosystem WUE using Eqn (5),

averaged and lagged values of D and atmospheric

pressure values were used to estimate u (the difference

in water vapor concentration), because a delay was

expected between the time of CO2 assimilation and

the time when the assimilated carbon was released as

CO2 by total ecosystem and autotrophic respiration.

Ekblad & Hogberg (2001) reported that the variations

of dR in a boreal mixed coniferous forest in Sweden

were best explained by the air relative humidity 1–4

days before the day of CO2 sampling. Based on the

same statistical approach, Bowling et al. (2002) found

peaks in the correlation coefficients between dR and D

at 5–10 days delay along a site gradient in Oregon.

McDowell et al. (2004a) calculated an average lag period

of one day for soil dR in a ponderosa pine forest in

Oregon, close to the 2–4 days lagged 14C activity peak

observed on radiolabeled Populus trees (Horwarth et al.,

1994; Mikan et al., 2000). Based on these previous

analyses, we chose to calculate u by using the daily

mean of the highest D values of the day (as described

above) and the daily mean atmospheric pressure both

averaged over a 5-day period before the date of the

atmospheric CO2 isotope sampling. Abnormally high

values of WUE were calculated on DOY 263 in the

Douglas-fir site, on DOY 265 in the aspen site and on

DOY 176 in the grassland site because of rain and very

low values of D observed over the 5-day period before

the air sampling. These obviously erroneous values

were discarded.

Collection and analysis of litter and soil samples

Surface litter and soil samples (free from roots) were

collected from soil pits dug at all sites. Five or six

replicate samples of surface litter, LFH horizon and

the mineral soil (0–10 and 10–20 cm depths) were col-

lected from randomly chosen locations in each study

site. Samples were dried at 65 1C, and ground to a fine

powder with a tissue grinder or a mortar and pestle.

The organic tissue samples (surface litter and LFH

layer) were prepared for measurements of carbon iso-

topic composition by combustion. A 1–2 mg subsample

of ground organic material was sealed in a tin capsule

and loaded into an elemental analyzer for combustion

(Carla Erba). The carbon dioxide generated from the

combustion was purified in a gas chromatographic

column and passed directly to the inlet of a gas isotope

ratio mass spectrometer (Delta Plus, Finnigan Mat, San

Jose, CA, USA). The mineral soil samples were reacted

with acid to remove any carbonate material before

analysis of the carbon isotope composition, as described

above with the exception that a higher range of sample

weights were used because of the generally lower

carbon content of the mineral soil samples.

Results

Seasonal variation in environmental conditions andecosystem gas exchange

The three sites used in our comparison span a large

range of vegetation types and environmental condi-

tions. When compared in the relative order Douglas-

fir, aspen, grassland, the sites demonstrate a progressive

decline in precipitation and a general increase in max-

imum air temperature and Dmax during the mid-sum-

mer (Fig. 1). In addition, there was a larger range of

variation observed between maximum and minimum

air temperatures at the grassland site than that observed

at the other two sites. All three sites showed a large

seasonal decline in soil moisture content that occurred

while air temperature increased. The longest extended

period of low soil moisture and high temperature

occurred at the grassland site (Fig. 1). The 2003 growing

season in western Canada (BC, Alberta, Saskatchewan)

was dry, with the precipitation recorded during May–

August lower than the 1971–2000 averages by 44% at

the Douglas-fir site, 36% at the aspen site and 40% at the

grassland site.

Maximum GPP was very similar in the two forest

sites, and only slightly lower in the grassland site,

although the seasonal pattern of variation in GPP con-

trasted strongly among sites (Fig. 2). The growing

season was considerably longer at the Douglas-fir site,

with GPP values always greater than zero even during

the winter months. There was a single peak in GPP near

DOY 180, with GPP values higher than 2 g C m�2 day�1

recorded as early as mid-February and as late as the end

of October. The aspen site showed a double peaked

pattern, with reduced GPP recorded during low soil

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moisture in late July (DOY 200–210) and some recovery

of GPP after precipitation on DOY 216 increased soil

moisture for a short time period. The grassland site

showed only a single peak of GPP and then a sharp

decline as vegetation senescence occurred in response

to drought.

The ecosystem ET rates showed similar seasonal

patterns to that observed for GPP, except with much

more scatter caused by low ET values during cool, wet

periods (Fig. 2). The highest ET values were recorded in

the grassland site and the lowest values were measured

at the Douglas-fir site, with the maximum ET values

approximately 2.7, 3.2 and 5 mm day�1 for the Douglas-

fir, aspen and grassland sites, respectively.

We made comparisons of ecosystem WUE measured

by eddy covariance at each site during the time when

Fig. 1 Seasonal variation in the daily maximum and minimum air temperatures, daily maximum atmospheric saturation deficit (Dmax),

daily total precipitation and daily mean soil moisture at the Douglas-fir, aspen and grassland study sites.

302 S . P O N T O N et al.

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the deciduous leaf species were near their peak photo-

synthetic activity. At the aspen site, the canopy was near

full GPP activity during DOY 150–250 (Fig. 2), and this

period of time was also used for comparison with the

Douglas-fir site. The grassland canopy was near full

GPP activity only during DOY 150–200 (Fig. 2), and so

calculations of WUE at the grassland site were restricted

to this shorter time period. WUE was low at the grass-

land, intermediate at the aspen site and highest at the

Douglas-fir site (Fig. 3). There was substantial day-to-

day variation in WUE, but there was no apparent

seasonal trend observed at the Douglas-fir and aspen

sites, with only a slight trend towards a seasonal decline

at the grassland site (Fig. 3). The average ( � SD) WUE

at the grassland site during DOY 150–200 was

2.6 � 0.7 mmol mol�1 (n 5 31), which was much lower

than the average values observed for the two other sites

during DOY 150–250 (Douglas-fir: 8.1 � 2.4 mmol

mol�1 (n 5 74), aspen: 5.4 � 2.3 mmol mol�1 (n 5 56)).

There was a strong negative correlation between WUE

and Dmax for all sites (Douglas-fir, r 5�0.703; aspen,

r 5�0.770; grassland, r 5�0.917). In addition there were

significant differences (unpaired t-tests, Po0.05) in the

slopes of linear regressions fitted to the WUE and Dmax

relationships, with the highest slope observed for the

Douglas-fir site and the lowest slope observed for the

grassland site (Fig. 4). The decline in the magnitude of

the slope was correlated with an increase in the Dmax

values observed at the different sites during the time of

peak GPP activity (Fig. 4).

Seasonal variation in the carbon isotope composition ofecosystem respired CO2

A consistent increase in dR was observed at the Dou-

glas-fir site during the course of the growing season,

with dR values changing from –27.7% on May 13 (DOY

133) to –24.5% at the peak on August 31 (DOY 243)

(Fig. 5). This pattern excluded the very negative dR

value of –29.6% that was recorded on August 11

(DOY 223) during a storm with very heavy rain

(35 mm/day). This dR value was discarded in subse-

quent analyses because we believe that it was strongly

influenced by this one strong rainstorm, while our other

data was collected during times of quite clear and dry

weather conditions.

At the aspen site, the dR increased from –26.2% on

July 2 (DOY 183) to –24.9% on September 3 (DOY 246),

after an initial decline from –25.3% to –26.2% recorded

in the early growing season (Fig. 5). A strong decrease

in dR (from –24.9% to –26.8%) was also observed at the

end of the growing season. A similar seasonal pattern

was observed at the grassland with an initial increase in

dR from –26.5% on May 27 (DOY 147) to�24.0% on July

Fig. 2 Seasonal variation in gross photosynthesis (GPP) and ecosystem evapotranspiration (ET) at the Douglas-fir, aspen and grassland

study sites.

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14 (DOY 195), followed by a decline to –26.4% on

August 5 (DOY 217).

The d13C values of the surface litter and LFH layer of

the soil profile were very similar among sites (Table 2).

The organic carbon became progressively more en-

riched in 13C with depth through the soil profile, and

this pattern was substantially stronger in the grassland

than the two forest sites. In particular, organic carbon

in the grassland mineral soil layers had much higher

d13C values than was observed for the two forest sites

(Table 2). We used the average d13C values of the surface

litter and LFH layer as a proxy for the stable isotope

composition of heterotrophic respiration in all sites, and

these values were very similar among sites (Douglas-fir,

�27.23%; aspen, –27.27%; grassland, �27.32%).

The pattern of increasing dR (and dRa) values ob-

served at the forest and grassland sites during the

middle of the growing season was consistent with a

Fig. 3 Comparison of seasonal variation in daily water-use

efficiency (WUE) calculated from the eddy-fluxes (ratio of GPP

and ET from Fig. 2) at the Douglas-fir, aspen and grassland

study sites.

Fig. 4 Relationship between water-use efficiency (WUE, calcu-

lated from the eddy-flux data) and maximum atmospheric

saturation deficit (Dmax) at the Douglas-fir, aspen and grassland

study sites. The solid lines represent fitted linear regressions:

Douglas-fir, y 5�2.686x 1 12.181; aspen, y 5�2.396x 1 10.419;

grassland, y 5�0.792x 1 4.776.

304 S . P O N T O N et al.

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drought-induced decline in stomatal conductance and

associated reduction in ci/ca. In order to test this hy-

pothesis we examined the relationship between changes

in soil moisture content and dR values (Fig. 6a). At all

three sites dR values were negatively correlated with soil

moisture content as expected (Douglas-fir, r 5�0.944,

P 5 0.005, n 5 6; aspen, r 5�0.742, P 5 0.26, n 5 4; grass-

land, r 5�0.978, P 5 0.13, n 5 3). However, because of

the very low number of observations these correlations

were not statistically significant in the aspen and grass-

land sites. Interestingly, the slopes of the relationships

between dR and available soil water content were very

similar (unpaired t-test, P40.05) in all three sites

(Fig. 6a). Identical correlation coefficients were ob-

served for the relationship between dRa values and soil

moisture, but the magnitude of the slope for the rela-

tionships was increased relative to that for dR and

available soil water content (Fig. 6b).

There was very good agreement between WUE

values calculated using eddy covariance flux data (EC

WUE) and that based on stable isotope measurements

(Eqn (5)) (Fig. 7). A strong positive correlation occurred

between EC WUE and WUE calculated based on dR

values (dR WUE) (r 5 0.912, n 5 14, Fig. 7a). A slightly

lower value was observed for the correlation coefficient

between EC WUE and WUE calculated based on dRa

values (dRa WUE) (r 5 0.869, n 5 14, Fig. 7b).

The large differences in WUE among sites were most

strongly determined by variation in D, as dR (and dRa

values) and associated ci/ca values were quite similar at

all sites. The average dR observed during the growing

season (excluding the declining phase at the end of the

growing season in the aspen and grassland sites) were

�25.9%, �25.3% and �25.1% for the Douglas-fir, the

aspen and the grassland sites, respectively. The corre-

sponding ci/ca values (Eqn (4)) were very similar in the

three different sites (0.59, 0.57 and 0.56, respectively).

The d13C values of the surface litter and LFH layer were

also consistent with this idea that leaf-level ci/ca values

were very similar among the three sites (Table 2).

Discussion

We observed distinct differences in WUE among the

three ecosystems compared. The grassland and aspen

Fig. 5 Seasonal variation of the carbon isotope composition of

CO2 respired by the ecosystem (dR) at the Douglas-fir, aspen and

grassland study sites. At the grassland site, the four chambers

that were sampled are represented by different symbols, and the

line links the daily average values calculated from the four

chambers (excluding one chamber during the third sampling

date (see text)). Errors bars represent uncertainties (standard

errors) in the calculation of the isotope ratio of respired CO2, as

described in ‘Materials and methods’.

Table 2 Comparison of the carbon isotope composition

(d13C, %) of organic carbon in surface litter and different soil

depths in the three study sites

Douglas-fir Aspen Grassland

Surface litter �27.68 � 0.49 �27.69 � 0.21 �27.41 � 0.40

LFH layer �26.79 � 0.34 �26.86 � 0.44 �27.23 � 0.25

Mineral soil

(0–10 cm)

�25.67 � 0.38 �25.79 � 0.17 �23.45 � 0.78

Mineral soil

(10–20 cm)

�25.06 � 0.59 �25.24 � 0.39 �21.27 � 0.49

n 5 5 6

The values in the table represent the mean � SD, n 5 sample

size.

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WUE (2.6 and 5.4 mmol mol�1, respectively) were typi-

cal of values measured at the leaf level for C3 vegeta-

tion, while the Douglas-fir (8.1 mmol mol�1) showed

very high WUE approaching values recorded for leaf

tissue of plants with the CAM photosynthetic pathway

(Osmond et al., 1982). For the grassland site very similar

WUE values were observed in previous years at the

same site (2.88–3.13 mmol mol�1; Wever et al., 2002).

Arain et al. (2002) reported similar WUE values of

4.5 mmol mol�1 for the same aspen site during 1994–

1999 when soil moisture availability was higher. In a

comparison among vegetation types, Law et al. (2002

and Addendum via personal communication with B.E.

Law) calculated ecosystem WUE as the slope of the

relationship between monthly values of GPP and ET

measured during an entire year. Similar values of the

GPP/ET slope (g CO2 kg�1 H2O) were observed for

grassland (3.4) and deciduous broadleaf forests (3.2),

with evergreen conifer forests having only slightly

higher values (4.2). The pattern of relatively similar

WUE among different vegetation types, reported by

Law et al. (2002), contrasts with the observations made

in this study. We have focused our study on the time

periods of active growth, which may have allowed us to

more readily detect differences in WUE among ecosys-

tems with contrasting dominant functional types.

When the effects of soil and surface water evapora-

tion are minimized, variation in WUE is controlled by

Fig. 6 (a) Relationship between the carbon isotope composition

of ecosystem respired CO2 (dR) and available soil moisture at the

Douglas-fir, aspen and grassland study sites. The solid lines

represent fitted linear regressions: Douglas-fir, y 5�4.038x–

24.333; aspen, y 5�4.717x–24.703; grassland, y 5�4.565x–

22.767. (b) Relationship between the carbon isotope composition

of CO2 respired by autotrophic (and rhizosphere) respiration

(dRa) and available soil moisture at the Douglas-fir, aspen

and grassland study sites. The solid lines represent fitted linear

regressions: Douglas-fir, y 5�8.076x–21.431; aspen, y 5�9.433x–

22.1331; grassland, y 5�9.129x–18.214. Errors bars represent

uncertainties (standard errors) in the calculation of the isotope

ratio of respired CO2 (dR), as described in the Methods. The error

bars are the same for both graphs (a, b). For the aspen site, the

first and last samples of the growing season were not considered

in this relationship as their dR values were interpreted to be

unrelated to variations in soil water content (see ‘Discussion’).

For the same reason, the last sampling was also discarded for the

grassland site.

Fig. 7 (a) Comparison of the relationship between water-use

efficiency (WUE) values calculated using eddy covariance flux

data (EC WUE) and that based on stable isotope measurements.

The y-axis values of WUE were calculated based on dR values (dR

WUE) using Eqn (5). (b) The y-axis values of WUE were

calculated based on dRa values (dRa WUE) using Eqn (5). The

lines shown in both panels represent the one-to-one comparison

lines.

306 S . P O N T O N et al.

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leaf gas exchange characteristics and environmental

factors (air temperature and humidity) (Eqn (1)). The

similar d13C values of the surface litter and LFH layer

suggest that spatially- and temporally-integrated leaf-

level ci/ca values were very similar among the three

sites (Table 2). However, air temperature and humidity

were systematically different among sites so that D was

progressively higher when the sites were compared in

the relative order Douglas-fir, aspen, grassland (Fig. 1).

So the pattern of variation in WUE among sites was

primarily controlled by differences in ET (Fig. 2), and

the higher ET was a result of variation in D, while GPP

and leaf-level ci/ca values were very similar among the

three sites. Consistent with this argument, and similar

to the patterns observed by Law et al. (2002), ecosystem

WUE declined strongly with increases in D at all three

sites (Fig. 4).

In contrast to the similar d13C of leaf litter and dR

values (and therefore ci/ca) among the different vegeta-

tion-types studied here, Bowling et al. (2002) observed a

quite large 2–3% range in dR between the wettest and

driest sites along a pronounced precipitation transect in

Oregon. However, the vegetation of the Oregon transect

was dominated by evergreen coniferous trees, although

there was a change in tree species composition and LAI

along the transect, while our study included changes

from evergreen coniferous trees, to deciduous broad-

leaf trees to herbaceous grasses. The shift in plant

functional types likely had a significant influence on

the observed similarity in ci/ca values among our study

sites. In comparisons among trees, broad leaf deciduous

species tend to have higher ci/ca values (lower leaf d13C)

than coniferous trees (Brooks et al., 1997). Similarly,

herbaceous plant species (forbs) also tend to have high-

er ci/ca values (lower leaf d13C) than trees species

(Brooks et al., 1997) and grasses tend to have higher

ci/ca values (lower leaf d13C) than forbs (Smedley et al.,

1991). So the expected trend toward lower ci/ca values

and higher dR as water availability decreased from

Douglas-fir to aspen to the grassland site, was counter-

acted by the shift in plant functional type toward

species with intrinsically higher ci/ca values and lower

d13C values.

In arid land habitats under constant environmental

conditions, shorter-lived species tend to have higher

ci/ca values (lower leaf d13C) than longer-lived species

(Ehleringer, 1993). In our study, plants from all func-

tional types had similar carbon isotope ratios while

there was significant variation in D among sites. In

other words the shorter-lived species (grasses) have

relatively high ci/ca values for the high D conditions

of their habitat. This is consistent with a strategy to

grow fast while moisture conditions are favorable for

growth and then enter dormancy during harsh condi-

tions. In contrast, the long-lived conifer species are

more conservative in water-use with lower ci/ca values

relative to their habitat D, but because of the moderate

D conditions in their coastal environment, high WUE

was obtained. This illustrates the interaction between

biological and environmental characteristics influen-

cing ecosystem-level WUE.

In the forest and grassland sites, our measurements

showed an increase in dR during time periods when soil

moisture declined (Fig. 6). These ecosystem-level re-

sponses were consistent with expectations for the nor-

mal physiological response of an individual plant,

specifically a reduction in stomatal conductance and

leaf ci/ca as soil moisture declined. Fessenden & Ehler-

inger (2003) have also observed significant increases in

dR as soil moisture declined in ponderosa pine ecosys-

tems in the state of Washington. Similarly, shifts in dR

were correlated with seasonal changes in precipitation

inputs in evergreen tropical forests in the Amazon Basin

(Ometto et al., 2002). Short-term variations in phloem

sugar d13C were strongly correlated with soil moisture-

induced changes in dR in an Italian beech forest (Scar-

tazza et al., 2004). McDowell et al. (2004b) observed

relationships among dR and climate variables (soil

moisture, vapor pressure deficit and temperature) and

canopy conductance that were consistent with predic-

tions based on stomatal regulation of ecosystem carbon

isotope discrimination.

Only the autotrophic (plant and rhizosphere) compo-

nent of ecosystem respiration (Ra) should be influenced

in the short-term (days) by changes in carbon isotope

discrimination during photosynthetic gas exchange

caused by reductions in soil moisture. The hetero-

trophic component (Rh) of ecosystem respiration should

have a carbon isotope composition that remains rela-

tively constant over the growing season, if not longer

time scales. We made calculations to estimate the mag-

nitude of the change in the isotopic composition of the

autotrophic component of ecosystem respiration that

would be required to cause the observed changes in dR

(Fig. 6). To do this, we needed an estimate of the relative

proportions contributed by autotrophic and hetero-

trophic respiration. Lacking this detailed information

we started with the simple assumption that 50% of

ecosystem respired CO2 was contributed by both auto-

trophic and heterotrophic respiration. This is likely an

underestimate of the contribution of autotrophic re-

spiration. Amthor & Baldocchi (2001) concluded from

a review of several studies that autotrophic respiration

was typically 50–70% of GPP. Assuming that most

ecosystems have a net accumulation of some carbon,

Ra/GPP values of 50–70% suggest that autotrophic

respiration is typically higher than heterotrophic

respiration. Our calculations made the additional

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assumption that the CO2 produced in heterotrophic

respiration has a 13C/12C composition that can be pre-

dicted by measurements of the isotopic composition of

litter and humus layers above the mineral soil. The

resulting data illustrated that the magnitude of the

calculated change in dRa values was higher than that

observed for dR as soil water content declined (Fig. 6),

but the calculated variation in dRa was still within a

realistic range for known physiological responses of C3

vegetation (Farquhar et al., 1989). We expect that the

magnitude of autotrophic respiration is actually higher

than 50% (but below that of 100%) of total ecosystem

respiration and so the real shift in dRa should be inter-

mediate to that illustrated in the two graphs shown in

Fig. 6.

We observed very good agreement between values of

ecosystem WUE calculated using eddy covariance data

with that calculated using ci/ca determined from the

carbon isotope ratio of ecosystem respired CO2. The

WUE calculated using dR values showed a stronger

correlation with WUE calculated using eddy covariance

than when dRa values were used for estimating

WUE (Fig. 7). This was also consistent with the idea

that autotrophic (plant and rhizosphere) respiration

represents a major fraction, larger than 50%, of

total ecosystem respiration. In addition, the strong

correlation between WUE calculated via independent

methods provides support for the use of dR as a

spatially-integrated estimate of short-term changes in

ecosystem ci/ca values.

As leaf intercellular CO2 concentration depends on

the ratio of photosynthetic rate to stomatal conductance,

change in either one (or both) of the later parameters

will cause changes to ci/ca and this should influence dR.

In both the grassland and aspen sites, the reduction in

photosynthesis associated with leaf senescence should

have resulted in increases to ci/ca and this may explain

the strong decline in dR values observed at the end of

the growing season. This pattern of change in dR has

been observed previously at the same aspen forest used

in this study and at another aspen forest in northern

Manitoba (Flanagan et al., 1996). The seasonal decline in

D at the end of the growing season in the aspen sites

would have lead to the same decreasing trend in dR

because of a reduction in the stomatal limitation of

photosynthesis. However, in the grassland site the

senescence-associated decrease in dR occurred in July

while the temperature, D and PPFD were still at their

maximum levels. In the grassland, leaf senescence was

associated with soil drought that caused the herbaceous

plants to enter dormancy.

The initial sampling date for dR in the aspen site (May

28, DOY 148) coincided with rapid leaf expansion and

the development of leaf photosynthetic competence

(Fig. 2). The relatively elevated dR value observed on

this first sample date may have been caused by the use

of stored carbohydrate for new leaf development (and

respiration) that was fixed during the previous year’s

growing season under conditions of water stress and

low ci/ca values. Repeated sampling over several years

with contrasting environmental conditions would be

necessary in order to test this speculation.

The strong correlation between dR and WUE occurred

despite the potential for a number of complicating

factors to disrupt such a relationship. Isotope fractiona-

tion during plant respiration, seasonal shifts in the

proportion of autotrophic and heterotrophic respiration,

temporal variation in the d13C of heterotrophic respira-

tion, isotope effects during microbial breakdown of litter

and humus, and significant lags between the time of CO2

fixation and the conversion of organic matter back to

CO2, are examples of some of the potential complicating

factors. Further research is required to completely under-

stand the role of these potential complicating factors.

However, based on all the results discussed above, we

conclude that, for the sites and conditions sampled, the

carbon isotope ratio of ecosystem-respired CO2 can be

used to estimate daytime ecosystem-scale WUE. This

suggests that measurements of dR represent a spatially

integrated estimate of ci/ca, and so provide an ecosys-

tem-scale tool to help study constraints to photosynthesis

and acclimation of an ecosystem to environmental stress.

One of the strong advantages of using stable isotope

measurements in this study was that mechanistic in-

sights were obtained into the separate contribution of

biological (ci/ca values) and environmental (air tempera-

ture and humidity) factors for their effects on WUE. A

complete ecological interpretation of patterns of varia-

tion in WUE requires insight into both the biological and

environmental controls.

Acknowledgements

This research was undertaken as part of the Fluxnet-Canadaresearch network and was funded by grants to LBF, TAB, AGBfrom NSERC, CFCAS and BIOCAP Canada. We thank Tiebo Caifor his assistance in the quality control of the eddy flux data fromthe Douglas-fir site, and Zoran Nesic for his technical assistancein maintaining the eddy covariance systems at the Douglas-firand aspen sites. Peter Carlson provided assistance with fieldand lab work and helped to construct and install the automaticflask sample systems.

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