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1 bs_bs_bnner Plnt, Cell nd Environment (2014) 37, doi: /pce Originl Article Vrition in the crbon nd oxygen isotope composition of plnt biomss nd its reltionship to wter-use efficiency t the lef- nd ecosystem-scles in northern Gret Plins grsslnd Lwrence B. Flngn 1 & Grhm D. Frquhr 2 1 Deprtment of Biologicl Sciences, University of Lethbridge, Wter & Environmentl Sciences Building, 4401 University Drive, Lethbridge, Albert T1K 3M4, Cnd nd 2 Reserch School of Biology, Austrlin Ntionl University, Acton, Austrlin Cpitl Territory 0200, Austrli ABSTRACT Mesurements of the crbon (δ 13 C m) nd oxygen (δ 18 O m) isotope composition of C 3 plnt tissue provide importnt insights into controls on wter-use efficiency. We investigted the cuses of sesonl nd inter-nnul vribility in wteruse efficiency in grsslnd ner Lethbridge, Cnd using stble isotope (lef-scle) nd eddy covrince mesurements (ecosystem-scle). The positive reltionship between δ 13 C m nd δ 18 O m vlues for smples collected during indicted tht vrition in stomtl conductnce nd wter stress-induced chnges in the degree of stomtl limittion of net photosynthesis were the mjor controls on vrition in δ 13 C m nd biomss production during this time. By comprison, the lck of significnt reltionship between δ 13 C m nd δ 18 O m vlues during 2002, 2003 nd 2006 demonstrted tht wter stress ws not significnt limittion on photosynthesis nd biomss production in these yers. Wteruse efficiency ws higher in 2000 thn 1999, consistent with expecttions becuse of greter stomtl limittion of photosynthesis nd lower lef c i/c during the drier conditions of Clculted vlues of lef-scle wter-use efficiency were 2 3 times higher thn ecosystem-scle wter-use efficiency, difference tht ws likely due to crbon lost in root respirtion nd wter lost during soil evportion tht ws not ccounted for by the stble isotope mesurements. Key-words: ecosystem productivity; eddy covrince; Fluxnet-Cnd Reserch Network; photosynthesis; precipittion; soil moisture; temperture. INTRODUCTION Wter-use efficiency, the rtio of crbon gin in net photosynthesis to wter loss during trnspirtion, is criticl to the survivl, productivity nd biologicl fitness of plnts, with implictions for ecosystem energy exchnge processes (Osmond, Winter & Ziegler 1982; Frquhr et l. 1988; Correspondence: L. B. Flngn. Fx: ; e-mil: lrry.flngn@uleth.c Frquhr, Ehleringer & Hubick 1989). Plnts from contrsting ecosystems hve different wter-use efficiencies becuse of genetic vrition in lef gs exchnge chrcteristics nd becuse of vrition in environmentl conditions mong hbitts (Frquhr et l. 1988). For exmple, plnts with different life history chrcteristics exhibit systemtic vrition in photosynthetic gs exchnge chrcteristics when grown under similr environmentl conditions. Wter-use efficiency nd the rtio of net photosynthesis to stomtl conductnce re consistently lower in grsses thn forbs, lower in nnuls thn perennils, nd lower in brod-lef deciduous trees thn in conifer trees (Smedley et l. 1991; Ehleringer 1993; Brooks et l. 1997; Ponton et l. 2006). Vrition in either or both stomtl conductnce nd photosynthetic cpcity cn result in ltertions in wter-use efficiency (Frquhr et l. 1988, 1989). Lower stomtl conductnce often leds to proportionlly lrger effects on trnspirtion thn on net photosynthesis, with ssocited increses in wter-use efficiency (Frquhr & Shrkey 1982). Alterntively, genetic vrition in photosynthetic cpcity or nutrient-stimulted increses in net photosynthesis cn lso be the primry cuse of vrition in wteruse efficiency (Brooks & Mitchell 2011). As consequence, ecosystems dominted by contrsting plnt functionl types such s evergreen conifers or brod-lef deciduous trees, exhibit importnt differences in ecosystem energy exchnge processes, such s the rtio of ltent to sensible het fluxes, becuse of vrition in lef gs exchnge chrcteristics (Bldocchi et l. 2001; Ponton et l. 2006; Bonn 2008). The lef-ir vpour pressure difference (VPD) strongly controls wter-use efficiency becuse it influences trnspirtion rte t constnt stomtl conductnce, nd becuse stomtl conductnces vry with chnges in VPD (Frquhr et l. 1988, 1989). The lef-ir VPD is not n independent environmentl vrible, but chnges with vrition in stomtl conductnce. A reduction in stomtl conductnce, trnspirtion nd ltent het flux mens tht more het must be lost by sensible het flux, with n ssocited increse in lef temperture nd chnges to the lef-ir vpour pressure grdient (Frquhr et l. 1989). The reltive mgnitudes of stomtl conductnce to lef/cnopy boundry lyer 2013 John Wiley & Sons Ltd 425

2 426 L. B. Flngn & G. D. Frquhr conductnce, nd the cpcity for photosynthesis, determine whether chnge in stomtl conductnce will hve equl or disproportionl effects on net photosynthesis nd trnspirtion with consequences for VPD nd wter-use efficiency (Cown 1988; Frquhr et l. 1988). Under well-wtered conditions with high ir temperture, dense cnopy (low boundry lyer conductnce for lef nd cnopy) of plnts with lrge cpcity for photosynthesis cn present specil conditions where n individul plnt with reltively high stomtl conductnce will hve greter wter-use efficiency thn n otherwise identicl plnt with reltively low stomtl conductnce (Cown 1988; Frquhr et l. 1988). However, this scenrio of higher stomtl conductnce cusing higher wter-use efficiency is not likely to occur in sprse plnt cnopies of C 3 species with low lef re index (LAI) nd high boundry lyer conductnce tht result in the plnts being well-coupled to the tmosphere. If differences in wter-use efficiency mong plnts in cnopy re result of vrition in photosynthetic cpcity rther thn stomtl conductnce,then the interction between boundry lyer chrcteristics nd lef-irvpd becomes much less importnt (Cown 1988). So identifiction of the cuse of vrition in wter-use efficiency, whether due to chnges in photosynthetic cpcity or stomtl conductnce nd the role of lef-ir VPDs, hs implictions for ecosystem energy exchnge processes, in ddition to plnt survivl, productivity nd fitness (Frquhr et l. 1989). Mesurements of the crbon isotope composition (δ 13 C m) of C 3 plnt tissue provide importnt insights into subtle differences in lef physiologicl chrcteristics like chnges in the rtio of net photosynthesis nd stomtl conductnce tht ffect the concentrtion of CO 2 in the lef intercellulr ir spces nd in the chloroplst (Frquhr et l. 1989). Crbon isotope techniques hve been importnt, therefore, in studies of the genetic nd environmentl controls on plnt wter-use efficiency (Frquhr et l. 1988, 1989). The oxygen isotope composition (δ 18 O m) of plnt orgnic mtter should reflect vrition in the lef-ir vpour pressure grdient nd chnges in stomtl conductnce (Frquhr et l. 1988; Frquhr, Brbour & Henry 1998; Brbour 2007). Simultneous mesurements of δ 13 C m nd δ 18 O m could provide, therefore, powerful informtion bout the physiologicl nd environmentl fctors tht control wter-use efficiency. However, no previous, explicit clcultions of lef-scle wter-use efficiency hve been mde using mesurements of δ 13 C m nd δ 18 O m. In ddition, since δ 18 O m is ffected by vrition in stomtl conductnce but is not ffected by chnges in photosynthetic cpcity, the oxygen isotope mesurements cn be used to identify if differences in δ 13 C m re primrily result of chnges in photosynthetic cpcity or stomtl conductnce (Frquhr et l. 1998; Brbour 2007). When the primry cuse of vrition in δ 13 C m is stomtl conductnce, there should be positive correltion between δ 18 O m nd δ 13 C m (Frquhr et l. 1998; Brbour & Frquhr 2000; Brbour et l. 2000; Scheidegger et l. 2000; Brbour, Wlcroft & Frquhr 2002; Grms et l. 2007; Sullivn & Welker 2007; Roden & Frquhr 2012).Alterntively,the reltionship between δ 18 O m nd δ 13 C m should be non-existent or illustrte negtive correltion when vrition in photosynthetic cpcity is the cuse of differences in δ 13 C m (Frquhr et l. 1998). These ptterns should be pprent when potentil complictions like vrition in the δ 18 O of wter tken up by plnts do not significntly lter the stomtl conductnce nd VPD signls recorded by δ 18 O m (Brbour 2007). Our objectives in this study were to investigte sesonl nd inter-nnul vrition in δ 13 C m, δ 18 O m nd to mke explicit clcultions of lef-scle wter-use efficiency using the stble isotope mesurements in the northern Gret Plins grsslnd ner Lethbridge, Albert, Cnd. Grsslnd ecosystems offer specil opportunities to study vrition in plnt nd ecosystem physiology becuse of the wide rnge of environmentl conditions tht cn occur within growing sesons nd mong study yers (Flngn & Adkinson 2011). The lrgest inter-nnul chnges in plnt production mong the mjor ecosystem types of the continentl USA occur in grsslnds, primrily result of vrition in summer precipittion inputs (Knpp & Smith 2001). Flngn (2009) hs previously nlysed the sesonl nd inter-nnul vrition in δ 13 C m tht occurs in this Lethbridge grsslnd. Inter-nnul vrition in pek biomss production nd lef δ 13 C m vlues were negtively correlted, nd this reltionship ws consistent with lower biomss production being predominntly controlled by vrition in summer precipittion nd reduced soil wter vilbility (Flngn 2009). In this pper, we test for significnt positive correltion between δ 13 C m nd δ 18 O m vlues, reltionship tht would be consistent with wter stressinduced vrition in stomtl conductnce being the primry cuse of vrition in δ 13 C m. In ddition, we use the δ 13 C m nd δ 18 O m mesurements long with other dt nd theory to clculte lef-level wter-use efficiency nd compre these vlues to estimtes of ecosystem-scle wter-use efficiency, which ws defined s the rtio of gross ecosystem photosynthesis (GEP) nd ecosystem evpotrnspirtion (ET) rtes determined using eddy covrince mesurements. It ws expected tht lef- nd ecosystem-scle wter-use efficiency would differ primrily becuse of crbon lost in root respirtion nd wter lost in soil evportion, processes tht were not ccounted for by the lef-scle stble isotope mesurements (Frquhr et l. 1988; Hubick & Frquhr 1989). MATERIALS AND METHODS Study site description nd meteorologicl mesurements The study site ws locted in ntive grsslnd (800 m 800 m re) pproximtely 1.5 km west of the city limits of Lethbridge, Albert, Cnd, nd hs been described in detil previously (Flngn, Wever & Crlson 2002; Flngn & Johnson 2005; Flngn & Adkinson 2011). The coordintes of the eddy covrince flux tower t the site re: Lt. N: ; Long. W: ; 951 m bove se level. The climte in the region is semi-rid continentl nd the men dily tempertures ( ) for Jnury nd July, mesured t the Lethbridge irport, re 7.8 nd 18.0 C, respectively (Environment Cnd 2013). Men nnul precipittion ( ) is mm, with 30% flling in My nd June.

3 δ 13 C nd δ 18 O in plnt biomss 427 The plnt community t the site ws dominted by the grsses Agropyron dsystchyum [(Hook.) Scrib.] nd Ag. smithii (Rydb.), which ccounted for pproximtely 50% of the plnt cover (Crlson 2000; Flngn & Johnson 2005). Other plnt species present included: Vici mericn (Nutt.), Artemesi frigid (Willd.), Koeleri cristt [(L.) Pers.], Crex filifoli (Nutt.), Stip comt (Trin. nd Rupr.), S. viridul (Trin.). The verge (± stndrd devition; SD) cnopy height from 2001 to 2006 ws 31.7 ± 7.4 cm, nd it rnged from 18.5 ± 3.4 cm in 2001, which ws very dry yer, to 34.3 ± 9.5 cm in 2002, reltively wet yer. The soil (orthic drk-brown chernozem; Cnd Soil Survey Committee, Subcommittee on Soil Clssifiction 1987; Flngn & Johnson 2005) ws underlin by thick glcil till with very low permebility nd no wter tble (Scrcek 1993; Berg 1997). The soil A horizon (0.09 m) ws cly lom (28.8% snd, 40% silt, 31.2% cly) nd the B horizon (0.16 m) hd cly texture (27.4% snd, 29.6% silt, 40% cly; Crlson 2000). The surfce soil horizon (top 10 cm) bulk density ws 1.24 g cm 3, nd the orgnic mtter content ws 5.2%. Meteorologicl instrumenttion to monitor environmentl conditions nd to mke eddy covrince flux mesurements of net ecosystem CO 2 exchnge nd ecosystem ET hs operted continuously t the site since it ws estblished in June 1998, nd the equipment nd procedures hve been described in detil previously (Flngn et l. 2002; Wever, Flngn & Crlson 2002; Flngn & Adkinson 2011). Precipittion dt were mesured by Environment Cnd t the Lethbridge irport, which is 14 km from the study site (Environment Cnd 2013). We used these environmentl dt in clcultions, s described further below. Net CO 2 exchnge is expressed s net ecosystem productivity (NEP),where positive vlues of NEP indicte uptke of CO 2 by the ecosystem. The Fluxnet-Cnd stndrd protocol ws used for gp-filling CO 2 flux dt nd prtitioning NEP into totl ecosystem respirtion (TER) nd GEP (Brr et l. 2004). Both component fluxes of NEP were considered to be positive, so tht GEP ws clculted s the sum of NEP nd TER (i.e. NEP = GEP TER).The hlf-hourly CO 2 flux dt were integrted to determine dily vlues of GEP (mmol m 2 dy 1 ). For ET, temperture nd other meteorologicl dt, smll gps ( 6 hlf-hour periods) were filled by liner interpoltion, while longer gps (> 6 hlf-hour periods) were filled with the men diurnl trend bsed on 10 dys of dt, using the 5 dys previous to nd 5 dys following the gp period (Wever et l. 2002; Flngn & Adkinson 2011). In few cses where the gp in ET dt ws longer thn 1 week, the men diurnl trend of the 7 dys previous to nd the 7 dys following the gp period ws used. The hlf-hourly wter flux dt were integrted to determine dily rtes of ET (mol m 2 dy 1 ). Ecosystem wter-use efficiency (mmol mol 1 ) s clculted from eddy covrince dt (ecosystem wter-use efficiency), ws determined from the rtio of dily GEP nd ET vlues. Sesonl vrition in ecosystem sensible het flux, ltent het flux nd Bowen rtio (sensible het flux/ltent het flux) were clculted bsed on the verge middy vlues. We used the verge of the eddy covrince mesurements from the five 30-min periods from 1200 h to 1400 h locl time for these clcultions. Plnt biomss collection nd stble isotope composition mesurements Replicte boveground biomss smples (n = 6) were collected t intervls during the growing seson (My September) by clipping vegettion within cm qudrts. The qudrts were plced in rndomly selected m subplots locted within two lrger m plots, one northest nd the other southest of the instrument hut. The live (green) biomss of the smples ws seprted from the ded biomss. The live biomss ws dried in n oven t 60 C for t lest 24 h nd then weighed (Mettler PJ400, Greifensee, Switzerlnd). Smples were ground to fine powder with mortr nd pestle nd coffee grinder or with tissue grinder (Retsch MM200, Hn, Germny). Subsmples of the biomss were nlysed for 13 C/ 12 C crbon isotope composition (expressed using delt-nottion, δ 13 C PDB, ) on CO 2 gs tht ws generted from combustion/ reduction of the dried plnt tissue in n elementl nlyser (NC2500, CE Instruments, Thermo-Quest Itli, Miln, Itly) nd quntified using gs isotope rtio mss spectrometer (Delt Plus, Finnign MAT, Sn Jose, CA, USA) t the University of Lethbridge (Ponton et l. 2006).The precision of the δ 13 C mesurements ws 0.1 bsed on the SD of repeted nlyses of two internl lbortory stndrds used during this study (UL-Mr1, δ 13 C = ; UL Rye, δ 13 C= ). These working stndrds hve been compred to the interntionl stndrd (Interntionl Atomic Energy Agency; IAEA), IAEA-CH-3 Cellulose (δ 13 C = ). Subsmples of the biomss were lso nlysed for 18 O/ 16 O oxygen isotope composition (expressed using delt-nottion, δ 18 O VSMOW, ) on CO gs tht ws generted from the dried plnt tissue in custom high temperture furnce-pyrolysis column nd quntified using gs isotope rtio mss spectrometer (Isochrom, Fisons, Middlewich, UK) t the Austrlin Ntionl University (ANU) (Sturt-Willims et l. 2008).The precision of the δ 18 O mesurements ws 0.5 bsed on the SD of repeted nlyses of two internl lbortory stndrds used during this study (ANU sucrose,δ 18 O = ; ANU NewBeet δ 18 O = ). There is no officil IAEA stndrd for δ 18 O in orgnic mtter, but ANU sucrose (lso known s IAEA-CH-6 sucrose [δ 13 C = ]) hs been used s n informl, interntionl reference mteril. Oxygen isotope composition of root nd soil wter, nd soil CO 2 During , we mesured the 18 O/ 16 O oxygen isotope composition of CO 2 purified from soil ir smples s proxy mesurement for the oxygen isotope composition of source soil wter. Evcuted glss flsks (250 ml) were used to collect soil ir smples t pproximtely 2-week intervls from four replicte sets of tubes buried in the soil in the erly spring of 1999.The bottom of ech soil tube ws connected to two 12 ml bgs (1.5 cm dimeter, 8 cm long) mde from fibre glss window screen tht contined glss beds, which were buried t 10 cm depth, with pproximtely 5 cm spce between the bgs. Crbon dioxide ws purified from the soil

4 428 L. B. Flngn & G. D. Frquhr ir smples using cryogenic vcuum extrction line nd seled in Pyrex tubes (Ehleringer, Roden & Dwson 2000). The purified CO 2 smples were nlysed in dul inlet mode on gs isotope rtio mss spectrometer (Delt Plus, Finnign MAT) t the University of Lethbridge. We clculted the expected δ 18 O VSMOW ( ) for soil wter bsed on the temperture-dependent frctiontion between wter nd CO 2, by ssuming tht soil CO 2 ws in complete isotopic equilibrium with soil wter (Botting & Crig 1969). In order to test the effectiveness of the soil CO 2 isotopic mesurements s proxy for the δ 18 O of soil wter, we lso nlysed the δ 18 O VSMOW of wter extrcted from soil nd root smples collected t intervls during the summer of Soil plugs (1.2 cm dimeter 10 cm long) were removed from the soil using smll coring device pushed into the soil fter the live boveground biomss nd litter hd been removed from the soil surfce. In ddition, the lrge root tubers of Trgopogon dubius were collected from the soil. Both the tubers nd soil plugs were immeditely seled in glss vils, returned to the lbortory nd stored in freezer. Wter ws subsequently extrcted from the soil plugs nd root tubers using cryogenic vcuum extrction (West, Ptrickson & Ehleringer 2006). The δ 18 O VSMOW of the wter ws determined, using technique similr to tht described by Fessenden et l. (2002), by nlysing pure CO 2 tht hd been equilibrted in the hed spce of vils contining the wter smples, fter the vils hd been incubted in controlledtemperture wter bth t 25 C. The equilibrted CO 2 smples were nlysed in continuous flow mode mking use of the gs chromtogrphic column in n elementl nlyser (NC2500, CE Instruments, Thermo-Quest Itli) tht ws connected to gs isotope rtio mss spectrometer (Delt Plus, Finnign MAT) t the University of Lethbridge. The smples were nlysed reltive to working wter stndrds (UL-BCGW, δ 18 O = ; UL-LTP, δ 18 O = ; UL-LMX, δ 18 O = ), tht were clibrted reltive to IAEA interntionl stndrds (VSMOW2, SLAP2, GISP). Reltionship between wter-use efficiency nd lef crbon nd oxygen isotope compositions Dytime lef-level wter-use efficiency (WUE) is the rtio of lef net CO 2 ssimiltion rte (A) nd trnspirtion rte (E), which in turn re dependent on the grdients for CO 2 nd H 2O diffusing into nd out of leves during photosynthetic gs exchnge (Frquhr et l. 1988): WUE A c ci 1 ( c ci) c = = = E ( e e ) e i 16. e i e where c is the prtil pressure of CO 2, e is the prtil pressure of wter vpour, nd subscripts refer to the tmosphere outside the lef () nd the lef intercellulr ir spces (i); 1.6 is the rtio of the diffusion coefficients for H 2O nd CO 2 in ir (Frquhr et l. 1988). The crbon isotope composition of lef orgnic mtter (δ 13 C m) is controlled by the isotope composition of source i (1) tmospheric CO 2 (δ 13 C ) nd frctiontion tht occurs during photosynthetic gs exchnge (Frquhr, O Lery & Berry 1982; Frquhr et l. 1989). The simplest nd most commonly used form of the model describing frctiontion of crbon isotopes during C 3 photosynthesis is (Frquhr et l. 1982): δ C i = δ C ( ) c b c m where is frctiontion during diffusion of CO 2 into lef (4.4 ), b is the net frctiontion during crboxyltion (27 ), nd the extent tht these two frctiontions get expressed depends on lef c i/c. Trnsfer of crbon dioxide from the intercellulr ir spces to the chloroplst (mesophyll conductnce, g i) nd photorespirtion re two dditionl processes tht cn influence the crbon isotope discrimintion (Δ 13 C m) during photosynthesis (Frquhr et l. 1982, 1989; Seibt et l. 2008): δ C δ C Δ 13 m Cm = δ Cm Δ 13 i C m = c c + c m ci c c c c + b c c (2) (3) Γ f * c (4) where m is the frctiontion during CO 2 trnsfer from intercellulr ir spces to the chloroplst (1.8 ; Frquhr et l. 1989), b is frctiontion during crboxyltion by ribulose-1,5- bisphosphte crboxylse (29 ; Frquhr et l. 1989), f is frctiontion during photorespirtion (16.2 ; Evns & von Cemmerer 2013), Γ * is the CO 2 compenstion point (μmol mol 1 ) in the bsence of drk respirtion (clculted from the temperture response: Γ * = (t-25) (t-25) 2, where t is temperture in C; Brooks & Frquhr (1985). In Eqn 4, we hve ssumed no frctiontion during dy respirtion, s hs been done previously (Evns & von Cemmerer 2013). In ddition, we did not consider the influence of post-crboxyltion frctiontion events s there is currently no forml theoreticl frmework to do so. Omitting these fctors dds somewht to the uncertinty of our finl wter-use efficiency clcultions, s will be discussed further below. Rerrngement of Eqn 2 shows tht mesurements of δ 13 C m cn be used to clculte lef c i/c ssuming tht δ 13 C is known. In ddition, Eqn 4 cn be used with some substitution nd rerrngement to obtin Eqn 5, which cn lso be used to clculte lef c i/c (Seibt et l. 2008): g Γ s Δ 13 Cm + ( b m) + f * ci 16. gi c = c g s b + ( b m ) 16. g i where g s is stomtl conductnce to CO 2 (mol m 2 s 1 ), nd g i is mesophyll conductnce (mol m 2 s 1 ) for trnsfer of CO 2 from intercellulr ir spces to the chloroplst. Cnopy conductnce ws clculted from eddy covrince mesurements of ecosystem trnspirtion rte using the inverted (5)

5 δ 13 C nd δ 18 O in plnt biomss 429 Penmn Monteith eqution s described in detil by Wever et l. (2002). Stomtl conductnce ws estimted from cnopy (surfce) conductnce by dividing by the green LAI of the cnopy, using LAI mesurements from Flngn et l. (2002). The pek LAI vlues in 1999 nd 2000 were 0.55 ± 0.05 nd 0.45 ± 0.03, respectively. We developed two scenrios with different estimtes of mesophyll conductnce bsed on clcultions of stomtl conductnce, g i = 1.5g s or g i =3g s (Wrren et l. 2003; Wrren & Dreyer 2006; Seibt et l. 2008), when using Eqn 5. The vlues of g s used in Eqn 5 were the dily GEP-weighted verge vlues clculted from the vilble screened conductnce dt for the periods, dys in 1999 nd dys in 2000 (see Tble 1). This ws done by first clculting men diurnl pttern for both GEP nd g s for the relevnt time periods in 1999 nd 2000, nd then clculting dily-verge, where the verge g s clcultion ws weighted by the ssocited diurnl vrition in GEP. The oxygen isotope composition of lef orgnic mtter (δ 18 O m) is controlled by the isotope composition of lef wter (δ 18 O lw), which in turn is influenced by wter tken up from the soil (δ 18 O sw, source wter), nd isotope effects tht occur during trnspirtion. Vrition in lef temperture, ir temperture nd tmospheric humidity cuse chnges to δ 18 O lw during trnspirtion, nd this gets recorded in the isotope composition of lef biomss (δ 18 O m). The mechnistic links between these sttements re illustrted in series of equtions shown below bsed on previous work by Frquhr nd collegues (Frquhr & Lloyd 2003; Frquhr et l. 1998; Frquhr, Cernusk & Brnes 2007). It is convenient to illustrte the linkges using some dditionl nottion tht expresses the enrichment of 18 O in lef wter reltive to tht of the source wter (Δ 18 O lw =(δ 18 O lw δ 18 O sw)/(1 + δ 18 O sw); Δ 18 O lw δ 18 O lw δ 18 O sw). Similrly, it is convenient to express the oxygen isotope composition of lef orgnic mtter reltive to tht of the source wter (Δ 18 O m δ 18 O m δ 18 O sw). Δ 18 O = Δ 18 O ( 1 ρ ρ )+ ε + ε (6) m lw ex x wc cm Tble 1. Comprison of wter-use efficiency (WUE), lef temperture nd other ssocited vlues tht were clculted using Eqns 1, 2, 5, 10, 11, nd the verge mesured crbon (δ 13 C m, ) nd oxygen isotope composition (δ 18 O m, ) of biomss smpled ner the pek of biologicl ctivity in 1999 (dy 166) nd 2000 (dy 164) δ 13 C m ( ) 28.1 ± ± 0.1 δ 18 O m ( ) 24.5 ± ± 1.1 δ 18 O sw ( ) δ 18 O v ( ) e (kp) ε + ( ) Air temperture ( C) g s (mol CO 2 m 2 s 1 ) c i/c (simple model) c i/c (with g i =3g s) c i/c (with g i = 1.5g s) Clcultions with L = m, ε cm = e /e i e i (kp) Lef temperture ( C) Isotope WUE (simple model) (mmol mol 1 ) Isotope WUE (with g i =3g s) (mmol mol 1 ) Isotope WUE (with g i = 1.5g s) (mmol mol 1 ) Clcultions with L = m, ε cm = e /e i e i (kp) Lef temperture ( C) Isotope WUE (simple model) (mmol mol 1 ) Isotope WUE (with g i =3g s) (mmol mol 1 ) Isotope WUE (with g i = 1.5g s) (mmol mol 1 ) EC WUE (mmol mol 1 ) where ρ ex is the proportion of oxygen toms in cellulose tht exchnge with wter t the site of cellulose synthesis (0.4, Cernusk, Frquhr & Pte 2005); ρ x is the proportion of wter t the site of cellulose synthesis tht is non-frctionted source wter (pproximtely 0.55 in grss plnts, Helliker & Ehleringer 2002); ε wc is the frctiontion fctor (27 ) for exchnge of oxygen toms between sucrose nd lef wter before sucrose is incorported in cellulose (Cernusk, Wong & Frquhr 2003); ε cm is the difference in frctiontion between cellulose nd whole lef orgnic mtter. A few studies hve shown tht whole lef tissue is less enriched in 18 O compred to the exchnging wter pool thn is cellulose, difference tht is vrible mong species (Frquhr, Henry & Styles 1997; Brbour & Frquhr 2000; Cernusk et l. 2005). We used ε cm s n djustble prmeter to fit Eqn 6 to mesured vlues of the oxygen isotope composition of plnt orgnic mtter nd the fitting procedure is described below. Helliker & Ehleringer (2002b) found tht the product, ρ exρ x, ws 0.25 for severl grss species, but the clculted ρ exρ x The c i/c clcultions ssume c ws P (385 μmol mol 1, tmospheric pressure in Lethbridge is 90 kp) nd δ 13 C ws 8. Both Eqn 2 (simple model) nd Eqn 5 were used to clculte c i/c; Eqn 5 ws used with either g i = 1.5g s or g i =3g s.the other inputs to Eqns 10 nd 11 (δ 18 O sw, δ 18 O v,, e, ε +, ir temperture) were ll GEPweighted verges for dys in 1999 nd dys in The wter-use efficiency vlues clculted from eddy covrince dt (EC WUE) represent GEP-weighted verges for dys in 1999 nd dys in 2000 (see Fig. 6). The vlues for δ 13 C m (n = 6) nd δ 18 O m (n = 4) represent verges (± SE). vlue ws lter corrected to 0.22 (B. Helliker, personl communiction), the vlue we used in our clcultions. Δ Olw = Δ Oes e The enrichment of 18 O in totl lef wter occurs becuse of frctiontion t the evportive sites of leves during trnspirtion (Δ 18 O es), explined further below (Flngn, (7)

6 430 L. B. Flngn & G. D. Frquhr Comstock & Ehleringer 1991). Wter enriched in 18 Otthe evportive sites diffuses bck into the lef mesophyll, but this process is countercted by dvection of unfrctionted source wter from the veins into the lef mesophyll. The blnce between these fctors is described by Péclet effect ( ) (Frquhr & Lloyd 2003), = EL CD which depends on the trnspirtion rte (E, mol m 2 s 1 ), the effective pth length for wter movement (L, m), the molr density of wter (C, 5.55 x 10 4 mol m 3 ), nd the diffusivity of H 2 18 O in wter (D, 2.43 x 10 9 m 2 s 1 t 25 C, with temperture dependence described by: D = exp(-627/(t- 137)), where T is the bsolute temperture (Cuntz et l. 2007)). Becuse of uncertinty in the vlue of the effective pth length (L), we conducted two sets of clcultions, one with L set t m nd one with L set t m. Trnspirtion rte ws estimted from eddy covrince mesurements nd mesured LAI. Isotopic frctiontion t the evportive sites in leves during trnspirtion is described by Δ O + = + + ( Δ O ) e ε ε ε e es k v k where ε + is the equilibrium frctiontion fctor during the phse chnge from liquid to vpour (9.1 t 25 C, with temperture dependence described by ε + = [ (10 3 / T) (10 3 /T) 2 ] (Botting & Crig 1969)); ε k is the kinetic frctiontion fctor for wter vpour diffusing through the stomtl pore (32, Cpp et l. 2003); Δ 18 O v is the δ 18 O of tmospheric wter vpour (δ 18 O v) expressed reltive to tht of the source wter (Δ 18 O v δ 18 O v δ 18 O sw), nd e /e i is the rtio of the prtil pressure of wter vpour in the tmosphere () nd the lef intercellulr ir spces (i). Equtions 6 9 cn be combined nd rerrnged to solve for e /e i: e e i ((( δ Om δ Osw εwc εcm) 128. ) ) + ε ε k ( 1 e ) = δ Ov δ O sw ε k i (8) (9) (10) where isotopic compositions (δ 18 O) re listed for lef orgnic mtter, source wter nd tmospheric wter vpour becuse it is these vlues tht re directly mesured, nd the following pproximtions re pplied in the derivtion of Eqn 10: Δ 18 O m δ 18 O m δ 18 O sw; Δ 18 O v δ 18 O v δ 18 O sw. The combined use of δ 13 C mesurements to estimte c i/c (Eqn2or5)ndδ 18 O mesurements to estimte e /e i (Eqn 10) llows lef wter-use efficiency to be clculted using Eqn 1, ssuming tht c nd e re known from tmospheric mesurements. In ddition, vlues of e /e i determined from Eqn 10 nd mesurements of e were used to clculte lef temperture (Helliker & Richter 2008; Song et l. 2011): T L ei ( ln ( )) = (11) ei ( ln ( ) ) where T L is lef temperture ( C), nd e i is expressed in units of kp (Buck 1981).Equtions 6 9 were first used to determine modelled δ 18 O m vlues on dily time-step by mking use of GEP-weighted dily verges for environmentl prmeters (e.g. ir temperture, RH, ET) or other prmeters tht were clculted using GEP-weighted dily verges (e.g. ε + clculted from dily verge temperture, or Péclet effect ( ) clculted from dily verge trnspirtion rte). The GEP vlues used in clculting these weighted dily verges were the 30-min vlues determined from eddy covrince mesurements. Dily vlues of δ 18 O sw were interpolted from the regressions fitted to δ 18 O sw mesured t regulr intervls during the growing sesons of 1999 nd 2000 (see Fig. 4). Similrly, dily vlues of δ 18 O v were interpolted from regression fitted to monthly verge δ 18 O v, which ws the δ 18 O of wter vpour clculted to be in isotopic equilibrium with monthly verge δ 18 O of precipittion (see Fig. 4), bsed on monthly verge ir temperture nd the temperture-dependent equilibrium frctiontion fctor (ε + ). We used the monthly verge δ 18 O of precipittion nd ir temperture clculted from mesurements mde during t Clgry, Albert, Cnd (200 km north of the Lethbridge study site, Peng et l. 2004) for these estimtes of δ 18 O v (see Fig. 4). Longer-term integrted vlues of modelled δ 18 O m were subsequently clculted over time intervl tht ws chosen to closely pproximte the time intervl tht mesured δ 18 O m vlues should reflect. For exmple, we compred mesured nd modelled δ 18 O m vlues (see Fig. 5) using the GEPweighted δ 18 O m vlues modelled for the period from dy 121 (in 1999, or dy 122 in 2000) until the dte tht the relevnt mesured δ 18 O m smple ws collected. The dte of My 1 (dy 121 or 122) ws chosen s strting vlue becuse it ws ner the beginning of the ctive photosynthetic period in both 1999 nd 2000 (see Fig. 6). Itertive djustments were mde to the vlue of ε cm in order to minimize the sum of squred differences between the eight pirs of mesured nd modelled δ 18 O m vlues for smples collected during dys in 1999 nd 2000 (see Fig. 5). The fitted vlue of ε cm ws 3 when L ws m, nd the fitted vlue of ε cm ws 0 when L ws m. RESULTS Vrition mong yers in biomss production nd orgnic mtter stble isotope composition We mde use of biomss smples collected during the growing sesons of nd 2006 to compre vrition in δ 13 C nd δ 18 O of plnt orgnic mtter. Flngn (2009) previously showed tht there ws wide rnge of vrition in δ 13 C m within nd mong these yers, with the δ 13 C m vrition correlted with the mount of precipittion nd mximum boveground biomss tht occurred in those different yers.

7 δ 13 C nd δ 18 O in plnt biomss 431 An exception ws lte seson pulse of precipittion in September 2000 tht resulted in second phse of plnt growth (re-greening) with ssocited reduction in δ 13 C m vlues shown on dy 262 (Fig. 2). There ws lso strong sesonl nd inter-nnul vrition pprent in the δ 18 O vlues of plnt orgnic mtter during (Fig. 2c), with significnt positive liner reltionship observed between the δ 13 C m nd δ 18 O m vlues (Fig 3). The drought of ws broken with high precipittion inputs in My 2002 (Flngn 2009), nd there ws n ssocited reduction in δ 13 C m vlues during the first prt of the 2002 growing seson (Fig. 2b). Very little other systemtic vrition occurred in either δ 13 C m or δ 18 O m vlues during 2002, 2003 nd 2006, nd there ws no significnt reltionship between the δ 13 C m nd δ 18 O m vlues in these 3 yers (Fig. 3b). Figure 1. Reltionship between pek boveground green biomss nd () the crbon isotope composition (δ 13 C m,, n = 6); nd (b) the oxygen isotope composition (δ 18 O m,, n =4)ofthe biomss smples collected from 1998 to 2003 nd 2006 t grsslnd ner Lethbridge, Albert, Cnd. Vlues represent verges ± SE. The lines re significnt liner regressions fitted to the dt: () y = x 24.98, r 2 = 0.872, P = 0.002; (b) y = x , r 2 = 0.658, P = We lso observed significnt negtive correltion between pek boveground biomss nd both δ 13 C m nd δ 18 O m (Fig. 1,b). Three yers ( ) hd reltively low pek biomss, primrily cused by reduced wter vilbility (Flngn 2009; Flngn & Adkinson 2011), with correspondingly high δ 13 C m nd δ 18 O m vlues. The other 4 yers (1998, 2002, 2003, 2006) hd high biomss production nd reltively low δ 13 C m nd δ 18 O m vlues. During , the grsslnd experienced progressive chnge from higher-thn-norml growing seson precipittion in 1998 to 2 yers ( ) with only bout 50% of norml growing seson precipittion (Flngn 2009; Flngn & Adkinson 2011). This chnge in moisture vilbility during ws recorded in the sesonl nd inter-nnul vrition in δ 13 C m vlues (Fig. 2; Flngn 2009). For exmple, hevy rinfll in lte My nd June (dys ) of 1998 stimulted reltively high biomss production nd n ssocited decline in δ 13 C m vlues (Fig. 2). Progressively lower precipittion during cused reduced biomss production nd lso incresed δ 13 C m vlues. Oxygen isotope composition of source soil wter We used mesurements of the δ 18 O of soil CO 2 to clculte the δ 18 O of soil wter in isotopic equilibrium with the CO 2. There ws strong liner reltionship between the δ 18 Oof source soil wter derived from mesurements of soil CO 2 (dependent vrible) nd the δ 18 O of wter extrcted from soil nd the root tubers of T. dubius (y = 0.49x-6.63, r 2 = 0.624, n = 15, P = ). In ddition, there ws significnt liner reltionship between the derived δ 18 O of source wter (dependent vrible) nd wter extrcted only from root tubers (y = 1.64x , r 2 = 0.723, n =9, P = ), nd ner significnt (P = 0.078, n = 6) liner reltionship between the derived δ 18 O of source wter nd wter extrcted only from soil smples. We conclude tht the δ 18 O of soil CO 2 cn be used s proxy to estimte the δ 18 Oof source soil wter. A polynomil regression fitted to the δ 18 O of source soil wter, derived from mesurements of soil CO 2 in My August 1999 (dys ), ws pproximtely 2.5 enriched in 18 O reltive to regression line fitted to the 10-yer record of monthly verge δ 18 O of precipittion mesured in Clgry, Albert (which is pproximtely 200 km north of the study site) (Peng et l. 2004; Fig. 4). While the clculted δ 18 O of source wter hd very similr vlues erly in the growing seson of both 1999 nd 2000, the δ 18 O vlues were consistently higher in 2000 thn in 1999 fter mid-seson (dy 180) when soil moisture content in 2000 reched low vlues (Fig. 4; Flngn & Adkinson 2011), nd the enrichment of 18 O in soil wter cused by soil evportion likely occurred to greter extent in 2000 thn in Comprison of mesured nd modelled δ 18 O vlues nd wter-use efficiency We observed good greement between verge mesured nd modelled δ 18 O m vlues, when ε cm ws set t 3 when L ws m (Fig. 5), nd similr good fits when ε cm ws set t 0 when L ws m (Fig. 5b). We clculted integrted dytime lef wter-use efficiency, c i/c, e /e i nd lef temperture for 1999 nd 2000 using Eqns 1, 2, 5, 10 nd 11 for both

8 432 L. B. Flngn & G. D. Frquhr Figure 2. Comprison of sesonl vrition in (, b) the crbon isotope composition (δ 13 C m,, n = 6); nd (c, d) the oxygen isotope composition (δ 18 O m,, n = 4) of green biomss smples collected from 1998 to 2003 nd 2006 t grsslnd ner Lethbridge, Albert, Cnd. Vlues represent verges ± SE. The legend shown in box lso pplies to box c. The legend shown in box b lso pplies to box d. sets of ε cm nd L vlues (Tble 1). The clcultions mde use of the verge mesured δ 13 C m nd δ 18 O m vlues for smples collected on dy 166 (June 15) in 1999 nd dy 164 (June 12) in These smple dys were ner the pek of the photosynthetic period in these yers (Fig. 6), nd, therefore, these orgnic smples should be representtive of the min growing seson in these yers. Clculted lef temperture ws lower thn the GEP-weighted verge ir temperture in both 1999 nd 2000, lthough the difference between lef nd ir temperture ws greter in 2000 thn in 1999 (Tble 1). Reltive to 1999, the stble isotope-bsed clcultions indicted slightly higher wter-use effiency in 2000 (Tble 1), which ws the drier yer (Flngn & Adkinson 2011). Higher vlues of wter-use efficiency were obtined with the stble isotope-bsed clcultions (lef-scle) thn were determined from ecosystem-scle CO 2 nd wter vpour fluxes s mesured by eddy covrince (Tble 1, Fig. 6c). DISCUSSION Sources of vrition in δ 13 C m nd δ 18 O m mesurements As expected, we observed positive reltionship between δ 13 C m nd δ 18 O m vlues for smples collected during , when vrition in precipittion inputs nd soil moisture strongly controlled plnt biomss production (Fig. 3). This indicted tht vrition in stomtl conductnce nd wter stress-induced chnges in the degree of stomtl limittion of net photosynthesis were the mjor controls on vrition in δ 13 C m during this time (Brbour 2007; Frquhr et l. 2007). For comprison, there ws no significnt reltionship between δ 13 C m nd δ 18 O m vlues during 2002, 2003 nd 2006 when there ws no significnt difference in biomss production (Fig. 1; Flngn 2009; Flngn & Adkinson 2011) nd only reltively smll vrition in δ 13 C m vlues (Fig. 3b). Similr ptterns of vrition in field studies of δ 13 C m nd

9 δ 13 C nd δ 18 O in plnt biomss 433 order to determine the contribution of chnges in the δ 18 Oof soil wter, we used mesurements of the δ 18 O of soil CO 2 s proxy for soil wter isotopic composition. In support of this proxy ppliction, we found tht there ws strong correltion between the δ 18 O of source soil wter derived from mesurements of soil CO 2 nd the δ 18 O of wter extrcted from soil nd the root tubers of T. dubius. Our proxy mesurements of the δ 18 O of source soil wter indicted tht this wter ws enriched in 18 O by pproximtely 2.5 reltive to the δ 18 O of verge precipittion inputs during 1999 (Fig. 4). In ddition, the δ 18 O vlues of source soil wter were consistently higher in 2000 thn in 1999 fter mid-seson (dy 180) when soil moisture content in 2000 reched low vlues (Fig. 4; Flngn & Adkinson 2011). These differences between the δ 18 O of precipittion nd soil wter reflect some enrichment of 18 O in soil wter ssocited with evportion of wter from the soil, nd the fct tht more evportive enrichment should occur in drier yer like Some previous studies hve ssumed tht the δ 18 O of soil wter ws equl to tht of precipittion inputs nd/or tht wter stress tretments would not significntly lter the δ 18 O of soil wter (Rmirez et l. 2009; Cbrer-Bosquet et l. 2011; Song et l. 2011): these re simplifying ssumptions tht should be mde with cution when using δ 18 O m vlues to estimte chnges resulting from vrition in stomtl conductnce nd VPD. For the 1.9 difference tht occurred between the verge pek seson mesurements of δ 18 O m in 1999 nd 2000 in this study, pproximtely 0.8 could be ttributed to vrition in the δ 18 O of soil wter tken up by plnts in the 2 yers (Tble 1). Figure 3. Reltionship between the crbon isotope composition (δ 13 C m, ) nd the oxygen isotope composition (δ 18 O m, ) of biomss smples collected during () , nd (b) 2002, 2003 nd The line in box represents significnt liner regression fitted to the dt: δ 18 O m = 1.51 δ 13 C m ; r 2 = 0.561, P = After excluding the point in box b mrked with the rrow, there ws no significnt liner reltionship between δ 18 O m nd δ 13 C m vlues. If the point lbelled with the rrow in box b ws included, there ws significnt negtive liner reltionship: δ 18 O m = 1.19 δ 13 C m 9.48; r 2 = 0.225, P = δ 18 O m nd their correltion with green lef re were observed in S. tencissim in semi-rid grsslnd in Spin (Rmirez, Querejet & Bellot 2009) nd in field-grown durum whet in Itly (Cbrer-Bosquet et l. 2011). In ddition, most of the vrition in lef δ 13 C mong field-grown bred whet cultivrs in Mexico ws result of vrition in stomtl conductnce nd so strong positive correltion ws observed between the δ 13 C nd δ 18 O of the whet plnt biomss (Brbour et l. 2000). By contrst, mesurements of δ 13 C m nd δ 18 O m in tree rings indicted tht increses in A/g s nd growth in fertilized field plots of Dougls-Fir were primrily result of higher photosynthetic cpcity (Brooks & Mitchell 2011). In ddition to chnges in stomtl conductnce, VPD nd other fctors ffecting the enrichment of 18 O in lef wter during trnspirtion, vrition in the δ 18 O of source soil wter tken up by plnts cn ffect δ 18 O m vlues (Brbour 2007). In Figure 4. Sesonl vrition in the verge (± SE, n = 4) oxygen isotope composition (δ 18 O, ) of soil wter clculted from mesurements of soil CO 2 mde during 1999 nd 2000 t Lethbridge Albert, Cnd. Also shown is the monthly verge δ 18 O of precipittion (± SE, n = 9 or 10) clculted from mesurements mde during t Clgry, Albert, Cnd (200 km north of the Lethbridge study site) s reported by Peng et l. (2004). The lines represent regressions fitted to the dt: (1999) y = x x , r 2 = 0.378, P < 0.05; (2000) y = x , r 2 = , P < 0.05; (Precipittion) y = x x , r 2 = 0.937, P < 0.05.

10 434 L. B. Flngn & G. D. Frquhr Figure 5. Reltionship between verge mesured nd modelled oxygen isotope composition of biomss smples (δ 18 O m, ) collected during 1999 nd 2000 for: () clcultions done with ε cm = 3 nd L = m; nd (b) clcultions done with ε cm = 0 nd L = m. The line represents the one-to-one reltionship. We did not hve mesurements of the δ 18 O of tmospheric wter vpour, nd so we estimted it by ssuming tht it would be in isotopic equilibrium with the δ 18 O of precipittion. This simplifying ssumption is supported by some recent studies (Lee, Smith & Willims 2006; Helliker & Griffiths 2007; Angert, Lee & Ykir 2008), but potentil inter-nnul vritions in the δ 18 O of precipittion nd tmospheric wter vpour could lso contribute to the observed differences in δ 18 O m mong study yers. This potentil contribution should be minor, however, becuse the influence of δ 18 O of tmospheric wter vpour on lef wter δ 18 O is reduced in environments with low tmospheric humidity, s is the cse in the semi-rid grsslnd ecosystem used in this study. Use of δ 13 C m nd δ 18 O m mesurements in clcultions of wter-use efficiency There re severl uncertinties nd complictions involved in clcultions of lef-scle wter-use efficiency bsed on mesurements of δ 13 C m nd δ 18 O m. First, estimtes of lef c i/c depend on the complexity of the crbon isotope frctiontion model used to interpret vrition in δ 13 C m vlues (Frquhr et l. 1989; Seibt et l. 2008). The simple 13 C frctiontion model (Eqn 2) ssumes tht c i is equl to c c nd tht no significnt isotopic frctiontion occurs during photorespirtion. This simple model will tend to underestimte lef c i/c vlues, prticulrly becuse it ssumes tht mesophyll conductnce is infinite, lthough this is countercted by the choice of b =27, rther thn the Rubisco frctiontion of 30. As frctiontion during photorespirtion ws included nd mesophyll conductnce ws reduced (clcultions done using Eqn 5), there ws systemtic increse in clculted lef c i/c nd corresponding reduction in clculted wteruse efficiency (Tble 1). As hs been done previously (Evns & von Cemmerer 2013), we ssumed no frctiontion during dy respirtion nd we did not consider the influence of post-crboxyltion frctiontion events. Omitting these fctors dds somewht to the uncertinty of lef c i/c clcultions. Secondly, the model we used for fctors controlling the isotopic composition of wter t the evportive sites in leves (Eqn 9) ssumes isotopic stedy stte conditions nd does not tke into ccount progressive isotope enrichment long grss leves nd the complexity of rdil nd longitudinl Péclet effects (Helliker & Ehleringer 2002; Gn et l. 2003; Cernusk et l. 2005; Cuntz et l. 2007; Ogée et l. 2007). In ddition, the effective pth length (L, Eqn 8) used in clcultions of the Péclet effect is not well chrcterized, cn vry significntly mong different plnt species (Wng, Ykir & Avishi 1998; Khmen et l. 2009) nd with environmentl conditions (Song et l. 2011; Ellsworth et l. 2013). We conducted clcultions with two vlues of effective pth length nd obtined similr results for lef-scle wter-use efficiency (Tble 1). The vlues of L used in our clcultions (0.005 nd m) were similr to vlues determined by Brbour & Frquhr (2000) nd Ripullone et l. (2008) for cotton (0.008 m), nd by Khmen et l. (2009) for Phseolus vulgris (0.017 m), Rizinus communis (0.025 m) nd Helinthus nnuus (0.011 m). The verge (± SE) effective pth length determined for rnge of species by Wng et l. (1998) ws ± m. Thirdly, bulk lef tissue hs been observed to be less enriched in 18 O compred to the exchnging wter pool thn is lef cellulose, nd this difference (ε cm, Eqn 6) is vrible mong species nd cn chnge with lef ge (Frquhr et l. 1997; Brbour & Frquhr 2000; Cernusk et l. 2005).We used ε cm s n djustble prmeter to fit Eqn 6 to mesured vlues of the oxygen isotope composition of plnt orgnic mtter nd our fitted ε cm (0 nd 3 ) hd bsolute vlues tht were smller thn the vlues determined in other studies (pproximtely 4 to 9 ; Frquhr et l. 1997; Brbour & Frquhr 2000; Cernusk et l. 2005). The uncertinty bout ll these fctors discussed bove complictes the interprettion of the bsolute vlues of clculted lef-scle wter-use efficiency (Frquhr et l. 1989; Brbour 2007; Seibt et l. 2008). Despite the uncertinties bout the bsolute vlues for clculted lef wter-use efficiency, we suggest tht comprtive use of such clcultions bsed on δ 13 C m nd δ 18 O m mesurements is vluble. For exmple, clculted dytime lef wter-use efficiency ws higher in 2000 thn 1999, consistent

11 δ 13 C nd δ 18 O in plnt biomss 435 Figure 6. Sesonl vrition in () gross ecosystem photosynthesis (GEP), (b) ecosystem evpotrnspirtion (ET), (c) ecosystem wter-use efficiency (EC WUE), (d) middy sensible het flux, (e) middy ltent het flux nd (f) middy Bowen rtio (sensible het flux/ltent het flux) t grsslnd site ner Lethbridge, Albert, Cnd during 1999 nd The vlues represent 5-dy verges bsed on eddy covrince mesurements. with expecttions becuse of greter stomtl limittion of photosynthesis nd lower lef c i/c during the drier conditions of 2000 (Tble 1). This higher clculted wter-use efficiency occurred despite the slightly greter clculted lef-ir vpour pressure (e i e ) difference in 2000 thn in 1999 (Tble 1). In ddition, the isotope-bsed clcultions suggested tht lef temperture ws reduced below ir temperture during photosynthesis nd tht the mgnitude of this pprent evportive cooling effect ws lrger in 2000 thn in 1999 (Tble 1). These re vluble insights bout whole plnt function gined from the temporlly integrted informtion provided by the δ 13 C m nd δ 18 O m mesurements tht would be very difficult to obtin using conventionl whole plnt gs exchnge mesurements in the field. The stble isotope clcultions of dytime verge lef temperture were lower thn verge ir temperture during the pek of the growing seson (Tble 1). This occurred despite the fct tht middy eddy covrince mesurements

12 436 L. B. Flngn & G. D. Frquhr of sensible het flux nd Bowen rtio indicted tht ecosystem surfce temperture ws wrmer thn ir temperture even when ltent het flux ws t its sesonl mximum (Fig. 6d,e,f). The reltively low mximum LAI ( ) in 1999 nd 2000 would llow for lef temperture to remin slightly below ir temperture while there ws positive sensible het flux, if the re on the ground surfce between plnts ws wrmer thn ir temperture. Imges of the ecosystem surfce tken with n infrred cmer were consistent with this suggestion (dt not shown). Comprison of wter-use efficiency cross sptil scles The clculted vlues of lef-scle wter-use efficiency from stble isotope mesurements were 2 3 times higher thn the ecosystem-scle wter-use efficiency bsed on eddy covrince mesurements, lthough the ltter lso showed greter vlues in 2000 thn 1999, s expected (Tble 1). It ws nticipted tht lef-scle wter-use efficiency would be higher thn corresponding vlues t the ecosystem-scle becuse of crbon lost in root respirtion nd wter lost during soil evportion tht were not ccounted for by the stble isotope mesurements. The wter-use efficiency of isolted plnts (WUE p) cn be described using modifiction of Eqn 1 (Frquhr et l. 1988; Hubick & Frquhr 1989): WUE p ( c ci) ( 1 φc) = 16. ( e e )( 1+ φ ) i w (12) where ϕ c is the proportion of crbon fixed in photosynthesis but lost s root respirtion nd exudtion of crbon to the rhizosphere during the dy, nd ϕ w is the wter lost during soil evportion nd cuticulr trnspirtion. During n entire 24-h period, plnts cn respire 30 50% of crbon cquired in photosynthesis (McCree 1986). Therefore, it my be possible tht root respirtion nd exudtion during the dytime releses 25% of photosynthetic crbon uptke. As noted bove, mximum LAI ws only during 1999 nd 2000, nd so the sprse plnt cnopy would llow for high solr rdition flux onto the soil surfce tht would promote soil evportion. It might be nticipted tht wter lost during soil evportion could be equl to wter lost during trnspirtion in sprse cnopy with n LAI ner 0.5. However, our study site hs not been grzed for t lest 30 yers nd there is lrge ccumultion (288 g m 2 )of ded plnt orgnic mtter on the soil surfce tht would ct s mulch nd help to reduce soil evportion (Flngn & Johnson 2005). If we conservtively ssume tht soil evportion nd cuticulr trnspirtion ws only 20% of wter lost through stomt (ϕ w = 0.2), nd tht crbon lost during dytime root respirtion/exudtion ws 25% of photosynthesis s noted bove (ϕ c = 0.25), then lef-scle isotope wter-use efficiency (with g i = 1.5g s) of 5.44 nd 6.38 mmol mol 1 (Tble 1) would be equl to WUE p vlues of 3.4 nd 4.0 mmol mol 1 for 1999 nd 2000, respectively. These estimtes of WUE p re much closer to vlues of ecosystem wter-use efficiency determined by eddy covrince mesurements, 2.67 nd 3.00 mmol mol 1 for 1999 nd 2000, respectively (Tble 1), nd typicl of vlues previously mesured for C 3 vegettion (Osmond et l. 1982; Lmbers, Chpin & Pons 2008). In ddition to the cvets discussed bove bout the complexity of interpreting bsolute vlues of wter-use efficiency clculted bsed on δ 13 C m nd δ 18 O m mesurements, it is importnt to note tht the stble isotope mesurements were mde on bulk biomss smples tht included severl ntive grss species nd some forbs species. There could hve been chnges in the reltive bundnce of the different species mong the study yers. In ddition, not ll plnt species showed the exct sme timing of plnt production nd the stble isotope mesurements should represent photosynthesis-weighted verges for species ctive t slightly different times, which my help to mximize the pprent clculted lef-scle wter-use efficiency of the plnt biomss smples. CONCLUSIONS The positive reltionship between δ 13 C m nd δ 18 O m vlues for smples collected during indicted tht vrition in stomtl conductnce nd wter stress-induced chnges in the degree of stomtl limittion of net photosynthesis were the mjor controls on vrition in δ 13 C m during this time. The lck of significnt reltionship between δ 13 C m nd δ 18 O m vlues during 2002, 2003 nd 2006 ws consistent with only reltively smll sesonl nd inter-nnul vrition in δ 13 C m vlues nd indictive of the fct tht wter stress ws not significnt limittion on photosynthesis nd biomss production in these yers. There ws strong correltion between the δ 18 O of source soil wter derived from mesurements of soil CO 2 nd the δ 18 O of wter extrcted from soil nd the root tubers of T. dubius, which supported our use of the δ 18 O of soil CO 2 s proxy for soil wter isotopic composition. Approximtely 50% of the difference tht occurred between the verge pek seson mesurements of δ 18 O m in 1999 nd 2000 could be ttributed to vrition in the δ 18 O of soil wter tken up by the plnts. Clculted lef-scle wter-use efficiency ws higher in 2000 thn 1999, consistent with expecttions becuse of greter stomtl limittion of photosynthesis nd lower lef c i/c during the drier conditions of In ddition, clculted vlues of lef-scle wter-use efficiency from stble isotope mesurements were 2 3 times higher thn ecosystem-scle wter-use efficiency bsed on eddy covrince mesurements. The difference ws likely due to crbon lost in root respirtion nd wter lost during soil evportion tht ws not ccounted for by the stble isotope mesurements. ACKNOWLEDGMENTS This reserch ws funded by the Nturl Sciences nd Engineering Reserch Council of Cnd nd the Albert-Isrel Wter Reserch Trust through grnts to L.B.F. We thnk Peter Crlson, Cher Emrick, Nicole Geske, Bruce Johnson,