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ContentslistsavailableatScienceDirect

Agricultural and Forest Meteorology

jo u r n al h om ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / a g r f o r m e t

Photosynthetic physiology of eucalypts along a sub-continental rainfall gradient in northern Australia

Lucas A. Cernusak

^a,∗

, Lindsay B. Hutley

^a,b

, Jason Beringer

^c

, Joseph A.M. Holtum

^d

, Benjamin L. Turner

^e

aSchoolofEnvironmentalandLifeSciences,CharlesDarwinUniversity,Darwin,NorthernTerritory0909,Australia

bSchoolofEnvironmentalResearch,CharlesDarwinUniversity,Darwin,NorthernTerritory0909,Australia

cSchoolofGeographyandEnvironmentalScience,MonashUniversity,Clayton,Victoria3800,Australia

dSchoolofMarineandTropicalBiology,JamesCookUniversity,Townsville,Queensland4811,Australia

eSmithsonianTropicalResearchInstitute,Balboa,Ancon,Panama

a r t i c l e i n f o

Articlehistory:

Received30July2010 Receivedinrevisedform 11November2010 Accepted4January2011

Keywords:

Carbon-isotopediscrimination Leafmassperarea

Photosyntheticcapacity Rainfallgradient Savanna

a b s t r a c t

Leaf-levelphotosyntheticparametersofspeciesinthecloselyrelatedgeneraEucalyptusandCorymbia wereassessedalongastrongrainfallgradientinnorthernAustralia.Bothinstantaneousgasexchange measurementsandleafcarbonisotopediscriminationindicatedlittlevariationinintercellularCO2con- centrationsduringphotosynthesis(ci)inresponsetoadecreaseinmeanannualprecipitationfrom

∼1700mmto∼300mm.Correlationbetweenstomatalconductanceandphotosyntheticcapacitycon- tributedtowardthemaintenanceofrelativelyconstantciamongthesampledleaves,whenassessedat ambientCO2concentrationandphotonirradiancesimilartofullsunlight.Leafmassperareawasthe mostplasticleaftraitalongtherainfallgradient,showingalinearincreaseinresponsetodecreasing meanannualprecipitation.ThemaximumRubiscocarboxylationvelocity,Vcmax,expressedonaleaf- areabasis,showedamodestincreaseinresponsetodecreasingrainfall.ThismodestincreaseinVcmax

wasassociatedwiththestronglyexpressedincreaseinleafmassperarea.Theseresultssuggestthat variationinecosystem-levelgasexchangeduringthedryseasoninnorth-Australiansavannaswilllikely bedominatedbychangesinleafareaindexinresponsetoincreasingaridity,ratherthanbychangesin photosyntheticperformanceperunitleafarea.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Understanding physiologicalresponsesof plants toenviron- mental resource gradients is important for predicting spatial variation in ecosystem processes and for predicting ecosystem responses to climatechange (Kochet al., 1995; Schulze et al., 1996).AstrongrainfallgradientexistsinnorthernAustralia(Table1 andFig.1),withhighestmeanannualprecipitation(MAP)occur- ringnearthecoastand decreasingfromnorthtosouth,roughly atarateof1mmkm⁻¹ (BowmanandConnors,1996;Cookand Heerdegen,2001;Kochetal., 1995).Thisprecipitationgradient isassociatedwithstructuralandfloristicchangesinlocallydomi- nantvegetationcommunities(BowmanandConnors,1996;Hutley etal.,2011;Williamsetal.,1996).Northofthe500mmyr⁻¹iso- hyet,dominantvegetationcommunitiestendtobesavannas,with adiscontinuoustreecanopyoverlyingagrassyunderstory.Euca- lypts,comprisingspeciesinthecloselyrelatedgeneraEucalyptus andCorymbia,dominatetheover-storiesofthesesavannas.The heightandabundanceoftheover-storyeucalyptsdecreasewith

∗Correspondingauthor.Tel.:+61889467630;fax:+61889466847.

E-mailaddress:lcernusak@gmail.com(L.A.Cernusak).

decliningMAPfromnorthtosouth(Hutley etal.,2011;Schulze et al.,1998;Williams etal.,1996).WhereMAPdeclinestoless than about 500mm, the eucalypt-dominated savannas tend to givewaytoAcacia-dominatedshrublands(BowmanandConnors, 1996).Eucalyptsarestillfoundinthesecommunities,butgener- allyoccurasscatteredindividuals,ratherthancanopy-dominant components.

Previousinvestigationsofleaf-levelresponsesofeucalyptsto variation in MAP in the Northern Territory of Australia have focussedonmeasurementsofstable carbonisotopediscrimina- tion(¹³C)inleafdrymatter(Milleretal.,2001;Schulzeetal., 1998).Leaveswerecollectedatsitesrangingfromapproximately 12^◦Sto25^◦Slatitude,encompassingMAPrangingfromapproxi- mately1800to200mm.Overmostofthisrange,littlevariation wasobservedinleaf¹³C,withamarkedresponseonlybecoming apparentwhenMAPdeclinedtobelowabout400mm(Milleretal., 2001;Schulzeetal.,1998).Asimilarlyflatresponseofeucalypt ¹³CtovariationinMAPhasalsobeenobservedinsouth-western Australia(Schulzeetal.,2006a;Turneretal.,2008).Theseresults contrastwitharecentglobalmeta-analysisoftreeleaf¹³C,which showedanapproximatelylinearincreasein¹³Cwithincreasing MAPbetween200and2000mm.Overthisrange,theaverage¹³C ofevergreenangiospermsincreasedbymorethan5‰(Diefendorf 0168-1923/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.agrformet.2011.01.006

Table1

Summarydataforthesixstudysites.Meanannualprecipitationestimatesarebasedontheyears2000to2007,andwerecomputedfrominterpolatedrainfallsurfaces.Mean annualtemperatureestimatesareaveragesofmeanminimumandmeanmaximumtemperaturesfortheyears1981–2009,usingdatafromthelongtermweatherstation nearesttoeachstudysite(http://www.bom.gov.au/index.shtml).

Site Location Species Elevation(m) Meanannual

precipitation(mm)

Meanannual temperature(^◦C) HowardSprings 12^◦2907S

131^◦0846E

Eucalyptusminiata Eucalyptustetrodonta

37 1714 27.8

AdelaideRiver 13^◦0437S 131^◦0704E

Eucalyptustectifica Corymbialatifolia

75 1532 27.0

DalyRiver 14^◦0933S 131^◦2317E

Eucalyptustetrodonta Corymbialatifolia

73 1170 27.2

DryCreek 15^◦1532S

132^◦2214E

Eucalyptustetrodonta Corymbiaterminalis

167 958 27.1

SturtPlains 17^◦0759S 133^◦1944E

Eucalyptuspruinosa Eucalyptuscoolabah

228 672 26.8

Boulia 22^◦5940S

139^◦5643E

Corymbiaterminalis Corymbiaaparrerinja

151 291 24.9

etal.,2010).Notably,thismeta-analysisdidnotincludedatafor anytreessampledinAustralia.

Analysis of ¹³C of leaf dry matter provides a convenient methodforinvestigatingleafphysiologybecauseitrelatestothe ratioof intercellularto ambientCO2 molefractions (c_i/ca)dur- ingphotosynthesis(Farquharetal.,1982,1989).Thus,the¹³C providesaproxymeasurementofanimportantleafgasexchange characteristic.Analysisofdrymatter¹³Chasthefurtheradvan- tagethatleavescanbecollected,dried,andstoredformeasurement atalatertime.InC₃plants,the¹³Crelatestoc_i/caaccordingtothe followingequation(Farquharetal.,1982;FarquharandRichards, 1984):

¹³C=a−d+(b−a)ci

ca, (1) whereaisthe¹³C/¹²Cfractionationthatoccursduringdiffusion ofCO₂throughthestomata(4.4‰),bisthediscriminationagainst

13Cbycarboxylatingenzymes(29‰forRubisco),anddisacom- positetermthatincludesfractionationscausedbydissolutionof CO₂,liquidphasediffusion,photorespiration,anddayrespiration

Fig.1. AmapshowingthelocationsofthestudysitesinnorthernAustralia.Addi- tionalinformationabouteachsiteisprovidedinTable1.

(Farquharetal.,1989).Thedwaspreviouslyestimatedtohavea meanvalueofabout3‰in15tropicaltreespecies(Cernusaketal., 2008),withotherestimatesintheliteraturerangingfromabout0 to4‰(Cernusaketal.,2007;Hubick,1990;HubickandFarquhar, 1989;Hubicketal.,1986).

Thec_i/caiscontrolledbythebalancebetweenphotosynthesis (A)andstomatalconductance(gs).Foragivengs,anincreaseinA willcauseadecreaseinc_i/ca.SimilarlyforagivenA,adecreaseings

willcauseadecreaseinc_i/c_a.Thus,whiletherelativelyflatresponse ofleaf¹³CtoMAPineucalyptsinnorthernAustraliasuggests relativelyconstantc_i/ca,thispatterndoesnotindicatewhetherA andgsarerelativelyconstantalongtheprecipitationgradient,or whetherthetwodeclineinconcertasMAPdeclines.Afurtherambi- guityassociatedwithinterpretingthe¹³Cofleafdrymatteris thatthesignalrecordedislikelydominatedbythecanopyc_i/caat thetimewhentheleafdrymatterwassynthesized.Therefore,itis unclearwhethertheobservedpatterninleafdrymatter¹³Cin north-Australianeucalyptsmightprovideinformationaboutc_i/ca

onlyduringfavourableconditionsinthewetseasonwhencanopy photosynthesisismostactive,orwhetheritprovidesinformation aboutyear-roundvariationinc_i/caalongtheprecipitationgradi- ent,includingduringthedryseasonwhendroughtstressismost severe.Althoughtheyrepresentonlyasnapshotintime,instan- taneousmeasurementsofleafgasexchangecanhelptoresolve theseambiguitiesbyprovidingcomplementaryinformationtothat providedbyleaf¹³C.

Leafphotosyntheticcapacityisanimportantparameterinbio- geochemicalmodelsthat predictcanopyC and water exchange (Baldocchiand Amthor,2001).Leaf-levelphotosynthesis canbe predictedaccordingtothebiochemicalmodelofFarquharetal.

(1980).Akeyparameterinthis modelisthemaximumRubisco carboxylation velocity (Vcmax). Thus, Vcmax provides a measure ofphotosyntheticcapacitythatcanbereadilyincorporatedinto canopyprocessmodels.Maximumleafphotosynthesisrates(Amax) aregenerallyrelatedtoleafNconcentrations(FieldandMooney, 1986),althoughtherelationshipbetweenthetwocanvaryamong taxaandamongregions(Wrightetal.,2005).Establishingglobal relationshipsbetweenphotosyntheticparameterslikeVcmaxand AmaxandmoreeasilymeasuredleaftraitslikeNconcentrationand leafmassperarea(LMA)offersapromisingavenuetowardparam- eterizationofglobalbiogeochemicalmodels(Reichetal.,2007).To preventregionalortaxon-specificbiasesinsuchglobalrelation- ships,itisimportanttoincludemeasurementsfromregionsand vegetationtypesthatareunder-representedtodate(Domingues et al., 2010). Photosynthetic capacity has not previously been assessedbyleafgasexchangeanalysisinnorth-Australianeuca- lyptsalongtheapproximately1500kmnorthtosouthgradientin MAP.

RainfallinourstudyregionofnorthernAustraliaishighlysea- sonal,witha pronounceddryseasonextendingfromMayuntil September/October.InsavannacommunitiesnearDarwin,atthe northern end of theprecipitation gradient, thesoil water that accumulatesduringthewetseasonissufficienttomaintaintranspi- rationratesatconstant,orevenhigher,ratesinsavannaeucalypts throughoutthedryseasoncomparedtoratesinthewetseason (Eamusetal.,1999,2000;Hutleyetal.,2000,2001;O’Gradyetal., 1999).Measurementsofpredawnwaterpotentialsuggestedthat thesetreeshaveaccesstoavailablewaterevenattheendofthe dryseason,presumablyfromrootsthatextendtoseveralmeters depthinthesoilprofile(Eamusetal.,2000;Hutleyetal.,2000).

Ontheotherhand,predawnwaterpotentialbecamemoreneg- ative during the dry season with increasing distance from the coast(Eamusetal.,2000),suggestingthatlatitudinalvariationin seasonaldrought stress alongthegradient in MAPin northern Australiashouldbemostpronouncedtowardtheendofthedry season.

Inthisstudy,wemeasuredleafgasexchangeinEucalyptusand CorymbiaspeciesatsixsitesinnorthernAustraliaranginginMAP fromabout1700mmtoabout300mm.Measurementswerecar- riedoutaspartofalargercampaigntoinvestigatebiogeochemical cyclinginnorth-Australiansavannas(Beringeretal.,2011).Most measurementstookplaceinSeptember,whenwe expectedlat- itudinalvariation indroughtstresstobemostpronounced.Our objectiveswere(1)toinvestigatetheapparentlackofvariation inc_i/caalongtheprecipitationgradient,assuggestedbyprevious measurementsofleafdrymatter¹³C,usinginstantaneousmea- surementsofleafgasexchangeand(2)toassessvariationinleaf photosyntheticcapacityalongtheprecipitationgradient.

2. Materialsandmethods

Leafgasexchangemeasurementsweremadeatsixsitesalonga sub-continentalrainfallgradientinnorthernAustralia(Fig.1).The sitescoveredalatitudinalrangefromapproximately12^◦Sto23^◦S, withcorrespondingMAPrangingfromapproximately1700mmto 300mm(Table1).Twocanopytreespeciescharacteristicofeach sitewereselectedfor measurements.Alloftheselectedspecies werefromthegenusEucalyptus,orthecloselyrelatedgenusCorym- bia(Table1).Twoindividualsofeachofthetwospeciesateachsite wereselectedformeasurements.Atotalofsixleavesweresampled fromeachspeciesateachsite.Canopyaccesswasachievedwitha 16melevatedworkplatformatthefourmorenortherlysites,and withladdersorfromthegroundatthetwomoresoutherlysites wheretreeswereshorter.

Two portable photosynthesis systems (Li-6400, LiCor Inc., Lincoln,NE,USA)wereusedtomeasuretheresponseofphotosyn- thesistointercellularCO₂ concentration(A–c_icurve)andtolight (lightresponsecurve).Thetwosystemswerecrosscalibrated,and gaveunbiasedresultswithrespecttoeachother.Themeasurement sequenceforeach sampledleafstartedwiththeA–c_icurveand thenproceededtothelightresponsecurve.TheCO₂concentration (␮molmol⁻¹)enteringtheleafgasexchangecuvettewasaltered inthefollowingsequence:400,280,230, 150,70,40, 230,400, 640,980,and1200.Ameasurementofphotosynthesiswaslogged approximately2minaftereachstepchangeinCO₂concentration enteringthecuvette.Irradiance duringtheA–c_icurvemeasure- mentswasset at2000␮molphotosynthetically activeradiation (PAR)m⁻²s⁻¹,suppliedbyanartificiallightsource(6400-02BLED, LiCorInc.).AttheconclusionoftheA–c_icurve,thelightresponse curvewasmeasuredonthesameleaf.TheCO2concentrationofair enteringthecuvettewasmaintainedat1200␮molmol⁻¹,andirra- diance(␮molPARm⁻²s⁻¹)wasalteredinthefollowingsequence:

2000,1500,1000,500,200,120,70,40,20,and0.Ameasurement

ofphotosynthesiswasloggedapproximately2minaftereachstep changeinirradiance.

Weendeavouredtomaintaintheleaf-to-airvapour pressure difference(D)andtheleaftemperature(T)constant acrosssites and within a site for each sample leaf during A–c_i and light responsemeasurements.ThetargetDwas2.5kPaandthetarget T was33^◦C. Meansite values (mean±1 SD)for T duringmea- surements rangedfrom32.3±0.6^◦C attheHowardSpringssite to33.9±2.0^◦C attheBouliasite.Meansite valuesduringmea- surementsforDrangedfrom2.1±0.2kPaattheHowardSprings site to3.1±0.7kPa attheBouliasite.Measurementsatallsites except theBouliasite tookplace between2and 15 September 2008.MeasurementsattheBouliasitetookplacebetween5and 8December2008.PrecipitationattheBouliasitedoesnotshow ashighlypronouncedaseasonalvariationasthemorenortherly sites. Acumulative precipitationof 56mmwasrecorded atthe Boulia Airport in the nine monthspreceding themeasurement campaign (http://www.bom.gov.au/index.shtml). Gas exchange measurementsweretakenbetween0800hand1700hlocaltime ateachsite.

Attheconclusionofthegasexchangemeasurements,eachleaf wascollectedandleafareawasdeterminedwithanimageanalysis system(Delta-TScan,Delta-TDevices,Cambridge,UK).Leaveswere driedat70^◦Ctoconstantmass,andleafdrymasswasdetermined tothenearestmg.Leaveswerethenground toa fine,homoge- neouspowder.Thenitrogenconcentrationandleafcarbonisotope ratioasubsampleofapproximately3mgleafmaterialweredeter- minedusingastableisotoperatiomassspectrometer(DeltaXP, FinniganMAT,Bremen,Germany),coupledtoanelementalanal- yser(ECS4010,CostechAnalyticalTechnologies,Valencia,CA,USA).

These analyseswereperformedin theStableIsotopeCore Lab- oratory,WashingtonStateUniversity,Pullman,WA,USA.Carbon isotopediscrimination(¹³C)inleafdrymatterwascalculatedas ¹³C=(␦¹³Ca−␦¹³Cp)/(1+␦¹³Cp),where␦¹³Cpis␦¹³Cofleafdry matter,and␦¹³CaisthatofatmosphericCO2.Weassumedavalue of−8‰for␦¹³Ca.Phosphorusconcentrationwasdeterminedby ashing100mgofleafdrymatterat550^◦C,followedbydissolution in1MH₂SO₄ andphosphatedetectionbyautomatedmolybdate colorimetry(LachatQuickchem8500,HachLtd,Loveland,CO,USA).

The A–c_i curves were analysed usingthespreadsheet utility (version2007.1)providedbySharkeyetal.(2007).Thespreadsheet utilityfitsthephotosynthesismodelofFarquharetal.(1980)to theobservedCO₂responsecurve.Estimatesaregeneratedofthe maximumRubiscocarboxylationrate,Vcmax(␮molCO₂m⁻²s⁻¹);

theelectrontransportrate,J(␮molelectronsm⁻²s⁻¹);triosephos- phateuse,TPU(␮moltriosephosphatem⁻²s⁻¹);dayrespiration, R_d (␮molCO₂m⁻²s⁻¹); and mesophyllconductance toCO₂,gm

(␮molm⁻²s⁻¹Pa⁻¹).Thespreadsheet utility providesestimates oftheseparametersnormalizedto25^◦Ctofacilitatecomparisons withmeasurementsmadeatotherleaftemperatures.

Thelightresponsecurveswereanalysedbyfittingtheobserved datatoanon-rectangularhyperbola:

A= I+Amax−

(I+Amax)²−4AmaxI

2 −RD (2)

where is theapparentquantumyield(molCO₂mol⁻¹ PAR),I is theirradiance(␮molPARm⁻²s⁻¹),Amaxis thelight-saturated gross photosynthetic rate(␮molCO2m⁻²s⁻¹), is the slope of the curve (dimensionless), and R_D is the dark respiration rate (␮molCO₂m⁻²s⁻¹). Values for and R_D were fitted first by linear regression for measurements made at I of 0, 20, and 40␮molPARm⁻²s⁻¹. The and Amax were then estimated by fitting Eq. (1) tothe light response curve using the non-linear regression routine in Systat 12 (Systat Software Inc., Chicago, IL,USA).Becausethelightresponsecurvesweremeasuredwith

the CO₂ concentration in air entering the leaf gas exchange cuvetteset at 1200␮molmol⁻¹,we assume that theAmax val- uesestimatedfromEq.(1)representbothCO₂andlightsaturated photosynthesis.

Wetestedforvariationamongsitesandbetweenspecieswithin a site in photosynthetic parameters using a nested analysis of variance.Posthoctestsforpair-wisedifferencesbetweenspecies werecarriedoutbyTukey’smethod.Relationshipsbetweencon- tinuousvariablesweretestedusingleast-squareslinearregression analyses.ResultswereconsideredsignificantatP<0.05.Statistical analyseswereperformedinSystat12(SystatSoftwareInc.).

3. Results

Leafmassperarea,LMA,increasedwithdecreasingMAPalong therainfallgradient(Fig.2A).TheMAPexplained60%ofvariation inLMAinalinearregressionanalysis(R²=0.60,P<0.001,n=81).

TherewasalsovariationinLMAbetweenspecieswithinseveral ofthesites(Fig.2A).Thus,speciesnestedwithinsite wasasig- nificanttermintheanalysisofvariance(P<0.001,n=81).There wassignificantvariationinmass-basedleafNconcentration(Nmass) amongsites andbetweenspecies withinsomeof thesites,but therewasnooveralltrendinthisparameterwithMAP(Fig.2B).

LeafNconcentrationexpressedonanareabasis(Narea)increased withdecreasingMAP(R²=0.35,P<0.001,n=81),duetothestrong trendinLMAwithMAP.Mass-basedleafPconcentration(Pmass) decreasedsignificantly with increasing MAP (R²=0.06, P=0.02, n=81),andvariedbetweenspeciesatsomeofthesites(Fig.2C).

ThedecreasingtrendsinPmassandLMAwithincreasingMAPalso resultedindecreasingarea-basedleafPconcentration(Parea)with increasingMAP(R²=0.40,P<0.001,n=81).Leafdrymatter¹³C wasnotrelatedtoMAP(P=0.36,n=81).Analysisofvarianceindi- catedthattherewassignificantvariationamongsomeofthesites andbetweenspecieswithinsomesitesforleaf¹³C,butthisvari- ationshowednooveralltrendwithMAP(Fig.2D).Leaf¹³Cdid notcorrelatewithNmass(P=0.38,n=81)orNarea(P=0.87,n=81), butdidcorrelatesignificantlywithbothPmass(R²=0.14,P<0.001, n=81)andParea(R²=0.20,P<0.001,n=81).

Instantaneous measurements of stomatal conductance (g_s), madeat ca ofapproximately 380␮molmol⁻¹ and irradiance of 2000␮molPARm⁻²s⁻¹,tendedtodecreasewithdecreasingMAP, butthetrendwasnotstatisticallysignificant(P=0.08,n=76).Mean valuesforgs foreach speciesateachsiteareshowninTable2.

Netphotosynthesismeasuredunderthesameconditionsshowed asimilartrend,withaweakdependenceonMAP(R²=0.06,P=0.03, n=76).Specieswithinasitedifferedfromeachotherattwoofthe sitesfornetphotosynthesisundertheseconditions(Table2).Vari- ationinnetphotosynthesiswascloselycorrelatedwithvariation ing_s whenmeasuredatc_a of380␮molmol⁻¹ andirradianceof 2000␮molPARm⁻²s⁻¹(Fig.3).Alinearrelationshipbetweennet photosynthesisandgswouldimplyaconstantc_i/caacrossthesetof measurements.Accordingly,c_i/cawasrelativelyconstantacrossthe sampledleaves,withonlyEucalyptustetrodontaatDalyRiverand CorymbiaaparrerinjaatBouliadifferingfromeachotherinthepair- wisecomparisons(Table2).Thec_i/cawasindependentofvariation inMAP(P=0.67,n=76).

Thec_i/cashowedalinearandrelativelyweakdependenceongs

forgasexchangemeasurementsmadeatcaof380␮molmol⁻¹and irradianceof2000␮molPARm⁻²s⁻¹ (R²=0.11,P=0.003,n=76), asshowninFig.4.ThedashedlineinFig.4showsthepredicted relationshipbetweenc_i/caandgsifVcmax,J,gm,andotherparam- etersintheFarquharetal.(1980)photosynthesismodelwereto remainconstantacrosstherangeofgsencounteredinthestudy.

ThedepartureofthesolidregressionlineinFig.4fromthepre- dicteddashedlinesuggeststhatc_i/cadeclinedlessatlowgsthan

Leaf mass per area (g m-2 ) 150 200 250 300

Eucalyptus miniata Eucalyptus tetrodonta Eucalyptus tectifica Corymbia latifolia Corymbia terminalis Eucalyptus pruinosa Eucalyptus coolabah Corymbia aparrerinja

Leaf N concentration (mg g-1 ) 2 4 6 8 10 12 14

Mean annual precipitation (mm) 2000 1500 1000 500

0 Leaf Δ13 C (‰)

18 20 22 24

A

D B

Leaf P concentration (mg g-1 ) 0.2 0.4 0.6

0.8

C

Fig.2. Leafmassperarea(A),leafnitrogenconcentration(B),leafphosphoruscon- centration(C),andcarbonisotopediscriminationinleafdrymatter(D)plottedas functionsofmeanannualprecipitation.Eachsymbolrepresentsthemeanofsixto eightleaves.Errorbarsareonestandarderror.

Table2 GasexchangeparametersforeucalyptleavesalongarainfallgradientinnorthernAustralia.Stomatalconductance,netphotosynthesis,andtheratiooftheinternaltoambientCO2molefractions(ci/ca)weremeasuredat irradianceof2000␮molphotosyntheticallyactiveradiation(PAR)m−2s−1andcaofapproximately380␮molmol−1.ThemaximalRubiscocarboxylationvelocity(Vcmax)andelectrontransportrate(J)weredeterminedat irradianceof2000␮molPARm−2s−1fromCO2responsecurves.Apparentquantumyield()andmaximumphotosyntheticrate(Amax)weredeterminedfromlightresponsecurvesatcaofapproximately1170␮molmol−1.Values shownarethemeanofsixtoeightleavesforeachspeciesateachsite±1standarddeviation.ValueswithinacolumnnotfollowedbyacommonletterdifferedfromeachotheratP<0.05. SiteSpeciesStomatal conductance (molm−2s−1) Net photosynthesis (␮molm−2s−1) ci/caVcmaxat25◦C(␮molm−2s−1)Jat25◦C(␮molm−2s−1)Apparent quantumyield, (molmol−1)

Amax (␮molm−2s−1) HowardSpringsEucalyptusminiata0.170±0.062a16.2±5.5a0.54±0.06a,b77.6±6.4a,b85.0±14.8a,b,c0.067±0.017a34.0±7.2a,b,c Eucalyptustetrodonta0.085±0.028a,b8.0±2.3b,c0.53±0.03a,b62.8±12.4b69.6±9.4b,c0.059±0.013a,b23.6±5.7b,c AdelaideRiverEucalyptustectifica0.160±0.032a14.4±3.0a,b0.57±0.03a,b84.1±15.9a,b103.3±20.3a,b0.057±0.012a,b39.5±9.2a,b Corymbialatifolia0.078±0.034a,b7.4±3.0b,c0.55±0.07a,b64.4±20.0b83.4±22.8a,b,c0.049±0.013a,b29.3±7.8a,b,c DalyRiverEucalyptustetrodonta0.151±0.049a11.9±3.2a,b0.59±0.04a,b93.4±14.3a,b98.0±12.0a,b0.060±0.013a,b44.0±8.9a Corymbialatifolia0.133±0.056a9.3±2.8a,b,c0.63±0.06a73.2±14.9b80.2±14.1a,b,c0.049±0.012a,b29.8±4.0a,b,c DryCreekEucalyptustetrodonta0.107±0.061a9.3±5.2b,c0.59±0.05a,b84.6±22.2a,b85.3±22.0a,b,c0.047±0.015a,b28.9±16.8a,b,c Corymbiaterminalis0.081±0.039a,b7.7±3.1b,c0.54±0.04a,b94.1±34.1a,b95.0±32.2a,b0.059±0.009a,b42.3±8.1a SturtPlainsEucalyptuspruinosa0.135±0.056a10.9±2.8a,b0.59±0.07a,b82.1±12.2a,b91.1±15.5a,b,c0.041±0.018b31.7±7.6a,b,c Eucalyptuscoolabah0.042±0.019b3.3±0.8c0.59±0.1a,b65.0±9.1b58.4±7.6c0.051±0.006a,b18.8±2.9c BouliaCorymbiaterminalis0.125±0.080a10.7±5.7a,b0.57±0.03a,b109.6±24.1a113.1±27.7a0.063±0.009a,b39.2±14.3a,b Corymbiaaparrerinja0.083±0.011a,b8.7±1.1b,c0.51±0.02b77.1±9.7a,b100.1±11.1a,b0.059±0.004a,b31.0±2.7a,b,c

Stomatal conductance to H

₂

O (mol m

^-2

s

^-1

)

0.3 0.2

0.1 0.0

Net photosynthesis (µmol CO

2

m

-2

s

-1

)

0 5 10 15 20 25

Eucalyptus miniata Eucalyptus tetrodonta Eucalyptus tectifica Corymbia latifolia Corymbia terminalis Eucalyptus pruinosa Eucalyptus coolabah Corymbia aparrerinja

Fig.3.Netphotosynthesisplottedagainststomatalconductanceforeucalyptleaves sampledalongarainfallgradientinnorthernAustralia.Measurementsweremade atexternalCO2concentrationof380␮molmol⁻¹andirradianceof2000␮molpho- tosyntheticallyactiveradiationm⁻²s⁻¹.

wouldbeexpectedinthecaseforinvariantphotosyntheticcapacity andgm.

TheV_cmaxnormalizedto25^◦Candexpressedonanareabasis showedaweakincreasingtrendwithdecreasingMAP(R²=0.07, P=0.02,n=79).ThisrelationshipwastheresultofincreasingLMA with decreasing MAP; when expressedon a mass basis, V_cmax showed an opposite trend of increasing with increasing MAP (R²=0.10, P=0.004,n=79).The Vcmax wasnot related toleafN concentration,neitherwiththetwoparametersexpressedonan areabasis(P=0.34,n=79),noronamassbasis(P=0.78,n=79).

Similarly,VcmaxwasnotrelatedtoleafPconcentration,neitherfor mass-based(P=0.86,n=79)norarea-based(P=0.44,n=79)com- parisons.However,area-basedV_cmaxwaspositivelycorrelatedwith LMA(Fig.5).Area-basedVcmaxalsocorrelatedwithgsmeasured atcaof380␮molmol⁻¹andPARof2000␮molm⁻²s⁻¹(R²=0.15, P<0.001,n=76).The electron transportrate,J,correlatedposi- tivelywithVcmax.Theregressionequationwiththetwoparameters normalized to25^◦C wasJ=0.91Vcmax+14.4(R²=0.76, P<0.001, n=79).Meanvalues ofVcmax and Jforeach speciesateachsite aregiveninTable2.

The apparent quantum yield, , estimated from the initial slopeofthelightresponsecurves,showedlittlevariationacross thesampledleaves(Table2).Thelight-saturatedgrossphotosyn- thetic rate, Amax, was positively correlated withVcmax (Fig. 6), suggestingthatthetwomeasurementsprovidedconsistentindi- cationsofvariation inphotosyntheticcapacity. Area-basedAmax

wasunrelatedtoMAP(P=0.90,n=74),whereasmass-basedAmax

increased withincreasing MAP (R²=0.16, P<0.001,n=74). The Amax showednosignificantrelationship withleafN concentra-

Stomatal conductance to H

₂

O (mol m

^-2

s

^-1

)

0.3 0.2

0.1 0.0

Instantaneous c / c

ia

0.0 0.2 0.4 0.6 0.8 1.0

Eucalyptus miniata Eucalyptus tetrodonta Eucalyptus tectifica Corymbia latifolia Corymbia terminalis Eucalyptus pruinosa Eucalyptus coolabah Corymbia aparrerinja

Fig.4. TheratioofintercellulartoambientCO2molefractionplottedagainststo- matalconductance.Thesolidlineisalinearregressionthroughthedata(R²=0.11, P=0.003,n=76).Thedashedlineisthepredictedrelationshipbetweenthetwo parametersifthemaximumRubiscocarboxylationvelocity,theelectrontransport rate,andmesophyllconductancewereassumedconstantovertheobservedrange ofstomatalconductance.

tion or with leaf P concentration.The Amax correlatedwith gs

measuredatc_aof380␮molmol⁻¹andPARof2000␮molm⁻²s⁻¹ (R²=0.39, P<0.001, n=69). The parameter, , representing the slope of thelight response curve,did not vary among sites or betweenspecieswithinasite.Themeanvalueacrossallsampled leaveswas0.17±0.06 (mean±1SE,n=74).Thedarkrespiration ratemeasuredduringthelightresponsecurvesvariedasafunction ofAmaxaccordingtotheregressionequationRD=0.028Amax+0.61 (R²=0.19,P<0.001,n=74).Thegrossphotosyntheticrateatirra- dianceof 2000␮molPARm⁻²s⁻¹ wason average 81% of Amax, suggestingthatphotosynthesisat2000␮molPARm⁻²s⁻¹wasnot light-saturated.MeanAmaxforeachspeciesateachsiteisgivenin Table2.

4. Discussion

Measurementsofleafdrymatter¹³Cforsavannaeucalypts inthisstudyweresimilartopreviousobservations,showinglit- tlevariationinresponsetoastrongrainfallgradientinnorthern Australia(Miller etal.,2001; Schulzeet al.,1998).Thispattern contrastswithrelativelystrongrelationshipsbetweenleafdrymat- ter¹³CandMAPfortreessampledinNorthAmericaandAsia (Diefendorfet al.,2010),and inAfrica(Midgleyetal.,2004).In thepresentstudy,wewereabletoconfirm,usinginstantaneous gasexchange measurements, that c_i/ca was nearly constant in savannaeucalyptsalongthestrongrainfallgradientinnorthern Australia.Thisobservationtookplaceattheendofthedry sea- son,whenweexpectedlatitudinalvariationindroughtstresstobe

Leaf mass per area (g m

^-2

)

350 300

250 200

150 100

V

cmax

at 25°C (µmol CO

2

m

-2

s

-1

)

0 20 40 60 80 100 120 140 160

Eucalyptus miniata Eucalyptus tetrodonta Eucalyptus tectifica Corymbia latifolia Corymbia terminalis Eucalyptus pruinosa Eucalyptus coolabah Corymbia aparrerinja

Fig.5.ThemaximumRubiscocarboxylationvelocityplottedagainsttheleafmass perareaforeucalyptleavessampledalongarainfallgradientinnorthernAustralia.

Thesolidlineisaregressionline:Vcmax=0.17LMA+45(R²=0.11,P=0.003,n=79).

mostpronounced.Thenearlyconstantc_i/caobservedinthisstudy resultedatleastpartlyfromacorrelationbetweenphotosynthetic capacityandstomatalconductance.Aclosecouplingbetweenpho- tosyntheticcapacityandstomatalconductancehasbeenpreviously observedinarangeofC₃andC₄plantspecies(BrodribbandFeild, 2000;CernusakandMarshall,2001;Hubbardetal.,2001;Wong etal.,1978,1979,1985).

Wefoundthree linesof evidence suggestingthat photosyn- thetic capacity in the leaves that we sampled was linked to stomatalconductance.Firstly,estimatesofVcmaxcorrelatedwith gs measured at ambient CO2 concentration and irradiance of 2000␮molPARm⁻²s⁻¹.Secondly,theAmaxestimatedfromlight responsecurves conductedatsaturating CO₂ concentrationcor- related with gs measured at ambient CO2 and irradiance of 2000␮molPARm⁻²s⁻¹.Andthirdly,theobservedc_i/c_adecreased less withdecreasinggs thanwasexpectedtheoretically for the caseofinvariantVcmax,J,andgm(Fig.4).Theseobservationssug- gestthat modellingapproachesforpredictingphotosynthesisin north-Australiansavannatreesmaybenefitbylinkingphotosyn- theticcapacitytogs.Severalauthorshaveformulatedmodelsthat applythisconcept(Balletal.,1987;Buckleyetal.,2003;Farquhar andWong,1984;JarvisandDavies,1998;Leuning,1990;Leuning, 1995),althoughthemechanisticbasisforcouplingbetweenpho- tosyntheticcapacityandgsisstilluncertain(vonCaemmereretal., 2004).

Acorrelationbetweengsandmesophyllconductance,gm,could havealsocontributedtotheapparentcorrelationbetweenpho- tosyntheticcapacityandgs,ifgmwerelowerinleaveswithlowgs

thaninleaveswithhighgs.Althoughthespreadsheetutilitythatwe usedtoestimateVcmaxalsoprovidesestimatesofgm(Sharkeyetal., 2007),thismethodforestimatinggmwasfoundtobelessreliable

V

cmax

at 25°C (nmol CO

₂

g

^-1

s

^-1

)

800 600

400 200

0

A

max

(nmol CO

2

g

-1

s

-1

)

0 50 100 150 200 250 300 350

Eucalyptus miniata Eucalyptus tetrodonta Eucalyptus tectifica Corymbia latifolia Corymbia terminalis Eucalyptus pruinosa Eucalyptus coolabah Corymbia aparrerinja

Fig.6. Maximumgrossphotosynthesisdeterminedfromlightresponsecurvesplot- tedagainstthemaximumRubiscocarboxylationvelocitydeterminedfromCO2

responsecurves.Bothparametersareexpressedonamassbasis.Thesolidlineisa linearregression:Amax=0.46Vcmax−21(R²=0.63,P<0.001,n=72).

thanmethodsthatapplychlorophyllflorescenceoron-line¹³C measurements(Pons etal.,2009).Wedidobserveacorrelation betweentheestimatedgmandgsmeasuredatambientCO2con- centrationand2000␮molPARm⁻²s⁻¹,buttherelationshipwas weak(R²=0.09,P=0.01,n=76).Themeanestimateofgmacross allleavesinthestudywas10␮molm⁻²s⁻¹Pa⁻¹.Thisestimateis considerablyhigherthanvaluesdeterminedpreviouslyforarange ofAustralianplantspecieswithsclerophyllousleaves(Niinemets etal.,2009).Consistentwithpreviousobservations(Flexasetal., 2008;Hassiotouetal.,2009),wealsoobservedanegativecorrela- tionbetweengmandLMA(R²=0.26,P<0.001,n=79).Thissuggests thatgmshoulddecreasewithdecreasingMAPduetotheincrease inLMA.Amoredetailedinvestigationofvariationingminsavanna eucalyptsalongthenorth-Australianrainfallgradientusingchloro- phyllflorescenceoron-line¹³Cmethodsincombinationwithgas exchangewouldbehelpfultoverifythistrend.

Therelativelyflatresponsesofleafdrymatter¹³Candinstan- taneous c_i/ca toa strongrainfall gradient innorthern Australia observedhereandpreviously(Milleretal.,2001;Schulzeetal., 1998)contrasts withresultsoftwo studiesineastern Australia (Stewartetal.,1995;Wrightetal.,2001).Thestudiesineastern Australiainvestigatedvariationsin¹³Candc_i/caofbroaderveg- etationcommunitiesinresponsetoMAP,whereasourstudyonly investigatedspecieswithinthecloselyrelatedgeneraofEucalyp- tusandCorymbia.However,thenorth-Australiantransectstudy of Schulze et al. (1998) included a broad taxonomic range of woodyplantspecies,andtheseauthorsstillonlyobservedaweak community-levelresponse of¹³C toMAP.Seasonalityofrain- fallcouldbe a featurecontributing tothesecontrasting results forresponsesof¹³Candc_i/catoMAPinnorthernversuseast-

ernAustralia(Milleretal.,2001;Schulzeetal.,1998).Rainfallin monsoonalnorthernAustraliashowsamuchstrongerseasonaldis- tributionthanrainfallineasternAustralia.Withineucalyptsmore generally,somespeciesappeartoshownoresponseof¹³Ctovari- ationinMAP,whereasotherspeciesshowamoreplasticresponse, with¹³C increasing in responseto increasingMAP(Cernusak etal.,2005;Milleretal.,2001;Schulzeetal.,2006a,b;Turneretal., 2010;Warrenetal.,2006).

ThemostplasticleaftraitinresponsetoMAPassessedinour studywastheleafmassperarea.TheLMAdecreasedwithincreas- ing MAPatarateof 70gleafdrymass m²leafaream⁻¹ mean annualprecipitation.BecauseleafNmasswasindependentofMAP, leafN_areaincreasedasMAPdecreasedduetothevariationinLMA.

The Vcmax,expressed perunit leaf area, also increasedas MAP decreased,althoughtheincrease wasnotaspronouncedasthat forleafN_area.ThischangeinLMA,leafN_area,andphotosynthetic capacitywithdecreasingMAPisconsistentwiththeoreticalpredic- tions(Farquharetal.,2002),andwithpreviousobservationsinthis andotherecosystems(Midgleyetal.,2004;Mooneyetal.,1978;

Schulzeetal.,1998;Wrightetal.,2001).

Leafphotosyntheticcapacityisoftenobservedtocorrelatewith leafNconcentration(Dominguesetal.,2010;FieldandMooney, 1986;Wrightetal.,2004).Thishasbeenexplainedonthebasisof theproportionallylargeallocationofNtophotosyntheticenzymes and light-harvesting pigments in leaves (Evans, 1989). In the presentstudywedidnotobserveasignificantrelationshipbetween leafNconcentrationandVcmax,Amax,orphotosynthesisatambi- entCO₂concentration.Thislackofcorrelationmostlikelyresulted fromtherelativelynarrowrangeofleafNconcentrationencoun- teredinthestudy(Fig.2B).LeafNconcentrationsintheleavesthat wesampledwerelowcomparedtotherangeofvaluestypically observedinangiospermtrees(ReichandOleksyn,2004),butsim- ilartoobservationsforothereucalypts(BellandWilliams,1997;

Prioretal.,2003;Schulzeetal.,1998).

In ordertocoveralargerrangeof leafN concentrations,we combined Vcmax data fromthepresent studywithobservations recently reportedfor seedlings ofnorth-Australian tree species grownina shadehouseontheCharlesDarwin Universitycam- pus (Orchardetal.,2010).Meanvaluesforeach speciesateach site weretakenasdatapoints.Asignificantcorrelationresulted between Vcmax at 25^◦C (nmolg⁻¹s⁻¹) and leaf Nmass (mgg⁻¹):

Vcmax=41.2Nmass+151(R²=0.67,P<0.001,n=22).Inthedataset, leafN_massrangedfrom5.7to33.7mgg⁻¹,andV_cmaxat25^◦Cfrom 301to1380nmolg⁻¹s⁻¹.Thisdemonstratesthateventhoughwe didnotobservearelationshipbetweenVcmaxandleafNmassinthe presentstudy,leafNmassisstillausefulpredictorofVcmax when a largerrange of leafNmass is taken intoaccount. TheVcmax–N slopeoftherelationshipobtainedfromthiscombinedanalysisof 41.2␮molCO2g⁻¹Ns⁻¹isinthemiddlerangeofVcmax–Nslopes observedinarangeofpreviousstudies(Kull,2002).

Ithasbeenpreviouslyobservedthatleafphotosyntheticcapac- itycorrelatedwithleafPconcentrationinsomeP-limitedtropical ecosystems(Dominguesetal.,2010;Meiretal.,2007),andthatleaf photosynthesis–NslopescanbeinfluencedbyleafPconcentrations (Reichetal.,2009).Wedidnotobserveanystrongrelationship betweenleafgasexchangeratesandleafPconcentrationsinour dataset.IncommonwiththeNcomparisons,thislikelyresulted fromalimitedrangeofvariationinPconcentrationsamongthe experimentalleaves(Fig.2C).Wedid,however,observeasignif- icantcorrelationbetweenleaf¹³CandleafP_area,whichhintsat thepossibilitythatleafPareacouldhavebeeninfluencingphotosyn- theticcapacityintheleavesthatwesampled.TheaverageN:Pratio acrossthedatasetwas16.4gg⁻¹.Accordingtoasimpleinterpreta- tionofleafN:Pratios,thiswouldbeconsistentwithco-limitationof productivityinover-storyeucalyptsalongtherainfallgradientby bothNandP(AertsandChapin,2000;Güsewell,2004;Koerselman