Glol Chnge Biology (28) 14, 234 2316, doi: 1.1111/j.1365-2486.28.1656.x Wrming nd drought lter C nd N concentrtion, lloction nd ccumultion in Mediterrnen shrulnd JORDI SARDANS, JOSEP PEÑUUELAS, MARC ESTIARTE ndpatricia PRIETO Ecophysiology nd Glol Chnge Unit CSIC-CEAB-CREAF, CREAF (Centre de Recerc Ecologic i Apliccions Forestls), Universitt Autònom de Brcelon, E-8193 Bellterr, Spin Astrct We investigted the effects of wrming nd drought on C nd N concentrtions, nitrogen use efficiency (NUE), nd C nd N ccumultion in different ecosystem comprtments. We conducted 6-yer (1999 25) field experiment to simulte the climte conditions projected y IPCC models for the coming decdes in Mediterrnen shrulnd. We studied the two dominnt species, Gloulri lypum nd Eric multiflor, nd n N-fixing species, Dorycnium pentphyllum, lso undnt in this shrulnd. Wrming (1 1C) decresed N lef concentrtions y 25% nd incresed N stem concentrtions y 4% in G. lypum. Although wrming chnged the ville mmonium in soil in some sesons, it did not increse totl soil N contents. Drought (19% verge reduction in soil moisture) decresed lef N concentrtions in the two dominnt shru species, E. multiflor nd G. lypum y 16% nd 19%, respectively, nd incresed stem N concentrtions y 56% nd 4%, respectively. Neither wrming nor drought chnged the lef N concentrtions in the N-fixing species D. pentphyllum, lthough wrming incresed stem N concentrtion y 9%. In G. lypum, the increse of stem N concentrtions contriuted to the oserved increse of N ccumultion in stem iomss in drought tretments with respect to control plots (8 kg N h 1 ). Neither wrming nor drought chnged NUE in the period 1999 25. Wrming incresed soil orgnic C reltive to drought. The effects of wrming nd drought on C nd N concentrtions, on N ccumultion nd on lef/stem N distriution were not the result of dilution or concentrtion effects produced y chnges in iomss ccumultion. Other fctors such s the chnges in soil N vilility, photosynthetic cpcity, nd plnt internl C nd N remoiliztion must e involved. These chnges which differed depending on the species nd the plnt tissue show tht the climte chnge projected for the coming decdes will hve significnt effects on the C nd N cycle nd stoichiometry, with prole implictions for ecosystem structure nd function, such s chnges in plnt herivore reltionships, decomposition rtes or community species composition. Keywords: C/N, climte chnge, Dorycnium pentphyllum, drought, Eric multiflor, Gloulri lypum, Mediterrnen shrulnd, N, NUE, wrming Received 25 July 27; revised version received 7 Jnury 28 nd ccepted 2 Mrch 28 Introduction Climte chnge generted y elevted tmospheric greenhouse gses hs incresed the Erth s surfce temperture y.74 1C in the pst century (IPCC, 27). Tempertures in the Mediterrnen region hve lso shown wrming trends (Peñuels et l., 22, 25; Correspondence: Jordi Srdns, tel. 1 34 93 581 29 34, fx 1 34 93 581 41 51, e-mil: j.srdns@cref.u.ct Peñuels & Bod, 23). Precipittion hs egun to exhiit either long-term downwrd trend, minly in the dry seson (Esten-Prr et l., 1998), or no significnt chnge (Piñol et l., 1998; Peñuels et l., 22, 25), lthough in ll cses rise in potentil evpotrnspirtion hs led to incresed ridity (Piñol et l., 1998; Peñuels et l., 25). Current glol circultion models (GCM) nd ecophysiologicl models such s Gotilw (Sté et l., 22; Peñuels et l., 25) predict further increses in wrming nd drought in the 234 Journl compiltion r 28 Blckwell Pulishing Ltd
CLIMATE CHANGE AND N ALLOCATION 235 Mediterrnen region in the coming decdes. Moreover, the min predictions of the most generl circultion models (GCMs) indicte tht over this century the men glol surfce temperture will increse y 1.1 6.4 1C depending on socioeconomic scenrios nd the resulting emissions of greenhouse gses (IPCC, 27). Such chnges in climte re expected to influence C cycling (Cox et l., 2). In ddition to C chnges, N vilility nd its dynmics nd stocks in ecosystems could lso e ffected. The effect of climte chnge on N stocks in different ecosystem comprtments could hve significnt impcts oth on ecosystem performnce nd on long-term C sequestrtion in terrestril ecosystems (Hungte et l., 23; Luo et l., 24). This is ecuse lef N concentrtion is one of the most importnt plnt trits tht determine photosynthetic C fixtion nd iomss ccumultion cpcity. Wrming nd/or drought cn chnge N plnt concentrtion minly y chnging iomss ccumultion nd N soil vilility, therey lso ffecting C/N concentrtion rtios, N use efficiency (NUE), N minerlomsses nd N reltive lloction to different tissues s oserved in temperte non-mediterrnen ecosystems (An et l., 25) nd in some rctic ecosystems (Tolvnen & Henry, 21; Weintru & Schimel, 25). The mgnitude of these effects depends on the functionl group (Lilley et l., 21) nd on the ecosystem climte type (Ll, 23; Luoml et l., 23), nd my e different mong different species in the sme ecosystem (Red & Morgn, 1996), leding to shifts in plnt community structure (Lilley et l., 21). The chnges in C nd N stoichiometry t plnt nd ecosystem levels hve een shown to ply n importnt role in trophic chins nd finlly in the species composition structure of ecosystems (Ngi & Jefferies, 24; Diehl et l., 25). Less is known out the impct of climte chnge on C nd N contents in Mediterrnen ecosystems. Nutrients re frequent limiting fctor in Mediterrnen ecosystems (Hnley & Fenner, 21; Srdns et l., 24, 25). Severl studies hve reported N limiting photosynthetic cpcity (De Lillis & Federici, 1993) nd iomss ccumultion cpcity (Rodríguez-Echeverri & Pérez-Fernández, 23; Villr-Slvdor et l., 24). By nlyzing herri specimens nd tree rings, Peñuels & Mtml (199) nd Peñuels & Estirte (1997) hve reported tht folir N concentrtions hve decresed in recent decdes in the Mediterrnen Bsin. N vilility my offset positive plnt growth responses to elevted CO 2 in Mediterrnen ecosystems (Cruz et l., 23), thus hving n importnt role in C sequestrtion cpcity. Wrming cn contriute to CO 2 fixtion in temperte nd high ltitude ecosystems during the growth sesons (Keeling et l., 1996) ecuse of its positive effects on iologicl processes in environments where tempertures re currently fr from optimum. In Mediterrnen ecosystems, however, it is uncler s to whether such wrming effect would occur. In Mediterrnen environments, the effects of wrming re insted strongly dependent on wrming s influence on wter vilility nd the efficiency of its use. Drought, on the other hnd, cn limit photosynthetic cpcity y decresing soil moisture. Growth decrese cn contriute to concentrting N in plnt tissues nd to chnges in lef photosynthetic cpcity, in trnspirtion fluxes nd in wter use efficiency (WUE), which hs consequences for C sequestrtion cpcity. These effects cn chnge the C nd N lef stem lloction. Drought limits plnt growth cpcity in Mediterrnen ecosystems (Ogy et l., 23) nd reduces soil microil ctivity (Srdns & Peñuels, 25) nd, therefore, cn led to decrese in the ecosystem s cpcity to ccumulte C nd N in plnt tissues. Susequently, there re chnges in the mounts nd chemicl forms of C nd N in soil. It is, thus importnt to gin knowledge out the response of plnt nd soil N to wrming nd drought, prticulrly in Mediterrnen plnts which re descried to hve gret cpcity for resource remoiliztion through the yer (Lloret et l., 1999; Filell & Peñuels, 23; Medivill & Escudero, 23). The response of the C nd N contents of Mediterrnen ecosystems to climte chnge is especilly importnt for dditionl resons. Some climtic models hve projected prtil loss of C from ecosystems to the tmosphere in southern Europen forests (White et l., 1999) nd on the contrry, others forecst n ccumultion in soil C stocks (Wessel et l., 24). Moreover, there is lck of experimentl dt on the direct nd indirect effects of wrming nd drought on C nd N concentrtions nd contents in the different comprtments in Mediterrnen ecosystems. In Mediterrnen regions, reduction in minerliztion rtes together with decrese in plnt iomss ccumultion nd N cpture would increse N ccumultion in soil. Furthermore, s result of the decrese in soil wter vilility, there would e n increse in oxidtion of the soil environment tht cn increse soil nitrifiction. The increse in wter soluility of soil N could result in greter N losses through leching fter more frequent torrentil rinflls, such s those projected y most models (IPCC, 27). This scenrio leds to n increse in the eutrophiction of the continentl wters nd to reduction in the N content of the ecosystem, thus compromising the future growth cpcity nd WUE of the ecosystem nd reducing the cpcity of cron sequestrtion. Finlly, humn ctivity hs incresed the nutrient inputs in nturl ecosystems in wide res of the Mediterrnen sin in recent Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
236 J. SARDANS et l. decdes (Peñuels & Filell, 21; Rodrigo & Avil, 22). Chnges in N ccumultion cpcity in terrestril ecosystems in new climte scenrio in the coming decdes would ffect the future N lnce. To test the hypothesis tht there will e significnt chnges in C nd N content in iomss nd soils in response to the wrming nd drought projected y GCM nd ecophysiologicl models such s GOTILWA in the Mediterrnen sin (Sté et l., 22; Peñuels et l., 25; IPCC, 27), we conducted field experiment simulting this predicted wrming nd drought in Mediterrnen shrulnd. We investigted the effects of 6 yers of experimentl wrming nd drought on (1) C nd N concentrtion nd ccumultion in the iomss of the two dominnt shru species, Gloulri lypum nd Eric multiflor, nd of n undnt N-fixing species Dorycnium pentphyllum, nd (2) C nd N concentrtions in litter nd soil. Mterils nd methods Study site The study ws crried out in nturl Mediterrnen clcreous shrulnd on south-fcing slope in the Grrf mountins in Ctloni (NE Spin) (41118 N, 1149 E). The site is locted on formerly cultivted terrces ndoned pproximtely century go with Petroclcic clcixerept soil lying on edrock of sedimentry limestone. During the study period (1999 25), the verge nnul temperture ws 15.1 1C (7.4 1C in Jnury nd 22.5 1C in July) nd the verge nnul rinfll ws 58 mm. The summer drought is pronounced nd usully lsts for 3 months. The totl vegettion cover is 75% nd consists of clcreous shrulnd with plnts out 1 m high dominted y the shrus G. lypum (L.), E. multiflor (L.), D. pentphyllum (Vill.), Rosmrinus officinlis (L.), Ulex prviflorus (Pourr.), nd Pistci lentiscus (L.). Aleppo pines Pinus hlepensis (Mill.) were ir-seeded fter the lst forest fire in 1994 nd re tody gining ground. The undergrowth is dominted y smll shrus such s Fumn ericoides (Cv.) nd Fumn thymifoli (L.). Experimentl design We conducted field-scle night-time wrming nd drought tretments (Beier et l., 24; Peñuels et l., 27) nd compred tretment plots with control plots. Ech of the three tretments (control, wrming nd drought) ws pplied in three plots. Ech plot occupied n re of 4 5m 2, lthough to void the effect of edge disturnce, smples were only tken from n internl re of 3 4m 2. Mnipultion strted in Mrch 1999 nd hs continued till present. The wrming tretment consisted in incresing nighttime tempertures y covering the vegettion with n luminum screen coiled on em nd connected to motor controlled y light sensors tht utomticlly covered the vegettion t night. This screen reflected long-wve infrred rdition ck into the vegettion, resulting in temperture increse in reltion to untreted plots. In order to void interfering with the hydrologicl cycle, this cover ws utomticlly removed when it rined. The wrming screen ws removed if the wind speed exceeded 1 m s 1 in order to void structurl dmge. The drought tretment reduced spring nd utumn rinfll input. This ws chieved y utomticlly covering vegettion with trnsprent plstic curtin during rin events y mens of utomtic rin sensors. Once the rin stopped, the screen ws utomticlly removed. As in the wrming tretment, the cover ws lso removed if wind speed exceeded 1 m s 1. In summer nd winter (outside the drought period), the tretment ws not pplied nd drought plots were llowed to develop under the sme conditions s control plots. The control plots were equipped with the sme scffolding s the tretment plots ut without the screen roof. Environmentl conditions were monitored in ll plots. Soil moisture ws mesured iweekly y the TDR (time domin reflectometry) model Tektronix 152C (Tektronix, Beverton, OR, USA), using three proes instlled in ech plot. The ir nd soil tempertures were recorded for every plot y temperture sensors (RTD Pt 1 1/3 DIN; Desin Instruments, Brcelon, Spin) locted t depths of 1 nd 5cm in the soil nd t 2 cm ove the soil. Air nd soil tempertures, s well s the correct functioning of the motorized screen (mgnetic sensors were instlled t the end of the screen trck), were recorded y dtlogger (Cmpell Scientific Inc., Logn, UT, USA). Precipittion ws mesured y stndrd rin guges nd ll wter entering the plots ws collected iweekly. Biomss growth In 1999 nd in 25, the oveground iomss ws estimted y mens of the pin point method (Peñuels et l., 24, 27). In ech plot, five permnent 3 m long trnsects seprted y 8 cm distnce were estlished. A verticl metl rod ws lowered every 5 cm within ech trnsect nd contcts etween the rod nd plnts were recorded for 35 points per plot. This llowed us to otin vlue of contcts nd mximum height per pin tht ws used to estimte eril iomss. Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
CLIMATE CHANGE AND N ALLOCATION 237 Pin-point mesurements were lso conducted in dditionl plots externl to the experiment. After pinpoint mesurements, the eril iomss of these externl plots ws cut nd rought to the lortory where it ws oven-dried t 75 1C to constnt weight. Regression nlysis etween eril iomss (AB, g m 2 ) nd mximum height (MH, cm) mesured y point trnsect resulted in the following equtions: E. multiflor AB 5 1 35.587 MH (R 2 5.91, Po.1); G. lypum AB 5 44.38 1 33.61 MH (R 2 5.98, Po.1). These regression equtions were used to estimte the eril iomss of experimentl plots. From these plnts tht were externl to the experiment, we lso otined llometric reltionships etween lef nd stem iomss nd plnt height for oth E. multiflor nd G. lypum plnt species. Equtions used were the following: for E. multiflor plnt lef iomss (y 5 2.58x 5.89, R 2 5.82) nd plnt stem iomss (y 5 3.95x 11.4, R 2 5.87), nd for G. lypum plnt lef iomss (y 5 3.4x 1.68, R 2 5.69) nd plnt stem iomss (y 5 5.33x 17.55, R 2 5.82), where y is the ln of iomss (g) nd x the ln of stem length (cm). Plnts within experimentl plots were mesured ( totl of 235 E. multiflor nd 268 G. lypum individuls) nd were used to otin lef to stem iomss rtio for 1999 nd 25. This rtio ws used to seprte the iomss per plot, otined y the pin-point method, into lef nd stem iomss for ech species. Smpling process Biomss. To nlyze C nd N concentrtions, we smpled the plnt iomss twice, once t the eginning of the experiment in Jnury 1999 nd then gin 6 yers lter in Jnury 25. Ech time we smpled 15 individul plnts per plot, five pertining to ech one of the two dominnt shru species, G. lypum nd E. multiflor, nd to the N-fixing species D. pentphyllum (this ltter species only in 25). Five rnches were smpled from ech plnt. Lef life is longer in E. multiflor thn in the other two species nd the lef popultion consisted mostly of current-yer leves nd some 1 yer old leves. Therefore, for this species we nlyzed the two different lef-yer cohorts (current yer nd 1 yer old). In the other two species, only current-yer leves were present during the smpling cmpign nd, therefore, only one cohort ws considered. Thus, two frctions of oveground iomss were considered in G. lypum nd D. pentphyllum (stem nd current-yer leves) nd three (stem, current-yer leves nd 1 yer old leves) in E. multiflor. For the root iomss, we took three smple cores (3 cm deep, f 5 5 cm) from ech plot. Becuse of the difficulty of distinguishing etween the roots of different species, we smpled ner G. lypum nd only collected roots of this species. In ech core, we selected the roots of fo1 mm nd the roots of f41mm nd nlyzed these two frctions seprtely. Litterfll. The litterfll of four to eight plnts of E. multiflor nd 9 12 plnts of G. lypum per plot ws monitored during the whole yer in 1999 nd 24. Plnt litterfll ws collected imonthly y mens of 19 open collectors (4.4 cm dimeter) in ech plot locted under ech selected plnt. Smples were dried to constnt weight nd fterwrds the lef litterfll ws selected from the rest, weighed nd nlyzed (lef litterfll represented 7% of the totl litter production). Soil. Three smple cores (1 cm deep, f 5 5 cm) from ech plot were tken in Jnury 25 for soil nlysis. Additionlly, for soil KCl-extrctle-mmonium nd soil KCl-extrctle-nitrte determintion, we repeted the soil smpling in July nd Octoer 24 nd in My 25 in order to detect the sesonl chnges of these soil vriles throughout the yer. Chemicl nlyses Smple preprtion. All the smples were tken to the lortory nd stored t 4 1C until nlysis egn. Soil smples were sieved nd the frction fo2 mm ws nlyzed. In order to nlyze only C nd N in the folir tissue, the leves were wshed with distilled wter s descried y Porter (1986). After ll smples hd een wshed, they were dried in n oven t 6 1C to constnt weight nd then ground in CYCLOTEC 193 (Foss Tector, Högnäs, Sweden) in the cse of the iomsses, or in FRITSCH Pulverisette (Rudolstdt, Germny) in the cse of the soils. Totl C, N nd P in iomss nd totl N in soil. C nd N were nlyzed in ll iomss nd soil smples. For C nd N concentrtion determintion in iomsses nd litter nd for N determintion in soils, 1 2 mg of finesieved smple of iomsses nd litter or 1 12 mg of soil plus 2 mg of V 2 O 5 (s oxidnt) were nlyzed y comustion coupled to gs chromtogrphy (Sté & Grci, 1994). We used Thermo Electron Gs Chromtogrph model NA 21 (C.E. Instruments- Thermo Electron, Milno, Itly). In order to ssess the ccurcy of the iomss digestion nd nlyticl procedures, we used stndrd certified iomss (DC73351). For soil nlyses, the nlyticl precision s verified y prllel nlyses to n interntionl (GSR- 6) stndrd ws etter thn 5%. P ws nlyzed in ll smples of current-yer leves, stems nd litter of E. multiflor nd G. lypum, in lef Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
238 J. SARDANS et l. nd stem of D. pentphyllum nd in roots of G. lypum,in order to clculte the tretment effects on N/P concentrtion rtes of these plnt tissues. The P concentrtions were mesured y ICP-OES (optic emission spectroscopy with inductively coupled plsm) y using model JOBIN IBON JY 38 (Longjumeu, HORIBA Join Ion S.A.S., Frnce). Before the iomss ICP-OES nlyses, n cid digestion of the smples ws crried out with n cid mixture of HNO 3 (6%) nd HClO 4 (6%) (2 : 1) in microwve oven (Smsung, TDS, Seoul, South Kore). Two milliliters of the mixed cid solution were dded to 1 mg of dry iomss for ech smple. The digested solutions were mde up to 1 ml finl volume. During the cid digestion process, two lnk solutions (2 ml of cid mixture without ny smple iomss) were lso nlyzed (Mteo & Sté, 1993). In order to ssess the ccurcy of iomss digestion nd nlyticl procedures, we used stndrd certified iomss (DC73351). Soil orgnic cron. To determine totl soil orgnic C in soil smples we used the Wlkley Blck method (Wlkley & Blck, 1934). Briefly, 1 ml of 1 N potssium dichromte 1 2 ml of concentrted H 2 SO 4 solution were dded to.1 g of sieved nd dried soil, mixed y gentle rottion for 1 min nd heted t 15 1C for 1 min nd fterwrds cooled t room temperture. Then the mixture ws diluted to 2 ml with deionized wter nd 1 ml of phosphoric cid,.2 g of mmonium fluoride nd 1 drops of diphenylmine indictor were dded. Finlly, the excess dichromte ws titrted with Morh slt solution (.5 N SO 4 FeNH 4 nd.1 N H 2 SO 4 ). Soil KCl-extrctle-mmonium nd soil KCl-extrctlenitrte determintion. The ville mmonium nd nitrte ws nlyzed in ll soil smples. We extrcted mmonium nd nitrte with 2 M KCl extrcting wter solution. Both mmonium nd nitrte were ssessed y colorimetric nlyses using spectrophotometer Spectronic 2 Genesys (Spectronic Instruments Inc., Rochester, NY, USA) ginst the regent lnk. We nlyzed mmonium in wter extrcts y modified Berthelot rection (Schinner et l., 1996) nd nitrte y cdmium reduction method (U.S. EPA, 1979). Dt nlyses The plot C nd N contents (PCC nd PNC, respectively) in stems, leves nd totl oveground iomss were clculted y multiplying the concentrtion in the iomss frction y its corresponding iomss per plot. We otined the solute ccumultion of C nd N in the oveground content of ech plot during the period 1999 25 y the difference of the oveground content in 25 nd in 1999 (i.e. in the cse of N, PNC 25 PNC 1999 ). The reltive ccumultion ws then otined, (i.e. in the cse of N y [(PNC 25 PNC 1999 )/ PNC 1999 ] 1). We lso clculted the rtio of C nd N contents in leves with respect to the contents in stems of ech species t the plot level. We estimted NUE in the two dominnt species E. multiflor nd G. lypum in the period 1999 25 y the reltionship etween the increse in iomss during this period (iomss 25 iomss 1999) nd the men nitrogen content during this period [(PNC 25 1 PNC 1999 )/2)] (Srdns et l., 25). To nlyze the results on concentrtions nd ccumultions, we used the STATVIEW 5.1 pckge (Acus concepts Inc., Berkeley, CA, USA). We conducted onewy ANOVAs using the men of ech plot of the mesured vriles, with the different tretments s fctors. We used the Bonferroni Dunn test for the post hoc comprisons. In the cse of C/N, N/P nd C- nd N-lef/stem rtios tht did not follow the ssumptions of ANOVA, we used nonprmetric Kruskl Wllis test. If significnt differences were detected, we conducted comprisons etween control nd drought nd etween control nd wrming tretments using the Wilcoxon Rnk Sum Test. Results Soil moisture nd temperture During the 6 yers of study (1999 25), the verge T increse in the wrming tretment ws 1 1C t2cm height nd.95 1C t soil depth of 5 cm. The drought tretment led to men reduction in soil moisture of 19% with respect to the control tretment nd of 17.9% with respect to the wrming tretment. The wrming tretment incresed the verge soil nd ir T during the 6 yers of study ut did not significntly chnge the soil moisture. Therefore, the wrming effects were minly due to the direct effects of temperture increse, more thn to possile indirect effects through decrese in moisture. A significnt decrese in soil moisture occurred in the drought plots in spring nd utumn riny sesons with the drought tretment running (Fig. 1). Regrding wrming, winter ws the period with greter effect (Fig. 2). In winter, the wrming plots hd soil tempertures 1.2.9 1C higher thn in control soils wheres in summer they were.77.8 1C higher (Fig. 2). Aoveground iomss ccumultion (1999 25) Neither wrming nor drought hd sttisticlly significnt effect on totl ccumulted oveground iomss Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
CLIMATE CHANGE AND N ALLOCATION 239 4 Soil moisture (%, v/v) 3 2 Control Drought Wrming 1 sp su u wi sp su u wi sp su u wi sp su u wi sp su u wi sp su u wi 1999 2 21 22 Yer nd seson 23 24 Fig. 1 Soil moisture (%, v/v) (men SE) during the period Mrch 1999 Jnury 25 for the different tretments. (n 5 3 mens of n 5 three replictes per plot). Asterisk () indictes significnt differences t Po.5 etween control nd drought tretment. Air temperture ( C) Soil temperture ( C t 5 cm) 27 18 9 35 3 25 2 15 1 5 wi Control Drought Wrming sp su u wi sp su u wi sp su u 22 23 24 Yer nd seson Fig. 2 Soil nd ir tempertures ( 1C) (men SE) during the period Jnury 22 Jnury 25 for the different tretments (n 5 three plots). Asterisk () indictes significnt differences t Po.5 etween control nd drought tretment. wi Tle 1 Aoveground iomss ccumultion (kg h 1, men SE, n 5 3 plots) of Eric multiflor nd Gloulri lypum in control, drought nd wrming plots during the period 1999 25 Species Tretment Leves Stems in the period 1999 25 in E. multiflor. However, there ws trend to decrese iomss ccumultion of E. multiflor in drought plots nd to increse iomss ccumultion of G. lypum in drought plots (Tle 1). C nd N concentrtions in iomss nd litter Aoveground E. multiflor Control 29 197 668 358 958 551 Drought 143 47 292 87 435 129 Wrming 381 236 166 563 1447 799 G. lypum Control 382 249 643 153 125 173 Drought 263 28 1712 49 1975 477 Wrming 61 164 123 446 183 61 There were no significnt differences etween tretments. C nd N concentrtions nd C/N concentrtion rtios in the E. multiflor nd G. lypum plnts of different plot tretments did not present differences in Jnury 1999 just efore tretments egn (dt not shown). In E. multiflor fter 6 yers of tretment ppliction, in Jnury 25, wrming hd not chnged N concentrtion in current-yer leves nd stems (Fig. 3) ut hd incresed the N/P rtio in lef litter (23%) (Fig. 3), Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
231 J. SARDANS et l. Concentrtion Accumultion (1999 25) N (%) C/N N/P C (kg h 1 ) N (kg h 1 ) 1.8.6.4.2 14 12 1 8 6 35 28 21 14 7 1 8 6 4 2 12 9 6 3 Leves Leves Leves Eric multiflor chnge tht ws relted to the increse in lef P concentrtions (Tle 2). Drought decresed N concentrtions y 16% in current-yer leves, incresing the corresponding C/N rtio y 17% (Fig. 3). On the contrry, drought incresed N concentrtions in stems y 56% nd s result, it decresed the C/N concentrtion rtio in stems y 25% (Fig. 3). Drought incresed the N/P rtio in stems y 13% (Fig. 3), ecuse of the increse in N concentrtion nd the decrese in P concentrtions (Tle 2). Neither wrming nor drought chnged significntly C nd N concentrtions Stem Control Drought Wrming Stem Biomss frctions Stems Lef-litter Lef-litter Fig. 3 N concentrtions (%, dw) nd C/N nd N/P concentrtions rtio in leves, stems nd lef litter, nd solute ccumultion (kg h 1 ) in leves nd stems (period 1999 25) of Eric multiflor (men SE) under control, drought or wrming tretments. Different letters indicte significnt sttisticl differences etween tretments (Po.5, post hoc Bonferroni Dunn test, ANOVA) (n 5 3 mens of n 5 five replictes per plot). in 1 yer old leves (dt not shown). The N concentrtion vlues in 1 yer old leves (.6.3% in control plnts) were similr to those of current-yer leves. In G. lypum fter 6 yers of tretment ppliction, in Jnury 25, wrming nd drought reduced N concentrtion in leves y 25% nd 19%, respectively, nd incresed N concentrtions in stems y 4% (Fig. 4). As result, wrming nd drought incresed the C/N concentrtion rtio in leves y 31% nd 33%, respectively, nd decresed the C/N concentrtion rtio in stems y 29% nd 27%, respectively (Fig. 4). Wrming incresed N concentrtions y 1% nd decresed the C/N concentrtion rtio y 52% nd 47% in roots (fine nd course, respectively) (dt not shown). Wrming incresed the N/P concentrtion rtio in roots y 165% nd 125% (fine nd course, respectively) nd in lef litter y 45%. However, with wrming, the N/P rtio ws decresed in leves y 43% (Fig. 4), in chnge tht ws relted to the decrese in P concentrtions in the lef litter (Tle 2). In D. pentphyllum fter 6 yers of tretment ppliction, in Jnury 25, wrming hd incresed N concentrtions in stems y 9% (Fig. 5) nd hd incresed the lef N/P rtio with respect to drought plots y 11% (Fig. 5). C nd N ccumultion in oveground iomss nd litter (period 1999 25) Neither wrming nor drought hd sttisticlly significnt effects on the solute ccumultion of C nd N in E. multiflor lthough drought tended to decrese C nd N ccumultion in this species (Fig. 3). In G. lypum, wrming decresed 3.3 kg h 1 the N ccumultion in leves (Fig. 4), ut incresed C nd N ccumultion in stems (y 12 nd 4 kg h 1, respectively) (i.e. y 92% nd 49%), reltive to the respective initil vlues for G. lypum in 1999. Drought incresed C ccumultion (563 kg h 1, 56% reltive to initil vlues) nd N ccumultion (8.1 kg h 1, 87% reltive to initil vlues) in stems of G. lypum (Fig. 4). Wrming nd drought did not significntly chnge the lef nd totl oveground iomss NUE oth in E. multiflor nd in G. lypum (dt not shown). C nd N in soil Wrming incresed (P 5.67) the concentrtion of extrctle mmonium in soil in winter 24 25, wheres it reduced it in spring 24 (Tle 3). No effects of tretments were oserved in soil extrctle mmonium in utumn nd summer 24 (Tle 3). Wrming incresed soil extrctle nitrte in summer nd in utumn 24, wheres no effects of tretments were Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
CLIMATE CHANGE AND N ALLOCATION 2311 Tle 2 P concentrtions (mg g 1 ) in current-yer leves nd lef-litter mesured in Jnury 25 in Eric multiflor nd Gloulri lypum for different experimentl tretments Tretments Species Frction Control Drought Wrming E. multiflor Current-yer leves.125.2.129.19.178.23 Stems.238.41.165.17.175.18 Lef litter.126.14.99.11.45.14 G. lypum Current-yer leves.418.32.331.31.429.23 Stems.256.6.281.11.27.17 Lef litter.336.44.261.23.225.23 Roots.476.7.52.236.392.78 Different letters indicte significnt (Po.5) differences etween tretments. They re itlic type. (n 5 3 mens of n 5 five replictes per plot). oserved in winter 24 nd in spring 25 (Tle 3). Neither wrming nor drought hd ny significnt effect on totl soil N content (Tle 3). Wrming decresed totl orgnic C in soil with respect to drought plots (2.9 vs. 2.1% [w/w], P 5.4) (Tle 3). Discussion N lloction to stems Folir N concentrtions nd totl lef N content decresed in drought conditions in oth dominnt species, G. lypum nd E. multiflor. It lso decresed in G. lypum in response to wrming. These decreses occurred in prllel to the increses in N lloction to stems, nd under wrming in G. lypum lso to roots. The N lloction to stems in the drought plots, oserved minly in G. lypum, cn e relted to mechnism of drought voidnce. The ccumultion of osmoprotectors rich in N such s nitrte, proline nd other -mino N compounds is typicl of shoots in plnts dpted to drought while non-n osmoprotectors re more frequent in leves (Ching & Dndekr, 1995; Bloch & Hoffmnn, 25). Villr-Slvdor et l. (1999) lso reported tht moderte drought resulted in more N concentrting in the shoots of P. hlepensis. The decrese in folir N concentrtions due to wrming hs lso een oserved in different temperte-type non-mediterrnen ecosystems (Tolvnen & Henry, 21; An et l., 25). In Fig. 4 N concentrtions (%, dw) nd C/N nd N/P concentrtion rtios in leves, stems nd lef litter, nd solute ccumultion (kg h 1 ) in leves nd stems (period 1999 25) of Gloulri lypum (men SE) under control, drought or wrming tretments. Different letters indicte significnt sttisticl differences etween tretments (Po.5, post hoc Bonferroni Dunn test, ANOVA) (n 5 3 mens of n 5 five replictes per plot). contrst, in colder regions some studies hve reported increses in N concentrtions in leves under wrming conditions due to the increse of physiologicl nd Concentrtion N (%) C/N N/P Accumultion (1999 25) N (kg h 1 ) C (kg h 1 ) 1.6 1.2.8.4 2 15 1 5 8 6 4 2 16 12 8 4 12 8 4 Leves Leves Gloulri lypum Stem Control Drought Wrming Stem Leves Stems Biomss frctions Lef-litter Lef-litter Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
2312 J. SARDANS et l. N (%) C/N N/P 2 1.5 1.5 6 4 2 8 6 4 2 Control Leves Dorycnium pentphyllum Drought Wrming Stems Leves Stems Biomss frctions Fig. 5 N concentrtions (%, dw) nd C/N nd N/P concentrtion rtios in leves nd stems of Dorycnium pentphyllum (men SE) under control, drought or wrming tretments. Different letters indicte significnt sttisticl differences etween tretments (Po.5, post hoc Bonferroni Dunn test, ANOVA) (n 5 3 mens of n 5 five replictes per plot). Tle 3 Cron nd nitrogen concentrtions in the soil: totl C (%, w/w), totl N (%, w/w), C/N rtio, totl extrctle (.1 M KCl) mmonium nd nitrte (mgg 1 ) Tretment Control Drought Wrming Totl orgnic 2.5.3 2.9.2 2.1.2 Cron Totl nitrogen.154.21.173.23.163.18 C/N rtio 17.6 1.4 2.6 4.1 14.9 2.3 1 Soil NH 4 Spring 1.4.1.94.17.73.19 Summer 1.5.35 1.2.21 1.64.2 Autumn 1.25.1 1.14.14 1.37.17 Winter 1.28.9() 1.51.1() 1.56.12() Soil NO 3 Spring 2.5 1.8 6.2 3.9 3.3 1.4 Summer 7.5 4.3 11.1 3.9 22.1 6.8 Autumn 8.5 2.3 4.1 1.7 18.4 4.2 Winter 17.8 3.5 25.9 6.1 24.8 2.9 Different letters indicte significnt sttisticl differences etween tretments (they re highlighted in itlics) (Po.5, post hoc Bonferroni Dunn test, ANOVA). (When etween rckets Po.1). (n 5 3 mens of n 5 five replictes per plot). photosynthetic ctivity nd of plnt N uptke cpcity (Soussn et l., 1996; Luoml et l., 23). Neither wrming nor drought hd significnt effects on the lef nd stem iomss ccumultion in the period 1999 25; therefore, the decreses in N lef concentrtions nd the increses in N stem concentrtions oserved minly in the drought plots seem not to e due to dilution or concentrtion effect. Other fctors such s chnges in photosynthetic cpcity, the different C nd N lloction etween lef nd stems or the synthesis nd ccumultion of osmoprotectors re the most likely cuses. The increse of N in stems with respect to leves in the two dominnt non-n-fixing species under drought nd of G. lypum under wrming my slow down the N turn-over, ecuse stem turn-over is slower thn lef turn-over. This my fvor the ccumultion of more N in totl plnt iomss. Moreover, this increse of N lloction to stems insted of to leves cn ffect the production cpcity nd plnt survivl of non-n-fixing dominnt species under long-term wrming nd/or drought conditions. In contrst with the lef N concentrtion decrese oserved in the other two shru species, wrming did not decrese lef N concentrtions in D. pentphyllum. This species is shru legume nd wrming could hve enhnced its N fixtion, thus incresing N cpture more thn in the other two species. N vs. P limittion We oserved similr vlues in lef N concentrtions in current-yer nd 1 yer old leves in E. multiflor nd higher vlues of N concentrtion in litterfll in oth species. Folir N concentrtion decresed in wrming conditions without decresing the plnt production cpcity, tht even tended to increse in oth species (Llorens et l., 23, ). All these results indicte tht N is proly not limiting in this ecosystem, nd tht other scrce nutrients, such s P, re proly more limiting thn N. In G. lypum, wrming decresed the N/P concentrtion rtio in leves nd incresed the N/ P concentrtion rtio in litterfll indicting greter ccumultion of P thn N in leves nd greter resorption of P thn N efore lef fll under wrming conditions. Moreover, E. multiflor lso presented higher N/P concentrtion rtio in lef litter. These results confirm tht P is proly the limiting nutrient in this ecosystem with low soil ph nd P vilility (Srdns et l., 26). Thus, the increse in photosynthetic cpcity in G. lypum (Llorens et l., 24) ws ssocited with n increse in P retrnsloction nd P uptke under wrming (Srdns et l., 28) nd to decrese in N/P rtio in leves, ut not to n increse in Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
CLIMATE CHANGE AND N ALLOCATION 2313 either N retrnsloction nor lef N concentrtions. Phosphorus concentrtion is relted to plnt photosynthetic cpcity nd is frequently limiting fctor for growth (Delzon et l., 25), prticulrly in Mediterrnen shrulnds (Hnley & Fenner, 21; Srdns et l., 24, 25, 26). Thus, it seems tht in these clcreous Mediterrnen shrulnds the ecosystem response to climte chnge cn e medited y P vilility more thn y N vilility. Incresing C/N rtio The increse in the C/N lef concentrtion rtio formed in the dominnt non-n-fixing species in response to wrming nd drought is widely oserved phenomenon (Groom & Lmont, 1999; Bussotti et l., 2). These effects were due to decrese in N concentrtions under wrming nd to n increse in C concentrtions nd decrese in N concentrtions under drought. This usully results from n increse in structurl compounds rich in cron nd poor in N, such s lignin, in order to protect lef tissues from wter deficit (Bussotti et l., 2). With further drought, n increse in lef C concentrtions nd stem N concentrtions nd content cn e expected in non-n-fixing shrus in order to improve the plnt s cpcity to withstnd drought. By incresing the lef C/N rtio, wrming nd drought contriuted to decresing the nutritionl qulity of the plnts, ut these chnges were different mong plnt tissues nd shru species of this community. These C/N stoichiometric chnges cn ffect trophic chins nd species community composition (Ngi & Jefferies, 24; Diehl et l., 25). The increses in the lef C/N concentrtion rtio my, for exmple, hve implictions in plnt herivore reltionships ecuse grzing pressure is correlted with folir N concentrtions (Estirte et l., 1994; Pptheodorou & Stmou, 24). Soil C nd N Wheres, in wrming plots there ws tendency to reduce soil C nd increse N vilility, in drought plots there ws tendency to increse soil C concentrtions. These results re relted to n incresed soil microe enzyme ctivity oserved in wrming plots nd to decrese of soil microe ctivity in drought plots (Srdns et l., 26, 28). The increses in soil enzyme ctivity in wrming plots re directly relted to the increse in soil tempertures during winter nd spring nd the decreses in soil enzyme ctivities in drought plots were direct consequence of the decrese in soil moisture throughout the yer (Srdns et l., 26, 28). Wrming incresed soil solule NH 4 1 in winter nd soil extrctle NO 3 in summer nd utumn in ccordnce with the oserved increse in soil enzyme ctivity. In winter, urese ws prticulrly ctive nd in generl there ws greter soil iologicl ctivity oserved in wrming plots (Srdns et l., 28). The decreses in soil extrctle NH 4 1 in spring oserved in wrming plots re relted to higher N plnt cpture. This ws due to the enhncement of growth oserved in wrming plnts in the growing seson (spring) of some yers during the period 1999 25 (Llorens et l., 24; Prieto, 27). The increses in soil extrctle NO 3 concentrtions in wrming plots in summer nd in utumn were expected, ecuse temperture is positively relted to nitrifiction in temperte non-mediterrnen ecosystems (Ryn et l., 1998; Wng et l., 26). C nd N iomss ccumultion 1999 25: differences etween species Neither wrming nor drought hd significnt effects on the NUE of E. multiflor nd G. lypum in the period 1999 25. The oserved results were not consistent with those oserved in some non-mediterrnen ecosystems. For exmple, An et l. (25) in wrming experiment in North Americn grsslnd oserved tht wrming decresed the NUE. The different ptterns of growth etween temperte grsses nd Mediterrnen shrus, the different experimentl wrming conditions (chieved y infrred heters) nd the different climte descried y An et l. (25) cn ccount for the disprity. Neither wrming nor drought chnged the C nd N ccumultion in the oveground iomss of E. multiflor s result of the counterlnce etween chnges in folir N concentrtion nd in iomss growth. In contrst, in G. lypum, wrming decresed N ccumultion in leves y sustntil decrese in lef N concentrtion, wheres drought incresed C nd N stem ccumultion y n increse in N concentrtion in stems nd y tendency to increse stem iomss ccumultion. In G. lypum, the effects of wrming on N concentrtions were ssocited to greter plsticity to llocte N to different plnt tissues nd not to n solute increse in N uptke nd ccumultion in oveground iomss, ecuse the sum of N ccumulted in lef iomss plus tht ccumulted in stem iomss ws similr in the control s in drought nd wrming plots. These different effects of wrming on C nd N ccumultion cpcity in the two dominnt non- N-fixing shru species were relted to the previously oserved differences in growth cpcity, lef net photosynthetic rtes nd retrnsloction in response to the tretments (Llorens et l., 23, ). Journl compiltion r 28 Blckwell Pulishing Ltd, Glol Chnge Biology, 14, 234 2316
2314 J. SARDANS et l. Regrding drought plots, G. lypum ccumulted more N in stems nd less N in leves in drought thn in control plots; however, these chnges were not oserved in E. multiflor. E. multiflor ws the most ffected species, ecuse drought reduced its photosynthetic cpcity nd stem length growth more when compred with G. lypum (Llorens et l., 24; Prieto, 27). G. lypum hs greter cpcity to resist lower wter potentils thn E. multiflor when wter is scrce, nd presents greter wter sorption, trnspirtion, photosynthetic rtes nd growth when wter is ville (Llorens et l., 23). Thus, these different species hve different cpcity to respond to wrming nd drought. This hs een oserved in similr experiments conducted in other ecosystems, minly in colder temperte climtes or in orel climte (Tolvnen & Henry, 21; Kudo & Suzuki, 23). Given the glol wrming scenrio, this cpcity to respond my end up chnging species community composition nd structure in the long term. In fct, chnges in the community species diversity in this Mediterrnen shrulnd re lredy strting to occur (Peñuels et l., 27; Prieto, 27). Acknowledgements This reserch ws supported y the Europen project NEU NITROEUROPE (GOCE17841), y the Spnish Government projects CGL24-142/BOS nd CGL26-425/BOS, the Ctln Government SGR 25-312, the Europen project ALARM (Contrct 56675) nd Fundción BBVA 24 grnt. References An Y, Wn S, Zhou X, Suedr AA, Wllce LL, Luo Y (25) Plnt nitrogen concentrtion, use efficiency, nd contents in tllgrss pririe ecosystem under experimentl wrming. Glol Chnge Biology, 11, 1733 1744. Beier C, Emmett B, Gundersen P et l. (24) Novel pproches to study climte chnge effects on terrestil ecosystems in the field: drought nd pssive nighttime wrming. Ecosystems, 7, 583 597. Bloch D, Hoffmnn C (25) Sesonl development of genotypic differences in sugr eet (Bet vulgris L.) nd their interction with wter supply. Journl of Agronomy nd Crop Science, 191, 263 272. 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