Contribution of Medicago sativa to the productivity and nutritive value of forage in semi-arid grassland pastures

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1 Received: 1 Septemer 2016 Revised: 24 Jnury 2017 DOI: /gfs ORIGINAL ARTICLE Contriution of Medicgo stiv to the productivity nd nutritive vlue of forge in semi-rid grsslnd pstures R. Kli 1,2 T. H. Bell 1,3 C. Hmel 2,4 A. Iws 2 M. P. Schellenerg 2 M. St-Arnud 1 1 Biodiversity Centre, Institut de Recherche en Biologie Vegetle, Universite de Montrel nd Jrdin otnique de Montrel, Montrel, Queec, Cnd 2 Swift Current Reserch nd Development Centre, Agriculture nd Agri-Food Cnd, Swift Current, SK, Cnd 3 School of Integrtive Plnt Science, Cornell University, Ithc, NY, USA 4 Queec Reserch nd Development Centre, Agriculture nd Agri-Food Cnd, Queec City, Queec, Cnd Correspondence M. St-Arnud, Biodiversity Centre, Institut de Recherche en Biologie Vegetle, Universite de Montrel nd Jrdin Botnique de Montrel, Montrel, Queec, Cnd. Emil: mrc.st-rnud@umontrel.c Funding informtion Nturl Sciences nd Engineering Reserch Council of Cnd; Agriculture nd Agri-Food Cnd Astrct The inclusion of legumes in semi-rid ntive grsslnds my promote the productivity nd nutritive vlue of forge. This study ws designed to ssess the effect of legumes (the introduced legume Medicgo stiv or the ntive legume Dle purpure) nd soil P fertility (ddition of 0, 50, or 200 P 2 O 5 kg/h t seeding) on the dry mtter nd nutrient content of ntive grsses mixtures, compred with the commonly used introduced forge grss Bromus ieersteinii grown with M. stiv. Plnt hrvests were performed in Septemer 2008, July 2009 nd Septemer Plnts nutrient content, d 15 N vlue nd dry mtter were nlysed. Results show tht the M. stiv enhnced the N nd P concentrtions of ntive grss mixtures erly in the summer, s well s the N concentrtion in Boutelou grcilis in lte summer of the driest yer, The higher AM fungl diversity promoted y M. stiv ws positively correlted with the dry mtter nd nitrogen uptke of M. stiv nd with the P concentrtion of ntive grsses, in erly summer. Overll, this study shows tht M. stiv promoted eneficil AM fungl tx nd improved forge production in the semi-rid priries. KEYWORDS AM fungl diversity, forge dry mtter, introduced grss, legumes, ntive grss mix, nitrogen, phosphorus, Priries 1 INTRODUCTION Ntive grsslnds ply importnt functionl roles in semi-rid regions. They provide high-qulity forge for livestock grzing, reduce soil erosion, enhnce soil orgnic cron sequestrtion nd limit the effects of drought on iomss production, in ddition to hosting iodiversity, while mintining hitt spce for ntive wildlife (Biley, McCrtney, & Schellenerg, 2010; Jefferson, McCughey, My, Woosree, & McFrlne, 2004; Muir, Pitmn, & Foster, 2011; Richrds, Chmers, & Ross, 1998; Weigelt, Weisser, Buchmnn, & Scherer-Lorenzen, 2009). In generl, ntive grss species yield less iomss nd fewer seeds thn high-yielding introduced grss species (Kilcher & Loomn, 1983; Lwrence & Rtzlff, 1989). Furthermore, their seeds re costly nd their vilility is limited. The ntive species lso tend to invest more energy in survivl thn in seed production (Smith & Smith, 1997). Additionlly, ntive species re chrcterized y lower seed viility nd germintion nd y slow rtes of estlishment, compred with introduced species (Kusler, 2009). In contrst, some studies hve shown tht monocultures of ntive grsses in semi-rid regions cn provide higher forge yield thn monocultures of introduced grsses (Jefferson, Wetter, & Wrk, 1999; Willms, Ellert, Jnzen, & Douwes, 2005). It ppers tht the generlly higher forge yield of introduced species my depend on soil fertility nd hrvesting time (Jefferson, Iws, Schellenerg, & McLeod, 2005). For exmple, the productivity of ntive species ws higher thn tht of introduced species in n unfertilized grsslnd, wheres introduced species produced more iomss when fertilizer ws dded (Jefferson et l., 2005; Johnston, Smith, Lutwick, & Grss Forge Sci. 2018;73: wileyonlinelirry.com/journl/gfs 2017 John Wiley & Sons Ltd 159

2 160 KLABI ET AL. Smolik, 1968; Knowles, 1987). Additionlly, some ntive species hve een shown to produce more herge thn introduced species in dry periods (Knowles, 1987), suggesting dpttion to the environmentl conditions rought y climte chnge (Kusler, 2009). Introduced species my displce ntive species nd reduce their locl iodiversity (Christin & Wilson, 1999; Wilson & Belcher, 1989). Such displcement cn e fcilitted y environmentl chnges or hitt disturnce fvouring the growth of introduced species over tht of ntive species (Gurevitch & Pdill, 2004; Vitousek, Antonio, Loope, & Westrooks, 1996). The introduced species my lso impir ecosystem stility nd nutrient cycling. Christin nd Wilson (1999) found similr short- nd long-term soil resource vilility in field seeded with ntive grsses nd in the undistured ntive pririe, wheres resources were less ville in the field seeded with the introduced grss Agropyron cristtum (crested whetgrss), suggesting negtive effect of A. cristtum on resource vilility. Consequently, oth privte nd pulic orgniztions tht re responsile for the mngement nd preservtion of ntive grsslnds in the Cndin priries hve restricted the use of introduced species, nd supported the re-seeding of distured res with ntive species (Asy, Horton, Jensen, & Plzzo, 2001; Jefferson et l., 2005). Over time, rising cttle on pstures seeded with ntive grsses is sustinle nd encroches less upon neighouring ntive priries thn do introduced species (Jefferson et l., 2004). Recently, severl studies hve indicted tht seeding ntive species in wellmnged systems cn produce lrge mounts of high-qulity forge for livestock grzing erly, ut lso lte, in the growing seson (Jefferson et l., 2002; Schellenerg, Biligetu, & Iws, 2012), s opposed to introduced species which re dormnt under the dry conditions of mid-summer (Knowles, 1987). In ddition, forge grsses tend to e more productive in mixture thn s monocultures (Schellenerg, 2008; Tilmn et l., 2001), especilly when the mixtures contin oth wrm- nd cool-seson grsses. Wrm- nd coolseson grsses rech mturity t different times in the growing seson nd mixed-grss pstures cn support longer grzing periods (Schellenerg et l., 2012). Cool-seson grsses re generlly seeded in forge pstures. Their productivity is high in spring, ut decreses over the summer months. In contrst, wrm-seson grsses rech mturity in summer (Aougendi, 1995) nd hve the potentil to increse the feeding vlue of ntive forge mixtures for lte-seson grzing (Schellenerg et l., 2012). Integrting N-fixing legumes into ntive grss mixtures my help to sustin nd even increse herge production in unfertilized pstures (Nyfeler et l., 2009; Temperton, Mwngi, Scherer-Lorenzen, Schmid, & Buchmnn, 2007). This prctice cn lso increse the totl protein nd nitrogen (N) content of grss legume mixtures, s well s the rte of digestion y grzing nimls (Fgererg, 1988; Frme & Hrkess, 1987; Schultz & Stuendieck, 1982; Suter et l., 2015). Grsses growing with neighouring legumes cn receive lrge mounts of fixed N, which often improves their N nutrition nd vigour (Boller & N oserger, 1987; Gusch et l., 2011; Høgh-Jensen & Schjoerring, 1997; Nyfeler, Huguenin-Elie, Suter, Frossrd, & L uscher, 2011). Legumes grown longside non-leguminous plnts fix more N 2 thn legumes grown in monoculture (Crlsson & Huss- Dnell, 2003; Corre-Hellou, Fustec, & Crozt, 2006; Fustec, Lesuffleur, Mhieu, & Cliquet, 2010). Soil N is preferentilly tken up y the grsses, nd legumes fix N for their needs (Temperton et l., 2007). In dry pririe soils, where nutrients re often limited, the ddition of phosphorus (P) to mixtures contining legumes nd grsses my promote N fixtion y legumes (Divito & Sdrs, 2014; Hyt, Ali, Siddique, & Chth, 2008; Isrel, 1987) nd fcilitte plnt estlishment, ecuse ntive species my estlish more slowly thn introduced species. In southwest Ssktchewn, the domesticted N-fixing legume Medicgo stiv L. is commonly included in forge systems, due to its high nutritive vlue nd rpid growth in semi-rid regions. This introduced legume hs mny dvntges nd, in prticulr, drought resistnce. The Cndin pririe is chrcterized y dry conditions in the second hlf of the growing seson (Biley et l., 2010). Most plnts re ffected y lte summer drought nd go into dormncy, ut due to its very deep root system, M. stiv cn remin ctive. Additionlly, M. stiv hs high potentil for tmospheric N fixtion nd cn increse the productivity nd qulity of seeded pstures (Popp, McCughey, Cohen, McAllister, & Mjk, 2000). Dle purpure Vent. is lso considered to e n importnt forge legume. This legume is ntive to the Cndin priries, nd it hs higher protein content thn M. stiv, s well s incresed pltility for grzing livestock (Schellenerg & Bnerjee, 2002). Dle purpure is wrm-seson plnt, mturing lter in the seson thn M. stiv nd potentilly providing more nutritious feed for cttle lte in the growing seson. Incorporting legumes in low-input forge systems reduces the need for expensive nd environmentlly dmging industril N-fertilizers (Deutsch, Khle, & Voss, 2006; Foley et l., 2005). In semi-rid grsslnds, the plnt roots nd surrounding soils re colonized y high diversity of fungi including the eneficil rusculr mycorrhizl (AM) fungi (Kli et l., 2015; Porrs-Alfro & Bymn, 2007; Porrs-Alfro, Herrer, Ntvig, Lipinski, & Sinsugh, 2011), which enhnce nutrient cquisition (e.g., P, N, Zn etc.; Smith & Red, 2008; Smith & Smith, 2012) nd improve plnt drought tolernce (Gupt & Kumr, 2000; Jeffries, Gininzzi, Perotto, Turnu, & Bre, 2003; Surmnin & Chrest, 1998). We recently found tht the diversity of AM fungi ws incresed y the ddition of legumes in semi-rid ntive grsslnd (Kli et l., 2015), which in turn my impct forge production. High AM fungi diversity cn hve positive influence on plnt diversity nd productivity (vn der Heijden, Boller, Wiemken, & Snders, 1998) ecuse plnts cn select the most eneficil AM fungi (Kiers et l., 2011). We conducted field experiment in the Cndin priries to determine the effect of P fertiliztion nd legume introduction (either M. stiv or D. purpure) on the dry mtter nd nutrient content of ntive grss mixture. The introduced grss Bromus ieersteinii Roem. & Schult., which is the commonly used domesticted forge species in semi-rid regions of Cnd, ws used s the point of reference. The inclusion of legumes in grss mixtures is n interesting

3 KLABI ET AL. 161 low-cost option for sustining the productivity of unfertilized pstures from yer to yer. We hypothesized tht including legumes within the ntive grss mixture, or long with the introduced B. ieersteinii, would increse forge production nd nutritionl content. Specificlly, we tested (i) whether N-fixing legumes contriute to incresed dry mtter, N concentrtion nd totl N uptke y the totl plnt community, nd in prticulr y the mix of ntive grsses, nd (ii) whether legumes increse the N concentrtion in the tissues of different species of ntive grss t the end of growing seson. Plnt growth prmeters were estimted t the end of the growing seson (Septemer) over two consecutive yers, s well s during the vegettive growth stge (July) of the second yer of the experiment. 2 MATERIALS AND METHODS 2.1 Site description The study took plce in 2008 nd 2009, in field experiment estlished in 2006 on lomy Brown Chernozem soil with ph of 6.8 t the South Frm of the Semirid Pririe Agriculturl Reserch Centre in Swift Current, Ssktchewn, Cnd ( N W). The soil contined 9.23 mg/kg of NO 3 -N, mg/kg of PO 4 -P nd mg/kg of K, on verge, t plnt estlishment. The experimentl site ws seeded with rley (Hordeum vulgre L.) in Totl nnul precipittion t the site ws mm in 2008, which ws higher thn the long-term verge, nd mm in 2009, which ws lower thn verge, nd the men nnul temperture ws C in 2008 nd C in Precipittion during the growing seson (1 April to 30 Septemer) of the study yers ws ove verge ( mm) in 2008 nd elow verge ( mm) in The men temperture ws C in 2008 nd C in 2009 ccording to wether sttion locted within 1000 m of the study site (Figure S1). All the wether dt were recorded y the wether sttion of the Swift Current Reserch nd Development Centre, Swift Current, SK. 2.2 Experimentl design The experimentl tretments were the fctoril comintion of four plnt mixtures (descried elow) nd three concentrtions of soil P ddition. Tretments were rrnged in four complete rndomized locks. In totl, there were forty-eight m plots, seprted y 1.83 m in ll directions. Plnt mixtures were (i) seven ntive grsses, (ii) the sme ntive grsses + the domesticted legume Medicgo stiv L. (lflf), (iii) the sme ntive grsses + the ntive legume Dle purpure Vent. (purple pririe clover) nd (iv) the introduced forge grss Bromus ieersteinii Roem. & Schult. (medow rome) + M. stiv. The seeds of legumes were coted with commercil inoculnt of their specific rhizoil symiont ccording to the mnufcturer s direction. Medicgo stiv received Nitrgin Gold nd D. purpure, Nitrgin type F, two pet-sed inoculnts (Monsnto Biog TM ). The ntive grss mix contined five cool-seson species nd two wrm-seson species. The scientific nd common nmes, life cycle nd growing seson of the different species re listed in Tle S1. The grss nd legume seeds were developed provided y Ducks Unlimited Cnd Inc. (Ssktchewn Provincil Office). The totl seeding rte ws 11.1 kg/h for the mixture of seven ntive grsses growing lone, 9.2 kg/h for the mixtures of seven ntive grsses growing with legumes nd 16.1 kg/h for the mixtures of B. ieersteinii nd M. stiv. The seeding rte ws determined sed on the trget seed density per unit re (kg/h), the numer of seeds per kg nd pure live seed (PLS) clculted y multiplying the percentge of germintion y the purity of the seed, nd these prmeters vry mong species nd plnt mixture used. The seeding rte nd the numer of seeds per kg for ech species in the plnt mixture re provided in Tle S1. Row spcing ws cm, nd the seeding depth ws ~0.5 cm. The seeds were sown in My 2006 using selfpropelled hydrosttic plot seeder (Semirid Pririe Agriculture Reserch Center, Design l, Swift Current, SK). At the time of seeding (i.e., 2 yers prior to initil smpling), plnt mixtures received 0, 50 or 200 kg/h of P 2 O 5 s triple super-phosphte, which ws rked mnully through the top 10 cm of soil. On July 2006, Roundup Renew (glyphoste) ws wick-pplied ove the young crop plnts for the control of tll weeds. Plots were not grzed ut were cut with hy conditioner t the end of July or eginning of August of ech yer, fter the qudrts were smpled. The stule remining ws ~10 cm tll to mimic the residul height under grzing. 2.3 Plnt smpling Plnts were smpled t the end of ech growing seson (i.e., the lst week of Septemer in 2008 nd 2009) nd in the first week of July 2009, which ws the time of ctive plnt growth in tht yer. Dry mtter ws determined y clipping the plnts t ground level from one 0.25 m 2 qudrt tht ws plced rndomly within ech plnt mixture. In 2008, the totl dry mtter ws mesured for plnt mixtures, wheres in 2009, the dry mtter of legumes nd grsses ws determined independently. The plnt mteril tken within these qudrts ws dried t 40 C for 3 dys nd weighed. Plnt dry mtter is expressed s kg/h. At the end of the growing seson in 2008 nd 2009, we determined visully the proportion of ech plnt species in the stnds except E. lnceoltus ssp. Lnceoltus nd P. smithii, which were considered together s it ws not possile to determine their individul contriutions ccurtely. The proportion of ech ntive grss species ws expressed s percentge of ll grsses, even in plots where legume ws lso present. In ddition, over the two consecutive yers, ech individul ntive grss, ech legume species nd B. ieersteinii were collected seprtely from ech plot nd dried t the end of the growing seson; the exceptions were E. lnceoltus ssp. lnceoltus nd P. smithii, which were collected together due to their similr morphology preventing ccurte seprtion. Dry plnt mterils were ground, nd

4 162 KLABI ET AL. susmples of ground plnt mteril were digested with H 2 SO 4 /Se/ N 2 SO 4 (Vrley, 1966). The concentrtion of N (Noel & Hmleton, 1976) nd P in plnt tissues (Milury, Stck, & Doll, 1970) ws determined using segmented flow uto nlyser (Technicon, AAII System, Trrytown, NY). The uptke of P nd N y plnts ws determined y multiplying plnt dry mtter yield y the concentrtion of ech nutrient in plnt tissues. A second susmple of ground plnt mteril ws finely ground to powder in ed miller nd g susmple ws plced in tin cpsules for isotopic 15 N/ 14 N nlyses using Crlo Er 17 NA1500 elementl CN nlyser coupled to n Optim isotope rtio mss spectrometer, to evlute the contriution of iologicl N 2 fixtion to herge N concentrtion nd N uptke. The nturl undnce of 15 N in plnt tissues cn e expressed s d 15 N vlue, which llows us to estimte the contriution of tmospheric N to the N nutrition of legumes nd grsses growing in mixture (Hogerg, 1997). The d 15 N vlue denotes the devition in & of the smple from the rtio of 15 N/ 14 N in tmospheric N 2 nd is clculted with the Eqution 1 (Roinson, 2001): d 15 N ¼ R smple R tm =Rtm 1000 (1) where R smple is the 15 N/ 14 N isotope rtio of the smple nd R tm is the 15 N/ 14 N rtio of tmospheric N 2 (R tm = ). The d 15 Nof tmospheric N 2 y definition is 0&. The d 15 N of plnt species cn vry depending on the surrounding plnt mixture. The d 15 N of N 2 -fixing legumes is usully much lower thn tht of other plnt species, s it is close to the tmospheric d 15 N (round 0&), s much of their N is derived from N fixtion (Hogerg, 1997). The d 15 N of grsses growing without legumes is usully higher, reflecting the higher d 15 N of the soil (Sherer & Kohl, 1986). Thus, the lower d 15 N of non-n2-fixing plnt (such s grss) when it is grown longside N 2 -fixing plnts (such s legumes) thn when it is grown lone indictes trnsfer of fixed N 2 from the N 2 -fixing plnts to the non-n2-fixing plnt (Temperton et l., 2007). The 15 N nturl undnce method of quntifiction of iologicl nitrogen fixtion (%BNF) in legumes hs limittions (Boddey, Peoples, Plmer, & Drt, 2000; Gehring & Vlek, 2004; Hogerg, 1997). The %BNF in legumes is clculted sed on the d 15 N of the N 2 -fixing legumes, the d 15 N of the non-n 2 -fixing plnts ville soil N nd the d 15 N tm of legumes when relying only on tmospheric N (Sherer et l., 1983). This method ssumes tht the non-n 2 -fixing plnts growing without legumes nd the N 2 -fixing legumes will cquire soil N from the sme source with the sme 15 N undnce. In this study, the legumes nd the non-n 2 -fixing plnts (mixes of ntive grsses species without legumes) hve different rooting depths nd thus my use soil N sources with different isotopic composition (Boddey et l., 2000). The d 15 N tm of legumes mesured in the study of Kli et l. (2014) ws higher thn the d 15 N of the N 2 - fixing legumes growing in the field in most of the cses, resulting in overestimtion of %BNF (more thn 100%). The use of single strin of rhizoium is lso importnt in ccurte determintion of % BNF, ut this cn e chllenging ecuse legumes plnted in the field for more thn 2 yers cn e nodulted y other strins of rhizoium (Boddey et l., 2000). Despite these cvets, plnt d 15 N provides indictions on the nitrogen-fixing ctivity of the legumes (Hogerg, 1997; Temperton et l., 2007), especilly when considered concurrently with plnt N concentrtion, nd llowed us to estimte the reltive contriution of tmospheric N to the N nutrition of legumes nd grsses growing in mixture. 2.4 Soil smpling Soil smples were collected in August 2009 to determine soil nutrient vilility. Smples were tken from two different soil depths (0 15 nd cm) using soil corer (5 cm Ø 9 15 cm length). Two core smples were rndomly tken from ech of the two soil lyers in ech plot nd pooled to produce single composite smple for ech depth nd plot. Plnt ville NO 3 -N ws determined y KCl extrction (Mynrd & Klr, 1993) nd plnt ville PO 4 -P ws determined y extrcting 2.5 g soil with NHCO 3 (Olsen, Cole, & Wtne, 1954). 2.5 AM fungl diversity The effect of plnt ssemlge nd soil P concentrtions on the communities of rusculr mycorrhizl fungi t the experimentl site ws nlysed nd reported in Kli et l. (2015). We used these AM fungl diversity dt in the present work to test the reltionships etween the Shnnon diversity of AM fungi nd plnt dry mtter nd nutrient content. Briefly, totl DNA ws extrcted from 0.26 g soil cores tken from the 0 to 15 cm soil depth of ll plots except those fertilized with 50 kg/h of P 2 O 5. A 18S rrna gene frgment ws mplified using the primer set AMV4.5NF nd AMDGR (Sto, Suym, Sito, & Sugwr, 2005) nd then sequenced using one hlf sequencing plte on the 454 GS FLX Titnium sequencing pltform (Roche, Brnford, CT, USA). Qulity processing of AM fungl 18S rrna gene ws performed in Mothur (v ) (Schloss, Gevers, & Westcott, 2011). The sequence dt generted were deposited in the NCBI Sequence Red Archive nd re ville under the SRA project numer SRP Shnnon diversity ws clculted to compre AM fungi diversity etween tretments. More detils re ville in Kli et l. (2015). 2.6 Sttisticl nlysis Sttisticl nlyses were conducted using JMP 9 softwre (SAS Institute Inc.). Tretment effects were nlysed seprtely t ech time point. The normlity of the residuls ws tested using the Shpiro Wilk test, nd non-norml dt were log-trnsformed prior to nlysis. The effect of plnt mixture, soil P ddition nd their interction on totl dry mtter, nutrient uptke nd nutrient concentrtion for the whole plnt iomss t ech smpling time were tested y twowy ANOVA. Blocks were included s rndom effect. ANOVAs were lso performed seprtely for grss nd legume dry mtter in One-wy ANOVA ws used to compre the d 15 N nd N concentrtion in the tissues of legume species to those in tissues of

5 KLABI ET AL. 163 ntive grss mixture grown without legumes t ech time of smpling in order to show whether the legumes were ctively fixing nitrogen. The effect of tretments on soil nutrient vilility, plnt nutrient concentrtions, d 15 N of plnt species nd of comined ntive grsses nd on the proportion of ntive grss species ws lso tested y two-wy ANOVA. Overll plnt dry mtter ws compred etween different smpling times using one-wy ANOVA. The significnce of differences etween mens ws tested with the Tukey s rnge test. The Student s t-test ws lso used to compre the men proportion of ech species mong yers. The d 15 N nd the nutrient concentrtion of the introduced grss B. ieersteinii mesured only in Septemer 2008 were compred with those of the ntive grsses growing lone, using the Student s t-test. Regression nlysis ws conducted to determine the reltionship etween plnt dry mtter nd nutrient content in different plnt mixtures with AM fungl diversity dt pulished in Kli et l. (2015) t ech smpling time. 3 RESULTS 3.1 Plnt dry mtter Totl dry mtter ws highest in July 2009 ( kg/h) nd ws higher t the end of the growing seson in 2008 ( kg/h) thn in 2009 ( kg/h) (Figure 1). Plnt dry mtter lso vried y plnt mixture, ut only in 2009 (Figure 1). At the end of the growing seson in 2008, much wetter yer, the dry mtter of plnt mixtures with legumes ws similr to the dry mtter of the ntive grss mix grown lone. In 2009, which ws the dry yer, the mixtures contining M. stiv hd the highest overll plnt dry mtter in July. However, y the end of the growing seson, the dry mtter of the ntive grss mix + M. stiv ws similr to ntive grss mixes growing without legumes, while the dry mtter of B. ieersteinii + M. stiv ws higher. When the dry mtter yield of grsses ws mesured lone, the dry mtter yield of the ntive grss mix ws not ffected y legumes in either July or Septemer of 2009, nd ws not significntly different from tht of B. ieersteinii (Figure 1). The growth of M. stiv ws lwys high nd significntly higher thn tht of D. purpure (Figure 1). There ws no effect of soil P fertility on forge dry mtter. 3.2 Species proportions The reltive undnce of some ntive grsses ws significntly ffected y yer (Tle S2). The proportions of the cool-seson grsses Nssell viridul nd the wrm-seson grss Boutelou grcilis were significntly higher in 2008 thn in They ccounted for ~30% nd ~20%, respectively, of ll the grsses in the ntive grss mixes in In ddition, the proportions of the wrm-seson species Schizchyrium scoprium nd D. purpure were generlly very low in 2008 nd even lower in In contrst, the cool-seson grss E. trchyculus susecundus nd the pool of the indissocile cool-seson grsses E. lnceoltus ssp. Lnceoltus nd P. smithii were significntly more undnt in 2009 thn They represented 44% nd 28% of ll the grsses in the ntive grsses mixes in 2009 nd 2008 respectively. The undnce of M. stiv, the introduced B. ieersteinii nd the cool-seson E. cndensis were similr cross yers. The reltive undnce of most ntive grsses ws not ffected y the presence of legume. The wrmseson grss S. scoprium, which ws the exception, ws reduced in the presence of M. stiv in Evidence of nitrogen fixtion The legume species growing in mixture hd higher tissue N concentrtion nd lower d 15 N (round 0&) vlue thn in tissues of ntive grsses growing without legumes (Tle S3), indicting tht the legumes were clerly different from grsses nd confirming ctive N fixtion y legumes (Hogerg, 1997; Temperton et l., 2007). Additionlly, soil P fertility differentilly ffected the d 15 N vlue of the two legume species in July 2009 (Tle S4). High soil P reduced the d 15 NofD. purpure compred with tht of M. stiv. In contrst, the d 15 NofD. purpure ws higher thn tht of M. stiv grown with B. ieersteinii t low soil P concentrtions. 3.4 N uptke, N concentrtion nd isotopic composition of d 15 N N concentrtion nd N uptke y the totl plnt dry mtter were influenced y plnt species mixture in oth yers nd were higher in the ntive grsses grown with M. stiv thn with D. purpure (Figure 1c, d). In 2009, the tissue N concentrtion nd iomss of M. stiv were higher thn tht of D. purpure t ech smpling time, leding to the highest N uptke y forge plnts over time. However, mixing grsses with legume did not generlly increse the totl N uptke y grsses, lthough M. stiv did contriute to incresed N concentrtion in the tissues of comined ntive grsses in July The d 15 N in ntive grss tissues ws not reduced y the presence of this legume. In contrst, in the tissues of introduced grsses grown with M. stiv, the d 15 N vlues were lower thn for the ntive grsses growing lone, ut only in Septemer 2008 nd July 2009 (Tle 1). At the end of the growing seson, most ntive grss species did not enefit from the presence of legumes, except for the cool-seson grss E. cndensis nd the wrm-seson grss B. grcilis; lthough this vried with yer, soil P fertility nd legume species (Tle 2). In 2008 nd t low soil P, the concentrtion of N in E. cndensis tissue ws enhnced in the presence of D. purpure, compred with this grss when grown without legumes. This effect ws not seen t the higher P levels or in In contrst, M. stiv enhnced the N concentrtion of B. grcilis tissues, irrespective of soil P fertility, in 2009 ut not in The d 15 N estimted for different species of ntive grsses ws not decresed y the presence of legumes t the end of the growing seson. Contrrily, M. stiv incresed the d 15 NofS. scoprium t the end of the growing seson in 2009 compred with tht of other ntive species of grsses (Tle 3).

6 164 KLABI ET AL. () 10,000 Dry mtter (kg/h) z Y X c c Septemer 2008 July 2009 Septemer 2009 () July 2009 Septemer 2009 July 2009 Septemer 2009 (c) N uptke (kg/h) (d) N concentrtion (kg/h) Totl dry mtter Grss dry mtter Legume dry mtter 160 Ntive grsses Ntive grsses + M. stiv 120 Ntive grsses + D. purpure B. ieersteinii + M. stiv Septemer 2008 July 2009 Septemer 2009 July 2009 Septemer 2009 July 2009 Septemer Septemer 2008 July 2009 Septemer 2009 Septemer 2008 July 2009 Septemer 2009 Septemer 2008 July 2009 Septemer 2009 Totl dry mtter Grss dry mtter Legume dry mtter FIGURE 1 () Totl dry mtter mesured in Septemer in 2008 nd 2009, nd once in July 2009, nd () the totl dry mtter of grsses or legumes mesured seprtely in 2009, s influenced y plnt mixtures t ech time of smpling, (c) N uptke nd (d) N concentrtion in totl dry mtter, mesured t different times of smpling nd in dry mtter of grsses or legumes mesured seprtely in 2009, s influenced y plnt mixtures t ech time of smpling. In 2008, the N concentrtion in B. ieersteinii nd legume species ws lso determined seprtely. Significnce ws tested y ANOVA. Vlues re mens SE. Within ech dte of smpling, significnt differences (p.05, n = 12) etween plnt mixtures re indicted y different letters over columns (,, c) nd significnt differences (p.05, n = 48) of totl dry mtter etween times of smpling re indicted y different letters over groups of columns (X, Y, Z) 3.5 P uptke nd P concentrtion In Septemer 2008, totl phosphorus uptke ws influenced y the interction etween soil P concentrtion nd plnt mixture (Tle 4). High soil P promoted totl P uptke y plnt mixtures tht contined M. stiv compred with tht of mixtures of ntive grsses growing without legumes, ut the concentrtion of P estimted in the tissues of those mixtures ws similr mong tretments. In July 2009, the highest P uptke nd concentrtion vlues were oserved in mixtures contining M. stiv, irrespective of soil P fertility, ut this effect disppered y the end of the growing seson in the sme yer. Among legume species, the P uptke y M. stiv ws lwys higher thn tht of D. purpure, nd the concentrtion of P in legume tissues ws influenced y tretments (Tle 4). In Septemer 2008, the P concentrtion in legumes growing with ntive grsses

7 KLABI ET AL. 165 TABLE 1 Nitrogen isotopic rtio (d 15 N), P concentrtion nd P uptke in grss tissues of the ntive grss mix or in B. ieersteinii s influenced y plnt mixtures t different times of smpling Grsses dry mtter d 15 N* P concentrtion in grsses tissues (g/kg)* P uptke y grsses (kg/h)* Sept July 2009 Sept Sept July 2009 Sept Sept July 2009 Sept Ntive grsses Ntive grsses + M. stiv Ntive grsses + D. purpure B. ieersteinii + M. stiv p vlue.008 < < < Sept.: Septemer. *Within ech column, mens re significntly different (p.05, ANOVA; n = 12) when followed y different letters (, ) ccording to Tukey s rnge tests or to the Student s t-test (B. ieersteinii vs ntive grsses in 2008). Bolded vlues re significnt. In 2008, the totl plnt dry mtter ws estimted, so the dry mtter of grsses or legumes lone is not ville, except for the d 15 N nd P concentrtion in B. ieersteinii tissues tht ws determined seprtely. TABLE 2 Nutrients concentrtion (g/kg) in the tissues of E. cndensis nd B. grcilis s influenced y the presence of legumes nd soil P concentrtion over 2 yers of smpling N concentrtion in E. cndensis* Septemer 2008 Low soil P Middle soil P High soil P Men No legumes 4.9 x 6.8 y 5.5 xy 5.7 M. stiv 5.0 x 5.4 x 5.7 x 5.4 D. purpure 6.6 xy 4.6 x 7.1 y 6.1 Men N concentrtion in B. grcilis* Septemer 2009 Low soil P Middle soil P High soil P Men No legumes M. stiv D. purpure Men P concentrtion in B. grcilis* Septemer 2009 Low soil P Middle soil P High soil P Men No legumes M. stiv D. purpure Men 0.9 x 1.0 xy 1.3 y *Mens re significntly different (p.05, ANOVA) when followed y different letters within column (, ) or within row (x, y), ccording to the Tukey s rnge test. ws higher thn tht of M. stiv grown with B. ieersteinii, nd the high soil P fertility incresed the P concentrtion in legume tissues. In contrst, the concentrtion of P in legume tissues ws ffected y the interction of soil P fertility nd plnt mixture in July 2009, ut not in Septemer of the sme yer. High soil P incresed the concentrtion of P in M. stiv when grown with the ntive grss mixture, while the concentrtion of P in D. purpure ws promoted when P ws dded t 50 kg/h. In Septemer 2009, the TABLE 3 Nitrogen isotopic rtio (d 15 N) of individul ntive grss species, s influenced y the presence of legumes t different times of smpling d 15 N* EL+PS ET NV EC SS BG Septemer 2008 No legumes M. stiv D. purpure p vlue Septemer 2009 No legume M. stiv D. purpure p vlue EL + PS = E. lnceoltus ssp. Lnceoltus + P. smithii; ET= E. trchyculus ssp. susecundus; NV= N. viridul, EC= E. cndensis; SS= S. scoprium; BG = B. grcilis. Bolded vlues re significnt. *Within ech column, mens re significntly different (p.05, ANOVA; n = 12) when followed y different letters (, ) ccording to Tukey s rnge tests. concentrtion of P in D. purpure tissues grown longside the ntive grss mixture ws higher thn tht of M. stiv grown longside B. ieersteinii. In July 2009, M. stiv enhnced the concentrtion of P in the grss tissues of the ntive grss mixture (Tle 1). In contrst, t the end of growing seson, the P concentrtion of different ntive grss species ws not ffected y tretments, except for tht of B. grcilis in 2009 (Tle 2). In the tissues of this grss species, high soil P improved the concentrtion of P, irrespective of legume tretment. The concentrtion of P in B. ieersteinii grown with M. stiv ws greter thn tht of the ntive grss mixture grown lone in Septemer 2008 nd in July 2009, ut not in Septemer 2009 (Tle 1).

8 166 KLABI ET AL. TABLE 4 Totl phosphorus uptke nd P concentrtion in whole species mixtures nd in legume tissues s influenced y the interction etween plnt mixtures nd soil P fertility t different times of smpling Totl P uptke (kg/h)* Totl P concentrtion* (g/kg) Species mixtures Low soil P Middle soil P High soil P Men Low soil P Middle soil P High soil P Men Sept Ntive grsses 2.1 x 3.6 y 2.5 x Ntive grsses + M. stiv 2.9 x 3.6 x 4.0 x Ntive grsses + D. purpure 3.1 x 2.3 x 2.7 x B. ieersteinii + M. stiv 3.3 x 2.9 x 4.0 x Men July 2009 Ntive grsses Ntive grsses + M. stiv Ntive grsses + D. purpure B. ieersteinii + M. stiv Men Sept Ntive grsses Ntive grsses + M. stiv Ntive grsses + D. purpure B. ieersteinii + M. stiv Men P uptke y legumes (kg/h)*, P concentrtion in legumes (g/kg)*, Sept Ntive grsses + M. stiv Ntive grsses + D. purpure B. ieersteinii + M. stiv Men 1.0 x 1.0 x 1.4 y July 2009 Ntive grsses + M. stiv x 1.3 xy 1.6 y 1.4 Ntive grsses + D. purpure x 1.7 y 1.2 x 1.4 B. ieersteinii + M. stiv x 1.1 x 1.3 x 1.2 Men Sept Ntive grsses + M. stiv Ntive grsses + D. purpure B. ieersteinii + M. stiv Men *Mens re significntly different (p.05, ANOVA, n = 4) when followed y different letters within column (, ) or within row (x, y), ccording to the Tukey s rnge tests. 3.6 Soil nutrient vilility Soil P vilility in the 0 15 cm soil lyer incresed with soil P ddition ut ws not ffected y plnt mixture. This creted three different soil P concentrtions which persisted over the experimentl period, ut this effect diminished with soil depth, nd ws not oserved t cm (Tle 5). In contrst, soil N vilility ws not ffected y tretment. nutrient content. A positive reltionship ws found etween the Shnnon index of AM fungl diversity nd the dry mtter nd nitrogen uptke y the entire plnt mixture, prticulrly y the legume M. stiv in July AM fungl diversity ws lso positively correlted with the P concentrtion in ntive grss tissues when grown with M. stiv during the sme period (Figure 2), ut those effects were not oserved in the mixture of ntive grsses nd D. purpure, or t the end of growing seson in 2008 nd in Reltionships etween plnt dry mtter, nutrient content nd AM fungl diversity As AM fungl diversity ws previously found to e higher in the mixture of legumes with the ntive grsses (Kli et l., 2015), we exmined the correltion etween this vrile nd plnt dry mtter nd 4 DISCUSSION Our results show tht the inclusion of M. stiv enhnced nitrogen nd phosphorus concentrtions in the tissues of the ntive grsses (ll grsses mesured together), erly in the growing seson of 2009.

9 KLABI ET AL. 167 TABLE 5 Soil nutrient vilility (PO 4 -P, NO 3 -N) mesured t two soil depths (from 0 to 15 cm nd from 30 to 45 cm) s influenced y plnt mixtures nd y soil P concentrtions in 2009 Soil depths 0 15 cm cm Soil nutrients PO 4 -P (mg/kg)* NO 3 -N (mg/kg)* PO 4 -P (mg/kg)* NO 3 -N (mg/kg)* Plnt mixtures Ntive grsses Ntive grsses + M. stiv Ntive grsses + D. purpure B. ieersteinii + M. stiv P vlue Soil P concentrtion Low soil P Middle soil P High soil P c p vlue < *Within ech column, mens re significntly different (p.05, ANOVA; n = 12) when followed y different letters (,, c), ccording to Tukey s rnge tests. Bolded vlues re significnt. This indictes tht M. stiv improved the nutritive vlue of the comined ntive grsses erly in the summer. Additionlly, this legume ppers to hve the ility to improve the nutritive vlue of forge lter in the summer y improving the N concentrtion in the wrm-seson grss B. grcilis tissues t the end of the drought yer, Our results confirm those of others studies, which showed tht the inclusion of legumes to ntive pstures my enhnce the nutritive vlue of forge (Posler, Lenssen, & Fine, 1993; Schellenerg & Bnerjee, 2002). Arusculr mycorrhizl fungl diversity ws positively relted with M. stiv dry mtter nd nitrogen uptke nd with the P concentrtion in ntive grsses, erly in the summer of 2009, suggesting tht this legume cn promote eneficil AM fungi with positive impcts on plnt nutrition nd growth under semi-rid conditions. The nitrogen rtio isotopic nlysis showed tht the N nutrition of ntive grsses grown with M. stiv ws lrgely dependent on soil N spring, ecuse the legumes were fixing much of their N. 4.1 Medicgo stiv enhnces overll plnt dry mtter, ut not tht of grsses The inclusion of the domesticted legume M. stiv to mixtures contining the ntive grss mix or with the introduced grss B. ieersteinii led to dry mtter yield vlues tht were similr to, or higher thn, grsses grown in the sence of the legume. At the end of 2008, the dry mtter yield of ntive grsses grown longside M. stiv ws similr to tht grown without legumes. In 2009, mixtures contining M. stiv were most productive erly in the growing seson, while the mixture of M. stiv nd B. ieersteinii produced the highest dry mtter t the end of the growing seson. Our findings concur with previous findings showing tht mixture of M. stiv nd either ntive forge shru (winterft [Krscheninnikovi lnt]) or nother shru species (Grdner s sltush [Atriplex grdneri]) yielded forge iomss tht ws higher thn, or similr to, those shru species grown lone or to M. stiv grown lone (Schellenerg & Bnerjee, 2002). The high dry mtter yield oserved in plnt mixtures contining M. stiv ppers to e minly ttriuted to the vigorous growth of this domesticted legume, ecuse the grss dry mtter yield ws similr in the presence or sence of M. stiv in 2009, when grsses nd legumes were mesured seprtely. Medicgo stiv ws found to e the most productive nd competitive species, essentilly due to its high drought tolernce nd gret dpttion to the soil nd to diverse environmentl conditions in semi-rid regions (Schellenerg & Bnerjee, 2002). Totl plnt dry mtter yield ws higher t the end of the growing seson in 2008 thn in In 2008, precipittion nd soil moisture levels were higher, which likely enefitted plnt growth. In ddition, plnt dry mtter ws greter erly in the growing seson of 2009 thn t the end of the growing seson. Extreme drought periods re common in the Cndin semi-rid priries. Typiclly, most precipittion occurs in the first hlf of the growing seson (from My to July), with the quntity nd frequency of precipittion vrying gretly mong yers, nd the second hlf is hot nd dry (Biley et l., 2010). Consequently, forge production peks in erly summer, when moisture is ville (Bron & Belnger, 2007), nd during the hot nd dry portion of summer the productivity nd feeding vlue of most species declines, prticulrly cool-seson species. However, the ntive cool-seson grsses nd introduced grsses tht re lso cool-seson species re fst-growing nd highly dominnt species in semi-rid regions. They initite their growth erly in the spring (April) nd mximum production occurs efore mid-summer, ut their productivity is reduced or they re dormnt in lte summer, when low soil moisture limits growth. In contrst, the wrm-seson species re slow-growing species nd generlly less productive, ut they cn e competitive under hot nd dry conditions, when the productivity of cool-seson species decreses. They initite their growth only in June nd re more productive during the hot summer months, nd their pek production occur y Septemer (Aougendi, 1995; Jefferson et l., 2005; Schellenerg et l., 2012). In the present study, the wrm-seson grsses (nd the wrm-seson legume D. purpure) did not help to sustin forge productivity t the end of the growing seson 3 yers fter estlishment, oviously due to the low productivity nd slow estlishment of the wrm-seson species in the mixture. This my lso e prtly explined y the extreme environmentl conditions tht previl in lte summer of the driest yer, Species dynmics mong yers The productivity of the plnts ws very low in 2006, the yer of seeding (Hmel, Iws, & Schellenerg, 2008). In 2007, the coolseson ntive species nd the introduced species B. ieersteini nd M. stiv estlished rpidly. The wrm-seson species were slower,

10 168 KLABI ET AL. Totl dry mtter (kg/h) y = 3308 x R² = 0.58 P =0.028 M. stiv dry mtter (kg/h) y = 3247 x 3411 R² = 0.71 P = Shnnon index of AM fungi diversity Totl N uptke (kg/h) y = x R² = 0.56 P =0.032 N uptke y M. stiv (kg/h) y = x R² = 0.55 P = Pconcentrtion in grsses tissues (g/kg) 2.0 Shnnon index of AM fungi diversity y = x R² = 0.59 P = Shnnon index of AM fungi diversity FIGURE 2 Significnt reltionships etween totl dry mtter, legume dry mtter, totl N uptke, N uptke y M. stiv nd P concentrtion in ntive grsses tissues, nd the Shnnon index of AM fungi diversity in the mixture contining the ntive grss mix nd M. stiv in July 2009; the dots represent the two concentrtions of soil P (low soil P nd high soil P) ut the wrm-seson grss B. grcilis grew rpidly in 2008 nd ecme the most undnt species, with the cool-seson grss N. viridul lte in the summer of However, the undnce of these species ws strongly reduced in the dry yer 2009, likely due to the rinfll scrcity nd distriution (Kilcher & Loomn, 1983; Kusler, 2009). The productivity of B. grcilis vries gretly, nd this species ws reported to produce well only once every 2 or 3 yers (Wyni, 2007). The cool-seson grsses E. lnceoltus ssp. lnceoltus, P. smithii nd E. trchyculus susecundus were the dominnt species in the dry yer of The productivity of these cool-seson grsses increses with time. The vilility of moisture in the utumn of the wetter yer 2008 my hve stimulted the vigorous growth of the cool-seson species erly in the spring of the following yer (Willms, McGinn, & Dormr, 1993). The undnce of the cool-seson species E. cndensis, B. ieersteinii nd M. stiv ws similr mong yers, which my indicte tht those species hd the ility to mintin their productivity nd they were dpted to different environmentl conditions. It seems tht the effects of climte chnge in semi-rid regions hve gret impct on the dynmics of most ntive species. A more diverse mixture could contriute to mitigte the negtive impct of drought on stnd productivity (Tilmn & Downing, 1996), ecuse ech species ppers to hve specific sesonl growth pttern. The undnce of the wrm-seson S. scoprium nd prticulrly D. purpure ws very low in lte summer of oth yers indicting tht these species did not estlish successfully. This my e due to these species eing disdvntged in the mixtures y the effects of high precipittion nd cool tempertures in 2006, the yer of seeding (Mischkolz, Schellenerg, & Lm, 2013). Moreover, the productivity of D. purpure is usully low compred with tht of other legumes, nd it is included in pstures for its high nutritive vlue rther thn its productivity (McGrw, Shockley, Thompson, & Roerts, 2004; Mischkolz et l., 2013). In ddition, in 2009, S. scoprium ws reduced y the presence of M. stiv. This my e due to the competition etween this grss nd M. stiv for wter or other resources (Tilmn et l., 1997). 4.3 Legume effects on nutrient uptke nd concentrtions in plnt mixes, in erly summer The plnt mixtures tht contined M. stiv hd the highest N uptke nd concentrtions over time. This ws mostly relted to the

11 KLABI ET AL. 169 higher N concentrtion within this legume, which ws ctively fixing N 2, ut lso to its higher iomss in comprison with D. purpure. Here, M. stiv lso incresed the P uptke y whole plnt mixtures ut only t the end of the growing seson in the wet yer (2008) when soil P ws high, nd t the vegettive growth stge in 2009, irrespective of soil P. This ws likely driven y higher M. stiv iomss production nd y the higher P uptke y this legume reltive to D. purpure, when soil moisture ws less limiting to plnt production. In contrst, M. stiv incresed the nutrient concentrtion of totl ntive grss tissues, compred with grsses grown without legumes, ut only erly in the growing seson of This increse in nutrient concentrtion did not result in n increse in totl nutrient uptke. It ppers tht erly in the seson, grss productivity ws not limited y N nd P vilility, ut rther y light, wter nd other soil nutrients. The higher nutrient concentrtions in the tissues of ntive grsses when M. stiv ws present in July 2009 contriuted to improving the nutritive vlue of ntive grss herge, when most grss species (cool-seson grsses) were ctive, erly in the summer. In ddition, the concentrtion of P ws higher in the tissues of the introduced grss thn those of the ntive grsses when grown lone t the end of the growing seson in 2008, nd lso t the vegettive growth phse of This my e due to higher nutrient demnd y this introduced grss, under conditions of moisture sufficiency, ecuse those effects were not oserved t the end of the growing seson in the drought yer of 2009, compred with the wetter yer of This my rely in prt on the fct tht this introduced species ws limited more y wter vilility thn soil nutrients under severely dry conditions typicl of lte summer. The higher P concentrtion of the ntive grss mixture nd of B. ieersteinii, in the presence of M. stiv, my hve lso contriuted to the incresed P concentrtion of the totl forge, in erly summer. However, it ws suggested tht secretion of cid phosphtse enzymes y the roots of N-fixing legumes to improve soil P vilility my lso enhnce the P nutrition of neighouring plnts (Houlton, Wng, Vitousek, & Field, 2008; Li et l., 2007). 4.4 Legume effects on nutrient uptke nd concentrtions of ntive grsses, in lte summer In generl, in semi-rid regions the productivity nd nutritive vlue of most ntive species decline drmticlly when the plnts mture or when environmentl conditions chnge (Wllce et l. 1961). We expected tht the inclusion of legumes might improve the nutritive vlue, in prticulr the nitrogen contents of different species of grsses lte in the growing seson, which is importnt for niml performnce. However, our results show tht the presence of legumes contriuted to incresed N contents in few individul ntive species, ut this vried depending on yer, legume species nd soil P fertility. Despite the low undnce of the wrm-seson legume D. purpure, this legume contriuted to incresing the N content in the tissues of the cool-seson grss E. cndensis in the wet yer (2008), while this effect ws not oserved t high soil P. It seems tht the low soil P vilility could reduce the nitrogen fixtion of D. purpure, which might increse the competition with E. cndensis for soil N source, s D. purpure might ecme more dependent on soil N uptke. A possile explntion is tht the wrm-seson D. purpure, perennil nd extremely slow-growing species, tends to invest more iomss in roots efore investing in shoots, prticulrly in semi-rid regions (Wever, 1958), mking it more competitive for soil N in species mixtures. Additionlly, D. purpure, wrm-seson species, ws proly more undnt during the summer months thn lte summer or erly utumn, which my explin its effect on the N nutrition of E. cndensis oserved lte in the summer. However, other studies hve indicted tht the mximum growth of wrm-seson species occurred during the summer nd then declined efore peking in production y lte summer or erly utumn (Bron & Belnger, 2007; Kusler, 2009). In ddition, the growth of cool-seson grss my hve reinitited in the utumn of the wet yer 2008, when soil moisture ecme ville, nd thus my hve led to increse its competition for soil N, in the mixtures. It hs een shown tht the effect of soil N vilility on plnt N nutrition depends on others fctors, such s soil P vilility nd plnt competition (Aerts & Chpin, 1999; Blnke, Bssin, Volk, & Fuhrer, 2012; G usewell, 2004). In the drought yer of 2009, the wrm-seson grss B. grcilis enefitted from incresed N concentrtion in its tissues when grown with M. stiv. This wrm-seson grss my then contriute to incresing the nutritive vlue of forge during the hottest periods of summer, when M. stiv ws present. This result is in greement with Schellenerg et l. (2012), who showed tht the inclusion of wrm-seson grsses in mixture contining cool-seson grsses cn enhnce the nutritive vlue of forge y incresing its protein content during lte summer periods in semi-rid regions. The wrmseson grss (B. grcilis) lso hd higher P concentrtion in its tissues, when soil P ws high. It seems tht the productivity of this wrm-seson grss ws more limited y nutrient vilility thn wter vilility, which my lso explin the lower undnce of this grss oserved in lte summer of the dry yer These two ntive grss species (E. cndensis nd B. grcilis) responded differently to the ddition of legumes or phosphorus t plnt mturity, nd this my depend on competitive interctions etween ntive species, legume identity, wter vilility nd/or climtic vrition in ntive grsslnds. 4.5 N concentrtion nd plnt d 15 N vlues Incresed N concentrtions in the tissues of ntive grss mixtures nd of B. grcilis, when M. stiv ws present, cn e ttriuted to different processes: (i) the higher vilility soil N tht ws not used y the legumes (clled soil N spring), ecuse they rely more on fixed N thn soil N when in competition with neighouring species (Temperton et l., 2007); (ii) the fixed N tht ws trnsferred from this legume to the grsses through root exudtion (Jlonen, Nygren, & Sierr, 2009) or relesed through orgnic mtter rekdown y slow decomposition of legume tissues from the previous yer