Evolutionary responses of invasive grass species to variation in precipitation and soil nitrogen

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1 Journl of Ecology 216, 4, doi:.1111/ Evolutionry responses of invsive grss species to vrition in precipittion nd soil nitrogen Monic A. Nguyen 1 *, Amy E. Orteg 1, Kurt Q. Nguyen 1, Srh Kimll 2, Michel L. Goulden 3 nd Jennifer L. Funk 1 1 Schmid College of Science nd Technology, Chpmn University, Ornge, CA, USA; 2 Center for Environmentl Biology, University of Cliforni, Irvine, CA, USA; nd 3 Deprtment of Erth System Science, University of Cliforni, Irvine, CA, USA Summry 1. Glol climte models suggest tht mny ecosystems will experience reduced precipittion over the next century nd the consequences for invsive plnt performnce re lrgely unknown. Annul invsive species my e le to quickly evolve trits ssocited with drought escpe or tolernce through rpid genetic chnges. 2. We investigted the influence of 5 yers of wter nd nitrogen mnipultions on trit vlues in southern Cliforni grsslnd system. Seeds from two nnul grss species (Aven rt nd Bromus mdritensis) were collected from experimentl plots nd grown in common environment over two genertions. We mesured 14 physiologicl, morphologicl, phenologicl nd reproductive trits. 3. Both species displyed phenotypic differences depending on the wter tretment from which they were collected, ut not depending on the nitrogen tretment. Both species displyed trit vlues chrcteristic of drought escpe (e.g. erlier flowering in A. rt nd B. mdritensis, lower wter-use efficiency in B. mdritensis) when grown from the seeds collected from plots tht experienced five yers of reduced precipittion. Furthermore, A. rt individuls grown from the seeds collected from drought plots hd higher reproductive output nd higher photosynthetic performnce thn individuls grown from wter ddition plots, with individuls grown from mient plots displying intermedite trit vlues. Notly, we found no phenotypic vrition mong tretments for six root trits. 4. Synthesis. Trit differences were oserved following two genertions in common grden, suggesting tht tretment differences were geneticlly sed. This suggests tht popultions were responding to selection over the 5 yers of wter mnipultions, remrkly short time period. The rpid evolutionry responses oserved here my help these two widespred invsive grss species thrive under reduced precipittion scenrios, which could hve importnt implictions for fire dynmics, invsive species mngement nd ntive plnt restortion in communities invded y nnul grsses. Key-words: nnul species, Aven rt, Bromus mdritensis, drought escpe invsion ecology, invsive species, phenology, photosynthesis, root trits, wter-use efficiency Introduction Glol chnge, including increses in temperture, tmospheric CO 2 concentrtion nd nitrogen (N) input from nthropogenic ctivities, is one of the most significnt threts to nturl ecosystems. Further, glol climte models hve predicted the ltertions in inter- nd intr-nnul precipittion in mny regions (Melillo, Richmond & Yohe 214). *Correspondence uthor: E-mil: nguyen.n.monic@gmil.com Biologicl invsion is nother importnt gent of glol chnge with significnt effects on glol iodiversity (Millennium Ecosystem Assessment, 25). A met-nlysis of 41 cses of invsion found tht lien invsive plnts decrese the undnce nd diversity of ntive resident plnt species y 43.5% nd 5.7%, respectively (Vil et l. 2). Significnt impcts on ntive resident species undnce nd diversity re more likely to occur if the invding species is n nnul grss (Pysek et l. 212). Refining our understnding of how invsive plnts respond to environmentl vrition will 216 The Authors. Journl of Ecology 216 British Ecologicl Society

2 98 M. A. Nguyen et l. improve our ility to predict their effects on nturl plnt communities in the fce of glol chnge. In this study, we explore the dptive responses of two nnul invsive grss species to declines in precipittion nd incresed N deposition in southern Cliforni grsslnd. There re two mjor strtegies plnt cn employ to cope with reduced wter vilility: drought tolernce nd drought escpe. Drought tolernce, chrcterized y low photosynthetic ctivity, high wter-use efficiency (WUE, the rte of cron ssimiltion reltive to trnspirtion) nd slow growth, llows n individul to conserve resources while continuing to e ctive during the drought period (Sherrrd & Mherli 26; Frnks 211). Conversely, drought escpe involves the completion of n individul s life cycle efore the drought reches its most extreme stte. Drought escpe is chrcterized y high photosynthetic rtes, low WUE nd erly flowering (Sherrrd & Mherli 26; Frnks 211). An increse in N lloction to photosynthetic enzymes nd chlorophyll content in leves my ct to increse light hrvesting (Tershim & Evns 1988; Evns 1989) nd, consequently, cron fixtion over shorter period of time. Thus, incresed N vilility resulting from humn ctivities my enhnce the growth in species displying drought escpe strtegy. Will invsive species enefit from projected rid, N-rich conditions? High soil N concentrtions cn increse invsive species performnce (Huenneke et l. 199; Dvis, Grime & Thompson 2; Ostertg & Verville 22; Gross, Mittelch & Reynolds 25); however, the effects of N ddition re complex nd will depend on soil wter vilility, competition nd the form of ville N (Everrd et l. 2; Ross, Ehrenfeld & Ptel 211; Eskelinen & Hrrison 214). Furthermore, n nlysis of severl U.S. dt ses suggests tht invsive species tend to initite leves nd flower erlier in the growing seson compred to ntive species (Wolkovich & Clelnd 211). This phenologicl pttern, consistent with drought escpe, suggests tht mny nnul invsive species my e well suited to incresingly rid conditions. Mny studies hve shown tht invsive species cn e phenotypiclly plstic in response to chnges in wter, light nd nutrient vilility (e.g. Funk 28; Dvidson, Jennions & Nicotr 211); however, fewer studies hve exmined the potentil for invsive species to dpt to environmentl vrition through genetic chnges. In study compring flowering times of nnul Brssic rp efore nd fter four-yer drought in southern Cliforni, Frnks, Sim & Weiss (27) found tht seeds collected from wet nd dry environments post-drought flowered significntly erlier thn those collected efore the drought, which is chrcteristic of drought escpe response. Further, Frnks (211) suggested tht popultions of B. rp escpe drought through reduced WUE, which llows for rpid development nd erlier flowering. A study of the nnul invsive grss Aven rt grown in wet nd dry environments lso found strong evidence tht erlier flowering ws dptive under drought (Sherrrd & Mherli 26). Despite evidence of rpid evolution in the flowering times of B. rp nd A. rt, dptive responses to climte chnge my e slower or lcking in other trits nd for other species (Frnks, Sim & Weiss 27). Antomicl nd physiologicl chrcteristics of roots will influence oth wter nd N cquisition, yet few studies hve exmined root dpttion long precipittion grdient. Studies using congeneric nd conspecific pirs occurring in high nd low rinfll sites suggest tht low specific root length (SRL, m g 1 ), lrge root dimeter or slow root elongtion rtes my e fvoured under drier conditions, reflecting n investment in thick, longer-lived roots tht more efficiently trnsport wter (Wright & Westoy 1999; Nicotr, Bick & Westoy 22; Heschel et l. 24). We serched for evolutionry responses to N nd wter mnipultions in two invsive grss species (A. rt nd Bromus mdritensis) tht re widespred throughout much of the west cost of North Americ. We sujected popultions of oth species to ltered precipittion nd N vilility for 5 yers, collected seeds from multiple mternl plnts nd grew them in common environment over two genertions. We mesured suite of ove- nd elow-ground trits tht re ssocited with wter nd N use to ddress this question: Did invsive species exhiit trit differences in response to the 5-yer environmentl mnipultions tht persisted fter two genertions in common environment? If so, our results would suggest tht the trit differences were genetic nd, thus, the result of rpid evolution in response to environmentl chnge. Answering this question is criticl first step in understnding how invsive species my respond to future climte chnge. Mterils nd methods STUDY SITE AND SEED SOURCE The study site ws locted in grsslnd community within the Irvine Rnch Conservncy in Ornge County, Cliforni (Potts et l. 212; Allison et l. 213; Kimll et l. 214). Ornge County hs Mediterrnen climte chrcterized y hot, dry summers nd cool, wet winters. We focused on two invsive grss species, A. rt nd B. mdritensis, which were undnt t our site. These species re ntive to the Mediterrnen Bsin nd, due to repeted introductions, hve widespred glol distriutions. In Cliforni, where our study ws conducted, A. rt nd B. mdritensis hve overlpping ut slightly different geogrphic distriutions, with B. mdritensis occurring in more rid environments including the Mojve Desert (DeFlco et l. 23; Steers, Funk & Allen 211). Twenty-four experimentl plots (6.7 m m) extending over n re roughly one cre in size were imposed on existing vegettion, with ech plot rndomly exposed to one of three precipittion tretments (mient, wter ddition or wter reduction, n = 8 per tretment, Fig. 1). Ech plot ws divided lengthwise with ech hlf receiving one of two N tretments (mient or N ddition). The wter reduction tretment received pproximtely 51% less wter thn the mient wter tretment, while the wter ddition tretment received pproximtely 33% more over the five-yer mnipultion period (Mrch 27 My 212) (Kimll et l. 214). Cler, retrctle roofs were deployed during suset of winter storms to control wter input into the reduction plots. Rinfll collected from reduction plots ws stored in opque polyethylene tnks nd used to supplement

3 Rpid evolution in two nnul exotic grsses 981 ddi on INITIAL POPULATION A. rt & B. mdritensis 8 plots 8 plots 8 plots mient reduc on 1-cre FIELD FIELD (5 yers) Tle 1. The numer of individuls of Aven rt nd Bromus mdritensis smpled for ech wter tretment. The numer of different plots tht mternl plnts originted from is given in prentheses. Becuse there ws no significnt effect of N on ny trit, individuls from the two N tretments were grouped within their respective wter tretments A. rt B. mdritensis Reduction 26 (7) 18 (8) Amient 21 (8) 15 (5) Addition 2 (8) 9 (3) F1 F2 F1 F2 COMMON GARDEN (2 yers) wter ddition plots through system of gsoline-powered pumps connected to drip tuing (Kimll et l. 214). Bseline N deposition t our study site is pproximtely 1.5 g m 2 yer 1 (Kimll et l. 214). Nitrogen ddition plots received 6 g N m 2 yer 1, vlue chosen to simulte site pproching N sturtion fter yers of continuous deposition (Kimll et l. 214). Supplementl N ws given in two pplictions: 2 g of quick-relese clcium nitrte following the first storm of the seson nd 4 g of slow-relese clcium nitrte following the first month of the growing seson (Kimll et l. 214). In My 212, the seeds were collected from rndomly selected mternl plnts of A. rt nd B. mdritensis nd trnsported to Chpmn University, which is locted 16 km from the field site nd hs similr wether conditions. To ensure the representtion of the entire popultion, nd minimize the effect of genetic drift, we tried to smple individuls from ll plots (n = 8) per wtering tretment. However, some plots did not contin individuls of oth species. When species ws present in given plot, we collected the seeds from one to eight mternl plnts in tht plot. Only seeds mture enough to e removed with gentle pull of the spikelet were collected. Seprte envelopes were used to ensure tht offspring from ech mother plnt remined isolted. On 3 Decemer 212, we germinted the seeds on moist filter pper t 4 C for 96 h. After germintion, only one seedling from ech mother plnt ws used; thus, ech replicte hd different mother. Becuse lter nlyses showed tht there ws no significnt effect of N on ny of the trits mesured, individuls from the two N tretments were grouped within ech wter tretment. We hd t lest nine replictes per wter tretment for B. mdritensis nd t lest 2 replictes per wter tretment for F1 F2 ABOVE & BELOWGROUND TRAIT SURVEY Fig. 1. Following 5 yers of wter mnipultions in the field (Irvine, CA), seeds were collected from experimentl plots nd grown in common environment over two genertions. Physiologicl, morphologicl nd phenologicl trits were mesured on the second genertion grown in common environment. A. rt (Tle 1). Following germintion, seedlings were trnsferred to 4.1-L pots filled with moistened potting soil (Sunshine Mix #1, Sun Gro Horticulture). Plnts were grown in full sun t Chpmn University nd wtered dily to sturtion. Becuse dpttion cnnot e ssumed sed on the phenotypes displyed in one genertion (Turner, Hufuer & Rieseerg 214), common grden experiments must occur over multiple genertions in order to differentite etween genetic nd epigenetic effects, including mternl effects (Morn & Alexnder 214). Mternl effects refer to sitution in which n individul s phenotype is determined not only y its genotype nd current environment, ut lso y the environment experienced y its mother (Lcey 1998). We controlled for mternl effects y growing seeds collected from the field (F1) nd their offspring (F2) in common environment (see Fig. 1). Any mternl effects present in the F1 genertion should hve een erdicted in the F2 genertion ecuse F1 plnts were grown under identicl environmentl conditions. Thus, tretment differences oserved in the F2 genertion should e the result of genetic differences rther thn mternl effects. We collected the seeds from ech F1 mother plnt nd germinted them s descried ove so tht ech F2 replicte hd different mother. Becuse of some mortlity during germintion nd estlishment, we hd slightly fewer replictes in the F2 genertion (n = 67 A. rt, n = 42 B. mdritensis) thn in the F1 genertion (n = 72 A. rt, n = 43 B. mdritensis) cross tretments. ABOVE-GROUND MEASUREMENTS Collection of physiologicl mesurements egn 7 dys fter germintion of the F2 genertion (Ferury 214). We conducted gsexchnge mesures with LI-64 portle photosynthesis system (LI-COR, Lincoln, NE, USA). We mintined constnt chmer conditions tht pproximted growing conditions, including CO 2 t 4 ll L 1, light t 18 lmol photon m 2 s 1, chmer temperture t 25 C nd reltive humidity t 52 62%. Mesures included photosynthetic rte, stomtl conductnce, trnspirtion rte nd instntneous WUE (photosynthetic rte/trnspirtion rte). When leves were too smll to fill the chmer, the lef re ws determined nd used to correct gs-exchnge dt. We mesured lef chlorophyll content with portle chlorophyll SPAD-52 meter (Spectrum Technologies, Plinfield, IL, USA). Three leves were hrvested following gs-exchnge nd chlorophyll mesures to determine lef dry mss per unit re (LMA) nd lef N concentrtion. The leves were scnned to determine totl lef re, dried t 6 C for 72 h nd weighed. The dried leves were then ground in Wiley mill with 4-mesh screen, nd lef N ws determined with n elementl nlyser (Costech 4, Pioltello, Itly). We ssessed the plnt size y mesuring cnopy cover prior to flowering, estimted s the product of plnt width long two

4 982 M. A. Nguyen et l. perpendiculr xes. We checked plnts dily nd recorded dte of flowering, defined s the first ppernce of florets. While oth species lrgely reproduce y selfing in nturl popultions (Johnsen- Morris & Ltt 26; Grossmn & Rice 214), outcrossing does occur; thus, plnts from different tretments were grown in the sme re ut with physicl rriers etween them to void cross-fertiliztion. Though sptilly seprted, ll tretments received similr light nd wter levels dily. From Mrch to June 214, mture seeds were collected, ir-dried nd weighed to determine totl seed iomss s metric of reproductive fitness. ROOT MEASUREMENTS To otin root mesures for the F2 genertion, seeds from suset of F1 plnts from the ove-ground trit survey were germinted using the procedure descried ove nd trnsferred to 4.1-L pots filled with moistened snd perlite vermiculite (1:1:1) mixture. Plnts were rndomly selected within plots nd, s with ove-ground mesures, we smpled from ll plots (n = 8) per wtering tretment where possile. We hd eight replictes per wtering tretment for ech species. Seedlings were grown for 5 weeks, receiving totl of.5 g N,.5 g P nd.5 g K (Mircle-Gro) over the first 2 weeks. The plnts were wtered dily to sturtion except on dys when pots received fertilizer. After 5 weeks, the plnts were hrvested nd the roots were wshed nd scnned using the WinRHIZO imge nlysis system (Regent Instruments Inc., Queec, QC, Cnd) to determine totl root length. Root growth rte (cm per dy), root length density (root length per volume of soil) nd fine root rtio [the rtio of fine root length (dimeter <.5 mm) to totl root length] were clculted. We seprted elow- nd ove-ground iomss, dried them t 6 C for 72 h nd weighed them to determine root-to-shoot iomss rtio (R: S) nd specific root length (SRL, m g 1 ). Biomss ws comined, ground nd nlysed for N concentrtion s descried ove. Plnt N uptke rte (g N per dy) ws clculted s [(plnt iomss 9 plnt N)/growing dy numer]. STATISTICAL ANALYSES Box Cox trnsformtions of the dt were used where necessry to meet the ssumptions of normlity for sttisticl nlyses. We performed nlysis of vrince (ANOVA) with wter nd N tretment s fixed effects nd plot s rndom effect to compre trit vlues etween tretments within ech species. The significnce of fixed effects ws ssessed using Stterthwite pproximted degrees of freedom. We used type III sums of squres ANOVA for mixed-effects model in the LME4 pckge in R ( v.3.2.). Following Morn (23), ll P-vlues re reported nd sequentil Bonferroni corrections for multiple sttisticl tests were not conducted. Post hoc nlyses were performed using Tukey s honestly significnt difference. Results There ws no significnt effect of N on ny trit; thus, dt from the two N tretments were grouped within wter tretment nd N s min effect ws excluded from ll susequent nlyses. vilility ffected severl ove-ground trits in A. rt s well s first flowering dte nd wter-use efficiency in B. mdritensis (Tle 2). With respect to Tle 2. F-vlues clculted from one-wy nov for second-genertion (F2) plnts of two invsive grss species with wter tretment s fixed fctor nd plot s rndom fctor. Numertor nd denomintor degrees of freedom for F-vlues re presented in prentheses. Significnt effects (P <.5) re in oldfce type Aven rt Bromus mdritensis F P F P Aove-ground trits Photosynthetic rte 3.82 (2,18) (2,4).645 Lef chlorophyll 5.9 (2,67) (2,41).84 WUE.18 (2,24) (2,4).8 Lef N content 2.62 (2,64) (2,41).224 LMA.23 (2,66) (2,42).36 Cnopy cover 3.32 (2,67) (2,42).235 Flowering dte 8.4 (2,67) < (2,41) <.1 Seed iomss 8.92 (2,67) < (2,42).7 Below-ground trits Root growth rte.49 (2,24) (2,42).586 R:S.3 (2,24) (2,24).339 Root length density.47 (2,24) (2,24).527 Fine root rtio.93 (2,12) (2,24).575 Specific root length.57 (2,17) (2,24).748 N uptke rte.62 (2,24) (2,24).583 WUE, -use efficiency; LMA, lef mss per re; R:S, root-to-shoot iomss rtio. physiologicl trits, photosynthetic rte nd lef chlorophyll content in A. rt differed cross wter tretments with trend towrds higher trit vlues in the wter reduction tretment reltive to the mient nd dded precipittion tretments (Tle 2, Fig. 2). Photosynthetic rte nd chlorophyll content did not differ cross tretments in B. mdritensis (Tle 2, Fig. 3). -use efficiency ws lower in plnts from the reduced precipittion plots in B. mdritensis compred to the mient nd wter ddition tretments (Tle 2, Fig. 3). There ws no difference in WUE cross the tretments in A. rt (Fig. 2). The pttern oserved in B. mdritensis ws driven y higher trnspirtion rtes in plnts from reduced precipittion plots (dt not shown). hd no significnt effect on lef N content in either species (Tle 2, Fig. 2). With respect to growth nd reproductive trits, flowering dte ws erlier in the wter reduction nd mient wter tretments reltive to the wter ddition tretment in oth A. rt nd B. mdritensis (Figs 2 nd 3; Tle 2). Plnts from the wter reduction plots in A. rt lso produced greter seed iomss reltive to the ddition plots, with plnts from the mient plots displying intermedite seed iomss (Fig. 2). There ws significnt effect of wter on cnopy cover in A. rt with higher cnopy cover in wter reduction plots reltive to wter ddition plots (Tle 2, Fig. 2). However, there ws no difference in seed iomss or cnopy cover cross wtering tretments in B. mdritensis (Fig. 3). LMA did not differ mong tretments for either species (Figs 2 nd 3). There ws no significnt effect of wter on root trits of either species in the F2 genertion (Tle 2, Fig. S1 in Supporting Informtion).

5 Rpid evolution in two nnul exotic grsses 983 Photosynthetic rte (µmol CO 2 per m 2 per s) 15 5 () () WUE (µmol C per mmol H 2 O) Lef N content (%) (c) (d) Lef chlorophyll content (SPAD units) (e) (f) First flowering dte (dy of yer) LMA (g per m 2 ) Fig. 2. Photosynthetic rte (), wter-use efficiency (), lef nitrogen content (c), lef chlorophyll content (d), first flowering dte (e), lef mss per re (f), seed iomss (g) nd cnopy cover (h) for Aven rt from three wtering tretments grown in common environment (F2 genertion). Mens nd stndrd errors of wter ddition, mient nd reduction tretments re shown. Different letters denote significnt differences mong mens s determined through post hoc Tukey s HSD tests. Where pplicle, significnt effects of wter re noted. Seed iomss (g per plnt) (g) Addition Amient Reduction (h) Addition Amient Reduction Cnopy cover (cm 2 per plnt) Discussion Our results suggest tht invsive species my exhiit rpid evolutionry chnge following 5 yers of glol chnge mnipultions. Trit differences occurred in response to ltered wter vilility, ut not in response to the chnges in ville N. Erlier flowering (A. rt nd B. mdritensis) nd lower WUE (B. mdritensis) in the reduced wter tretment re in line with drought escpe response s found in previous work (Sherrrd & Mherli 26; Frnks, Sim & Weiss 27; Frnks 211). Aven rt from wter reduction plots lso hd significntly higher totl reproductive iomss thn individuls from wter ddition plots, nd photosynthetic rte nd lef chlorophyll content showed trends towrds higher vlues in wter reduction plots thn in mient wter plots, suggesting tht these physiologicl responses my e dvntgeous in drought-prone regions. High lef chlorophyll content my llow for incresed light hrvesting over shorter periods of time, s evidenced y higher photosynthetic rtes, nd thus enle n incresed reproductive output. Higher reproductive output my lso hve resulted from n erlier flowering time or shift in lloction from vegettive to reproductive structures (Wolfe & Tonsor 214) nd could increse the numer of successful offspring in rid environments, where estlishment is difficult. Notly, we found tht field mnipultions did not lter root trits, which is lrgely unstudied outside of model orgnisms (reviewed in Coms et l. 213). Previous studies of root dpttion in nturl systems hve focused on conspecific or congeneric comprisons of species from different rinfll environments, which reflect evolution over potentilly long time-scles (Wright & Westoy 1999; Nicotr, Bick & Westoy 22; Heschel et l. 24). It is possile tht our

6 984 M. A. Nguyen et l. Photosynthetic rte (µmol CO 2 per m 2 per s) 15 5 () () WUE (µmol C per mmol H 2 O) Lef N content (%) (c) (d) Lef chlorophyll content (SPAD units) First flowering dte (dy of yer) (e) (f) LMA (g per m 2 ) Seed iomss (g per plnt) 15 5 (g) Addition Amient Reduction (h) Addition Amient Reduction Cnopy cover (cm 2 per plnt) Fig. 3. Photosynthetic rte (), wter-use efficiency (), lef nitrogen content (c), lef chlorophyll content (d), first flowering dte (e), lef mss per re (f), seed iomss (g) nd cnopy cover (h) for Bromus mdritensis from three wtering tretments grown in common environment (F2 genertion). Mens nd stndrd errors of wter ddition, mient nd reduction tretments re shown. Different letters denote significnt differences mong mens s determined through post hoc Tukey s HSD tests. Where pplicle, significnt effects of wter re noted. five-yer experiment ws too short to oserve the differences in root trits. In the sence of rpid genetic responses, plsticity in root trits my e n importnt mechnism for responding to environmentl chnges on short time-scles. In response to the reductions in wter vilility, individul plnts cn increse root iomss, root elongtion rte nd SRL to mximize wter cquisition (e.g. Willims & Blck 1994; Pdill et l. 29; Drenovsky, Khsnov & Jmes 212; Lrson & Funk 216). Severl of these responses (rpid elongtion, high SRL) re contrry to strtegies found in species dpted to low rinfll environments (Wright & Westoy 1999; Nicotr, Bick & Westoy 22). More dt re needed to understnd the reltive role of plsticity nd genetic dpttion in elow-ground trit responses to the vrition in wter vilility. In contrst to wter mnipultion, nitrogen ddition over five-yer period did not result in detectle trit differences. Previous work from these field plots suggested tht N ddition only incresed grsslnd productivity when comined with wter ddition, nd only in some yers (M. L. Goulden, unpul. dt). It is possile tht the proximity of the suplots (mient nd N ddition within single plot) my explin why N ddition filed to lter trits in either species. Crosspollintion or seed dispersl my hve contriuted to gene flow mong suplots. While mny studies hve found tht exotic nnul grsses respond positively to N ddition (e.g. Huenneke et l. 199; Dvis, Grime & Thompson 2; Gross, Mittelch & Reynolds 25), growth rtes my sturte or decline t high soil N (e.g. Pdgett & Allen 1999; Mttingly & Reynolds 214). Our site is reltively fertile (.19% N nd mg P kg 1 ; J. L. Funk, unpul. dt), likely resulting from historic cttle grzing nd N deposition in the Ornge County re. Thus, N mnipultions my yield different results if conducted in more N-limited environments.

7 Rpid evolution in two nnul exotic grsses 985 One novel impliction of our study is tht it my e possile for invsive plnt species to dpt to environmentl chnge over just 5 yers or five genertions. Although there re exmples of rpid evolution in other weed species (Frnks, Sim & Weiss 27; Frenck et l. 213; Sultn et l. 213; Grossmn & Rice 214), 5 yers is remrkly short time period sed on dt from other studies. In review of evolutionry responses of invsive species to novel environmentl conditions, Morn & Alexnder (214) suggested tht significnt evolutionry chnges in relevnt trits re not likely to e oserved in <25 genertions. For exmple, Turner, Hufuer & Rieseerg (214) confirmed tht rpid evolution in growth nd reproductive output hs occurred in popultions of Centure diffus in the invded rnge, which were seprted from ntive popultions y yers, or pproximtely 5 genertions. Similrly, Dlugosh & Prker (28) found evidence for rpid evolution of growth rte, dte of first flowering nd size of Hypericum cnriense in wht they predicted to e 25 genertions following introduction. Our results suggest tht popultions of nnul invsive grsses cn show significnt chnges in response to environmentl conditions in s little s five genertions. Higher rtes of cron ssimiltion, erlier phenology nd higher reproductive output in weedy species under conditions of reduced precipittion could hve importnt implictions t the community level. In Mediterrnen climte ecosystems, such s our southern Cliforni site, rinfll occurs lmost exclusively during the winter months (Novemer to My) nd drought often rings reduced rinfll nd/or shorter rinfll period. Invsive species tht re le to complete their life cycle during shorter wet seson while mintining or, in our cse, enhncing reproductive output (A. rt) will hve n dvntge over species tht hve longer life cycles nd re dversely ffected y drought. Although A. rt my e more evolutionrily responsive to wter mnipultion thn B. mdritensis (displyed more phenotypic responses), the differences in solute trit vlues suggest tht B. mdritensis ws etter t executing drought escpe strtegy thn A. rt (lower LMA nd WUE, higher totl reproductive iomss nd lef N content compred to A. rt; Figs 2 nd 3). Consequently, B. mdritensis ws one of the most common species in reduced precipittion plots fter 5 yers (Goulden et l., unpul. dt). While we did not mesure trit vlues in co-occurring ntive species, the plot-level community composition dt suggest tht ntive grsslnd species my hve limited potentil to respond to drought on short time-scles. Future reserch should test for dptive evolution in co-occurring ntive nd invsive species to ssess the potentil impct tht rpid evolution my hve on community-level processes nd ecosystem function. Acknowledgements We thnk Julie Lrson nd Dine Cmpell for comments on the mnuscript. 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