Genetic and environmental interactions determine plant defences against herbivores

Transcription

1 Journl of Ecology 2011, 99, doi: /j x Genetic nd environmentl interctions determine plnt defences ginst herbivores Dniel J. Bllhorn 1,2 *, Stefnie Kutz 2,3, Mnfred Jensen 2, Imke Schmitt 1,4,5, Mrtin Heil 2,6 nd drin D. Hegemn 7 1 Deprtment of Plnt Biology, University of Minnesot, 250 Biologicl Sciences Center, 1445 Gortner ve., St. Pul, MN 55108, US; 2 Deprtment of Generl Botny Plnt Ecology, Universit t Duisburg-Essen, Universit tsstrße 5, Essen, Germny; 3 Deprtment of Zoology, Field Museum of Nturl History, 1400 South Lke Shore Drive, Chicgo, IL 60605, US; 4 Biodiversity nd Climte Reserch Centre BiK-F,Senckenbergnlge 25, Frnkfurt m Min, Germny; 5 Goethe University Frnkfurt m Min, Siesmyerstr. 70, Frnkfurt m Min, Germny; 6 Deprtmento de Ingenierı Gene tic, CINVESTV - Irputo Km. 9.6 Librmiento Norte, Crreter Irputo-Leo n, Irputo, Gunjuto, Mexico; nd 7 Deprtment of Horticulturl Science, University of Minnesot, 290 ldermn Hll, 1970 Folwell venue, St. Pul, MN 55108, US Summry 1. Plnts express multiple defensive trits, but little is known bout the genetic stbility nd phenotypic plsticity of these trits in nture. To investigte sources of vrition nd their potentil ecologicl consequences for herbivores, we combined field observtions of cynogenic lim ben with lbortory experiments. 2. Field studies in South Mexico reveled distinct vribility of cynogenic trits within nd mong wild lim ben popultions. To differentite mong genetic vrition nd the impct of mbient conditions on plnt phenotypes, we used seed-grown plnts s well s clones propgted from high- (HC) nd low-cynogenic (LC) wild type plnts. 3. In growth chmber experiments, we cultivted plnts under three intensities ech of drought nd slt stress, nutrient supply, nd light vilbility. We consecutively quntified cynogenesis nd totl phenolics s chemicl defences, soluble proteins s mesure of lef qulity, nd lef mss per re nd tissue hydrtion s physicl defence-ssocited trits. 4. Plnt trits were geneticlly stble, s clones propgted from the HC- nd LC-mother plnts mintined their cynogenic fetures nd lso showed constncy of other lef prmeters tested. 5. In contrst to geneticlly heterogeneous seed-grown plnts, cynogenesis nd soluble protein concentrtion in clonl plnts showed significnt vrition in response to reduced wter supply, incresed soil slinity, nd fertiliztion. The other lef trits, however, showed no significnt phenotypic plsticity depending on tretment. 6. mong ll trits nlysed, genetic nd phenotypic vrition in cynogenesis proved the best predictor of herbivore behviour nd development, s LC-plnts were preferred by dult Mexicn ben beetles nd llowed for fster lrvl development. 7. Synthesis. We demonstrte tht (i) functionl nlyses of plnt responses to biotic fctors require methodicl seprtion of genotypic vribility nd phenotypic plsticity, (ii) different biotic prmeters quntittively ffect the plnts chemicl phenotype nd tht (iii) chnges of plnt phenotype cn hve strong impcts on nturl herbivores. Our results suggest tht genetic vribility nd phenotypic plsticity of plnt nti-herbivore defences llow plnt popultions to rpidly respond to chnging environmentl conditions. Key-words: cynogenesis, Epilchn vrivestis, genotype, globl chnge, Phseolus luntus, phenolics, phenotypic plsticity, plnt herbivore interctions, resource vilbility hypothesis, slinity *Correspondence uthor. E-mil: bllhorn@umn.edu Ó 2010 The uthors. Journl of Ecology Ó 2010 British Ecologicl Society

2 314 D. J. Bllhorn et l. Introduction Plnts cnnot escpe when ttcked by pthogens nd herbivores or when exposed to unfvourble mbient conditions. However, they re fr from being pssive orgnisms. In the lst decdes, reserch hs demonstrted how plnts cn ctively up-regulte wide rry of defensive trits when ttcked by herbivores or pthogens or being subjected to biotic stress fctors (e.g. Hrtley et l. 2001; Bllhorn, Heil & Lieberei 2006; Sdtmnd et l. 2008). Until now, little is known of the interply between genetic control nd phenotypic plsticity of plnt defence trits, lthough both fctors re importnt for short-term dpttions nd the evolution of plnt defensive syndromes (grwl & Fishbein 2006; grwl, Ljeunesse & Fishbein 2008). Phytochemicl chnges in response to vrition of biotic conditions my hve significnt implictions for plnt pltbility nd defence (e.g. Hughes & Bzzz 1997; Stevens, Wller & Lindroth 2007). The extent to which biochemicl plnt responses to chnging biotic conditions hve quntittive effects on nturl plnt herbivore interctions is, however, still poorly understood (e.g. Tikknen & Julkunen-Tiitto 2003; Thylinkis et l. 2008). Given the rpidly ccelerting environmentl chnges on locl nd globl scles, there is strong demnd for better understnding of the consequences of plnts plsticity on higher trophic levels. nlysing the impct of externl fctors on plnt trits, nd consequently on plnt herbivore interctions, is often complicted by the high genotypic vribility of plnt trits in nturl popultions (e.g. Donldson & Lindroth 2007). Thus, functionl nlyses require experimentl seprtion of genetic vribility nd phenotypic plsticity to ddress sources of vrition. Lim ben (Fbcee: Phseolus luntus L.) hs been used s n ecologicl model to study indirect defences vi tritrophic interctions (Heil 2008). The elicittion of the production of herbivore-induced voltile orgnic compounds (VOCs) nd extrflorl nectr (EFN) benefits this species under nturl growing conditions (Heil 2004; Yi et l. 2009). In ddition to indirect defences, lim ben expresses direct defensive trits such s cynogenesis. To dte, defensive function ginst herbivores could be unmbiguously demonstrted for cynogenesis (Bllhorn & Lieberei 2006; Bllhorn et l. 2009) nd EFN secretion (Kost & Heil 2008), but not for the relese of VOCs (but see Heil 2004). Voltile orgnic compounds re likely to ct minly s within-plnt signlling fctors in this species nd prime rther thn induce defences (Heil & Silv Bueno 2007). Interestingly, the nti-herbivore defence of lim ben is subject to vrious trde-offs, tht is, negtive correltions mong different defensive trits. For exmple, we found tht cynogenesis is negtively correlted with the relese of VOCs (Bllhorn et l. 2008,b) nd the plnts resistnce to pthogens vi polyphenol oxidse ctivity (PPOs; enzymes tht oxidise phenolic compounds to rective quinones) (Bllhorn, Pietrowski & Lieberei 2010b). In the present study, we combined quntittive nlyses of the cynogenic fetures of wild lim ben t nturl sites in South Mexico with lbortory experiments. We investigted the cuses (i.e. genetic vribility or phenotypic plsticity) nd consequences (on prticulr herbivore plnt interction) of the observed defensive trit vrition. To nlyse the impct of biotic fctors tht purportedly ffect plnt phenotypes, we experimentlly ltered wter vilbility, soil slinity, nutrient supply nd light intensity under growth chmber conditions. Plnts derived from seed mteril collected in the field (thus exhibiting nturl genetic vribility) s well s clonl plnts propgted from stem cuttings from one high (HC) nd one low cynogenic (LC) mother plnt were ech included in the nlyses. Plnts grown from seedlings enbled the ssessment of lim ben s responses to chnging biotic conditions t the intrspecific level, wheres clonl plnts llowed for the exclusion of genotypic vribility nd, thus, quntittive estimtions of phenotypic plsticity. To ddress the vrition of plnt defences t nturl sites, we focussed on cynogenic potentil (HCNp; concentrtion of cynogenic precursors) s chrcteristic nd efficient ntiherbivore defence of wild lim ben (Bllhorn et l. 2008c, 2009). In lbortory experiments, we included n rry of dditionl trits tht potentilly ffect the pltbility to herbivores. Besides HCNp, we quntified totl phenolics s further chemicl defensive trit (Bllhorn et l. 2007). While cynogenesis represents nitrogen-bsed direct plnt defence ginst herbivores (Møller & Seigler 1999), phenolics re crbon-bsed compounds tht inhibit the digestion of protein by mny herbivores (e.g. Nomur & Itiok 2002; Mtsuki, Sno & Koike 2004). Both defences re quntittively ffected by biotic fctors, such s soil moisture (e.g. Foulds 1982), slinity (e.g. Keymer & Ellis 1978) s well s nutrient nd light vilbility (e.g. Schreiner, Nfus & Pimentel 1984; Frehner et l. 1997). In ddition to quntittive or qulittive expression of plnt defences, overll ttrctiveness of plnts to herbivores is determined by the plnts nutritive chrcteristics (e.g. wmck & Lether 2002). To consider this point we mesured the concentrtion of soluble (nd esily digestible) proteins s n importnt mrker for the nutritive vlue of lef tissue (Mttson 1980; Gnzhorn 1992). Previous experiments suggest tht the concentrtion of soluble proteins is correlted with the overll nitrogen content in lim ben leves (D.J. Bllhorn, unpublished dt). Furthermore, we quntified lef mss per re (LM) nd tissue hydrtion of plnts exposed to different tretments, since these fctors mutully ffect ttrctiveness to herbivores (e.g. Goodger, Choo & Woodrow 2007). Herbivore response to plnt defence ws quntified using the Mexicn ben beetle (Coccinellide: Epilchn vrivestis Muls.), which is nturl herbivore of lim ben. This experimentl system hs proven to be highly efficient for nlysing quntittive effects of chemicl plnt trits on herbivores (Bllhorn & Lieberei 2006; Bllhorn et l. 2007, 2008c; Bllhorn, Kutz & Lieberei 2010). To our knowledge, this is the first comprtive pproch to nlyse the quntittive impct of multiple biotic fctors on different defensive nd nutritive plnt fetures t the genotypic nd phenotypic level, nd to ssess quntittive effects

3 biotic fctors ffect plnt resistnce to herbivores 315 on performnce nd choice behviour of nturl insect herbivore. 120 b c Mterils nd methods VRIBILITY OF LIM BEN CYNOGENESIS IN NTURE Different nturl lim ben popultions in South Mexico (stte Oxc) were screened for their HCNp in October 2007 (for nlysis of HCNp see below). We compred four different popultions (Fig. 1). Two popultions were locted in the vicinity of Puerto Escondido (site PTE1: N, W, 8 m.s.l. nd PTE2: N, W, 20 m.s.l.) ner the Pcific cost lmost t se level, nother costl popultion (site RG: N, W, 106 m.s.l.) ws locted on hill ner the villge Rio Grnde, wheres the fourth popultion (site MR) ws locted close to Mtis Romero in the Isthmus of Tehuntepec ( N, W, 202 m.s.l.). t sites PTE1 nd PTE2, lim ben plnts were prtly shded by surrounding shrubs nd trees, wheres site RG ws locted on dry hill with sprse nd smll vegettion. Sixteen to 26 plnt individuls per site were included in the nlyses (for number of plnts included in this study see Fig. 2). We selected young, fully unfolded leves (N = 1 trifolite lef per plnt) inserting three positions down from the pex of the min shoot to reduce uncontrolled vrition of HCNp due to ontogenetic vribility of leves (Bllhorn, Heil & Lieberei 2006). HCNp (μmol HCN g 1 fresh wt.) PTE1 PTE2 RG MR Lim ben sites Men Men ± SE Men ± SD Outliers Extremes Fig. 2. Cynogenic potentil in different lim ben popultions. Lim ben plnts t four sptilly seprted popultions (PTE1, PTE2, RG, nd MR) were screened for cynogenic fetures of defined lef developmentl stges. Letters in the upper prt of the figure indicte significnt differences of plnt trits mong popultions [ccording to lest significnt difference (LSD) post hoc nlysis (P < 0.05) fter one-wy nov; PTE1:N = 22, PTE2: N =26, RG: N =17,ndMR:N = 24 plnts per site]. PLNTS ND INSECTS In controlled growth chmber experiments, we used seed-grown nd clonl lim ben plnts. Seed mteril ws collected t site PTE1 from lrge number of mother plnts (>50 plnts). The test of seeds ws scrtched with rzor blde to fcilitte wter bsorption nd to ensure homogenous germintion of seeds. Plnts were cultivted in growth chmber (Thermotec-Weilburg GmbH & Co.KG., Weilburg, Germny) djusted to resemble conditions t nturl sites in Mexico s recorded for September to October 2007 with light regime of 13:11 L:D under photon flux density of lmol photons m )2 s )1 t tble height. Light in the chmber ws provided by combintion Oxc 179 Mtis Romero Site MR Site RG Slin Cruz 200 Rio Grnde Puerto Escondido 200 Costline Rod 200 City/villge Collection site Site PTE1 Site PTE2 Pcific Ocen Fig. 1. Loclity of wild lim ben popultions investigted in South Mexico. Four popultions of wild lim ben plnts were nlysed for their cynogenic potentil (HCNp). Three popultions (RG, PTE1, PTE2) were locted t the pcific cost of South Mexico, while the fourth (MR) ws situted in the Isthmus of Tehuntepec ner Mtis Romero.

4 316 D. J. Bllhorn et l. (1:1) of HQI-BT 400 W (Osrm) nd RNP-T LR 400 W (Rdium) lmps. Temperture ws 30:23 C (i.e. temperture of 30 C in the light period nd 23 C in the drk period) nd we mintined n ir humidity of 70 80%. Plnts were grown in continers (one plnt per pot) with 12 cm in dimeter in 1:1 rtio of stndrd substrte (TKS Ò -1-Instnt, Florgrd Ò, Oldenburg, Germny) nd snd (grin size mm). For the production of clonl plnts, we used one high cynogenic (HC) nd one low cynogenic (LC) plnt derived from the nturl popultion t PTE1. Mother HC- nd LC-plnts ech hd developed 6 brnch shoots ech t the time of propgtion. For propgtion, plnts were cut into segments with one lef ech. These segments were rooted in plstic trys (40 60 cm) contining 1:1 mixture of stndrd substrte nd snd. Cuttings were shded nd kept t n mbient temperture of 23 C under constnt wter supply. When cuttings hd developed roots (fter c. 10 dys), individul cuttings were trnsferred to plnt continers (12 cm dimeter) nd plced in the growth chmber. For ech group of experimentl plnts (seed-grown plnts, HC- nd LC-clones), we cultivted N = 63 individuls. Plnts of ll three groups were cultivted simultneously in the growth chmber nd the position of plnts ws chnged every 3 dys to void ny position effects. Experiments were conducted when plnts hd developed five secondry leves (fter 4 weeks in the growth chmber). We used young, fully unfolded leves (N = 1 trifolite lef per plnt) of the sme ontogenetic stge s those smpled in field studies. Mexicn ben beetles (Coccinellide: Epilchn vrivestis Muls.) were mintined on non-cynogenic snp ben (Fbcee: Phseolus vulgris vr. Sx) to prevent development of ny preference for low or high cynogenic lim bens. Beetles were held under the sme growth chmber conditions s plnts. In choice experiments, we used dult beetles in rndom sex-rtios, wheres in experiments on insect performnce, we used freshly htched lrve. PLNT TRETMENTS Experiments were crried out in the growth chmber under mbient conditions s mentioned bove. Nine tretment groups of seed-grown nd HC- nd LC-clonl plnts (G1 G9; N = 7 plnts per tretment, N = 189 plnts in totl) were fertilized with 50 ml of 0.1% queous solution of Flory-3 Ò (NPK-Fertilizer, EUFLOR GmbH, Munich, Germny) twice week nd wtered dily with 100 ml tp wter per plnt (with the exception of G2 nd G3, which were supplied with reduced mounts of wter, i.e nd 12.5 ml per dy, respectively). In ddition, to this bsic tretment, experimentl groups of plnts were treted with different concentrtions of queous NCl solutions [G4, G5: 0.3 nd 0.6% NCl solution (50 ml) twice week] nd two levels of nutrient supply [G6, G7: 0.3 nd 0.6% Flory-3 solution (50 ml) insted of regulr fertiliztion twice week]. Two groups of plnts (G8, G9) were shded by single- nd doublelyer net (Schttenleinen; Herwig GmbH, Zittu, Germny) tht let pss c. 190 nd c. 70 lmol photons m )2 s )1, respectively. Group (G1) served s control nd ws held under bsic conditions (totl of 100 ml wter per dy of which 50 ml were substituted with 50 ml of 0.1% fertilizer twice week; full light exposure). Solutions dropping from plnt continers were collected in bsins under the continers (tht hd no contct to collected solutions) nd removed dily. To minimize potentil concentrtion effects of slt- nd fertilizer-treted plnts in the soil, substrtes of ll plnts of these tretment groups (G4 G7) were flushed with pure wter (1 L) once week directly before tretment with the respective slt nd fertilizer solutions. Quntittive fluctutions of slt nd fertilizer concentrtion (nd potentil effects on plnt trits) due to flushing were ssumed to be low, since desired concentrtions of slt nd fertilizer in the soil were immeditely re-djusted. SMPLING OF LEVES Cynogenic potentil, totl phenolics, soluble protein concentrtions, LM nd tissue hydrtion were nlysed for individul leves of seed-grown nd HC- nd LC-clonl plnts used in feeding trils. Thus, responses of herbivores could be directly linked to lef trits. One leflet of ech trifolite lef ws rndomly selected nd removed for biochemicl nd physicl nlyses. second leflet ws removed for feeding choice experiments, wheres the third leflet remined ttched to the plnt for ssessing herbivore performnce. Chemicl chrcteristics of the two leflets used in the different trils (choice nd performnce) were ssumed to be the sme s in the leflet nlysed, since previous studies showed high quntittive homogeneity in biochemicl prmeters mong leflets of individul trifolite leves (Bllhorn et l. 2007, 2009). We dditionlly mesured totl mounts of cynide, phenolics nd protein produced per complete plnt to investigte bsolute investment in these trits. ll leves were cut-off fter the feeding experiments, nd pooled per plnt individul. To clculte the bsolute mount of these plnt compounds per plnt individul, vlues from defined leves used in feeding trils nd the pooled leves were dded. Primry leves were excluded from the nlyses, since these were only present in seed-grown plnts nd possess different chemicl chrcteristics thn secondry leves (Bllhorn et l. 2008, 2008b, 2008c). PLNT CYNOGENIC POTENTIL Lef cynogenic potentil ws nlysed by complete enzymtic degrdtion nd subsequent quntittive mesurement of relesed cynide ccording to Bllhorn, Lieberei & Gnzhorn (2005). For hydrolysis of cynogenic glycosides in lef extrcts, we used b-glucosidse isolted from rubber tree (Euphorbicee: Heve brsiliensis) ccording to Bllhorn, Lieberei & Gnzhorn (2005). fter 20 min of incubtion t 30 C in gs-tight glss vessels (Thunberg-vessels) relesed cynide ws spectrophotometriclly mesured t 585 nm (Genesys 20, Thermo Spectronic, Mdison, WI, US) using the Spectroqunt Ò cynide test (Merck, Drmstdt, Germny). Single leves nd folige of entire plnts were both hrvested between 10:00 nd 10:45 m to exclude diurnl effects. TOTL PHENOLICS Totl phenolics were nlysed following Tikknen & Julkunen-Tiitto (2003). Homogentes of leves were extrcted three times for 15 min in 5 ml (individul leves) or 50 ml (lef mteril of entire plnts) cetone diluted with distilled wter (60:40). fter ech extrction, smples were incubted in n ultrsonic bth (3 min) nd finlly centrifuged for 10 min t 5000 g. The superntnt ws trnsferred to 2 ml (20 ml in cse of totl plnts) concentrted cetic cid (Merck KG, Drmstdt, Germny), cetone ws removed under vcuum (60 mbr) t 40 C, nd the residue ws quntittively trnsferred by using distilled wter. Smples were diluted with 2.5% cetic cid, nd 1 ml of this solution ws mixed with 0.5 ml Folin-Cioclteus phenol regent (Merck). fter dding 2 ml 20% N 2 CO 3,the solution ws dded to 10 ml with distilled wter. Smples were incubted t 70 C nd, fter cooling, spectrophotometriclly quntified t 730 nm ginst blnk contining wter insted of smple. Epicthechin (Sigm, Deisenhofen, Germny) t different concentrtions served s stndrd.

5 biotic fctors ffect plnt resistnce to herbivores 317 SOLUBLE PROTEIN CONCENTRTION Concentrtion of soluble proteins in leves nd plnts ws quntified ccording to Brdford (1976) with modifictions following Bllhorn et l. (2007). Brdford regent (Biord Lbortories, Munich, Germny) ws diluted 1:5 with ddh 2 Ond20lL ofechhomogenized plnt smple ws combined with 1 ml of diluted Brdford solution. Bovine serum lbumin (BS; Fluk Chemie G, Buchs, Switzerlnd) t different concentrtions served s stndrd. fter 5 min incubtion time, concentrtion of protein ws spectrophotometriclly mesured t 595 nm. We used the sme individul plnt extrcts for protein mesurements s for HCNp nlyses, thus, both prmeters could be quntittively ttributed to the sme smple. PHYSICL LEF TRITS ND MORPHOLOGICL PRMETERS Lef mss per re nd tissue wter content of individul leves of seed-grown nd clonl plnts used in feeding trils were mesured to ssess potentil effects of lef toughness (deduced from lef dry weight per re) nd wter content on performnce nd food plnt selection of the Mexicn ben beetles. Lef smples of defined re (lef discs, 1.2 cm in dimeter) were cut from the lef tissue weighed to the nerest g nd dried t 45 C to constnt weight. Lef smples were consecutively weighed for determintion of dry mtter. In ddition, we considered totl folige biomss, plnt height nd number of leves. ll leves per plnt were counted t the end of the experiment nd totl lef biomss (fresh weight) ws determined. Mesuring dry weight of totl lef biomss ws not possible, since fresh lef mteril ws required for chemicl nlyses. Plnt height ws determined by cutting plnts off t the substrte surfce nd mesuring the outstretched shoot. CHOICE BEHVIOUR OF HERBIVORES ttrctiveness of differently treted plnts ws nlysed in experiments where dult Mexicn ben beetles were offered choice between lef mteril from seed-grown nd clonl plnts of ll tretments (N = 9 lef discs per feeding tril, N = 7 feeding trils per group of experimentl plnts, i.e. seed-grown, HC- or LC-plnts). We used lef discs (16 mm in dimeter) to exclude ny effects on the beetles preference resulting from lef size or shpe. fter cutting the lef discs with cork borer, we wited for 30 min to llow evportion of gseous cynide from the cutting edges. For feeding trils, beetles were deprived of food for 2 h prior to the experiment nd then lef discs (one disc from one plnt of ech tretment) were offered to individul beetles for 2 h. Feeding trils were crried out in Petri dishes (14 cm in dimeter) lined with slightly moist filter pper. Order of lef discs derived from plnts of the different tretment groups (G1 G9) ws set t rndom. fter the experiment, lef discs were scnned on scle nd the missing lef re ws quntified using nlysis Ò softwre (Olympus, Hmburg, Germny). LRVL GROWTH ND PERFORMNCE Freshly htched first lrvl instrs of the Mexicn ben beetle were used for quntittive nlyses of herbivore biomss ccumultion nd performnce on differently treted seed-grown nd clonl plnts. Individul lrve were plced on one leflet, which ws then bgged with guze. Ech lrv ws weighed to the nerest g before nd fter the experiment (4 d) nd the bsolute increse in body weight ws considered. In ddition, to lrvl biomss ccumultion per time, ecdysis of lrve ws recorded to evlute differences in herbivore performnce. STTISTICL NLYSES To test for significnt differences in HCNp mong nturl lim ben popultions we pplied one-wy novs followed by lest significnt difference (LSD) post hoc nlyses. To nlyse effects of plnt tretment on plnt trits, we pplied one-wy nov followed by LSD post hoc nlyses for ech trit within ech group of plnts (i.e. seedlings, HC- or LC-clonl plnts). dditionlly, we used multivrite generlized liner model (GLM) design to nlyse effects of Cynogenic type (i.e. HC or LC), Tretment nd Cynogenic type Tretment on plnt trits. nlyses were crried out exclusively using clonl plnts nd seprtely for the respective tretments (i.e. reduced wter supply, slt tretment, fertiliztion nd shding) nd for plnt mteril (i.e. ontogeneticlly defined leflets nd totl plnts). For the nlysis of leflets, we set Cynogenic type, Tretment, nd Cynogenic type Tretment s fctors, while HCNp, protein concentrtion, concentrtion of totl phenolics, LM nd tissue hydrtion served s vribles. To nlyse effects on whole plnts, we set totl mount of cynide, phenolics, soluble proteins s well s totl biomss, plnt height nd lef number s vribles. To nlyse effects on choice behviour of dult beetles s well s on lrvl growth prmeters (body mss ccumultion nd ontogenetic development), we included lef consumption, lrvl weight gin nd lrvl performnce s vribles into GLM. To test for trde-offs between cynogenesis nd totl phenolics nd between cynogenesis nd protein concentrtion in untreted HC- nd LC-plnts (control groups; G1), we pplied Person s correltions. In ddition, to test for correltions of both trits mong plnts with nturl genetic vribility (seed-grown plnts), we pplied Person s correltion for dt on cynide nd totl phenolics. ll sttisticl nlyses were crried out using spss 13 (SPSS for Windows, SPSS Inc., Chicgo, US). Results VRIBILITY OF CYNOGENIC POTENTIL IN NTURE Cynogenic potentil (HCNp) of young leves showed substntil quntittive vribility within nd mong different lim ben popultions. In the popultions t sites PTE1 nd PTE2, HCNp rnged from 5.05 to nd 8.83 to lmol HCN g )1 fresh wt., t site RG vlues vried between nd 99.38, while HCNp t site MR rnged from 4.36 to lmol HCN g )1 fresh wt. (Fig. 2). mong popultions, we found significnt differences in HCNp (ccording to one-wy nov: F 3,85 = 7.946, P < 0.001). Post hoc nlysis (LSD, P < 0.05) reveled tht HCNp of lim ben plnts t site RG ws significntly higher compred to sites PTE1 nd PTE2, wheres HCNp t site MR ws significntly lower thn in the three other popultions (Fig. 2). Lim ben plnts t sites PTE1ndPTE2showednosignificntdifferencesinHCNp. VRIBILITY OF TRITS IN SEED-GROWN ND CLONL PLNTS Seed-grown lim ben plnts cultivted under growth chmber conditions showed similrly high vribility of HCNp

6 318 D. J. Bllhorn et l. HCNp (μmol HCN g 1 fresh wt.) Totl phenolics (mg g 1 fresh wt.) Soluble protein (mg g 1 fresh wt.) () B (b) (c) G1: control C B G2: wter D CD G3: wter γ B BC G4: slt + C B Seed-grown plnts HC clonl plnts LC clonl plnts CD D G5: slt ++ B B E G6: fertilizer + Tretment of plnts Fig. 3. Responses of seed-grown nd clonl plnts to vrition of biotic prmeters. Nine experimentl groups (G1 G9; N = 7 plnts per group) of seed-grown nd clonl (HC = high-cynogenic nd LC = low-cynogenic) lim ben plnts were cultivted under different conditions. Defined lef stges of plnts were nlysed for cynogenic potentil (HCNp) (), concentrtion of totl phenolics (b) nd soluble proteins (c). Vlues shown re mens±sd. Smll white circles represent extremes. mong tretments, differences in lef prmeters were nlysed seprtely for ech plnt type (seed-grown, HC, LC) nd significnt differences re indicted by different letters (lower-cse letters for seed-grown plnts, cpitl letters for HCplnts, nd Greek letters for LC-plnts) t the box plots [ccording to lest significnt difference post hoc nlysis (LSD; P < 0.05) fter one-wy nov]. γ F G7: fertilizer ++ δ G8: light E G9: light (Fig. 3) s plnts mesured in the field (Fig. 2). In the controls (G1), HCNp in young leves rnged from to lmol HCN g )1 fresh wt. (N = 7 plnts). Likewise, concentrtion of totl phenolics nd soluble protein in young leves of untreted seed-grown plnts showed substntil vrition (Fig. 3b,c). Concentrtion of phenolics rnged from 0.19 to 0.35 mg g )1 fresh wt., nd protein concentrtion rnged from to mg g )1 fresh wt. In contrst to seed-grown plnts, clonl propgtes exhibited consistent HCNp, totl phenolics nd protein concentrtions (Fig. 3 c). Compred to their respective mother plnt, men HCNp of clones (G1, control group) differed by only 3.39 ± 3.43% (men ± SD; HC-plnts) nd 2.42 ± 3.79% (LC-plnts). The mother plnts (one HC- nd one LC-plnt) showed HCNp of ± 2.14 nd ± 1.93 lmol HCN g )1 fresh wt., respectively (men ± SD; N = 6 leves per plnt ech derived from one brnch shoot). Concentrtion of totl phenolics in the mother plnts rnged between 0.19 ± 0.01 (HC-plnt) nd 0.29 ± 0.01 mg g )1 fresh wt. (LC-plnt; men±sd; N = 6 leves per plnt) nd showed differences of 0.60 ± 5.47% for HC- nd 0.28 ± 4.23% for LCplnts. lso, men protein concentrtions in young leves of HC- nd LC-clones ws highly similr to those of their mother plnts (HC-plnt: ± 0.13; LC-plnt: ±0.05 mg g )1 fresh wt.; men ± SD; N = 6 leves per plnt) nd showed only smll differences compred to the mother plnts (0.41 ± 0.76% for HC- nd 0.49 ± 0.90% for LC-plnts). COVRITION OF TRITS IN UNTRETED PLNTS mong ll tretment groups of seed-grown plnts, HCNp of young leves ws significntly negtively correlted to concentrtion of totl phenolics (ccording Person s correltion: r = )0.867, P < 0.001) nd to soluble protein concentrtion (r = )0.776, P < 0.001). Concentrtion of both phenolics nd protein ws higher in LC- thn in HC-plnts (Fig. 3b,c). Generlized liner model (GLM) reveled significnt effects of Cynogenic type (i.e. HC or LC) on phenolics nd protein in young leves (see Tble S1 in Supporting Informtion). Consequently, phenolics concentrtion ws significntly correlted to concentrtion of proteins (r = 0.700, P < 0.001). Ptterns observed for young leves held true t the entireplnt level (see Tble S2). Totl production of cynide ws negtively correlted with totl mounts of phenolics (r = )0.338, P < 0.01) nd protein (r = )0.309, P <0.05) per plnt, while phenolics nd protein were significntly correlted (r = 0.559, P < 0.001). In contrst to covrition of chemicl trits, physicl lef trits showed no significnt correltion with the lef chemicl prmeters. Lef mss per re nd tissue hydrtion were homogenous mong seed-grown nd clonl plnts (see Tble S3). However, morphologicl prmeters such s totl plnt biomss, plnt height nd lef number were ffected by Cynogenic type (ccording to GLM; see Tble S4). mong ll tretments, LC-plnts showed higher vlues thn HCplnts for ll three prmeters (Tble 1).

7 biotic fctors ffect plnt resistnce to herbivores 319 Tble 1. Morphologicl plnt prmeters. Plnt s morphologicl chrcteristics were mesured mong the different tretment groups (G1 G9; N = 7 plnts per group). Vlues shown re mens±sd. Smll-typed letters indicte significnt differences in plnt prmeters mong tretments per plnt type [i.e. seed-grown plnts, high cynogenic (HC) or low cynogenic (LC) clonl plnts] ccording to lest significnt difference post hoc nlysis (LSD; P < 0.05) fter one-wy nov. Plnt group Tretment group Tretment Plnt biomss [g )1 fresh wt.] Plnt height [cm] Lef number Seed-grown G1 Control 5.91± ± ±0.5 - G2 H 2 O[)] 5.86± ± ±1.3 - G3 H 2 O[))] 5.71± ± ±1.0 - G4 NCl [+] 5.90± ± ±1.0 - G5 NCl [+ +] 5.86± ± ±0.8 - G6 Fertilizer [+] 6.02± ± ±0.5 - G7 Fertilizer [+ +] 6.01± ± ±0.5 - G8 Light [)] 5.92± ± ±0.8 - G9 Light [))] 5.90± ± ±0.5 HC-clones G1 Control 5.88± ± ±0.4 - G2 H 2 O[)] 5.79± ± ±0.4 - G3 H 2 O[))] 5.79± ± ±0.5 - G4 NCl [+] 5.82± ± ±0.5 - G5 NCl [+ +] 5.76± ± ±0.5 - G6 Fertilizer [+] 5.93± ± ±0.0 - G7 Fertilizer [+ +] 6.03± ± ±0.5 - G8 Light [)] 5.87± ± ±0.4 - G9 Light [))] 5.93± ± ±0.4 LC-clones G1 Control 6.18± ± ±0.4 - G2 H 2 O[)] 6.15± ± ±0.7 - G3 H 2 O[))] 6.10± ± ±0.4 - G4 NCl [+] 6.15± ± ±0.5 - G5 NCl [+ +] 6.10± ± ±0.5 - G6 Fertilizer [+] 6.21± ± ±0.5 - G7 Fertilizer [+ +] 6.21± ± ±0.8 - G8 Light [)] 6.19± ± ±0.6 - G9 Light [))] 6.18± ± ±0.5 TRETMENT EFFECTS ON SEED-GROWN PLNTS In seed-grown plnts, we found no significnt tretment effects on chemicl nd physicl trits neither t the level of young leves nor t the whole-plnt level [ccording to one-wy nov: (HCNpofleves):F 8,54 = 0.367, P = 0.934; (totl cynide in plnts): F 8,54 = 0.220, P = 0.986; (phenolic concentrtion in leves): F 8,54 = 2.054, P = 0.057; (mount of phenolics in plnts): F 8,54 = 0.191, P = 0.991; (protein concentrtion in leves): F 8,54 =0.444,P = 0.889; (totl protein per plnt): F 8,54 =1.896, P = 0.058; (lef mss per re): F 8,54 = 0.300, P = 963; (tissue hydrtion): F 8,54 = 0.156, P = 0.995]. TRETMENT EFFECTS ON CLONL PLNTS In clonl plnts significnt tretment effects were observed (Fig. 3). Vrition of wter supply, slinity, nd nutrient vilbility quntittively ffected HCNp nd protein concentrtion in both HC- nd LC-plnts s well s in young leves nd in whole plnts. Vrition of light vilbility, in contrst, hd no significnt effect on ny of the plnt prmeters mesured. With focus on HCNp of young leves, seprte nlysis of HC- nd LC-plnts reveled significnt differences mong tretment groups [ccording to one-wy nov (HC-plnts): F 8,54 = 7.32, P < 0.001; (LC-plnts): F 8,54 =6.77, P < 0.001; Fig. 3). Reduced wter supply (G3), incresed slinity (G5), or enhnced nutrient supply (G7) resulted in significntly incresed HCNp in young leves of both clonl lines (Fig. 3). For LC-plnts, even slightly reduced wter vilbility resulted in significntly incresedhcnp(g2)[ccording to LSD post hoc nlysis (P < 0.05) fter one-wy nov]. Plnt cynide-production responses t the level of young leves were lso confirmed for entire plnts [ccording to one-wy nov (HC-plnts): F 8,54 = 3.532, P = 0.002; (LC-plnts): F 8,54 = 4.077, P < 0.001; see Tble S2]. For both clonl lines, GLM reveled significnt tretment effects on the totl mount of cynide ccumulted per plnt (see Tble S4). Strictly reduced wter supply (G3), high slt tretment (G5) nd incresed nutrient supply (G7) significntly enhnced totl cynide in HC- nd LC-clonl plnts compred to the controls [ccording to post hoc nlysis (LSD, P < 0.05) fter one-wy nov;seetbles2]. Soluble protein concentrtions in young leves of HC- nd LC-plnts showed significnt differences mong tretment groups [ccording to one-wy nov (HC-clones): F 8,54 = , P < 0.001; (LC-clones): F 8,54 = , P < 0.001; Fig. 3c]. In HC-clones, protein concentrtion significntly decresed in response to strictly reduced wter supply (G3) nd slt tretment t higher concentrtion (G5),

8 320 D. J. Bllhorn et l. wheres it significntly incresed in both nutrient-supplied groups (G6 7). In leves of LC-plnts, protein concentrtion decresed significntly in response to the lower slt concentrtion tretment (G4) nd incresed in both nutrient-supplied groups (G6 7) [ccording to LSD post hoc nlysis (P < 0.05) fter one-wy nov; Fig. 3c]. t the level of whole plnts protein ccumultion lso showed significnt differences depending on tretment [ccording to one-wy nov (HC-plnts): F 8,54 = 2.429, P = 0.025; (LC-plnts): F 8,54 = 2.170, P = 0.044; see Tble S2]. The mount of protein produced per plnt incresed in response to nutrient supply (G6 7) nd decresed in plnts growing under wter or slt stress. However, with exception of incresed protein production following the high nutrient tretment (G7), these differences were not significnt compred to the controls [ccording to post hoc nlysis (LSD, P < 0.05) fter one-wy nov;seetble S2]. None of the tretments hd significnt effect on totl phenolics in either leves or entire plnts (ccording to GLM; see Tbles S1 nd S4). mong ll tretment groups of HCnd LC-plnts, no significnt differences in totl phenolics concentrtion in young leves were detected [ccording to one-wy nov (HC-plnts): F 8,54 = 1.845, P = 0.085; (LC-plnts): F 8,54 = 2.054, P = 0.057; Fig. 3]. For entire plnts, no differences in bsolute ccumultion of phenolics were observed [ccording to one-wy nov; HC-plnts: F 8,54 = 0.301, P = 0.999; LC-plnts: F 8,54 = 0.191, P = 0.991; see Tble S2]. Furthermore, for both clonl lines no tretment effects on normlized LM nd tissue hydrtion were observed (ccording to GLM; see Tble S1). mong ll tretment groups, we found no significnt differences in LM [ccording to one-wy nov (HC-plnts): F 8,54 = 0.829, P =0.580; (LC-plnts): F 8,54 =1.387, P = 0.223] or tissue hydrtion [(HC-plnts): F 8,54 = 0.894, P = 0.528; (LC-plnts): F 8,54 = 0.280, P = 0.952]. MORPHOLOGICL PLNT PHENOTYPE mong ll plnt types (seed-grown, HC- nd LC-plnts) evlution of totl lef biomss, plnt height nd number of leves reveled the highest vlues for fertilized, seed-grown nd clonl plnts (G6 7), wheres wter-deprived nd slt-treted plnts tended to show lower vlues (Tble 1). However, per plnt type,tretmentsppliedhereresultedinnosignificntdifferences of plnt morphology for ll considered prmeters t the time of nlysis (Tble 1). CHOICE BEHVIOUR OF BEETLES The ttrctiveness of lef mteril from seed-grown plnts to dult Mexicn ben beetles (i.e. lef consumption per time) ws different mong plnt tretments (Fig. 4). However, differences in lef re consumption were not significnt (ccording to one-wy nov: F 8,54 = 1.347, P = 0.241). mong plnts of ll tretments, (N = 63) lef consumption by beetles ws significntly negtively correlted to HCNp (ccording to two-tiled Person s correltion: r = )0.868, P < 0.001) nd Consumed lef re (cm 2 ) Weight gin of lrve (mg) () (b) B G1: control BC G2: wter γ Seed-grown plnts HC clonl plnts LC clonl plnts B C G3: wter γ C D G4: slt + γ positively correlted to lef protein concentrtion (r = 0.695, P < 0.001). For clonl plnts, Cynogenic type hd significnt effect on beetle-lef consumption (ccording to GLM, see Tble S5). In ddition, mong ll tretment groups with exception of plnts growing under reduced light vilbility Tretment of plnts significntly ffected feeding of insects (see Tble S5). For both clonl lines, the mount of consumed lef mteril ws significntly different mong the tretment groups [ccording to one-wy nov (HC-clones): F 8,54 = 2.624, B C B CD G5: slt ++ γ B G6: fertilizer + Tretment of plnts Fig. 4. Feeding choice behviour nd weight gin of herbivores. Lef re consumption of dult Mexicn ben beetles () ws nlysed in free-choice feeding trils. Lef mteril of ll nine tretment groups (G1 G9) from seed-grown nd high- (HC) nd low- cynogenic (LC) clonl lim ben plnts (N = 7 feeding trils per tretment) ws offered simultneously to the insects. In ddition, lrvl biomss ccumultion (b) ws mesured over time period of 2 dys on differently treted seed-grown, HC- nd LC-plnts (N = 7 lrve per tretment nd plnt type). Vlues shown for lrvl body mss nd lef consumption of dult beetles re given s men±sd. Smll white circles represent extremes. mong tretments, differences in lef re consumption nd lrvl biomss ccumultion were nlysed seprtely for ech plnt type (seed-grown, HC, LC) nd significnt differences re indicted by different letters (lower-cse letters for seed-grown plnts, cpitl letters for HC-plnts, nd Greek letters for LC-plnts) t the box plots [ccording to lest significnt difference post hoc nlysis (LSD; P < 0.05) fter one-wy nov]. γ δ D G7: fertilizer ++ B G8: light C G9: light

9 biotic fctors ffect plnt resistnce to herbivores 321 P = (LC-clones): F 8,54 = 5.450, P < 0.001]. Wterdeprived (G2 3), slt-treted (G4 5) nd extensively fertilized plnts (G7) of both clonl lines were significntly less consumed thn the controls (Fig. 4). Lef consumption ws negtively correlted to HCNp [ccording to two-tiled Person s correltion (HC-plnts): r = )0.313, P < 0.05; (LC-plnts):r = )0.385, P < 0.05, ech N = 63 feeding trils] but showed no significnt correltion to ny of the other lef prmeters tested. LRVL BIOMSS CCUMULTION Lrve developing on wter-deprived nd slt-treted seedgrown plnts (G2 5) s well s on extensively fertilized seedgrown plnts (G7) showed lower biomss ccumultion thn lrve feeding on control (G1) or shded plnts (G8 9) (Fig. 4b). mong plnt tretments, however, differences in lrvl weight gin were not significnt (ccording to one-wy nov: F 8,54 = 1.256, P = 0.286). Insect biomss ccumultion mong ll tretments (N = 63) ws significntly negtivelycorreltedtohcnpinleves(ccordingtotwotiled Person s correltion: r = )0.766, P < 0.001; N =63 feeding trils) nd positively correlted to lef proteins (r = 0.621, P < 0.001). For clonl plnts, GLM reveled significnt effect of Cynogenic type on lrvl biomss ccumultion (see Tble S5). mong ll tretments, weight gin of lrve feeding on HC-clones ws lower thn tht of those feeding on LCclones. In ddition, to Cynogenic type, Tretment of plnts quntittively ffected feeding of insects with the exception of experimentlly vried light intensity (see Tble S5). Differences in lrvl weight gin were highly significnt mong plnt tretments [ccording to one-wy nov (HC-plnts): F 8,54 = , P < 0.001; (LC-plnts): F 8,54 = , P <0.001; Fig. 4b]. For both HC- nd LC-clonl lines, lrve tht were feeding on strictly wter-deprived (G3) nd slt-treted plnts (G4 5) showed significntly lower biomss ccumultion thn on the controls, wheres slightly incresed nutrient supply (G6) resulted in enhnced lrvl weight gin tht ws significnt for LC-plnts (Fig. 4b). Slightly reduced wter supply (G2) nd extensive fertiliztion (G7) resulted in reduced yet not significnt biomss ccumultion. Within clonl lines, lrvl weight gin ws significntly negtively correlted to HCNp [ccording to Person s correltion (HC-plnts): r = )0.552, P < 0.001; (LC-plnts, HCNp): r = )0.570, P < 0.001], nd positively correlted to protein concentrtion in HC-plnts (r = 0.458,P < 0.001) yet not in LC-plnts (r = 0.157, P =0.220;N =63).For both clonl lines, biomss ccumultion of lrve nd lef consumption of dult beetles in choice-feeding trils were significntly correlted (HC-clones: r = 0.255, P < 0.05; LC-clones: r = 0.510, P < 0.001). LRVL DEVELOPMENT mong plnt tretment groups of seed-grown plnts, we observed differences in lrvl development (Tble 2). On Tble 2. Ontogenetic development of lrve. Numbers of moulting lrve were counted mong different tretments (G1 G9; N = 7 lrve per group) nd percentge of moulting individuls ws clculted. Smll-typed letters indicte significnt differences of moulting incidents mong tretments per plnt type [i.e. seed-grown plnts, high cynogenic (HC) or low cynogenic (LC) clonl plnts] ccording to lest significnt difference post hoc nlysis (LSD; P < 0.05) fter one-wy nov. Plnt type Tretment group Tretment Number of moulting lrve Seed-grown G1 Control G2 H 2 O[)] G3 H 2 O[))] G4 NCl [+] G5 NCl [+ +] G6 Fertilizer [+] G7 Fertilizer [+ +] G8 Light [)] G9 Light [))] HC-clones G1 Control G2 H 2 O[)] G3 H 2 O[))] G4 NCl [+] G5 NCl [+ +] G6 Fertilizer [+] G7 Fertilizer [+ +] G8 Light [)] G9 Light [))] LC-clones G1 Control 3 b G2 H 2 O[)] 3 b G3 H 2 O[))] G4 NCl [+] 2 b G5 NCl [+ +] G6 Fertilizer [+] 5 b G7 Fertilizer [+ +] G8 Light [)] 4 b G9 Light [))] 3 b 42.9 Percentge of moulting lrve seed-grown plnts, ll lrve feeding on strictly wter-deprived (G3) nd slightly slt-treted plnts (G4) filed to develop pst the first lrvl stge, while those feeding on slightly wterdeprived (G2) s well s on strongly slt-treted nd nutrientsupplied plnts (G5, G7) reched the second instr (t frequency of one in seven lrve). On extensively shded plnts nd on the controls (G9, G1) two lrve ech reched the second stge, while on slightly nutrient-supplied nd shded plnts three individuls ech reched this stge (G6, G8). For seed-grown plnts, differences in lrvl development mong tretment groups were not significnt (ccording to one-wy nov: F 8,54 = 1.113, P = 0.369). Independent of plnt tretment, lrvl development ws significntly negtively correlted with plnt HCNp (ccording to Person s correltion: r = )0.664, P < 0.0), while it ws positively correlted with soluble protein concentrtion (r = 0.657, P < 0.001). For clonl plnts, GLM reveled significnt effect of Cynogenic type nd Tretment on lrvl development (see Tble S5). While Tretment ws source of vrition exclusively in differently nutrient-supplied plnts, ll other

10 322 D. J. Bllhorn et l. tretments showed no significnt effects (ccording to GLM, see Tble S5). Limited tretment effects on lrvl development s predicted by GLM were due to different performnce on HC- nd LC-clonl plnts. This cn be explined by the lck of developmentl progression beyond the first developmentl stge of ll lrve feeding on HC-plnts of ll tretment groups (Tble 2). For LC-plnts, differences in development were significnt mong tretment groups [ccording to post hoc nlysis (LSD, P < 0.05) fter one-wy nov; Tble 2]. Lrve feeding on plnts exposed to reduced wter supply (G3), high slt tretment (G5) nd fertiliztion (G7) showed significntly slower development thn lrve feeding on plnts fertilized t lower intensity (G6). These differences were not significnt, however, when compred to the controls (G1). For ll tretment groups during the experimentl period, 23, 13 nd 0 lrve reched the second instr on LC-clones, seedgrown plnts nd HC-clones, respectively (Tble 2). mong LC-plnts, development of lrve ws significntly negtively correlted to HCNp (ccording to Person s correltion: r = )0.570, P < 0.001), while the other lef prmeters tested showed no significnt correltion to the performnce of lrve. Discussion In nturl systems, nlyses of plnt responses to biotic conditions t smll nd lrger scles nd nlyses of the consequences of these responses for higher trophic levels re complicted by vrious fctors (Bllhorn et l. 2009b). mong these, overlpping effects of the plnts genetic vribility (i.e. genotypic vrition), ontogenetic vribility (vrition depending on developmentl stges of plnts nd or specific plnt orgns) nd phenotypic plsticity (here defined s vrition depending on externl fctors) re mjor sources of vrition tht hve to be considered nd seprted in functionl nlyses of plnt trits (Schlichting & Pigliucci 1998; Busk & Møller 2002; Bllhorn, Kutz & Rkotorivelo 2009c; Bllhorn et l. 2009b). Lim ben plnts in nture (Fig. 2) s well s seed-derived experimentl plnts (Fig. 3) showed high genetic vribility in ll trits investigted. t the sme time, the vrition of trits ws geneticlly bsed s clonl propgtes retined the sme trits s the mother plnts. Genetic bsis of cynogenesis hs lso been found for other plnt species, s for exmple tropicl Prunus turnerin (Miller, Gledow & Woodrow 2004). The nturl vrition of HCNp mong lim ben individuls ws prticulrly extensive, with the highest vlues being 16.8 fold higher thn the lowest vlues. Under controlled growth chmber conditions, experimentlly chnged biotic fctors such s nutrient supply, drought stress, slinity nd light hd no significnt effects on plnt trits in seed-grown plnt mteril (Fig. 3). By contrst, highly significnt tretment effects on plnt trits such s HCNp nd soluble protein were found once genetic vribility hd been excluded by using clones. Interestingly, the differentil effect of wter nd slt stress nd fertiliztion on HC- nd LC-lines incresed with incresing stress intensity, pointing towrds dose-dependency in the complex interply mong biotic fctors nd plnt resistnce expression (Fig. 3). ll these effects becme pprent only when using clonl plnts s they were msked by the genetic vribility in the wild plnt popultions (Fig. 3). Chnging chemicl trits in plnts growing under environmentl stress conditions hve been ttributed to concentrtion effects resulting from reduced plnt size or weight (e.g. Rice & Bzzz 1989; Korichev 1999) rther thn to incresed production of the chemicl in question. In this study, physicl lef trits (LM nd tissue hydrtion; see Tble S3) nd morphologicl phenotypes (totl plnt fresh weight, plnt height, nd lef number; Tble 1) were not significntly ffected by the tretments when compred within the geneticlly defined plnt groups (HC nd LC clones). Investments in different chemicl components were similrly ffected in entire plnts nd in individul leves (Fig. 3; see Tble S2), demonstrting tht trits such s HCNp nd protein concentrtion cn be quntittively up- or down-regulted t the phenotypic level in response to mbient conditions. Cn we regrd the chnges in plnt phenotypes observed in this study s being dptive? First, the observed chnges in biochemicl trits clerly ffected the development of the herbivore used in this study: lrve of Mexicn ben beetle grew fster on the high-protein nd low-cynogenic lines thn on the low-protein nd highly cynogenic lines (Fig. 4). The biochemicl responses to ltered biotic conditions (Fig. 3) were generlly reflected in the behviourl nd developmentl responses of the herbivore. Thus, protein content nd cynogenic potentil showed both geneticlly fixed nd phenotypiclly plstic components, nd ffected herbivore performnce. Consequently, herbivores cn exhibit selective pressure on these trits. Severl theories hve been formulted to predict dptive ptterns of plnt investment in resistnce. The resource-vilbility hypothesis (RH) ssumes tht plnt species dpted to grow t resource-poor sites re chrcterized by inherently slow growth rtes, low mximum photosynthetic rtes nd low turnover rtes, nd predicts tht these species should be intensively defended by metboliclly irreversible, quntittive defences such s lignin or tnnins (Coley, Brynt & Chpin 1985; Herms & Mttson 1992; rendt 1997). By contrst, species dpted to grow t resource-rich sites should possess flexible responses to pulses in resource supply nd thus should exhibit high morphologicl nd biochemicl plsticity (Brynt, Chpin & Klein 1983; Coley, Brynt & Chpin 1985; Lim & Turner 1996). lthough we compred plnts of single species showing different degrees of genetic vribility rther thn different species, our findings re consistent with the bsic ssumptions of the RH. Lim ben possesses the chrcteristics of the ltter group of plnts, showing low investment in immobile defences (Bllhorn et l. 2007) but extensive lloction of resources to mobile defences such s cynogenic precursors (Budoin, Brthelemy & Ndungo 1991), VOCs nd EFN (Heil 2004), defensive trits which due to their inducibility show prticulrly high phenotypic plsticity. Wild type lim ben ppers dpted to high-nitrogen conditions, becuse the seeds germinte fter locl fires (thus, t sites rich in minerl

11 biotic fctors ffect plnt resistnce to herbivores 323 nutrients) nd most individuls re colonized by nitrogenfixing bcteri (rhizobi) under nturl conditions (D.J. B. nd M. H., personl observtion). Strins of these bcteri (GenBnk ccession numbers EU , EU , EU842048) isolted from lim ben roots t nturl sites were foundtobehighlyefficientinnitrogenfixtion(d.j.bllhorn, unpublished dt). In the present study, the plnts were cultivted in the bsence of rhizobi nd responded strongly to experimentl increse in nutrient vilbility. This observtion is in ccordnce with ssumptions of the RH. Our findings suggest tht within nturl lim ben popultions different genotypes exhibit different investment strtegies. This is exemplified by the fct tht low-cynogenic plnts showed higher growth rtes (i.e. biomss production, plnt height nd number of leves) nd higher soluble protein concentrtionthnhigh-cynogenic plnts (Tble 1,S2). Such geneticlly prtly fixed vribility of investment in defence or growth should substntilly enhnce the plsticity by which lim ben popultions respond to vritions in externl fctors. lthough cynogenesis ppers to be the mjor direct defence of lim ben (Bllhorn et l. 2008c, 2009), defence-ssocited phenolic compounds lso vried depending on genotype. Low-cynogenic nd fst-growing plnts showed higher concentrtions of phenolics thn highcynogenic (nd slower-growing) plnts. Thus, concerning these crbon-bsed defence compounds we observed no constrints in lloction of resources in growth or defence s predicted by theory (Herms & Mttson 1992; Goodger, Gledow & Woodrow 2006). This finding suggests tht genetic vrition of cynogenesis is ssocited with constrints on growth, while enhnced production of crbon-bsed phenolics in low-cynogenic genotypes did result in growth-ffecting lloction costs. The pprent trde-off between nitrogen-bsed defence (cynogenesis) nd crbon-bsed defence (phenolics) indictes the existence of considerble vrition in the lim ben s overll defence. Different investment strtegies nd or trde-offs betweenlimbentritsththvebeenobservedinthelbortory for cynogenesis nd relese or herbivore-induced VOCs (Bllhorn et l. 2008, 2008b) s well s for cynogenesis nd polyphenol oxidse ctivity (Bllhorn, Pietrowski & Lieberei 2010b), which suggests complex defence syndrome in this plnt species. There is continuously growing literture on the impct of specific environmentl fctors on quntittive expression of cynogenesis. However, studies showed different responses of plnt species towrds chnging environmentl conditions (Pederson, Firbrother & Greene 1996). In our study, wter stress resulted in incresed cynide production. This is in line with dt on Mnihot esculent plnts, which showed incresed cynide concentrtions when subjected to drought (Bokng, Eknyke & Dixon 1994; ikmn et l. 1996). Furthermore, Crdus et l. (1990) found tht the frequency of cynogenic Trifolium repens ws higher in res with low rinfll in New Zelnd. However, Foulds & Grime (1972) found the opposite being true in study of T. repens in Englnd. In lim ben, soil slinity t higher intensities resulted in incresed cynide levels (Fig. 3). Similr results hve been reported for Lotus cornicultus nd Lotus ustrlis (Keymer & Ellis 1978; Foulds 1982), but it remins elusive whether slt directly impcts on plnt cynogenesis or whether sltmedited chnges in wter vilbility cused the observed effects (Stockml & Oleszek 1997). In our study we found no significnt effects of light vilbility on cynide concentrtion (Fig. 3). This finding is supported by our recent study on lim ben cynogenesis t nturl sites in Mexico, in which no effects of shde on cynide ccumultion in lim ben could be observed (Bllhorn et l. 2009). However, s for wter vilbility nd soil slinity, previous studies of this issue hve yielded conflicting results (Burns, Gledow & Woodrow 2002). Studies on brcken fern Pteridium quilinum (Schreiner, Nfus & Pimentel 1984) nd on T. repens (Vickery, Wheeler & Mulchy 1987) showed tht plnt individuls from res with low light vilbility were more cynogenic thn those growing in open sites, wheres Niedz wiedz -Siegien & Giersimiul (2001) found decrese in cynogenic glycoside content with shde in flx (Linum usittissimum). Webber & Woodrow (2009) found no effects of light on cynogenesis in the tropicl rinforest tree Rypros kurrngii. There is incresing evidence tht there re intercting effects of different environmentl conditions on cynogenesis. Supporting the importnce of nitrogen vilbility for plnt investment in cynogenic compounds demonstrted in our present study (Fig. 3), studies on the highly cynogenic tree Euclyptus cldoclyx showed tht wter stress enhnces cynogenesis by 30% under nitrogen-limited conditions, but resulted in n increse of 70% when plnts were grown under enhnced nitrogen vilbility (Gledow & Woodrow 2002). Furthermore, Gledow & Woodrow (2002) showed tht different co-occurring plnt defences re ffected in different wys by the sme biotic fctors s, in contrst to cynogenic glycosides, totl phenolics nd condensed tnnins in E. cldoclyx decresed with incresing nitrogen supply while they were unffected by wter stress. In this line, experimentl shding of E. cldoclyx effected decrese in the concentrtion of cynogenic glycosides, while crbon-bsed secondry metbolites (totl phenolics nd condensed tnnins) were little or not effected by shding (Burns, Gledow & Woodrow 2002). From n ecologicl perspective, the ctive up-regultion of cynogenic glycosides under stress conditions such s reduced wter vilbility nd soil slinity we report here cn be interpreted in the light of the resource-vilbility hypothesis (Coley, Brynt & Chpin 1985) s plnts under stressful environmentl conditions re limited in replcing leves lost due to herbivory. Considering drmticlly chnging environmentl conditions on locl nd globl scles, functionl studies on the ecologicl consequences of vrition of toxic nd nutritive plnt trits re of gret ecosystemic importnce (reviewed by Bidrt-Bouzt & Imeh-Nthnil 2008). In the present study we showed tht the tretment-dependent up-regultion of HCNp significntly ffects nturl herbivores when genetic vribility ws excluded from the experimentl plnt system (Fig. 4). In geneticlly heterogeneous seed-grown plnts the individul plnts HCNp ws the most importnt predictor