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1 Reserch Phytologist Vrition nd fitness costs for tolernce to different types of herbivore dmge in Boecher strict genotypes with contrsting glucosinolte structures Antonio J. Mnzned 1,2, Ksvjhl V. S. K. Prsd 1 nd Thoms Mitchell-Olds 1 1 Institute for Genome Sciences nd Policy, Deprtment of Biology, Duke University, PO Box 9338, Durhm, NC 2778, USA; 2 Deprtmento de Biologí Animl, Biologí Vegetl y Ecologí, Universidd de Jén, Prje ls Lgunills s n, 2371, Jén, Spin Summry Author for correspondence: Antonio J. Mnzned Tel: Emil: mvil@ujen.es Received: 19 April 21 Accepted: 9 June 21 Phytologist (21) 188: doi: /j x Key words: constitutive glucosinoltes, generlist herbivores, genetic vrition, herbivory, induced glucosinoltes, plnt defenses, specilist herbivores, tolernce. Anlyses of plnt tolernce in response to different modes of herbivory re essentil to n understnding of plnt defense evolution, yet re still scrce. Alloction costs nd trde-offs between tolernce nd plnt chemicl defenses my influence genetic vrition for tolernce. However, vrition in defenses lso occurs for the presence or bsence of discrete chemicl structures; yet, the effects of intrspecific polymorphisms on tolernce to multiple herbivores hve not been evluted. Here, in glsshouse experiment, we investigted the vrition for tolernce to different types of herbivore dmge, nd direct lloction costs, in 1 genotypes of Boecher strict (Brssiccee), wild reltive of Arbidopsis, with contrsting folir glucosinolte chemicl structures (methionine-derived glucosinoltes vs glucosinoltes derived from brnched-chin mino cids). We found significnt genetic vrition for tolernce to different types of herbivore. Structurl vritions in the glucosinolte profile did not influence tolernce to dmge, but predicted plnt fitness. Levels of constitutive nd induced glucosinoltes vried between genotypes with different structurl profiles, but we did not detect ny cost of tolernce explining the genetic vrition in tolernce mong genotypes. Trde-offs between plnt tolernce to multiple herbivores my not explin the existence of intermedite levels of tolernce to dmge in plnts with contrsting chemicl defensive profiles. Introduction Plnts possess rich diversity of defensive dpttions ginst herbivores, which my enble resistnce to herbivore ttck or tolernce to dmge. In the first cse, plnts rely on trits tht reduce herbivore dmge on plnt tissues, such s trichomes, spines or toxic secondry compounds (reviewed in Struss & Zngerl, 22). Alterntively, some plnt genotypes my be less susceptible to negtive impcts when herbivore dmge occurs (Struss & Agrwl, 1999). In both cses, plnt defensive trits re typiclly geneticlly complex quntittive trits (Rosenthl & Kotnen, 1994; Weinig et l., 23; Agrwl & Fishbein, 26; Schrnz et l., 29), nd often show heritble vrition within nd mong popultions (e.g. Fornoni & Nuñez-Frfán, 2; Kliebenstein et l., 21; Windsor et l., 25; Løe et l., 27). In prticulr, plnt tolernce to dmge (the bility to regrow nd reproduce fter herbivory; Struss & Agrwl, 1999) is geneticlly vrible in mny species (reviewed by Struss & Agrwl, 1999; Fornoni et l., 23; Nuñez-Frfán et l., 27; but see Ivey et l., 29), nd therefore my evolve in response to nturl selection. However, the ecologicl nd genetic bsis of tolernce vrition is still not well understood, especilly mong popultions (Fornoni et l., 23). The expression of plnt tolernce to dmge vries mong environments (Wise & Abrhmson, 27). Resource vilbility, the timing nd mgnitude of dmge, nd the type of herbivore dmge re importnt ecologicl fctors influencing the evolution of tolernce (e.g. Mschinski & 464 Phytologist (21) 188: Journl compiltion Ó Phytologist Trust (21)

2 Phytologist Reserch 465 Whithm, 1989; Struss & Agrwl, 1999; Tiffin, 22; Steven et l., 27; Suw & Mherli, 28). In prticulr, tolernce to dmge is often dependent on the type of tissue dmged tht is, picl vs folir dmge, dmge on young leves vs mture ones, or dmge to roots vs leves (e.g. Houle & Simrd, 1996; Stinchcombe, 22; Bolt & Lehtilä, 27; but see Tiffin & Rusher, 1999). However, these studies simulte dmge minly through mnul clipping, which my be poor surrogte for genuine herbivory (Struss & Agrwl, 1999). In ddition, little is known bout how different kinds of herbivore dmge influence tolernce to herbivory cross nturlly vrying genotypes (but see Agrwl et l., 1999; Tiffin & Rusher, 1999; Pilson, 2). However, to understnd the evolution of tolernce, it is essentil to determine whether conspecific genotypes show differentil tolernce to herbivore dmge. Furthermore, plnts re often ttcked by multiple herbivore species, nd so we must exmine diverse generlist nd specilist herbivores feeding on different prts of the plnt. In ddition, plnts fce different types of herbivore cross their rnges, which my result in different ecologicl nd evolutionry outcomes (Thompson, 1988; Stinchcombe & Rusher, 22; Struss & Irwin, 24). The influence of quntittive intrspecific vrition in plnt chemicl defenses on tolernce to dmge is frequently studied in the context of ecologicl trde-offs nd direct costs of tolernce (Struss et l., 22; Leimu & Korichev, 26). Alloction costs occur when there re significnt negtive genetic correltions between tolernce nd resistnce, or between tolernce nd fitness in the bsence of herbivores. Such costs re thought to mintin the existing levels of genetic vrition in tolernce within species (Struss & Agrwl, 1999; Struss et l., 22), but they re not lwys detected (Leimu & Korichev, 26). Both tolernce to dmge nd the mgnitude nd significnce of tolernce costs my depend on phenotypic plsticity, such s differences in trit expression through ontogeny (Boege et l., 27; Brton, 28) or in levels of defense induction (Agrwl, 1998, 1999). However, genetic vrition in chemicl defenses occurs not merely t the quntittive level, but lso with regrd to the presence or bsence of discrete chemicl structures (e.g. Schrnz et l., 29), nd differentil lloction costs could rise mong genotypes with different chemicl compositions, especilly if geogrphicl structure underlies such vrition. Recent studies hve shown tht constitutive structurl polymorphism in chemicl plnt defenses ffects plnt resistnce to herbivores nd influences herbivore communities (ton et l., 29; Schrnz et l., 29); yet, to our knowledge, the effects of intrspecific chemicl polymorphism on plnt fitness hve not been evluted in the context of herbivore tolernce. However, if distinct defensive compounds hve different biosynthetic costs, such structurl polymorphisms in plnt chemicl defenses my lso influence tolernce or its costs (Korichev, 22 nd references therein). Here, we investigte genetic vrition in tolernce to different types of dmge nd the lloction costs of tolernce to dmge in genotypes of Boecher strict (Brssiccee) with contrsting folir glucosinolte chemicl structures. Tolernce is defined here s the difference in fitness between dmged nd undmged plnts (Struss & Agrwl, 1999). In B. strict, heritble nturl polymorphism exists in liphtic glucosinoltes within nd mong popultions (Schrnz et l., 29). Although Arbidopsis, Brssic nd most other crucifers produce lef glucosinoltes lrgely derived from the mino cids methionine or tryptophn, some genotypes of B. strict synthesize glucosinoltes from the brnched-chin mino cids (BCAA) vline, leucine or isoleucine (Windsor et l., 25; Schrnz et l., 29). Although there is currently little informtion bout the ecologicl role of BCAA-derived liphtic glucosinoltes, recent quntittive trit locus (QTL) mpping hs shown tht heritble resistnce to the lrve of the generlist lepidoptern Trichoplusi ni (Noctuide) vries significntly between genotypes with contrsting glucosinolte profiles (Schrnz et l., 29). In prticulr, lines producing methioninederived glucosinoltes were significntly more resistnt nd suffered less lef herbivory thn lines producing predominntly BCAA-derived glucosinoltes (Schrnz et l., 29). In ddition, biosynthesis of BCAA nd methionine-derived glucosinoltes is controlled by different genes using different metbolic pthwys (Mikkelsen & Hlkier, 23), which my ffect tolernce if differences in the biosynthetic costs of such compounds vry. To our knowledge, ours is the first investigtion of the effects of nturl structurl polymorphism in plnt chemicl defenses on tolernce to different herbivores. Specificlly, we ddress the following questions. Is there genetic vrition in B. strict for tolernce to herbivory? If so, does this vrition depend on the type of herbivore dmge or the glucosinolte structurl profile (glucosinoltes derived from methionine vs BCAA). Do lloction costs explin the observed genetic vrition in tolernce to dmge in B. strict?, Is the mgnitude nd significnce of tolernce costs determined by the type of herbivore dmge, the structurl glucosinolte profile or glucosinolte induction? We seek to determine the ecologicl nd evolutionry significnce of nonmethionine-derived liphtic glucosinoltes in plnt defense to herbivory. Mterils nd Methods Study system Boecher strict (Grhm) (previously Arbis drummondii)is morphologiclly nd geneticlly well-defined, monophyletic, short-lived perennil herb distributed cross diverse Journl compiltion Ó Phytologist Trust (21) Phytologist (21) 188:

3 466 Reserch Phytologist hbitts in western North Americ (Mitchell-Olds, 21; Song et l., 26). Boecher strict is predominntly selffertilizing, sexul diploid, nd is ttcked by wide rry of specilist (e.g. the pierid Ponti spp.) nd generlist (e.g. noctuids, grsshoppers, fle beetles nd weevils) insect herbivores. In the field, levels of individul plnt dmge rnge between % nd 1% (verge dmge per lef, 8.8%; verge proportion of leves dmged, 13.1%), nd there is substntil vrition in the verge herbivore dmge mong popultions nd yers (T. Mitchell-Olds, unpublished). Plnts produce between one nd five inflorescences in lte spring, nd both fruit mturtion nd seed set tke plce in June July. Like other members of the Brssiccee, B. strict produces glucosinoltes, which constitute primry chemicl defense ginst herbivores (Hopkins et l., 29). There is extensive nturl genetic vrition for type nd quntity of glucosinoltes within nd mong nturl popultions (Windsor et l., 25; Schrnz et l., 29). The glucosinolte polymorphism controls lloction to BCAA- vs methionine-derived glucosinoltes nd predicts levels of herbivory (Schrnz et l., 29). Becuse lrge portion of genetic polymorphism in B. strict is distributed mong popultions (Song et l., 26), we exmined one genotype from ech popultion, in order to mximize genetic vrition for given smple size. We considered nine genotypes from our study res in the Northern Rocky Mountins (see lter nd Supporting Informtion Fig. S1). One of these genotypes (Lost Tril, Montn) hs been used s prent for QTL mpping of insect resistnce (Schrnz et l., 29); hence, the other prent (Tylor River, Colordo) ws lso included for comprison. Experimentl procedure Mture seeds of 1 genotypes were collected from 1 different popultions locted in the Rocky Mountins in the western USA (in Montn, Idho nd Colordo, see Fig. S1 nd Tble S1 for detils). These popultions re diverse in terms of ecologicl conditions nd lso differ in the levels of dmge received by the plnts. Genotypes included in the experiment were selected on the bsis of their chemicl bckground: four genotypes produce minly BCAA-derived glucosinoltes, nd six genotypes produce methioninederived glucosinoltes (Tble S1). We minimized potentil mternl effects by using seeds from second genertion of self-fertilized, glsshouse-grown plnts. In November 27, we plced 12 self-sib seeds genotype into Petri dishes t 4 C for 6 wk of cold strtifiction. Once germinted, 11 seeds per genotype were individully plnted nd grown on stndrd soil (Ffrd 4p mix; Ffrd Inc., Agwm, MA, USA) in rndomized complete block design, with the blocks consisting of one try of 4 (5 5 6cm 3 ) pots, distributed rndomly within the try. The trys were plced in the Duke University glsshouse under controlled growth conditions. After c. 6 wk, plnts were moved to growth chmber (22 C, 16 h light nd 8 h drk) nd rndomly ssigned to the following four tretments: (1) undmged control; (2) specilist herbivore tretment, with 33% of plnt lef re dmged by cged second-instr Pieris rpe lrv (Lepidopter: Pieride); (3) generlist herbivore tretment, with 33% of plnt lef re dmged by cged second-instr Trichoplusi ni lrv (Lepidopter: Noctuide); (4) mnul clipping tretment, with 33% of ech lef clipped nd removed using scissors. Therefore, ech of the 1 genotypes hd 11 individuls (replicted in blocks) in ech of the four tretments, for totl of 44 plnts. The insect species used here re not ntive enemies of B. strict, but hve been used extensively to investigte plnt functionl responses to herbivory by specilist nd generlist insects (e.g. Agrwl, 1999, 2; Jones et l., 26; Schrnz et l., 29). Pieris rpe is ble to detoxify glucosinoltes but Trichoplusi ni is not, which ffects the feeding behvior of the herbivores nd the pttern of plnt dmge cused by ech type of herbivore (see Notes S1 nd Fig. S2). Second-instr T. ni lrve were ordered from Benzon Reserch Inc. (Crlisle, PA, USA) nd fed on n rtificil diet. Second-instr P. rpe lrve were obtined from colony mintined in the lbortory on fresh Rphnus stiv leves, originting from eggs provided by Crolin Biologicl Supply (Burlington, NC, USA). In both of the insect tretments, single lrv without ny previous strvtion period ws plced on top of ech plnt. To gurntee single insect on ech plnt, ech plnt lrv pir ws enclosed using cylindricl tube (dimeter, 5 cm; height, 14 cm) mde of cette (3MÒ) with both ends open. One end ws inserted into the soil nd the other ws covered by loose-weve fbric. Lrve were removed from the plnts once 33% of the lef re hd been consumed (fter h nd h, for specilist nd generlist insects, respectively). The size of the treted plnts (estimted from the plnt bsl dimeter) rnged between 3.8 nd 11.4 cm. Plnts were checked for dmge 24 h fter the infesttion, nd then every 8 h. In every census, we recorded both the proportion of leves with herbivore dmge nd estimted the percentge of tissue removed per lef (tken by the sme person nd rnging from 1% to 1%; see Schrnz et l., 29 for similr procedure) to clculte the percentge of plnt dmge. After 72 h, the mjority of the plnts (368 plnts, 87.7%) in both insect tretments hd the trget level of dmge, wheres 72 plnts (12.3%) did not rech the level of plnt dmge desired or hd excess dmge. These plnts were not included in the sttisticl nlysis. More detils on the feeding behvior of ech insect species nd the wy tht insects dmged the plnts re given in Notes S1. Phytologist (21) 188: Journl compiltion Ó Phytologist Trust (21)

4 Phytologist Reserch 467 Like most perennil species, Boecher requires cold vernliztion period to induce flowering nd seed production. For this reson, 1 wk fter the herbivore tretments we moved the plnts to cold room (4 C, 16 h light nd 8 h drk) for 6 wk, in order to initite flowering nd reproduction. This lbortory tretment provides n effective simultion of winter vernliztion becuse plnts perceive vernliztion cues only when tempertures re bove freezing, rther thn the long periods of below-freezing tempertures tht occur in the wild. Subsequently, plnts were moved bck to the glsshouse ( C, 16 h light nd 8 h drk) until the end of the experiment (in My 28). Reproductive mesurements For ech plnt, we recorded both the flowering time (the number of dys from germintion until the opening of the first flower) nd severl correltes of mternl fitness: totl number of flowers, seed set (totl number of seeds totl number of flowers) nd reproductive biomss [the weight of one individul seed rndomly chosen (fresh weight using Mettler ToledoÒ xs15 precision scle, Columbus, OH, USA) multiplied by the totl number of seeds]. Boecher strict is self-comptible nd highly inbred (Song et l., 26); thus, differences in fitness re unlikely to reflect inbreeding depression. Anlysis of constitutive nd induced glucosinoltes Concurrent with the tolernce experiment, we grew 44 dditionl plnts from ech of the sme 1 genotypes under the sme conditions for the nlysis of constitutive nd induced glucosinoltes. The experimentl procedure nd methods for glucosinolte extrction, isoltion, purifiction nd quntifiction follow our previous methods (Schrnz et l., 29) nd re given in Notes S2. Sttisticl nlyses To nlyze the effect of herbivory on multiple fitness components, we conducted both multivrite nlyses nd generl liner mixed models with mximum likelihood estimtes, using JMP 7..1 (SAS Institute Inc., Cry, NC, USA). As fitness components were correlted (Tble S2), we first performed MANOVA to test the effects of tretment, genotype nd their interction on overll fitness. A significnt interction between genotype nd tretment shows the existence of genetic vrition in tolernce (i.e. difference in fitness between the dmge tretments nd the undmged control, see the next prgrph) for overll plnt fitness. Second, we conducted principl component nlysis (PCA) to obtin independent fctors (fter vrimx normlized rottion) ccounting for plnt fitness trits. Fctor scores correlted significntly with fitness were included s dependent vribles in seprte mixed models fitted to test the fixed effects of tretment, genotype nd their interction. We included plnt size s covrite nd block s rndom effect in these models. The interction between plnt size nd genotype ws nonsignificnt (not shown) nd ws removed from the models. Reproductive vlues were log-trnsformed to improve normlity nd homoscedsticity. Tolernce ws estimted for ech genotype nd fitness component (i.e. principl component) s the difference in fitness between the dmge tretments (either specilist herbivore, generlist herbivore or clipping) nd the undmged control (Struss & Agrwl, 1999). Higher nd positive vlues depict greter tolernce to dmge thn smller or negtive vlues. Dmge levels nd fitness components were on the sme multiplictive scle (Wise & Crr, 28). For ech of the fitness components, genetic vrition in tolernce ws inferred from the significnce of genotype by tretment interction term in the liner mixed models described bove. When significnt interction between genotype nd herbivore tretment ws detected, we crried out tests of simple min effects using the SLICE option in JMP, which llows the effects of given fctor to be explored t ech level of the other fctors (Schbenberger et l., 2). In the context of this study, this test llowed us to determine, for ech genotype, wht type of herbivore dmge hd significnt effect on fitness components. To nlyze whether tolernce to dmge is ffected by the type of glucosinolte profile, we grouped the 1 genotypes into two ctegories: methionine-derived glucosinoltes or BCAA-derived glucosinoltes (see Tble S1). Differences in tolernce mens between these two groups were estimted using nonprmetric Kruskll Wllis test with genotype s the unit of repliction. In ddition, to test the effect of the chemicl bckground of the genotypes nd herbivory on plnt fecundity, for ech fitness component, we fitted generl liner mixed model including tretment, the glucosinolte group (BCAA-derived vs methionine-derived) nd their interction s fixed fctors, nd block s rndom fctor. As plnts with high proportion of BCAA-derived glucosinoltes were significntly lrger (bsl rdius men ± 1SE: BCAA-derived, ± 1.98 mm; methionine-derived, 7.77 ± 1.89 mm; ANOVA: F 1,46 = 2.45, P =.15), plnt size ws included s covrite. When significnt genetic vrition ws found, we estimted the lloction costs of tolernce to dmge. We nlyzed the genetic correltion mong genotype fitness mens (in ll cses, mens re model-djusted LS-MEANS) for dmged nd undmged plnts using correltion nlyses in JMP. A significnt negtive correltion between the fitness of dmged nd undmged plnts indictes cost of tolernce (Struss & Agrwl, 1999). In ddition, we nlyzed the genetic correltion mong genotype men tolernce in ech of the herbivore dmge tretments (i.e. Journl compiltion Ó Phytologist Trust (21) Phytologist (21) 188:

5 468 Reserch Phytologist tolernce to generlist vs tolernce to specilist dmge; tolernce to generlist vs tolernce to clipping dmge; nd tolernce to specilist vs tolernce to clipping dmge). Similrly, significnt negtive correltion between tolernce to different types of dmge would indicte cost of tolernce. Becuse ll dmged plnts hd equl percentge of lef re removed (see the section entitled Experimentl Procedure ), we could not infer directly heritble vrition for resistnce mong our plnt genotypes. However, given the defensive role plyed by glucosinoltes (t lest ginst generlist herbivores, e.g. Hopkins et l., 29), for ech type of dmge, we lso nlyzed tolernce chemicl defense trde-offs by regressing the genotype tolernce mens obtined for ech type of dmge nd the genotype totl glucosinolte concentrtion mens t both the constitutive nd induced level. A significnt negtive genetic reltion between tolernce nd the concentrtion of defensive metbolite indictes the presence of n lloction trde-off between tolernce nd resistnce (Leimu & Korichev, 26). Becuse the totl concentrtion of constitutive nd induced glucosinoltes ws ffected significntly by the chemicl bckground of the genotypes (see the Results section), we explored the covrince between tolernce nd resistnce through both groups. Becuse our dt did not stisfy the ssumptions of ANOVA (e.g. smll smple size, non-norml errors nd presence of outliers), we used robust nlysis of covrince bsed on M estimtion (Chen, 22). Robust ANCOVAs were then performed using the procedure ROBUSTREG in SAS 9.2 (SAS Institute Inc., Cry, NC, USA). In these nlyses, genotypic men tolernce ws lwys the dependent vrible nd the glucosinolte group ws the grouping fctor. We included s covrites the men genotype totl glucosinolte concentrtion in the undmged tretment (constitutive resistnce) or the difference in the men totl glucosinolte concentrtion fter 24 h of herbivory between dmged nd undmged plnts (induced resistnce). The sttisticl significnce of ANCOVAs cme from Rn 2 robust Wld s liner test (Chen, 22). Results Effects of herbivore tretments on overll plnt fitness A MANOVA conducted on ll fitness components did not show significnt min effect of herbivore tretment (Tble 1). However, there were significnt effects of genotype on overll fitness, nd the genotype by herbivore interction (Tble 1), which suggests tht the mgnitude nd or sign of the herbivore tretment on fitness depended on genotype. Anlyses conducted seprtely on ech of the fitness components (see the Results section) reveled tht herbivory ffected the erly fitness components, lthough its Tble 1 MANOVA to test the effects of genotype, herbivore tretment nd their interction on four fitness components of Boecher strict Source Wilks k df F P Genotype , <.1 Herbivory , Genotype herbivory , Plnt size 4, <.1 Block.51 4, <.1 Significnt vlues (P <.5) re in bold. effect ws dependent on genotype. Block nd plnt size lso influenced plnt fitness (Tble 1). Fitness components nd genetic vrition in tolernce Tken together, three independent fctors ccounted for 98.6% of the vrition in reproductive trits in our PCA (Tble S3). The first fctor (PC1) depicts lte plnt fecundity, becuse it is relted to seed set nd reproductive biomss (Tble S3). The second fctor (PC2) indictes erly fecundity, nd is closely relted to the numbers of flowers (Tble S3), providing the opportunity for reproductive fitness vi mle function. Both fctors re relted to vrition in fecundity, where higher nd positive vlues indicte higher levels of seed nd flower production. The third fctor (PC3) depicts flowering time vrition (Tble S3). In this cse, negtive nd smller vlues denote erly flowering times, nd positive nd higher vlues depict lte flowering times. Herbivory, s min effect, did not influence the PC3 fitness component relted to flowering time PC3 (Tble 2). However, there ws significnt effect of genotype, nd the genotype by herbivore tretment interction, on the PC3 fitness component (Tble 2). This result suggests tht the consequences of different types of herbivory on the PC3 fitness component re genotype dependent, nd tht there is significnt genetic vrition in tolernce when fitness components correlted with flowering time re considered (Fig. 1). Tests of min effects showed tht six of 1 genotypes were not ffected by herbivore tretment (Tble 3). However, ID7 nd MT49 genotypes flowered more rpidly in response to insect dmge, wheres MT55 nd SAD12 genotypes flowered more slowly in response to dmge (Fig. 1). Erly fitness component PC2 differed significntly mong genotypes nd showed only mrginl effects of herbivore tretment; however, the genotype by herbivore tretment interction ws significnt (Tble 2). This indictes tht the fitness consequences of herbivore tretments depend on genotype, nd there is significnt genetic vrition in tolernce for erly fitness components (Fig. 2). Tests of min effects showed tht most of the genotypes (seven of Phytologist (21) 188: Journl compiltion Ó Phytologist Trust (21)

6 Phytologist Reserch 469 Tble 2 Summry results of the generl liner mixed model testing the effects of herbivore tretment, genotype nd their interction on three principl components summrizing fitness-relted vrition in Boecher strict PC1 PC2 PC3 Fixed effects df F P df F P df F P Genotype 9, <.1 9, <.1 9, <.1 Herbivory 3, , , Genotype Herbivory 27, , , Significnt P vlues (P <.5) re in bold. PC1 is lte fitness component, PC2 is n erly fitness component nd PC3 is n indictor of flowering time (see text for detils). Tolernce (PC3) *.8 * b * * b * * Dely 1) were ble to compenste for herbivore dmge, but three of 1 did not (Tble 3, Fig. 2). Thus, herbivore dmge to the ID14, ID7 nd MT55 genotypes hd negtive impct on fitness (Fig. 2), lthough the mgnitude nd impct of ech type of herbivore dmge lso vried mong these genotypes (Fig. 2). For the remining genotypes, the type of dmge (i.e. tretment) hd no detectble effect on PC2, n indictor of erly fecundity nd flower number. MT55 dmged plnts hd lower PC2 fitness compred with undmged plnts regrdless of the type of dmge (Fig. 2). For the ID7 genotype, only plnts subjected to the specilist tretment hd significntly reduced fitness reltive to undmged controls. Furthermore, for the ID14 genotype, plnts in both the insect specilist nd generlist tretments hd significnt lower fitness thn undmged control plnts. Finlly, plnt size hd significnt positive effect on fitness component PC2 (F = 31.96, df = 1, 323, P <.1;.22 ±.4, estimte ± 1SE vlue). The lte fitness component PC1 (which is correlted with seed production) ws ffected significntly by genotype nd mrginlly by herbivore tretment (Tble 2). Thus, plnts * Erlier Fig. 1 Vrition in tolernce, estimted s the verge (djusted LS-MEANS ± 1SE) difference in fitness (fitness component PC3, correlted with flowering time) between the dmged tretments nd the undmged controls (the zero line in the grph), for three types of herbivore dmge mong 1 different genotypes of Boecher strict. Significnt differences (P <.5) from undmged control plnts re depicted with n sterisk bsed on tests of simple min effects (see Mterils nd Methods section for detils). For ech genotype, the letters bove the brs not identified by the sme letter re significntly different (P <.5). Tretments: open brs, Pieris rpe (specilist); gry brs, Trichoplusi ni (generlist); blck brs, clipped. Tble 3 Tests of simple min effects (interction slices) for the effect of herbivore tretment on fitness components correlted with erly fitness flower number (PC2) nd flowering time (PC3) for ech Boecher strict genotype Effect Genotype df PC2 PC3 F P F P Herbivory ID14-76A 3, Herbivory ID7-1A 3, Herbivory ID86-38A 3, Herbivory ID87-31A 3, Herbivory ID88-2A 3, Herbivory ID89-5A 3, Herbivory LTM 3, Herbivory MT49-18B 3, Herbivory MT55-9B 3, < * Herbivory SAD12 3, Significnt vlues (P <.5) re in bold. *, Interction with the specilist nd generlist levels is significnt t P <.5. Also see Fig. 1. in the mnul clipping tretment hd lower fitness (PC1 LSMEANS ± 1SE, ).16 ±.11) thn undmged control plnts (.7 ±.11; contrst test: F 1,331 = 3, 59, P =.59) or plnts subjected to the insect specilist tretment (.17 ±.12; contrst test: F 1,332 = 6, 62, P =.1), but not significntly different from plnts in the generlist tretment (.13 ±.11; contrst test: F 1,331 =1, 9, P =.16). Among genotypes, PC1 vlues rnged between.57 ±.18 for genotype MT55 to ).77 ±.16 for genotype ID7. The genotype by herbivore interction ws not significnt (Tble 2), indicting tht the effect of the type of herbivore dmge on the lte fitness component PC1 ws constnt for ll genotypes. Further, this nonsignificnt interction term lso indictes tht there is no detectble genetic vrition in tolernce to dmge when lte fitness components re considered. Effect of glucosinolte polymorphism on tolernce to dmge nd plnt fecundity The type of glucosinolte profile ws unrelted to tolernce to dmge for ny of the fitness components or types of dmge considered (see Tble S4). However, vrition in Journl compiltion Ó Phytologist Trust (21) Phytologist (21) 188:

7 47 Reserch Phytologist Tolernce (PC2) * * * * c bc * Fig. 2 Vrition in tolernce, estimted s the verge (djusted LS-MEANS ± 1SE) difference in fitness (erly fitness component PC2) between the dmged tretments nd the undmged controls (the zero line in the grph), for three types of herbivore dmge mong 1 different genotypes of Boecher strict. Significnt differences (P <.5) from undmged control plnts re depicted with n sterisk bsed on tests of simple min effects (see Mterils nd Methods section for detils). For ech genotype, letters bove the brs not identified by the sme letter re significntly different (P <.5). Tretments: open brs, Pieris rpe (specilist); gry brs, Trichoplusi ni (generlist); blck brs, clipped. the constitutive glucosinolte profile ws ssocited with differences in ll plnt fitness components, irrespective of herbivore tretment, s indicted by the nonsignificnt interction between glucosinolte type nd herbivore tretment (Tble 4). Thus, plnts with high proportions of BCAA-derived glucosinoltes hd significntly lower fitness thn plnts with methionine-derived glucosinoltes (Fig. 3). Similrly, plnts with high proportion of BCAA-derived glucosinoltes flowered lter thn plnts with methioninederived glucosinoltes (Fig. 3). Finlly, there ws significnt positive effect of plnt size on fitness components PC2 nd PC3 (.22 ±.31,.27 ±.4; estimte ± 1SE vlues for PC2 nd PC3 respectively, see Tble 4). Alloction costs of tolernce Becuse significnt genetic vrition ws only detected for the PC2 nd PC3 fitness components, we nlyzed the costs of tolernce only for these trits. PC2 genetic mens on dmged nd undmged plnts were not significntly correlted (Fig. 4). However, genotype PC3 fitness mens for dmged nd undmged plnts showed significnt positive correltion (Fig. 4), which suggests tht genotypes with lte flowering times in the undmged tretment lso showed lte times to flower in ll the dmge tretments (Fig. 4). For both fitness components PC2 nd PC3, genotypic men tolernce to dmge in ech of the tretments ws positive nd significntly correlted in most cses (Fig. 5). This suggests tht plnts of given genotype show similr levels of tolernce to different herbivore species. Trde-offs between tolernce nd chemicl defenses The constitutive concentrtion of lef glucosinoltes vried significntly mong the 1 genotypes (ANOVA result for genotype fctor: F 9,82 = 1.61, P <.1), nd lso between plnts with different chemicl profiles (ANOVA: F 1,9 = 5.33, P =.23; Fig. S4). However, overll, we did not detect significnt genetic correltion between the totl concentrtion of constitutive glucosinoltes nd tolernce to dmge (Tble 5). We detected significnt effect, dependent on the glucosinolte group, of the constitutive glucosinolte concentrtion on tolernce (PC2) to clipping (Tble 5). Tolernce nd defensive metbolite concentrtions were significntly nd positively geneticlly correlted in plnts with methionine-derived glucosinoltes (pirwise correltion coefficient: n = 6, r =.93, P =.7; Fig. S5), but not in plnts with BCAA-derived glucosinoltes (n =4,r =.54, P =.45). These results provide no evidence for trde-off between tolernce nd resistnce for ech type of dmge. The totl concentrtion of lef constitutive glucosinoltes fter 24 h of dmge vried significntly mong genotypes nd herbivore tretments (Figs 6, S6, Tble S5), but genetic vrition in glucosinolte induction ws nonsignificnt (the interction of genotype nd tretment ws not significnt; Tble S5). The induction of glucosinoltes ws significntly Tble 4 Results of generl liner mixed models nlyzing the effect of the glucosinolte profile nd herbivore tretment on three plnt fitness components of Boecher strict Fitness components PC1 PC2 PC3 Source df F P df F P df F P Herbivory 3, , , Glucosinolte profile (GS) 1, <.1 1, , <.1 Herbivory GS 3, , , Covrite Plnt size 1, , <.1 1, <.1 Significnt P vlues (P <.5) re in bold. PC1 is lte fitness component, PC2 is n erly fitness component nd PC3 depicts flowering time. See text for detils. Phytologist (21) 188: Journl compiltion Ó Phytologist Trust (21)

8 Phytologist Reserch 471 Fitness PC1 PC2 PC3.3.1 relted to the genotypic chemicl profile in the specilist herbivore tretment only (Kruskll Wllis ANOVA: df = 1, 1, v 2 = 3.68, P =.5; P >.5 for the rest of the tretments; see lso Fig. S7). Overll, glucosinolte induction did not ffect tolernce to herbivore dmge (Tble S6). Only in one cse did glucosinolte induction significntly ffect tolernce (PC3 component) to specilist dmge (Tble S6), but this correltion ws positive (pirwise correltion: n = 1, r =.65, P =.43). Discussion BCAA derived Met derived Fig. 3 Vrition in plnt fitness components mong Boecher strict plnts with contrsting structurl glucosinolte profiles: high proportion of brnched-chin mino cid (BCAA)-derived glucosinoltes (drk gry brs) vs low proportion of BCAA-derived glucosinoltes (light gry brs). Met, methionine. PC1 is n indictor of lte fitness seed production, PC2 is correlted with flowering number erly fitness nd PC3 is ssocited with vrition in flowering time. Vlues re djusted LS-MEANS ± 1SE. Our experiments show significnt genetic vrition for tolernce to different types of herbivore dmge mong genotypes of B. strict, wild reltive of Arbidopsis. We found genetic heterogeneity in flowering responses to dmge by different herbivore tretments. Different types of dmge hd heterogeneous effects on flowering time (PC3) nd the number of flowers (PC2), nd these responses vried mong genotypes. By contrst, the influence of dmge tretments on lter fitness components (PC1) ws geneticlly homogeneous. Vrition in the structurl glucosinolte profile did not influence tolernce to dmge, lthough it is ssocited with spects of plnt fitness. Plnts with high proportion of BCAA-derived folir glucosinoltes hd significntly lter flowering times nd lower fitness thn plnts contining methionine-derived glucosinoltes. We did not find significnt negtive genetic correltion between tolernce nd fitness in the bsence of herbivores, or between tolernce to different types of herbivore dmge. Finlly, we did not detect ny trde-off between tolernce nd resistnce tht might explin the genetic vrition in tolernce mong these genotypes. Although this study does not include ntive herbivores of B. strict, results from our experiment re relevnt becuse we nlyzed the effect on tolernce to dmge by different types of herbivory, nd lso exmined genotypes with contrsting constitutive chemicl bckground, which re understudied spects in the context of plnt tolernce to herbivore dmge. Genetic vrition for tolernce nd effects of types of herbivory on plnt fitness Effects of herbivory on plnt fitness rnge from mortlity to overcompenstion (reviewed by Agrwl, 2b; Hwkes & Sullivn, 21; Wise & Abrhmson, 27). Within host species, responses to herbivory re determined by the degree of genetic vrition, environmentl effects nd their complex interctions (Struss & Agrwl, 1999; Fornoni et l., 23; Nuñez-Frfán et l., 27). Our results support this view. Lef herbivory ffected fitness-relted trits in B. strict, lthough both the mgnitude nd direction of these effects vried mong genotypes, s well s mong different fitness components nd types of herbivore dmge. Despite progress in this field, nlyses of tolernce in response to different insects nd modes of herbivory re still scrce (Agrwl et l., 1999; Jones et l., 26). The type of herbivory nd the distribution of dmge within the plnt re believed to influence tolernce nd its expression cross genotypes (Rosenthl & Kotnen, 1994; Agrwl et l., 1999). This view is grounded in two wellknown fcts: (1) different herbivores feed on different plnt tissues nd structures, which my ffect plnt performnce differently ccording to the fitness vlue of the tissue (e.g. Krbn & Struss, 1993; Zngerl & Rutledge, 1996; Anderson & Agrell, 25; Brto & Cipollini, 25); (2) induced resistnce in plnts is herbivore specific, s re the Journl compiltion Ó Phytologist Trust (21) Phytologist (21) 188:

9 472 Reserch Phytologist PC2 specilist dmge PC3 specilist dmge 1 r = 8, P =.43 1 r =.47, P = PC2 generlist dmge r = 1, P = PC2 undmged PC2 undmged PC2 undmged PC3 generlist dmge 3 r =.91,P P =.2 r =.95, P <.1 3 r =.96, P < PC3 undmged PC3 undmged PC3 undmged PC2 clipping dmge PC3 clipping dmge Fig. 4 Genetic correltions between men fitness components correlted with erly fecundity PC2 (bove) nd flowering time PC3 (below) between dmged Boecher strict plnts nd their undmged controls. Vlues in the figure re the genetic correltion coefficients. A significnt negtive correltion between the fitness of dmged nd undmged plnts would indicte cost of tolernce. However, no evidence for such costs is detectble. Tolernce specilist Tolernce specilist PC2.4 r =.79, P =.6.4 r =.66, P =.37.6 r =.7, P = Tolernce generlist Tolernce clipping Tolernce clipping Tolernce specilist PC3.8 r =.78, P =.7.6 r =.58, P =.8.8 r =.73, P = Tolernce generlist Tolernce clipping Tolernce clipping Tolernce specilist Tolernce generlist Tolernce generlist Fig. 5 Genetic correltions for tolernce to different dmge tretments. For ech Boecher strict genotype (ech point in the grphs), tolernce is estimted s the verge (djusted LS-MEANS) difference in fitness between the dmged tretments nd the undmged controls. Ech pnel indictes the genetic correltion nd its sttisticl significnce. The erly fitness component PC2 is in the top row, nd the flowering time fitness component PC3 is below. A significnt negtive correltion between the tolernce of different types of dmge would indicte cost of tolernce. However, no evidence for such costs is detectble. consequences for plnt fitness (Agrwl, 1998, 1999, 2). In our experiment, the mount of dmge imposed ws constnt cross plnt genotypes nd herbivore tretments, yet the distribution of this dmge within the plnt differed mong tretments. Pieris rpe lrve fed mostly on the upper prts of the plnt (primrily on young nd recently mture upper leves), wheres T. ni lrve fed on lower leves nd voided feeding on the picl prt of the plnt (Fig. S3). By contrst, in the clipping tretment, the dmge ws homogeneously distributed within the plnt (see the Mterils nd Methods section). In ddition, we found tht induced resistnce vried significntly mong herbivore tretments, nd this vrition ws homogeneous mong genotypes (Figs 6, S6 nd Tble S5). In prticulr, we found tht induced glucosinolte responses were higher in plnts ttcked by P. rpe lrve thn in other herbivore tretments. However, lthough the herbivore tretments differed consistently cross genotypes in terms of the loction of the dmge nd the pttern of induced response, the effect of the type of dmge on tolernce ws heterogeneous mong genotypes (Tble 2). MANOVA detected significnt genetic vrition on overll fitness trits, with significnt responses to herbivory t erlier stges of plnt reproduction (i.e. on flowering time nd the totl number of flowers). Two of the genotypes flowered erlier when exposed to insect dmge, nd insect herbivory cused two other genotypes to dely flowering in comprison with their undmged controls (Fig. 1). Similrly, herbivory hd significnt impct on the totl number of flowers produced per plnt, yet the mgnitude of these effects were genotype specific nd dependent on the type of dmge. When significnt (in three of 1 genotypes), herbivory lwys hd Phytologist (21) 188: Journl compiltion Ó Phytologist Trust (21)

10 Phytologist Reserch 473 Tble 5 Results of the robust ANCOVA nlyzing trde-offs between constitutive resistnce (genotype men glucosinolte concentrtion, GS ) nd tolernce to three types of herbivore dmge cross 1 Boecher strict genotypes with two contrsting constitutive glucosinolte profiles (BCAA-derived glucosinoltes vs methionine-derived glucosinoltes) Tolernce PC2 Tolernce PC3 Effect df Specilist Generlist Clipping Specilist Generlist Clipping R 2 n P R 2 n P R 2 n P R 2 n P R 2 n P R 2 n P [GS ] 1, GS profile 1, < GS profile [GS ] 1, < Significnt P vlues (P <.5) re in bold. [Glucosinoltes fter 24 h] (µmol g ) b negtive effect, resulting in fewer flowers thn in the undmged controls. The mrginlly significnt effect of the type of dmge on lter fitness components (PC1) is lso noticeble, which suggests tht plnts dmged by the specilist insect tended to overcompenste, wheres clipped plnts tended to undercompenste, nd this ws constnt for ll the genotypes (s indicted by the nonsignificnce of the herbivory by genotype interction for the component PC1, see Tble 2). Overcompenstion following P. rpe dmge hs been shown previously in the Brssiccee fmily, is relted to the induced response to such dmge, nd is thought to be dptive (i.e. plnts hve enhnced fitness when induced by specilist insects, nd lower fitness when undmged or clipped; see Agrwl, 1998, 1999). Similrly, overcompenstion is common when plnts re exposed to picl dmge (Wise & Abrhmson, 28, nd references therein), which ws the min type of dmge inflicted by P. rpe lrve in our experiment. Our results show tht, lthough B. strict cn compenste for herbivory (prticulrly dmge cused by the specilist insect), herbivory still reduces overll flower production nd bc Control Generlist Clipped Specilist Fig. 6 Totl Boecher strict lef glucosinolte concentrtion (djusted LS-MEANS ± 1SE) fter 24 h of dmge mong four different herbivore tretments. Different letters depict significnt differences (P <.5). *, Mrginlly significnt (P =.7). c c * lters flowering for some genotypes. The mechnisms involved in compenstion for herbivory re beyond the scope of this pper, but differentil lloction of resources to other plnt tissues, vrition in flowering time nd chnges in growth rtes re known mechnisms (Rosenthl & Kotnen, 1994; Agrwl et l., 1999; see lso Pilson & Decker, 22) which might explin the observed vrition in tolernce to herbivory mong B. strict genotypes. Clerly, the expression of tolernce my be influenced by other ecologicl fctors not considered in our study. For exmple, the levels of interspecific competition experienced in the field (Jones et l., 26) nd the timing of dmge my be importnt fctors determining the tolernce of B. strict to herbivory. We dmged the plnts fter 6 wk of vegettive growth, which my correspond to the midsummer period when most herbivores re ctively feeding on B. strict plnts in nturl popultions (T. Mitchell- Olds, pers. obs.). However, lter herbivory is still possible if herbivore outbrek occurs lte in the seson, or if plnt phenology is ltered by locl climtic conditions. The lter in the seson tht the dmge occurs, the shorter the compenstion period will be, nd hence dmge my lso ffect lte fecundity (see Mschinski & Whithm, 1989; Tiffin, 22). Glucosinolte polymorphism nd plnt fecundity A first step to understnding the significnce underlying the polymorphism of plnt chemicl defenses is the determintion of whether such vrition hs ecologicl implictions. Previously, in our system, we found tht chemicl polymorphism ffected plnt resistnce to generlist herbivores (Schrnz et l., 29). By contrst, we hve shown here tht tolernce to herbivore dmge does not depend on the structurl glucosinolte profile. In the current study, the chemicl polymorphism ws ssocited with plnt fitness regrdless of herbivore dmge. Thus, plnts with high proportion of BCAA-derived glucosinoltes in their leves flowered significntly lter nd hd significntly lower fitness thn plnts contining minly methionine-derived Journl compiltion Ó Phytologist Trust (21) Phytologist (21) 188:

11 474 Reserch Phytologist glucosinoltes (Fig. 3). Previous studies hve shown negtive effects of chemicl defenses on plnt fecundity in severl systems, suggesting the existence of direct lloction costs of resistnce (Struss et l., 22). However, to our knowledge, no work hs described the existence of direct costs of resistnce derived from nturl nd discrete vrition in the structurl chemicl profile within plnt species. Although this effect on fitness might lso reflect other genotypic effects (i.e. linkge disequilibrium), severl rguments suggest tht discrete vrition in the chemicl profile of B. strict genotypes might truly ffect other components of plnt fitness. First, mong plnt species, the mgnitude nd significnce of constitutive resistnce costs t the quntittive level vry mong types of defensive compounds, becuse distinct defensive compounds hve different biosynthetic costs (Korichev, 22 nd references therein). Interestingly, in our system, we lso found significnt negtive genetic correltion between the totl concentrtion of glucosinoltes nd fitness components mong genotypes with different chemicl profiles, suggesting direct cost of resistnce. Although the totl concentrtion of glucosinoltes ws negtively correlted with erly plnt fecundity (PC2), it ws strongly nd negtively correlted with lte plnt fecundity (PC1) in genotypes with high proportion of BCAA-derived glucosinoltes (Tble S7, Fig. S8). Second, QTL mpping nlyses recently conducted on B. strict mpping popultion hve reveled significnt QTL predicting survivl nd reproduction in the BCMA chromosoml region, which controls the synthesis of BCAA- vs methionine-derived glucosinoltes in B. strict (Schrnz et l., 29; J. Anderson & T. Mitchell-Olds, unpublished). Future work using trnsgenic lines will clrify whether BCMA genes hve significnt effects on plnt fitness trits. One limittion of our study, however, is tht fitness estimtes were obtined under glsshouse conditions, which my differ from fitness mesurements in nturl popultions, especilly if vrition in the chemicl profile in B. strict is the result of locl dpttion or nother form of blncing selection. The ltter possibility is currently being exmined in nturl popultions of B. strict. Costs of tolernce Plnt defense theory proposes tht lloction costs mintin genetic vrition for tolernce, preventing the fixtion of lleles for mximl tolernce mong individuls within nd mong popultions (Struss & Agrwl, 1999; Struss et l., 22). However, empiricl evidence for such costs is limited, despite multiple ttempts to ddress this question (reviewed by Korichev, 22; Leimu & Korichev, 26). Here, we investigted two different types of lloction costs of tolernce. However, we did not find significnt negtive genetic correltion between tolernce nd fitness in the bsence of herbivores, or between tolernce to different types of herbivore dmge. Indeed, tolernce to different types of dmge showed significnt positive genetic correltions. These results indicte tht lloction costs do not constrin the evolution of tolernce (hence genetic vrition for tolernce my be mintined by other ecologicl or evolutionry forces) nd genotypes showed positive genetic correltions in their tolernce to different herbivore tretments. This result is concordnt with the few existing studies exmining tolernce to different enemies, which found no evidence for trde-offs between tolernce to different types of dmge (Tiffin & Rusher, 1999; Pilson, 2). These results suggest tht the evolution of tolernce to multiple herbivores my not ccount for the existence of intermedite levels of tolernce to lef dmge on plnts (Nuñez- Frfán et l., 27). In ddition, we did not find ny trde-off between tolernce nd investment in defensive metbolites, s suggested by the lck of significnt negtive genetic reltionship between constitutive or induced glucosinoltes nd tolernce to different types of dmge. On the contrry, we found tht the concentrtion of constitutive glucosinoltes nd tolernce to clipping were significntly nd positively correlted, t lest in terms of the production of flowers. This suggests tht tolernt plnts my lso be more resistnt to some types of dmge. This ws especilly true for genotypes with methionine-derived glucosinoltes (Tble 5, Fig. S5). Thus, the evolution of resistnce nd tolernce in B. strict does not seem to be limited by genetic constrints nd, for genotypes with methionine-derived glucosinoltes, resistnce nd tolernce will evolve jointly (i.e. the lloction of resources simultneously to both tolernce nd resistnce; Nuñez-Frfán et l., 27). Although we did not detect ny lloction costs of tolernce in our study, there is growing evidence tht the existence of lloction costs is dependent on complex interctions with the biotic nd or biotic environment (Korichev, 22; Siemens et l., 29), which we did not mnipulte in our glsshouse experiment. Therefore, future work will need to tke into ccount the environmentl vrition tht exists under nturl conditions. In short, our study hs shown tht structurl vritions in the chemicl profile of plnt defenses do not influence the bility of B. strict to compenste for different types of herbivore dmge, lthough this chemicl vrition is correlted with plnt fitness in our smple. In ddition, our study demonstrtes the importnce of considering jointly both intrinsic plnt fctors nd extrinsic ecologicl fctors to understnd the evolution of plnt tolernce to dmge nd its costs. Acknowledgements We thnk three nonymous reviewers nd J. Anderson for helpful comments on the mnuscript, nd K. Springer, S. Phytologist (21) 188: Journl compiltion Ó Phytologist Trust (21)