Early resistance of alien and native pines against two native generalist insect herbivores: no support for the natural enemy hypothesis

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1 Functionl Ecology 212, 26, doi: /j x Erly resistnce of lien nd ntive pines ginst two ntive generlist insect herivores: no support for the nturl enemy hypothesis Ampro Crrillo-Gvilán*,1, Xoquín Moreir 2, Rfel Zs 3, Montserrt Vilà 1 nd Luis Smpedro 2 1 Estción Biológic de Doñn (EBD-CSIC), Avd. Américo Vespucio s n, Isl de l Crtuj, 4192 Sevill, Spin; 2 Centro de Investigción Forestl de Lourizán Unidd Asocid MBG-CSIC, Apdo. 127, 368 Pontevedr, Spin; nd 3 Misión Biológic de Glici (MBG-CSIC), Apdo. 28, 368 Pontevedr, Spin Summry 1. The nturl enemy hypothesis (NEH) predicts tht lien plnt species might receive less pressure from nturl enemies thn do relted coexisting ntive plnts. However, most studies to dte re sed on pirs of ntive nd lien species, nd the results remin inconclusive. The level of ttck y ntive generlist herivores cn vry considerly etween plnt species, depending on defensive trits nd strtegies. Plnt defences include preformed constitutive nd induced defences tht re ctivted s plstic responses to herivore ttck. However, the efficcy of induced defences could e ltered when lien species entering n re re exposed to ntive enemies. 2. We tested the NEH for severl closely relted lien nd ntive pines to Europe y exmining erly nti-herivore resistnce to dmge y two generlist ntive insect herivores (Hyloius ietis nd Thumetopoe pityocmp); the differences in constitutive nd inducile chemicl defences (i.e. non-voltile resin nd totl phenolics in the stem nd needles); nd whether consumption preferences shift fter induced defences hve een triggered y rel herivory. 3. We did not find lien pines to e less dmged y two generlist herivores thn ntive pines were. The constitutive concentrtion of chemicl defences significntly differed mong pine species. The concentrtion of constitutive totl phenolics in the stem ws greter in ntive thn in lien pines. The opposite trend ws found for constitutive totl phenolics in the needles. The concentrtion of chemicl defences (non-voltile resin nd totl phenolics) in the stem significntly incresed fter herivory y H. ietis. Moreover, the induction of totl phenolics y H. ietis dmge ws significntly greter in ntive pine species thn in lien pines. On the other hnd, only concentrtions of non-voltile resin in needles significntly incresed fter herivory y T. pityocmp, ut without significnt differences in induciility etween lien nd ntive pines. In cfeteri iossys, H. ietis consumed the twigs from lien more thn those from ntive species irrespective of prior exposure to the insect. Menwhile, no differences mong rnge origin were found in the T. pityocmp cfeteri iossys. 4. Overll, we found no support for the NEH in lien pines to Europe. This suggests tht lien pines, in regions where they coexist with ntive congeners, my e controlled y ntive generlist herivores, this eing one reson tht invsion y lien pines is not frequent in Europe. Key-words: condensed tnnins, Hyloius ietis, induced defences, non-voltile resin, plnt invsions, phenolics, plnt herivore interctions, Thumetopoe pityocmp *Correspondence uthor. E-mil: mprocg@ed.csic.es Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society

2 284 A. Crrillo-Gvil n et l. Introduction The success of lien plnts in their new rnge depends prtilly on the interction with the ntive iot, prticulrly with herivores tht could dmge estlishing seedlings (Mron & Vil` 21; Levine, Adler & Yelenik 24). The impct of ntive herivores on lien plnts is determined y the interply etween their ility to recognize the lien plnt s food resource (Crpenter & Cppuccino 25), the effectiveness of the lien plnt defences ginst herivores in the new rnge (Stowe et l. 2) nd the ility of herivores to overcome plnt defences of the lien (Rusher 21). All these mechnisms might influence the success or filure of n lien plnt to invde new re. The nturl enemies hypothesis (NEH) predicts tht lien plnt species re successful invders in the new rnge ecuse of lck of specilist nturl enemies, which were left ehind in their ntive rnge (Mron & Vilà 21). Severl studies hve shown how introduced plnt popultions in new re re less dmged y ntive phytophges nd pthogens thn re popultions in the ntive rnge (DeWlt, Denslow & Ickes 24; Vilà, Mron & Mrco 24; Rogers & Siemnn 25). Nevertheless, there re contrdictory results on whether ntive herivores cuse more dmge to ntive thn to lien plnt species (Chun, vn Kleunen & Dwson 21). The level of dmge inflicted ginst lien species nd its influence on plnt fitness is not lwys lower thn ginst coexisting ntive plnts, even when the species re txonomiclly relted (Agrwl & Kotnen 23). The ssumption of less dmge in lien species in new rnge hs een ttriuted to freedom from specilist herivores. However, it is not lwys possile to ttriute dmge to prticulr herivore species, nd generlist herivores would e the first to hve the ility to choose etween host species. A recent metnlysis compring pirwise ntive nd lien trees noted tht the mjority of ntive insect species tht colonize lien trees re generlist herivores (Bertheu et l. 21). In ddition, reduced resistnce of lien plnts leding to rpid coloniztion y ntive generlist herivores ws found to occur when congener ntive plnt species live in the introduced rnge (e.g. Goßner et l. 29). However, most studies hve compred lien ntive species pirs, nd, s suggested y Chun, vn Kleunen & Dwson (21), more thn two species should e considered for testing-relted lien nd ntive species. The level of ttck y herivores cn vry considerly etween plnt species, depending on their defensive strtegies (Agrwl et l. 25). Plnt chemicl defences include constitutive defences, which re permnently expressed irrespective of the plnt exposure to nturl enemies, nd induced defences tht enhnce the sl defence cpcity s result of plstic responses to nturl enemy ttck (Krn & Myers 1989). Induced strtegies, rther thn constitutive, re expected to e fvoured when herivore pressure vries sptilly nd temporlly, nd initil ttcks re relile cues of further ttcks (Krn 211). Closely relted species my differ in their defensive strtegy, depending on the iotic nd iotic environment where they hve evolved (Orins & Wrd 21). Production of effective induced defences requires the plnt to quickly recognize the herivore dmge s potentil risk, signlling the dnger cross tissues nd susequently triggering defences (Heil 21). There is emerging evidence tht induced responses to herivory cn show high specificity regrding oth the dmging herivore (i.e. specificity of elicittion) nd the efficcy of the induced responses (i.e. specificity of the effect) (Agrwl 211). The efficcy of the induced defensive strtegies could thus e notly ltered when lien plnts re exposed to new enemies in the introduced re. Ntive plnts shring n evolutionry ssocition with ntive generlist enemies my hve evolved greter inducile defence ginst such enemies thn lien plnts hve (Joshi & Vrieling 25). Recognition mismtch y the lien plnt could hinder induced responses nd, consequently, reduce the fitness of the lien plnt. Although constitutive s well s induced defences cn contriute to effective resistnce to herivory, the interply etween the two defensive strtegies hs een rrely considered when explining invsiveness of lien species (Orins & Wrd 21). Pines (Pinus spp.) constitute clssic model in the study of trits ssocited with plnt-invsion potentil (i.e. invsiveness) (Richrdson 26). Pine invsiveness hs een ssocited with smll seed mss, short juvenile period, short intervl etween lrge seed crops nd fst reltive erly growth rte (Rejmánek & Richrdson 1996; Grotkopp, Rejm nek & Rost 22). However, there is pronounced geogrphicl vrition in invsion success within species. For exmple, the North Americn Pinus rdit is invsive in mny regions of the Southern Hemisphere (Lvery & Med 1998), while in Europe, little evidence of invsion hs een reported (Crrillo-Gvilán & Vilà 21). Differences in invsiveness mong iogeogrphicl regions hve een suggested to e relted to different propgule pressure (Pyšek & Jrosˇik 25), ut might lso depend on other fctors such s llelopthy (Hierro & Cllwy 23) or differences in the interction with nturl enemies (Richrdson & Higgins 1998). For exmple, in Europe, P. rdit is ttcked y mny generlist pests tht lso dmge ntive pines species (Lomrdero, Vázquez-Mejuto & Ayres 28), while in the Southern Hemisphere ntive pines nd hence their ssocited specilist nd generlist herivores re sent. In ddition, s survivl t erly stges is key for future fitness of pine trees, erly resistnce of pine seedlings is likely determinnt for invsiveness. In this study, we investigted the erly resistnce to generlist herivores in seedlings of lien nd ntive pine species to Europe within the context of the predictions of the NEH. The specific ojectives were s follows: (i) to compre the dmge cused y two generlist ntive insect herivores in lien nd ntive pine species, (ii) to evlute quntittive differences etween lien nd ntive pine species in constitutive chemicl defences nd in those expressed fter exposure to herivory y those insects nd (iii) to ssess whether feeding preferences on lien nd ntive pines shift fter defences hve een induced. We expected tht: (i) generlist herivores would cuse lower dmge to lien thn to ntive pine species s predicted y the NEH; (ii) lien plnts exposed to generlist Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society, Functionl Ecology, 26,

3 Resistnce lien nd ntive pines to herivores 285 ntive herivores would induce weker defences thn would ntive pines ecuse lck of shred evolutionry history nd finlly (iii) herivory y ntive herivores would decrese further herivore consumption more in ntive thn in lien pines. Mterils nd methods To test the ove-mentioned ojectives, we mesured the whole-plnt dmge cused y two insect generlist herivores to living seedlings, evluted the differences in constitutive nd inducile chemicl defences fter the tretments of exposure to herivores nd ssessed through cfeteri iossys whether feeding preferences of oth insects shifted fter plnt defences hve een induced y exposure to the herivores. STUDY SPECIES Nine pine species elonging to the Pinus clde (Eckert & Hll 26), which re rodly plnted world-wide, were used for this study: Pinus cnriensis C. Sm. (CAN), Pinus hlepensis Mill. (HAL), Pinus pine L. (PIN), Pinus pinster Ait. (PTR) nd Pinus sylvestris L. (SYL) re ntive to Europe; while Pinus coulteri D. Don (COU), P. rdit D. Don (RAD), Pinus roxurghii Srg. (ROX) nd Pinus sinin Dougls ex D.Don (SAB) re lien to Europe (DAISIE 29). Seeds were purchsed in the following nurseries: Intersemills, Spin ( Sheffield s Seed Co., USA ( nd Les Semences Du Puy, Frnce ( For more informtion on the ntive distriution nd seed geogrphicl provennces of Pinus species ssyed, see Tle 1 of Appendix S1 (Supporting informtion). The herivores ssyed were the lrge pine weevil, Hyloius ietis L. (Coleopter: Curculionide) nd the pine processionry cterpillr, Thumetopoe pityocmp Dennis nd Schiff (Lepidopter: Thumetopoeide) (Hyloius nd Thumetopoe, respectively, herefter). Both herivores were chosen ecuse they ttck vriety of coniferous species nd thus re considered generlist conifer herivores. Ech herivore feeds on conifer tissues with contrsting fitness vlue: Hyloius ttcks stem phloem while Thumetopoe feeds on needles. Their ntive distriutionl rnge is within the geogrphicl limits of Europe nd concurs with ntive Europen Pinus species hving high proility to ttck them ecuse of their extensive distriution re. Hyloius occurs nturlly in Europe nd northern Asi (Scott & King 1974) where it is one of the most hrmful ntive pests ffecting the regenertion of mnged coniferous forests in mny prts of Europe (Långström & Dy 24). This pine weevil is polyphgous herivore tht feeds on the rk nd phloem of young conifer seedlings (Wllertz 25), minly ffects Pinus nd Pice species, leding to high seedling mortlity (Nordlnder et l. 23). Hyloius cn cuse extensive tree mortlity t young ges nd mjor growth losses ecuse of leder loss (Lieutier et l. 24). As n exmple, the pine weevil hs een estimted to cuse the deth of up to 8% of coniferous seedlings plnted following cler-cutting (von Sydow & Birgersson 1997; Orlnder & Nilsson 1999; Nordlnder, Nordenhem & Hellqvist 28). Thumetopoe, the pine processionry, is defolitor nturlly distriuted in the Mediterrnen Bsin nd southern Europe nd my eventully feed on severl coniferous gener. It ttcks minly plnts of the genus Pinus (Devkot & Schimidt 199). Its outreks inflict serious economic losses (Ho dr, Zmor & Cstro 22). Lrve feed in conifer needles of juvenile nd dult pines cusing them to dry nd fll. Dmge y the pine processionry wekens nd reduces the growth of juvenile trees (Lieutier et l. 24), lowers the reproductive cpcity of dult ones (Ho dr, Cstro & Zmor 23) nd even cuses deth when trees re hevily defolited. The ility of Thumetopoe to develop vries widely depending on the pine species nd its living conditions (Devkot & Schimidt 199; Msutti & Bttisti 199; Hódr, Zmor & Cstro 22). Adult weevils were trpped in the field following the method descried y Moreir et l. (28), stored in culture chmers t 15 C nd fed fresh twigs for mximum 2 weeks efore the experiment egn. Entire Thumetopoe cterpillr nests were collected directly from infested trees, lelled nd mintined s ove. GREENHOUSE EXPERIMENTAL DESIGN We conducted two-fctoril greenhouse experiment with pine species nd induction of plnt defensive responses y exposing living seedlings to two insect herivores s the min fctors (herivoryinduction tretments, herefter). The experiment followed rndomized split-plot design replicted in 1 locks, with herivory-induction (three levels: control nd herivory y Hyloius nd y Thumetopoe) s the whole fctor nd pine species (nine levels) s the split fctor. However, ecuse of lck of enough pine seedlings, only six pine species (CAN, PTR, SYL, COU, RAD nd ROX) were induced with Thumetopoe. In totl, we grew 24 pine seedlings, corresponding to 1 locks two tretments (control nd herivory y Hyloius) nine species, plus 1 locks one tretment (herivory y Thumetopoe) six species. In Octoer 28, t the Forestry Reserch Centre of Lourizán (Pontevedr, Spin), pine seeds were individully sown in 2-L pots filled with mixture of perlite nd pet (1 : 1 v:v), fertilized with 12 g of slow-relese fertilizer (Multicote Ò N:P:K 15:15:15, Hif Chemicls Ltd., Isrel) nd covered with 1- to 2-cm lyer of sterilized snd. Pots were plced in greenhouse with controlled light (12 h light) nd temperture (1 25 C t night nd dy, respectively) nd dily irrigtion. Plnts were supplemented monthly with solution of micronutrients to void nutritionl deficiencies ccording to previous experience (see complete fertiliztion tretment in Tle 2 of Appendix S2, Supporting informtion from Smpedro, Moreir & Zs 211). Folir nutrition ws pplied monthly using chelted iron. Before the herivory-induction tretment egn, weevils were kept individully without food for 48 h in lelled Petri dishes with moist filter pper (15 C, drk) nd then weighed. Similrly, Thumetopoe nests were crefully opened, 2nd-instr lrve rndomly seprted into groups of 1 cterpillrs nd strved nd weighed s ove. In Septemer 29, we pplied the herivory-induction tretments (herivory y Hyloius nd y Thumetopoe). In the herivory y Hyloius, one pre-weighed weevil ws plced on ech pine seedling, llowed to feed for 5 dys nd then removed nd weighed gin. Dmge inflicted y the weevil ws evluted independently in every 1 5 stem sections s the reltive derked re using four-level scle ( = undmged; 1 = 1 25% dmged; 2 = 26 5% dmged; 3 = >5% dmged), nd the sum of vlues for the five sections per seedling (i.e. 15 score) ws considered to e the derked re. For the herivory y Thumetopoe, s cterpillrs re gregrious nd move little from the plce where they re deposited, we plced one pre-weighed group of 1 cterpillrs in the top section nd other in the ottom section of ech living pine seedling. Becuse of the smller size of P. sylvestris seedlings, only one group of 1 lrve ws plced on this species. Cterpillrs were llowed to feed on the needles for 6 dys nd then removed, counted nd weighed. Folir dmge ws Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society, Functionl Ecology, 26,

4 286 A. Crrillo-Gvil n et l. evluted for the whole plnt in three-level scle: = undmged leves, 1 = dmged leves <5, 2 = dmged leves >5 (i.e. 2 score). Ech plnt of the three herivory-induction tretments ws crefully covered with nylon mesh to void either herivore escpe or interference mong tretments. One week fter the ppliction of herivory-induction tretments, ll pine seedlings were hrvested nd directly weighed to determine the totl ove-ground plnt iomss. Just fter hrvesting, two sl 2Æ5-cm-long stem sections nd two susmples (1Æ5 g ech) of needles rndomly from the whole pool of needles of ech plnt were collected, lelled nd immeditely stored in ice-coolers to e used in the cfeteri iossys. CHEMICAL ANALYSES In the current pper, we nlysed the concentrtion of non-voltile resin nd totl phenolics in the stem nd needles. Additionlly, we nlysed the concentrtion of condensed tnnins in needles fter herivory y Thumetopoe. Long-lived plnts such s pine trees, which must cope with multiple enemies simultneously, re likely to employ quntittive defences (sensu Feeny 1976) tht offer incresed resistnce in higher concentrtions. Although the relevnce of different types of chemicl defences ginst diverse herivores in conifers is not fully known, it is cler tht these re involved in direct resistnce ginst pine defolitors nd stem orers (see reviews y Frnceschi et l. 25; Mumm & Hilker 26). Indeed, in previous reserch with Mediterrnen pine species, we found tht non-voltile resin nd phenolics re inducile, responsive to herivore dmge nd confer resistnce to weevil feeding (Moreir, Smpedro & Zs 29; Smpedro, Moreir & Zs 211,; Zs, Moreir & Smpedro 211; Moreir, Zs & Smpedro 211). The concentrtion of non-voltile resin in stems nd needles ws estimted grvimetriclly following the procedure proposed y Smpedro, Moreir & Zs (211) nd expressed s mg of non-voltile resin per grm plnt tissue dry weight (d.w.). A 5-cm-long section of the low prt of the stem or 2 g of fresh needles of ech plnt ws collected, weighed, immeditely frozen nd stored t )3 C. Plnt mteril ws cut in smll sections, nd resin compounds were quntittively extrcted twice with n-hexne in n ultrsonic th, fter which the plnt mteril ws recovered y filtrtion (Whtmn GFF, Whtmn, Kent, UK), the solvent in the tues ws evported to dryness, nd the mss of the non-voltile resin residue ws determined grvimetriclly with precision scle. This grvimetric determintion of non-voltile resin ws well correlted with the concentrtion of the diterpenoids frction s quntified y gs chromtogrphy in previous trils (r =Æ921, P <Æ1; Smpedro, Moreir & Zs 211). The totl phenolics in the needles nd stem (nd condensed tnnins in the needles) were extrcted nd nlysed s descried y Smpedro, Moreir & Zs (211). A susmple of needles (c. 2 g dry weight) or of stem (c. 3 mg dry weight) ws lso weighed, ovendried (45 C to constnt weight) nd then mnully ground in mortr with liquid nitrogen. Briefly, needles were extrcted with queous methnol (1 : 1 vol : vol) in n ultrsonic th, nd totl phenolics in the methnolic extrct were nlysed y the Folin-Cioclteu method, with colorimetric determintion in microplte reder t 74 nm using tnnic cid s stndrd. The concentrtion of totl phenolics is thus expressed s mg of tnnic cid equivlents per grm plnt tissue dry weight. Needle-condensed tnnins in the queous methnol extrcts were determined y the procynidine method s in Brz et l. (24). The methnolic extrct ws mixed with cidified utnol nd ferric mmonium sulphte solution, llowed to rect in oiling wter th for 5 min nd then cooled rpidly on ice. The concentrtion of condensed tnnins in this solution ws determined colorimetriclly in Biord 65 microplte reder t 55 nm, using s stndrd purified condensed tnnins of quercho (Schinopsis lnse Engl.; Droguerí Modern, Vigo, Spin). Needle concentrtion of condensed tnnins ws thus expressed s mg of quercho equivlents per grm needle dry weight. Becuse of lck of plnt mteril, P. sylvestris ws not included in the chemicl nlyses of non-voltile resin in needles nd totl phenolics in stem. CAFETERIA BIOASSAYS Independent cfeteri iossys were conducted with Hyloius nd with Thumetopoe in six pine species: CAN, PTR, SYL, COU, RAD nd ROX. In the Hyloius iossy, one weevil (strved nd weighed s explined ove) ws llowed to feed on 2Æ5-cm-long section of sl stem for 48 h in Petri dish with moist filter pper (drk, 18 C). The volume of tissue ingested y the weevil (mm 3 ) ws estimted fter mesuring the derked re y the weevil with millimeter grid, nd the depth of the gnwing with n electronic guge. We conducted two replictes per plnt, except for P. sylvestris, for which only one replicte ws mde ecuse of insufficient plnt mteril. In the Thumetopoe iossy, groups of ten 2nd- or 3rd-instr cterpillrs (strved nd weighed s ove) were llowed to feed on fresh needles for 7 dys in Petri dishes s descried ove. A prllel series of needles ws immeditely dried t 65 Ctoconstntweighttodetermine percentge of dry mtter. The specific ingestion of needles (g needles d.w.) y the cterpillrs ws clculted s the dry food ingested. The percentge of cterpillr survivl t the end of iossys ws lso registered s n indictor of plnt defences. As in the Hyloius iossy, two replictes were performed per seedling except for P. sylvestris, for which we included only one replicte. In totl, 33 Petri dishes were used in the iossy (five species three tretments 1 plnts two replictes, nd one species (SYL) with three tretments 1 plnts one replicte). STATISTICAL ANALYSES Differences mong pine species in whole-plnt dmge to seedlings, mesured s derked re y Hyloius nd folir dmge y Thumetopoe, were nlysed in treted plnts using generlized liner model: Y jk = l +SP j + B k + e jk, where l is the generl men, SP j nd B k re the min effects of the species j (SP j = 1 9 nd SP j = 1 6 in herivory for Hyloius nd y Thumetopoe tretments, respectively) nd lock k (B k = 1 1), respectively. This nlysis ws performed with the PROC-GLIMMIX procedure of the SAS System (Littell et l. 26) with norml error nd n identity link function, while differences mong pine species in constitutive chemicl defences were nlysed in control untreted plnts using the sme eqution model s efore ut using PROC-MIXED procedure of the SAS System. The effects of herivory-induction tretments nd pine species on chemicl defences were nlysed with the following mixed model: C ijk = l + T i +SP j + B k + T i *SP j + T i *B k + e ijk, where l is the overll men, T i nd SP j re the min effects of induction tretments i (T i =, 1, 2), species j (SP j = 1 9 nd SP j = 1 6 in herivory for Hyloius nd y Thumetopoe tretments, respectively), T i SP j nd B k T i re the corresponding interctions nd e ijk is the experimentl error, while lock k (B k = 1 1) nd the B k T i interction were considered rndom fctors to nlyse the min fctor T with the pproprite error terms (Littell et l. 26). Becuse of the different numer of pine species included in the Hyloius nd Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society, Functionl Ecology, 26,

5 Resistnce lien nd ntive pines to herivores 287 Thumetopoe herivory-induction tretments, the effects of induction were nlysed independently for ech tretment together with the control. Plnt iomss ws previously checked not to covrite with the response vriles nd thus ws not included in the model. The cfeteri iossys were nlysed with n hierrchicl mixed model in which we included the B k T i SP j interction s rndom effect (split-split design), to ccount for the utocorreltion of the two Petri dishes used for ech pine seedling (Littell et l. 26), nd to nlyse the species fctor with the pproprite degrees of freedom. Insect weight ws included s covrite in the nlyses. Reltionships etween dmge in cfeteri iossys nd chemicl defences were crried out cross pine species (n =6). When there were significnt differences etween species, we nlysed whether ntive pine species differed from lien species y including the fctor Rnge (lien or ntive) nd nesting the fctor Species within the fctor Rnge in the mixed models. All nlyses were performed with the PROC-MIXED procedure of the SAS System. When needed, normlity ws chieved y log-trnsforming the originl vriles. Equlity of residul vrince cross tretments ws tested in ll cses, nd residul heterogeneity vrince models were used when significnt devitions were found (Littell et l. 26). Dt re shown s lest squre mens ± stndrd error of the men (SEM). When min effects or differences etween species were significnt, pirwise differences were tested for significnce using the LSMEANS sttement. Results SEEDLING DAMAGE Seedling dmge y Hyloius nd Thumetopoe fter herivory-induction tretments significntly differed mong pine species (Hyloius: F 8,7 =2Æ66, P <Æ5; Thumetopoe: F 5,45 =3Æ4, P <Æ5) (Fig. 1). The reltive resistnce of the pine species to oth herivore species ws not consistent. For exmple, the lest dmged pine y Thumetopoe ws P. cnriensis, while stems of this pine species showed intermedite resistnce to Hyloius (Fig. 1). Additionlly, herivore dmge did not significntly differ etween lien nd ntive pine species (Hyloius: F 1,7 =Æ19, P =Æ66; Thumetopoe: F 1,45 =1Æ19, P =Æ28). CONSTITUTIVE AND INDUCED CHEMICAL DEFENCES Constitutive concentrtion of most of chemicl defences (undmged seedlings) vried significntly mong pine species (non-voltile resin in stem: F 8,71 =18Æ88, P <Æ1, Fig. 2; non-voltile resin in needles: F 7,61 =13Æ71, P <Æ1, Fig. 2; totl phenolics in needles: F 8,72 =2Æ17, () Fig. 1. Dmge in Europen ntive (lck rs) nd lien (grey rs) pine species fter exposure of living seedlings to rel herivory y () the pine weevil Hyloius ietis nd () the pine processionry Thumetopoe pityocmp. Brs re LS mens ± SEM. Different letters ove columns indicte significnt differences (P <Æ5) mong pine species or etween rnge (ntive vs. lien). Species revitions: Pinus cnriensis (CAN), Pinus hlepensis (HAL), Pinus pine (PIN), Pinus pinster (PTR) nd Pinus sylvestris (SYL) s ntive species, nd Pinus coulteri (COU), Pinus rdit (RAD), Pinus roxurghii (ROX) nd Pinus sinin (SAB) s lien species. Folir dmge ( 3 score) Derked re ( 15 score) () ntive lien Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society, Functionl Ecology, 26,

6 288 A. Crrillo-Gvil n et l. STEM NEEDLES 6 () Control Hyloius 6 () Control Thumetopoe Non-voltile resin (mg g 1 d.w.) c cd c c d c c Totl Phenolics (mg tnnic cid equiv g 1 d.w.) (c) CAN HAL PIN PTR SYL COU RAD ROX SAB ntive lien Control Hyloius Control Thumetopoe CAN PTR SYL COU RAD ROX ntive lien ntive lien ntive lien (d) Fig. 2. Effects of the herivory-induction tretments on the concentrtion of () non-voltile resin nd (c) totl phenolics in the stem in plnts exposed to herivory y the phloem feeder Hyloius ietis; nd () non-voltile resin nd (d) totl phenolics in the needles in plnts exposed to herivory y the defoliting cterpillr Thumetopoe pityocmp. The species re grouped into ntive nd lien species in Europe. Brs re LS mens ± SEM. Different letters ove columns indicte significnt differences (P < Æ5) mong pine species or etween rnge (ntive vs. lien). = non-significnt differences. See Fig. 1 legend for species revitions. P =Æ4, Fig. 2d; condensed tnnins in needles: F 8,7 =7Æ46, P <Æ1). Pinus sinin nd P. sylvestris were the species with the gretest concentrtion of non-voltile resin in the stem (Fig. 2). Pinus roxurghii showed the highest concentrtion non-voltile resin in the needles (Fig. 2) while P. rdit nd P. roxurghii showed the highest concentrtion oth of totl phenolics (Fig. 2d) nd of condensed tnnins in the needles. Only constitutive concentrtion of totl phenolics in the stem did not significntly vry mong pine species (F 7,63 =1Æ92, P =Æ8; Fig. 2c). Alien pine species registered significntly greter constitutive concentrtion of totl phenolics nd condensed tnnins in the needles thn did ntive pine species (totl phenolics: F 1,72 =6Æ22, P <Æ5, Fig. 2d; condensed tnnins: F 1,7 =4Æ8, P < Æ5). Contrrily, ntive pine species showed significntly greter constitutive concentrtion of stem totl phenolics thn lien pine species (F 1,63 =4Æ76, P <Æ5; Fig. 2c). However, lien nd ntive pines did not differ in their constitutive concentrtion of stem nd needle non-voltile resin (stem non-voltile resin: F 1,71 =1Æ79, P =Æ18, Fig. 2; needle non-voltile resin: F 1,61 =Æ78, P =Æ38; Fig. 2). The exposure to rel herivore y Hyloius ffected the concentrtion of non-voltile resin nd totl phenolics in the stem (Tle 1). Overll, the concentrtion of non-voltile resin in seedlings exposed to Hyloius ws 25% greter thn those in undmged control seedlings (Fig. 2). Moreover, the induction of stem non-voltile resin fter herivory y Hyloius differed mong the pine species, s indicted y the significnt species tretment interction (Tle 1, Fig. 2). Non-voltile resin concentrtions in the stem of P. pinster, P. sylvestris, P. coulteri nd P. roxurghii significntly incresed fter exposure to the weevil, while no significnt chnges were detected in the other pine species (Fig. 2). We found no differences etween lien nd ntive pine species (F 1,137 =1Æ21, P = Æ27). The concentrtion of totl phenolics in the stem differed etween tretments nd mong species (Tle 1). Overll, the concentrtion of totl stem phenolics in seedlings exposed to Hyloius ws 23% greter thn those in undmged control seedlings (Fig. 2c). Pinus hlepensis nd P. rdit hd the highest nd the lowest concentrtion of totl phenolics in stem, respectively. Moreover, ntive pines showed significntly greter totl phenolics concentrtion thn lien pines (F 1,12 =5Æ53, P <Æ5, Fig. 1). After rel herivory y Thumetopoe, only non-voltile resin in needles significntly differed etween tretments nd mong species (Tle 1, Fig. 2,d). Pine species exposure to herivory y Thumetopoe hd greter non-voltile resin concentrtion in needles thn control seedlings, nd P. roxurghii nd P. pinster showed the gretest nd lowest induction of non-voltile resin concentrtion in needles, Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society, Functionl Ecology, 26,

7 Resistnce lien nd ntive pines to herivores 289 Tle 1. Results of the mixed models with species nd herivoryinduction tretment s fixed fctors, nd concentrtion of nonvoltile resin nd of totl phenolics s dependent vriles. Anlyses were performed independently to compre control plnts nd () pine seedlings with induced herivory from the lrge pine weevil Hyloius ietis nd () pine seedlings with induced herivory from cterpillrs of the pine processionry moth Thumetopoe pityocmp. Significnt P-vlues (P <.5) re given in old () Nine pine species in control nd fter herivory-induction y Hyloius Non-voltile resin in the stem Totl phenolics in the stem Tle 2. Results of the mixed model for the cfeteri iossys with species nd herivory-induction tretment s fixed fctors. Anlyses were performed independently to compre control plnts nd () the consumed volume of stem y the lrge pine weevil Hyloius ietis nd () the specific ingestion of needles nd cterpillr survivl of the pine processionry Thumetopoe pityocmp s dependent vriles. Nine pine species were tested with the weevil, nd six were tested with the cterpillr, so tht independent nlyses were performed for ech insect iossy. Significnt P-vlues (P <.5) re given in old () Hyloius cfeteri iossy Consumed volume of stem Source d.f. F P d.f. F P Source d.f. F P Block 9, 9 Æ43 Æ885 9, 9 1Æ23 Æ383 Tretment (T) 1,9 12Æ55 Æ6 1, 9 9Æ23 Æ14 Species (SP) 8, Æ33 <Æ1 7, 12 2Æ12 Æ46 T*SP 8, 137 2Æ46 Æ15 7, 12 Æ42 Æ891 () Six pine species in control nd fter herivory-induction y Thumetopoe Block 9, 9 5Æ34 Æ1 Tretment (T) 1,9 Æ23 Æ645 Species (SP) 5, 89 8Æ71 <Æ1 T*SP 5, 89 Æ51 Æ766 Insect weight 1, 94 6Æ36 Æ13 () Thumetopoe cfeteri iossy Non-voltile resin in the needles Totl phenolics in the needles Specific ingestion of needles Cterpillr survivl Source d.f. F P d.f. F P Source d.f. F P d.f. F P Block 9, 9 1Æ93 Æ17 9, 9 2Æ66 Æ8 Tretment (T) 1,9 4Æ72 Æ58 1, 9 Æ31 Æ59 Species (SP) 4, 68 8Æ52 <Æ1 5, 85 2Æ28 Æ53 T*SP 4, 68 1Æ5 Æ21 5, 85 Æ52 Æ759 Block 9, 9 4Æ51 Æ17 9, 9 Æ71 Æ69 Tretment (T) 1,9 Æ1 Æ938 1, 9 Æ31 Æ593 Species (SP) 5, 89 12Æ9 <Æ1 5, 9 7Æ65 <Æ1 T*SP 5, 9 1Æ42 Æ225 5, 91 Æ67 Æ643 Insect weight 1, 64 1 Æ32 1, 65 27Æ35 <Æ1 respectively (Fig. 2). However, we found no differences etween lien nd ntive pine species (non-voltile resin in needles: F 1,68 =Æ42, P =Æ51). The concentrtion of condensed tnnins in the needles ws not ffected y Thumetopoe tretment (F 1,9 =Æ1, P =Æ76). In ddition, chemicl defences in needles ginst Hyloius herivory nd in stem ginst Thumetopoe herivory showed no significnt chnges fter exposure to oth generlist herivores (see Tle 2 nd Fig. 1 of Appendix S2, Supporting informtion). CONSUMPTION PREFERENCES IN THE CAFETERIA BIO- ASSAYS Pine species differed significntly in the consumed volume of stem y Hyloius in the cfeteri iossys (Tle 2). Pinus sylvestris ws the lest dmged while P. coulteri nd P. cnriensis were the most (Fig. 3). We found mrginlly significnt negtive reltionship (t the species level) etween the mount of tissue consumed y Hyloius in the cfeteri iossys nd the concentrtion of non-voltile resin in the stem (r 2 =Æ46, P =Æ6, n = 6). Consumption y Hyloius ws not significntly ffected y the herivory-induction tretment (Tle 2). Specificlly, stems from seedlings previously exposed to herivory y Hyloius were consumed s much s were those from control seedlings (Fig. 3). However, lien nd ntive pine species significntly differed in consumption (F 1,89 =7Æ69, P <Æ5), with lien pines on verge were consumed more thn ntive pine species. Pine species lso differed significntly in the ingestion of needles y Thumetopoe cterpillrs (Tle 2). Less-consumed needles hd higher concentrtions of condensed tnnins (r 2 =Æ5, P <Æ5, n = 6). Specificlly, P. pinster nd P. coulteri were the most consumed pine species (Fig. 3). These two pine species lso showed the gretest cterpillr survivl (Fig. 3c). Specific ingestion of needles nd cterpillr survivl were not significntly ffected y herivory-induction tretment (Tle 2; Fig. 3,c). Also, specific ingestion of needles did not differ etween ntive nd lien pine species (F 1,89 =2Æ96, P =Æ8). Discussion Given tht lien pine seedlings were not less dmged thn ntive pines when exposed to ntive generlist herivores, our results do not support the predictions of the NEH. Our oservtions gree with recent met-nlysis tht found no differences in the dmge cused y ntive herivores with respect to coexisting lien nd ntive species (Chun, vn Kleunen & Dwson 21). Hyloius hs een documented to feed on severl conifer species, ut there re no ville studies tht simultneously Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society, Functionl Ecology, 26,

8 29 A. Crrillo-Gvil n et l. Consumed volume of stem (mm 3 ) Specific ingestion of needles (g needles d.w.) Cterpillr survivl (%) () () (c) c c Control Thumetopoe compre severl lien nd ntive pine species with regrd to the preference of this insect. Under field conditions, Hyloius significntly feeds more on lien P. rdit thn on ntive P. pinster seedlings (Zs et l. 26, 28). However, studies evluting the dmge tht other insect species cuse to lien nd ntive pines showed opposite results. For exmple, oservtionl field studies hve shown tht the rk eetle Tomicus piniperd L. (Coleopter: Curculionide) ttcks P. pinster more thn P. rdit (Lomrdero, Vázquez-Mejuto & Ayres 28). With respect to herivory y Thumetopoe, it reportedly feeds on severl pine species cross Europe, ut the results on c Control Control Hyloius Thumetopoe CAN PTR SYL COU RAD ROX ntive lien ntive lien Fig. 3. Results of the cfeteri iossys showing the feeding ctivity of Hyloius ietis (), nd feeding ctivity () nd performnce (c) of Thumetopoe pityocmp when feeding on pine tissues from control seedlings (white rs) nd seedlings previously induced y these insects (lck nd grey rs). Pine species re grouped from ntive to lien species in Europe. Brs re LS mens ± SEM. Different letters ove columns indicte significnt differences (P < Æ5) mong pine species or etween rnge (ntive vs. lien). See Fig. 1 legend for species revitions. Insect weight ws included s covrite in the models. the preference of this insect for different species re lso contrdictory. For exmple, in Greece, Avtzis (1986) found different resistnce ginst this cterpillr mong five pine species, P. rdit nd P. pine eing the most nd the lest consumed, respectively. On the contrry, in Itly, Thumetopoe cterpillrs showed no preferences etween different lien nd ntive pine species (Tieri et l.1999). Our results show tht constitutive concentrtion of chemicl defences differed mong pine species, with greter concentrtions of constitutive totl phenolics in the stems of ntive pine species. The opposite trend ws oserved for constitutive phenolic compounds in the needles. Herivory y Hyloius lrgely incresed the concentrtion of non-voltile resin nd totl phenolics in the stem of some species. Moreover, s we hypothesized, ntive pines showed significntly greter concentrtion of totl phenolics in stems thn lien pines. However, some lien pine species surprisingly showed quntittive induction of chemicl defences in the stem fter herivory y Hyloius (see Fig. 2,c), especilly for non-voltile resin concentrtion. Incresing induced responses in those lien pines might e for severl resons. First, the coexistence of other insect herivores elonging to the sme feeding guild s Hyloius, such s Pissodes spp. in North Americ, might explin the induction of stem non-voltile resin y the weevil in the Americn origin P. coulteri. Secondly, the resource vilility hypothesis (Coley, Brynt & Chpin 1985) ssumes tht fst-growing species will e less defended ecuse they invest more in growth thn do slow-growing species, which invest more in defences under the sme environmentl conditions (Blumenthl 26). According to the pine clssifiction of Rejm nek & Richrdson (1996) nd Grotkopp, Rejmánek & Rost (22), only P. rdit is considered n invsive nd fst-growing species in mny prts of the world, while the lien pines studied here re considered minly non-invsive species nd slow-growing species. Thus, non-invsive species might e etter defended thn P. rdit, which might invest more in growth thn in defences. On the other hnd, the Thumetopoe tretment only significntly incresed the concentrtion of non-voltile resin in needles, without significnt differences in induciility etween lien nd ntive pines. However, the increse of needle non-voltile resin fter the Thumetopoe tretment ws pprent in only few species (see Fig. 2). The wek quntittive induction of chemicl defences y Thumetopoe tretment might e due to severl non-exclusive fctors. First, our pine species might hve not een dmged long enough y herivores to provoke defensive response (Grdner & Agrwl 22; Underwood, Anderson & Inouye 25). Second, lthough it is known tht mesurle responses to Hyloius y ntive pines cn e found within 48 h (Smpedro, Moreir & Zs 211), the response of pines to Thumetopoe cterpillrs might e slower, requiring greter lg time etween the dmge nd the induced response (Underwood, Anderson & Inouye 25). Furthermore, there is lso lg time from the trnsloction of the signl from dmged to undmged leves (Howe & Schller 28); the ctivtion of defensive response in undmged tissues might require more time thn Ó 211 The Authors. Functionl Ecology Ó 211 British Ecologicl Society, Functionl Ecology, 26,

9 Resistnce lien nd ntive pines to herivores 291 the length of our experiment (6 dys) for quntittive chnges in secondry chemicl metolites to e detected. Besides, the induction of defences might involve qulittive chnges in secondry compounds rther thn quntittive chnges (Petrkis et l. 25; Smpedro et l. 21). We expected tht prior exposure of the seedlings to herivores would influence the consumption of oth generlist herivores in the iossys. When compring lien vs. ntive pines, only in the Hyloius cfeteri iossys were lien pine species consumed more thn ntive species were, while no differences mong rnge origin were found in the Thumetopoe cfeteri iossys. This difference in the Hyloius iossys might e explined y the greter induction of totl stem phenolics in ntive pine species fter rk weevil ttck. Perhps inducile defences in ntive pines re more responsive to dmge y ntive generlist enemies thn those in lien pines, ecuse of shred evolutionry history (Joshi & Vrieling 25; Zs, Moreir & Smpedro 211). Cfeteri iossys with insects provide useful informtion, ut it should e noted tht some other fctors could ffect the ccurcy of the results. In other studies, cfeteri iossys using Hyloius in P. rdit nd P. pinster hve registered significnt differences in consumption etween species nd induction tretments (Moreir, Smpedro & Zs 29; Zs, Moreir & Smpedro 211). However, our results from iossys performed Thumetopoe were not conclusive, ecuse, lthough differences etween some species were found, the herivores showed little sensitivity to chnges in the nutritionl qulity or other physicl or chemicl properties of the tissues offered, s reported in previous studies (Hódr, Zmor & Cstro 22). However, very little is known out the ility of pine trees to produce induced responses ginst those insects, s reflected y the present work. Our study reports erly plnt resistnce nd response to generlist herivores in severl lien nd ntive woody species. Overll, our results did not support the ssumption sed on the NEH tht ntive herivores would void lien plnts ecuse of lck of recognition. Results from other studies re somewht contrdictory. Some show either no evidence tht generlist herivores voided lien plnts (Morrison & Hy 211) or, y contrst, they show tht generlist herivores hve greter impct on ntive thn on lien species (Schffner et l. 211). Our findings gree with recent metnlysis tht found no differences in the dmge cused y ntive herivores with respect to coexisting lien nd ntive species (Chun, vn Kleunen & Dwson 21). Our results suggest tht inducile defences do not protect lien pines, in regions where they coexist with ntive congeners. This grees with Lind & Prker (21), who compred chemicl deterrents in severl invsive nd co-occurring ntive plnt species nd found tht lien species did not show higher deterrence to ntive generlist herivores thn did ntive species. Ntive generlist herivores might e one reson why there is low incidence of invsion y lien pines in Europe (Crrillo-Gvil n & Vilà 21). Acknowledgements We thnk B. Sntos, D. Blnco, A. Grcı, L. Mrtı nez, A. Pzos nd E. Dios for greenhouse nd lortory ssistnces; S. Pin eiro nd M. J. Villnuev for field ssistnce nd mintennce of insects; nd P. Gonz lez, E. Mnzno, A. Montero nd A. Montesinos for their comments on previous drft of the mnuscript. Reserch ws prtilly founded y the Spnish Ministerio de Cienci e Innovción, projects Consolider-Ingenio MONTES (CSD28-4), COMPROPIN (AGL ) nd CSIC (project 284I153). References Agrwl, A.A. (211) Current trends in the evolutionry ecology of plnt defence. Functionl Ecology, 25, Agrwl, A.A. & Kotnen, P. (23) Herivores nd the success of exotic plnts: phylogeneticlly controlled experiment. Ecology Letters, 6, Agrwl, A.A., Kotnen, P., Mitchell, C., Power, A.G., Godsoe, W. & Klironomos, J. (25) Enemy relese? An experiment with congeneric plnt pirs nd diverse ove- nd elowground enemies. Ecology, 86, Avtzis, N. (1986) Development of Thumetopoe pityocmp (Den. nd Schiff.) in reltion to food consumption. Forest Ecology nd Mngement, 15, Brz, E., Gómez, J., Hódr, J. & Zmor, R. (24) Herivory hs greter impct in shde thn in sun: response of Quercus pyrenic seedlings to multifctoril environmentl vrition. Cndin Journl of Botny, 82, Bertheu, C., Brockerhoff, E.G., Roux-Morito, G., Lieutier, F. & Jctel, H. (21) Novel insect-tree ssocitions resulting from ccidentl nd intentionl iologicl invsions: met-nlysis of effects on insect fitness. Ecology Letters, 13, Blumenthl, D.M. (26) Interctions etween resource vilility nd enemy relese in plnt invsion. Ecology Letters, 9, Crpenter, D. & Cppuccino, N. (25) Herivory, time since introduction nd the invsiveness of exotic plnts. Journl of Ecology, 93, Crrillo-Gvil n, M.A. & Vil`, M.(21) Little evidence of invsion y lien conifers in Europe. Diversity nd Distriutions, 16, Chun, Y.J., vn Kleunen, M. & Dwson, W. (21) The role of enemy relese, tolernce nd resistnce in plnt invsions: linking dmge to performnce. Ecology Letters, 13, Coley, P.D., Brynt, J.P. & Chpin, F.S. (1985) Resource vilility nd plnt ntiherivore defense. Science, 23, DAISIE (29) Hndook of Alien Species in Europe. Springer, Dordrecht. Devkot, B. & Schimidt, G.H. (199) Lrvl development of Thumetopoe pityocmp (Den. nd Schiff.) (Lepidopter: Thumetopoeide) from Greece s influenced y different host plnts under lortory conditions. Journl of Applied Entomology, 19, DeWlt, S.J., Denslow, J.S. & Ickes, K. (24) Nturl enemy relese fcilittes hitt expnsion of the invsive tropicl shru Clidemi hirt. Ecology, 85, Eckert, A.J. & Hll, B.D. (26) Phylogeny, historicl iogeogrphy, nd ptterns of diversifiction for Pinus (Pincee): phylogenetic tests of fossil-sed hypotheses. Moleculr Phylogenetics nd Evolution, 4, Feeny, P. (1976) Plnt Apprency nd chemicl defense. Biologicl Interctions etween Plnts nd Insects (eds J.W. Wllce & R.L. Nnsel), pp Recent Advnces in Phytochemistry, 1. Plenum Press, New York. Frnceschi, V.R., Krokene, P., Christinsen, E. & Krekling, T. (25) Antomicl nd chemicl defenses of conifer rk ginst rk eetles nd other pests. New Phytologist, 162, Grdner, S.N. & Agrwl, A.A. (22) Induced plnt defence nd the evolution of counter-defences in herivores. Evolutionry Ecology Reserch, 4, Goßner, M.M., Cho, A., Biley, R.I. & Prinzing, A. (29) Ntive fun on exotic trees: phylogenetic conservtism nd geogrphic contingency in two lineges of phytophges on two lineges of trees. The Americn Nturlist, 173, Grotkopp, E., Rejmánek, M. & Rost, T.L. 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