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Journal of Ecology 20181062043ndash2057 wileyonlinelibrarycomjournaljec emsp|emsp2043copy 2018 The Authors Journal of Ecology copy 2018 British Ecological Society
Received21June2017emsp |emsp Accepted30January2018DOI 1011111365-274512956
R E S E A R C H A R T I C L E
Anti- herbivore defences and insect herbivory Interactive effects of drought and tree neighbours
Bastien Castagneyrol1 emsp|emspHerveacute Jactel1emsp|emspXoaquiacuten Moreira2
1BIOGECOINRAUniversityofBordeauxCestasFrance2MisioacutenBioloacutegicadeGalicia(MBG-CSIC)PontevedraGaliciaSpain
CorrespondenceBastienCastagneyrolEmailbastiencastagneyrolinrafr
Funding informationFrenchMinistryofEcology
HandlingEditorMatthewHeard
Abstract1 HowmuchaplantisattackedbyinsectherbivoreslikelydependsonitsapparencyandabilitytoproducedefensivetraitswhichmaybemodifiedbyneighbouringplantsandabioticconditionsYethowmuchthedirectandtrait-mediatedeffectsofneighboursonherbivoryismodifiedbyabioticfactorsisstillunknown
2 ByusingatreediversityexperimentinSWFrancewemeasuredleafinsecther-bivory(chewersandminers)nutritionalquality(watercontentCNratiosugarandstarchcontent)andchemicaldefences (totalpolyphenolicsandcondensedtannins) on birch (Betula pendula) trees growing inmonocultures andmixtureswithoakpineorbothspeciesWealleviatedwaterstressbyirrigatingtreesinhalfoftheplotswhiletreesremainedunwatered(iedrought-stressed) intheotherhalf
3 Overallinsectherbivorywashigheramongheterospecificneighbourswhichcor-respondstoassociationalsusceptibilityConsistentwiththisfinding leaveshadlower amount of anti-herbivore defences among heterospecific neighbours Inturninsectscausedmoredamageindrought-stressedconditionsbutsucheffectwasindependentofleafchemistryWealsofoundthattheeffectoftreespeciesdiversityonherbivorywascontingentofdroughtconditionsasassociationalsus-ceptibilityonlyoccurredindrought-stressedtreesTheindependentandinterac-tive effects of neighbour diversity and irrigation on leaf herbivory remainedsignificant after accounting for birch apparency and leaf chemistry suggestingthatunmeasuredplanttraitsorsomeothermechanismsnotassociatedwithplanttrait variation and apparency might be involved in the observed herbivorypatterns
4 SynthesisBydemonstratingthatassociationaleffectsarecontingentuponabioticconstraintswebringnewinsightsintoourunderstandingofthemechanismsdriv-ingdiversitymdashresistancerelationshipsacrossclimaticgradients
K E Y W O R D S
associationaleffectsBetula pendulabiodiversitychewersforestinsectherbivoryminersORPHEEexperiment
2044emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1emsp |emspINTRODUC TION
Ecologicalresearchconductedoverthelasttwodecadeshasshownthat plant species diversity has substantial effects on ecosystemprocessessuchasproductivity(Cardinaleetal2011)andalsoin-fluence arthropod community structure and overall species rich-nessathigher trophic levels (Barbosaetal2009Cardinaleetal2011MoreiraAbdala-RobertsRasmannCastagneyrolampMooney2016Scherberetal2010)Inparticularitiswell-establishedthatagreaterdiversityofplantspeciesreducesherbivoreattackbyde-creasingtheconcentration(ienumberofindividuals)andfrequency(ierelativeabundance)ofherbivoresrsquohosts(ldquotheresourceconcen-tration hypothesisrdquo Hambaumlck Inouye Andersson amp Underwood2014 Root 1973 Underwood Inouye amp Hambaumlck 2014) or bydecreasing plant apparency through an interferencewith physicaland chemical cues used by insect herbivores to detect and reachtheirhostplant(CastagneyrolGiffardPeacutereacuteampJactel2013JactelBirgerssonAnderssonampSchlyter2011)Inadditionagrowingnum-berofstudieshasreportedthatplantspeciesdiversitycanindirectlyaffectherbivoryonafocalplantbymodifyingitsnutritionalqualityandanti-herbivoredefences(egphysicaltraitsandsecondaryme-tabolites KostenkoMulderCourboisampBezemer 2017MoreiraAbdala-Roberts Parra-Tabla amp Mooney 2014 Mraja UnsickerReicheltGershenzonampRoscher2011)independentlyofresourceconcentration and hostndashplant apparency However observed pat-terns arehighly inconsistent and themechanismsunderlying suchpatterns are poorly understood For example some studies havereported increased investment in anti-herbivore defences amongheterospecificneighbours(Moreiraetal2014Mrajaetal2011)while others have reported the opposite (Glassmire etal 2016Kostenkoetal2017)generatinganegative(Glassmireetal2016)or a neutral effect (Kos Bukovinszky Mulder amp Bezemer 2015Moreiraetal2014)oninsectherbivores
Alikelysourceofinconsistenciesamongstudiesaddressingplantdiversityeffectsonplantndashherbivoreinteractionsisthelackofcon-siderationof abiotic factors It iswell known that abiotic stressessuchasdroughtdirectlyand indirectlyalterplantndashherbivore inter-actions (HubertyampDenno2004 Jacteletal2012Walteretal2011)Herbivory consumption rates and fitness aswell as insectherbivore performance may drastically increase with increaseddroughtstressbecausedroughtmay indirectlymodifyplantnutri-tional quality and investment in anti-herbivore defences (HubertyampDenno2004Jacteletal2012Walteretal2011White19742009) Inthissensetheplantstresshypothesispositsthatabioticstressesonplantssuchaslackofwaterenhancethenutritionalqual-ityoffoliagefor insectherbivoresbyalteringbiochemicalsourcendashsink relationships and foliar chemistry (Walter etal 2011White19742009)ForinstanceXimeacutenez-EmbuacutenOrtegoandCastantildeera(2016)reportedanincreaseinaminoacidandsugarconcentrationsin leavesofdrought-stressed tomatoplants resulting in increasedperformanceofthemiteTetranychus evansi
Todatetheoryonplantdiversityanddroughteffectsonplantndashherbivoreinteractionshavebeendevelopedindependentlyforthe
mostpartbutrecentstudiesaddressingtheeffectofabioticfactorsonassociationalresistancesuggestthatthereareimportantinsightstobegainedfromconsideringthetwodriverstogether (Glassmireetal 2016 Grettenberger amp Tooker 2016 Kambach KuumlhnCastagneyrolampBruelheide2016Walteretal2011)Indeeditisincreasingly acknowledged that plant neighbourhood compositioncan modulate the way plants respond to water stress (ForresterTheiveyanathanCollopyampMarcar2010Klausetal2016Otienoetal 2012) For instance in accordancewith the stress gradienthypothesis predicting reduced competition and increased facilita-tionunderhighstress(CallawayampWalker1997)Foreyetal(2016)foundthatbeechtreesgrowinginmixedstandsincreasesoilnutri-entacquisitionincomparisontothosetreesgrowinginmonospecificstandsinparticularinwater-stressedsoilsItisthereforelikelythatthe effects of plant diversity on leaf quality and resulting herbiv-oryaremodifiedbydrought(GrettenbergerampTooker2016Walteretal2011)
Inthepresentstudywetestedforindependentandinteractiveeffectsoftreespeciesdiversityanddroughtoninsectleafherbiv-ory (miners and chewers) and leaf chemical traits associatedwithdefence and nutritional quality (phenolic compounds CN ratiowatercontentandcarbohydrates) in silverbirch trees (Betula pen-dulaBetulacae)acommonbroadleavedspeciesinthestudyareaInadditionweinvestigatedifthemeasuredtreetraitsaswellastreeapparency (measured as how much individual birches were tallerthantheirneighboursegCastagneyroletal2013)underliedtheeffects of neighbourhooddiversity anddrought on leaf herbivoryinbirchtreesTotestfortheseeffectsweusedafactorialfieldex-perimentwherewemanipulatedbothhostndashplant speciesdiversity(threelevelsbirchmonoculturestwo-speciesmixturesassociatingbirchwiththepedunculateoakQuercus roburormaritimepinePinus pinasterandthree-speciesmixturewithpedunculateoakthemar-itimepineandbirch)andwateravailability(twolevelsirrigatedvsnon-irrigated)Byaddressingthesegoalsthisstudybuildstowardsabettermechanisticunderstandingofthecombinedeffectsofplantspeciesdiversityandabioticconditionsonplantndashherbivoreinterac-tionsandplanttraitsassociatedwithresistancetoherbivory
2emsp |emspMATERIAL S AND METHODS
21emsp|emspNatural history
Silver birch (B pendula Roth Betulacae) is a broadleaved woodyspecies with wide climatic and edaphic tolerance Its distributionrangecoversallEurope(Atkinson1992)BeingacommonpioneerspeciesinnorthernandwesternEuropethesilverbirchisnaturallypresentinthestudyareawhereitisfrequentlyfoundinassociationwithmaritimepineandvariousoakspeciesBetula pendulaisintoler-anttodrought(Atkinson1992)whichcausesprematureleafdropinsummer(BCastagneyrolpersobs)Betula pendula isattackedbyalargecommunityofinsectherbivoresespeciallyleaf-chewersandminers such asOperophtera fagata Hemichroa crocea or Eriocrania spp (Atkinson 1992 Kunca Csokaamp Zubrik 2013) Even at low
emspensp emsp | emsp2045Journal of EcologyCASTAGNEYROL ET AL
damagelevelstheseherbivorescancausesignificantgrowthlossonbirch(ZverevaZverevampKozlov2012)
22emsp|emspExperimental design
221emsp|emspTree diversity experiment
We conducted the present study at the ORPHEE Tree DiversityExperiment (httpssitesgooglecomvieworpheeexperiment) inSWFrance(44deg440N00deg460W)along-termexperimentdesignedtotesttheeffectsoftreespeciesdiversityonproductivityandas-sociatedfaunas(seeCastagneyroletal2013forarepresentationof the experimental design) The experiment was established in2008onasandysoil(podzol)formerlyplantedwithmaritimepineItconsistsofeightblockscovering12hawith32plotsineveryblockcorrespondingto the31possiblecombinationsofoneto five treespecies (B pendula Q robur Quercus pyrenaica Quercus ilex and
P pinaster)withanadditionalreplicateofthefive-speciesplotEachplotis400m2(20times20m)andcontains10rowsof10treesplanted2m apart (100 trees) Tree speciesmixtureswere established ac-cordingtoasubstitutivedesignkeepingtreedensityequalacrossplotswithequalproportionofeachtreespeciesWithinplots in-dividualtreesfromdifferentspecieswereplantedinaregularalter-natepatternsuchthatatreefromagivenspecieshadatleastoneneighbourfromeachoftheotherspecies
In thepresent studywe focusedonbirchmonocultures two-speciesmixturesassociatingbirchwiththepedunculateoakQ robur orthemaritimepineP pinasterandthemixturesofpedunculateoakmaritimepine andbirch for a total of four compositions In totalforthisstudyweused32plotscorrespondingto4plotstimes8blocksTheseplotswerechoseninordertosystematicallyassociatebirchwithabroadleavedandaconiferspeciesandtospanalargegradi-entofvariabilityintreeheightcanopyclosureandbirchapparency(Figure1)Birchapparency representshowmuch ()an individual
F IGURE 1emspComparisonofmeanbirchheight(a)variabilityoftreeheight(b)plotcanopyclosure(c)andbirchapparency(d)betweentreatments(a)Heightofthethreerandomlyselectedbirchesperplotatthebeginningofthe2016growingseason(b)Coefficientofvariationofheightofthe36innermosttreesperplotatthebeginningofthe2016growingseason(c)CanopyclosureestimatedinearlyJune(d)Apparencyquantifieshowmuchbircheswereonaveragetaller(positivevalues)orsmaller(negativevalues)thantheirneighbours(Damienetal2016)InallplateslightanddarkgreysareforcontrolandirrigatedplotsrespectivelyBBirchmonoculturesBQBirchndashOakmixturesBPBirchndashPinemixturesBQPBirchndashOakndashPinemixturesBoxesindicatethefirstandthirdquartilesDotsandthickhorizontallinesareformeanandmedianrespectivelyContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystars
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irch
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B BQ BP BQP
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2046emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
birch is taller or smaller than its neighbours (Castagneyrol etal2013)andwascalculatedas100times(HbirchminusHplot)HplotwhereHbirch represented theheightof the focalbirch andHplot themean treeheightofthecorrespondingplot(averagedacrossthe36innermosttrees)NegativeandpositivevaluesindicatethatfocalbircheswereonaveragesmallerandtallerthantheirneighboursCanopyclosurewasestimatedinearlyJuneasthemeanpercentageofpixelsoccu-piedbycanopyonphotosWetook fourphotosperplotat15mfromthegroundwithsmartphoneswith8Mpxresolution Imageswere analysed with the CanopyDigi software (Goodenough ampGoodenough2012)andpercentagecanopyclosurewasaveragedattheplotlevelBirchheight(meanandCV)canopyclosureandbirchapparencywereallstronglycorrelatedwitheachother(FigureS1)
222emsp|emspIrrigation treatments
FromMay toOctober2015 and2016 half of theblockswere ir-rigatedbysprinklingc 42 m3pernightandperblockcorrespond-ingtoc3mmpernightperplotThisvolumewascalculatedbasedonregionalclimaticdata(evapotranspiration)Wesupplied60ofyearly precipitations during the five summer months (FigureS2)whichenabledtoavoidanysoilwaterdeficitintheirrigatedblocksduringtheentiregrowingseason
Weusedthreeapproachestoconfirmthatwaterstresswasalle-viatedinirrigatedblocks(FigureS3Castagneyroletal2017)Firstwe quantified soil gravimetric water content in late August 2016by sampling two soil cores between20 and40cm in eachof theselected32plotsWepooled the twosamples inhermeticplasticbags and stored them into portable fridges before being broughtto the laboratorywhere sampleswere immediatelyweightedWedried samplesat105degC for72hr andweighted themGravimetricwater content was estimated as the proportion of water in freshsamples Secondwemeasured predawn leafwater potential (ψw)inearlySeptember2016ononebirchtakenclosetothecentreofeachstudiedplotψwwasusedasaproxyofthespatiallyintegratedwaterpotentialofthesoilexploredbyrootsWeusedaScholander-typepressurechamber (DGmeca typeNP100model0-100bars)tomeasureψwononeleafpertreeLeavesweresampledaftersappressurere-equilibratedbetween430and630amlocaltime(be-fore sunrise)with clippers connected to extension loppers Thirdwe compared birch relative growth rate (RGR) between irrigatedandnon-irrigatedplotsInearly2015(C2015)andearly2016(C2016)wemeasuredtreecircumferenceat130mfromgroundlevelonarandomsampleof sevenbirchesperplot (provided theywere tallenough)inallirrigatedandnon-irrigatedplotsThisresultedin852measurementsRGRwasestimatedasthepercentageofcircumfer-enceincrement(100times(C2016minusC2015)C2015)duringthefirstyearofirrigationandwasusedtoconfirmthatbirchesintegratedtheeffectofirrigationthroughthewholegrowingseason
InFrancesummer2016wasoneofthedriestofthe1959ndash2016period (wwwmeteofrancefr) During the 2016 growing seasonin addition to natural precipitations irrigation brought c 550 mm waterperplotwhichrepresentshalfoftotalprecipitationsreceived
that year (FigureS2) In the absence of irrigation thewater-tablewas 50cmbelow-ground level in early season andwent down tominus150minOctoberInSeptember2016soilwatercontentbetween20and40cmwas26 timeshigher in irrigatedplots than innon-irrigatedplots(F130 = 2622 plt001FigureS3)Soilwatercontentwasindependentoftreespeciescomposition(F327 = 150 p = 237 FigureS3) or compositiontimesirrigation interaction (F324 = 222 p=112FigureS3)Birchψwwas38 times lower (iemorewaterstressed) in control plots (M plusmn SE minus163plusmn008MPa) than in irri-gatedplots(minus043plusmn009MPa)Togetherwiththeobservationthatbirch growth ratewas higher in irrigated than non-irrigated plots(F156789 = 2664 p=003FigureS3)theseresultsconfirmthatirri-gationcreatedtwocontrastedtreatmentsoneinwhichtreeswerewaterstressedandanotheroneinwhichwaterwasnotlimiting
23emsp|emspMeasurements of insect herbivory
WemeasureddamagecausedbytwoguildsofleafinsectherbivoreschewersandminersWithintheselected32plots(4plotstimes8blocks)werandomlysampledthreeindividualbirchtreesoutofthe36in-nermosttreesofeachplot(resultinginatotalof96birches)Damagecausedby the twoguildswasassessed twice to test for a seasoneffect inearlyJune(earlysummerinthestudyarea)andlateJuly2016(midsummerinthestudyarea)byvisualinspectionof50leavespersampledbirch(JohnsonBertrandampTurcotte2016)Becauseofpossibleverticalstratificationofattackswithincanopywerandomlyselectedleavesonfourrandomlychosenbranchestwoat2mandtwoat5mhigh
Thepercentageofleafarearemoved(LAR)byleaf-chewerswasestimatedoneachsampledleafbyauniqueobserverallalongtheex-periment(BC)usingsevenclasses(01ndash56ndash1516ndash2526ndash5051ndash75andgt75LAR)andthenaveragedpersampledtree using the mid-point of each damage class (eg Castagneyroletal 2013) Leaf-miner damagewasmeasured as the proportionof leaveswith at leastonemine (leaf-miner incidencehenceforthBarton etal 2014 Kozlov Skoracka Zverev Lewandowski ampZvereva 2016) While chewing damage could be made by eithergeneralistorspecialistherbivoresleafminersfoundonbirchwerestrictlyspecialistherbivoresinthestudyarea
24emsp|emspChemical analyses
Wequantified constitutive totalpolyphenolic andcondensed tan-ninscontentasproxiesforchemicaldefencesWequantifiedcon-stitutive leaf dry matter content (LDMC) CN ratio starch andsolublesugarconcentrationsasproxies for leafnutritionalqualityThese constitutive plant traits arewidely recognized as herbivorefeeding stimulant and deterrents and to confer resistance againstinsect herbivores inmanyplant taxa (Abdala-Roberts etal 2016ForknerMarquisampLill2004Lorangeretal2013Pearse2011Schoonhoven2005)
In late July 2016 we collected five to six undamaged leavesin each of the 96 sampled trees Leaves were directly put into
emspensp emsp | emsp2047Journal of EcologyCASTAGNEYROL ET AL
hermeticplasticbagsfilledwithhumidifiedabsorbentpaperTheywerestoredatdarkintoportablefridgebeforebeingbroughtbackto the laboratorywhere theywere kept at 4degC to rehydrate for48hratdark(Peacuterez-Harguindeguyetal2013)Leaveswerethendried for 48hr at 55degC and weighted again to estimate LDMC(mgdrygfresh Peacuterez-Harguindeguyetal 2013) Then leaveswerefinelygroundedwithliquidnitrogentoquantifytheconcentrationof constitutive total polyphenolics condensed tannins starchsoluble sugars andCN ratio Low LDMCCN ratio and concen-trationsofpolyphenolicsandcondensedtanninsontheonehandandhighconcentrationsofsugarsandstarchontheotherhandarecommonlyassociatedwithgoodleafqualitytoherbivores(Abdala-Roberts etal 2016 Forkner etal 2004 Loranger etal 2013Pearse2011Schoonhoven2005)
Leaf totalphenolicswereextractedandanalysedasdescribedbyMoreiraMooneyZasandSampedro (2012)andMoreiraZasandSampedro (2012)Briefly theywereextracted from20mgofplanttissuewith70methanolinanultrasonicbathfor15minfol-lowedbycentrifugationandsubsequentdilutionofthemethanolicextractTotalphenoliccontentwasdeterminedcolorimetricallybytheFolinndashCiocalteumethodinaBiorad650microplatereader(Bio-RadLaboratoriesPAUSA)at740nmusingtannicacidasstandardCondensed tannins in theaqueousmethanolextractsweredeter-minedbytheprocyanidinemethodasinSampedroMoreiraandZas(2011)Themethanolicextractwasmixedwithacidifiedbutanolanda ferric ammoniumsulphate solution allowed to react inaboilingwaterbathfor50minandthencooledrapidlyoniceTheconcentra-tionofcondensedtanninsinthissolutionwasdeterminedcolorimet-ricallyinaBiorad650microplatereaderat550nmusingasstandardpurifiedcondensedtanninsofquebracho(Schinopsis balansaeEnglDrogueriacuteaModernaVigoSpain)Weexpressedphenoliccompoundconcentrationsinmggtissueonadryweightbasis
Theconcentrationsofsolublesugarsandnonstructuralcarbohy-dratereserves(starch)intheleaveswereanalysedbytheanthronemethod (Hansen amp Moslashller 1975) Soluble sugars were extractedfromfinelygroundedleaves(50mg)withaqueousethanol(80vv)Starchwasextractedwith11hydrochloricacidinawaterbathat100degCfor30minfollowedbycentrifugationandsubsequentdilu-tion of the extract Soluble sugars and starch concentrationweredetermined colorimetrically in a Biorad 650 microplate reader at630nmusingglucoseandpotatostarchrespectivelyasstandards
LeafCcontent()andtotalfoliarNcontent()weremeasuredwith an Elemental AnalyserGas Analyser - Isotope-Ratio MassSpectrometry (Carlo Erba Elementar Finnigan Isoprime BremenGermany)
25emsp|emspStatistical analyses
251emsp|emspGeneral modelling approach
TheORPHEE experiment followed a randomized split-plot designreplicatedineightblockswithIrrigation(fourreplicatesoftwolev-els irrigatedvs non-irrigatedwith irrigationhomogenizinghydric
conditions among plots and across time) as thewhole factor andneighbour identity (henceforth Neighbours four levels monocul-turesofbirch two-two-speciesmixtures includingbirchwithpineoroaktreesandone-three-speciesmixturesofbirchoakandpinetrees)asthesplitfactorThesplit-plotdesignrequiresadaptingthecalculationofdegreesoffreedomandmeansumofsquaresofre-siduals(AltmanampKrzywinski2015)Thiswasachievedusinglinearmixedeffectmodels(LMM)withBlock and BlocktimesIrrigationasran-domfactors (1|BlockIrrigation in Rsyntax)For leafminerdataweusedGeneralizedlinearmixedeffectmodels(GLMM)withbinomialerrordistributionand logit-linkHerbivoryand leaf traitdatawereanalysedatthetree levelNon-independenceof individualbircheswithinplotswasaccountedforbydefiningPlotasarandomfactornestedwithintheBlockfactor(SchielzethampNakagawa2013)
Foreachtest(seebelow)wefirstbuiltafullmodelincludingallfixed effects and their interactions The fullmodelwas then sim-plifiedbysequentiallydroppingnon-significanttermsstartingwithhighest-orderinteractionsModelparameterswerefinallyestimatedon themost parsimonious obtainedmodel using restrictedmaxi-mum likelihood forLMMWeestimatedmarginal (R2
m) andcondi-tional(R2
c)R2followingNakagawaandSchielzeth(2013)Whenever
necessaryresponsevariableswerelog-transformedtomeetmodelassumptions
Allanalysesweredone inrversion331 (RCoreTeam2016)usingthefollowingpackageslmerTest car multcomp car and MuMIn (Bartoń 2016 Fox etal 2016Hothorn etal 2016 KuznetsovaBrockhoffampChristensen2015)
252emsp|emspInsect herbivory
Insect herbivory was measured in early June (early season) andlate July (mid season)We first built a full LMM includingSeason Irrigation and Diversity (iemonoculturevsmixture)orNeighbours asexplanatoryfactorsandalltwo-andthree-waysinteractionsWeaddedindividualtreeidentitynestedwithinPlotfactorasarandomfactortoaccountforvariancearisingforrepeatedmeasurementsonthesameindividualtrees
253emsp|emspPlant defensive traits
WefirstanalysedallleaftraitsseparatelyusingthesamemodellingapproachwithbirchapparencytreespeciesdiversityorneighbouridentityirrigationandIrrigationtimesDiversity(orIrrigationtimesNeighbours)and IrrigationtimesApparencyinteractionsasfixedeffectsinLMM
Becausesomeleaftraitsarehighlycorrelated(FigureS4)wefur-therusedaPCAtosummarizeinformationcontainedinthesixleaftraitsWeextractedcoordinatesonthetwo-firstPCaxes(PC1andPC2respectively)andtestedwhethertheywerepredictedbytreeheight irrigation and composition using the same LMMdescribedabove The first and second principal component (PC) axis of themultivariate principal component analysis explained 443 (PC1)and 183 (PC2) of variability in leaf traits respectively PC1wasdrivenbychemicaldefencespositivevaluesbeingassociatedwith
2048emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
highconcentrationsofbothtotalpolyphenolicsandcondensedtan-nins(Figure3a)PC2wasdrivenbynutritionalqualityrelatedtraitsPositivevalueswereassociatedwithhighLDMCandCNcontent(ielowwaterandlowNcontent)whilenegativevaluescorrespondedtohighconcentrationsofstarchandsolublesugars(Figure3a)Negativevaluesthereforeindicategreaternutritionalleafquality
254emsp|emspPlant traits underlying effects of irrigation and tree diversity or neighbour identity on leaf herbivory
We finally used a multiple regression approach and included leaftraits (PC1 PC2 and PC1timesPC2 interaction) and birch apparencyinto a fullmodel testing the effects of irrigation and tree speciesdiversityorneighbouridentityonleafherbivoryThiswaslimitedtomidseasondatawhentraitsweremeasuredWethenappliedthesimplificationprocedureexplainedaboveBecausebirchapparency(Figure1)andleaftraits(seeSection3)werenotindependentoftreediversityorneighbouridentitythemultipleregressionapproachal-lowstestingwhethersomevarianceremainedtobeexplainedbythelatterwhenaccountingfortheformerandconverselyWeexpectedthat ifdefencesmediateeffectsof irrigationandtreediversityonleaf herbivory then significant effects of any of these factors (ortheirinteraction)shouldbecomenon-significantoncesuchtraitsareaccounted for in themodel If irrigationand treediversityeffectsremainsignificantafterincludingthesetraitsthissuggeststhatirri-gationandtreediversityinfluenceherbivorythroughotherunmeas-uredplanttraitsorviasomeothermechanismnotassociatedwithplanttraitvariation
3emsp |emspRESULTS
31emsp|emspTree apparency
Treeapparencywasstronglydeterminedbythespecificcomposi-tionofexperimentalplots(Figure1)whichreflectedvariabilityintreeheightat theplot scale (Figure1)Notablybirchapparencywasthehighestinbirchndashoakmixtures(Figure1)asaresultofthesmallersizeofoakscomparedtobirches (seefigure2 inDamienetal 2016) At the opposite birch apparency was minimal inbirchndashpinemixtures(Figure1)wherebircheswerepartiallyhiddenbytallerpineneighboursBirchapparencycorrelatedwithotherstand-level variables describing tree growth and absolute andrelativeheight(FigureS1)Henceforthwefocusedonapparencyinsteadof anyother variables as it integrates informationaboutboththefocalbirchesandtheirneighboursheight
32emsp|emspLeaf insect herbivory
321emsp|emspLeaf-chewer damage
Leaf-chewer damage ranged from 034 to 1580 (mean (M) 408plusmn231) The season of sampling plant species diversity and
irrigationtreatmentsignificantlyaffectedleaf-chewerdamage(Table2)Specificallyleaf-chewerdamagewas15timesgreaterinmidsummerthan inearly summer Itwas significantlyhigher inmixtures than inmonocultureplotsanditwas15timeshigherinnon-irrigatedthaninirrigatedplots(Figure2)AssociationaleffectsdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigation interactions (Table1) The effects of diversityandcompositiononleaf-chewerdamageweresignificantonlyinnon-irrigatedplotswhereleaf-chewerdamagewasgreaterinspeciesmix-tures than in speciesmonocultures (Figure2) Leaf-chewer damagewashigherinbirchndashoakmixtureslowerinmonoculturesandinterme-diateinmixturescontainingpine(Figure2)WedidnotfindsignificanteffectsofSeasontimesDiversity SeasontimesNeighbour or SeasontimesIrrigation interactionsonleaf-chewerdamage(Table1)
322emsp|emspLeaf- miner incidence
Leaf-minerincidence(leaveswithatleastonemine)rangedfrom0 to24 (mean (M) 500plusmn426) The seasonof sampling andplant species diversity significantly affected leaf-miner incidence(Table2)Specificallyleaf-minerincidencewas15timesgreaterinmidsummerthaninearlysummerand15timeshigherinmixturesthan inmonocultureplots (ie associational susceptibility) and13timeshigherincontrolthaninirrigatedplots(Figure3)
TherewasasignificanteffectoftheSeasontimesDiversitytimesIrrigation (χ2 = 1148 df = 1 plt001) and SeasontimesDiversitytimesNeighbour (χ2 = 1776 df = 3 p=006)three-waysinteractionsonleaf-minerin-cidencesuchthatearlyandmidseasondatawereanalysedseparately(Table1)Overall inbothseasonsleaf-minerincidencewaslowerinmonocultures intermediate in two-species mixtures and higher inthree-speciesmixtures (ie associational susceptibility)However indetailsthiseffectdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigationinter-actions(Table1)Inearlyseasonassociationalsusceptibilityonlyoc-curredinnon-irrigatedplotswhiletherewasnoeffectoftreediversityoridentityinirrigatedplots(Figure3)Atthecontraryinlateseasonassociationalsusceptibilityonlyoccurredinirrigatedwhileleaf-minerincidencedidnotdiffersignificantlyamongcompositiontreatmentsinnon-irrigatedplots(Figure3)
33emsp|emspPlant defensive traits
Tree species diversity (ie monocultures vs mixtures) and neigh-bour identity (ie birch monocultures birchndashoak birchndashpine andbirchndashoakndashpine mixtures) had qualitatively similar effects on leaftraits (TableS1) Concentration of total polyphenolics was signifi-cantly12-foldhigherintreesgrowinginmonoculturesthanintreesgrowing inmixtures (TableS1 FigureS5) Specifically the highestconcentration of total polyphenolics was found in trees growinginmonoculturesthe lowest intreesgrowinginthree-speciesmix-tures and intermediate in trees growing in two-species mixtures(Table2)Noneofothertraits individuallyrespondedtotreespe-cies diversity or neighbour identity (Table2 TableS1 FigureS5)
emspensp emsp | emsp2049Journal of EcologyCASTAGNEYROL ET AL
Starch concentrationwas 11-fold higher in trees growing in non-irrigated plots than in trees growing in irrigated plots (FigureS5Table2)Individuallynoneofothertraitsweresignificantlyaffectedbyirrigation(Table2)FinallyLDMCwastheonlytraitbeingsignifi-cantlyinfluencedbybirchapparencyandwassignificantlyhigherinmore apparent birches (slopeplusmnSE 41times10minus4plusmn11times10minus4 Table2FigureS5)indicatingthatleaveshadalowerwatercontentinmoreapparentthaninlessapparentbirches
There was a significant effect of tree species diversity andneighbour identity on PC1 (Table2 TableS1) with chemical
defences being greater in trees growing inmonocultures than intreesgrowinginanyothermixture(Figure4b)CoordinatesonPC1increased with tree apparency (Table2 FigureS5 slopeplusmnSE 36times10minus2plusmn15times10minus2) indicating that investment in chemicaldefencesincreasedasbirchesweremoreapparentDespiteaten-dencytowardshigherleafnutritionalquality(ieleaveswithhigherwatersugarstarchandnitrogencontent)intreesgrowinginnon-irrigatedplots(Figure4c)therewasnoeffectofbirchapparencyirrigation or tree species diversity or neighbour identity on PC2axis
F IGURE 2emspEffectsofseason(a)diversity(b)andplotspecificcomposition(c)andirrigationonleaf-chewerdamageIn(b)and(c)contrastsamongcompositionswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
0
2
4
6
8
10
12
Def
olia
tion
( L
AR
)
Earlyseason
Midseason
(a)
(96)
(96)0
2
4
6
8
10
12
Monocultures Mixtures
ControlIrrigated
(b)
a
b
(24) (24)(72)
(72) 0
2
4
6
8
10
12
B BQ BP BQP
(c)
a
bab
ab
(24) (24) (24) (24)(24) (24)
(24) (24)
TABLE 1emspSummaryofLMM(forleaf-chewers)andGLMM(forleaf-miners)testingtheeffectsofirrigationandtreespeciesdiversity(monoculturevsmixture)orneighbouridentity(birchoakpineoak+pine)onleaf-chewerdamageandleaf-minerincidence
Season Predictors
Leaf- chewers Leaf- miners
F (df) p- value R2m (R2
c) χ2 (df) p- value R2m (R2
c)
Early Irrigation 350(192) 065 014(014) 154(1) 215 005(007)
Diversity 004(192) 847 150(1) 220
IrrigationtimesDiversity 406(192) 047 1040(1) 001
Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)
Diversity 187(122) 185 426(1) 039
IrrigationtimesDiversity 508(122) 035 044(1) 509
Early Irrigation 1155(188) 001 020(020) 202(1) 156 005(007)
Neighbours 122(388) 306 597(3) 113
IrrigationtimesNeighbours 293(388) 038 1338(3) 004
Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)
Neighbours 159(321) 223 806(3) 045
IrrigationtimesNeighbours 208(318) 138 852(3) 036
LMMlinearmixedeffectmodelsGLMMGeneralizedlinearmixedeffectmodelsAlthoughforleaf-chewersthethree-waysSeason times Irrigation times DiversityandSeason times Irrigation times Neighboursinteractionswerenotsignificantwereportresultsforearlyandmidseasonseparatelytobeconsistentwithleaf-minersandtoshowthattheinteractiveeffectsofirrigationandneighbourdiversityoridentitywereconsistentacrossseasonsBoldcharac-tersindicatesignificantpredictorsatα = 005 R2werereportedforthesimplifiedmodel(ieaftertheinteractionwasremovedifnotsignificant)R2
m and R2
ccorrespondtomarginal(ieaccountingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
Early season
ControlIrrigated
Monoculture Mixture
Mid season
B BQ BP BQP
a
ab b
b
Composition
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
ControlIrrigated
B BQ BP BQP
A AB
AB
B
Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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Altman N amp KrzywinskiM (2015) Points of significance Split plotdesign Nature Methods 12 165ndash166 httpsdoiorg101038nmeth3293
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
AtsattPRampOrsquodowdDJ (1976)PlantdefenseguildsScience 193 24ndash29httpsdoiorg101126science193424724
Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
BantildeuelosM-JampKollmannJ (2011)Effectsofhost-plantpopulationsizeandplantsexonaspecialistleaf-minerActa Oecologica 37 58ndash64httpsdoiorg101016jactao201011007
Barbosa P Hines J Kaplan I Martinson H Szczepaniec A ampSzendreiZ (2009)Associational resistanceandassociational sus-ceptibilityHavingrightorwrongneighborsAnnual Review of Ecology Evolution and Systematics 401ndash20httpsdoiorg101146annurevecolsys110308120242
BartońK(2016)MuMIn Multi-model inferenceRetrievedfromhttpR-ForgeR-projectorgprojectsmumin
BartonKEValkamaEVehvilaumlinenHRuohomaumlkiKKnightTMampKorichevaJ(2014)Additiveandnon-additiveeffectsofbirchge-notypicdiversityonarthropodherbivoryinalong-termfieldexperi-mentOikos 124 697ndash706
BernaysEBrightKGonzalezNampAngelJ(1994)DietarymixinginageneralistherbivoreTestsoftwohypothesesEcology 75 1997ndash2006httpsdoiorg1023071941604
Callaway R M amp Walker L R (1997) Competition and facilita-tion A synthetic approach to interactions in plant communitiesEcology 78 1958ndash1965 httpsdoiorg1018900012-9658(1997) 078[1958CAFASA]20CO2
CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
2044emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1emsp |emspINTRODUC TION
Ecologicalresearchconductedoverthelasttwodecadeshasshownthat plant species diversity has substantial effects on ecosystemprocessessuchasproductivity(Cardinaleetal2011)andalsoin-fluence arthropod community structure and overall species rich-nessathigher trophic levels (Barbosaetal2009Cardinaleetal2011MoreiraAbdala-RobertsRasmannCastagneyrolampMooney2016Scherberetal2010)Inparticularitiswell-establishedthatagreaterdiversityofplantspeciesreducesherbivoreattackbyde-creasingtheconcentration(ienumberofindividuals)andfrequency(ierelativeabundance)ofherbivoresrsquohosts(ldquotheresourceconcen-tration hypothesisrdquo Hambaumlck Inouye Andersson amp Underwood2014 Root 1973 Underwood Inouye amp Hambaumlck 2014) or bydecreasing plant apparency through an interferencewith physicaland chemical cues used by insect herbivores to detect and reachtheirhostplant(CastagneyrolGiffardPeacutereacuteampJactel2013JactelBirgerssonAnderssonampSchlyter2011)Inadditionagrowingnum-berofstudieshasreportedthatplantspeciesdiversitycanindirectlyaffectherbivoryonafocalplantbymodifyingitsnutritionalqualityandanti-herbivoredefences(egphysicaltraitsandsecondaryme-tabolites KostenkoMulderCourboisampBezemer 2017MoreiraAbdala-Roberts Parra-Tabla amp Mooney 2014 Mraja UnsickerReicheltGershenzonampRoscher2011)independentlyofresourceconcentration and hostndashplant apparency However observed pat-terns arehighly inconsistent and themechanismsunderlying suchpatterns are poorly understood For example some studies havereported increased investment in anti-herbivore defences amongheterospecificneighbours(Moreiraetal2014Mrajaetal2011)while others have reported the opposite (Glassmire etal 2016Kostenkoetal2017)generatinganegative(Glassmireetal2016)or a neutral effect (Kos Bukovinszky Mulder amp Bezemer 2015Moreiraetal2014)oninsectherbivores
Alikelysourceofinconsistenciesamongstudiesaddressingplantdiversityeffectsonplantndashherbivoreinteractionsisthelackofcon-siderationof abiotic factors It iswell known that abiotic stressessuchasdroughtdirectlyand indirectlyalterplantndashherbivore inter-actions (HubertyampDenno2004 Jacteletal2012Walteretal2011)Herbivory consumption rates and fitness aswell as insectherbivore performance may drastically increase with increaseddroughtstressbecausedroughtmay indirectlymodifyplantnutri-tional quality and investment in anti-herbivore defences (HubertyampDenno2004Jacteletal2012Walteretal2011White19742009) Inthissensetheplantstresshypothesispositsthatabioticstressesonplantssuchaslackofwaterenhancethenutritionalqual-ityoffoliagefor insectherbivoresbyalteringbiochemicalsourcendashsink relationships and foliar chemistry (Walter etal 2011White19742009)ForinstanceXimeacutenez-EmbuacutenOrtegoandCastantildeera(2016)reportedanincreaseinaminoacidandsugarconcentrationsin leavesofdrought-stressed tomatoplants resulting in increasedperformanceofthemiteTetranychus evansi
Todatetheoryonplantdiversityanddroughteffectsonplantndashherbivoreinteractionshavebeendevelopedindependentlyforthe
mostpartbutrecentstudiesaddressingtheeffectofabioticfactorsonassociationalresistancesuggestthatthereareimportantinsightstobegainedfromconsideringthetwodriverstogether (Glassmireetal 2016 Grettenberger amp Tooker 2016 Kambach KuumlhnCastagneyrolampBruelheide2016Walteretal2011)Indeeditisincreasingly acknowledged that plant neighbourhood compositioncan modulate the way plants respond to water stress (ForresterTheiveyanathanCollopyampMarcar2010Klausetal2016Otienoetal 2012) For instance in accordancewith the stress gradienthypothesis predicting reduced competition and increased facilita-tionunderhighstress(CallawayampWalker1997)Foreyetal(2016)foundthatbeechtreesgrowinginmixedstandsincreasesoilnutri-entacquisitionincomparisontothosetreesgrowinginmonospecificstandsinparticularinwater-stressedsoilsItisthereforelikelythatthe effects of plant diversity on leaf quality and resulting herbiv-oryaremodifiedbydrought(GrettenbergerampTooker2016Walteretal2011)
Inthepresentstudywetestedforindependentandinteractiveeffectsoftreespeciesdiversityanddroughtoninsectleafherbiv-ory (miners and chewers) and leaf chemical traits associatedwithdefence and nutritional quality (phenolic compounds CN ratiowatercontentandcarbohydrates) in silverbirch trees (Betula pen-dulaBetulacae)acommonbroadleavedspeciesinthestudyareaInadditionweinvestigatedifthemeasuredtreetraitsaswellastreeapparency (measured as how much individual birches were tallerthantheirneighboursegCastagneyroletal2013)underliedtheeffects of neighbourhooddiversity anddrought on leaf herbivoryinbirchtreesTotestfortheseeffectsweusedafactorialfieldex-perimentwherewemanipulatedbothhostndashplant speciesdiversity(threelevelsbirchmonoculturestwo-speciesmixturesassociatingbirchwiththepedunculateoakQuercus roburormaritimepinePinus pinasterandthree-speciesmixturewithpedunculateoakthemar-itimepineandbirch)andwateravailability(twolevelsirrigatedvsnon-irrigated)Byaddressingthesegoalsthisstudybuildstowardsabettermechanisticunderstandingofthecombinedeffectsofplantspeciesdiversityandabioticconditionsonplantndashherbivoreinterac-tionsandplanttraitsassociatedwithresistancetoherbivory
2emsp |emspMATERIAL S AND METHODS
21emsp|emspNatural history
Silver birch (B pendula Roth Betulacae) is a broadleaved woodyspecies with wide climatic and edaphic tolerance Its distributionrangecoversallEurope(Atkinson1992)BeingacommonpioneerspeciesinnorthernandwesternEuropethesilverbirchisnaturallypresentinthestudyareawhereitisfrequentlyfoundinassociationwithmaritimepineandvariousoakspeciesBetula pendulaisintoler-anttodrought(Atkinson1992)whichcausesprematureleafdropinsummer(BCastagneyrolpersobs)Betula pendula isattackedbyalargecommunityofinsectherbivoresespeciallyleaf-chewersandminers such asOperophtera fagata Hemichroa crocea or Eriocrania spp (Atkinson 1992 Kunca Csokaamp Zubrik 2013) Even at low
emspensp emsp | emsp2045Journal of EcologyCASTAGNEYROL ET AL
damagelevelstheseherbivorescancausesignificantgrowthlossonbirch(ZverevaZverevampKozlov2012)
22emsp|emspExperimental design
221emsp|emspTree diversity experiment
We conducted the present study at the ORPHEE Tree DiversityExperiment (httpssitesgooglecomvieworpheeexperiment) inSWFrance(44deg440N00deg460W)along-termexperimentdesignedtotesttheeffectsoftreespeciesdiversityonproductivityandas-sociatedfaunas(seeCastagneyroletal2013forarepresentationof the experimental design) The experiment was established in2008onasandysoil(podzol)formerlyplantedwithmaritimepineItconsistsofeightblockscovering12hawith32plotsineveryblockcorrespondingto the31possiblecombinationsofoneto five treespecies (B pendula Q robur Quercus pyrenaica Quercus ilex and
P pinaster)withanadditionalreplicateofthefive-speciesplotEachplotis400m2(20times20m)andcontains10rowsof10treesplanted2m apart (100 trees) Tree speciesmixtureswere established ac-cordingtoasubstitutivedesignkeepingtreedensityequalacrossplotswithequalproportionofeachtreespeciesWithinplots in-dividualtreesfromdifferentspecieswereplantedinaregularalter-natepatternsuchthatatreefromagivenspecieshadatleastoneneighbourfromeachoftheotherspecies
In thepresent studywe focusedonbirchmonocultures two-speciesmixturesassociatingbirchwiththepedunculateoakQ robur orthemaritimepineP pinasterandthemixturesofpedunculateoakmaritimepine andbirch for a total of four compositions In totalforthisstudyweused32plotscorrespondingto4plotstimes8blocksTheseplotswerechoseninordertosystematicallyassociatebirchwithabroadleavedandaconiferspeciesandtospanalargegradi-entofvariabilityintreeheightcanopyclosureandbirchapparency(Figure1)Birchapparency representshowmuch ()an individual
F IGURE 1emspComparisonofmeanbirchheight(a)variabilityoftreeheight(b)plotcanopyclosure(c)andbirchapparency(d)betweentreatments(a)Heightofthethreerandomlyselectedbirchesperplotatthebeginningofthe2016growingseason(b)Coefficientofvariationofheightofthe36innermosttreesperplotatthebeginningofthe2016growingseason(c)CanopyclosureestimatedinearlyJune(d)Apparencyquantifieshowmuchbircheswereonaveragetaller(positivevalues)orsmaller(negativevalues)thantheirneighbours(Damienetal2016)InallplateslightanddarkgreysareforcontrolandirrigatedplotsrespectivelyBBirchmonoculturesBQBirchndashOakmixturesBPBirchndashPinemixturesBQPBirchndashOakndashPinemixturesBoxesindicatethefirstandthirdquartilesDotsandthickhorizontallinesareformeanandmedianrespectivelyContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystars
500
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900
(9)
(9)
(12)
(11)
(12)
(11)
(12)(11)
Indi
vidu
al b
irch
heig
ht (c
m)
B BQ BP BQP
02
04
06
08
10
(12)
(12)
(12)(12)
(12) (12)
(12)(12)
CV
of t
ree
heig
ht
B BQ BP BQP
a b a c
30
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(12)
(12)
(12)
(12)
(12)
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(12)(12)
Can
opy
clos
ure
()
B BQ BP BQP
a a b aA B C A
minus20
0
20
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(9)
(9)
(12)
(11)
(12)(11)
(12) (11)
Birc
h ap
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ncy
()
B BQ BP BQP
a b c d
(a) (b)
(c) (d)
2046emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
birch is taller or smaller than its neighbours (Castagneyrol etal2013)andwascalculatedas100times(HbirchminusHplot)HplotwhereHbirch represented theheightof the focalbirch andHplot themean treeheightofthecorrespondingplot(averagedacrossthe36innermosttrees)NegativeandpositivevaluesindicatethatfocalbircheswereonaveragesmallerandtallerthantheirneighboursCanopyclosurewasestimatedinearlyJuneasthemeanpercentageofpixelsoccu-piedbycanopyonphotosWetook fourphotosperplotat15mfromthegroundwithsmartphoneswith8Mpxresolution Imageswere analysed with the CanopyDigi software (Goodenough ampGoodenough2012)andpercentagecanopyclosurewasaveragedattheplotlevelBirchheight(meanandCV)canopyclosureandbirchapparencywereallstronglycorrelatedwitheachother(FigureS1)
222emsp|emspIrrigation treatments
FromMay toOctober2015 and2016 half of theblockswere ir-rigatedbysprinklingc 42 m3pernightandperblockcorrespond-ingtoc3mmpernightperplotThisvolumewascalculatedbasedonregionalclimaticdata(evapotranspiration)Wesupplied60ofyearly precipitations during the five summer months (FigureS2)whichenabledtoavoidanysoilwaterdeficitintheirrigatedblocksduringtheentiregrowingseason
Weusedthreeapproachestoconfirmthatwaterstresswasalle-viatedinirrigatedblocks(FigureS3Castagneyroletal2017)Firstwe quantified soil gravimetric water content in late August 2016by sampling two soil cores between20 and40cm in eachof theselected32plotsWepooled the twosamples inhermeticplasticbags and stored them into portable fridges before being broughtto the laboratorywhere sampleswere immediatelyweightedWedried samplesat105degC for72hr andweighted themGravimetricwater content was estimated as the proportion of water in freshsamples Secondwemeasured predawn leafwater potential (ψw)inearlySeptember2016ononebirchtakenclosetothecentreofeachstudiedplotψwwasusedasaproxyofthespatiallyintegratedwaterpotentialofthesoilexploredbyrootsWeusedaScholander-typepressurechamber (DGmeca typeNP100model0-100bars)tomeasureψwononeleafpertreeLeavesweresampledaftersappressurere-equilibratedbetween430and630amlocaltime(be-fore sunrise)with clippers connected to extension loppers Thirdwe compared birch relative growth rate (RGR) between irrigatedandnon-irrigatedplotsInearly2015(C2015)andearly2016(C2016)wemeasuredtreecircumferenceat130mfromgroundlevelonarandomsampleof sevenbirchesperplot (provided theywere tallenough)inallirrigatedandnon-irrigatedplotsThisresultedin852measurementsRGRwasestimatedasthepercentageofcircumfer-enceincrement(100times(C2016minusC2015)C2015)duringthefirstyearofirrigationandwasusedtoconfirmthatbirchesintegratedtheeffectofirrigationthroughthewholegrowingseason
InFrancesummer2016wasoneofthedriestofthe1959ndash2016period (wwwmeteofrancefr) During the 2016 growing seasonin addition to natural precipitations irrigation brought c 550 mm waterperplotwhichrepresentshalfoftotalprecipitationsreceived
that year (FigureS2) In the absence of irrigation thewater-tablewas 50cmbelow-ground level in early season andwent down tominus150minOctoberInSeptember2016soilwatercontentbetween20and40cmwas26 timeshigher in irrigatedplots than innon-irrigatedplots(F130 = 2622 plt001FigureS3)Soilwatercontentwasindependentoftreespeciescomposition(F327 = 150 p = 237 FigureS3) or compositiontimesirrigation interaction (F324 = 222 p=112FigureS3)Birchψwwas38 times lower (iemorewaterstressed) in control plots (M plusmn SE minus163plusmn008MPa) than in irri-gatedplots(minus043plusmn009MPa)Togetherwiththeobservationthatbirch growth ratewas higher in irrigated than non-irrigated plots(F156789 = 2664 p=003FigureS3)theseresultsconfirmthatirri-gationcreatedtwocontrastedtreatmentsoneinwhichtreeswerewaterstressedandanotheroneinwhichwaterwasnotlimiting
23emsp|emspMeasurements of insect herbivory
WemeasureddamagecausedbytwoguildsofleafinsectherbivoreschewersandminersWithintheselected32plots(4plotstimes8blocks)werandomlysampledthreeindividualbirchtreesoutofthe36in-nermosttreesofeachplot(resultinginatotalof96birches)Damagecausedby the twoguildswasassessed twice to test for a seasoneffect inearlyJune(earlysummerinthestudyarea)andlateJuly2016(midsummerinthestudyarea)byvisualinspectionof50leavespersampledbirch(JohnsonBertrandampTurcotte2016)Becauseofpossibleverticalstratificationofattackswithincanopywerandomlyselectedleavesonfourrandomlychosenbranchestwoat2mandtwoat5mhigh
Thepercentageofleafarearemoved(LAR)byleaf-chewerswasestimatedoneachsampledleafbyauniqueobserverallalongtheex-periment(BC)usingsevenclasses(01ndash56ndash1516ndash2526ndash5051ndash75andgt75LAR)andthenaveragedpersampledtree using the mid-point of each damage class (eg Castagneyroletal 2013) Leaf-miner damagewasmeasured as the proportionof leaveswith at leastonemine (leaf-miner incidencehenceforthBarton etal 2014 Kozlov Skoracka Zverev Lewandowski ampZvereva 2016) While chewing damage could be made by eithergeneralistorspecialistherbivoresleafminersfoundonbirchwerestrictlyspecialistherbivoresinthestudyarea
24emsp|emspChemical analyses
Wequantified constitutive totalpolyphenolic andcondensed tan-ninscontentasproxiesforchemicaldefencesWequantifiedcon-stitutive leaf dry matter content (LDMC) CN ratio starch andsolublesugarconcentrationsasproxies for leafnutritionalqualityThese constitutive plant traits arewidely recognized as herbivorefeeding stimulant and deterrents and to confer resistance againstinsect herbivores inmanyplant taxa (Abdala-Roberts etal 2016ForknerMarquisampLill2004Lorangeretal2013Pearse2011Schoonhoven2005)
In late July 2016 we collected five to six undamaged leavesin each of the 96 sampled trees Leaves were directly put into
emspensp emsp | emsp2047Journal of EcologyCASTAGNEYROL ET AL
hermeticplasticbagsfilledwithhumidifiedabsorbentpaperTheywerestoredatdarkintoportablefridgebeforebeingbroughtbackto the laboratorywhere theywere kept at 4degC to rehydrate for48hratdark(Peacuterez-Harguindeguyetal2013)Leaveswerethendried for 48hr at 55degC and weighted again to estimate LDMC(mgdrygfresh Peacuterez-Harguindeguyetal 2013) Then leaveswerefinelygroundedwithliquidnitrogentoquantifytheconcentrationof constitutive total polyphenolics condensed tannins starchsoluble sugars andCN ratio Low LDMCCN ratio and concen-trationsofpolyphenolicsandcondensedtanninsontheonehandandhighconcentrationsofsugarsandstarchontheotherhandarecommonlyassociatedwithgoodleafqualitytoherbivores(Abdala-Roberts etal 2016 Forkner etal 2004 Loranger etal 2013Pearse2011Schoonhoven2005)
Leaf totalphenolicswereextractedandanalysedasdescribedbyMoreiraMooneyZasandSampedro (2012)andMoreiraZasandSampedro (2012)Briefly theywereextracted from20mgofplanttissuewith70methanolinanultrasonicbathfor15minfol-lowedbycentrifugationandsubsequentdilutionofthemethanolicextractTotalphenoliccontentwasdeterminedcolorimetricallybytheFolinndashCiocalteumethodinaBiorad650microplatereader(Bio-RadLaboratoriesPAUSA)at740nmusingtannicacidasstandardCondensed tannins in theaqueousmethanolextractsweredeter-minedbytheprocyanidinemethodasinSampedroMoreiraandZas(2011)Themethanolicextractwasmixedwithacidifiedbutanolanda ferric ammoniumsulphate solution allowed to react inaboilingwaterbathfor50minandthencooledrapidlyoniceTheconcentra-tionofcondensedtanninsinthissolutionwasdeterminedcolorimet-ricallyinaBiorad650microplatereaderat550nmusingasstandardpurifiedcondensedtanninsofquebracho(Schinopsis balansaeEnglDrogueriacuteaModernaVigoSpain)Weexpressedphenoliccompoundconcentrationsinmggtissueonadryweightbasis
Theconcentrationsofsolublesugarsandnonstructuralcarbohy-dratereserves(starch)intheleaveswereanalysedbytheanthronemethod (Hansen amp Moslashller 1975) Soluble sugars were extractedfromfinelygroundedleaves(50mg)withaqueousethanol(80vv)Starchwasextractedwith11hydrochloricacidinawaterbathat100degCfor30minfollowedbycentrifugationandsubsequentdilu-tion of the extract Soluble sugars and starch concentrationweredetermined colorimetrically in a Biorad 650 microplate reader at630nmusingglucoseandpotatostarchrespectivelyasstandards
LeafCcontent()andtotalfoliarNcontent()weremeasuredwith an Elemental AnalyserGas Analyser - Isotope-Ratio MassSpectrometry (Carlo Erba Elementar Finnigan Isoprime BremenGermany)
25emsp|emspStatistical analyses
251emsp|emspGeneral modelling approach
TheORPHEE experiment followed a randomized split-plot designreplicatedineightblockswithIrrigation(fourreplicatesoftwolev-els irrigatedvs non-irrigatedwith irrigationhomogenizinghydric
conditions among plots and across time) as thewhole factor andneighbour identity (henceforth Neighbours four levels monocul-turesofbirch two-two-speciesmixtures includingbirchwithpineoroaktreesandone-three-speciesmixturesofbirchoakandpinetrees)asthesplitfactorThesplit-plotdesignrequiresadaptingthecalculationofdegreesoffreedomandmeansumofsquaresofre-siduals(AltmanampKrzywinski2015)Thiswasachievedusinglinearmixedeffectmodels(LMM)withBlock and BlocktimesIrrigationasran-domfactors (1|BlockIrrigation in Rsyntax)For leafminerdataweusedGeneralizedlinearmixedeffectmodels(GLMM)withbinomialerrordistributionand logit-linkHerbivoryand leaf traitdatawereanalysedatthetree levelNon-independenceof individualbircheswithinplotswasaccountedforbydefiningPlotasarandomfactornestedwithintheBlockfactor(SchielzethampNakagawa2013)
Foreachtest(seebelow)wefirstbuiltafullmodelincludingallfixed effects and their interactions The fullmodelwas then sim-plifiedbysequentiallydroppingnon-significanttermsstartingwithhighest-orderinteractionsModelparameterswerefinallyestimatedon themost parsimonious obtainedmodel using restrictedmaxi-mum likelihood forLMMWeestimatedmarginal (R2
m) andcondi-tional(R2
c)R2followingNakagawaandSchielzeth(2013)Whenever
necessaryresponsevariableswerelog-transformedtomeetmodelassumptions
Allanalysesweredone inrversion331 (RCoreTeam2016)usingthefollowingpackageslmerTest car multcomp car and MuMIn (Bartoń 2016 Fox etal 2016Hothorn etal 2016 KuznetsovaBrockhoffampChristensen2015)
252emsp|emspInsect herbivory
Insect herbivory was measured in early June (early season) andlate July (mid season)We first built a full LMM includingSeason Irrigation and Diversity (iemonoculturevsmixture)orNeighbours asexplanatoryfactorsandalltwo-andthree-waysinteractionsWeaddedindividualtreeidentitynestedwithinPlotfactorasarandomfactortoaccountforvariancearisingforrepeatedmeasurementsonthesameindividualtrees
253emsp|emspPlant defensive traits
WefirstanalysedallleaftraitsseparatelyusingthesamemodellingapproachwithbirchapparencytreespeciesdiversityorneighbouridentityirrigationandIrrigationtimesDiversity(orIrrigationtimesNeighbours)and IrrigationtimesApparencyinteractionsasfixedeffectsinLMM
Becausesomeleaftraitsarehighlycorrelated(FigureS4)wefur-therusedaPCAtosummarizeinformationcontainedinthesixleaftraitsWeextractedcoordinatesonthetwo-firstPCaxes(PC1andPC2respectively)andtestedwhethertheywerepredictedbytreeheight irrigation and composition using the same LMMdescribedabove The first and second principal component (PC) axis of themultivariate principal component analysis explained 443 (PC1)and 183 (PC2) of variability in leaf traits respectively PC1wasdrivenbychemicaldefencespositivevaluesbeingassociatedwith
2048emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
highconcentrationsofbothtotalpolyphenolicsandcondensedtan-nins(Figure3a)PC2wasdrivenbynutritionalqualityrelatedtraitsPositivevalueswereassociatedwithhighLDMCandCNcontent(ielowwaterandlowNcontent)whilenegativevaluescorrespondedtohighconcentrationsofstarchandsolublesugars(Figure3a)Negativevaluesthereforeindicategreaternutritionalleafquality
254emsp|emspPlant traits underlying effects of irrigation and tree diversity or neighbour identity on leaf herbivory
We finally used a multiple regression approach and included leaftraits (PC1 PC2 and PC1timesPC2 interaction) and birch apparencyinto a fullmodel testing the effects of irrigation and tree speciesdiversityorneighbouridentityonleafherbivoryThiswaslimitedtomidseasondatawhentraitsweremeasuredWethenappliedthesimplificationprocedureexplainedaboveBecausebirchapparency(Figure1)andleaftraits(seeSection3)werenotindependentoftreediversityorneighbouridentitythemultipleregressionapproachal-lowstestingwhethersomevarianceremainedtobeexplainedbythelatterwhenaccountingfortheformerandconverselyWeexpectedthat ifdefencesmediateeffectsof irrigationandtreediversityonleaf herbivory then significant effects of any of these factors (ortheirinteraction)shouldbecomenon-significantoncesuchtraitsareaccounted for in themodel If irrigationand treediversityeffectsremainsignificantafterincludingthesetraitsthissuggeststhatirri-gationandtreediversityinfluenceherbivorythroughotherunmeas-uredplanttraitsorviasomeothermechanismnotassociatedwithplanttraitvariation
3emsp |emspRESULTS
31emsp|emspTree apparency
Treeapparencywasstronglydeterminedbythespecificcomposi-tionofexperimentalplots(Figure1)whichreflectedvariabilityintreeheightat theplot scale (Figure1)Notablybirchapparencywasthehighestinbirchndashoakmixtures(Figure1)asaresultofthesmallersizeofoakscomparedtobirches (seefigure2 inDamienetal 2016) At the opposite birch apparency was minimal inbirchndashpinemixtures(Figure1)wherebircheswerepartiallyhiddenbytallerpineneighboursBirchapparencycorrelatedwithotherstand-level variables describing tree growth and absolute andrelativeheight(FigureS1)Henceforthwefocusedonapparencyinsteadof anyother variables as it integrates informationaboutboththefocalbirchesandtheirneighboursheight
32emsp|emspLeaf insect herbivory
321emsp|emspLeaf-chewer damage
Leaf-chewer damage ranged from 034 to 1580 (mean (M) 408plusmn231) The season of sampling plant species diversity and
irrigationtreatmentsignificantlyaffectedleaf-chewerdamage(Table2)Specificallyleaf-chewerdamagewas15timesgreaterinmidsummerthan inearly summer Itwas significantlyhigher inmixtures than inmonocultureplotsanditwas15timeshigherinnon-irrigatedthaninirrigatedplots(Figure2)AssociationaleffectsdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigation interactions (Table1) The effects of diversityandcompositiononleaf-chewerdamageweresignificantonlyinnon-irrigatedplotswhereleaf-chewerdamagewasgreaterinspeciesmix-tures than in speciesmonocultures (Figure2) Leaf-chewer damagewashigherinbirchndashoakmixtureslowerinmonoculturesandinterme-diateinmixturescontainingpine(Figure2)WedidnotfindsignificanteffectsofSeasontimesDiversity SeasontimesNeighbour or SeasontimesIrrigation interactionsonleaf-chewerdamage(Table1)
322emsp|emspLeaf- miner incidence
Leaf-minerincidence(leaveswithatleastonemine)rangedfrom0 to24 (mean (M) 500plusmn426) The seasonof sampling andplant species diversity significantly affected leaf-miner incidence(Table2)Specificallyleaf-minerincidencewas15timesgreaterinmidsummerthaninearlysummerand15timeshigherinmixturesthan inmonocultureplots (ie associational susceptibility) and13timeshigherincontrolthaninirrigatedplots(Figure3)
TherewasasignificanteffectoftheSeasontimesDiversitytimesIrrigation (χ2 = 1148 df = 1 plt001) and SeasontimesDiversitytimesNeighbour (χ2 = 1776 df = 3 p=006)three-waysinteractionsonleaf-minerin-cidencesuchthatearlyandmidseasondatawereanalysedseparately(Table1)Overall inbothseasonsleaf-minerincidencewaslowerinmonocultures intermediate in two-species mixtures and higher inthree-speciesmixtures (ie associational susceptibility)However indetailsthiseffectdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigationinter-actions(Table1)Inearlyseasonassociationalsusceptibilityonlyoc-curredinnon-irrigatedplotswhiletherewasnoeffectoftreediversityoridentityinirrigatedplots(Figure3)Atthecontraryinlateseasonassociationalsusceptibilityonlyoccurredinirrigatedwhileleaf-minerincidencedidnotdiffersignificantlyamongcompositiontreatmentsinnon-irrigatedplots(Figure3)
33emsp|emspPlant defensive traits
Tree species diversity (ie monocultures vs mixtures) and neigh-bour identity (ie birch monocultures birchndashoak birchndashpine andbirchndashoakndashpine mixtures) had qualitatively similar effects on leaftraits (TableS1) Concentration of total polyphenolics was signifi-cantly12-foldhigherintreesgrowinginmonoculturesthanintreesgrowing inmixtures (TableS1 FigureS5) Specifically the highestconcentration of total polyphenolics was found in trees growinginmonoculturesthe lowest intreesgrowinginthree-speciesmix-tures and intermediate in trees growing in two-species mixtures(Table2)Noneofothertraits individuallyrespondedtotreespe-cies diversity or neighbour identity (Table2 TableS1 FigureS5)
emspensp emsp | emsp2049Journal of EcologyCASTAGNEYROL ET AL
Starch concentrationwas 11-fold higher in trees growing in non-irrigated plots than in trees growing in irrigated plots (FigureS5Table2)Individuallynoneofothertraitsweresignificantlyaffectedbyirrigation(Table2)FinallyLDMCwastheonlytraitbeingsignifi-cantlyinfluencedbybirchapparencyandwassignificantlyhigherinmore apparent birches (slopeplusmnSE 41times10minus4plusmn11times10minus4 Table2FigureS5)indicatingthatleaveshadalowerwatercontentinmoreapparentthaninlessapparentbirches
There was a significant effect of tree species diversity andneighbour identity on PC1 (Table2 TableS1) with chemical
defences being greater in trees growing inmonocultures than intreesgrowinginanyothermixture(Figure4b)CoordinatesonPC1increased with tree apparency (Table2 FigureS5 slopeplusmnSE 36times10minus2plusmn15times10minus2) indicating that investment in chemicaldefencesincreasedasbirchesweremoreapparentDespiteaten-dencytowardshigherleafnutritionalquality(ieleaveswithhigherwatersugarstarchandnitrogencontent)intreesgrowinginnon-irrigatedplots(Figure4c)therewasnoeffectofbirchapparencyirrigation or tree species diversity or neighbour identity on PC2axis
F IGURE 2emspEffectsofseason(a)diversity(b)andplotspecificcomposition(c)andirrigationonleaf-chewerdamageIn(b)and(c)contrastsamongcompositionswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
0
2
4
6
8
10
12
Def
olia
tion
( L
AR
)
Earlyseason
Midseason
(a)
(96)
(96)0
2
4
6
8
10
12
Monocultures Mixtures
ControlIrrigated
(b)
a
b
(24) (24)(72)
(72) 0
2
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6
8
10
12
B BQ BP BQP
(c)
a
bab
ab
(24) (24) (24) (24)(24) (24)
(24) (24)
TABLE 1emspSummaryofLMM(forleaf-chewers)andGLMM(forleaf-miners)testingtheeffectsofirrigationandtreespeciesdiversity(monoculturevsmixture)orneighbouridentity(birchoakpineoak+pine)onleaf-chewerdamageandleaf-minerincidence
Season Predictors
Leaf- chewers Leaf- miners
F (df) p- value R2m (R2
c) χ2 (df) p- value R2m (R2
c)
Early Irrigation 350(192) 065 014(014) 154(1) 215 005(007)
Diversity 004(192) 847 150(1) 220
IrrigationtimesDiversity 406(192) 047 1040(1) 001
Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)
Diversity 187(122) 185 426(1) 039
IrrigationtimesDiversity 508(122) 035 044(1) 509
Early Irrigation 1155(188) 001 020(020) 202(1) 156 005(007)
Neighbours 122(388) 306 597(3) 113
IrrigationtimesNeighbours 293(388) 038 1338(3) 004
Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)
Neighbours 159(321) 223 806(3) 045
IrrigationtimesNeighbours 208(318) 138 852(3) 036
LMMlinearmixedeffectmodelsGLMMGeneralizedlinearmixedeffectmodelsAlthoughforleaf-chewersthethree-waysSeason times Irrigation times DiversityandSeason times Irrigation times Neighboursinteractionswerenotsignificantwereportresultsforearlyandmidseasonseparatelytobeconsistentwithleaf-minersandtoshowthattheinteractiveeffectsofirrigationandneighbourdiversityoridentitywereconsistentacrossseasonsBoldcharac-tersindicatesignificantpredictorsatα = 005 R2werereportedforthesimplifiedmodel(ieaftertheinteractionwasremovedifnotsignificant)R2
m and R2
ccorrespondtomarginal(ieaccountingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
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20
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Early season
ControlIrrigated
Monoculture Mixture
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Leaf
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er in
cide
nce
()
0
5
10
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B BQ BP BQP
A AB
AB
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Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
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PC1
PC
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Polyphenolics
Tannins
Starch
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Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
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KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
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KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
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MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
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Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
emspensp emsp | emsp2045Journal of EcologyCASTAGNEYROL ET AL
damagelevelstheseherbivorescancausesignificantgrowthlossonbirch(ZverevaZverevampKozlov2012)
22emsp|emspExperimental design
221emsp|emspTree diversity experiment
We conducted the present study at the ORPHEE Tree DiversityExperiment (httpssitesgooglecomvieworpheeexperiment) inSWFrance(44deg440N00deg460W)along-termexperimentdesignedtotesttheeffectsoftreespeciesdiversityonproductivityandas-sociatedfaunas(seeCastagneyroletal2013forarepresentationof the experimental design) The experiment was established in2008onasandysoil(podzol)formerlyplantedwithmaritimepineItconsistsofeightblockscovering12hawith32plotsineveryblockcorrespondingto the31possiblecombinationsofoneto five treespecies (B pendula Q robur Quercus pyrenaica Quercus ilex and
P pinaster)withanadditionalreplicateofthefive-speciesplotEachplotis400m2(20times20m)andcontains10rowsof10treesplanted2m apart (100 trees) Tree speciesmixtureswere established ac-cordingtoasubstitutivedesignkeepingtreedensityequalacrossplotswithequalproportionofeachtreespeciesWithinplots in-dividualtreesfromdifferentspecieswereplantedinaregularalter-natepatternsuchthatatreefromagivenspecieshadatleastoneneighbourfromeachoftheotherspecies
In thepresent studywe focusedonbirchmonocultures two-speciesmixturesassociatingbirchwiththepedunculateoakQ robur orthemaritimepineP pinasterandthemixturesofpedunculateoakmaritimepine andbirch for a total of four compositions In totalforthisstudyweused32plotscorrespondingto4plotstimes8blocksTheseplotswerechoseninordertosystematicallyassociatebirchwithabroadleavedandaconiferspeciesandtospanalargegradi-entofvariabilityintreeheightcanopyclosureandbirchapparency(Figure1)Birchapparency representshowmuch ()an individual
F IGURE 1emspComparisonofmeanbirchheight(a)variabilityoftreeheight(b)plotcanopyclosure(c)andbirchapparency(d)betweentreatments(a)Heightofthethreerandomlyselectedbirchesperplotatthebeginningofthe2016growingseason(b)Coefficientofvariationofheightofthe36innermosttreesperplotatthebeginningofthe2016growingseason(c)CanopyclosureestimatedinearlyJune(d)Apparencyquantifieshowmuchbircheswereonaveragetaller(positivevalues)orsmaller(negativevalues)thantheirneighbours(Damienetal2016)InallplateslightanddarkgreysareforcontrolandirrigatedplotsrespectivelyBBirchmonoculturesBQBirchndashOakmixturesBPBirchndashPinemixturesBQPBirchndashOakndashPinemixturesBoxesindicatethefirstandthirdquartilesDotsandthickhorizontallinesareformeanandmedianrespectivelyContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystars
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2046emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
birch is taller or smaller than its neighbours (Castagneyrol etal2013)andwascalculatedas100times(HbirchminusHplot)HplotwhereHbirch represented theheightof the focalbirch andHplot themean treeheightofthecorrespondingplot(averagedacrossthe36innermosttrees)NegativeandpositivevaluesindicatethatfocalbircheswereonaveragesmallerandtallerthantheirneighboursCanopyclosurewasestimatedinearlyJuneasthemeanpercentageofpixelsoccu-piedbycanopyonphotosWetook fourphotosperplotat15mfromthegroundwithsmartphoneswith8Mpxresolution Imageswere analysed with the CanopyDigi software (Goodenough ampGoodenough2012)andpercentagecanopyclosurewasaveragedattheplotlevelBirchheight(meanandCV)canopyclosureandbirchapparencywereallstronglycorrelatedwitheachother(FigureS1)
222emsp|emspIrrigation treatments
FromMay toOctober2015 and2016 half of theblockswere ir-rigatedbysprinklingc 42 m3pernightandperblockcorrespond-ingtoc3mmpernightperplotThisvolumewascalculatedbasedonregionalclimaticdata(evapotranspiration)Wesupplied60ofyearly precipitations during the five summer months (FigureS2)whichenabledtoavoidanysoilwaterdeficitintheirrigatedblocksduringtheentiregrowingseason
Weusedthreeapproachestoconfirmthatwaterstresswasalle-viatedinirrigatedblocks(FigureS3Castagneyroletal2017)Firstwe quantified soil gravimetric water content in late August 2016by sampling two soil cores between20 and40cm in eachof theselected32plotsWepooled the twosamples inhermeticplasticbags and stored them into portable fridges before being broughtto the laboratorywhere sampleswere immediatelyweightedWedried samplesat105degC for72hr andweighted themGravimetricwater content was estimated as the proportion of water in freshsamples Secondwemeasured predawn leafwater potential (ψw)inearlySeptember2016ononebirchtakenclosetothecentreofeachstudiedplotψwwasusedasaproxyofthespatiallyintegratedwaterpotentialofthesoilexploredbyrootsWeusedaScholander-typepressurechamber (DGmeca typeNP100model0-100bars)tomeasureψwononeleafpertreeLeavesweresampledaftersappressurere-equilibratedbetween430and630amlocaltime(be-fore sunrise)with clippers connected to extension loppers Thirdwe compared birch relative growth rate (RGR) between irrigatedandnon-irrigatedplotsInearly2015(C2015)andearly2016(C2016)wemeasuredtreecircumferenceat130mfromgroundlevelonarandomsampleof sevenbirchesperplot (provided theywere tallenough)inallirrigatedandnon-irrigatedplotsThisresultedin852measurementsRGRwasestimatedasthepercentageofcircumfer-enceincrement(100times(C2016minusC2015)C2015)duringthefirstyearofirrigationandwasusedtoconfirmthatbirchesintegratedtheeffectofirrigationthroughthewholegrowingseason
InFrancesummer2016wasoneofthedriestofthe1959ndash2016period (wwwmeteofrancefr) During the 2016 growing seasonin addition to natural precipitations irrigation brought c 550 mm waterperplotwhichrepresentshalfoftotalprecipitationsreceived
that year (FigureS2) In the absence of irrigation thewater-tablewas 50cmbelow-ground level in early season andwent down tominus150minOctoberInSeptember2016soilwatercontentbetween20and40cmwas26 timeshigher in irrigatedplots than innon-irrigatedplots(F130 = 2622 plt001FigureS3)Soilwatercontentwasindependentoftreespeciescomposition(F327 = 150 p = 237 FigureS3) or compositiontimesirrigation interaction (F324 = 222 p=112FigureS3)Birchψwwas38 times lower (iemorewaterstressed) in control plots (M plusmn SE minus163plusmn008MPa) than in irri-gatedplots(minus043plusmn009MPa)Togetherwiththeobservationthatbirch growth ratewas higher in irrigated than non-irrigated plots(F156789 = 2664 p=003FigureS3)theseresultsconfirmthatirri-gationcreatedtwocontrastedtreatmentsoneinwhichtreeswerewaterstressedandanotheroneinwhichwaterwasnotlimiting
23emsp|emspMeasurements of insect herbivory
WemeasureddamagecausedbytwoguildsofleafinsectherbivoreschewersandminersWithintheselected32plots(4plotstimes8blocks)werandomlysampledthreeindividualbirchtreesoutofthe36in-nermosttreesofeachplot(resultinginatotalof96birches)Damagecausedby the twoguildswasassessed twice to test for a seasoneffect inearlyJune(earlysummerinthestudyarea)andlateJuly2016(midsummerinthestudyarea)byvisualinspectionof50leavespersampledbirch(JohnsonBertrandampTurcotte2016)Becauseofpossibleverticalstratificationofattackswithincanopywerandomlyselectedleavesonfourrandomlychosenbranchestwoat2mandtwoat5mhigh
Thepercentageofleafarearemoved(LAR)byleaf-chewerswasestimatedoneachsampledleafbyauniqueobserverallalongtheex-periment(BC)usingsevenclasses(01ndash56ndash1516ndash2526ndash5051ndash75andgt75LAR)andthenaveragedpersampledtree using the mid-point of each damage class (eg Castagneyroletal 2013) Leaf-miner damagewasmeasured as the proportionof leaveswith at leastonemine (leaf-miner incidencehenceforthBarton etal 2014 Kozlov Skoracka Zverev Lewandowski ampZvereva 2016) While chewing damage could be made by eithergeneralistorspecialistherbivoresleafminersfoundonbirchwerestrictlyspecialistherbivoresinthestudyarea
24emsp|emspChemical analyses
Wequantified constitutive totalpolyphenolic andcondensed tan-ninscontentasproxiesforchemicaldefencesWequantifiedcon-stitutive leaf dry matter content (LDMC) CN ratio starch andsolublesugarconcentrationsasproxies for leafnutritionalqualityThese constitutive plant traits arewidely recognized as herbivorefeeding stimulant and deterrents and to confer resistance againstinsect herbivores inmanyplant taxa (Abdala-Roberts etal 2016ForknerMarquisampLill2004Lorangeretal2013Pearse2011Schoonhoven2005)
In late July 2016 we collected five to six undamaged leavesin each of the 96 sampled trees Leaves were directly put into
emspensp emsp | emsp2047Journal of EcologyCASTAGNEYROL ET AL
hermeticplasticbagsfilledwithhumidifiedabsorbentpaperTheywerestoredatdarkintoportablefridgebeforebeingbroughtbackto the laboratorywhere theywere kept at 4degC to rehydrate for48hratdark(Peacuterez-Harguindeguyetal2013)Leaveswerethendried for 48hr at 55degC and weighted again to estimate LDMC(mgdrygfresh Peacuterez-Harguindeguyetal 2013) Then leaveswerefinelygroundedwithliquidnitrogentoquantifytheconcentrationof constitutive total polyphenolics condensed tannins starchsoluble sugars andCN ratio Low LDMCCN ratio and concen-trationsofpolyphenolicsandcondensedtanninsontheonehandandhighconcentrationsofsugarsandstarchontheotherhandarecommonlyassociatedwithgoodleafqualitytoherbivores(Abdala-Roberts etal 2016 Forkner etal 2004 Loranger etal 2013Pearse2011Schoonhoven2005)
Leaf totalphenolicswereextractedandanalysedasdescribedbyMoreiraMooneyZasandSampedro (2012)andMoreiraZasandSampedro (2012)Briefly theywereextracted from20mgofplanttissuewith70methanolinanultrasonicbathfor15minfol-lowedbycentrifugationandsubsequentdilutionofthemethanolicextractTotalphenoliccontentwasdeterminedcolorimetricallybytheFolinndashCiocalteumethodinaBiorad650microplatereader(Bio-RadLaboratoriesPAUSA)at740nmusingtannicacidasstandardCondensed tannins in theaqueousmethanolextractsweredeter-minedbytheprocyanidinemethodasinSampedroMoreiraandZas(2011)Themethanolicextractwasmixedwithacidifiedbutanolanda ferric ammoniumsulphate solution allowed to react inaboilingwaterbathfor50minandthencooledrapidlyoniceTheconcentra-tionofcondensedtanninsinthissolutionwasdeterminedcolorimet-ricallyinaBiorad650microplatereaderat550nmusingasstandardpurifiedcondensedtanninsofquebracho(Schinopsis balansaeEnglDrogueriacuteaModernaVigoSpain)Weexpressedphenoliccompoundconcentrationsinmggtissueonadryweightbasis
Theconcentrationsofsolublesugarsandnonstructuralcarbohy-dratereserves(starch)intheleaveswereanalysedbytheanthronemethod (Hansen amp Moslashller 1975) Soluble sugars were extractedfromfinelygroundedleaves(50mg)withaqueousethanol(80vv)Starchwasextractedwith11hydrochloricacidinawaterbathat100degCfor30minfollowedbycentrifugationandsubsequentdilu-tion of the extract Soluble sugars and starch concentrationweredetermined colorimetrically in a Biorad 650 microplate reader at630nmusingglucoseandpotatostarchrespectivelyasstandards
LeafCcontent()andtotalfoliarNcontent()weremeasuredwith an Elemental AnalyserGas Analyser - Isotope-Ratio MassSpectrometry (Carlo Erba Elementar Finnigan Isoprime BremenGermany)
25emsp|emspStatistical analyses
251emsp|emspGeneral modelling approach
TheORPHEE experiment followed a randomized split-plot designreplicatedineightblockswithIrrigation(fourreplicatesoftwolev-els irrigatedvs non-irrigatedwith irrigationhomogenizinghydric
conditions among plots and across time) as thewhole factor andneighbour identity (henceforth Neighbours four levels monocul-turesofbirch two-two-speciesmixtures includingbirchwithpineoroaktreesandone-three-speciesmixturesofbirchoakandpinetrees)asthesplitfactorThesplit-plotdesignrequiresadaptingthecalculationofdegreesoffreedomandmeansumofsquaresofre-siduals(AltmanampKrzywinski2015)Thiswasachievedusinglinearmixedeffectmodels(LMM)withBlock and BlocktimesIrrigationasran-domfactors (1|BlockIrrigation in Rsyntax)For leafminerdataweusedGeneralizedlinearmixedeffectmodels(GLMM)withbinomialerrordistributionand logit-linkHerbivoryand leaf traitdatawereanalysedatthetree levelNon-independenceof individualbircheswithinplotswasaccountedforbydefiningPlotasarandomfactornestedwithintheBlockfactor(SchielzethampNakagawa2013)
Foreachtest(seebelow)wefirstbuiltafullmodelincludingallfixed effects and their interactions The fullmodelwas then sim-plifiedbysequentiallydroppingnon-significanttermsstartingwithhighest-orderinteractionsModelparameterswerefinallyestimatedon themost parsimonious obtainedmodel using restrictedmaxi-mum likelihood forLMMWeestimatedmarginal (R2
m) andcondi-tional(R2
c)R2followingNakagawaandSchielzeth(2013)Whenever
necessaryresponsevariableswerelog-transformedtomeetmodelassumptions
Allanalysesweredone inrversion331 (RCoreTeam2016)usingthefollowingpackageslmerTest car multcomp car and MuMIn (Bartoń 2016 Fox etal 2016Hothorn etal 2016 KuznetsovaBrockhoffampChristensen2015)
252emsp|emspInsect herbivory
Insect herbivory was measured in early June (early season) andlate July (mid season)We first built a full LMM includingSeason Irrigation and Diversity (iemonoculturevsmixture)orNeighbours asexplanatoryfactorsandalltwo-andthree-waysinteractionsWeaddedindividualtreeidentitynestedwithinPlotfactorasarandomfactortoaccountforvariancearisingforrepeatedmeasurementsonthesameindividualtrees
253emsp|emspPlant defensive traits
WefirstanalysedallleaftraitsseparatelyusingthesamemodellingapproachwithbirchapparencytreespeciesdiversityorneighbouridentityirrigationandIrrigationtimesDiversity(orIrrigationtimesNeighbours)and IrrigationtimesApparencyinteractionsasfixedeffectsinLMM
Becausesomeleaftraitsarehighlycorrelated(FigureS4)wefur-therusedaPCAtosummarizeinformationcontainedinthesixleaftraitsWeextractedcoordinatesonthetwo-firstPCaxes(PC1andPC2respectively)andtestedwhethertheywerepredictedbytreeheight irrigation and composition using the same LMMdescribedabove The first and second principal component (PC) axis of themultivariate principal component analysis explained 443 (PC1)and 183 (PC2) of variability in leaf traits respectively PC1wasdrivenbychemicaldefencespositivevaluesbeingassociatedwith
2048emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
highconcentrationsofbothtotalpolyphenolicsandcondensedtan-nins(Figure3a)PC2wasdrivenbynutritionalqualityrelatedtraitsPositivevalueswereassociatedwithhighLDMCandCNcontent(ielowwaterandlowNcontent)whilenegativevaluescorrespondedtohighconcentrationsofstarchandsolublesugars(Figure3a)Negativevaluesthereforeindicategreaternutritionalleafquality
254emsp|emspPlant traits underlying effects of irrigation and tree diversity or neighbour identity on leaf herbivory
We finally used a multiple regression approach and included leaftraits (PC1 PC2 and PC1timesPC2 interaction) and birch apparencyinto a fullmodel testing the effects of irrigation and tree speciesdiversityorneighbouridentityonleafherbivoryThiswaslimitedtomidseasondatawhentraitsweremeasuredWethenappliedthesimplificationprocedureexplainedaboveBecausebirchapparency(Figure1)andleaftraits(seeSection3)werenotindependentoftreediversityorneighbouridentitythemultipleregressionapproachal-lowstestingwhethersomevarianceremainedtobeexplainedbythelatterwhenaccountingfortheformerandconverselyWeexpectedthat ifdefencesmediateeffectsof irrigationandtreediversityonleaf herbivory then significant effects of any of these factors (ortheirinteraction)shouldbecomenon-significantoncesuchtraitsareaccounted for in themodel If irrigationand treediversityeffectsremainsignificantafterincludingthesetraitsthissuggeststhatirri-gationandtreediversityinfluenceherbivorythroughotherunmeas-uredplanttraitsorviasomeothermechanismnotassociatedwithplanttraitvariation
3emsp |emspRESULTS
31emsp|emspTree apparency
Treeapparencywasstronglydeterminedbythespecificcomposi-tionofexperimentalplots(Figure1)whichreflectedvariabilityintreeheightat theplot scale (Figure1)Notablybirchapparencywasthehighestinbirchndashoakmixtures(Figure1)asaresultofthesmallersizeofoakscomparedtobirches (seefigure2 inDamienetal 2016) At the opposite birch apparency was minimal inbirchndashpinemixtures(Figure1)wherebircheswerepartiallyhiddenbytallerpineneighboursBirchapparencycorrelatedwithotherstand-level variables describing tree growth and absolute andrelativeheight(FigureS1)Henceforthwefocusedonapparencyinsteadof anyother variables as it integrates informationaboutboththefocalbirchesandtheirneighboursheight
32emsp|emspLeaf insect herbivory
321emsp|emspLeaf-chewer damage
Leaf-chewer damage ranged from 034 to 1580 (mean (M) 408plusmn231) The season of sampling plant species diversity and
irrigationtreatmentsignificantlyaffectedleaf-chewerdamage(Table2)Specificallyleaf-chewerdamagewas15timesgreaterinmidsummerthan inearly summer Itwas significantlyhigher inmixtures than inmonocultureplotsanditwas15timeshigherinnon-irrigatedthaninirrigatedplots(Figure2)AssociationaleffectsdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigation interactions (Table1) The effects of diversityandcompositiononleaf-chewerdamageweresignificantonlyinnon-irrigatedplotswhereleaf-chewerdamagewasgreaterinspeciesmix-tures than in speciesmonocultures (Figure2) Leaf-chewer damagewashigherinbirchndashoakmixtureslowerinmonoculturesandinterme-diateinmixturescontainingpine(Figure2)WedidnotfindsignificanteffectsofSeasontimesDiversity SeasontimesNeighbour or SeasontimesIrrigation interactionsonleaf-chewerdamage(Table1)
322emsp|emspLeaf- miner incidence
Leaf-minerincidence(leaveswithatleastonemine)rangedfrom0 to24 (mean (M) 500plusmn426) The seasonof sampling andplant species diversity significantly affected leaf-miner incidence(Table2)Specificallyleaf-minerincidencewas15timesgreaterinmidsummerthaninearlysummerand15timeshigherinmixturesthan inmonocultureplots (ie associational susceptibility) and13timeshigherincontrolthaninirrigatedplots(Figure3)
TherewasasignificanteffectoftheSeasontimesDiversitytimesIrrigation (χ2 = 1148 df = 1 plt001) and SeasontimesDiversitytimesNeighbour (χ2 = 1776 df = 3 p=006)three-waysinteractionsonleaf-minerin-cidencesuchthatearlyandmidseasondatawereanalysedseparately(Table1)Overall inbothseasonsleaf-minerincidencewaslowerinmonocultures intermediate in two-species mixtures and higher inthree-speciesmixtures (ie associational susceptibility)However indetailsthiseffectdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigationinter-actions(Table1)Inearlyseasonassociationalsusceptibilityonlyoc-curredinnon-irrigatedplotswhiletherewasnoeffectoftreediversityoridentityinirrigatedplots(Figure3)Atthecontraryinlateseasonassociationalsusceptibilityonlyoccurredinirrigatedwhileleaf-minerincidencedidnotdiffersignificantlyamongcompositiontreatmentsinnon-irrigatedplots(Figure3)
33emsp|emspPlant defensive traits
Tree species diversity (ie monocultures vs mixtures) and neigh-bour identity (ie birch monocultures birchndashoak birchndashpine andbirchndashoakndashpine mixtures) had qualitatively similar effects on leaftraits (TableS1) Concentration of total polyphenolics was signifi-cantly12-foldhigherintreesgrowinginmonoculturesthanintreesgrowing inmixtures (TableS1 FigureS5) Specifically the highestconcentration of total polyphenolics was found in trees growinginmonoculturesthe lowest intreesgrowinginthree-speciesmix-tures and intermediate in trees growing in two-species mixtures(Table2)Noneofothertraits individuallyrespondedtotreespe-cies diversity or neighbour identity (Table2 TableS1 FigureS5)
emspensp emsp | emsp2049Journal of EcologyCASTAGNEYROL ET AL
Starch concentrationwas 11-fold higher in trees growing in non-irrigated plots than in trees growing in irrigated plots (FigureS5Table2)Individuallynoneofothertraitsweresignificantlyaffectedbyirrigation(Table2)FinallyLDMCwastheonlytraitbeingsignifi-cantlyinfluencedbybirchapparencyandwassignificantlyhigherinmore apparent birches (slopeplusmnSE 41times10minus4plusmn11times10minus4 Table2FigureS5)indicatingthatleaveshadalowerwatercontentinmoreapparentthaninlessapparentbirches
There was a significant effect of tree species diversity andneighbour identity on PC1 (Table2 TableS1) with chemical
defences being greater in trees growing inmonocultures than intreesgrowinginanyothermixture(Figure4b)CoordinatesonPC1increased with tree apparency (Table2 FigureS5 slopeplusmnSE 36times10minus2plusmn15times10minus2) indicating that investment in chemicaldefencesincreasedasbirchesweremoreapparentDespiteaten-dencytowardshigherleafnutritionalquality(ieleaveswithhigherwatersugarstarchandnitrogencontent)intreesgrowinginnon-irrigatedplots(Figure4c)therewasnoeffectofbirchapparencyirrigation or tree species diversity or neighbour identity on PC2axis
F IGURE 2emspEffectsofseason(a)diversity(b)andplotspecificcomposition(c)andirrigationonleaf-chewerdamageIn(b)and(c)contrastsamongcompositionswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
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TABLE 1emspSummaryofLMM(forleaf-chewers)andGLMM(forleaf-miners)testingtheeffectsofirrigationandtreespeciesdiversity(monoculturevsmixture)orneighbouridentity(birchoakpineoak+pine)onleaf-chewerdamageandleaf-minerincidence
Season Predictors
Leaf- chewers Leaf- miners
F (df) p- value R2m (R2
c) χ2 (df) p- value R2m (R2
c)
Early Irrigation 350(192) 065 014(014) 154(1) 215 005(007)
Diversity 004(192) 847 150(1) 220
IrrigationtimesDiversity 406(192) 047 1040(1) 001
Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)
Diversity 187(122) 185 426(1) 039
IrrigationtimesDiversity 508(122) 035 044(1) 509
Early Irrigation 1155(188) 001 020(020) 202(1) 156 005(007)
Neighbours 122(388) 306 597(3) 113
IrrigationtimesNeighbours 293(388) 038 1338(3) 004
Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)
Neighbours 159(321) 223 806(3) 045
IrrigationtimesNeighbours 208(318) 138 852(3) 036
LMMlinearmixedeffectmodelsGLMMGeneralizedlinearmixedeffectmodelsAlthoughforleaf-chewersthethree-waysSeason times Irrigation times DiversityandSeason times Irrigation times Neighboursinteractionswerenotsignificantwereportresultsforearlyandmidseasonseparatelytobeconsistentwithleaf-minersandtoshowthattheinteractiveeffectsofirrigationandneighbourdiversityoridentitywereconsistentacrossseasonsBoldcharac-tersindicatesignificantpredictorsatα = 005 R2werereportedforthesimplifiedmodel(ieaftertheinteractionwasremovedifnotsignificant)R2
m and R2
ccorrespondtomarginal(ieaccountingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
Early season
ControlIrrigated
Monoculture Mixture
Mid season
B BQ BP BQP
a
ab b
b
Composition
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
ControlIrrigated
B BQ BP BQP
A AB
AB
B
Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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Altman N amp KrzywinskiM (2015) Points of significance Split plotdesign Nature Methods 12 165ndash166 httpsdoiorg101038nmeth3293
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
AtsattPRampOrsquodowdDJ (1976)PlantdefenseguildsScience 193 24ndash29httpsdoiorg101126science193424724
Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
BantildeuelosM-JampKollmannJ (2011)Effectsofhost-plantpopulationsizeandplantsexonaspecialistleaf-minerActa Oecologica 37 58ndash64httpsdoiorg101016jactao201011007
Barbosa P Hines J Kaplan I Martinson H Szczepaniec A ampSzendreiZ (2009)Associational resistanceandassociational sus-ceptibilityHavingrightorwrongneighborsAnnual Review of Ecology Evolution and Systematics 401ndash20httpsdoiorg101146annurevecolsys110308120242
BartońK(2016)MuMIn Multi-model inferenceRetrievedfromhttpR-ForgeR-projectorgprojectsmumin
BartonKEValkamaEVehvilaumlinenHRuohomaumlkiKKnightTMampKorichevaJ(2014)Additiveandnon-additiveeffectsofbirchge-notypicdiversityonarthropodherbivoryinalong-termfieldexperi-mentOikos 124 697ndash706
BernaysEBrightKGonzalezNampAngelJ(1994)DietarymixinginageneralistherbivoreTestsoftwohypothesesEcology 75 1997ndash2006httpsdoiorg1023071941604
Callaway R M amp Walker L R (1997) Competition and facilita-tion A synthetic approach to interactions in plant communitiesEcology 78 1958ndash1965 httpsdoiorg1018900012-9658(1997) 078[1958CAFASA]20CO2
CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
2046emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
birch is taller or smaller than its neighbours (Castagneyrol etal2013)andwascalculatedas100times(HbirchminusHplot)HplotwhereHbirch represented theheightof the focalbirch andHplot themean treeheightofthecorrespondingplot(averagedacrossthe36innermosttrees)NegativeandpositivevaluesindicatethatfocalbircheswereonaveragesmallerandtallerthantheirneighboursCanopyclosurewasestimatedinearlyJuneasthemeanpercentageofpixelsoccu-piedbycanopyonphotosWetook fourphotosperplotat15mfromthegroundwithsmartphoneswith8Mpxresolution Imageswere analysed with the CanopyDigi software (Goodenough ampGoodenough2012)andpercentagecanopyclosurewasaveragedattheplotlevelBirchheight(meanandCV)canopyclosureandbirchapparencywereallstronglycorrelatedwitheachother(FigureS1)
222emsp|emspIrrigation treatments
FromMay toOctober2015 and2016 half of theblockswere ir-rigatedbysprinklingc 42 m3pernightandperblockcorrespond-ingtoc3mmpernightperplotThisvolumewascalculatedbasedonregionalclimaticdata(evapotranspiration)Wesupplied60ofyearly precipitations during the five summer months (FigureS2)whichenabledtoavoidanysoilwaterdeficitintheirrigatedblocksduringtheentiregrowingseason
Weusedthreeapproachestoconfirmthatwaterstresswasalle-viatedinirrigatedblocks(FigureS3Castagneyroletal2017)Firstwe quantified soil gravimetric water content in late August 2016by sampling two soil cores between20 and40cm in eachof theselected32plotsWepooled the twosamples inhermeticplasticbags and stored them into portable fridges before being broughtto the laboratorywhere sampleswere immediatelyweightedWedried samplesat105degC for72hr andweighted themGravimetricwater content was estimated as the proportion of water in freshsamples Secondwemeasured predawn leafwater potential (ψw)inearlySeptember2016ononebirchtakenclosetothecentreofeachstudiedplotψwwasusedasaproxyofthespatiallyintegratedwaterpotentialofthesoilexploredbyrootsWeusedaScholander-typepressurechamber (DGmeca typeNP100model0-100bars)tomeasureψwononeleafpertreeLeavesweresampledaftersappressurere-equilibratedbetween430and630amlocaltime(be-fore sunrise)with clippers connected to extension loppers Thirdwe compared birch relative growth rate (RGR) between irrigatedandnon-irrigatedplotsInearly2015(C2015)andearly2016(C2016)wemeasuredtreecircumferenceat130mfromgroundlevelonarandomsampleof sevenbirchesperplot (provided theywere tallenough)inallirrigatedandnon-irrigatedplotsThisresultedin852measurementsRGRwasestimatedasthepercentageofcircumfer-enceincrement(100times(C2016minusC2015)C2015)duringthefirstyearofirrigationandwasusedtoconfirmthatbirchesintegratedtheeffectofirrigationthroughthewholegrowingseason
InFrancesummer2016wasoneofthedriestofthe1959ndash2016period (wwwmeteofrancefr) During the 2016 growing seasonin addition to natural precipitations irrigation brought c 550 mm waterperplotwhichrepresentshalfoftotalprecipitationsreceived
that year (FigureS2) In the absence of irrigation thewater-tablewas 50cmbelow-ground level in early season andwent down tominus150minOctoberInSeptember2016soilwatercontentbetween20and40cmwas26 timeshigher in irrigatedplots than innon-irrigatedplots(F130 = 2622 plt001FigureS3)Soilwatercontentwasindependentoftreespeciescomposition(F327 = 150 p = 237 FigureS3) or compositiontimesirrigation interaction (F324 = 222 p=112FigureS3)Birchψwwas38 times lower (iemorewaterstressed) in control plots (M plusmn SE minus163plusmn008MPa) than in irri-gatedplots(minus043plusmn009MPa)Togetherwiththeobservationthatbirch growth ratewas higher in irrigated than non-irrigated plots(F156789 = 2664 p=003FigureS3)theseresultsconfirmthatirri-gationcreatedtwocontrastedtreatmentsoneinwhichtreeswerewaterstressedandanotheroneinwhichwaterwasnotlimiting
23emsp|emspMeasurements of insect herbivory
WemeasureddamagecausedbytwoguildsofleafinsectherbivoreschewersandminersWithintheselected32plots(4plotstimes8blocks)werandomlysampledthreeindividualbirchtreesoutofthe36in-nermosttreesofeachplot(resultinginatotalof96birches)Damagecausedby the twoguildswasassessed twice to test for a seasoneffect inearlyJune(earlysummerinthestudyarea)andlateJuly2016(midsummerinthestudyarea)byvisualinspectionof50leavespersampledbirch(JohnsonBertrandampTurcotte2016)Becauseofpossibleverticalstratificationofattackswithincanopywerandomlyselectedleavesonfourrandomlychosenbranchestwoat2mandtwoat5mhigh
Thepercentageofleafarearemoved(LAR)byleaf-chewerswasestimatedoneachsampledleafbyauniqueobserverallalongtheex-periment(BC)usingsevenclasses(01ndash56ndash1516ndash2526ndash5051ndash75andgt75LAR)andthenaveragedpersampledtree using the mid-point of each damage class (eg Castagneyroletal 2013) Leaf-miner damagewasmeasured as the proportionof leaveswith at leastonemine (leaf-miner incidencehenceforthBarton etal 2014 Kozlov Skoracka Zverev Lewandowski ampZvereva 2016) While chewing damage could be made by eithergeneralistorspecialistherbivoresleafminersfoundonbirchwerestrictlyspecialistherbivoresinthestudyarea
24emsp|emspChemical analyses
Wequantified constitutive totalpolyphenolic andcondensed tan-ninscontentasproxiesforchemicaldefencesWequantifiedcon-stitutive leaf dry matter content (LDMC) CN ratio starch andsolublesugarconcentrationsasproxies for leafnutritionalqualityThese constitutive plant traits arewidely recognized as herbivorefeeding stimulant and deterrents and to confer resistance againstinsect herbivores inmanyplant taxa (Abdala-Roberts etal 2016ForknerMarquisampLill2004Lorangeretal2013Pearse2011Schoonhoven2005)
In late July 2016 we collected five to six undamaged leavesin each of the 96 sampled trees Leaves were directly put into
emspensp emsp | emsp2047Journal of EcologyCASTAGNEYROL ET AL
hermeticplasticbagsfilledwithhumidifiedabsorbentpaperTheywerestoredatdarkintoportablefridgebeforebeingbroughtbackto the laboratorywhere theywere kept at 4degC to rehydrate for48hratdark(Peacuterez-Harguindeguyetal2013)Leaveswerethendried for 48hr at 55degC and weighted again to estimate LDMC(mgdrygfresh Peacuterez-Harguindeguyetal 2013) Then leaveswerefinelygroundedwithliquidnitrogentoquantifytheconcentrationof constitutive total polyphenolics condensed tannins starchsoluble sugars andCN ratio Low LDMCCN ratio and concen-trationsofpolyphenolicsandcondensedtanninsontheonehandandhighconcentrationsofsugarsandstarchontheotherhandarecommonlyassociatedwithgoodleafqualitytoherbivores(Abdala-Roberts etal 2016 Forkner etal 2004 Loranger etal 2013Pearse2011Schoonhoven2005)
Leaf totalphenolicswereextractedandanalysedasdescribedbyMoreiraMooneyZasandSampedro (2012)andMoreiraZasandSampedro (2012)Briefly theywereextracted from20mgofplanttissuewith70methanolinanultrasonicbathfor15minfol-lowedbycentrifugationandsubsequentdilutionofthemethanolicextractTotalphenoliccontentwasdeterminedcolorimetricallybytheFolinndashCiocalteumethodinaBiorad650microplatereader(Bio-RadLaboratoriesPAUSA)at740nmusingtannicacidasstandardCondensed tannins in theaqueousmethanolextractsweredeter-minedbytheprocyanidinemethodasinSampedroMoreiraandZas(2011)Themethanolicextractwasmixedwithacidifiedbutanolanda ferric ammoniumsulphate solution allowed to react inaboilingwaterbathfor50minandthencooledrapidlyoniceTheconcentra-tionofcondensedtanninsinthissolutionwasdeterminedcolorimet-ricallyinaBiorad650microplatereaderat550nmusingasstandardpurifiedcondensedtanninsofquebracho(Schinopsis balansaeEnglDrogueriacuteaModernaVigoSpain)Weexpressedphenoliccompoundconcentrationsinmggtissueonadryweightbasis
Theconcentrationsofsolublesugarsandnonstructuralcarbohy-dratereserves(starch)intheleaveswereanalysedbytheanthronemethod (Hansen amp Moslashller 1975) Soluble sugars were extractedfromfinelygroundedleaves(50mg)withaqueousethanol(80vv)Starchwasextractedwith11hydrochloricacidinawaterbathat100degCfor30minfollowedbycentrifugationandsubsequentdilu-tion of the extract Soluble sugars and starch concentrationweredetermined colorimetrically in a Biorad 650 microplate reader at630nmusingglucoseandpotatostarchrespectivelyasstandards
LeafCcontent()andtotalfoliarNcontent()weremeasuredwith an Elemental AnalyserGas Analyser - Isotope-Ratio MassSpectrometry (Carlo Erba Elementar Finnigan Isoprime BremenGermany)
25emsp|emspStatistical analyses
251emsp|emspGeneral modelling approach
TheORPHEE experiment followed a randomized split-plot designreplicatedineightblockswithIrrigation(fourreplicatesoftwolev-els irrigatedvs non-irrigatedwith irrigationhomogenizinghydric
conditions among plots and across time) as thewhole factor andneighbour identity (henceforth Neighbours four levels monocul-turesofbirch two-two-speciesmixtures includingbirchwithpineoroaktreesandone-three-speciesmixturesofbirchoakandpinetrees)asthesplitfactorThesplit-plotdesignrequiresadaptingthecalculationofdegreesoffreedomandmeansumofsquaresofre-siduals(AltmanampKrzywinski2015)Thiswasachievedusinglinearmixedeffectmodels(LMM)withBlock and BlocktimesIrrigationasran-domfactors (1|BlockIrrigation in Rsyntax)For leafminerdataweusedGeneralizedlinearmixedeffectmodels(GLMM)withbinomialerrordistributionand logit-linkHerbivoryand leaf traitdatawereanalysedatthetree levelNon-independenceof individualbircheswithinplotswasaccountedforbydefiningPlotasarandomfactornestedwithintheBlockfactor(SchielzethampNakagawa2013)
Foreachtest(seebelow)wefirstbuiltafullmodelincludingallfixed effects and their interactions The fullmodelwas then sim-plifiedbysequentiallydroppingnon-significanttermsstartingwithhighest-orderinteractionsModelparameterswerefinallyestimatedon themost parsimonious obtainedmodel using restrictedmaxi-mum likelihood forLMMWeestimatedmarginal (R2
m) andcondi-tional(R2
c)R2followingNakagawaandSchielzeth(2013)Whenever
necessaryresponsevariableswerelog-transformedtomeetmodelassumptions
Allanalysesweredone inrversion331 (RCoreTeam2016)usingthefollowingpackageslmerTest car multcomp car and MuMIn (Bartoń 2016 Fox etal 2016Hothorn etal 2016 KuznetsovaBrockhoffampChristensen2015)
252emsp|emspInsect herbivory
Insect herbivory was measured in early June (early season) andlate July (mid season)We first built a full LMM includingSeason Irrigation and Diversity (iemonoculturevsmixture)orNeighbours asexplanatoryfactorsandalltwo-andthree-waysinteractionsWeaddedindividualtreeidentitynestedwithinPlotfactorasarandomfactortoaccountforvariancearisingforrepeatedmeasurementsonthesameindividualtrees
253emsp|emspPlant defensive traits
WefirstanalysedallleaftraitsseparatelyusingthesamemodellingapproachwithbirchapparencytreespeciesdiversityorneighbouridentityirrigationandIrrigationtimesDiversity(orIrrigationtimesNeighbours)and IrrigationtimesApparencyinteractionsasfixedeffectsinLMM
Becausesomeleaftraitsarehighlycorrelated(FigureS4)wefur-therusedaPCAtosummarizeinformationcontainedinthesixleaftraitsWeextractedcoordinatesonthetwo-firstPCaxes(PC1andPC2respectively)andtestedwhethertheywerepredictedbytreeheight irrigation and composition using the same LMMdescribedabove The first and second principal component (PC) axis of themultivariate principal component analysis explained 443 (PC1)and 183 (PC2) of variability in leaf traits respectively PC1wasdrivenbychemicaldefencespositivevaluesbeingassociatedwith
2048emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
highconcentrationsofbothtotalpolyphenolicsandcondensedtan-nins(Figure3a)PC2wasdrivenbynutritionalqualityrelatedtraitsPositivevalueswereassociatedwithhighLDMCandCNcontent(ielowwaterandlowNcontent)whilenegativevaluescorrespondedtohighconcentrationsofstarchandsolublesugars(Figure3a)Negativevaluesthereforeindicategreaternutritionalleafquality
254emsp|emspPlant traits underlying effects of irrigation and tree diversity or neighbour identity on leaf herbivory
We finally used a multiple regression approach and included leaftraits (PC1 PC2 and PC1timesPC2 interaction) and birch apparencyinto a fullmodel testing the effects of irrigation and tree speciesdiversityorneighbouridentityonleafherbivoryThiswaslimitedtomidseasondatawhentraitsweremeasuredWethenappliedthesimplificationprocedureexplainedaboveBecausebirchapparency(Figure1)andleaftraits(seeSection3)werenotindependentoftreediversityorneighbouridentitythemultipleregressionapproachal-lowstestingwhethersomevarianceremainedtobeexplainedbythelatterwhenaccountingfortheformerandconverselyWeexpectedthat ifdefencesmediateeffectsof irrigationandtreediversityonleaf herbivory then significant effects of any of these factors (ortheirinteraction)shouldbecomenon-significantoncesuchtraitsareaccounted for in themodel If irrigationand treediversityeffectsremainsignificantafterincludingthesetraitsthissuggeststhatirri-gationandtreediversityinfluenceherbivorythroughotherunmeas-uredplanttraitsorviasomeothermechanismnotassociatedwithplanttraitvariation
3emsp |emspRESULTS
31emsp|emspTree apparency
Treeapparencywasstronglydeterminedbythespecificcomposi-tionofexperimentalplots(Figure1)whichreflectedvariabilityintreeheightat theplot scale (Figure1)Notablybirchapparencywasthehighestinbirchndashoakmixtures(Figure1)asaresultofthesmallersizeofoakscomparedtobirches (seefigure2 inDamienetal 2016) At the opposite birch apparency was minimal inbirchndashpinemixtures(Figure1)wherebircheswerepartiallyhiddenbytallerpineneighboursBirchapparencycorrelatedwithotherstand-level variables describing tree growth and absolute andrelativeheight(FigureS1)Henceforthwefocusedonapparencyinsteadof anyother variables as it integrates informationaboutboththefocalbirchesandtheirneighboursheight
32emsp|emspLeaf insect herbivory
321emsp|emspLeaf-chewer damage
Leaf-chewer damage ranged from 034 to 1580 (mean (M) 408plusmn231) The season of sampling plant species diversity and
irrigationtreatmentsignificantlyaffectedleaf-chewerdamage(Table2)Specificallyleaf-chewerdamagewas15timesgreaterinmidsummerthan inearly summer Itwas significantlyhigher inmixtures than inmonocultureplotsanditwas15timeshigherinnon-irrigatedthaninirrigatedplots(Figure2)AssociationaleffectsdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigation interactions (Table1) The effects of diversityandcompositiononleaf-chewerdamageweresignificantonlyinnon-irrigatedplotswhereleaf-chewerdamagewasgreaterinspeciesmix-tures than in speciesmonocultures (Figure2) Leaf-chewer damagewashigherinbirchndashoakmixtureslowerinmonoculturesandinterme-diateinmixturescontainingpine(Figure2)WedidnotfindsignificanteffectsofSeasontimesDiversity SeasontimesNeighbour or SeasontimesIrrigation interactionsonleaf-chewerdamage(Table1)
322emsp|emspLeaf- miner incidence
Leaf-minerincidence(leaveswithatleastonemine)rangedfrom0 to24 (mean (M) 500plusmn426) The seasonof sampling andplant species diversity significantly affected leaf-miner incidence(Table2)Specificallyleaf-minerincidencewas15timesgreaterinmidsummerthaninearlysummerand15timeshigherinmixturesthan inmonocultureplots (ie associational susceptibility) and13timeshigherincontrolthaninirrigatedplots(Figure3)
TherewasasignificanteffectoftheSeasontimesDiversitytimesIrrigation (χ2 = 1148 df = 1 plt001) and SeasontimesDiversitytimesNeighbour (χ2 = 1776 df = 3 p=006)three-waysinteractionsonleaf-minerin-cidencesuchthatearlyandmidseasondatawereanalysedseparately(Table1)Overall inbothseasonsleaf-minerincidencewaslowerinmonocultures intermediate in two-species mixtures and higher inthree-speciesmixtures (ie associational susceptibility)However indetailsthiseffectdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigationinter-actions(Table1)Inearlyseasonassociationalsusceptibilityonlyoc-curredinnon-irrigatedplotswhiletherewasnoeffectoftreediversityoridentityinirrigatedplots(Figure3)Atthecontraryinlateseasonassociationalsusceptibilityonlyoccurredinirrigatedwhileleaf-minerincidencedidnotdiffersignificantlyamongcompositiontreatmentsinnon-irrigatedplots(Figure3)
33emsp|emspPlant defensive traits
Tree species diversity (ie monocultures vs mixtures) and neigh-bour identity (ie birch monocultures birchndashoak birchndashpine andbirchndashoakndashpine mixtures) had qualitatively similar effects on leaftraits (TableS1) Concentration of total polyphenolics was signifi-cantly12-foldhigherintreesgrowinginmonoculturesthanintreesgrowing inmixtures (TableS1 FigureS5) Specifically the highestconcentration of total polyphenolics was found in trees growinginmonoculturesthe lowest intreesgrowinginthree-speciesmix-tures and intermediate in trees growing in two-species mixtures(Table2)Noneofothertraits individuallyrespondedtotreespe-cies diversity or neighbour identity (Table2 TableS1 FigureS5)
emspensp emsp | emsp2049Journal of EcologyCASTAGNEYROL ET AL
Starch concentrationwas 11-fold higher in trees growing in non-irrigated plots than in trees growing in irrigated plots (FigureS5Table2)Individuallynoneofothertraitsweresignificantlyaffectedbyirrigation(Table2)FinallyLDMCwastheonlytraitbeingsignifi-cantlyinfluencedbybirchapparencyandwassignificantlyhigherinmore apparent birches (slopeplusmnSE 41times10minus4plusmn11times10minus4 Table2FigureS5)indicatingthatleaveshadalowerwatercontentinmoreapparentthaninlessapparentbirches
There was a significant effect of tree species diversity andneighbour identity on PC1 (Table2 TableS1) with chemical
defences being greater in trees growing inmonocultures than intreesgrowinginanyothermixture(Figure4b)CoordinatesonPC1increased with tree apparency (Table2 FigureS5 slopeplusmnSE 36times10minus2plusmn15times10minus2) indicating that investment in chemicaldefencesincreasedasbirchesweremoreapparentDespiteaten-dencytowardshigherleafnutritionalquality(ieleaveswithhigherwatersugarstarchandnitrogencontent)intreesgrowinginnon-irrigatedplots(Figure4c)therewasnoeffectofbirchapparencyirrigation or tree species diversity or neighbour identity on PC2axis
F IGURE 2emspEffectsofseason(a)diversity(b)andplotspecificcomposition(c)andirrigationonleaf-chewerdamageIn(b)and(c)contrastsamongcompositionswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
0
2
4
6
8
10
12
Def
olia
tion
( L
AR
)
Earlyseason
Midseason
(a)
(96)
(96)0
2
4
6
8
10
12
Monocultures Mixtures
ControlIrrigated
(b)
a
b
(24) (24)(72)
(72) 0
2
4
6
8
10
12
B BQ BP BQP
(c)
a
bab
ab
(24) (24) (24) (24)(24) (24)
(24) (24)
TABLE 1emspSummaryofLMM(forleaf-chewers)andGLMM(forleaf-miners)testingtheeffectsofirrigationandtreespeciesdiversity(monoculturevsmixture)orneighbouridentity(birchoakpineoak+pine)onleaf-chewerdamageandleaf-minerincidence
Season Predictors
Leaf- chewers Leaf- miners
F (df) p- value R2m (R2
c) χ2 (df) p- value R2m (R2
c)
Early Irrigation 350(192) 065 014(014) 154(1) 215 005(007)
Diversity 004(192) 847 150(1) 220
IrrigationtimesDiversity 406(192) 047 1040(1) 001
Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)
Diversity 187(122) 185 426(1) 039
IrrigationtimesDiversity 508(122) 035 044(1) 509
Early Irrigation 1155(188) 001 020(020) 202(1) 156 005(007)
Neighbours 122(388) 306 597(3) 113
IrrigationtimesNeighbours 293(388) 038 1338(3) 004
Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)
Neighbours 159(321) 223 806(3) 045
IrrigationtimesNeighbours 208(318) 138 852(3) 036
LMMlinearmixedeffectmodelsGLMMGeneralizedlinearmixedeffectmodelsAlthoughforleaf-chewersthethree-waysSeason times Irrigation times DiversityandSeason times Irrigation times Neighboursinteractionswerenotsignificantwereportresultsforearlyandmidseasonseparatelytobeconsistentwithleaf-minersandtoshowthattheinteractiveeffectsofirrigationandneighbourdiversityoridentitywereconsistentacrossseasonsBoldcharac-tersindicatesignificantpredictorsatα = 005 R2werereportedforthesimplifiedmodel(ieaftertheinteractionwasremovedifnotsignificant)R2
m and R2
ccorrespondtomarginal(ieaccountingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
Early season
ControlIrrigated
Monoculture Mixture
Mid season
B BQ BP BQP
a
ab b
b
Composition
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
ControlIrrigated
B BQ BP BQP
A AB
AB
B
Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
Abdala-Roberts L Hernandez-Cumplido J Chel-Guerrero LBetancur-Ancona D Benrey B ampMoreira X (2016) Effects ofplant intraspecificdiversity across three trophic levelsUnderlyingmechanismsandplanttraitsAmerican Journal of Botany 103 1810ndash1818httpsdoiorg103732ajb1600234
Altman N amp KrzywinskiM (2015) Points of significance Split plotdesign Nature Methods 12 165ndash166 httpsdoiorg101038nmeth3293
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
AtsattPRampOrsquodowdDJ (1976)PlantdefenseguildsScience 193 24ndash29httpsdoiorg101126science193424724
Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
BantildeuelosM-JampKollmannJ (2011)Effectsofhost-plantpopulationsizeandplantsexonaspecialistleaf-minerActa Oecologica 37 58ndash64httpsdoiorg101016jactao201011007
Barbosa P Hines J Kaplan I Martinson H Szczepaniec A ampSzendreiZ (2009)Associational resistanceandassociational sus-ceptibilityHavingrightorwrongneighborsAnnual Review of Ecology Evolution and Systematics 401ndash20httpsdoiorg101146annurevecolsys110308120242
BartońK(2016)MuMIn Multi-model inferenceRetrievedfromhttpR-ForgeR-projectorgprojectsmumin
BartonKEValkamaEVehvilaumlinenHRuohomaumlkiKKnightTMampKorichevaJ(2014)Additiveandnon-additiveeffectsofbirchge-notypicdiversityonarthropodherbivoryinalong-termfieldexperi-mentOikos 124 697ndash706
BernaysEBrightKGonzalezNampAngelJ(1994)DietarymixinginageneralistherbivoreTestsoftwohypothesesEcology 75 1997ndash2006httpsdoiorg1023071941604
Callaway R M amp Walker L R (1997) Competition and facilita-tion A synthetic approach to interactions in plant communitiesEcology 78 1958ndash1965 httpsdoiorg1018900012-9658(1997) 078[1958CAFASA]20CO2
CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
emspensp emsp | emsp2047Journal of EcologyCASTAGNEYROL ET AL
hermeticplasticbagsfilledwithhumidifiedabsorbentpaperTheywerestoredatdarkintoportablefridgebeforebeingbroughtbackto the laboratorywhere theywere kept at 4degC to rehydrate for48hratdark(Peacuterez-Harguindeguyetal2013)Leaveswerethendried for 48hr at 55degC and weighted again to estimate LDMC(mgdrygfresh Peacuterez-Harguindeguyetal 2013) Then leaveswerefinelygroundedwithliquidnitrogentoquantifytheconcentrationof constitutive total polyphenolics condensed tannins starchsoluble sugars andCN ratio Low LDMCCN ratio and concen-trationsofpolyphenolicsandcondensedtanninsontheonehandandhighconcentrationsofsugarsandstarchontheotherhandarecommonlyassociatedwithgoodleafqualitytoherbivores(Abdala-Roberts etal 2016 Forkner etal 2004 Loranger etal 2013Pearse2011Schoonhoven2005)
Leaf totalphenolicswereextractedandanalysedasdescribedbyMoreiraMooneyZasandSampedro (2012)andMoreiraZasandSampedro (2012)Briefly theywereextracted from20mgofplanttissuewith70methanolinanultrasonicbathfor15minfol-lowedbycentrifugationandsubsequentdilutionofthemethanolicextractTotalphenoliccontentwasdeterminedcolorimetricallybytheFolinndashCiocalteumethodinaBiorad650microplatereader(Bio-RadLaboratoriesPAUSA)at740nmusingtannicacidasstandardCondensed tannins in theaqueousmethanolextractsweredeter-minedbytheprocyanidinemethodasinSampedroMoreiraandZas(2011)Themethanolicextractwasmixedwithacidifiedbutanolanda ferric ammoniumsulphate solution allowed to react inaboilingwaterbathfor50minandthencooledrapidlyoniceTheconcentra-tionofcondensedtanninsinthissolutionwasdeterminedcolorimet-ricallyinaBiorad650microplatereaderat550nmusingasstandardpurifiedcondensedtanninsofquebracho(Schinopsis balansaeEnglDrogueriacuteaModernaVigoSpain)Weexpressedphenoliccompoundconcentrationsinmggtissueonadryweightbasis
Theconcentrationsofsolublesugarsandnonstructuralcarbohy-dratereserves(starch)intheleaveswereanalysedbytheanthronemethod (Hansen amp Moslashller 1975) Soluble sugars were extractedfromfinelygroundedleaves(50mg)withaqueousethanol(80vv)Starchwasextractedwith11hydrochloricacidinawaterbathat100degCfor30minfollowedbycentrifugationandsubsequentdilu-tion of the extract Soluble sugars and starch concentrationweredetermined colorimetrically in a Biorad 650 microplate reader at630nmusingglucoseandpotatostarchrespectivelyasstandards
LeafCcontent()andtotalfoliarNcontent()weremeasuredwith an Elemental AnalyserGas Analyser - Isotope-Ratio MassSpectrometry (Carlo Erba Elementar Finnigan Isoprime BremenGermany)
25emsp|emspStatistical analyses
251emsp|emspGeneral modelling approach
TheORPHEE experiment followed a randomized split-plot designreplicatedineightblockswithIrrigation(fourreplicatesoftwolev-els irrigatedvs non-irrigatedwith irrigationhomogenizinghydric
conditions among plots and across time) as thewhole factor andneighbour identity (henceforth Neighbours four levels monocul-turesofbirch two-two-speciesmixtures includingbirchwithpineoroaktreesandone-three-speciesmixturesofbirchoakandpinetrees)asthesplitfactorThesplit-plotdesignrequiresadaptingthecalculationofdegreesoffreedomandmeansumofsquaresofre-siduals(AltmanampKrzywinski2015)Thiswasachievedusinglinearmixedeffectmodels(LMM)withBlock and BlocktimesIrrigationasran-domfactors (1|BlockIrrigation in Rsyntax)For leafminerdataweusedGeneralizedlinearmixedeffectmodels(GLMM)withbinomialerrordistributionand logit-linkHerbivoryand leaf traitdatawereanalysedatthetree levelNon-independenceof individualbircheswithinplotswasaccountedforbydefiningPlotasarandomfactornestedwithintheBlockfactor(SchielzethampNakagawa2013)
Foreachtest(seebelow)wefirstbuiltafullmodelincludingallfixed effects and their interactions The fullmodelwas then sim-plifiedbysequentiallydroppingnon-significanttermsstartingwithhighest-orderinteractionsModelparameterswerefinallyestimatedon themost parsimonious obtainedmodel using restrictedmaxi-mum likelihood forLMMWeestimatedmarginal (R2
m) andcondi-tional(R2
c)R2followingNakagawaandSchielzeth(2013)Whenever
necessaryresponsevariableswerelog-transformedtomeetmodelassumptions
Allanalysesweredone inrversion331 (RCoreTeam2016)usingthefollowingpackageslmerTest car multcomp car and MuMIn (Bartoń 2016 Fox etal 2016Hothorn etal 2016 KuznetsovaBrockhoffampChristensen2015)
252emsp|emspInsect herbivory
Insect herbivory was measured in early June (early season) andlate July (mid season)We first built a full LMM includingSeason Irrigation and Diversity (iemonoculturevsmixture)orNeighbours asexplanatoryfactorsandalltwo-andthree-waysinteractionsWeaddedindividualtreeidentitynestedwithinPlotfactorasarandomfactortoaccountforvariancearisingforrepeatedmeasurementsonthesameindividualtrees
253emsp|emspPlant defensive traits
WefirstanalysedallleaftraitsseparatelyusingthesamemodellingapproachwithbirchapparencytreespeciesdiversityorneighbouridentityirrigationandIrrigationtimesDiversity(orIrrigationtimesNeighbours)and IrrigationtimesApparencyinteractionsasfixedeffectsinLMM
Becausesomeleaftraitsarehighlycorrelated(FigureS4)wefur-therusedaPCAtosummarizeinformationcontainedinthesixleaftraitsWeextractedcoordinatesonthetwo-firstPCaxes(PC1andPC2respectively)andtestedwhethertheywerepredictedbytreeheight irrigation and composition using the same LMMdescribedabove The first and second principal component (PC) axis of themultivariate principal component analysis explained 443 (PC1)and 183 (PC2) of variability in leaf traits respectively PC1wasdrivenbychemicaldefencespositivevaluesbeingassociatedwith
2048emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
highconcentrationsofbothtotalpolyphenolicsandcondensedtan-nins(Figure3a)PC2wasdrivenbynutritionalqualityrelatedtraitsPositivevalueswereassociatedwithhighLDMCandCNcontent(ielowwaterandlowNcontent)whilenegativevaluescorrespondedtohighconcentrationsofstarchandsolublesugars(Figure3a)Negativevaluesthereforeindicategreaternutritionalleafquality
254emsp|emspPlant traits underlying effects of irrigation and tree diversity or neighbour identity on leaf herbivory
We finally used a multiple regression approach and included leaftraits (PC1 PC2 and PC1timesPC2 interaction) and birch apparencyinto a fullmodel testing the effects of irrigation and tree speciesdiversityorneighbouridentityonleafherbivoryThiswaslimitedtomidseasondatawhentraitsweremeasuredWethenappliedthesimplificationprocedureexplainedaboveBecausebirchapparency(Figure1)andleaftraits(seeSection3)werenotindependentoftreediversityorneighbouridentitythemultipleregressionapproachal-lowstestingwhethersomevarianceremainedtobeexplainedbythelatterwhenaccountingfortheformerandconverselyWeexpectedthat ifdefencesmediateeffectsof irrigationandtreediversityonleaf herbivory then significant effects of any of these factors (ortheirinteraction)shouldbecomenon-significantoncesuchtraitsareaccounted for in themodel If irrigationand treediversityeffectsremainsignificantafterincludingthesetraitsthissuggeststhatirri-gationandtreediversityinfluenceherbivorythroughotherunmeas-uredplanttraitsorviasomeothermechanismnotassociatedwithplanttraitvariation
3emsp |emspRESULTS
31emsp|emspTree apparency
Treeapparencywasstronglydeterminedbythespecificcomposi-tionofexperimentalplots(Figure1)whichreflectedvariabilityintreeheightat theplot scale (Figure1)Notablybirchapparencywasthehighestinbirchndashoakmixtures(Figure1)asaresultofthesmallersizeofoakscomparedtobirches (seefigure2 inDamienetal 2016) At the opposite birch apparency was minimal inbirchndashpinemixtures(Figure1)wherebircheswerepartiallyhiddenbytallerpineneighboursBirchapparencycorrelatedwithotherstand-level variables describing tree growth and absolute andrelativeheight(FigureS1)Henceforthwefocusedonapparencyinsteadof anyother variables as it integrates informationaboutboththefocalbirchesandtheirneighboursheight
32emsp|emspLeaf insect herbivory
321emsp|emspLeaf-chewer damage
Leaf-chewer damage ranged from 034 to 1580 (mean (M) 408plusmn231) The season of sampling plant species diversity and
irrigationtreatmentsignificantlyaffectedleaf-chewerdamage(Table2)Specificallyleaf-chewerdamagewas15timesgreaterinmidsummerthan inearly summer Itwas significantlyhigher inmixtures than inmonocultureplotsanditwas15timeshigherinnon-irrigatedthaninirrigatedplots(Figure2)AssociationaleffectsdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigation interactions (Table1) The effects of diversityandcompositiononleaf-chewerdamageweresignificantonlyinnon-irrigatedplotswhereleaf-chewerdamagewasgreaterinspeciesmix-tures than in speciesmonocultures (Figure2) Leaf-chewer damagewashigherinbirchndashoakmixtureslowerinmonoculturesandinterme-diateinmixturescontainingpine(Figure2)WedidnotfindsignificanteffectsofSeasontimesDiversity SeasontimesNeighbour or SeasontimesIrrigation interactionsonleaf-chewerdamage(Table1)
322emsp|emspLeaf- miner incidence
Leaf-minerincidence(leaveswithatleastonemine)rangedfrom0 to24 (mean (M) 500plusmn426) The seasonof sampling andplant species diversity significantly affected leaf-miner incidence(Table2)Specificallyleaf-minerincidencewas15timesgreaterinmidsummerthaninearlysummerand15timeshigherinmixturesthan inmonocultureplots (ie associational susceptibility) and13timeshigherincontrolthaninirrigatedplots(Figure3)
TherewasasignificanteffectoftheSeasontimesDiversitytimesIrrigation (χ2 = 1148 df = 1 plt001) and SeasontimesDiversitytimesNeighbour (χ2 = 1776 df = 3 p=006)three-waysinteractionsonleaf-minerin-cidencesuchthatearlyandmidseasondatawereanalysedseparately(Table1)Overall inbothseasonsleaf-minerincidencewaslowerinmonocultures intermediate in two-species mixtures and higher inthree-speciesmixtures (ie associational susceptibility)However indetailsthiseffectdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigationinter-actions(Table1)Inearlyseasonassociationalsusceptibilityonlyoc-curredinnon-irrigatedplotswhiletherewasnoeffectoftreediversityoridentityinirrigatedplots(Figure3)Atthecontraryinlateseasonassociationalsusceptibilityonlyoccurredinirrigatedwhileleaf-minerincidencedidnotdiffersignificantlyamongcompositiontreatmentsinnon-irrigatedplots(Figure3)
33emsp|emspPlant defensive traits
Tree species diversity (ie monocultures vs mixtures) and neigh-bour identity (ie birch monocultures birchndashoak birchndashpine andbirchndashoakndashpine mixtures) had qualitatively similar effects on leaftraits (TableS1) Concentration of total polyphenolics was signifi-cantly12-foldhigherintreesgrowinginmonoculturesthanintreesgrowing inmixtures (TableS1 FigureS5) Specifically the highestconcentration of total polyphenolics was found in trees growinginmonoculturesthe lowest intreesgrowinginthree-speciesmix-tures and intermediate in trees growing in two-species mixtures(Table2)Noneofothertraits individuallyrespondedtotreespe-cies diversity or neighbour identity (Table2 TableS1 FigureS5)
emspensp emsp | emsp2049Journal of EcologyCASTAGNEYROL ET AL
Starch concentrationwas 11-fold higher in trees growing in non-irrigated plots than in trees growing in irrigated plots (FigureS5Table2)Individuallynoneofothertraitsweresignificantlyaffectedbyirrigation(Table2)FinallyLDMCwastheonlytraitbeingsignifi-cantlyinfluencedbybirchapparencyandwassignificantlyhigherinmore apparent birches (slopeplusmnSE 41times10minus4plusmn11times10minus4 Table2FigureS5)indicatingthatleaveshadalowerwatercontentinmoreapparentthaninlessapparentbirches
There was a significant effect of tree species diversity andneighbour identity on PC1 (Table2 TableS1) with chemical
defences being greater in trees growing inmonocultures than intreesgrowinginanyothermixture(Figure4b)CoordinatesonPC1increased with tree apparency (Table2 FigureS5 slopeplusmnSE 36times10minus2plusmn15times10minus2) indicating that investment in chemicaldefencesincreasedasbirchesweremoreapparentDespiteaten-dencytowardshigherleafnutritionalquality(ieleaveswithhigherwatersugarstarchandnitrogencontent)intreesgrowinginnon-irrigatedplots(Figure4c)therewasnoeffectofbirchapparencyirrigation or tree species diversity or neighbour identity on PC2axis
F IGURE 2emspEffectsofseason(a)diversity(b)andplotspecificcomposition(c)andirrigationonleaf-chewerdamageIn(b)and(c)contrastsamongcompositionswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
0
2
4
6
8
10
12
Def
olia
tion
( L
AR
)
Earlyseason
Midseason
(a)
(96)
(96)0
2
4
6
8
10
12
Monocultures Mixtures
ControlIrrigated
(b)
a
b
(24) (24)(72)
(72) 0
2
4
6
8
10
12
B BQ BP BQP
(c)
a
bab
ab
(24) (24) (24) (24)(24) (24)
(24) (24)
TABLE 1emspSummaryofLMM(forleaf-chewers)andGLMM(forleaf-miners)testingtheeffectsofirrigationandtreespeciesdiversity(monoculturevsmixture)orneighbouridentity(birchoakpineoak+pine)onleaf-chewerdamageandleaf-minerincidence
Season Predictors
Leaf- chewers Leaf- miners
F (df) p- value R2m (R2
c) χ2 (df) p- value R2m (R2
c)
Early Irrigation 350(192) 065 014(014) 154(1) 215 005(007)
Diversity 004(192) 847 150(1) 220
IrrigationtimesDiversity 406(192) 047 1040(1) 001
Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)
Diversity 187(122) 185 426(1) 039
IrrigationtimesDiversity 508(122) 035 044(1) 509
Early Irrigation 1155(188) 001 020(020) 202(1) 156 005(007)
Neighbours 122(388) 306 597(3) 113
IrrigationtimesNeighbours 293(388) 038 1338(3) 004
Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)
Neighbours 159(321) 223 806(3) 045
IrrigationtimesNeighbours 208(318) 138 852(3) 036
LMMlinearmixedeffectmodelsGLMMGeneralizedlinearmixedeffectmodelsAlthoughforleaf-chewersthethree-waysSeason times Irrigation times DiversityandSeason times Irrigation times Neighboursinteractionswerenotsignificantwereportresultsforearlyandmidseasonseparatelytobeconsistentwithleaf-minersandtoshowthattheinteractiveeffectsofirrigationandneighbourdiversityoridentitywereconsistentacrossseasonsBoldcharac-tersindicatesignificantpredictorsatα = 005 R2werereportedforthesimplifiedmodel(ieaftertheinteractionwasremovedifnotsignificant)R2
m and R2
ccorrespondtomarginal(ieaccountingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
Early season
ControlIrrigated
Monoculture Mixture
Mid season
B BQ BP BQP
a
ab b
b
Composition
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
ControlIrrigated
B BQ BP BQP
A AB
AB
B
Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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Altman N amp KrzywinskiM (2015) Points of significance Split plotdesign Nature Methods 12 165ndash166 httpsdoiorg101038nmeth3293
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
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Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
BantildeuelosM-JampKollmannJ (2011)Effectsofhost-plantpopulationsizeandplantsexonaspecialistleaf-minerActa Oecologica 37 58ndash64httpsdoiorg101016jactao201011007
Barbosa P Hines J Kaplan I Martinson H Szczepaniec A ampSzendreiZ (2009)Associational resistanceandassociational sus-ceptibilityHavingrightorwrongneighborsAnnual Review of Ecology Evolution and Systematics 401ndash20httpsdoiorg101146annurevecolsys110308120242
BartońK(2016)MuMIn Multi-model inferenceRetrievedfromhttpR-ForgeR-projectorgprojectsmumin
BartonKEValkamaEVehvilaumlinenHRuohomaumlkiKKnightTMampKorichevaJ(2014)Additiveandnon-additiveeffectsofbirchge-notypicdiversityonarthropodherbivoryinalong-termfieldexperi-mentOikos 124 697ndash706
BernaysEBrightKGonzalezNampAngelJ(1994)DietarymixinginageneralistherbivoreTestsoftwohypothesesEcology 75 1997ndash2006httpsdoiorg1023071941604
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CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
2048emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
highconcentrationsofbothtotalpolyphenolicsandcondensedtan-nins(Figure3a)PC2wasdrivenbynutritionalqualityrelatedtraitsPositivevalueswereassociatedwithhighLDMCandCNcontent(ielowwaterandlowNcontent)whilenegativevaluescorrespondedtohighconcentrationsofstarchandsolublesugars(Figure3a)Negativevaluesthereforeindicategreaternutritionalleafquality
254emsp|emspPlant traits underlying effects of irrigation and tree diversity or neighbour identity on leaf herbivory
We finally used a multiple regression approach and included leaftraits (PC1 PC2 and PC1timesPC2 interaction) and birch apparencyinto a fullmodel testing the effects of irrigation and tree speciesdiversityorneighbouridentityonleafherbivoryThiswaslimitedtomidseasondatawhentraitsweremeasuredWethenappliedthesimplificationprocedureexplainedaboveBecausebirchapparency(Figure1)andleaftraits(seeSection3)werenotindependentoftreediversityorneighbouridentitythemultipleregressionapproachal-lowstestingwhethersomevarianceremainedtobeexplainedbythelatterwhenaccountingfortheformerandconverselyWeexpectedthat ifdefencesmediateeffectsof irrigationandtreediversityonleaf herbivory then significant effects of any of these factors (ortheirinteraction)shouldbecomenon-significantoncesuchtraitsareaccounted for in themodel If irrigationand treediversityeffectsremainsignificantafterincludingthesetraitsthissuggeststhatirri-gationandtreediversityinfluenceherbivorythroughotherunmeas-uredplanttraitsorviasomeothermechanismnotassociatedwithplanttraitvariation
3emsp |emspRESULTS
31emsp|emspTree apparency
Treeapparencywasstronglydeterminedbythespecificcomposi-tionofexperimentalplots(Figure1)whichreflectedvariabilityintreeheightat theplot scale (Figure1)Notablybirchapparencywasthehighestinbirchndashoakmixtures(Figure1)asaresultofthesmallersizeofoakscomparedtobirches (seefigure2 inDamienetal 2016) At the opposite birch apparency was minimal inbirchndashpinemixtures(Figure1)wherebircheswerepartiallyhiddenbytallerpineneighboursBirchapparencycorrelatedwithotherstand-level variables describing tree growth and absolute andrelativeheight(FigureS1)Henceforthwefocusedonapparencyinsteadof anyother variables as it integrates informationaboutboththefocalbirchesandtheirneighboursheight
32emsp|emspLeaf insect herbivory
321emsp|emspLeaf-chewer damage
Leaf-chewer damage ranged from 034 to 1580 (mean (M) 408plusmn231) The season of sampling plant species diversity and
irrigationtreatmentsignificantlyaffectedleaf-chewerdamage(Table2)Specificallyleaf-chewerdamagewas15timesgreaterinmidsummerthan inearly summer Itwas significantlyhigher inmixtures than inmonocultureplotsanditwas15timeshigherinnon-irrigatedthaninirrigatedplots(Figure2)AssociationaleffectsdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigation interactions (Table1) The effects of diversityandcompositiononleaf-chewerdamageweresignificantonlyinnon-irrigatedplotswhereleaf-chewerdamagewasgreaterinspeciesmix-tures than in speciesmonocultures (Figure2) Leaf-chewer damagewashigherinbirchndashoakmixtureslowerinmonoculturesandinterme-diateinmixturescontainingpine(Figure2)WedidnotfindsignificanteffectsofSeasontimesDiversity SeasontimesNeighbour or SeasontimesIrrigation interactionsonleaf-chewerdamage(Table1)
322emsp|emspLeaf- miner incidence
Leaf-minerincidence(leaveswithatleastonemine)rangedfrom0 to24 (mean (M) 500plusmn426) The seasonof sampling andplant species diversity significantly affected leaf-miner incidence(Table2)Specificallyleaf-minerincidencewas15timesgreaterinmidsummerthaninearlysummerand15timeshigherinmixturesthan inmonocultureplots (ie associational susceptibility) and13timeshigherincontrolthaninirrigatedplots(Figure3)
TherewasasignificanteffectoftheSeasontimesDiversitytimesIrrigation (χ2 = 1148 df = 1 plt001) and SeasontimesDiversitytimesNeighbour (χ2 = 1776 df = 3 p=006)three-waysinteractionsonleaf-minerin-cidencesuchthatearlyandmidseasondatawereanalysedseparately(Table1)Overall inbothseasonsleaf-minerincidencewaslowerinmonocultures intermediate in two-species mixtures and higher inthree-speciesmixtures (ie associational susceptibility)However indetailsthiseffectdependedonirrigationtreatmentasdemonstratedbythesignificantDiversitytimesIrrigation and NeighbourtimesIrrigationinter-actions(Table1)Inearlyseasonassociationalsusceptibilityonlyoc-curredinnon-irrigatedplotswhiletherewasnoeffectoftreediversityoridentityinirrigatedplots(Figure3)Atthecontraryinlateseasonassociationalsusceptibilityonlyoccurredinirrigatedwhileleaf-minerincidencedidnotdiffersignificantlyamongcompositiontreatmentsinnon-irrigatedplots(Figure3)
33emsp|emspPlant defensive traits
Tree species diversity (ie monocultures vs mixtures) and neigh-bour identity (ie birch monocultures birchndashoak birchndashpine andbirchndashoakndashpine mixtures) had qualitatively similar effects on leaftraits (TableS1) Concentration of total polyphenolics was signifi-cantly12-foldhigherintreesgrowinginmonoculturesthanintreesgrowing inmixtures (TableS1 FigureS5) Specifically the highestconcentration of total polyphenolics was found in trees growinginmonoculturesthe lowest intreesgrowinginthree-speciesmix-tures and intermediate in trees growing in two-species mixtures(Table2)Noneofothertraits individuallyrespondedtotreespe-cies diversity or neighbour identity (Table2 TableS1 FigureS5)
emspensp emsp | emsp2049Journal of EcologyCASTAGNEYROL ET AL
Starch concentrationwas 11-fold higher in trees growing in non-irrigated plots than in trees growing in irrigated plots (FigureS5Table2)Individuallynoneofothertraitsweresignificantlyaffectedbyirrigation(Table2)FinallyLDMCwastheonlytraitbeingsignifi-cantlyinfluencedbybirchapparencyandwassignificantlyhigherinmore apparent birches (slopeplusmnSE 41times10minus4plusmn11times10minus4 Table2FigureS5)indicatingthatleaveshadalowerwatercontentinmoreapparentthaninlessapparentbirches
There was a significant effect of tree species diversity andneighbour identity on PC1 (Table2 TableS1) with chemical
defences being greater in trees growing inmonocultures than intreesgrowinginanyothermixture(Figure4b)CoordinatesonPC1increased with tree apparency (Table2 FigureS5 slopeplusmnSE 36times10minus2plusmn15times10minus2) indicating that investment in chemicaldefencesincreasedasbirchesweremoreapparentDespiteaten-dencytowardshigherleafnutritionalquality(ieleaveswithhigherwatersugarstarchandnitrogencontent)intreesgrowinginnon-irrigatedplots(Figure4c)therewasnoeffectofbirchapparencyirrigation or tree species diversity or neighbour identity on PC2axis
F IGURE 2emspEffectsofseason(a)diversity(b)andplotspecificcomposition(c)andirrigationonleaf-chewerdamageIn(b)and(c)contrastsamongcompositionswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
0
2
4
6
8
10
12
Def
olia
tion
( L
AR
)
Earlyseason
Midseason
(a)
(96)
(96)0
2
4
6
8
10
12
Monocultures Mixtures
ControlIrrigated
(b)
a
b
(24) (24)(72)
(72) 0
2
4
6
8
10
12
B BQ BP BQP
(c)
a
bab
ab
(24) (24) (24) (24)(24) (24)
(24) (24)
TABLE 1emspSummaryofLMM(forleaf-chewers)andGLMM(forleaf-miners)testingtheeffectsofirrigationandtreespeciesdiversity(monoculturevsmixture)orneighbouridentity(birchoakpineoak+pine)onleaf-chewerdamageandleaf-minerincidence
Season Predictors
Leaf- chewers Leaf- miners
F (df) p- value R2m (R2
c) χ2 (df) p- value R2m (R2
c)
Early Irrigation 350(192) 065 014(014) 154(1) 215 005(007)
Diversity 004(192) 847 150(1) 220
IrrigationtimesDiversity 406(192) 047 1040(1) 001
Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)
Diversity 187(122) 185 426(1) 039
IrrigationtimesDiversity 508(122) 035 044(1) 509
Early Irrigation 1155(188) 001 020(020) 202(1) 156 005(007)
Neighbours 122(388) 306 597(3) 113
IrrigationtimesNeighbours 293(388) 038 1338(3) 004
Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)
Neighbours 159(321) 223 806(3) 045
IrrigationtimesNeighbours 208(318) 138 852(3) 036
LMMlinearmixedeffectmodelsGLMMGeneralizedlinearmixedeffectmodelsAlthoughforleaf-chewersthethree-waysSeason times Irrigation times DiversityandSeason times Irrigation times Neighboursinteractionswerenotsignificantwereportresultsforearlyandmidseasonseparatelytobeconsistentwithleaf-minersandtoshowthattheinteractiveeffectsofirrigationandneighbourdiversityoridentitywereconsistentacrossseasonsBoldcharac-tersindicatesignificantpredictorsatα = 005 R2werereportedforthesimplifiedmodel(ieaftertheinteractionwasremovedifnotsignificant)R2
m and R2
ccorrespondtomarginal(ieaccountingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
Early season
ControlIrrigated
Monoculture Mixture
Mid season
B BQ BP BQP
a
ab b
b
Composition
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
ControlIrrigated
B BQ BP BQP
A AB
AB
B
Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
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HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
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JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
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KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
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abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
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KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
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Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
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Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
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Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
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SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
emspensp emsp | emsp2049Journal of EcologyCASTAGNEYROL ET AL
Starch concentrationwas 11-fold higher in trees growing in non-irrigated plots than in trees growing in irrigated plots (FigureS5Table2)Individuallynoneofothertraitsweresignificantlyaffectedbyirrigation(Table2)FinallyLDMCwastheonlytraitbeingsignifi-cantlyinfluencedbybirchapparencyandwassignificantlyhigherinmore apparent birches (slopeplusmnSE 41times10minus4plusmn11times10minus4 Table2FigureS5)indicatingthatleaveshadalowerwatercontentinmoreapparentthaninlessapparentbirches
There was a significant effect of tree species diversity andneighbour identity on PC1 (Table2 TableS1) with chemical
defences being greater in trees growing inmonocultures than intreesgrowinginanyothermixture(Figure4b)CoordinatesonPC1increased with tree apparency (Table2 FigureS5 slopeplusmnSE 36times10minus2plusmn15times10minus2) indicating that investment in chemicaldefencesincreasedasbirchesweremoreapparentDespiteaten-dencytowardshigherleafnutritionalquality(ieleaveswithhigherwatersugarstarchandnitrogencontent)intreesgrowinginnon-irrigatedplots(Figure4c)therewasnoeffectofbirchapparencyirrigation or tree species diversity or neighbour identity on PC2axis
F IGURE 2emspEffectsofseason(a)diversity(b)andplotspecificcomposition(c)andirrigationonleaf-chewerdamageIn(b)and(c)contrastsamongcompositionswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
0
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Def
olia
tion
( L
AR
)
Earlyseason
Midseason
(a)
(96)
(96)0
2
4
6
8
10
12
Monocultures Mixtures
ControlIrrigated
(b)
a
b
(24) (24)(72)
(72) 0
2
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6
8
10
12
B BQ BP BQP
(c)
a
bab
ab
(24) (24) (24) (24)(24) (24)
(24) (24)
TABLE 1emspSummaryofLMM(forleaf-chewers)andGLMM(forleaf-miners)testingtheeffectsofirrigationandtreespeciesdiversity(monoculturevsmixture)orneighbouridentity(birchoakpineoak+pine)onleaf-chewerdamageandleaf-minerincidence
Season Predictors
Leaf- chewers Leaf- miners
F (df) p- value R2m (R2
c) χ2 (df) p- value R2m (R2
c)
Early Irrigation 350(192) 065 014(014) 154(1) 215 005(007)
Diversity 004(192) 847 150(1) 220
IrrigationtimesDiversity 406(192) 047 1040(1) 001
Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)
Diversity 187(122) 185 426(1) 039
IrrigationtimesDiversity 508(122) 035 044(1) 509
Early Irrigation 1155(188) 001 020(020) 202(1) 156 005(007)
Neighbours 122(388) 306 597(3) 113
IrrigationtimesNeighbours 293(388) 038 1338(3) 004
Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)
Neighbours 159(321) 223 806(3) 045
IrrigationtimesNeighbours 208(318) 138 852(3) 036
LMMlinearmixedeffectmodelsGLMMGeneralizedlinearmixedeffectmodelsAlthoughforleaf-chewersthethree-waysSeason times Irrigation times DiversityandSeason times Irrigation times Neighboursinteractionswerenotsignificantwereportresultsforearlyandmidseasonseparatelytobeconsistentwithleaf-minersandtoshowthattheinteractiveeffectsofirrigationandneighbourdiversityoridentitywereconsistentacrossseasonsBoldcharac-tersindicatesignificantpredictorsatα = 005 R2werereportedforthesimplifiedmodel(ieaftertheinteractionwasremovedifnotsignificant)R2
m and R2
ccorrespondtomarginal(ieaccountingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
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20
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Early season
ControlIrrigated
Monoculture Mixture
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B BQ BP BQP
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minusmin
er in
cide
nce
()
0
5
10
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B BQ BP BQP
A AB
AB
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Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
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PC1
PC
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QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
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PC1
PC
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QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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Altman N amp KrzywinskiM (2015) Points of significance Split plotdesign Nature Methods 12 165ndash166 httpsdoiorg101038nmeth3293
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
AtsattPRampOrsquodowdDJ (1976)PlantdefenseguildsScience 193 24ndash29httpsdoiorg101126science193424724
Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
BantildeuelosM-JampKollmannJ (2011)Effectsofhost-plantpopulationsizeandplantsexonaspecialistleaf-minerActa Oecologica 37 58ndash64httpsdoiorg101016jactao201011007
Barbosa P Hines J Kaplan I Martinson H Szczepaniec A ampSzendreiZ (2009)Associational resistanceandassociational sus-ceptibilityHavingrightorwrongneighborsAnnual Review of Ecology Evolution and Systematics 401ndash20httpsdoiorg101146annurevecolsys110308120242
BartońK(2016)MuMIn Multi-model inferenceRetrievedfromhttpR-ForgeR-projectorgprojectsmumin
BartonKEValkamaEVehvilaumlinenHRuohomaumlkiKKnightTMampKorichevaJ(2014)Additiveandnon-additiveeffectsofbirchge-notypicdiversityonarthropodherbivoryinalong-termfieldexperi-mentOikos 124 697ndash706
BernaysEBrightKGonzalezNampAngelJ(1994)DietarymixinginageneralistherbivoreTestsoftwohypothesesEcology 75 1997ndash2006httpsdoiorg1023071941604
Callaway R M amp Walker L R (1997) Competition and facilita-tion A synthetic approach to interactions in plant communitiesEcology 78 1958ndash1965 httpsdoiorg1018900012-9658(1997) 078[1958CAFASA]20CO2
CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
2050emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
Response Predictor F- value (df) p- value R2m (R2
c)
LDMC Apparency 1367(1341) 001 015(032)
Neighbours 084(3261) 485
Irrigation 081(158) 404
ApparencytimesIrrigation 103(1335) 318
NeighbourstimesIrrigation 051(3234) 676
Totalpolyphenolics Apparency 233(179) 131 012(037)
Neighbours 420(3178) 021
Irrigation 049(158) 513
ApparencytimesIrrigation 038(1745) 538
NeighbourstimesIrrigation 086(3712) 466
Condensedtannins Apparency 348(1788) 066 minus(039)
Neighbours 223(318) 120
Irrigation 051(156) 505
ApparencytimesIrrigation 018(1271) 672
NeighbourstimesIrrigation 031(3207) 819
CN Apparency 202(1564) 160 minus(06)
Neighbours 103(3266) 395
Irrigation 126(16) 304
ApparencytimesIrrigation 078(166) 379
NeighbourstimesIrrigation 112(3171) 367
Starch Apparency 064(1755) 426 008(046)
Neighbours 088(3274) 462
Irrigation 430(1304) 047
ApparencytimesIrrigation 027(1748) 604
NeighbourstimesIrrigation 282(3242) 060
Sugars Apparency 142(1761) 238 minus(032)
Neighbours 104(3218) 394
Irrigation 016(16) 702
ApparencytimesIrrigation 005(1751) 825
NeighbourstimesIrrigation 195(3184) 157
PC1 Apparency 536(1796) 023 020(038)
Neighbours 345(325) 032
Irrigation 085(155) 395
ApparencytimesIrrigation 030(1293) 591
NeighbourstimesIrrigation 066(3225) 586
PC2 Apparency lt001(1734) 978 minus(032)
Neighbours 016(3206) 923
Irrigation 392(161) 094
ApparencytimesIrrigation 118(1732) 281
NeighbourstimesIrrigation 174(3177) 196
F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2
m and R2c correspond tomarginal (ieac-
countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2
mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor
TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
Early season
ControlIrrigated
Monoculture Mixture
Mid season
B BQ BP BQP
a
ab b
b
Composition
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
ControlIrrigated
B BQ BP BQP
A AB
AB
B
Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
Abdala-Roberts L Hernandez-Cumplido J Chel-Guerrero LBetancur-Ancona D Benrey B ampMoreira X (2016) Effects ofplant intraspecificdiversity across three trophic levelsUnderlyingmechanismsandplanttraitsAmerican Journal of Botany 103 1810ndash1818httpsdoiorg103732ajb1600234
Altman N amp KrzywinskiM (2015) Points of significance Split plotdesign Nature Methods 12 165ndash166 httpsdoiorg101038nmeth3293
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
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Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
BantildeuelosM-JampKollmannJ (2011)Effectsofhost-plantpopulationsizeandplantsexonaspecialistleaf-minerActa Oecologica 37 58ndash64httpsdoiorg101016jactao201011007
Barbosa P Hines J Kaplan I Martinson H Szczepaniec A ampSzendreiZ (2009)Associational resistanceandassociational sus-ceptibilityHavingrightorwrongneighborsAnnual Review of Ecology Evolution and Systematics 401ndash20httpsdoiorg101146annurevecolsys110308120242
BartońK(2016)MuMIn Multi-model inferenceRetrievedfromhttpR-ForgeR-projectorgprojectsmumin
BartonKEValkamaEVehvilaumlinenHRuohomaumlkiKKnightTMampKorichevaJ(2014)Additiveandnon-additiveeffectsofbirchge-notypicdiversityonarthropodherbivoryinalong-termfieldexperi-mentOikos 124 697ndash706
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CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
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Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
emspensp emsp | emsp2051Journal of EcologyCASTAGNEYROL ET AL
34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory
341emsp|emspLeaf-chewer damage
The effects of irrigation plant diversity or neighbour identityand their interaction on leaf chewer damage remained significant
(IrrigationtimesDiversity F122 = 508 p = 035 IrrigationtimesNeighbours F3755 = 554 p=002) after including birch apparency and plant de-fensivetraits(PC1andPC2axes)ascovariatesinthestatisticalmodelLeaf-chewerdamageincreasedwithcoordinatesonPC2axis(slopeplusmnSE 015plusmn006) suggesting greater consumption of leaves of low qual-ity This effect was however significant only in non-irrigated plots(PC2timesIrrigation F18094 = 586 p=017FigureS6)Birchapparencyhad
F IGURE 3emspEffectsofirrigationandtreespeciesdiversity(ab)orcomposition(cd)onleaf-minerincidenceinearly(ac)andmidseason(bd)ContrastsamongdiversityandcompositionlevelswereestimatedforeachlevelofirrigationseparatelySamelettersabovebarsindicatenon-significantdifferencesamongcompositionsLowercaseanduppercaselettersarefornon-irrigatedandirrigatedtreatmentsrespectivelyTheeffectofirrigationwastestedwithineachlevelofdiversityandcompositionseparatelyanditssignificanceisindicatedbystarsLegendsarethesameasinFigure1
Monoculture Mixture
a
b
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
Early season
ControlIrrigated
Monoculture Mixture
Mid season
B BQ BP BQP
a
ab b
b
Composition
Leaf
minusmin
er in
cide
nce
()
0
5
10
15
20
25
ControlIrrigated
B BQ BP BQP
A AB
AB
B
Composition
F IGURE 4emspMultivariateanalysisofleaftraits(a)CorrelationcircleshowingcorrelationsamongleaftraitsandbetweenleaftraitsandPCaxes(bc)ProjectionsofindividualtreesonPCaxesaccordingto(b)treespeciescompositionand(c)irrigationtreatmentThickgreyarrowspointtowardsgreaterleafquality(ielowerdefencesandgreaternutritionalquality)[Colourfigurecanbeviewedatwileyonlinelibrarycom]
minus10 minus05 00 05 10
minus10
minus05
00
05
10
PC1
PC
2
LDMC
Polyphenolics
Tannins
Starch
Sugars
CN
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
BBQ
BPBQP
minus4 minus2 0 2 4
minus3
minus2
minus1
0
1
2
PC1
PC
2
QUALITY
Control
Irrigated
(b)(a) (c)
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
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KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
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Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
2052emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency
342emsp|emspLeaf- miner incidence
Theeffectsofirrigationplantdiversityandtheirinteractiononleaf-miner incidence remained significant after including birch appar-encyandplantdefensivetraitsascovariatesinthestatisticalmodel(χ2 = 484 df = 1 p=028)withgreaterleaf-minerincidenceinmix-turesthaninmonocultures(Figure3)Thissuggeststhateffectsofirrigationandtreespeciesdiversityonleaf-minerincidencewerenotmediatedbythestudiedplanttraitsnorbytreeapparency
Althoughneitherleaftraits(PC1χ2 = 004 df = 1 p = 839 PC2 χ2 = 006 df = 1 p=799) nor birch apparency (χ2 = 011 df = 1 p=741)hadasignificanteffectonleaf-minerincidencetheeffectoftreeneighbouridentitywasnolongersignificant(χ2 = 631 df = 3 p=097)whenintroduced inthesamemodelsuggestingthat leaftraitsandbirchapparencycouldpartiallyaccountforsomepartofvariabilityinleaf-minerincidenceamongtreespeciescompositions
4emsp |emspDISCUSSION
Despitedecadesofindependentinvestigationsonplantdiversityordroughteffectsoninsectherbivoryresultsremaininconsistentandacomprehensiveunderstandingofmechanismsatplayisstillmiss-ing In this senseour study reveals thatpartof such inconsisten-ciesmay result from theoverlookedeffectsof abiotic constraintsontreediversityeffectsandviceversa(seeFigure5foragraphicalsummaryofourresults)Inparticularweshowthatinsectherbivorywashigheramongheterospecificneighboursthanamongconspecif-icswhichcorrespondstoanassociationalsusceptibilityeffectbutthesepatternswerecontingentuponabioticenvironmentandoc-curredonlyinwater-stressedtrees
Our results revealed that the interactive effect of neighboursanddroughtoninsectherbivorywaspartlyinfluencedbyneighbour-mediated changes in plant nutritional quality and production ofconstitutivedefencesThisresult isconsistentwithpreviousstud-ies showing thatplant ability todefendagainstherbivores canbeaffectedbyabioticconditionssuchaswaterornutrientavailability(Herms amp Mattson 1992 Jactel etal 2012 Walter etal 2011White 1974) andwithmore recent studies reporting that the di-versityandidentityofplantrsquosneighbourscanmodifyitsproductionofanti-herbivoredefences(Kostenkoetal2017MoreiraGlauserampAbdala-Roberts2017Moreiraetal2014Mrajaetal2011)Itisthuspossiblethatthestrengthanddirectionofassociationalef-fects ismodulatedbyabioticfactors(Kambachetal2016)partlybecausebothcontributetocontrolleafnutritionalqualityandanti-herbivoredefences
Insect herbivory on birch was greater among heterospecificneighbours(ieassociationalsusceptibilityWhiteampWhitham2000Barbosaetal2009)Threeclassicalmechanismshavebeenproposedtoexplainassociationalsusceptibilitysuchas(1)thespill-overofher-bivores fromneighbouringspeciesontobirches (WhiteampWhitham2000)(2)thebiasedlandingrateofherbivoresonthemostapparenthosttrees (Hambaumlcketal2014) (3) theconcentrationofspecialistherbivores on the fewer birches available in mixtures (Bantildeuelos ampKollmann2011Damienetal2016OtwayHectorampLawton2005)oracombinationofthesethreemechanismsBecauseourtreediver-sityexperimentfollowsareplacementdesignbirchconcentration(ienumber of individuals per plot) is correlatedwith its frequency (ierelativenumberofbirchesandassociatedspecies)Pureassociationaleffectscannotbethereforeseparatedfromconcentrationanddilutioneffects(Damienetal2016)Allthesehypothesesassumethatassoci-ationalsusceptibilityresultsfromdifferentialcolonizationofhosttreesamongdifferentneighboursAlternatively (4) thedietarymixinghy-pothesis(BernaysBrightGonzalezampAngel1994HaumlgeleampRowell-Rahier1999)proposesthatassociationalsusceptibilitycouldbeduetogeneralistherbivoresbenefitingfromfeedingonvariousresources
F IGURE 5emspSummaryofresultsEachpathwayreferstoaspecificfigureortablePathwaysrepresentourinitialhypothesesBlackarrowsrepresentstrongsupportofourresultstothepathwaywhereasgreysolidanddashedarrowsrepresentpartialandabsenceofsupportrespectivelySunsindicatethatthecorrespondingpathwaywasonlyobservedinstressedtree
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
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CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
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abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
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emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
emspensp emsp | emsp2053Journal of EcologyCASTAGNEYROL ET AL
differinginnutritionalqualityorlevelsoftoxicity(LefcheckWhalenDavenport StoneampDuffy 2013) Previous studies using the sameexperimental design suggested that tree apparency and host con-centrationhypotheseswerethemainfactorsdrivinginsectherbivory(Castagneyroletal20132017Damienetal2016)Howeverwhiletheapparencyhypothesiscouldexplainthegreaterherbivoryinbirchndashoakmixtures(birchesweretallerthanoaks)itcannotaccountforher-bivory in birchndashpinemixtureswhere birch treeswere less apparent(birchessmallerthanpines)thaninpurebirchstandsOurresultsdonotsupporteitherthedietarymixinghypothesisasbothleaf-chewers(likely consisting in both generalists and specialists) and leafminers(mostlyspecialistspecies)similarlyrespondedtotreediversitytreat-mentsConsequentlynoneoftheabovementionedhypothesescouldindividuallyexplainourobservedpatterns
Bycontrastourfindingspartlysupportafifthalternativehypoth-esis (5) associational effects would result from neighbour-inducedchanges in leaf chemistry In particular birch leaves had greateramounts of constitutive defences in species monocultures than inspecies mixtures and this led to concomitant patterns of reducedherbivore damage in monocultures for both chewers and minersHoweveronceothersourcesofvariation(namelytreeapparencyandneighbour diversity and identity)were controlled for anti-herbivoredefenceshadnosignificanteffectonherbivoryBecauseneighbouridentity and diversity partially modified leaf chemistry this resultsuggeststhatbyitselfleafchemistryonlyexplainsasmallamountofvariation inherbivoryand thatotherunmeasured traits (egvolatileorganic compounds induced defences Zakir etal 2013) or otherprocessesindependentofleaftraits(egtopndashdowncontrolbypreda-torsEsquivel-GoacutemezAbdala-RobertsPinkus-RendoacutenampParra-Tabla2017 Moreira Mooney etal 2012 Muiruri Rainio amp Koricheva2016)couldaccountfortheeffectofneighbourdiversityandidentityoninsectherbivoryThisisinaccordancewiththeresultsofaprevi-ous study using the same experimental design but focusing onoak(Q roburCastagneyroletal2017)whichshowedthatdespitecleareffectsoftreeneighboursonleaftraitstraitspoorlyexplainedvaria-tioninleafinsectherbivory(Castagneyroletal2017)
Thereisgrowingevidencethatbelow-andabove-groundinter-actionsamongplantscanaltertheexpressionofdirectandindirect(ieenemy-mediated)anti-herbivoredefencesandtraitsdeterminingleafnutritionalquality(Glassmireetal2016Kostenkoetal2017Moreiraetal20142017Mrajaetal2011Nitschkeetal2017Walteretal2011)Howeverwhetherneighbour-inducedchangesinleafchemistrymediatesassociationaleffectsonlyreceivedpartialsupportintheliteratureForinstanceMoreiraetal(2014)reportedthatgreaterproductionofanti-herbivorechemicaldefencesinmix-turesof tree speciesandgenotypesofmahogany (comparedwithmonocultures)didnot lead to concomitantpatternsof insecther-bivoryLikewiseKostenkoetal(2017)reportedthattheeffectsofneighboursonarthropodsassociatedtoJacobaea vulgaris were inde-pendentofchangesinplantchemistryContrarilyneighbourdiver-sityhadindirectpositiveeffectsonherbivorediversityviareducedinvestment in anti-herbivore defences on pepper trees (Glassmireetal 2016) By addressing the direct- and indirect-traitmediated
effectsofneighboursourresultsaddtothegrowingbodyofknowl-edgeonmechanismscontrollingforinsectdamageonplants
Insects causedmore damage in stressful conditions indepen-dentofleafchemistryOurresultsshowedthatdefoliationincreasedindrought-stressedplantsThesefindingsarepartiallyinaccordancewiththeplantstresshypothesiswhichpredictsgreaterperformanceoffoliage-feedingherbivoresinstressedplants(Jacteletal2012White1974)Howeverbecauseirrigationhadnoeffectonindivid-ualleaftraits(exceptedstarchcontent)orcoordinatesonPCaxesourresultsdonotsupportthepremiseoftheplantstresshypothesisthatincreasedherbivoryinstressedplantsresultsfromchangesinleaf chemistry and in particular from the increased concentrationof soluble sugars and secondary metabolites (Walter etal 2011White1974Ximeacutenez-Embuacutenetal2016)Wecannotexcludethatotherunmeasuredtraitssuchasconcentrationoffreeaminoacidsinducedsecondarymetabolitesoremissionofvolatileorganiccom-poundsmayhavebeenthecausallinkbetweendroughtandinsectherbivoryForinstanceGutbrodtDornandMody(2011)reportedthatdroughthadnoeffectoninduceddefencesinappletreeswhileconstitutivedefenceswere reduced inmoderately stressedplantsand increased inhighlystressedplantsBecauseweonly targetedundamagedleavesfortraitanalyseswearenotabletoteaseaparttheresponseofconstitutivevsinduceddefences
AssociationaleffectswerecontingentuponwaterstressLeaf-chewingdamageandleaf-minersincidenceinearlyseasonwereonaverage higher in mixtures than in monocultures but only understressful conditions Importantly this pattern remained unalteredwhenleaftraitswereincludedinthestatisticalmodelItisimport-ant to note thatwewere able to detect significant effects of theinteractionbetweentreespeciesdiversityandwatertreatmentsonherbivorywithourrathersmallsamplesize(N=96)suggestingthattheseeffectswerestrong
The state ofORPHEE experiment at the timewe performed thisstudywassuchthatbirchconcentrationfrequencyandapparency(sensuCastagneyroletal2017)werecomparablebetweenirrigatedandnon-irrigatedplotswhichmakesitunlikelythatprocessesrelatedtobirchac-cessibilitycouldexplaintheobservedinteractionbetweendroughtandtreediversityonherbivoryItispossiblethatdroughtincreasedspecies-specificdifferencesinplantcues(egemissionofvolatileorganiccom-pounds)usedbyherbivorestolocateasuitablehostwithinexperimentalplots (including trees andunderstoryvegetation)Thiswould result instrongerrelativeattractivenessofthefewerbirchesinnon-irrigatedmix-turesascomparedtomonocultures(Hambaumlcketal2014)
Becausetheirrigationtreatmentwasinitiatedonly1yearbeforeourmeasurementsacriticalgapofthisstudywouldbethatwecannotaddresstemporaldynamics intheassociationaleffects Inparticularand although the irrigation treatment affects insect herbivory (thisstudy) avian predation (Castagneyrol etal 2017) and understoreyvegetation(BCastagneyrolunpubldata)itmaytakelongertohavedetectableeffectsontreeheightandapparencyHereweshowthatmoreapparentbircheshadleaveswithlowerquality(lowerwatercon-tenthigherlevelsofanti-herbivoredefencesFigureS5)Yetgiventheknowntrade-offsbetweengrowthanddefences(HermsampMattson
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
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GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
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HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
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KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
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abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
2054emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
1992)itispossiblethattherelationshipbetweendefencetraitsandapparencywillbeaffectedbyirrigationtreatmentsinthelongterm
Finallyitisimportanttonotethatwedidnotcontrolwaterstressdirectly the irrigation treatment aimed at alleviating water stresscausedbysummerdroughtHencethedifferenceinwaterstressin-tensitybetween irrigatedandnon-irrigatedplots is thereforehighlydependentonclimaticconditionsYetsummer2016wasoneofthedriestoverthelast60yearsandweprovidedseverallinesofevidencethat the experimental set up created two contrasted situations intermsofwateravailabilityImportantlydroughtalleviationwasinde-pendentoftreespeciescompositionandaffectednotonlybirchbutalsooaksandpinesplantedintheORPHEEexperiment(Castagneyrolunpublisheddata)Thusweareconfidentthatourresultsdidshowtheinteractiveeffectsofwaterstressandneighbourson leaftraitsandinsectherbivoryHoweverthereisgrowingevidencethattheeffectsofwaterstressonherbivoryaremorecomplexthanthatinitiallysug-gestedbytheplantstresshypothesisInparticularstressfrequency(Banfield-ZaninampLeather2014HubertyampDenno2004)andstressintensity (Mody Eichenberger amp Dorn 2009 Sconiers amp Eubanks2017)emergedaskeydriversofinsectresponseItwillbepossibletotesttheseeffectswithourexperimentinthefuturebycomparingtherelativestrengthofdiversityandirrigationeffectsoninsectherbivoryacrossyearsvaryingintheamountofsummerrainfalls
5emsp |emspCONCLUSIONS AND PERSPEC TIVES
Alongheldviewinecologyholdsthatinsectherbivoryonagivenplantdependsonthediversityandidentityofitsneighbours(Atsattamp Orsquodowd 1976) However despite dozens of empirical and ex-perimental studies reporting evidence for associational resistanceandsusceptibility(reviewedbyBarbosaetal2009Moreiraetal2016)predictingthestrengthanddirectionofassociationaleffectsremains challengingHerebydemonstrating thatassociationalef-fects are contingent upon abiotic constraints we bring anotherdegree of complexity into our understanding of the mechanismscontrollingforinsectherbivoryamongdifferentneighbours
Altogether our results suggest that neighbours have indirecttrait-mediated effects on insect herbivory but neighboursrsquo effectsdifferentially affect two key components of leaf chemistry namelyleafnutritionalqualityandanti-herbivoredefencesImportantlytrait-mediatedeffectsonlypartiallyaccountedforthevariability in insectherbivory on trees among different neighbours suggesting that un-measuredfactorsotherthanchangesinleafqualityorhostavailability(ieconcentrationfrequency)andapparencymayhavecontributedtoexplaintheeffectofneighbourdiversityandidentityonleafherbivory
While previous studies on birch reported greater resistanceto insects inmixed forest (MuiruriMilliganMorathampKoricheva2015 SetiawanVanhellemont BaetenDillenampVerheyen 2014VehvilaumlinenKorichevaRuohomaumlkiJohanssonampValkonen2006)wefoundtheoppositepatternYettheORPHEEexperimentislo-cated at the South-Westmargin ofB pendula geographical rangewhere it is more likely to suffer from summer heat and drought
(Atkinson1992)Togetherwithrecentstudies (Haaseetal2015Kambachetal2016)ourresultschallengetheviewthatassocia-tionalresistancetoinsectherbivoresisapervasivephenomenoninforestecosystemsAtthecontrarytheywarnthatabioticstresses(eg drought temperature Kambach etal 2016 Ratcliffe etal2017)are likelymodifiersofassociationaleffectsthatmustbeac-countedfortobetteranticipatethefutureofplantndashplantndashinsectin-teractionsinthefaceofclimatechange
ACKNOWLEDG EMENTS
The research inORPHEEwas funded by the GIP-ECOFOR pro-gramme from theFrenchMinistryofEcology under theprojectBIOPICCECOFOR-2014ndash15CollaborationbetweenBCandXMwaspermittedbyagrantfromthe iLINK+CSICProgram(I-LINK1221)We thank CeacutelineMeredieu for helpful comments on cli-maticandgrowthdataWealsothanktwoanonymousreviewersfortheirinsightfulcommentsonearlierversionofthismanuscriptWearegrateful toall peoplewhohelped in the fieldand in thelaboratory Angelina Ceballos-Escalera Martine Martin-ClotteacuteEdithReuzeauBegontildeaGarrido-DiazSilvanaPoceiroandPatriciaToledo for their help with field and plant trait analyses CeacutelineMeredieu IngevanHalderRaphaeumllSeacuteguraandFabriceVeacutetillardforhavingmeasuredwaterpotentialsoearlyinthemorningTheORPHEE experiment is managed by INRA Experimental UnitForecirct-PierrotonandinparticularbyBernardIssenhuthThanks
AUTHORSrsquo CONTRIBUTIONS
BCandHJconceivedtheexperimentBCandXMacquiredthedata BC analysed the data andwrote the first draft whichwascommentededitedandimprovedbyHJandXM
DATA ACCE SSIBILIT Y
Data available from the Dryad Digital Repository httpsdoiorg105061dryad73md8h4(Castagneyrol2018)
ORCID
Bastien Castagneyrol httporcidorg0000-0001-8795-7806
Xoaquiacuten Moreira httporcidorg0000-0003-0166-838X
R E FE R E N C E S
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Altman N amp KrzywinskiM (2015) Points of significance Split plotdesign Nature Methods 12 165ndash166 httpsdoiorg101038nmeth3293
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
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Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
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CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
emspensp emsp | emsp2055Journal of EcologyCASTAGNEYROL ET AL
Atkinson M D (1992) Betula pendula Roth (B Verrucosa Ehrh) andB pubescens Ehrh Journal of Ecology 80 837ndash870 httpsdoiorg1023072260870
AtsattPRampOrsquodowdDJ (1976)PlantdefenseguildsScience 193 24ndash29httpsdoiorg101126science193424724
Banfield-ZaninJAampLeatherSR(2014)FrequencyandintensityofdroughtstressaltersthepopulationsizeanddynamicsofElatobium abietinumonSitka spruceAnnals of Applied Biology 165 260ndash269 httpsdoiorg101111aab12133
BantildeuelosM-JampKollmannJ (2011)Effectsofhost-plantpopulationsizeandplantsexonaspecialistleaf-minerActa Oecologica 37 58ndash64httpsdoiorg101016jactao201011007
Barbosa P Hines J Kaplan I Martinson H Szczepaniec A ampSzendreiZ (2009)Associational resistanceandassociational sus-ceptibilityHavingrightorwrongneighborsAnnual Review of Ecology Evolution and Systematics 401ndash20httpsdoiorg101146annurevecolsys110308120242
BartońK(2016)MuMIn Multi-model inferenceRetrievedfromhttpR-ForgeR-projectorgprojectsmumin
BartonKEValkamaEVehvilaumlinenHRuohomaumlkiKKnightTMampKorichevaJ(2014)Additiveandnon-additiveeffectsofbirchge-notypicdiversityonarthropodherbivoryinalong-termfieldexperi-mentOikos 124 697ndash706
BernaysEBrightKGonzalezNampAngelJ(1994)DietarymixinginageneralistherbivoreTestsoftwohypothesesEcology 75 1997ndash2006httpsdoiorg1023071941604
Callaway R M amp Walker L R (1997) Competition and facilita-tion A synthetic approach to interactions in plant communitiesEcology 78 1958ndash1965 httpsdoiorg1018900012-9658(1997) 078[1958CAFASA]20CO2
CardinaleB JMatulichK LHooperDUByrnes JEDuffyEGamfeldtLhellipGonzalezA(2011)Thefunctionalroleofproducerdiversity in ecosystems American Journal of Botany 98 572ndash592 httpsdoiorg103732ajb1000364
CastagneyrolB(2018)DatafromAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursDryad Digital Repositoryhttpsdoiorg105061dryad73md8h4
CastagneyrolBBonalDDamienMJactelHMeredieuCMuiruriEWampBarbaroL(2017)Bottom-upandtop-downeffectsoftreespecies diversity on leaf insect herbivoryEcology and Evolution 7 3520ndash3531httpsdoiorg101002ece32950
Castagneyrol B Giffard B Peacutereacute C amp Jactel H (2013) Plant ap-parency an overlooked driver of associational resistance to in-sect herbivory Journal of Ecology 101 418ndash429 httpsdoiorg1011111365-274512055
Damien M Jactel H Meredieu C Reacutegolini M van Halder I ampCastagneyrolB(2016)PestdamageinmixedforestsDisentanglingtheeffectsofneighboridentityhostdensityandhostapparencyatdifferent spatial scalesForest Ecology and Management 378 103ndash110httpsdoiorg101016jforeco201607025
Esquivel-Goacutemez L Abdala-Roberts L Pinkus-Rendoacuten M amp Parra-TablaV(2017)EffectsoftreespeciesdiversityonacommunityofweaverspidersinatropicalforestplantationBiotropica 49 63ndash70 httpsdoiorg101111btp12352
Forey E Langlois E Lapa G Korboulewsky N Robson T M ampAubertM(2016)Treespeciesrichnessinducesstrongintraspecificvariabilityofbeech(Fagus sylvatica)leaftraitsandalleviatesedaphicstressEuropean Journal of Forest Research 135707ndash717httpsdoiorg101007s10342-016-0966-7
Forkner R E Marquis R J amp Lill J T (2004) Feeny revisitedCondensedtanninsasanti-herbivoredefencesin leaf-chewingher-bivorecommunitiesofQuercus Ecological Entomology 29 174ndash187 httpsdoiorg101111j1365-231120040590x
ForresterDITheiveyanathanSCollopyJJampMarcarNE(2010)Enhancedwater use efficiency in amixed Eucalyptus globulus and
Acacia mearnsii plantation Forest Ecology and Management 259 1761ndash1770httpsdoiorg101016jforeco200907036
Fox JWeisberg SAdlerDBatesDBaud-BovyG Ellison ShellipR-Core (2016) Car Companion to applied regression Retrievedfromhttpsmranmicrosoftcompackagecar
GlassmireAEJeffreyCSForisterMLParchmanTLNiceCC Jahner J P hellip Dyer L A (2016) Intraspecific phytochemicalvariationshapescommunityandpopulationstructureforspecialistcaterpillarsNew Phytologist 212208ndash219httpsdoiorg101111nph14038
GoodenoughAEampGoodenoughAS(2012)Developmentofarapidand precise method of digital image analysis to quantify canopydensity and structural complexity International Scholarly Research Notices 2012 e619842
Grettenberger I M amp Tooker J F (2016) Inter-varietal interactionsamong plants in genotypically diverse mixtures tend to decreaseherbivore performance Oecologia 182 189ndash202 httpsdoiorg101007s00442-016-3651-0
GutbrodtBDornSampModyK (2011)Droughtstressaffectscon-stitutive but not induced herbivore resistance in apple plantsArthropod- Plant Interactions 6 171ndash179
HaaseJCastagneyrolBCornelissenJHCGhazoulJKattgeJKorichevaJhellipJactelH(2015)Contrastingeffectsoftreediver-sityonyoungtreegrowthandresistancetoinsectherbivoresacrossthreebiodiversityexperimentsOikos 1241674ndash1685httpsdoiorg101111oik02090
HaumlgeleBFampRowell-RahierM (1999)Dietarymixing in threegen-eralist herbivores Nutrient complementation or toxin dilutionOecologia 119 521ndash533
HambaumlckPA InouyeBDAnderssonPampUnderwoodN (2014)Effects of plant neighborhoods on plantndashherbivore interactionsResourcedilutionandassociationaleffectsEcology 95 1370ndash1383 httpsdoiorg10189013-07931
Hansen J amp Moslashller I (1975) Percolation of starch and soluble car-bohydrates from plant tissue for quantitative determinationwith anthrone Analytical Biochemistry 68 87ndash94 httpsdoiorg1010160003-2697(75)90682-X
HermsDAampMattsonWJ(1992)ThedilemmaofplantsTogrowordefendThe Quarterly Review of Biology 67283ndash335httpsdoiorg101086417659
HothornTBretzFWestfallPHeibergerRMSchuetzenmeisterAampScheibeS(2016)Multcomp Simultaneous inference in general parametric models Retrieved from httpscranr-projectorgwebpackagesmultcompindexhtml
HubertyAFampDennoRF(2004)Plantwaterstressanditsconse-quencesforherbivorousinsectsAnewsynthesisEcology 85 1383ndash1398httpsdoiorg10189003-0352
Jactel H Birgersson G Andersson S amp Schlyter F (2011) Non-host volatilesmediate associational resistance to the pine proces-sionary moth Oecologia 166 703ndash711 httpsdoiorg101007s00442-011-1918-z
JactelHPetitJDesprez-LoustauM-LDelzonSPiouDBattistiAampKorichevaJ(2012)Droughteffectsondamagebyforestin-sects and pathogens A meta-analysis Global Change Biology 18 267ndash276httpsdoiorg101111j1365-2486201102512x
JohnsonMTJBertrandJAampTurcotteMM(2016)Precisionandaccuracy in quantifying herbivory Ecological Entomology 41 112ndash121httpsdoiorg101111een12280
KambachSKuumlhnICastagneyrolBampBruelheideH(2016)Theim-pactoftreediversityondifferentaspectsofinsectherbivoryalonga global temperature gradient ndash A meta-analysis PLoS ONE 11 e0165815httpsdoiorg101371journalpone0165815
KlausVHHoumllzelNPratiDSchmittBSchoumlningISchrumpfMhellipKleinebeckerT(2016)PlantdiversitymoderatesdroughtstressingrasslandsImplicationsfromalargereal-worldstudyon13Cnatural
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
2056emsp |emsp emspenspJournal of Ecology CASTAGNEYROL ET AL
abundances Science of the Total Environment 566ndash567 215ndash222 httpsdoiorg101016jscitotenv201605008
Kos M Bukovinszky T Mulder P P J amp Bezemer T M (2015)Disentangling above- and below-ground neighbor effects on thegrowth chemistry and arthropod community on a focal plantEcology 96164ndash175httpsdoiorg10189014-05631
KostenkoOMulderPPJCourboisMampBezemerTM (2017)Effects of plant diversity on the concentration of second-ary plant metabolites and the density of arthropods on focalplants in the field Journal of Ecology 105 647ndash660 httpsdoiorg1011111365-274512700
KozlovMVSkorackaAZverevVLewandowskiMampZverevaE L (2016) Two birch species demonstrate opposite latitudinalpatterns in infestation by gall-makingmites inNorthernEuropePLoS ONE 11 e0166641 httpsdoiorg101371journalpone0166641
KuncaACsokaGampZubrikM(2013)Insects and diseases damaging trees and shrubs of Europe A colour atlasVerrieres-le-buissonFranceNAPEditions
KuznetsovaABrockhoffPBampChristensenRHB(2015)lmerTestTestsinlinearmixedeffectsmodelsRetrievedfromhttpCRANR-projectorgpackage=lmerTest
Lefcheck J S Whalen M A Davenport T M Stone J P ampDuffy J E (2013) Physiological effects of diet mixing on con-sumer fitness A meta-analysis Ecology 94 565ndash572 httpsdoiorg10189012-01921
Loranger JMeyer ST ShipleyBKattge J LorangerHRoscherChellipWeisserWW(2013)PredictinginvertebrateherbivoryfromplanttraitsPolyculturesshowstrongnon-additiveeffectsEcology 941499ndash1509httpsdoiorg10189012-20631
Mody K Eichenberger D amp Dorn S (2009) Stress magnitudematters Different intensities of pulsed water stress producenon-monotonic resistance responses of host plants to insectherbivores Ecological Entomology 34 133ndash143 httpsdoiorg101111j1365-2311200801053x
MoreiraXAbdala-RobertsLParra-TablaVampMooneyKA(2014)Positive effects of plant genotypic and species diversity on anti-herbivoredefensesinatropicaltreespeciesPLoS ONE 9 e105438 httpsdoiorg101371journalpone0105438
Moreira X Abdala-Roberts L Rasmann S Castagneyrol B ampMooney K A (2016) Plant diversity effects on insect herbivoresandtheirnaturalenemiesCurrentthinkingrecentfindingsandfu-turedirectionsCurrent Opinion in Insect Science 141ndash7httpsdoiorg101016jcois201510003
MoreiraXGlauserGampAbdala-RobertsL(2017)InteractiveeffectsofplantneighbourhoodandontogenyoninsectherbivoryandplantdefensivetraitsScientific Reports 74047httpsdoiorg101038s41598-017-04314-3
MoreiraXMooneyKAZasRampSampedroL (2012)Bottom-upeffectsofhost-plantspeciesdiversityandtop-downeffectsofantsinteractively increase plant performance Proceedings of the Royal Society of London B Biological Sciences 2794464ndash4472httpsdoiorg101098rspb20120893
Moreira X Zas R amp Sampedro L (2012) Differential allocation ofconstitutiveandinducedchemicaldefensesinpinetreejuvenilesAtestoftheoptimaldefensetheoryPLoS ONE 7e34006httpsdoiorg101371journalpone0034006
Mraja A Unsicker S B Reichelt M Gershenzon J amp Roscher C(2011) Plant community diversity influences allocation to directchemical defence in Plantago lanceolata PLoS ONE 6 e28055 httpsdoiorg101371journalpone0028055
Muiruri E W Milligan H T Morath S amp Koricheva J (2015)Moose browsing alters tree diversity effects on birch growth andinsect herbivory Functional Ecology 29 724ndash735 httpsdoiorg1011111365-243512407
MuiruriEWRainioKampKoricheva J (2016)Dobirdssee the for-estforthetreesScale-dependenteffectsoftreediversityonavianpredation of artificial larvaeOecologia 180 619ndash630 httpsdoiorg101007s00442-015-3391-6
Nakagawa S amp Schielzeth H (2013) A general and simple methodfor obtaining R2 from generalized linear mixed-effects mod-els Methods in Ecology and Evolution 4 133ndash142 httpsdoiorg101111j2041-210x201200261x
NitschkeNAllanEZwoumllferHWagnerLCreutzburgSBaurHhellip Weisser W W (2017) Plant diversity has contrasting effectson herbivore and parasitoid abundance in Centaurea jacea flowerheadsEcology and Evolution 79319ndash9332httpsdoiorg101002ece33142
OtienoDKreyling JPurcellAHeroldNGrantKTenhunenJhellip Jentsch A (2012) Drought responses of Arrhenatherum ela-tius grown in plant assemblages of varying species richnessActa Oecologica- International Journal of Ecology 39 11ndash17 httpsdoiorg101016jactao201110002
Otway S JHector Aamp Lawton JH (2005) Resource dilution ef-fects on specialist insect herbivores in a grassland biodiversityexperiment Journal of Animal Ecology 74 234ndash240 httpsdoiorg101111j1365-2656200500913x
Pearse I S (2011) The role of leaf defensive traits in oaks onthe preference and performance of a polyphagous herbivoreOrgyia vetusta Ecological Entomology 36 635ndash642 httpsdoiorg101111j1365-2311201101308x
Peacuterez-Harguindeguy N Diacuteaz S Garnier E Lavorel S Poorter HJaureguiberryPhellipCornelissenJHC(2013)Newhandbookforstandardised measurement of plant functional traits worldwideAustralian Journal of Botany 61 167ndash234 httpsdoiorg101071BT12225
RCoreTeam(2016)R A Language and environment for statistical comput-ingViennaAustriaRFoundationforStatisticalComputing
Ratcliffe S Wirth C Jucker T van der Plas F Scherer-LorenzenMVerheyenKhellipBaeten L (2017)Biodiversity andecosystemfunctioningrelationsinEuropeanforestsdependonenvironmentalcontext Ecology Letters 20 1414ndash1426 httpsdoiorg101111ele12849
RootRB(1973)Organizationofaplant-arthropodassociationinsim-ple and diverse habitats The fauna of collards (Brassica Oleracea)Ecological Monographs 4395httpsdoiorg1023071942161
Sampedro LMoreira X amp Zas R (2011) Costs of constitutive andherbivore-induced chemical defences in pine trees emerge onlyunder low nutrient availability Journal of Ecology 99 818ndash827 httpsdoiorg101111j1365-2745201101814x
Scherber C Eisenhauer N Weisser W W Schmid B Voigt WFischerMhellipTscharntkeT (2010)Bottom-upeffectsofplantdi-versity on multitrophic interactions in a biodiversity experimentNature 468553ndash556httpsdoiorg101038nature09492
SchielzethHampNakagawaS(2013)NestedbydesignModelfittingandinterpretationinamixedmodeleraMethods in Ecology and Evolution 414ndash24httpsdoiorg101111j2041-210x201200251x
SchoonhovenLM (2005) Insect-plant biology (2nded)OxfordNewYorkNYOxfordUniversityPress
SconiersWBampEubanksMD (2017)NotalldroughtsarecreatedequalTheeffectsofstressseverityoninsectherbivoreabundanceArthropod- Plant Interactions 11 45ndash60 httpsdoiorg101007s11829-016-9464-6
SetiawanNNVanhellemontMBaetenLDillenMampVerheyenK (2014) The effects of local neighbourhood diversity on pestanddiseasedamageoftreesinayoungexperimentalforestForest Ecology and Management 334 1ndash9 httpsdoiorg101016jforeco201408032
UnderwoodN InouyeBDampHambaumlck PA (2014)A conceptualframework for associational effects When do neighbors matter
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956
emspensp emsp | emsp2057Journal of EcologyCASTAGNEYROL ET AL
andhowwouldweknowThe Quarterly Review of Biology 89 1ndash19 httpsdoiorg101086674991
VehvilaumlinenHKorichevaJRuohomaumlkiKJohanssonTampValkonenS(2006)Effectsoftreestandspeciescompositiononinsectherbiv-oryofsilverbirchinborealforestsBasic and Applied Ecology 7 1ndash11 httpsdoiorg101016jbaae200505003
WalterJHeinRAugeHBeierkuhnleinCLoumlfflerSReifenrathKhellip JentschA (2011)Howdoextremedrought andplant commu-nitycompositionaffecthostplantmetabolitesandherbivoreperfor-manceArthropod- Plant Interactions 6 15ndash25
WhiteTC (1974)Ahypothesis toexplainoutbreaksof loopercater-pillarswithspecialreferencetopopulationsofSelidosema suavis in aplantationofPinus radiatainNewZealandOecologia 16 279ndash301 httpsdoiorg101007BF00344738
WhiteTCR (2009)PlantvigourversusplantstressA falsedichot-omy Oikos 118 807ndash808 httpsdoiorg101111j1600-0706 200917495x
WhiteJAampWhithamTG(2000)Associationalsusceptibilityofcot-tonwoodtoaboxelderherbivoreEcology 811795ndash1803httpsdoiorg1018900012-9658(2000)081[1795ASOCTA]20CO2
Ximeacutenez-Embuacuten M G Ortego F amp Castantildeera P (2016) Drought-stressed tomato plants trigger bottomndashup effects on the inva-sive Tetranychus evansi PLoS ONE 11 e0145275 httpsdoiorg101371journalpone0145275
Zakir A Sadek M M Bengtsson M Hansson B S Witzgall Pamp Anderson P (2013) Herbivore-induced plant volatiles pro-vide associational resistance against an ovipositing herbivoreJournal of Ecology 101 410ndash417 httpsdoiorg1011111365- 274512041
Zvereva E L Zverev V amp Kozlov M V (2012) Little strokes fellgreatoaksminorbutchronicherbivorysubstantiallyreducesbirchgrowth Oikos 121 2036ndash2043 httpsdoiorg101111j1600- 0706201220688x
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle
How to cite this articleCastagneyrolBJactelHMoreiraXAnti-herbivoredefencesandinsectherbivoryInteractiveeffectsofdroughtandtreeneighboursJ Ecol 20181062043ndash2057 httpsdoiorg1011111365-274512956