Anti‐herbivore defences and insect herbivory: Interactive ... · CASTAGNEYRO ET AL. Journal of Ecolog y | 2045 damage levels, these herbivores can cause significant growth loss

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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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


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


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)


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)


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



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


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


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)





Condensedtannins Apparency 348(1788) 066 minus(039)





CN Apparency 202(1564) 160 minus(06)





Starch Apparency 064(1755) 426 008(046)





Sugars Apparency 142(1761) 238 minus(032)





PC1 Apparency 536(1796) 023 020(038)





PC2 Apparency lt001(1734) 978 minus(032)





F-valuesdegreesoffreedom(numeratordenominator)aregiventogetherwithsignificanceBoldcharacters indicatesignificantpredictorsatα = 005 R2

m and R2c correspond tomarginal (ieac-

countingforfixedeffectsonly)andconditional(accountingforbothfixedandrandomeffects)R2 R2

mwascalculatedonlywhenthefinalmodelretainedatleastonepredictor

TABLE 2emspSummaryoflinearmixedeffectmodels(LMM)testingtheeffectsofbirchapparencyirrigationandtreeneighbouridentityonleafnutritionalqualityanddefensivetraits


34emsp|emspPlant traits underlying effects of tree species diversity and irrigation on leaf herbivory


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)


noeffecton leaf-chewerherbivoryAltogether these results suggestthateffectsofirrigationandtreespeciesdiversityonchewerherbivorywerenotmediatedbythestudiedplanttraitsnorbytreeapparency


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


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


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


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


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


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


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









500

600

700

800

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

40

50

60

70

80

(12)

(12)

(12)

(12)

(12)

(12)

(12)(12)

Can

opy

clos

ure

()

B BQ BP BQP

a a b aA B C A

minus20

0

20

40

60

(9)

(9)

(12)

(11)

(12)(11)

(12) (11)

Birc

h ap

pare

ncy

()

B BQ BP BQP

a b c d

(a) (b)

(c) (d)





































3emsp |emspRESULTS

















0

2

4

6

8

10

12

Def

olia

tion

( L

AR

)

Earlyseason

Midseason

(a)

(96)

(96)0

2

4

6

8

10

12


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)


Season Predictors




c)


Diversity 004(192) 847 150(1) 220


Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)

Diversity 187(122) 185 426(1) 039



Neighbours 122(388) 306 597(3) 113


Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)

Neighbours 159(321) 223 806(3) 045



m and R2




c)

LDMC Apparency 1367(1341) 001 015(032)






























PC1 Apparency 536(1796) 023 020(038)





















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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S































































































500

600

700

800

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

40

50

60

70

80

(12)

(12)

(12)

(12)

(12)

(12)

(12)(12)

Can

opy

clos

ure

()

B BQ BP BQP

a a b aA B C A

minus20

0

20

40

60

(9)

(9)

(12)

(11)

(12)(11)

(12) (11)

Birc

h ap

pare

ncy

()

B BQ BP BQP

a b c d

(a) (b)

(c) (d)





































3emsp |emspRESULTS

















0

2

4

6

8

10

12

Def

olia

tion

( L

AR

)

Earlyseason

Midseason

(a)

(96)

(96)0

2

4

6

8

10

12


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)


Season Predictors




c)


Diversity 004(192) 847 150(1) 220


Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)

Diversity 187(122) 185 426(1) 039



Neighbours 122(388) 306 597(3) 113


Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)

Neighbours 159(321) 223 806(3) 045



m and R2




c)

LDMC Apparency 1367(1341) 001 015(032)






























PC1 Apparency 536(1796) 023 020(038)





















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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S



























































































































3emsp |emspRESULTS

















0

2

4

6

8

10

12

Def

olia

tion

( L

AR

)

Earlyseason

Midseason

(a)

(96)

(96)0

2

4

6

8

10

12


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)


Season Predictors




c)


Diversity 004(192) 847 150(1) 220


Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)

Diversity 187(122) 185 426(1) 039



Neighbours 122(388) 306 597(3) 113


Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)

Neighbours 159(321) 223 806(3) 045



m and R2




c)

LDMC Apparency 1367(1341) 001 015(032)






























PC1 Apparency 536(1796) 023 020(038)





















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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S














































































































3emsp |emspRESULTS

















0

2

4

6

8

10

12

Def

olia

tion

( L

AR

)

Earlyseason

Midseason

(a)

(96)

(96)0

2

4

6

8

10

12


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)


Season Predictors




c)


Diversity 004(192) 847 150(1) 220


Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)

Diversity 187(122) 185 426(1) 039



Neighbours 122(388) 306 597(3) 113


Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)

Neighbours 159(321) 223 806(3) 045



m and R2




c)

LDMC Apparency 1367(1341) 001 015(032)






























PC1 Apparency 536(1796) 023 020(038)





















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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S



























































































3emsp |emspRESULTS

















0

2

4

6

8

10

12

Def

olia

tion

( L

AR

)

Earlyseason

Midseason

(a)

(96)

(96)0

2

4

6

8

10

12


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)


Season Predictors




c)


Diversity 004(192) 847 150(1) 220


Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)

Diversity 187(122) 185 426(1) 039



Neighbours 122(388) 306 597(3) 113


Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)

Neighbours 159(321) 223 806(3) 045



m and R2




c)

LDMC Apparency 1367(1341) 001 015(032)






























PC1 Apparency 536(1796) 023 020(038)





















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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S




























































































0

2

4

6

8

10

12

Def

olia

tion

( L

AR

)

Earlyseason

Midseason

(a)

(96)

(96)0

2

4

6

8

10

12


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)


Season Predictors




c)


Diversity 004(192) 847 150(1) 220


Mid Irrigation 376(1869) 086 025(046) 090(1) 342 002(007)

Diversity 187(122) 185 426(1) 039



Neighbours 122(388) 306 597(3) 113


Mid Irrigation 1003(16) 019 018(045) 094(1) 333 003(007)

Neighbours 159(321) 223 806(3) 045



m and R2




c)

LDMC Apparency 1367(1341) 001 015(032)






























PC1 Apparency 536(1796) 023 020(038)





















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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S

























































































c)

LDMC Apparency 1367(1341) 001 015(032)






























PC1 Apparency 536(1796) 023 020(038)





















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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S





























































































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



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)




























ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S


















































































































ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S








































































































ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S































































































ACKNOWLEDG EMENTS






ORCID



R E FE R E N C E S







































































































































































































































Documents

Anti‐herbivore defences and insect herbivory: Interactive ... · CASTAGNEYRO ET AL. Journal of Ecolog y | 2045 damage levels, these herbivores can cause significant growth loss