Improved Deschampsia cespitosa growth by nitrogen fertilization jeopardizes Quercus petraea regeneration through intensification of competition

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fertilization jeopardizes Quercus petraea regeneration

through intensification of competition

Antoine Vernay, Philippe Malagoli, Marine Fernandez, Thomas Perot, Thierry

Ameglio, Philippe Balandier

To cite this version:

Antoine Vernay, Philippe Malagoli, Marine Fernandez, Thomas Perot, Thierry Ameglio, et al..

Im-proved Deschampsia cespitosa growth by nitrogen fertilization jeopardizes Quercus petraea

regenera-tion through intensificaregenera-tion of competiregenera-tion. Basic and Applied Ecology, Elsevier, 2018, 31, pp.21-32.

�10.1016/j.baae.2018.06.002�. �hal-01893947�

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BasicandAppliedEcologyxxx(2017)xxx–xxx

Improved

Deschampsia

cespitosa

growth

by

nitrogen

fertilization

jeopardizes

Quercus

petraea

regeneration

through

intensification

of

competition

Antoine

Vernay

,

Philippe

Malagoli

,

Marine

Fernandez

,

Thomas

Perot

,

Q1

Thierry

Améglio

,

Philippe

Balandier

a_{UniversitéClermontAuvergne,INRA,PIAF,63000Clermont-Ferrand,France}

b_{Irstea,ResearchUnitonForestEcosystems(EFNO),DomainedesBarres,45290Nogent-sur-Vernisson,France}

Received10October2017;accepted17June2018

Abstract

Plant–plantinteractionsshowdifferentialresponsestodifferentcombinationsofavailableresources thathasbeen under-explored.

Theshort-termfunctionalresponseofQuercuspetraeaseedlingsandDeschampsiacespitosatuftsgrownaloneorinmixture wasmonitoredincontrastingcombinationsofsoilinorganicnitrogen×lightavailabilitiesinagreenhouseexperiment.Growth, biomassallocation,functionaltraitsandresourceacquisitionwerequantified.Intensityandimportanceofinteractionswere calculatedbyorganbiomass-basedindices.

CompetitionexertedbyD.cespitosaonoakwasprimarilydrivenbylightavailabilityandsecondly,for eachlight level, bynitrogensupply,leadingtoastronghierarchyofresourcecombinationsforeachconsideredplantorgan.Underhighlight,

oakpreferentially allocatedbiomass to the roots, underliningthe indirectrole of light on the belowground compartment.

Unexpectedly,Deschampsiacespitosagrewbetterinthepresenceofoakseedlingsunderhighnitrogensupplywhateverthe

lightavailability.

Oakshort-term nitrogen storage instead of investment ingrowth might bea long-term strategyto survive D.cespitosa

competition. Why Deschampsia hada higher biomass inthe presence of oak under nitrogenfertilization is an intriguing

question.Theroleofrootexudatesorchangeinbalancebetweenintra-vsinterspecificinteractionsmayholdtheanswer.There maybeanactivemechanismofcompetitionratherthanonlycompetitiveresourceexploitation.

Forestmanagers sometimes practice addingnitrogen fertilizer toimprove oak seedlinggrowth inplantationsor natural

regeneration.Here,the higherbiomass inmixture tothe benefitofthe competitorclearlyquestions thispractice:oakmay provideextranitrogentocompetitorsduringtheearlyperiodofplant–plantinteractionoritmayinfluencethebalancebetween intra-vsinterspecificinteractions.Theidentificationandquantificationofactivecompetitionmayresultinnewpracticesfora broaddiversityofplant–plantinteractionssuchastreeregeneration,intercropmanagementandweedcontrolinagriculture. ©2018PublishedbyElsevierGmbHonbehalfofGesellschaftf¨ur ¨Okologie.

Keywords: Competition;Functionaltraits;Light;Plantinteractions;Regeneration;Soilinorganicnitrogen;Intra/interspecificinteractions

∗_{Correspondingauthor.}

E-mailaddress:philippe.malagoli@uca.fr(P.Malagoli).

https://doi.org/10.1016/j.baae.2018.06.002

1439-1791/©2018PublishedbyElsevierGmbHonbehalfofGesellschaftf¨ur ¨Okologie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Introduction

Plantabilitytocompeteforresourceshaslongbeen

stud-ied over a wide range of species, but no unifying theory

has yet emerged to explain all plant responses to biotic

interactionsindifferentabioticcontexts.Grime(1974)first

proposedathree-determinanttriangle—competition,stress,

disturbance—to classifyplant species on a site according

to their behavior to cope with resource availability and

stress/disturbances in a given environment. Based on his

ownobservations, Grimeconcluded thatcompetition grew

strongerwithhighersoilfertility(Grime1974).In another

approach,Tilman(1987)focusedontheprocessesinvolved

incompetitionandsuggestedthatcompetitionwasstrongest forsoilresourcesinanunfertileenvironmentandstrongest for light inafertile environment.However,neither theory satisfactorilyaccountsforeveryobservedplantresponseto thecombinedeffectsofcompetitionandfluctuatingresource availability(Craine2005).Nevertheless, morerecent stud-ieshavemanagedtoreconcilethesetheories,asbothwould predict survival of thespecies withthe lowestR*, i.e.the lowestresource level allowing the plantto survive, tothe detrimentofspecieswithhigherR*.Thedifferencebetween thetwotheoriesresidesintheintensity ofthedisturbances studied,i.e.arelativelylowdisturbanceintensityforTilman andhigherintensityforGrime(Grime2007;Jabot&Pottier 2012).Plantgrowthandfunctionalresponsesremainunclear inseveralcasesofresourcelimitations.PugnaireandLuque (2001),using an environmental gradient,showed stronger

competition in the most fertile environment, as predicted

by Grime, but they also found that belowground organs

underwentstrongercompetition inthemoststressful envi-ronmentthaninthemostfertileone,thusendorsingTilman’s

theory (Pugnaire & Luque 2001). They demonstrated a

dynamicbalancebetweenfacilitationandcompetitionalong the environmental gradient. This is relevant to the facili-tation process (broadlydefined as at least positive impact

of plant A on plant B) which is positively correlated to

stressintensity(Bertness&Callaway1994)untilfacilitation collapsesunderthehigheststressoruntilcompetition inten-sityovertakesfacilitationintensity(Verwijmeren,Rietkerk, Wassen, & Smit 2013). However, conclusions strongly

depend on experimental design and/or environmental

contexts.

Q2

Interactionscanbecharacterizedbytwovariables:

impor-tance and intensity (Welden & Slauson 1986; Corcket,

Liancourt,Callaway,&Michalet2003).Intensityisdefined

as the absolute effect of plant A on plant B, commonly

measuredbycomparingaperformanceindexsuchas plant

biomasswithorwithoutaneighbor.Importance isdefined

as therelative negativeimpactof competition onplant fit-nesstraitscomparedwithenvironmentalconstraints(Welden & Slauson 1986; Brooker et al. 2005). This concept of importancewasintroducedtoassessthecontributionofthe interactioneffectrelativetotheenvironmenteffectin

reduc-ing the performance of a given plant. How intensity and

importancevaryamongdifferentmulti-resourceavailabilities isstilllargelyunknown(Pugnaire&Luque2001;Liancourt, Corcket,&Michalet2005;Pugnaire,Zhang,Li,&Luo2015).

When several species are competing for the same

resources,plantscanalsoacclimateinresponsetonew

envi-ronmental conditions with fewer resources (Violle et al.

2007). According to a plant’s phenotypic plasticity, plant

traits can be adjusted to optimize the growth of organs

involvedinresourcecapturesoastobettercopewith com-petitive neighbors, and with greater efficiency (Casper & Jackson1997).Thispatternisconsistentwithforaging the-ory,whichstatesthatwhenaresourceisrare,captureorgans

can acclimate to become more efficient and favor higher

growth.Incontrast,intheconservativestrategy,nutrientsand carbohydrates arepreferentiallystoredinperennial organs for laterre-useinamorefavorableenvironmentalcontext, reducingriskofsurvivalfailure(Valladares,Martinez-Ferri, Balaguer,Perez-Corona,&Manrique2000;Yan,Wang,& Huang2006).

Mostearlierstudiesonplant–plantinteractionshaveonly consideredoneresource.Veryfewstudieshaveaccountedfor crossedavailabilitiesinaerialandsoilresources,including soilinorganic nitrogen(Nsoil)(Davisetal.1999; Siemann &Rogers 2003),andmost ofthem weredesigned

incom-pletely for all of the factors combinations or with only

partial control of factors studied. Here, we studied how

light andnitrogen availabilityand their interactions could

influence plant responses to biotic interactions in terms

of growth and functional traits. These two factors would

enable to separate aboveground competition from

below-groundcompetitionintermsofimportanceandintensityof

interaction.

Our experimentaimedtomeasureearly plantresponses

of sessileoak(Quercus petraea)seedlingsand

Deschamp-siacespitosainamixture,intermsofgrowthandresource acquisitioninfournitrogen×lightcombinations.Thesetwo

species arewidespreadandcommonlyoccur ininteraction

throughout temperateEuropean forests (Davy 1980).

Cur-rent silviculturalpractices thataim toreducestandingtree density (Puettmann et al. 2015) will increase light in the understory,thusfavoringcolonizationbytheherbaceousD. cespitosa. Weexpectedtofind amitigatedcompetition by grassesinashadedenvironmentassociatedwithlowergrass performanceintermsofgrowthandfunctioning.Weexpected

oak seedlingstoshow higher investmentto the root

com-partmentinunfertilizedplaces(higherrootbiomass,specific rootlength(SRL),allocationofresourcestotherootsystem)

andhigherinvestmentforaboveground organsinashaded

environment (highergrowth rate,preferentialallocation of

resources to leaves). We expected to find that the

under-ground foragingbehavior ofoakwouldcounteractthe fast

D.cespitosagrowth.Theexperimentalsetupwasdesigned (i)todeterminehowgrowthofoak/D.cespitosawasaffected

bythecombinationofabioticenvironmentsonashort-term

scale andhow functional traitsallow bothplants to

accli-mate or respond toresource combinationsof resources in

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A.Vernayetal./BasicandAppliedEcologyxxx(2017)xxx–xxx 3

Fig.1. Experimentaldesignofallcrossedtreatmentcombinations.Dc=D.cespitosa,Qp=Quercuspetraea,N=nitrogen,n=numberof replicates(seetextfordetails).

termsofresourceacquisitionstrategy,and(ii)todetermine theimportanceandintensityofinteractions(positiveor neg-ative), and (iii) to elucidate the plant response strategies employedtodealwiththeseinteractionsinallthetreatment combinations.

Materials

and

methods

Experimental

setup

The experiment was conducted in a greenhouse at

the INRA UMR PIAF research unit in Clermont-Ferrand

(Auvergne, France, 45◦45N 3◦07E, altitude 394m a.s.l)

frommid-December2014toJune2015.Atotalof120

one-year-oldbare-rootoakseedlings[Q.petraea(Matt.)Leibl.; 149±20gfreshweightonaveragepertree]sourcedfroma localtreenurserywereplantedonDecember15,2014in 20-Lpotsfilledwithalocalsandy-claysoil(clay20.3%,loam

22.8%, sand56.9%; pH6.15, totalNcontent 1.45gkg−1,

totalCcontent14.6gkg−1)beforebudbreak.D.cespitosa

(L.)tufts(abovegroundparts+roots)werecarefullycollected undernaturalforestconditionsatParay-le-Frésil(Auvergne, France;46◦39N3◦36E)andthentransplantedintothepots

onDecember16,2014.Oakseedlingsweregrown(i)

with-outD.cespitosa[solespecies;40pots(oneseedlingperpot)] or(ii)withthreesurroundingtufts[mixedspecies;80pots,

0.97±0.02gperfreshtuftmatterofD.cespitosa],andthe lasttreatmentwas(iii)D.cespitosa(3tuftsperpot)without oakseedlings(40pots).Mixturedensitywassettobeasclose aspossibletospeciesabundanceinrealfieldconditions,in termsofrelativeabundance.Halfofthepotswereexposedto

59%ofthephotonfluxdensity(PFD)inthephotosynthetic

activeradiationrange(PAR)reachingthetopofthe green-house(i.e.resultingfromgreenhousestructureinterception), andmimickinganappreciableforestgapunderinsitu condi-tions,treatmentL59.Theotherhalfwassetundernetshelters

(Hormasem®,50%extinction),exposingpotsto27%ofthe

PFDmeasuredabovethegreenhousei.e.closeto%PFD

val-uesfrequentlyrecordedunderanopennatural oakcanopy,

treatmentL27 (Fig.1).Ournetsheltersgavesunprotection

withnoinfluenceonthered-to-far-redratioofthePFD,so light quality wasthe same outside andunder netshelters. Finally,forthetwoirradiances,halfthepotsweresupplied witheitheraddedNH4NO3solutioncorrespondingtoa

fer-tilizationrate of89kgha−1year−1(924mgofinorganicN Q3

perpotor0.42gkg−1,treatmentN89)ornoNH4NO3

addi-tion,treatment N0.ForN89,fertilizationwasappliedthree

timesatanaveragerateof26kgNha−1year−1(0.14gkg−1) inMarch,AprilandMay,evenlyspreadwithabottleonthe potsurface.N0correspondedtonativeNsoil (Fig.1).Light

treatment was constantover thegrowth period(December

2014–June2015)whereasfertilizationwasappliedinthree

pulses. Because no statistical effect of single fertilization

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pulseswasrecordedonthegrowthcurves(datanotshown), thedatacollected attheendof theexperimentwere inter-pretedfromanintegratedresponseoveralltheperiod.Mean

temperatureovertheexperimentwas21±4◦C(±SD;min.

14◦C,max.30◦C).Meanairhumidityovertheexperiment

was 63±8% (±SD; min.42%, max. 82%). Any

undesir-ablespeciesappearing inpots weremanuallyweededout.

Fortypallets(consideredhereassubplots)gatheredsixpots fortechnicalconvenience,with15subplotsshaded.Allother

treatments (N andbiotic interactions) were randomly

dis-tributed among subplots, in equal numbers in each light

treatment.

Growth

measurement

Height of oak seedlings, highest D. cespitosa leaf, and

diameteratthe stembaseof oakseedlingsweremeasured

every10days throughout the experiment.Relative growth

rate(RGR)wascalculatedfordiameterandheightwiththe formula: Q4 RGR=ln  xt2  −lnxt1  t2−t1 (1) wherexisplantheightordiameter,t2isdateofharvest,and

t1isdateofplanting.

15

_N

_labeling

15_NO

315NH4(20mgof15Ndissolvedin500mLofwater)

wasevenlysuppliedatthesurfaceof eachpotonJune05,

2015toassesshowNuptakeduringthevegetativeseasonwas distributedbetweenandwithineachspecies.TotalNcontent and15Nisotopicabundanceweredeterminedbyisotope-ratio

massspectrometryatthePTEFOC081(Nancy)functional

ecologyplatform.Labelingmethodsandassociated calcula-tionsare detailed inVernay, Balandier,Guinard,Améglio, andMalagoli(2016).

Plant

harvesting

Plants were harvested on June 22, 2015. Aboveground

partsandrootswerecollectedinbothspecies.Foroak, above-groundpartswereseparatedintowoodypartsandleavesand driedat60◦Cforatleast48hbeforedryweight determina-tion,androotswereseparatedintofine(diameter<2mm)and coarse(includingtaproot,diameter>2mm).ForD.cespitosa,

nodiameterdistinctionwasmade(diameteralways<2mm).

Soilandstones leftaround the rootwerethenwashedout

withtapwater.Asub-sampleofroots(oneperspecies)for eachharvestedpotwascollected,wrappedinmoistpaper,and storedat−20◦Cformorphologicalanalysis.Theremaining partwas driedat60◦Cforatleast 48hbefore dryweight determination.

Root

trait

measurements

Frozensub-samplesoffinerootswerethawedandscanned

(Epson scanner, professional mode, 16 bits, dpi 600,

pic-tures inTIF format). D.cespitosa roots were pre-colored

with methylene blue to improve contrasts. Pictures were

thenanalyzedwithWinRHIZO® software(V2005a,Regent

Instruments, Canada) to measure root length, surface and

diameter. Specific root length (SRL) was expressed in

cmg−1.

Intensity

and

importance

of

competition:

calculation

of

indices

Intensity and importance of competition were assessed

for bothspeciesusing twoindices,i.e.Iint andIimp,where

I for index refersto the neighborhoodeffect(Díaz-Sierra, Verwijmeren,Rietkerk,deDios,&Baudena2016).Wechose theseindicesastheyarestandardizedandsymmetrical,with finitelimits,andthusallowunbiasedcomparisons. Calcula-tionsweredoneasfollows:

Iint=2× P P_−N+|P| (2) Iimp=2× P 2MP_−N−P_−N+|P| (3)

where P_−N isplant performancewithout neighbor, P is

the difference between plant performance with and

with-out neighbor, and MP_−N is maximum plant performance

among all treatment combinations(MP_−N was reached in

L59/N89 for abovegroundorgansandinL59/N0for

below-groundorgans).Indiceswerecalculatedforeachorganwith drybiomassastheperformancevariable.ValuesofIintand

Iimp rangebetween−1 and+2andbetween−1and+2/3,

respectively.Anegativeorpositivevaluemeansacompetitive orafacilitativeinteraction,respectively.

Statistics

Toanalyzetheeffectsoflightintensity,nitrogen

availabil-ity and biotic interactionson plant growth,we performed

analysesof variance withlinear mixedeffects models.All

analyzeddatawerebasedonthevariablesmeasuredat

har-vestattheendofthisexperiment,i.e.inJune2015,andthus quantifiedintegratedplantresponsesfromDecember2014to

June2015.

Allfactorsandfactor–factorinteractionswereincludedin

the model simultaneously. Full modelswere simplifiedby

removinginsignificanthigher-orderinteractions.Toaccount forthespatialstructureofourexperimentaldesign,we

intro-ducedasubplotrandomeffectinthemodels.Finalmodels

werefittedusingtherestrictedmaximumlikelihoodmethod

(REML) to betterestimate variance components(Pinheiro

& Bates2000).The lme functionof the nlme package(R

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A.Vernayetal./BasicandAppliedEcologyxxx(2017)xxx–xxx 5

Table1. Modelofnitrogen,lightandbioticinteractionforabovegroundbiomassforoakseedlings(leavesandstem)andD.cepsitosa(shoots). Onlyresultsfromsignificanttermsareshown.Df=degreeoffreedom(Num=numeratorandDen=denominator),N=numberofreplicates, N=nitrogen,L=light,BI=bioticinteraction,DW=dryweight,SRL=specificrootlength.

Oakseedlings D.cespitosa

N NumDf DenDf F-values p-Values N NumDf DenDf F-values p-Value

Light 119 1 36 15.3 <0.001 119 1 38 64.5 <0.001

Nitrogen 119 1 75 9.9 0.002 119 1 74 145.9 <0.001

Bioticinteraction 119 1 75 104.2 <0.001

L×BI 119 1 75 21.9 <0.001

N×BI 119 1 75 11.5 0.001

Fig.2. Abovegrounddryweightinsole-grownandmixed-grownoakandD.cespitosaunderacrossedcombinationoftwolevelsoflight (L59andL27)andNsoilavailability(N89andN0;seeMaterialsandmethodsforfurtherdetails).Valuesarereportedasmeans±SE(n=10for

sole-grown(SSp),n=20formixed-grownplants(MSp),degreeoffreedom=50).Forstatisticalrelevance,datawerelog10-transformed,but

forreadability,untransformedvaluesaregiveninthefigure.Differentlettersresultfrommultiplepairwisecomparisons(Tukey’sHSDtest) betweeneachtreatmentcombinationatp<0.05.

software) was used to fit the linear mixed effect models

(Pinheiro,Bates,DebRoy,&Sarkar2016).Theconditional

F-test given by the anova function of the nlme package

was used to assess the significance of the different terms ofthemodels.Todeterminewhichtreatmentsdifferedfrom

each other, we conducted multiple pairwise comparisons

(Tukey’sHSDtest)usingthelsmeanspackage(Lenth2016).

Becausethree-way interactionswere neversignificant, we

didnotpresentthem inourdata.Comparisonof 15N

allo-cation (%) between sole-grown species and mixed-grown

species was assessed witha Student’s t-testin each plant compartment.

RGR was measured via regular growth measurements

enabling pot to also be included as a random factor for

thesevariables.Preliminaryanalysisshowednoeffectof spa-tialpositionofeachpotinthegreenhouse.Somevariables weretransformedbyalog10functiontomeetnormalityand

homoscedasticityrequirements.

AllanalyseswereconductedwiththeRsoftwareversion

3.3.2(RCoreTeam2016).

Results

Plant

responses

to

biotic

interactions

under

different

resource

combinations

Only N×biotic interactions and L×biotic interactions

had significant effects on aboveground oak seedling dry

weight(leafdryweightandstemdryweight,Table1).Our

data showed disordinal interactions (Doove, Van Buuren,

& Dusseldorp 2014), making simple factor interpretation

irrelevant between sole and mixed grown oaks.

Without-neighbor data clearly showed a higher aboveground oak

biomass when light and/or nitrogen werehighly available

(Fig.2).L59/N89producedsignificantlyhigheraboveground

biomass than other treatment combinations. These

posi-tive effects were cancelled in mixed cultures, producing

significant interactions between L×biotic interaction and N×biotic interaction (Fig. 2, Table 1). This pattern was

observedformostoftheoakvariablesstudied(AppendixA

inSupplementarymaterial)exceptforwholeplantbiomass

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Fig.3. Relationshipbetweenimportance(Iimp)andintensity(Iint)ofinteractionbetweenD.cespitosaandoak.Indices,basedonoakbiomass,

werecalculatedforallcrossedlight×Nsoilavailabilitycombinationsbasedondryweightinfineroots(black-filled),coarseroots(white-filled),

stem(light-grey-filled)andleaves(dark-grey-filled).Valuesarereportedasmeans(n=10formonoculture,n=20inmixtures).Regression equationsandcoefficientsforeachcompartmentarelistedinthefigure.

whichwasonlydependentonbioticinteractionsandwasnot significantlysensitivetofactorinteractions(AppendixAin Supplementarymaterial).Shoot/root,finerootarea,leafdry weightandtotalabovegrounddryweightwereallaffectedby L×bioticinteractionandN×bioticinteraction(AppendixA inSupplementarymaterial),withlowervaluesinMSp

treat-mentsthaninSSpandnovisibleeffectofLandNinMSp

(datanotshown).However,rootlengthandstemdryweight

were onlysensitive toL×biotic interaction (Appendix A

inSupplementarymaterial)whereasrootdiameterwasonly

neagtivelyaffectedbyN×bioticinteraction(AppendixAin

Supplementarymaterial).

Dryweightsoffineandcoarserootsinoakwerenot statis-ticallydifferentamongalltreatmentcombinationsandwere onlydependentonthe simpleeffectsof lightand/orbiotic

interaction(AppendixBinSupplementarymaterial).

In contrast, aboveground biomass in mixed-grown D.

cespitosa was unchanged compared with sole-grown D. cespitosa, exceptforL59/N89 whereaboveground biomass

wasgreaterinthemixture(Fig.2B).Abovegroundbiomass

(mainlycomposedofleaves)wasonlyaffectedbylightand

nitrogenavailability,increasingaerialbiomass,withnoeffect ofinteractingfactors(Table1).Onlytotalplantdryweight wassensitivetofactorinteractionswiththesignificanteffect

of N×LandN×bioticinteractions(AppendixAin

Sup-plementarymaterial).Apositiveeffectoflightwasobserved

onrootlength,rootdiameter,rootarea,finerootdryweight, andbioticinteractionsinfluencedtheSRLtraitinD.

cespi-tosa (AppendixesAandC inSupplementary material).In

conclusion,D.cespitosaperformancewasmainlydependent

on simpleeffectsof each factor(exceptfortotalplantdry

weight, AppendixAinSupplementarymaterial)withlittle

effect of bioticinteraction whereas oakseedlingsstrongly sufferedfrombioticinteractioncancellingallpositiveeffects ofhigherLandNavailability.

Intensity

(I

)

and

importance

(I

)

of

interaction

with

neighbor

species

Considering the effectof D.cespitosaonoakseedlings

(Fig. 3), for every light×Nsoil combination, Iint and Iimp

valueswerenegativeforalloakorgans,indicatingthat the

interaction was always competitive.Iimp was highest (low

competition)forL27×N0andlowest(highcompetition)for

L59×N89 (Fig. 3).Moreover, for agiven N supply,both

indices showed lower negative values inL59 thanin L27.

WithineachLtreatment,indexvaluesweremorenegativein N89 thaninN0(Fig.3).Thispatternwasobservedforeach

organ,pointingtoacommonimpactofD.cespitosaonthe

wholeoakplant.Consideringeachoakorgan,respectively,in

abovegroundandbelowgroundcompartments(MP_−Nvalue

wasnotthesameaccordingtoaerialorbelowgroundorgans,

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A.Vernayetal./BasicandAppliedEcologyxxx(2017)xxx–xxx 7

Fig.4. Relationshipbetweenimportance(Iimp)andintensity(Iint)ofinteractionbetweenoakandD.cespitosa.Indices,basedonD.

cespi-tosabiomass,werecalculatedforallcrossedlight×Nsoilavailabilitycombinationsbasedondryweightinaboveground(white-filled)and

belowground(black-filled)compartments.Valuesarereportedasmeans(n=10formonoculture,n=19inmixtures).Regressionequations andcoefficientsforeachcompartmentarelistedinthefigure.

hinderingcomparison),leavesandfinerootshadmore nega-tivevaluesforbothindicesthan,inorder,stemandcoarseroot (exceptforL27×N89,whereindiceswerelowerinstemthan

inleaves).Theseresultsshowthatcompetitionwasstronger in capture organs(i.e. leaves and fine roots) than storage organs(i.e.stemandcoarseroots).

Thepositiveeffectof oakonD.cespitosa,inL59×N89

treatment, suggest two types of interaction: antagonistic

facilitationunderN89 (positiveindicesforD.cespitosabut

negativeindices for oak seedlings)andcompetition under

N0(negativeindices,Fig.4).Theamplitudeoftheeffectwas

muchgreaterforbelowgroundorgans(verypositiveinN89

andvery negativeinN0)thanaerialorgans(closetozero,

meaninganeutralinteraction,Fig.4).

Nitrate

and

ammonium

amounts

in

soil

at

harvest

At the beginningof the experiment, amounts of nitrate

and ammonium measured in pots were 0.032gkg−1 and

0.0013gkg−1,respectively.After6monthsofgrowth,there

weremuch largeramounts of soilnitrate leftinpots with

sole-grownoakthaninpotswitheithersole-grownD. cespi-tosatuftsorthemixture(Fig.5).Amountsofsoilammonium showednostatisticaldifferenceaccordingtomixturedesign orlight×Nsoilcombination(Fig.5).

Intra-

and

inter-specific

allocation

of

soil

inorganic

_N

Of20mgof15Nappliedperpot7mg±0.32mg(n=238)

was taken upby themixture of which98% was allocated

toD.cespitosa.Insole-grownoakseedlings,15Nwas pref-erentially allocated to leaves (Fig. 6). In contrast, when mixed-grownwithD.cespitosa, the15Nallocationpattern

changed: 15N allocationto oakleaves was loweredto the

benefit of coarse and fine roots (Fig. 6), with no change

in the stem, which was not simply due to differences in

biomass growth (AppendixB inSupplementary material).

Insole-grownandmixed-grownD.cespitosatufts,15Nwas

mainlyallocatedtoabovegroundparts(Fig.6).This

differ-ence was notdue tobiomassdifference. Allocationto the

abovegroundpartswashigherinthemixture,attheexpense

ofbelowgroundparts.

Discussion

Do

light

×

soil

inorganic

N

modulate

plant

interactions?

Overall, increased availability in at least one of the

two combined resources (L and/or Nsoil) led toa reduced

374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418

Fig.5. Soilnitrate(NO3−)andammonium(NH4+)contents(gkg−1)atharvestinsole-grownoak (whitebars)and D.cespitosa(black

bars)orinmixtures(greybars)underallcrossedlight×Nsoilcombinations.Valuesarereportedasmeans±SE(n=3formonoculture,n=6

formixtures, degreesoffreedom=27).Differentletterscorrespondtostatisticallysignificantdifferencesbetweensole-grownplantsand mixed-grownplantsatp<0.05,aftermultiplepairwisecomparisons(Tukey’sHSDtest).

Fig.6. Relativeallocationof15_{Namongleaves,stems,coarseroots(CR)andfineroots(FR)in oakseedlingsandD.cespitosaamong}

aboveground(AG)andbelowground(BG)plantpartsinD.cespitosawhensole-grown(SSp)ormixed-grown(MSp).Valuesarereportedas means±SE.·,*,**,***correspondtop<0.1,0.05,0.01and0.001,respectively,afterStudent’st-testforeachorgan;degreesoffreedom=27.

abovegroundbiomass inmixed-grown oakseedlingswhen

comparedtothelowlevelsoftheresourcesstudied.

Deciphering combined effects of light and soil N on

mixed-grownoakseedlingsisnotstraightforward.Actually, neighbor-effect indices demonstratedaprevalence of light impact.First, thesizedifference (infavorof thetalleroak seedlings)makesdirectcompetitionforlightunlikelyunder ourstudyset-up.Second,foragivenamountoflight,adding theNsoilresourceincreasedboththeintensityandimportance

ofcompetitiononoak.Thiswouldsuggestthatgreaterlight

availabilitymayleadtohighercarbongainbyD.cespitosa

(Vernayetal.2016).Thisextraamountofcarbonwould

indi-rectlypromoterootsystemgrowthandthuspre-emptionof

Nsoil.ThestrongabilityofD.cespitosatocaptureNsoil led

to asubsequent bypass of extraavailable resources to the

detriment of oakgrowth (Freschetet al.2017).This abil-itywouldexplainthedisordinalinteractionobserved(Doove et al. 2014). Actually, only sole-grown oaks significantly respondedtoadditionalresourceamount.Indeed,some stud-ieshavereportedthatbelowgroundresourcesplayakeyrole

419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438

A.Vernayetal./BasicandAppliedEcologyxxx(2017)xxx–xxx 9

indrivingthecompetitionrelationship:infertilesoil, compet-itiveexclusionoccurs,enhancedbyhigherbiomassallocation toabovegroundorgans,switchingcompetitionfromnutrients tolight(Newman1973; Hautier,Niklaus,&Hector2009;

DeMalach & Kadmon 2017). However, these conclusions

mainlyresultfromstudiesongrasslandcommunities,which

shareverysimilarecologicalstrategies.Here,perennialsand

ligneousspeciesbehaveddifferentlyandresponded to

dif-ferentneeds,whichcouldexplainwhylight would havea

strongerinfluence.However,thetwo resourcesdid notact independently(Rajaniemi2002),ashighlightedby neighbor-effectindices.Takingintoaccountcrossedcombinationsof Nsoil×Lthusbringsfine-tuningelementsthathaveseldom

beeninvestigatedtogether(butseePugnaire&Luque2001). TheonlysituationwhenD.cespitosahadnoeffectonoak

growthand associated traitswas under low levels of both

resources(i.e.L27andN0).Thisisconsistentwithcommon

findings inthe literature (Baribault& Kobe2011; Vernay etal.2016) reporting weakercompetition under low light andnutrientavailability,ascompetitivespeciesfreeuptheir spaceforstress-tolerantspecies(Grime1974;Pierretetal. 2016).

How

to

explain

the

positive

effect

of

oak

seedling

on

D.

cespitosa

biomass?

Antagonisticfacilitation(i.e.whenspeciesAhasapositive effectonspeciesBbutBanegativeeffectonA)ofD. cespi-tosabyoakseedlings,intheN89treatmentswhateverthelight

level,wasanunexpectedandsurprisingfinding(Stachowicz 2001;Schöb,Prieto,Armas,&Pugnaire2014).

Twoprocessesmaybe proposedtoexplainthispositive

effect on D. cespitosa. First, oak seedlings could have a

higherrhizodepositioninfertilizedpotswithoutanybiomass change (Karst, Gaster, Wiley, & Landhausser2016). This

supplementarynitrogensupplymightofferanextrasoilN

source,rapidlyabsorbedbyD.cespitosa.Asaperspective, identifyingandthenquantifyingsuchfluxeswouldbehugely informativetohelpgainarefinedunderstandingofthe

under-lyingmechanisms.Second,interspecificcompetitioncould

beamplifiedinN89/L59,becomingstrongerthanintraspecific

grasscompetition (Vernayetal.2018).Thisprocesscould

befostered byexudates whichwouldact as signalsinthe

rhizosphere,allowingself-recognitioninaplantcommunity (Delory,Delaplace,Fauconnier,&duJardin2016).Exudates comingfromotherspeciesmaytriggerpositivefeedbackon

root length and root density of D. cespitosa(Semchenko,

Saar,&Lepik2014).

N

depletion

to

the

benefit

of

D.

cespitosa

Morethan90% of 15Nappliedwas massivelyabsorbed

by D. cespitosatufts, in line with previous studies (Coll, Balandier,&Picon-Cochard2004;Vernayetal.2016).

AccordingtoTilman’stheory,thiswouldsuggestthatthe competitiverelationshipwasduetoalowR*ofD.cespitosa, i.e. ahighgrowthpotentialatvery lowlevelsofresources (Tilman1982).Suchbehaviorraisesquestionsoverthe sus-tainability of the grass’slife cycle.On the onehand, it is legitimatetoquestionwhether thestrategyof D.cespitosa

involvesacontinuousdepletionof resources atthe riskof

notbeingabletomaintainthewholeorganismlaterondue

toexcessivegrowth(Hardin1968;Gersani,Brown,O’Brien, Maina,&Abramsky2001).Ontheotherhand,“game the-ory”(trade-offbetweensurvivalatthecommunityleveland growthattheplantlevel)wouldpredictatrade-offbetween

resource depletionfor individual D.cespitosagrowth and

the cost of individual maintenance induced by its growth

(McNickle&Dybzinski2013).

In

planta

_N

_allocation:

_a

_conservative

_strategy

for

oak

Oakseedlingsinthemixtureallocatedmuchmore15Nto coarse andfine rootsto thedetriment of leaves than

sole-grownseedlings.Thisphenomenonwasobservedinavery

shorttime(only6months ofinteraction)whichhasrarely

been quantifiedinliterature.Indeed, thisstudy showsthat plant–plantinteractionsandtheirresponsesintermsoflife strategyoccurveryrapidly.WesuggestthathigheroakN

allo-cationtobelowgroundcompartmentsmayfeedanNstorage

pool(Vizosoetal.2008)insteadofusingitforprospection andresourcecapture,associatedwithlowinvestmentin

tis-sue creation (fine root dry weightwas constant despite N

allocationchange).Oakstrategyisthereforeconservative. Nitrogenresourcecanbetakenupandassimilatedquickly (Uscola,Villar-Salvador,Oliet,&Warren2014;Gao,Chen, Yuan,Zhang,&Mi2015).However,fewstudieshaveshown anearlypreferentialNdistributiontotherootsystem,ashas beendoneforcarbon(Kaiseretal.2015).

Foster

oak

regeneration

in

practice

Becausethepresenceofoakhadanunexpectedpositive

effectonD.cespitosagrowthwhenNfertilizerwasadded, fieldfertilizationcannotberecommended(Colletal.2004;

Salifu,Jacobs,&Birge2009).UseofpreliminaryN-loaded

oak seedlingscoming from anursery would allow oakto

benefitfrom itsown internalN-reserve, improving its sur-vival anditsresistance tograss-drivenN-depletion (Salifu &Timmer2001;Villar-Salvador etal.2012;Vernayetal. 2018).Another solutionwould betoconsiderfoliar fertil-ization, allowing to targetoak seedlingsmore specifically without fertilizing understory species (Gagnon &Deblois 2014). All suggested solutions will not be efficient

with-out grassmanagementreducing grassdensity.Thiscanbe

achievedbydecreasinglightavailabilitywhenpossible.

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537

Conclusion

Asexpected,D.cespitosacompeted withoakseedlings

andtothedetrimentofoak.Thiscompetitionarosewhenever

resources became moreavailable (59% PFD for light and

89kgha−1Nsupply).Thisstudyshows originalresponses ofplant–plantinteractionsindifferentresourcecombination (antagonisticfacilitationofD.cespitosabyoakseedlingsand indirectinfluenceoflight).Thisfurtherarguesfor consider-ingcrossedfactorsinsteadofoneresource.Neighbor-effect indicesindicatedthatlightwasaprimaryfactordrivingplant response,butthiseffectwasindirectasdrivenbyimproved Nsoil uptake.Eachspeciesexhibited acontrastingresponse

strategytocompetitionandNsoil×lightcombinations:a

con-servative strategy for oak, and a capture strategy for D.

cespitosa.Finally,D.cespitosagrowthwasenhancedbythe presenceofoakunderhighNsoil.

Investigation of functional mechanisms of antagonistic

facilitation and intra- vs interspecific interaction balance offersinterestingperspectivesforfurtherstudies:Nstorage inoakmightplayapivotalroleincopingwithNsoildepletion

byD.cespitosa.Othersoilresources,suchaswateror phos-phorus,alsowarrantattention.Finally,itwouldbeofgreat interesttotestwhether suchobservationsalsooccur under naturalconditions.

Authors’

contributions

AV,PM,TAandPBconceivedtheideasanddesignedthe

methodology;AVandMFcollectedthedata;AV, TP,PM,

TAandPBanalyzedthedata;AV,PM,TAandPBwrotethe

manuscript.Alltheauthorscontributedcriticallytothedrafts andgavefinalapprovalforpublication.

Acknowledgments

Thisworkreceivedfinancial supportfromtheEuropean

Q5

AgriculturalFundforRuralDevelopment(EAFRD‘Leader’

programme), the French Ministry of Agriculture, the

Auvergne Region Directorate for Agriculture (DRAAF),

and the Allier department (CG 03). The authors thank

André Marquier, Christophe Serre, Aline Faure, Patrice

Chaleil,BrigitteSaint-Joanis,MarcVandame,PascalWalser

and Pierre Conchon for their invaluable help in

prepar-ing the greenhouse setup, managing daily pot watering

andshading, weekly measurements of plantgrowth, plant

andsoilharvesting,datacollectionandsampleprocessing.

Wealso thankDr.Catherine Picon-Cochardfor her

exper-tiseinrunning theWinRHIZO® software,andDr.Pascale

Maillard forexpedientlyshipping theisotope.The authors

thank the certified facility in functional ecology (PTEF

OC081) from UMR 1137EEF andUR1138 BEF atthe

INRANancy-Lorraineresearchcenterforitscontributionto

isotopic analysis. The PTEF facility is supported by the

FrenchNationalResearchAgencythroughtheARBRE

Lab-oratory of Excellenceprogram(ANR-11-LABX-0002-01).

Antoine Vernay was supported by a French Ministry of

Researchgrant.Finally, theauthorsthankthethree

anony-mousreviewersfortheiradviceandcomment.

Appendix

A.

Supplementary

data

Supplementary data associated with this article can be

found, in the online version, at https://doi.org/10.1016/

j.baae.2018.06.002.

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