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Journal of Ecology. 2019;00:1–16. | 1wileyonlinelibrary.com/journal/jec
Received:9July2019 | Accepted:10September2019DOI: 10.1111/1365-2745.13292
R E S E A R C H A R T I C L E
The impact of elevated temperature and drought on the ecology and evolution of plant–soil microbe interactions
Pil U. Rasmussen1 | Alison E. Bennett2 | Ayco J. M. Tack1
1DepartmentofEcology,EnvironmentandPlantSciences,StockholmUniversity,Stockholm,Sweden2DepartmentofEvolution,EcologyandOrganismalBiology,TheOhioStateUniversity,Columbus,OH,USA
CorrespondencePilU.RasmussenEmail:[email protected]
Funding informationMajandTorNesslingfoundation,Grant/AwardNumber:2014211;ResearchCouncilVetenskapsrådet,Grant/AwardNumber:2015-03993
HandlingEditor:BrajeshSingh
Abstract1. Climatechangeisshiftingthedistributionofspecies,andmayhaveaprofoundim-pactontheecologyandevolutionofspeciesinteractions.However,weknowlittleabouttheimpactofincreasingtemperatureandchangingrainfallpatternsontheinteractionsbetweenplantsandtheirbeneficialandantagonisticrootsymbionts.
2. Here,weusedareciprocalmultifactorialgrowthchamberexperimentwithseedsand soilmicrobial communities from three origins to investigate the impact oftemperatureandsoilmoistureonthegrowth,arbuscularmycorrhizal(AM)fungalcolonizationandroot‐associatedfungalcommunityofaperennialherb.Moreover,wetestedwhetherplantsandAMfungiperformedbetterorworsewhenplantsweregrownwith their local soil biota, for example, due toplant adaptationorchangesinthegeneticorspeciescompositionofthesoilmicrobialcommunity.
3. Temperatureandsoilmoisturegenerally increasedplantgrowth,whereas tem-peraturebutnotsoilmoistureincreasedAMfungalcolonization.Thestrengthanddirectionoftheplants'responsetotemperatureweredependentonsoilmoistureanddifferedamongplantpopulations, andAMfungal colonizationwas furtheraffectedbytheoriginofthesoilmicrobialcommunity.Theroot‐associatedfungalcommunitystructurewasimpactedbytemperature,soilmoistureandthesoilmi-crobialorigin,withinteractiveeffectsbetweenthemicrobialoriginandtheabioticenvironment.Plantbiomasswaslowerwhenplantsweregrownwiththeirlocalsoilmicrobes,potentiallyduetointraspecificnegativeplant–soilfeedbacks.
4. Synthesis.Ourfindingsindicatethat,beyondarelativelyuniformincreaseofplantgrowthandarbuscularmycorrhizal(AM)fungalcolonizationwithincreasingtem-perature, plants and root‐associated fungi of different originswill vary in theirresponsetoclimatechange(i.e.elevatedtemperatureandshiftsinrainfall).Thismaycreatepronounced,butdifficulttopredict,spatialandtemporalvariationintheecologyandevolutionofplant–microbeinteractionswithachangingclimate.
K E Y W O R D S
abioticandbioticfactors,arbuscularmycorrhizalfungi,localadaptation,Plantago lanceolata,plant–soil(below‐ground)interactions,plant–soilfeedback,root‐associatedfungi,soilmoisture
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2019TheAuthors.Journal of EcologypublishedbyJohnWiley&SonsLtdonbehalfofBritishEcologicalSociety.
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1 | INTRODUC TION
Soil communities canhavea large impactonplant communityas-sembly,biodiversityandecosystemfunctioning(Bardgett&vanderPutten,2014;vanderHeijden,Bardgett,&vanStraalen,2008).Overthenextcentury,wemayexpecttoseeelevatedtemperaturesandchangesinrainfallregimes(IPCC,2014)changingthedistributionofsoilmicrobes and plants andmodifying the outcomeof plant–soilmicrobeinteractions.Increasingourfundamentalknowledgeoftheimpactofclimateontheecologyandevolutionofplant–soilmicrobeinteractionsmay allow us to predict the consequences of climatechangeforplant–microbeinteractionsandthesurroundingecosys-tem,andprovideavenuestomitigatetheconsequencesofclimatechangeinnaturalandappliedsystems.
Thedistributionofplantsandroot‐associatedfungi,suchasar-buscularmycorrhizal (AM) fungi and pathogens, is to a large partdriven by spatial variation in abiotic factors, such as climate, soilphysical and chemical properties and the biotic environment (e.g.Chaudhary, Lau, & Johnson, 2008; Rasmussen et al., 2018; Vályi,Mardhiah,Rillig,&Hempel,2016).Spatialheterogeneityintheabi-otic and biotic environment can also result inwithin‐species spa-tial genetic structure and local adaptation, where genotypes areadapted to their local environment (Blanquart, Kaltz, Nuismer, &Gandon,2013;Hoeksema&Forde,2008).Inthisregard,plantsareknowntoadaptbothtotheabiotic(Brady,Kruckeberg,&Bradshaw,2005;Maceletal.,2007)andbioticsoilenvironment(Crémieuxetal.,2008;Johnson,Wilson,Bowker,Wilson,&Miller,2010),andan-tagonisticandbeneficialmicrobesareknowntoadapttothe localplantgenotypes(Johnsonetal.,2010;Tack,Thrall,Barrett,Burdon,&Laine,2012).Plantsmayalsoperformbetterorworseintheirlocalsoilenvironmentduetochangesinthegeneticandspeciescompo-sitionof the localmicrobialcommunity in response to thegeneticor species composition of the local plant community (Kulmatiski,Beard,Stevens,&Cobbold,2008;vanderPuttenetal.,2013).Suchplant–soilfeedbacksmaybeeitherpositive,forexample,leadingtoincreasedmutualist densities, or negative, for example, leading toincreasedpathogendensities(vanderPuttenetal.,2013).
The ecological outcome of species interactions, and patterns oflocaladaptation,maybeimpactedbyclimatechange,bothinthecur-rentdistributionalrangeandintheexpandingrange(Bergetal.,2010).Within the existing range, the ecological outcome of plant–soilmi-crobeinteractionsdependsnotonlyonplantgenes,microbialgenesandmicrobialspeciescompositionbutalsoontheenvironmentalcon-text (Hoeksema&Forde,2008; Laine,2009;Thompson,2005).Forexample, soil moisture and temperature have been shown to alterfungal communities (Deveautour, Donn, Power, Bennett, & Powell,2018;Rasmussenetal.,2018)andmaytherebychangefeedbacksbe-tweenplantsandfungi,forexample,ifcommunitieschangefromonemore dominated by beneficialmicrobes to onemore dominated byantagonisticmicrobes orviceversa.As such, environmental changecan disrupt both plant local adaptation and plantmaladaptation tothelocalsoilmicrobialcommunitywithinthepresentrange.Climatechangemayalsoshifttherangesofspecies,whichwillresultinnew
associationsbetweenplants and their soilmicrobeswithin thenewrange.Ifplantsandsoilmicrobesshifttheirrangeinspaceandtimein a similar fashion, patterns of local (mal)adaptationmay be re‐es-tablishedwithinashorttimespan.Incontrast,ifplantswillassociatewithnewmicrobialspeciesorgenotypesinthenewrange,theinterac-tionmaybefundamentallychanged.Asanexample,ifplantsperformworsewhengrowingwith their local soil community in theexistingrange,plantperformancemaybehigherthanexpectedintheexpand-ingrangeduetotheabsenceofcertainantagonisticmicrobes,thelackoftimeforadaptationofantagonisticmicrobestothelocalplantgen-otypesortheadaptationtoandbybeneficialmicrobes.
We used a reciprocal multifactorial climate chamber experi-ment to investigate the impactof climatechange (elevated tem-peratureanddrought)ontheecologyandevolutionofplant–soilmicrobeinteractions.Forthis,weusedPlantago lanceolataplantsandsoilmicrobialcommunitiesoriginatingfromthreedifferentlo-cations (acoastal, forestandmeadowsite),planted in reciprocalcombinationswithtwotemperatureandtwosoilmoisturetreat-ments.We then assessed plant growth, AM fungal colonizationandthecompositionoftheroot‐associatedmicrobialcommunity.Weaimedtoanswerthefollowingspecificquestions:
1. What is the impact of temperature, soil moisture, plant originandsoilmicrobialoriginonplantgrowth,AMfungalcolonizationandthecompositionoftheroot‐associatedmicrobialcommunity?
2. DoplantsandAMfungiperformbetterorworsewhenplantsaregrownwiththeirlocalsoilbiota?Ifthereisevidenceforlocal(mal)adaptation,isthispatterninfluencedbytheabioticenvironment?
We expected that increased temperature and high moisture levelswould generally lead to an increase in plant growth, increase anddecrease inAMfungalcolonization,respectively,andaltertheroot‐associated community composition (Augé, 2001; Compant, van derHeijden,&Sessitsch,2010;Rustadetal.,2001),butthattheresponseto the abiotic environmentwill differ among plant populations andsoil biotic communities (e.g.Al‐Karaki,McMichael,&Zak,2004;Anetal.,2010).Forplantandfungal localadaptation,empiricalstudieshaveshownthatP. lanceolatacanbothbelocallyadaptedtoitslocalsoilcommunity(Mursinoff&Tack,2017)orperformworseinitslocalsoil due to negative plant–soil feedbacks (Bever, 2002; Harrison &Bardgett, 2010).Therefore,wemay expect to see either a positiveoranegativeresponse,dependingonwhichsoilmicrobialfunctionalgroupsaredominatingtheresponse.Finally,Laine(2008)showedthattemperatureaffectedpatternsoflocaladaptationofafoliarpathogentoP. lanceolata,andwethereforeexpectthattheabioticenvironmentmaylikewisemediatepatternsoflocaladaptationtothesoilbiota.
2 | MATERIAL S AND METHODS
2.1 | Study system
Plantago lanceolata is a widespread perennial herb that occurs ina wide range of habitats, for example, dry grasslands, hayfields,
| 3Journal of EcologyRASMUSSEN Et Al.
roadsidesanddisturbedareas(Cavers,Bassett,&Crompton,1980).The plant produces rosettes and reproduces by seed productionand clonal propagation through side rosettes (Cavers et al., 1980;Mook,Haeck,vanderToorn,&vanTienderen,1992;Ross,1973).P. lanceolata associates with many soil organisms (e.g. De Deyn,Raaijmakers,vanRuijven,Berendse,&vanderPutten,2004),includ-ingAM(e.g.Rasmussenetal.,2018)andotherroot‐associatedfungi,andhasoftenbeenusedinlocaladaptationstudies(e.g.Crémieuxetal.,2008;Mursinoff&Tack,2017)andstudiesonplant–soilfeed-backs(e.g.Bever,2002;Brandt,deKroon,Reynolds,&Burns,2013;Harrison&Bardgett,2010).Thereisevidenceforgeneticvariationinbothplant(e.g.Azcon&Ocampo,1981;Graham&Eissenstat,1994;Hetrick,Wilson,&Cox,1992)andmycorrhizalfungi(Burgess,Dell,&Malajczuk,1994;Kochetal.,2004)fortheoutcomeoftheplant–mycorrhizalinteraction,asmeasuredby,forexample,thenumberoffungalstructureswithinplantrootsorthepercentageofAMfungalcolonizedplantroots.Inthisexperiment,weusedplantgrowthandAMfungalcolonizationasmeasurestoassessplantandAMfungalperformance.
2.2 | Experimental design
To investigatehowabiotic (temperatureandsoilmoisture)andbi-otic (soil biota and plant population of origin) factors affect plantgrowthtraits,AMfungalcolonization,root‐associatedfungalcom-munitystructureandadaptation,weusedareciprocalmultifactorialgrowthchamberexperiment.Seedsoriginatingfromthreelocationswerereciprocallyplantedinacombinationwithawholesoilmicro-bial inoculumor a sterile control. These12 combinations of plantoriginandsoilmicrobialoriginwerethensubjectedtofourabioticenvironments: (a) low temperature and low soil moisture; (b) lowtemperature and high soilmoisture; (c) high temperature and lowsoilmoisture;and(d)hightemperatureandhighsoilmoisture.Thesefactorsmayrepresentsomeofthekeyabioticfactorsaffectedbycli-matechange.Eachcombinationoffactorswasreplicatedfourtimes.
2.2.1 | Plant origin
Inordertoinvestigatehowplantgeneticvariationinfluencesplantgrowth,AMfungalcolonization,root‐associatedfungalcommunitystructureandlocaladaptation,wecollectedseeds(27–103seedsperplant) fromsixplant individualsateachof threeplantpopulationsincentralSweden.ThesethreeSwedishplantpopulationsarepartof the global plant demographic networkPlantPopNet (www.plantpopnet.com):ÖstraRyd is a rocky seashore surroundedby forest(hereafterreferredtoascoast site),Tjuvstigenisameadow‐likeroad-sidesurroundedbyforest (hereafterreferredtoas forest site),andTullgarnnäsisasemi‐openmeadowclosetotheseashoregrazedbycattle(hereafterreferredtoasmeadow site;seeTableS1fordetailedabiotic and biotic measurements from these three populations).Seedsfromthesamemotherplantwererandomlyallocatedamongtreatmentcombinations,withtwoseedsfromthesamemotherplantgrownineachpot(withtheexceptionof10pots,whereonlyasingle
seedwasplanted). Ifboth seedsgerminated,oneof the seedlingswasremoved.
To inform the abiotic treatments and allow comparison be-tweenthefieldandgrowthchamber,wealsoassessedtheabioticandbioticconditionsattheP. lanceolatapopulationsfromwhichtheseedsoriginated.Morespecifically,werecordedabove‐groundandbelow‐ground temperature using iButton data loggers (DS1922L;MaximIntegrated,SanJose,CA,USA),measuredsoilmoistureusinga soilmoisturemeter (HH2,SM300;Delta‐T,Cambridge,UK) andassessedplantgrowthtraitsandAMfungalcolonization(seeTableS1forfurtherdetails).Threesoilsamplesweretakenateachfieldsitelocationandfrozenat−20°Cforlatermolecularidentificationofthefungalcommunity.
2.2.2 | Soil microbial origin
Toinvestigatetheeffectofthesoilmicrobialcommunitiesonplantgrowth,AMfungalcolonization,root‐associatedfungalcommunitiesandadaptation,wecollectedsoilfromthesamelocationsaswecol-lectedseeds.Soilwascollectedateachlocationtoadepthof15cm,pooledandpassedthrougha1cmsieveandthenhomogenizedthor-oughly.Carewastakentosterilizesurfacesandavoidcrosscontami-nationbetweensoilsthroughouttheexperiment.
Our main aim was to investigate the impact of the bioticsoilcommunity.To isolatetheeffectofthethreesoilmicrobialcommunities fromdifferences in thesoilphysicalandchemicalpropertiesinwhichtheywereembedded,andatthesametimereducebias in responsetoapotential spike innutrientsduetosoil sterilization (Troelstra,Wagenaar, Smant, & Peters, 2001),wetookthefollowingapproach(assummarizedvisuallyinFigureS1). First, we filled 560ml potswith c. 300ml 3:1mixture ofsterile potting soil (Plugg‐ och såjord, SWHorto,Hammenhög,Sweden) and sand (Specialsand,Rådasand, Lidköping, Sweden).Soil sterilizationwas conducted by double autoclaving the soilmixtureat121°Cfor1hr.Wethen inoculatedthepots (exceptthecontroltreatment)withamixof56mlofsoilfromeachsoilorigin (one live and two sterile). The sterile control treatmentreceived3×56mlofsterilesoilfromeachlocation.Lastly,thepotsweretoppedwithc.50mlofthesterilepottingsoilmixture.Taken together, this approachallowedus to isolate theeffectsof the soil microbial community from concurrent differencesin the abiotic soil environment, such as soil pH, chemistry andphysical structure.Using a commercial soil inoculatedwithmi-crobeshas frequentlybeenused ina rangeof local adaptation(e.g.Hoeksema&Thompson,2007;Lankau,Wheeler,Bennett,&Strauss,2011)andplant–soil feedbackstudies (e.g.Callaway,Thelen,Rodriguez,&Holben,2004;Packer&Clay,2000),eventhough themethod has potential caveats regarding the estab-lishmentandfunctioningoftheexperimentalsoilcommunities.Tovalidatewhetherthesoilcommunitiesatthefieldsitesestab-lishedthemselveswithintheexperimentalsetting,wecomparedthefungi inthesoilattheoriginalsiteswiththefungithathadcolonizedtherootsintheendoftheexperiment.
4 | Journal of Ecology RASMUSSEN Et Al.
2.2.3 | Temperature treatment
Plantsweregrown inclimatechambersateither loworhigh tem-perature(15°Cand25°C)andadaylengthof16hr.Thedifferenceintemperaturebetweenthelowandhightreatmentswasbasedonapproximatedifferencesinsoiltemperaturerecordedinthefieldatthethreefocalplantpopulations (TableS1),andsuchtemperaturegenerallyfollowsthelong‐termexpectationintemperatureincreaseof theworst‐caseemissionsclimatechangescenario (IPCC,2014).Werandomizedtheplacementofplantswithineachclimatecham-berevery2weeks.
2.2.4 | Soil moisture treatment
For the first 2 weeks after seed sowing, pots in all treatmentswere given 100mlwater three times aweek. For the following3 weeks, the high soil moisture treatment continued to receive100ml,whereasthe lowsoilmoisturetreatmentreceived50mlwater,threetimesaweek.Hereaftersoilmoisturewasmaintainedatamaximumof40%and10%watervolume,asmeasuredonaTypeHH2moisturemeterwithaSM300sensor(Delta‐TDevicesLtd,Cambridge,UK),forthehighandlowsoilmoisturetreatmentrespectively.ThesevaluesmatchedthevariationinsoilmoisturebetweenP. lanceolatapopulationsatthefieldsites(TableS1)andfollow the expectation of an increasing frequency of summerdroughtinEurope(IPCC,2014).Apilotstudyshowedthatthelowwater treatmentallowedplants tosurvive,butat thesame timegavevisualsignsofdroughtstress.
2.3 | Measurements
We recorded seedling emergence for each pot. Every fortnight,starting19daysafter sowing,we recorded thenumberof leavesandthelengthandwidthofthelongestleaf.Fromthesemeasures,wecalculatedleafsize(leaflength×leafwidth),totalleafarea(leaflength × leaf width × number of leaves) and leaf allometry (leafwidth/leaflength)(Rasmussenetal.,2017).Weadditionallymeas-uredplantrosetteshape(flatorhigh)74daysaftersowing.Plantswere harvested after 80 days, and leaves and roots were sepa-rated. Leaf biomasswas assessed byweighing leaves before andafteroven‐dryingat60°C,while rootswerewashed,cut to2cmpieces,mixed thoroughly, frozen and then later freeze‐dried andweighed.Wealsocalculatedtheroottoshootratio.Theresultsforthefullsetofplantmeasurescanbefound intheSupplementaryInformation,whileonlykeyplantgrowthmeasuresarepresentedinthemaintext.
To determine AM fungal root colonization, dried roots weretransferredtotissuecassettes,clearedfor5minin3%KOH,acid-ifiedfor30minin2%HClandstainedfor20minin0.05%trypanblue solution (Koske & Gemma, 1989; Phillips & Hayman, 1970).RootswerethenscoredforAMfungalcolonizationusingthegridlineintersectmethodat100 intersectionsperroot (McGonigle,Miller,Evans,Fairchild,&Swan,1990).
2.4 | Molecular methods and bioinformatics
Todeterminethefungalcommunitycompositionwithin treatmentroots (excluding the sterile soil treatment) and from soil taken atthefieldsites,DNAwasextractedfromc.25mgfreeze‐driedrootmaterialor fromc.250mgfrozensoilusingNucleoSpinPlantandSoil kits (Macherey‐Nagel, Düren, Germany). Extracted DNAwassenttoMcGillUniversityandGénomeQuébecInnovationCentre,Montréal,Canada,where fungalDNAwas sequencedonaMiSeqplatform (Illumina Inc. San Diego, CA, USA) using primers fITS7(Ihrmarketal.,2012)andITS4(White,Bruns,Lee,&Taylor,1990),whichtargeta250–450bpfragmentencompassingtheentireITS2region with flanking sequences in the 5.8 and LSU genes. Theseprimers cover most of the root‐associated fungal community andhavebeenchosenastheuniversalDNAbarcodeforfungi(Schochetal.,2012).Itisworthnotingthough,thattheseprimersdonotpickupallfungalgroupsequally(e.g.Schadt&Rosling,2015;Schochetal.,2012).However,Lekbergetal.(2018)showedthatwhileprimerstargetingtheITSorSSUregiongeneratedslightlydifferentcommu-nities,thecommunitiesrespondedsimilartotheenvironmentalpa-rameterstested.Furthermore,insufficientgeneticvariabilitywithintheITSregionsmeansthatspecies‐levelassignmentscanbeunre-liableornon‐valid.Assuch,species‐level identificationsshouldbeinterpretedwithcaution.
Primers were removed using CutAdapt (Martin, 2011). WeusedtheDADA2ITSpipelinetofilterthereadsusingthestandardDADA2 filtering parameters (Callahan et al., 2016). TheDADA2algorithmuses a parametric errormodelwhich is trainedon theentire dataset. This model is then applied to correct and groupsequences into amplicon sequence variants (ASVs) (Callahan,McMurdie,&Holmes,2017;Callahanetal.,2016).Chimeraswereremoved,andlastly,taxonomywasassignedusingtheUNITEdata-base(Abarenkovetal.,2010;Kõljalgetal.,2005),wheretheDADA2pipelineusesanativeimplementationofthenaiveBayesianclas-sifiermethodfortaxonomicassignment(Wang,Garrity,Tiedje,&Cole,2007).
After removal of plant reads (c. 40%), 2,209,193 reads wereobtained from the116 root samples and436,500 reads from thenine soil samples collected at the original sites. Species accumu-lation curves showed that the sequencing effort inmost sampleswassufficient(FigureS2).Fromthesereads,atotalof3,580fungalASVswererecorded.Forfurtheranalyses,weusedthesetof200mostcommonASVsmakingup90%of thetotalnumberof reads,excludingonesamplewithtoofewreads(<500reads),henceforthreferredtoasroot‐associatedfungi.ThesetofmostcommonASVsdidnot includeAM fungi,whichwereonly found inc. halfof thesamples(n=57).AnAMfungalASVtable,whichincluded118AMfungalASVsmakingup0.2%ofthetotalamountofreads,wasana-lysedseparatelyanddetailsonanalysisandresultscanbefoundintheSupplementaryInformation(AnalysisS1).DNAsequenceshavebeendeposited atNCBIunder accessionnumbersPRJNA564044andPRJNA564041 for root samples from theexperimentand fortheoriginalsoilsamplesrespectively.
| 5Journal of EcologyRASMUSSEN Et Al.
2.5 | Statistical analyses
To investigate theeffectof theabiotic andbioticenvironmentonplant growth, AM fungal colonization, observed root‐associatedfungalrichness,root‐associatedfungalShannondiversity(Shannon,1948) and local adaptation, we used generalized linear mixed ef-fectsmodelswithnormaldistributionsusingthelme4 and carpack-ages inr v.3.4.2 (Bates,Maechler,Bolker,&Walker,2014;Fox&Weisberg,2011;RCoreTeam,2017),whileseedlingemergenceandrosetteshapewereanalysedwithabinomialdistributionand logitlinkfunction.Non‐significantthree‐andfour‐wayinteractionswereexcludedfromthemodels.ThesignificanceofrandomeffectswastestedusingtheMASSpackage(Venables&Ripley,2002),andsig-nificantplantandsoilmicrobialoriginmaineffectswereassessedbyposthocTukeytests.
To test the effect of the abiotic and biotic environment onthe root‐associated fungal community composition, we usedPERMANOVAasimplementedinthefunctionadonisinther‐pack-agevegan(Oksanenetal.,2015).CCAwasusedtovisualizehowfun-galcommunitytreatmentsdifferedamongtreatmentlevelsandhowcommunity composition at theoriginal field sitesoverlappedwiththetreatmentcommunities.Weusedbothrelativeabundancesandpresence–absencedataforallthestatisticalanalyses.AllcommunitydatawereHellingerpre‐transformed(Legendre&Gallagher,2001).
2.5.1 | The effect of environmental factors on seedling emergence and plant growth
Toinvestigatetheimpactoftheabioticandbioticenvironmentonseedlingemergenceandplantgrowth,wemodelledseedlingemer-gence,leafnumber,leaflength,leafwidth,leafsize,totalleafarea,leaf allometry, rosette shape, leaf fresh and dry weight, root dryweightandtheroottoshootratioasafunctionofplant origin,soil microbial origin,temperature,soil moistureandtheir interactions.Toaccountforvariationamongsiblingsfromdifferentmotherplants,we added mother plantnestedwithinplant originasarandomeffect.Weusedarepeatedmeasuresanalysisby includingdate and plant IDinmodelswherewemeasuredgrowthtraitsrepeatedly,includingtwo‐way interactions betweendate and the experimental factors.Forrosetteshape,weexcludedtheinteractionsfromthemodeldueto problems with model convergence. When the effect of treat-mentsdifferedbetweendates,wealsoconductedseparatemodelsfor eachdate.We further testedwhetherAM fungal colonizationmediatedtheeffectsoftheabioticandbioticenvironmentonplantgrowthbyaddingitasacovariateinthemodel(AnalysisS2).
2.5.2 | The effect of environmental factors on root‐associated fungi
Toinvestigatetheimpactoftheabioticandbioticenvironment,wemodelled AM fungal colonization and root‐associated fungal rich-nessanddiversityasa functionofplant origin,soil microbial origin,temperature, soil moisture and their interactions. To account for
variation among siblings from different mother plants, we addedmother plantnestedwithinplant originasarandomeffect.Toachievehomogeneousresiduals,diversitywasln+1transformed.Wealsotestedwhetherplantbiomassmediatedtheimpactoftheenviron-mentalfactorsonAMfungalcolonizationbyaddingitasacovariateinthemodel(AnalysisS2).
Toinvestigatewhetherroot‐associatedfungalcommunitieswereinfluencedbytheabioticandbioticenvironment,wemodelledfun-galcommunitycompositionasafunctionofplant origin,soil microbial origin,temperature, soil moistureandtheirinteractions,inadditiontomother plantnestedwithinplant origin.
2.5.3 | The effect of abiotic factors on plant and AM fungal adaptation
To investigatewhether plants and AM fungi performed better orworsewhengrowninlocalornon‐localcombinations,andhowtheabioticenvironmentmayinfluencepatternsoflocaladaptation,weassessed seedling emergence, plant growth traits and AM fungalcolonization in local (sympatric) and non‐local (allopatric) combi-nationsofplantsandsoilbiota.Specifically,wemodelledseedlingemergence,plantgrowthandAMfungalcolonizationasafunctionofplant origin,soil microbial origin,temperature, soil moisture and sym-patry(Blanquartetal.,2013;Laine,2005;Mursinoff&Tack,2017).Sympatryiscategorizedaseitherlocalornon‐localcombinationsofplantandsoilmicrobialorigin,whichcapturesthevariationbetweenlocalandnon‐localconditionsafteraccountingforthemaineffectsofplantandmicrobialorigin(Blanquartetal.,2013)andtheabiotictreatmentlevels.Totestwhethertheabioticenvironmentmightin-fluencepatternsof local (mal)adaptionofplantsandAMfungi,wealsoaddedtheinteractionssympatry × temperature,sympatry × soil moisture and sympatry × temperature × soil moisture.Forthoseplantgrowthtraitsthatweremeasuredeveryfortnight,weranrepeatedmeasuresanalysesaddingtheeffectsofdateanditsinteractionwithsympatry, temperature and soil moisture as listed above. The ran-domeffectplant IDwasalsoaddedto themodels.As thesemod-elsshowedthatthepatternofsympatrychangedovertime,weranmodelsforeachindividualtimepoint.Thesterilesoiltreatmentwasnotincludedinthesemodels.
3 | RESULTS
3.1 | The impact of climate on the ecology of plants and root‐associated fungi
3.1.1 | The effect of environmental factors on seedling emergence and plant growth
Inmostpots,eitheroneortwoseedlingsemerged,exceptfor34potswherenoseedlingsemerged.Emergenceofseedlingswasnotinfluencedbythemaineffectsinvestigated(Table1).Insteadseed-ling emergencewas influenced by the interaction between plantoriginandtemperature,whereseedlingemergencewashigherfor
6 | Journal of Ecology RASMUSSEN Et Al.
seedlingsfromthemeadowsiteinthehightemperaturetreatment,unlikeseedlingsfromthecoastalandforestsites(Figure1).Asig-nificanteffectoftheinteractionbetweensoilmicrobialoriginandsoilmoisturewas found, but this interactionwas driven only bydifferencesbetweenplantsinthesterilesoiltreatmentandplantsinoculated with live soil microbial communities (cf. Table 1 andTableS2).
Highertemperatureandsoilmoistureledtoanincreaseinmostof the plant growth traits (Figure 2a, b, Table 1, and Tables S2–S4), and severalplantgrowth traitswereaffectedbyplantorigin(Figure2d–fandFigureS3,Table1,andTablesS2–S4).Soilmicrobialoriginhadastrongeffectonshootandrootweight,aswellastheroottoshootratio(Figure2g–i,andFigureS3),butthispatternwas–unlike themaineffectsof temperature, soilmoistureandplantorigin–mostlydrivenbythesteriletreatment(cf.Table1andTableS2).Plantsgrowninsterilesoilwerelarger,inparticularintermsofrootbiomass(Figure2h,TableS4).Theroottoshootratioofplantsgrowninthesterilesoilwasmuchlargerthanforplantsgrownwithlivemicrobial communities (Figure2i, TableS4), indicating a rela-tively larger investment in roots than shoots.Therewere severalsignificant two‐way interactions, which all included temperature(Figure2,Table1andTableS3).Theimpactoftemperatureonleaflengthandroottoshootratio,butnotrootbiomass,differedamongplantpopulations(Figure2d–f),whereastheimpactofsoilmoisturedifferedbetweenthetemperaturetreatmentsforrootbiomassandthe root to shoot ratio (Figure 2b, c). Temperature alsomodifiedtheeffectof thesoilmicrobialorigin (Figure2g–i,Table1),but–likethesignificantmaineffectofsoilmicrobialorigin–thispatternwasdrivenbydifferentresponsesofplantsgrowingintheliveandsterile soil (cf.Table1andTableS2).Notably,while theeffectoftemperaturewasalreadyapparentinthenewlyemergingseedlings,theeffectofplantoriginbecamemoreapparenttowardstheendoftheexperiment(TablesS3andS5).Foranin‐depthcomparison
TAB
LE 1
Theimpactofplantorigin,soilmicrobialorigin,temperature,soilmoisture,theirtwo‐wayinteractionsandmotherplantonseedlingemergenceandplantgrowthtraitsof
Plan
tago
lanc
eola
ta
Pl
ant o
rigin
(P)
Soil
mic
robi
al
orig
in (M
ic)
Tem
pera
ture
(T
)So
il m
oist
ure
(Moi
)P
× M
icP
× T
P ×
Moi
Mic
× T
Mic
× M
oiT
× M
oiM
othe
r pl
ant
Seedlingemergence(n=192)
0.483
0.286
0.387
0.61
00.
966
0.00
30.
064
0.27
70.
042
0.21
3—
Leafnumber(
n=948)
<0.0
010.
172
<0.0
010.
144
0.850
0.00
10.
333
0.26
20.
114
0.00
50.
06
Leaflength(n=948)
<0.0
010.
029
<0.0
010.
346
0.25
7<0
.001
0.42
50.
019
0.69
90.378
0.00
2
Leafwidth(n=948)
0.00
20.
219
<0.0
010.
004
0.31
70.
207
0.51
30.
691
0.32
90.
397
0.8
Leafallometry(n=948)
<0.0
010.
130
0.02
70.
244
0.24
00.
002
0.05
50.
043
0.228
0.52
70.
03
Shootdw(n=158)
0.46
40.108
<0.0
01<0
.001
0.14
30.
966
0.811
0.854
0.25
3<0
.001
0.4
Rootdw(n=158)
0.42
2<0
.001
<0.0
01<0
.001
0.588
0.07
70.886
<0.0
010.
226
<0.0
010.8
Roottoshootratio(n=158)
0.32
1<0
.001
0.04
90.
003
0.876
0.00
50.852
<0.0
010.814
0.04
61.
0
Not
e: Shownare
p‐values,withsignificantvaluesinbold.
Forp‐valueswhenexcludingthesterilesoiltreatment,seeTableS2.
Forp‐valuesonallplantgrowthmeasuresand
p‐valuesofdate,andinteractionsbetweendate,plantorigin,soilmicrobialoriginandsoilmoisture,seeTableS3.
Abbreviations:fw=freshweight,dw=dryweight.
F I G U R E 1 Theimpactoftemperatureandplantoriginonnumberofseedlingsemerged(n=192).Shownisaninteractionplotwherethelinesconnectthemeanvaluesforeachtreatmentcombination
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| 7Journal of EcologyRASMUSSEN Et Al.
ofplantsinthefieldsiteandplantgrowthwithintheexperimentalsetting,seeAnalysisS3.
3.1.2 | The effect of environmental factors on root‐associated fungi
We detected a large diversity of fungi in all treatment combina-tions(Figure3).Themajorityoffungiwerepresentatrelativelylowabundances,withtheexceptionfortwoparticularlyabundantfungalspecies (Figure3).TheplantpathogencomplexOlpidium brassicae,likelyOlpidium virulentus (Lay,Hamel,&St‐Arnaud,2018),had thehighestrelativeabundance inall treatmentcombinations,whereas
the secondmost abundant species, the yeastApiotrichum xylopini was only abundant in the low temperature and low soil moisturetreatments and inplants and soil originating from the coastal site(Figure3).
Themaindriversoftheroot‐associatedfungalcommunityweretemperature and the origin of the soil microbial community. AMfungalcolonizationwashigher in thehigh temperature treatment,whereas root‐associated fungal richnesswas lower at higher tem-peratures(Figure4a,b,Tables2andS6).Temperaturealsoimpactedthe composition of the fungal community (Table 2, Figure 5). AMfungal colonization and root‐associated fungal richnesswere low-est when plants were grown with soil biota from the forest site,
F I G U R E 2 Theimpactof(a–c)temperatureandsoilmoisture,(d–f)temperatureandplantoriginand(g–i)temperatureandsoilmicrobialoriginonleaflength(n=158)atharvest,rootbiomass(n=158)andtheroottoshootratio(n=158)inPlantago lanceolata.Shownareinteractionplotswherethelinesconnectthemeanvaluesforeachtreatmentcombination.Non‐significantinteractionsareindicatedbyNS
6
8
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20Le
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ngth
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(a)
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NS
(b) (c)
(d) (e) (f)
(g) (h) (i)
8 | Journal of Ecology RASMUSSEN Et Al.
F I G U R E 3 Therelativeproportionofroot‐associatedfungalspeciesineachtreatmentcombinationdependingonplantorigin,soilmicrobialorigin,temperatureandsoilmoisture.Plant=plantorigin,Soil=soilmicrobialorigin,Temp=temperature,Moisture=soilmoisture,H=high,L=lowandCoast,ForestandMeadowrefertothethreesites
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H L H L
SpeciesOlpidium brassicae complexApiotrichum xylopiniChalara sp.Conlarium sp.Didymellaceae sp.Drechslera sp.Fusarium oxysporumGibberella tricinctaGlarea sp.Guehomyces pullulansLachnum asiaticumLeotia lubricaLoramyces macrosporusMelanommataceae sp.Microdochium phragmitisPyrenochaetopsis leptosporaSaccharicola sp.Trichocladium opacumUnknownRemaining ASVs (<0.02 each)
F I G U R E 4 Theimpactof(a–b)temperatureand(c–d)soilmicrobialoriginonarbuscularmycorrhizal(AM)fungalcolonizationandroot‐associatedfungalrichnessintherootsofPlantago lanceolata plants(n=116).Shownareboxplots,wherethethickhorizontallineshowsthemedian,boxesrepresentthefirstandthirdquantileandwhiskersrepresenteithertheminimumandmaximumvalueor1.5timestheinterquartilerangeofthedata(whicheverissmaller).Significantdifferences(p<.05)amongsoilmicrobialorigins,basedonposthocTukeytests,areindicatedbydifferentletters
AM
fung
al c
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ion
(%)
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al c
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ion
(%)
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t−as
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| 9Journal of EcologyRASMUSSEN Et Al.
intermediate when plants were grown with soil biota from thecoastalsiteandhighestwhenplantsweregrownwithsoilbiotafromthemeadowsite(Figure4c,d,Table2andTableS6).Soilmicrobialorigin also strongly influenced root‐associated fungal communitycomposition(Figure5,Table2).Notably,thefungalcommunitycom-positionofsoilfromthecoastalandmeadowsitesoverlappedwiththecommunitycompositionattheoriginalfieldlocations,whereasthecommunitycompositionofsoilfromtheforestsitedifferedfromthesoilattheoriginalfieldlocation(Figure5).
Soil moisture affected the root‐associated fungal communitycomposition both directly and interactively (i.e. as mediated bythe origin of themicrobial community and temperature; Table 2).Diversityandcomposition,butnotrichness,differedamongplantsfromdifferentorigins(Table2).
3.2 | The impact of climate on plant and AM fungal adaptation
Plantgrowthwasreducedwhenplantsweregrownwiththeirlocalcompared to non‐local soilmicrobial communities (Figure 6a, b; asignificant effect of ‘sympatry’ in Table S7).Overall, the effect ofsympatrywasnot (or onlyweakly) affectedby the environmentalconditions (Table S7), indicating that patterns of (mal)adaptationwerenotaffectedbyclimaticconditions.
AMfungalcolonization levelswereaffectedbythe interactionbetweenplantandsoilmicrobialorigin(Figure6c,Table2andTableS2).However,wedidnotdetectadifferenceinAMfungalcoloniza-tionbetweenplantsgrownwiththeirlocalornon‐localsoilmicrobialcommunity(Figure6c,TableS7).
4 | DISCUSSION
We investigated the environmental drivers of plant growth,AMfungal colonization, fungal community structure andpatternsoflocal adaptation. Plant growth was influenced by climate (tem-peratureandsoilmoisture)aswellastheoriginoftheplantandsoil microbial community. As predicted, the response of plantsto changes in temperature was conditional on plant origin, soilmicrobial community and soil moisture. AM fungal colonizationand root‐associated fungal community structure were stronglyaffected by temperature, soilmicrobial origin and soilmoisture.Plantsperformedworsewhengrownwiththeirlocalsoilmicrobialcommunity,whichmaybeduetointraspecificnegativeplant–soilfeedbacks. Our findings illustrate that genetic variation amongplantpopulationsand themicrobial communitywillplayamajorrole in the response of plant growth and plant–microbial inter-actionstoachangingclimate,butalsothatthe localplantgeno-typesdo–potentiallyduetonegativeplant–soilfeedbacks–notalwaysperformbestwhengrowingwiththeirlocalsoilmicrobialcommunity.Incontrasttoourexpectation,wefoundnoorweakeffectsofclimateonpatternsofplantandAMfungallocaladapta-tion.GiventhevariableresponsesofplantsofdifferentorigintoTA
BLE
2 Theimpactofplantorigin,soilmicrobialorigin,temperature,soilmoisture,theirtwo‐wayinteractionsandmotherplantonAMfungalcolonizationandroot‐associatedfungal
richness,diversityandcommunitycompositionofP
lant
ago
lanc
eola
ta
Pl
ant o
rigin
(P
)So
il m
icro
bial
or
igin
(Mic
)Te
mpe
ratu
re (T
)So
il m
ois‐
ture
(Moi
)P
× M
icP
× T
P ×
Moi
Mic
× T
Mic
× M
oiT
× M
oiM
othe
r pla
nt
AMfungi
Colonization(n=116)
0.805
<0.0
010.
024
0.21
50.
062
0.13
00.
461
0.29
50.
620
0.27
61.
000
Root‐associatedfungi
Richness(n=116)
0.868
0.01
10.
036
0.14
60.
157
0.13
10.
127
0.086
0.72
10.580
0.987
Diversity(n=116)
0.00
40.
004
0.06
6<0
.001
0.75
30.
210
0.34
30.
213
0.831
0.75
01.
000
Communitycomposition
(relabund)(
n=115)
0.01
00.
001
0.00
20.
001
0.21
40.
355
0.49
50.
014
0.00
90.
016
0.04
4
Communitycomposition
(presabs)(n=115)
0.22
20.
001
0.00
10.
001
0.29
70.
404
0.49
20.
001
0.01
70.
002
0.483
Not
e: Shownare
p‐values,withsignificantvaluesinbold.
Abbreviations:relabund=relativeabundance,presabs=presenceabsence.
10 | Journal of Ecology RASMUSSEN Et Al.
temperature, in combinationwith negative plant–soil feedbacks,we predict complex but profound effects of climate change ontheecologyandevolutionofplants and soilmicrobes innaturalsystems.
4.1 | The impact of climate on the ecology of plants and root‐associated fungi
4.1.1 | The effect of environmental factors on seedling emergence and plant growth
Plantgrowthandrelativeinvestmentinabove‐groundbiomasswasconsistentlyhigherinthewarm(25°C)comparedtothecold(15°C)temperaturetreatment,consistentwithstudiesperformedinboththe lab and field, including studies onP. lanceolata (Clemmensen& Michelsen, 2006; Heinemeyer, Ineson, Ostle, & Fitter, 2006;
Olsrud et al., 2004). Low soilmoisture,mimicking low rainfall ordroughtconditions,ledtoreducedplantgrowthandhigherbelow‐groundbiomass.Asdroughteventsincreaseandprecipitationlev-elschangeasaconsequenceofglobalchange(IPCC,2014),wemayexpecttoseeanegativeimpactonplantgrowthandperformanceandhigher investmentbelow‐ground. Interestingly, the increasedroot toshoot ratio in response to increasingdroughteventsmaybecounteractedbyasimultaneousdecrease intheroottoshootratio in response to increased temperature. Drought stress haspreviouslybeenshowntoincreaserootcolonizationbyAMfungi(Jayne&Quigley,2014), andAM fungihave thereforebeenpro-posedasanimportantmechanismforplantsandecosystemstoal-leviatetheeffectsofdrought(Mohanetal.,2014).However,inourstudy,wefoundnodifferencesinAMfungalcolonizationbetweentwostronglydivergentmoisturetreatments.Moreover,therewasnodifferenceinplantresponsestodroughtbetweenplantsgrown
F I G U R E 5 Theimpactof(a)temperature,(b)soilmoisture,(c)plantoriginand(d)soilmicrobialoriginontheroot‐associatedfungalcommunitycompositionofPlantago lanceolata(n=115)asbasedoncanonicalcorrespondenceanalysis(CCA).Colouredcirclesrepresentdispersionellipsesforeachgroupusingthestandarddeviationofpointscores(usingtheordiellipsefunctioninthepackageveganinR).Thefirsttwoaxesexplained2.5%and2%ofthevariationrespectively.Largersymbolsin(d)representtheroot‐associatedfungalcommunitiesfoundinthesoilattheoriginallocations
–4 –3 –2 –1 –4 –3 –2 –10 1 2 3 0 1 2 3
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| 11Journal of EcologyRASMUSSEN Et Al.
in sterileor inoculated soil.Oneexplanation for thisdiscrepancymay lie in the fact that previous studieshave focusedononeora few species of AM fungi (e.g. Al‐Karaki et al., 2004; Porcel &Ruiz‐Lozano,2004;Wu&Xia,2006),whileweusedamixedfieldinoculumwhichcontainsadiversemixofbothbeneficialandan-tagonisticsoilmicrobes.Hence,thebenefitsofincreasedcoloniza-tion levelsbybeneficialmicrobes in response todroughtmaybe
absentwithin thenaturalcontext.Thishighlights the importanceof comparing the effect of beneficialmicrobes not only in isola-tionbutalsoembeddedwithintheirnaturalmicrobialcommunity,therebyobtainingamorerealisticpictureoftheresilienceofnatu-ralsystemstoglobalchange.Interestingly,soilmoisturefrequentlychangedplantresponsestotemperature,illustratingthattheeffectofelevatedtemperaturecanbeeitherexacerbatedoralleviatedbychangesinrainfallpatternsanddrought.
Notably,theoriginoftheplantsmediatedtheresponseofplantstotemperature.Suchdependencyoftheresponseofplantstotheabiotic environment on plant genetics may be a common featureofnaturalsystems(DeLongetal.,2019;Franks,Weber,&Aitken,2014; Jump & Penuelas, 2005). For example, Al‐Karaki and Al‐Raddad(1997)demonstratedthattheresponseofwheattodroughtdiffered among plant genotypes. Variable responses among plantpopulations to changes in climatic conditions may thereby play amajorroleinmaintainingspatialandtemporalheterogeneityinplanttraitsanddemography.
4.1.2 | The effect of environmental factors on root‐associated fungi
root‐associated fungal communities differed among the low andhigh temperature treatment, andAM fungal colonization increasedwith temperature while root‐associated fungal richness decreased.IncreasedAMfungalcolonization is frequently reported inobserva-tionalandexperimentalstudiesinresponsetoincreasedwarming(e.g.Compantetal.,2010;Staddon,Heinemeyer,&Fitter,2002);however,effectsofwarmingonroot‐associatedfungal richnessare lessclear,withreportsofno(Fujimura,Egger,&Henry,2008;Gemletal.,2015),increased(Gemletal.,2015)ordecreased(Gemletal.,2015;Morgadoetal.,2015)richness,likelydependingonthetaxonomicorfunctionalgroup (Gemletal.,2015).Root‐associated fungicanhavemany im-portantecosystemfunctions,suchascarbonstorage(Clemmensenetal.,2013)andimprovingsoilstructure(Rillig&Mummey,2006).Giventhestrongdependenceofroot‐associatedfungalspeciesrichnessandcommunitycompositionontemperature,wemayalsoseeshiftsinim-portantecosystemfunctionsandservicesastheclimatechanges.
We found that AM fungal colonization was not significantlyinfluencedbysoilmoisture.However,aspredicted,theroot‐asso-ciatedfungalcommunitycompositionwasaffectedbothdirectlyand interactively (as mediated by soil microbial origin and tem-perature)by soilmoisture.Previous studieshave similarly foundthatAMfungalcolonizationisnotalwaysaffectedbychangesinprecipitation (e.g.Hawkes et al., 2011), but that the communitycomposition can be (Barnes, van derGast,McNamara, Rowe, &Bending, 2018; Deveautour et al., 2018; Hawkes et al., 2011).Like inourstudy,Gemletal. (2015), investigatingarctic fungi inAlaska,foundaninteractiveeffectofmoistureandtemperature,wheretheeffectoftemperaturedifferedbetweendryandmoisttussocks.
Perhaps not surprisingly, both AM fungal colonization androot‐associated fungal community structure differed among
F I G U R E 6 Plantago lanceolata(a)shootbiomass,(b)rootbiomassand(c)AMfungalcolonizationwhenplantsweregrownwiththeirlocal(n=39)andnon‐local(n=79)soilmicrobialcommunity.Shownin(a–b)areboxplots,wherethethickhorizontallineshowsthemedian,boxesrepresentthefirstandthirdquantileandwhiskersrepresenteithertheminimumandmaximumvalueor1.5timestheinterquartilerangeofthedata(whicheverissmaller),and(c)showsabarplotwithmeans±SE
0.0
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12 | Journal of Ecology RASMUSSEN Et Al.
plantsgrown insoiloriginating fromthe threenaturalP. lanceo-latapopulations.Otherstudieshavesimilarlyfoundthatroot‐as-sociatedfungalcommunitiesdifferamongthesameplantspeciesgrown at different locations (Ji et al., 2013; Rasmussen et al.,2018). Root‐associated fungi were to some degree influencedbyplantorigin,matchingthecommonlyreportedeffectsofhostidentityandgeneticvariationonfungalcommunitystructure:Forexample,Anetal.(2010)demonstratedthatAMfungalcoloniza-tion levels differed amongmaize plants of different genotypes,andBecklin,Hertweck,andJumpponen (2012) showedthatdif-ferentalpinetreespecieshosteddifferentAMandnon‐AMfungalcommunities.
4.2 | The impact of climate on plant and AM fungal adaptation
Plant performancewas lowerwhen plantswere grownwith theirlocal soil microbial community, suggesting negative intraspecificplant–soil feedbacks,whichmaybedue to anaccumulationof lo-callyadaptedpathogenicmicrobesinthesoil(Felker‐Quinn,Bailey,&Schweitzer,2011;Lankauetal.,2011;vanderPuttenetal.,2013;Wagg,Boller,Schneider,Widmer,&vanderHeijden,2015).Thiscor-respondstofindingsofastronglynegativeplant–soilfeedbackinP. lanceolatabyHarrisonandBardgett(2010),whoshowedthatplantsperformedworsewhen grown in soil conditioned byP. lanceolata thaninsoilpreviouslyconditionedbyamixedplantcommunity.Assuch,ourfindingscontrasttostudiesdetectinglocaladaptationofplantstotheirlocalAMfungalcommunity(Johnsonetal.,2010;Rúaetal.,2016).Interestingly,negativeintraspecificplant–soilfeedbackscanalsobecausedbyachangeintheAMfungalcommunity.AsanexampleofnegativefeedbackmediatedbyAMfungi,Bever(2002)foundthatAMfungiassociatedwithP. lanceolatawerepoorgrowthpromoters,andP. lanceolataplantsgrewbetterwithAMfungifromsoilpreviouslyoccupiedbyanotherplantspecies.Notably,thispat-ternofnegativeplant–soilfeedbackswasnotaffectedbytheabioticenvironmentalconditions(temperatureandsoilmoisture).Thissug-geststhatclimatechangemaynotaffectpatternsof localadapta-tionwithinthecurrentrange,unlesstheindividualspeciesshiftoutoftheirpresentrange.Theoverallnegativeimpactofthelocalsoilcommunityinourstudymayindicatethattemporalchangesinpath-ogencommunitycomposition,orgeneticchangeswithinindividualpathogenspecies,overridechangesinthegeneticsandcommunitycompositionofbeneficialsoilmicrobes.
We detected no signal of AM fungal adaptation to local plantgenotypes,asinferredbythelackofdifferencesinrootcolonizationbetweenplantsgrownintheir localandnon‐localsoil. Incontrast,Johnson et al. (2010) found that AM fungi producedmore fungalstructures when grown in their local compared to non‐local soil,and that locally adaptedmycorrhizasweremoremutualisticwhenresourceswere limited. Inaccordancewith this,Revillini,Gehring,and Johnson (2016) argue that local adaptation of soil mutualistshappenswhenresourcesarescarce, therebypromotingplant–mu-tualistinteractionsinordertoameliorateresourcelimitation.When
resources areplentiful, it promotesopportunistic plantpathogensrelative to commensal and mutualist microbes. The patterns ob-served here for AM fungi could therefore be due to the lack ofresourcescarcitywithintheexperiment,despitetheuseofalow‐nu-trientbackgroundsoil,andsuggestthatwithincreasednutrientlev-elsinnaturalenvironments,pathogensmaybecomeanincreasinglydominantforcecomparedtobeneficialmicrobesinthestructureandevolutionofplantcommunities.Asthereisnoclearcutconsensusonhowtomeasurefungalfitness(Bennett&Bever,2009;Pringle&Taylor,2002),futurestudiesmayincludemultiplepotentialcompo-nentsoffungalfitness,including,forexample,numberofarbusculesandamountofextraradicalhyphae.
To disentangle the effects of the local soil abiotic environ-ment from that of the local soil microbial community, we useda background soil inoculated with live and sterile inocula fromeach location, an approach frequently used in studies on localadaptation and plant–soil feedbacks (e.g. Callaway et al., 2004;Holah&Alexander,1999;Lankauetal.,2011).Weverifiedthatinoculations were successful by assessing whether the fungalcommunitiesthatestablishedwithintheexperimentweresimilarto thosepresent in theoriginal field sites.We found thatwhilesoil at the original costal andmeadow communities overlappedwell with the communities found in treatment roots, the com-munitycompositionfromtheoriginal forestsitediffered inonedimensionof themultivariateordinationspace fromthatof thecommunitiesintheexperiment.Notallsoilfungiwillestablishinpottingsoilorcolonizeroots,andthismaybebehindthediscrep-ancybetweenthefungalcommunitycompositionintherootsoftreatmentplantsandthesoilatoneoftheoriginal locations (asfurthersupportedbythehigherspeciesrichnessinthesoilattheoriginallocations).Overall,acomparisonbetweenthesoilattheoriginalsitesandthefungicolonizingtherootsintheexperimentillustrates that the soil biota that establishwithin experimentalsettingsmayretainahighsimilaritytothesoilbiotaintheorigi-nalsoil,butmayalsodeviatefromtheoriginalsoilbiotainsomeaspects. A frequently overlooked caveat of soil inoculations iswhetherestablishedmicrobesretaintheirfunctioningwithintheexperimentalenvironment, liketheirroleintheexchangeofnu-trientsbetweenplantsandbeneficialmicrobes,andthe(severityof)attackbyharmfulmicrobes.Whilebothlocaladaptationandplant–soilfeedbackstudiesrelyonthefactthatthefunctioningofmicrobesisretainedinexperimentalsettings,thisassumptionis rarely tested, and therefore would be an important area forfutureresearch.
5 | CONCLUSION
Our findings imply that climatic changes (elevated temperatureanddrought)mayhavealargeimpactonplantgrowthandroot‐associated fungal community structure, but that the directionand strength of the response will differ throughout the land-scape due to spatial variation in plant genes and soilmicrobial
| 13Journal of EcologyRASMUSSEN Et Al.
communities.Thissuggeststhatelevatedtemperaturesmayhavevariableoutcomesonplant–soilmicrobe interactions in naturalsystems.Plantsperformedworsewith their local soilmicrobialcommunity,apatternthatmaybedrivenbynegativeintraspecificplant–soilfeedbacks.Interestingly,wedetectednosignsthatcli-mate(i.e.temperatureordrought)willaffectthispatternof(mal)adaptation;however,whenplantsandmicrobesshifttheirdistri-bution intoanewrange,plantperformancemaybehigherthanexpectedduringtheinitialcolonizationstage.Futurestudiesmayfurtherinvestigatetheimportanceof,andbalancebetween,theforcesthatshapeplantadaptationtothelocalsoilmicrobialcom-munity,andadaptationofthebeneficialandpathogenicsoilmi-crobialcommunitytothelocalplantgenotypes.Forthis,wemaycombinedetailedgrowthchamberexperiments focusingon theunderlyingmechanismswithexperimentalfieldmanipulationstoobserve the realized outcome of the ecological and evolution-ary dynamics in natural populations. Such knowledgemay leadtoadvances in theeffectivenessof restorationand sustainablemanagement,aswellasincreaseourabilitytomitigatethecon-sequencesofanthropogenicenvironmentalchangeonplantsandsoilmicrobes.
ACKNOWLEDG EMENTS
WethankLinneaStrömforhelpinthelab,JohanEhrlénforadvice,aswell as seeds fromanddataon, twoof thePlantago lanceolata populationsandMassimoPerniceandAndreasNovotnyforadviceon thebioinformatics.Theauthorsacknowledge funding fromtheMaj and Tor Nessling foundation (2014211 to A.J.M.T.) and theResearch Council Vetenskapsrådet (2015‐03993 to A.J.M.T.). Theauthorsdeclarenoconflictofinterest.
AUTHORS' CONTRIBUTIONS
P.U.R.andA.J.M.T.conceivedanddesignedtheexperiment.P.U.R.conductedtheempiricalandmolecularworkandanalysedthedata.P.U.R.wrotethefirstdraft,andP.U.R.,A.E.B.andA.J.M.T.allcon-tributedtothefinalmanuscript.
DATA AVAIL ABILIT Y S TATEMENT
Dataassociatedwiththisstudyaredeposited intheDryadDigitalRepository: https://doi.org/10.5061/dryad.dt7bh4b (Rasmussen,Bennett, & Tack, 2019). DNA sequences are deposited at NCBIunderBioProjectsPRJNA564044andPRJNA564041.
ORCID
Pil U. Rasmussen https://orcid.org/0000‐0003‐0607‐4230
Alison E. Bennett https://orcid.org/0000‐0002‐1037‐0713
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Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.
How to cite this article:RasmussenPU,BennettAE,TackAJM.Theimpactofelevatedtemperatureanddroughtontheecologyandevolutionofplant–soilmicrobeinteractions.J Ecol. 2019;00:1–16. https://doi.org/10.1111/1365‐2745.13292
