Multiscale climate change impacts on plant diversity in the Atacama Desert
Francisca P. Díaz1,2 | Claudio Latorre3,4 | Gabriela Carrasco‐Puga1,2 | Jamie R. Wood5 | Janet M. Wilmshurst5,6 | Daniela C. Soto1,2 | Theresa L. Cole5,7 | Rodrigo A. Gutiérrez1,2
AbstractComprehendingecologicaldynamicsrequiresnotonlyknowledgeofmoderncom‐munities but also detailed reconstructions of ecosystem history. Ancient DNA(aDNA)metabarcodingallowsbiodiversityresponsestomajorclimaticchangetobeexploredatdifferentspatialandtemporalscales.WeextractedaDNApreservedinfossilrodentmiddenstoreconstructlateQuaternaryvegetationdynamicsinthehy‐peraridAtacamaDesert. By comparing our paleo‐informedmillennial recordwithcontemporaryobservationsofinterannualvariationsindiversity,weshowlocalplantcommunitiesbehavedifferentiallyatdifferenttimescales.Intheinterannual(yearstodecades) time frame, only annual herbaceous expand and contract their distribu‐tional ranges (emerging from persistent seed banks) in response to precipitation,whereasperennialsdistributionappearstobeextraordinarilyresilient.Incontrast,atlongertimescales(thousandsofyears)manyperennialspeciesweredisplacedupto1,000mdownslopeduringpluvialevents.Givenongoingandfuturenaturalandan‐thropogenically inducedclimatechange,our resultsnotonlyprovidebaselines forvegetationintheAtacamaDesert,butalsohelptoinformhowtheseandotherhighmountainplantcommunitiesmayrespondtofluctuationsofclimateinthefuture.
Climate has a major role in the distribution of ecosystems andspecies range on Earth. The last few decades have revealed
overwhelming evidence that climate is changing in the world (Pachauri etal., 2014; Root etal., 2003). How climate change isaffectingspecies,biodiversityandecosystemstructure,functionandservices,bothnowandinthefuture,arequestionsofutmost
importance (Root etal., 2003). Moreover, how existing adapta‐tionscouldpotentiallybufferplantsandotherspeciesfromongo‐ingandfutureclimatechangeremainsunresolved.Understandingthe impact of climate change is particularly relevant in arid re‐gions, where ecosystems are often vulnerable to natural or an‐thropogenic ecosystem changes in water availability (Diaz,Grosjean, &Graumlich, 2003).Moreover, plants in such regionsdisplayremarkableadaptationstosuchdemandingenvironmentalconditions,andlocalcommunitiescanbesurprisinglydiverseanduniqueprovidingrichgeneticresourcesofglobalinterest.
On the western slopes of the Andes, the ecosystems of theAtacamaDesert (whichrangefromhyperarid tohighAndean)canbefoundacrossanelevationalrangespanningmorethan6,000mwhich encompasses pronounced precipitation and temperaturegradients over relatively short distances (~300km). Elevationalgradientsare important tools forunderstandingclimaticandevenevolutionary controls on biodiversity (Arroyo, Squeo, Armesto,&Villagran, 1988; Lenoir,Gégout,Marquet,DeRuffray,&Brisse,2008;Whittaker,Willis,&Field,2001).TheAtacamaisthusanex‐cellent model with which to study biodiversity dynamics and their climatic controls. On interannual timescales and during the late Holocene, Atacama climate variability is drivenmainly by ElNiñoSouthernOscillation(ENSO)(Vuille,Bradley,&Keimig,2000).Afterlarge precipitation events, annual blooms are frequent, increasingplantrichnessandcover,andsoilmicrobialactivity(Schulze‐Makuchetal.,2018).AncientDNA(aDNA)ofplantpathogensalsoincreasedin abundance and diversity in the Atacama during wetter events in thepast (Woodetal.,2018).Onmuch longer (millennial) times‐cales,theAtacamahasexperiencedmajorhydroclimatevariations.Airtemperaturesstartedtowarmfromglacialtointerglacialcondi‐tionsat~19,000calyearbpcoupledwithac.4°Cincreaseinregionalseasurfacetemperature (SST) (Kaiser,Schefuss,Lamy,Mohtadi,&Hebbeln,2008)alongwitha6°Cincreaseinterrestrialtropicaleco‐systems (Thompson,2000).Moistureorprecipitationchanges fol‐lowed,withaperiodofincreasedprecipitation(byaround500mm),originallydescribedastheCentralAndeanPluvialEvent (CAPE) intwodistinctintervals(CAPEI:15,900–13,800andCAPEII:12,700–9,700calyearbp) (Quadeetal.,2008).Here,wefollowtherecom‐mendationofarecentrevisionofdifferentpaleoclimaterecords(DePorras,Maldonado,DePol‐Holz,Latorre,&Betancourt,2017)fromthe central‐southern Atacama Desert, which defines widespreadwetconditionsfrom~17,500–13,800(CAPEI)and12,700–8,500calyear bp(CAPEII).
As each plant species currently inhabits a preferred tempera‐ture and precipitation range (determined mainly by elevation inthis environment), the exact identificationof the plants that livedin thedesert throughdifferent timeperiods canprovide informa‐tionaboutpastclimatechange(Díaz,Latorre,Maldonado,Quade,&Betancourt,2012;Latorre,Betancourt,Rylander,&Quade,2002).DNAmetabarcodingisamoleculartechniqueforidentifyingspeciesusingmolecularmarkers(barcodes)obtainedfromasamplecontain‐ingDNAofmorethanoneorganism(Hebert,Cywinska,&Ball,2003;Taberlet,Coissac,Pompanon,Brochmann,&Willerslev,2012)and
combinedwithaDNAcanbeusedtoreconstructpastdiversityfromancient or fossil samples like lake sediments andother substrates(Alsosetal.,2016;Murrayetal.,2012;Willerslevetal.,2003).
TherecoveryofaDNAorpaleoenvironmentalDNAfromavari‐etyoforganismsisatechniquethathasbeengreatlyimprovedduringthe lastdecade (andnowoften termedpaleogenomics) (Rawlenceetal., 2014;Willerslev&Cooper, 2005). The highest success rateforaDNAextractionhasbeenattainedwithorganismsfromfrozensites(Gould,León,Buffen,&Thompson,2010)andgenerallyfromdry or cold environments, rather than hot or humid sites (Grealyetal., 2016; Haouchar etal., 2014; Poinar etal., 1998; Rawlenceetal.,2014).TheAtacamaDesert,duetoitshyperaridity,thereforeoffers a potentially perfect environment to preserve aDNA (Kuchetal.,2002),butthehighdiurnaltemperaturesandstrongradiationcould also limit this.
Pastvariationsinclimateandvegetationcanbereconstructedbyanalyzing the contents of fossilmiddens of herbivorous rock‐dwell‐ing rodents (Betancourt & Saavedra, 2002; Pearson & Betancourt,2002),which are found in deserts around theworld (Betancourt&Davis,1984;Gil‐Romera,Scott,Marais,&Brook,2007;Murrayetal.,2012).Middensareamalgamationsofplant,animal,andotherorganicremains, embedded in a matrix of crystallized urine (Betancourt &Saavedra,2002).TheycanbepreservedfortensofmillenniainthearidAtacama(withtheoldestsamplesdatingto>50,000yearsbp),offeringexceptional spatiotemporal and taxonomic resolution.Middens pro‐videauniquesourceofancientmaterialpreservedinidealconditions,astheyareusuallyfoundinrockcreviceswhichoffersomeprotectionagainsttheeffectsofhighdiurnaltemperaturesanddirectradiation.
SequenceinformationfromaDNAcanbeusedtoidentifytaxathatareoftennot resolvedbymorphological analyses (plantmac‐rofossils, cuticles, or pollen) and has the potential of providing acomplementaryviewofbiodiversityovertimeascomparedtomoretraditionalmethodologies(Rawlenceetal.,2014;Woodetal.,2012).Although aDNA from one 11,700‐year‐old fossil rodent middenfromtheAtacamadeserthaspreviouslybeenusedtoreconstructpast rodent distributions (Kuch etal., 2002), we present the firstapplicationofDNAmetabarcodingandnext‐generationsequencingforentireplantcommunities,fromatimeseriesofmiddensspanning27,590–200yearsbeforepresent.
Ourgoal istounderstandhowdifferentmagnitudesofclimatechangeaffectplantcommunities in theAndeanAtacama.To inte‐grate spatial and temporal changes in plant biodiversity from an‐nual tomillennial timescales,wecompareaDNAdatafromrodentmiddens with contemporary plant biodiversity information overthecourseofeightconsecutiveyearsacrossanaltitudinalgradient(from2,500to4,500ma.s.l.)withstrongprecipitationdifferences(from10to300mm/year).Plantcommunitycompositionshouldbeaffectedinproportiontothemagnitudeofclimatechange,butthechallengebehindthishypothesis is todefine thesensitivityof thesystem.Forinstance,howhasvegetationrespondedtothelastde‐cadeofclimatechangeincontrasttomuchlongertimescaleswithmuch greater magnitudes of climate change (i.e., across the LastGlacial–interglacialtransition)(Nolanetal.,2018).
F I G U R E 1 Climatechangeoverthedifferenttimescalespresentinourstudy.(a)Last30,000calyearbp.Blackline:ModeledprecipitationratesfortheBolivianAltiplanoat3,700ma.s.l.(m)(Placzek,Quade,&Patchett,2013).Rainfallscale(0–900mm/year)isshowninblackontheleft.Orangedots:MinimumMeanannualrainfall(MAR)estimatedfromfossilvegetationat3,100m(Latorreetal.,2006).Rainfallscale(0–140mm/year)isshowninorangeontheleft.Blueline:TemperatureestimatedfromtheAntarcticaEpicaDomeC(temperaturedifferencefromtheaverageofthelast1,000years)(Jouzel&Masson‐Delmotte,2007).Grayboxes:CentralAndeanPluvialEvent(CAPE)IncludesCAPEI(17,500–13,800calyearbp)andCAPEII(12,700–8,500calyearbp)(DePorrasetal.,2017,modifiedfromQuadeetal.,2008).(b)Last40years.Blackline:Averagetotalannualrainfall(Dataobtainedfrom20DGAstationsat22–24°latSand2,300–4,400m,Antofagastaregion).Uppergraylimitrepresentsaverageprecipitationat4,400–4,000mandlowergraylimitrepresentsaverageprecipitationat2,300–2,700m.Redline:AnnualMultivariateENSOIndex(Dataobtainedfromwww.esrl.noaa.gov)(Wolter&Timlin,2011).CorrelationbetweenannualaveragerainfallandMEIisnotsignificant(r2=0.04,p>0.05).(c)Last8years.Blacklineandgraylimits:SameasinB.Greenline:Totalplantrichnessobservedinthestudyareaaftertherainyseason(Talabre‐Lejíatransect,2,500–4,500m).Correlationbetweenaveragerainfallandplantrichnessisr2=0.21,p>0.05.CorrelationbetweenplantrichnessandMEIisr2=0.39,p>0.05
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InnorthernChile's regionofAntofagasta lie thedriestareas in theworldasitcorrespondstothemaximumpenetrationoftheAtacamaDesert into the interior from the Pacific coast. Seasonal timing ofprecipitation determines a single season productivity pulse in theAtacama(aftersummer,duringMarchtoApril).Meanannualrainfall,obtainedfrom20DirecciónGeneraldeAguas,Chile(DGA)weatherstations locatedat22–24° latSand2,300–4,400m is54mm/year(Figure1b). The elevation gradient and their close interactionwithincreasing rainfall and decreasing temperatures (among other fac‐torssuchasgrazingorhumanimpacts)giverisetodistinctvegeta‐tion beltswhich can be consistently recognized across the region,based on overall plant physiognomy (Figure2a). However, species
Present relationships between environmental variables andplant distributions were established with vegetation surveys per‐formedduring the firstweekofAprilovereight consecutiveyears
F I G U R E 2 VegetationbeltsintheAndeanAtacamaDesertandcomparisonacrossdifferenttimescalesshowingmajorelevationalrangesofplanttaxainthestudysites.(a)PhotographsofvegetationbeltsintheAndeanAtacama.(b)Boxplotsshowtheinterannualvariabilityofrangedistributionsofannualandperennialtaxaobservedduringthe8yearsofvegetationsurveys.Elevationchangeisshownasannualvariationsalongtheupperandlowerlimitsofdistributionsforeachspecies,comparedtotheaveragepresent‐dayelevation(averagedoverthe8yearsofthestudy).Outliersrepresentspecieswithlowcoverage(onlyfoundinsomeyears).(c)Long‐termrangechangescomparedtopresent‐dayranges(metersbeloworabovetheircurrentdistribution).ZerohereisdefinedasthemaximumdistributionrangeestimatedfromA.Forinstance,ifaspecieswasfoundina10,000calyearbpmiddencollectedat3,000mandthatspeciesgrowstodaybetween4,000–4,200m,itwillappearasapointat−1,000m(theactualelevationalchange).Asmostmiddenswerecollectedalongthelowerlimitofthevegetation,theselimitstendtodescendinthepast,whichgivesagoodestimateoftheirpastlowerlimitofdistribution
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(2011–2018)andfollowingevery100mofelevation(22sites)acrossan elevation gradient from approximately 2,500 to 4,500m a.s.l.(Talabre‐Lejíatransect,TLT)(seeDíaz,Frugone,Gutiérrez,&Latorre,2016formoredetails).Atotalof61plantspecieswerecollectedandfrozentoconstructalocalDNAreferencedatabase(Atacama data‐base)acrossthisgradient(TableS1).Plantspeciesrichnesswasmea‐sured using two 250 m2plotsateachofthe22sitesoveralltheyearsof the study (Figure1c). Species observed permanence (i.e., howmanyyearsthesamespeciesappearedateachsite)wascalculatedforeachsiteandalongeachspeciesdistributionrange(minandmaxaltitude) aswell as for the averageupper and lower limit of distri‐butionofeachplantspecies in the last8years’surveys.Additionaldata regardingperennialspeciesdistribution (usingasimilarcollec‐tionmethod)wereavailablefromMay1998alongthesametransect(Latorre, 2002). Simple linearmodels were used for assessing anypossiblecorrelationswithPearson'scorrelationcoefficient,analyseswereperformedintherProgrammingLanguage(RCoreTeam,2018).
2.2 | Barcodes database
Using local DNA barcode databases can significantly increasethe resolutionorcapacity to identify taxa fromacomplexsample(Taberletetal.,2007).Toimproveresolutionandsubsequentanaly‐sisofaDNAreconstruction,weconstructedalocalbarcodeAtacama database using the species collectedduring the plant surveys andsomeadditionalspeciesthatwecollectedpreviouslyfromloweral‐titudesites(TableS1).Theseplantswereidentified,andspecimenswere deposited at the Herbarium at Universidad de Concepción,Chile (“Herbariode laUniversidaddeConcepciónCONC”;specieslistandcodesinTableS1).WeextractedDNAfromthesespecimensusingthePureLinkPlanttotalDNApurificationkit(Invitrogen)fol‐lowing themanufacturer's protocol.We amplified the chloroplasttrnLregionusingprimerscanddasdescribedbyTaberlet(Taberletetal.,2007).Amplificationwasperformedin25μl containing 0.4 μl of Phusion High‐Fidelity DNA Polymerase (Thermo Scientific,EEUU);8.0μlofenzymebuffer;0.2μlofeach10mMdNTP(ThermoScientific,EEUU);2.0μl fromeach10mMprimerand4.0μlofex‐tracted DNA (between 10 and 50 ng/μl). Following an activationstepof30s at95°C for theenzyme, thePCRmixtureunderwent25cyclesof5sat95°C,30sat55°Cand5minat72°Cona2720ThermalCycler(AppliedBiosystems,EEUU).
WeobtainedtrnLsequencesorbarcodesfromthose61speciesto construct the Atacama database (GenBank accession numbersMH115328–MH115388). The resolution of the selected barcode
2.3 | Fossil rodent middens collections and radiocarbon dating
All middens (N:26) were previously collected in Cerros de Aiquina(Antofagastaregion,northernChile,from2,414to3,380ma.s.l.)andradiocarbondatedbyC.Latorreandcollaborators(Latorre,Betancourt,Rylander,Quade,&Matthei,2003)(seeTableS2fordetails).Middensaredepositedepisodically andmostlydiscontinuously, neverthelessouragesdistributioncoversthedifferentclimaticeventsdescribedinFigure1a.Aftercollection,middenswereprocessedtoidentifymac‐rofossilsandpollen(resultspublishedinDePorrasetal.,2017;Latorreetal.,2003,comparisonsinFigure3)andanunprocessedsubsampleofeachmiddenwasretainedasavoucherspecimen(usedheretoex‐tractaDNA).FigureS1showsphotographsandaworkflowsummaryfortheaDNArecoveryandanalyses.Acceleratormassspectrometryradiocarbondatingwasperformedatdifferent facilities, seedetailsinTableS2.RadiocarbondatesforthemiddenswerecalibratedusingShCal13(Hoggetal.,2013)inOxCalv.4.3.2(Ramsey,1995)andme‐dianageswereusedforanalyses(TableS1).Twoofthemiddensweredeterminedtobemodern(after14Cdating)andtheothersarefrom49,600to20014C year bp.However, in thisstudyweconsideronlysamplesfrom27,590to200calyearbp,becausewecouldnotamplifyplantDNAfromtheoldersample(seeSection3.2inResults).
2.4 | Midden subsampling, aDNA extraction and amplification
Torecover thedamagedaDNAandavoidmodernDNAcontamina‐tion,subsampling,extractionandamplificationwereperformedatthespecializedaDNAlaboratoryatManaakiWhenua‐LandcareResearchLong‐TermEcologyLaboratory,Lincoln,NewZealand(FigureS1).First,weisolatedaninnerpieceofmaterialfrom24fossilrodentmiddens,following the protocol for subsampling late Quaternary coprolites(detailedinWood&Wilmshurst,2016).Thesubsamplingprocedurewascarriedoutinasterilizedsubsamplinghoodandconsistedof:ir‐radiatingthesamplewithUVC,removingtheoutersurface,irradiat‐ingagain,bisectingthesampleandtakingasampleforaDNAanalysisfromtheinteriorofonehalf.Toolsandthesamplingboxweresteri‐lizedbetweensamplesusingbleach,ethanol,andUVC.
F I G U R E 3 Numberoftaxaidentifiedfromthesamefossilrodentmiddens(CerrosdeAiquinaorCDAsites)usingdifferenttechniques.Macrofossildatawereobtainedfrom(Latorreetal.,2003)andpollendatawereobtainedfrom(DePorrasetal.,2017)
SubsamplingwasundertakeninsidethephysicallyisolatedaDNAlaboratorywhereportionsweighing5gwererehydratedinH2Ofor24hr.Tocheckforpossiblecontamination(Cooper&Poinar,2000;Rawlence etal., 2014;Wood&Wilmshurst, 2016),we carried outextractionblanksalongsideactualtemplatesamples.TheaDNAex‐tractionwasperformedusingthePowerMaxsoilDNAisolationkit(MoBio).Fromtwomiddens,wedidextractionreplicatestodetectheterogeneity inside themiddenand insamplesize.Sincesuitablemetabarcodes foraDNAstudies shouldbeno longer than300bp,but typicallyaround~130bpforplants (Rawlenceetal.,2014),weamplifiedasmallersectionoftrnL(163–228bp),usingprimerscandh(includingtheP6loop)describedin(Taberletetal.,2007).Assomeinconsistent results betweenPCRsof the same samplehavebeenshown in amplifications from environmental or complex samples(Ficetolaetal.,2015),wedidtwoindependentamplificationsusingthesameprimersforeachsample.Wemodifiedtheprimersaddingalinkersequence.Toavoidincorrectdetectionofclustersofourlownucleotidediversitylibrariesduringnext‐generationsequencing,weusedamixedsetoffourdifferentlengthprimerswiththesametar‐get(trnLc‐h),butwith0,1,2,or3nucleotidespacersbetweenthetrnLprimersandlinkersequence.TheaDNAPCRprotocolforonereaction (25 μl)was:BSA2.5μl,Buffer10×2.5μl,MgSO4 1 μl,dNTP0.2 μl,Fprimer0.5μl,Rprimer0.5μl,TaqPlatinumHiFi0.25,H2O 15.3 μl and DNA 2 μl(PCRprotocol:94°3:00then50–55cycles[94°0:30;55°0:30;68°0:45]and68°10:00).WealsofollowedaShrimpDNAaseprotocol (0.25μl:DNAasediluted10times,15minat37°and15minat65°)toremovepossiblecontaminantDNAfrommastermixbeforeaddingtheaDNA.ExtractioncontrolsandPCRnegativecontrols were included in all PCR reactions. We checked the ex‐tractionandamplificationsuccessbyvisualizingtheampliconsinanagarosegelelectrophoresis.DNAamplificationandalldownstreamprocedures (quantification,purification,andsequencing)wereper‐formedinlaboratoriesphysicallyseparatedfromtheaDNAlabora‐tory.Wesequencedbothamplificationreplicatesfromeachsample.Tocontrolforpossiblecontamination,wealsosequencedthreeex‐tractionblanks,whichweredoneinparallelwiththeextractedsam‐ples.AsweusedPCRtoamplify,itwasnotpossibletoinvestigatethequalityofDNApreservationbecausetheendsofDNAmoleculesarenotrevealed.ButwefollowothercriteriaofauthenticityforaDNA(Paaboetal.,2004)suchasthementionedextractioncontrols,PCRcontrolsandrepeatedamplificationsfromallextracts.
2.5 | Libraries preparation and sequencing
Topreparethelibraries,wefollowedtheprotocol“16SMetagenomicSequencing Library Preparation” (Illumina, 2013), adapted to ourmarker trnL (c‐h). We did a PCR clean‐up using magnetic beads(AgencourtAMPureXP),cleaningbothsizeextremes.Weranasec‐ondamplificationusing Illuminaadaptorswith the Illumina indices(NexteraXT)andweranasecondClean‐upusingthesamemethod.WequantifiedthelibrariesusingtheIlluminaLibraryQuantificationKit, Universal qPCR Mix (KAPA Biosystems) and real‐time PCRand validate them through capillary electrophoresis. Finally, we
Togeneratethedatabasesandanalyzethesequencingresults,weusedtheOBIToolspackage (Boyeretal.,2016).Weprocessedthedata, constructing a local Atacama database with our sequencesobtainedfromthevegetationsurveysandaglobalDDBJdatabaseperforminganinsilicoPCRtoDDBJ(Mashimaetal.,2016)(mirrorofGenBank)withourprimersusingecoPCRtool(Ficetolaetal.,2010).TheaDNAsequenceswereprocessedpairingforwardandreversereads,removing“joined”pairs.Then,wedereplicatedthereads,andfiltered thembycountnumber (more than5 reads) andby length(between60and240bp).Finally,we identifiedand removedpos‐siblePCRerrorsanddidthetaxonomicassignation(topscoretaxon,morethan95%ofidentity)tobothdatabasesusingNCBItaxonomydatabaseasreference(Federhen,2011).TheAtacama database was usedtoidentifyinthefirstplace.TheDDBJdatabasewasusedtoidentify missing taxa, higher taxonomic levels and contaminants(TableS4).Threelow‐qualitysamples(withmorethan10%ofcon‐taminants)werenotincludedinposterioranalyses.Weselected29(outof73)ofthemostinformativeplanttaxatoconstructtheresultsdiagraminFigure4(mostlyidentifiedtospeciesorgenuslevelandrepresentativeofAtacamavegetationbelts).
Consideringthatourmiddenseriesarenotcontinuouslydepos‐ited,andhavearelativelylowsamplesize,wewerenotabletodotime‐seriesstatisticalanalyses.Resultsarepresentedastaxaabun‐dancechartsandtaxaelevationdisplacementcomparedtothepres‐ent‐dayranges(metersbeloworabovetheircurrentdistribution).TocomparePCRandextractionreplicates,wecalculatedaBray–Curtisdissimilarity index, and then compared groups using an ANOVAfollowedbyaTukeymeandifferencestest(FigureS2).AllanalysesandchartswereperformedintheRProgrammingLanguage(RCoreTeam,2018),usingpackagerioja(Juggins,2007).
2.7 | Data availability statement
Sequencedata(localplantbarcodes)thatsupportthefindingsofthisstudy have been deposited inGenBankwith the accession codesMH115328–MH115388. Unprocessed reads from each middensample (.fastq) of this study are available from the correspondingauthoruponreasonablerequest.
3 | RESULTS
3.1 | Perennial plant species in high Andean Atacama exhibit resilience to short‐term climate changes
How vegetation adjusts to short‐term and interannual environ‐mentalchangesisrelevantforthedevelopmentofcomprehensive
diversitystudiesinaglobalchangescenario(Figure1).Thespeciesaccumulationcurvefromourstudyareashowsthatittookatleast6yearsofsurveystofindthebaselineofplantbiodiversity(>90%ofobservedspeciesandcurvesaturation).Asexpected,precipitationseemstobethemaindriverof interannualproductivityandplantspecies richness (Figure1c). Although other factors, such as tim‐ingofprecipitationandtemperaturechanges,werealsoimportantforgerminationofsomespecies.Forinstance,weobservedanim‐portantannualbloomofLupinus(reachingalmost50%ofsoilcoverin somesites)onlyduringone “LaNiña”year in2012 (Figure1b).Thisspecieswasnotregisteredinotheryearswithcomparablepre‐cipitation (2017)or another “LaNiña”year in2018, indicatinganinteractionofenvironmentalfactorsisrequiredformajorchangesin Lupinusgerminationrates.TheseresultsunderscoreasurprisingpersistenceofLupinusseedbanks,andalsotheimportanceofsus‐tainedeffortsovertimetounderstandbiodiversity,particularlyinextreme environments.
distribution during the 8years of surveys. In addition, perennialplantsshowedthesamedistributioninthelastyearsascomparedtoa survey from1998 (Villagránetal.,1998). Incontrast, annualherbaceous species contracted and expanded their distributionrangeeachyear(probablygerminatingfromapersistentsoilseedbank),accordingtointerannualweatherconditions(Figures1cand2b) and their permanence at each sitewas generally low (on av‐erageeachannualwasseeninthreeoutofthe8years).Theout‐comeofoursurveysunderscoresasurprisingpersistenceofseedbanksforsomeannuals (perhapsfordecades,andnot justyears)inahyperariddesert.Theseresultsindicatedistributionofperen‐nialplants isresilienttoshort‐term(annualanddecadal)environ‐mentalchangesandannualplantsshowedmoreflexibilityintheirobserveddistributionaccordingtoenvironmentalparameters,butprobablyalsoasaproductofaresilientsoilseedbank.Thesere‐sultsalsosuggestthattoproducesignificantcommunitychangesinperennialspecies,themagnitudeofclimatechangeshouldbemoreextensive in time or magnitude than what we have observed in the last two decades.
F I G U R E 4 Selectedplanttaxaidentifiedin21middensusingaDNA.Barsrepresentreadabundance(normalizedfrom1to10)andcolorshadowsbehindbarsareexaggeratedcurvestoshowtendencies.(*)Highlightnewtaxa,notpreviouslyidentifiedbymacrofossilorpollenanalyses.ConstrainedIncrementalSumsofSquaresclusteranalysis(CONISS)indicatesthesimilarityamongmiddenswithinsimilargroups(redlines).Allmiddenswerecollectedaround3,100ma.s.l.(from3,000to3,400ma.s.l.),excepttheoldestmidden(27,590calyearbp)whichwascollectedat2,414ma.s.l
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3.2 | Using DNA barcodes increases taxonomic resolution and identification of plant species in samples as old as 27,590 years
Tounderstanddiversity changesover longerperiodsof time thanispossibletoobservebystandardfieldsurveys,weobtainedhigh‐quality aDNA, amplified and sequenced the trnL barcode in fossilsamples (dated200–27,590calyearbp)andfrommodernmiddensusing Illumina technology (see Figure S1 formidden photographsand a workflow summary of aDNA recovery and analyses). Fivelow‐quality samples were not included in posterior analyses (seeMaterialsandMethodsfordetails).Forinstance,theoldestmidden(49,60014C year bp)had92%of reads identifiedascontamination(assigned as Pottiaceae), and due to the absence of Pottiaceae inthe study region (and itspresence in thenegativecontrol)wedidnotincludethissampleinthefinalanalyses.Forothersamples,ex‐traction and amplification success were independent of age. Weobtainedameanof115,000readspermidden/samplefor21mid‐dens (seedetails inTableS2).Wecompared replicates anddiffer‐ent samplesusing theBray–Curtisdissimilarity index (BCI) (FigureS2).ExtractionreplicatesfromtwomiddensrevealsimilaridentifiedtaxawithameanBCIof0.3.PCRreplicatesforallsamplesyieldedsimilar results (mean BCI of 0.3) and were significantly differentcompared to independent samples (meanBCI:0.8).Therefore,weaveraged and combined PCR replicates for posterior analysis.WesequencedthreePCRnegativecontrols,obtaininglessthan70reads(mostly Asteraceae, Pottiaceae, Pooideae), except for one samplefromwhich3,000readswereobtained(mostlyTriticeaetribe),andremovedthesetaxafromposterioranalyses.Ofthesequences,94%weretaxonomicallyassignedusingbothplantdatabases.Ofthetotalassignedreads,99%wereclassifiedasAtacamataxaand1%ascon‐taminants (mainly fromvegetationoutside the laboratoryor food,seeTableS4).Weidentifiedatotalof73Atacamaplanttaxa(25spe‐cies,46genera,and26families)(TableS3)(Figures3and4).
We compared our aDNA record with pollen (De Porras etal.,2017) andmacrofossil (Latorreetal., 2003) reconstructionsprevi‐ouslyanalyzedinthesesamemiddens(Figure3).Thetotalnumber
of taxa was highest for aDNA (n=73), followed by macrofossils(n=63)andfinallypollen(n=24).Atotalof29plantfamilieswereidentifiedbyallthreemethods,butninewereidentifiedexclusivelybyaDNA(Figure3).Usingpaleogenomics,wewereabletoidentify14 species that were not previously recorded in either pollen ormacrofossil records.WebelievetheuseofaDNAandbarcodese‐quencingprovidesandaccurate,complementaryand,insomecases,morecompleteassessmentofbiodiversityovertimeinthesefossilsamplescomparedwithwhatcanbeobtainedwithtraditionalexper‐imentalapproachesalone.
3.3 | Major ecosystem responses to climate change in the past
To assess biodiversity in Atacama plant communities over the last27,590calyearbp,weusedourbarcodesequencedatafromaDNAandcomparedspeciesoccurrencetocurrentplantdistributionsandplantassemblagesfrommodernmiddensforwhichweusedthesameDNAbarcodes.Figure4showsselectedplanttaxa(29of73)andtheirap‐pearancesovertime.Asplanttolerancedefinesthealtitudeatwhichwe finda species,wepresent the results in relationship toexistingvegetationbelts (i.e.,Prepuna,PunaorSteppe,Figures2aand5) tofacilitateinterpretations.Wecomparedshort‐termdistributionrangeofeachplantspecieswith long‐termpastdistributions (Figure2b,c).DuringtheLastGlacial,at27,590calyearbp richness is very low and justafewPrepunaspecieswereidentified.Themostdramaticchangesoccurred from 17,000 to 7,000cal year bp, when Puna and Steppeplantspeciesoccurredalmost1,000mlowerthantoday(Figure2b).Nevertheless,Prepunaspecieswerealsopresentduringthatperiod,generating mixed and novel plant communities (Figure5b). Duringthelast7,000calyearbpplantspeciesrichnessislowanddominatedbyPrepunaspecies,exceptforthe200calyearbp midden which has morePunaelements,indicativeofmorehumidconditionsthantoday.
Atmillennialtimescales,ourresultsshowthat largemagnitudeclimatechanges(inlengthorintensity)triggeredbothperennialsandannualstomigratedownslopewherewaterlimitationandtempera‐tures are extreme today.
F I G U R E 5 Present‐daydistributionvs.majorpastvegetationchangeintheAtacamaDesert.(a)Currentvegetationbeltsat23°24′lat.S,AtacamaDesert,Chile.Aselevationandprecipitationincrease,temperaturedecreases.Whiteregionsindicateabsenceofplants.(b)Pastdownslopeplantmovementsduringpluvialeventsprovokedamixingofdifferentvegetationbeltsor“nonanalogcommunities”from2,500to3,500m.Thequestionmarkabove4,000mrepresentsourlackofmiddensfromhighaltitudes(allmiddensitesarefrom2,414to3,380m,seeTableS2fordetails)
(a) Present day (late-Holocene) (b) Pluvial events (late-Pleistocene and early Holocene)
Relative Distance0 km 25 km Relative Distance0 km 25 km
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4 | DISCUSSION
Long‐termpaleoecologicalstudieslinkedtoshort‐termbiodiversitystudiesarefundamentaltounderstandingvegetationresponsestoongoing (anthropogenicallydriven) climatechange. InAndeanaridecosystems, we are severely data‐limited on both of these time‐scales. Fossil rodent middens are unique archives of biodiversityandanimportantsourceofinformationfromthepast.SequencingaDNAfrommiddensisnowpossible(Kuchetal.,2002;Murrayetal.,2012),andthisapproachoffersgreatopportunitiestoaddressques‐tionsofchangesinbiodiversityoverlongperiodsoftime.Thisareaofresearch,attheintersectionofgenomicsandpaleobotany,shouldbecomeanimportantareaofdiscoveriestounderstandvegetationresponsestoclimatechange.
4.1 | An aDNA record from fossil rodent middens
Weuse radiocarbondated fossil rodentmiddens andplant aDNAmetabarcoding to describe plant community changes up to27,590yearsbeforepresent.Onlyonepreviousstudy (Kuchetal.,2002)hassuccessfullyextractedDNAfromanAtacamarodentmid‐denbutusedSangersequencingfromindividualclones.WecompareouraDNArecordwithpollenandmacrofossilsreconstructions,andconcludethatdifferentexperimentalapproacheshavestrengthsandweaknessesandshouldthereforebeconsideredascomplementary(Figure3)(Parduccietal.,2015;Woodetal.,2012,2013).Thenum‐berofgenerawassimilarbetweenaDNAandmacrofossils,andbothrecordsarecomplementaryintermsofidentification.Morethanhalfof thegenerawere identified in justonetypeof record (aDNAormacrofossils).Plantmacrofossilsstillprovidethehighestnumberofidentifiedspecies(37),butaDNAwasabletoidentify14newspecies(includingsomekeyindicatorsspeciesfromtheSteppebelt,seetaxamarkedwithanasteriskinFigure4)notfoundasmacrofossilsorpol‐len(Figure3).Thecombineduseofpollenandmacrofossilsformid‐denanalysesistime‐consumingandinvolvesahighlevelofexpertisetoidentifylocalflorasfromthefragmentaryplantremainsfoundinmiddens. Incontrast,aDNArequiresspecialized facilities forDNAextractionfromfossilsamplesandPCRamplification,butifDNAiswell‐preserved taxonomic resolutiononly requiresa localbarcodedatabase.The fact thatouraDNAanalyseswereable to replicateandoftenexpandonthetotaldiversityindicatesthistechnologyasan importantcomplement to standardmiddenanalysis.Moreover,ourstudyprovidesareferencedatabaseforfuturestudiesofthesespecies.
Aswithotherproxies,theuseofaDNAtoreconstructbiodiver‐sityislimitedbytheresolutionofthemolecularmarkersorbarcodesobtained frommodern floras. ThedevelopmentofDNAbarcodesfromlocalflorasisafundamentalsteptoimprovetheresolutionofanyaDNArecord.Theserecordscanalsobefurtherbiasedbytheuniquetaphonomicenvironmentandageofthemidden.Therefore,more than establishing comparisonswith the entiremodern plantcommunities we use specific plants as key indicators of past cli‐mate change. DNA originates from different sources, including
pollenfrombothlocalandregionalsources(DePorras,Maldonado,Zamora‐Allendes,&Latorre,2015),particularlyinthecaseofwind‐pollinatedspecies.Sincetheephemeralappearanceofsomespeciesand the limitationsmentionedbefore for identifyingspeciesusingDNAbarcodes,somelocalflorafromthemiddencollectionsitesdonotappearintheDNAfrommodernmiddens,butalsoDNArevealspeciesthatwedidnotobserveinthefield.Nevertheless,DNAfrommodern middens shows that the reconstructed communities are similartopresent‐daycommunities,indicatingthatregionalsourcesofDNAlikelycontributeverylittletotherecord.Middensaccumu‐latedoverafewyearsoradecadeandcouldreflectawidertemporalframethanaone‐timepointsurvey.
4.2 | Integrating multiscale climate change with biodiversity dynamics
Plantcommunitiesandwhathasbeendefinedasvegetationbeltsaretemporaryagglomerationsofspeciesconstantlyrespondingtoclimatic,biologic,andanthropogenicfactors.Moreover, thosecur‐rentassemblagesareephemeralatthemillennialtimescalealthoughtheirpersistenceat shorter timescales is still unknown.Withpro‐nounced rainfall seasonality and interannual variability, thedesertvegetation of the Atacama can be challenging to study regardingshort‐termecologicalresponses.Annualsexpandandcontracttheirdistributionalranges inresponsetoprecipitationintheshortterm(yearstodecades)andtheirpermanenceateachsiteisgenerallylow.Thesespeciescanrespondquickly,andtheseedbanksareabundantandpersistentenoughtobridgeacrossscarceprecipitationevents.Such strategies allow plants to remain sensitive to environmentalchange of relatively little magnitude, such as ENSO variations. Incontrast, the elevational distribution of perennials did not changeover the entire sampling period (2011–2018) and did not changeevenwhenweincludedasurveyfrom1998(Villagránetal.,1998).Thisabsenceofchangeisunmistakableasareductioninmeanan‐nual precipitation (Sarricolea,Meseguer Ruiz, & Romero‐Aravena,2017)andanincreaseinmeanannualtemperature(Bennett,New,Marino,&Sillero‐Zubiri,2016)hasoccurredover the lastdecadesinAtacamaDesert.Thissuggests thatneitherENSOvariationnorrecentclimatechangehasbeenofsufficientmagnitudeoverthelast20yearstoinduceanysignificantelevationalmigrationinperenni‐alsorplantcommunitychanges.Thisresiliencecouldbeexplainedby theuniqueadaptations tovariablearidandhyperaridAtacamaconditionsorcouldbetheexpressionofanextinctiondebtor“ex‐tinctionlag,”ashasbeendescribedinotherhigh‐altitudeplantcom‐munities(Alexanderetal.,2018;Dullingeretal.,2012).
Incontrast,over longer timescales (thousandsofyears)stress‐resistantperennialherbaceousandshrubs(e.g.,Arroyoetal.,1988)appear to respond to larger or sustained climate change, such asexperiencedbyplantsduringtheLastGlacialperiodorthepluvialeventsatthePleistocene‐Holocenetransition (e.g.,CAPEIandII).Thebiggestchangescomparedtothepresentoccurredfrom17,000to 7,000cal bp, when plant species were found up to a 1,000mbelowtheircurrentdistribution(Figure2c),likelydrivenbyincreases
10 | DÍAZ et Al.
inprecipitationduringmajorpluvialevents(e.g.,Latorreetal.,2002,2003).ThevegetationidentifiedusingaDNArevealssomeimport‐ant differences between CAPE I and CAPE II. Diverse perennialshrubsandfewSteppegrassesarepartofCAPEIassemblages,asfoundinourmiddensdatedfrom16,000to15,000calyearBP.Incontrast, middens dated between 10,500 and 8,500cal year BP(CAPEII)showdominanceofParastrephia/Baccharis (both high ele‐vationPunashrubs)andSteppegrasses.ThesedifferencesbetweenthetwowetphasesofCAPEshowthatCAPEIIwaspossiblywetterandlikelywarmerthanCAPEI.
SuchmovementswerenotlimitedtothePleistocene,however,andourdatashowthatafewtaxadescended~800moverthelast3,000calbp,indicatingthatplantdistributionsrespondedtoshortertermclimatevariationsduringthelateHoloceneandoncentennialtimescalesduringtheHistoricPluvialEventattheendoftheLittleIceAge(c.1,400–1,900CE,seeLima,Christie,Santoro,&Latorre,2016; Mujica etal., 2015). Since plant diversity and productivityhave strong bottom‐up effects (Scherber etal., 2010), major pastplantchangeswouldbefeltthroughouttheentireAndeanAtacamaDesert ecosystem (Marquet etal., 1998). As interannual and sea‐sonal climate variability have strong effects on short‐lived herbi‐vores,suchasrodents(Meserve&Glanz,1978),climatevariabilityonlengthiertimescalesshouldimpactlong‐livedherbivoresandcar‐nivoressuchasfoxesandsouthAmericancamelids(Marquetetal.,1998).
Althoughtotalspeciesrichnessincreasedduringpluvialevents,vegetationbeltswerenotcompletelydisplaceddownslope.Instead,Prepuna communities were enriched by species moving in fromhigher elevations (Puna and Steppe) (Figure5b), generatingmixednovelplantcommunitiesor“nonanalogcommunities”(e.g.,Williams& Jackson, 2007). Temperature changes, although important inexplaining the presence of extreme xerophytes found at higherelevationswhere theygrowtoday (Latorre,Betancourt,&Arroyo,2006)weremostlikelynotimportantinexplainingthesedownslopeshifts. Indeed, colder temperatures during the Pleistocene (~6°colder;Thompson,2000)wouldhave forcedmanySteppespeciesdownslopefromtheAltiplano(whereitwouldhavebeentoocold;Mujica etal., 2015) but the presence of such species inwhat aretodayPrepunaenvironments,requiresincreasesinprecipitationasthecurrentenvironmentwouldhavebeentoodrytosupportthem(Latorreetal.,2006).Indeed,aspreviouslyshowninplantmacrofos‐silrecords,aDNAfloraduringtheverycoldtemperaturesoftheLastGlacialMaximumweremostlycomposedofjustafewPrepunaspe‐cies(Figure4).Clearly,thehigherelevationbelt(Steppe)exhibitsthelargestelevationalshifts.Inpart,this isduetowherethemiddenswerecollected(inlowerelevationbelts)butanothercausecouldre‐sideinthefactthathighAndeanvegetationrequiresmorefrequentand largeramountsofwater (rainfall)which inturnmakes itmuchmoresensitivetopastand(mostlikely)futureclimatechange.
Vegetation belts at our study sites (and in general for theAtacamaDeserthighlands) arehighlydynamic and their composi‐tionappears tobemostlydrivenbypast climatechangealthoughtheextentofhumanimpacts(bothhistoricalandinprehistory)has
notbeenfullyaddressedintheseecosystemsandremainsanopenquestion.PreviousstudiesintheAtacamashowhowrecentclimatefluctuationsoverthepast600yearsappeartobedrivingchangesinrichness (Mujicaetal.,2015)andthatevenhumanpopulationsre‐spondtothesesamechanges(Limaetal.,2016).Here,weevaluatedtheeffectofrecentclimatechange(overthelasttwodecades)andobservedthat theperennialplantdistributionshavechangedverylittlefrom1998topresent.AlthoughpasthumanoccupationsintheAtacamapeakedaround1,000yearago(Gayo,Latorre,&Santoro,2015),thehyperaridnatureofthedesertpreservespastlandscapeuse.Thus,humanpopulationdensityaroundoursiteswaslikelyverylowinthepastandisevenlowertoday,thusminimizingimpactonthenatural landscapewherewechoseoursites.Furthermore,oursites are close to a small road, but away fromanyevidenthumanperturbationsuchassettlements,majorminingcompanies,farmingterraces, fences, or garbage. The onlymajor human impact todaycouldbeattributedtodomesticatedllamasroamingthelandscape,buttheirpopulationdensityislowandthepressuretheseherdscur‐rentlyexertcouldbeofthesamemagnitudeastheherbivorouspres‐suresexertedbywildmegafauna(includingguanacos)inthepast.
Howvegetationadjuststoshiftingenvironmentalconditions,andthespeciesrichnessandcommunitydiversitypatternsthatcanarisefromsuchrestructuring,isrelevantforthedevelopmentofmorecom‐prehensivediversitystudiesandmodels(Whittakeretal.,2001).Forexample,regionalclimatemodelsfornorthernChilepredictincreasesin temperature (4°Chigher than today) anddecreases inprecipita‐tion (10%–30% lower than today)over thenext century (Minvielle&Garreaud, 2011; Thibeault, Seth,&Wang, 2012). SuchpotentialfutureclimatechangeissimilarinmagnitudetothatwhichoccurredduringtheLastGlacial–interglacialtransitionandoveratimescaleofseveralthousandsofyears.Inafast‐changingworld,Atacamaplantcommunitieswillmostlikelyundergoprofoundchangesasperenni‐alsaswellasannualsadjustaccordingly.Suchchangeswouldseeanupwardshiftof theupperboundaryof theabsolutedesertaspre‐cipitationdecreases,togetherwithaninvasionofhigh‐altitudecoldenvironmentsastemperatureincreases.Along‐termperspectiveandwidespreadintegrationofpaleoecologyandconservationbiologywillneedtobeadoptedifthevibrantAndeanAtacamaecosystemsaretobeconservedforthefuture(Barnoskyetal.,2017).Moreover,ourre‐sultsprovideimportantelementstoconsidermodelsofbiodiversityto climate change in other desert or high mountain ecosystems.
ACKNOWLEDG EMENTS
ThisresearchwasfundedbyFONDECYTPostdoctorado#3150616toF.P.D.,MillenniumInstituteforIntegrativeBiology(iBio)IniciativaCientífica Milenio‐MINECON, Fondo de Desarrollo de AreasPrioritarias (FONDAP)Center forGenomeRegulation (15090007)and EvoNet grant (DE‐FOA‐0001207) from Department ofEnergy,UnitedStatesofAmerica.C.L.acknowledgesfundingfromCONICYT'sAportedeFinanciamientoBasal (AFB‐170008). J.R.W.and J.M.W. acknowledge Strategic Science Investment Fundingfor Crown Research Institutes from theNew ZealandMinistry of
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Business, Innovation and Employment's Science and InnovationGroup, and Manaaki Whenua – Landcare Research for hostingF.P.D.'svisittotheLandcareResearchLong‐TermEcologyLaboratorywhere the aDNA extractions were undertaken. We thank AliciaMarticorenaandRosaMonterofortheirhelpintheHerbariodelaUniversidad de Concepción (CONC). We appreciate helpful com‐mentsandconstructivecriticismsfromthereviewersandeditorofourmanuscript.Wewouldalsoliketoacknowledgethoughtfulcom‐mentsandsuggestionsbyDr.JulioBetancourtthatgreatlyhelpedinimprovingourmanuscript.
ORCID
Francisca P. Díaz https://orcid.org/0000‐0002‐1100‐7801
Jamie R. Wood http://orcid.org/0000‐0001‐8008‐6083
Janet M. Wilmshurst http://orcid.org/0000‐0002‐4474‐8569
Daniela C. Soto http://orcid.org/0000‐0002‐6292‐655X
Theresa L. Cole http://orcid.org/0000‐0002‐0197‐286X
Rodrigo A. Gutiérrez http://orcid.org/0000‐0002‐5961‐5005
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Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.
How to cite this article:DíazFP,LatorreC,Carrasco‐PugaG,etal.Multiscaleclimatechangeimpactsonplantdiversityinthe Atacama Desert. Glob Change Biol. 2019;00:1–13. https://doi.org/10.1111/gcb.14583
