Phenological Variation in Ambrosia artemisiifolia L. Facilitates Near Future Establishment at ...

15 Pages·2016·1.2 MB·English

RESEARCHARTICLE
Phenological Variation in Ambrosia
artemisiifolia L. Facilitates Near Future
Establishment at Northern Latitudes
RomainScalone1,AndreasLemke2,EditaSˇtefanić3,Anna-KarinKolseth1,SandaRasˇić3,
LarsAndersson1*
1 SwedishUniversityofAgriculturalSciences,DepartmentofCropProductionEcology,Uppsala,Sweden,
2 TechnischeUniversita¨tBerlin,DepartmentofEcology,PlantEcologyandEcosystemScience,Berlin,
Germany,3 UniversityofJosipJurajStrossmayer,FacultyofAgriculture,Osijek,Croatia
a11111 *[email protected]
Abstract
TheinvasiveweedAmbrosiaartemisiifolia(commonragweed)constitutesagreatthreatto
publichealthandagricultureinlargeareasoftheglobe.Climatechange,characterizedby
OPENACCESS
highertemperaturesandprolongedvegetationperiods,couldincreasetheriskofestablish-
Citation:ScaloneR,LemkeA,SˇtefanićE,Kolseth mentinnorthernEuropeinthefuture.However,asthespeciesisashort-dayplantthat
A-K,RasˇićS,AnderssonL(2016)Phenological
requireslongnightstoinducebloomformation,itmightstillfailtoproducematureseeds
VariationinAmbrosiaartemisiifoliaL.Facilitates
NearFutureEstablishmentatNorthernLatitudes. beforetheonsetofwinterinareasatnorthernlatitudescharacterizedbyshortsummer
PLoSONE11(11):e0166510.doi:10.1371/journal. nights.Tosurveythegeneticvariationinfloweringtimeandstudytheeffectoflatitudinalori-
pone.0166510
ginonthistrait,areciprocalcommongardenexperiment,includingelevenpopulationsofA.
Editor:SylvainDelzon,INRA-Universityof artemisiifoliafromEuropeandNorthAmerica,wasconducted.Theexperimentwascon-
Bordeaux,FRANCE
ductedbothoutsidetherangelimitofthespecies,inSwedenandwithinitsinvadedrange,
Received:August17,2015 inCroatia.Ourmainhypothesiswasthatthephotoperiodic-thermalrequirementsofA.arte-
Accepted:October31,2016 misiifoliaconstituteabarrierforreproductionatnorthernlatitudesand,thus,haltsthenorth-
ernrangeshiftdespiteexpectedclimatechange.Resultsrevealedthepresenceofanorth-
Published:November15,2016
southgradientinfloweringtimeatbothgardensites,indicatingthatcertainEuropeanpopu-
Copyright:©2016Scaloneetal.Thisisanopen
lationsarepre-adaptedtophotoperiodicandthermalconditionsatlatitudesupto,atleast,
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which 60˚N.Thiswasconfirmedbyphenologicalrecordingsperformedinaregionclosetothe
permitsunrestricteduse,distribution,and northernrangelimit,thenorthofGermany.Thus,weconcludethatthereexistsahighrisk
reproductioninanymedium,providedtheoriginal
forestablishmentandspreadofA.artemisiifoliainFennoScandinaviainthenearfuture.
authorandsourcearecredited.
Therangeshiftmightoccurindependentlyofclimatechange,butwouldbeacceleratedbyit.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.
Funding:TheworkwasfundedbySvenska
ForskningsrådetFormas(http://www.formas.se),
grantnumber217-2012-994.LAandAKreceived Introduction
thefunding.Thefunderhadnoroleinstudy
design,datacollectionandanalysis,decisionto Rangeshiftsofplantsareexpectedasaconsequenceofclimatechange,characterizedbyhigher
publish,orpreparationofthemanuscript. temperatures,changesinprecipitationpatternsandprolongedvegetationperiods[1].Distri-
butionbeyondthecurrentrangelimitmightoccurgraduallyastheclimatechangesandnew
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. areascomewithinthespeciesnichelimit[2].Severalattemptshavebeenmadetopredictthe
PLOSONE|DOI:10.1371/journal.pone.0166510 November15,2016 1/15
PhenologicalVariationinA.artemisiifolia
rangeshiftofsoutherlyspeciestonorthernlatitudes[1,3,4],basedonassumptionsofachange
inclimate.Thegeographicshiftofspeciesovertimeinresponsetocontemporaryclimate
changehasbeenestimatedto1.69kmyear-1towardsnorthernlatitudes,andisingeneral
sufficienttotracktemperaturechanges[5].Ithas,however,beenquestionedwhetherthiscon-
clusionisvalidforlowlandterrestrialplants[6,7].ABritishstudy[8]revealednoclearfinger-
printofclimatechangeonpolewardmovementofplants,andanexpectedlagindistribution
attheleadingedgewassuggested.Alagphaseiscommonlyobservedininvasivespecies,
whichmakesitdifficulttopredictthetimingofthedifferentprocessesrelatedtoinvasion[9].
Thelagphaseispoorlyunderstoodbutitmaybepartlyexplainedasthetimeneededforthe
speciestoevolveandadapttothenewhabitat[10].Consequently,Whitney&Gabler[11]con-
cludedthatevolutionarypotentialshouldbeincorporatedintheassessmentoftheinvasiveness
ofaspecies,andmentionedshortgenerationtimeasoneoftenkeytraits.Onereasonforthis
couldbearequirementforchangesinfloweringtimesincemanyplantspecies(i.e.short-day
plants)requirelongnightsforinductionofflowering[12].Thus,fornorthernrangeexpansion
tooccurinshort-dayspeciesanadaptationtonewphotoperiodic-thermalconditions(i.e.pho-
toperiod,temperatureandtheirinteraction)isrequired,ashasbeenobservedintheshort-day
grassMicrostegiumvinimeum[13].Severalstudieshavereportedearlierfloweringinpopula-
tionssampledatthenorthernrangemarginscomparedtothesoutherlycounterparts[14–16].
Theinvasiveshort-dayplantAmbrosiaartemisiifoliaL.(commonragweed)isconsideredto
beoneofthemostnoxiousplantsinEurope[17].Thisrankingisbasedbothonitsnegative
effectsonpublichealthanditsreductiveimpactsontheyieldsofseveralmajorcrops.Thisdra-
maticsituationhasledtointernationalcooperationamongscientiststocontrolthespeciesina
sustainableway(COSTActionFA1203“SustainableManagementofA.artemisiifoliain
Europe”[18]).HealthproblemscausedbyA.artemisiifoliaincludedermatitisandeczemaafter
directskincontact[19],andrespiratoryproblemsandasthmaduetothereleaseofabundant
quantitiesofhighlyallergenicpollengrains[3].Moreover,thelatefloweringtimeduetoits
short-dayplanttraitextendstheperiodofhayfeverforallergysufferersuntillateautumn[20,
21].ThepollengrainproductionfromasingleA.artemisiifoliaplanthasbeenestimatedtosev-
eralbillionperseason[22]anditsdispersalviawindtoseveralhundredkilometres[23,24].In
additiontoimpactonhumanhealthA.artemisiifoliaisrecognizedasahighlycompetitive
weed,withhighinfestationofrowcropssuchassunflowerandmaize,incroppingsystemsin
Western,CentralandEasternEurope[25,26].InHungary,forexample,70%ofagricultural
fieldsarereportedtobeinfested[21].
OriginatingfromNorthAmerica,withaknowndistributionfromlatitude31˚Nto52˚N,
severalgenotypicstudieshaveshownthatthesuccessfulworldwideinvasionandrangeexpan-
sionofA.artemisiifoliaistheresultofmultipleintroductionsfromdifferentregionsofthe
nativerange[25,27–31].ItwasfirstspreadtoEuropeinthemiddleofthe19thcenturyandhas
sincebeenrepeatedlyintroducedtoEurope.ThemainareaofinfestationisCentralEurope,
butitisnowestablishedinlargepartsofthecontinent,withanorthernrangelimitsituated
southoftheScandinaviancountriesandtheUK,andthesouthernrangelimitinmidItaly,the
IberianPeninsulaandGreece[1,4](Fig1).Withclimatechange,thenorthernlimit,basedon
environmentalfactors,ofcommonragweedrangeistheonemostlikelytobeextended.
Thespecieshasbeenreportedtorequireaminimumnightlengthofeighthourstoinduce
flowering[34].Nightlength,definedasthetimedifferencebetweensunsetandsunrise,never
fallsbeloweighthoursinBudapest(latitude47˚30´N),butdoesnotexceedeighthoursuntil
mid-AugustinStockholm(latitude59˚51´N)ormid-JulyinBerlin(latitude52˚31´N)[35].
PhotoperiodicconditionsinnorthernEuropemightthusdelaytheonsetoffloweringuntil
autumn.Indeed,accordingtoArtportalen,theSwedishSpeciesInformationCentre[36],the
earliestdayreportedforfloweringofA.artemisiifoliabetween2004and2013was21August
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PhenologicalVariationinA.artemisiifolia
Fig1.DistributionofAmbrosiaartemisiifoliainA)Europe(modifiedfromSmithetal.2013[32])and
B)NorthAmerica(modifiedfromGentonetal.2005[33]).Starsmarkthelocationofpopulationssampled
fortheexperiment,andarrowsindicatethelocationofthecommongardensitesinUppsalaandOsijek,
respectively.Attribute,Europemap:Ssolbergj-https://commons.wikimedia.org/w/index.php?curid=4203715.
Attribute,NorthAmericamap:AlanRockefeller-https://commons.wikimedia.org/wiki/File%3ANorth_
america_blank_range_map.png.
doi:10.1371/journal.pone.0166510.g001
(daylength:15h4min./nightlength:8h56min.).Thisappearstobetoolatetoenablepro-
ductionofviableseedsbeforeplantsarekilledbyfrost,whichcouldexplainwhythereare,to
ourknowledge,noconfirmedestablishedpopulationsinSweden.Sincethephotoperiodisa
stablefactor,unaffectedbyclimatechange[12],itmightserveasabarrieragainstestablish-
mentofA.artemisiifoliainthenorthofEuropedespiteimprovedclimaticconditions.There
are,however,twofactors,whichmightoverrulethepreventativeeffectofashortsummerpho-
toperiod.First,theremayexistpopulationsatthenorthernrangemarginofA.artemisiifolia,
whicharepre-adaptedtonorthernphotoperiodic-thermalconditions.Thesepopulations
couldgraduallyexpandtheirrangelimit,aidedbyon-goingchangesinclimate.Secondly,the
commercialtradeinbirdseedinfestedwithseedsofA.artemisiifoliaisthemainsourceof
introductioninScandinavia,whichmakesprivategardensandmunicipalsoildepositscom-
moncasualhabitats.ContinuousintroductionofA.artemisiifoliaseedsinScandinaviamight
contributetotheprocessofselectionofearly-floweringindividuals,whichwouldsubsequently
enabletheestablishmentofviablepopulations.Althoughgeneflowmaycounteractlocaladap-
tation,multipleintroductionshavebeenshowntoenhanceinvasivesuccessbyintroducing
novelallelesandincreasinggeneticvariation[37].Whenaspecieshassimilarecologicaland
climaticnichesinbothinvasiveanddomesticpopulations,invasionstypicallyfailwithoutpre-
adaptationandtherateofmigrationofsuitableallelesalongtheselectivegradientbecomes
crucialforadaptation[38].Underthesecircumstancesseedsofpre-adaptedgenotypeshitch-
hikingwithcontaminatedbirdseedcanalsobeimportant.Allscenariosareinlinewithcon-
clusionsbyClements&DiTommaso[39]whoarguedthatevolutionofinvasiveweedsmight
increasetherangeshiftmorethanforecastedandfasterthantheprocessofclimatechange.
ForthetwoscenariosofrangeshifttoberealisticforA.artemisiifolia,thephotoperiodic-ther-
malrequirementsforfloweringshouldcontaingeneticvariation.Recentworkusingcommon
gardenexperimentsandgenomictoolshasconfirmedadifferentiationoflife-historytraits
betweennativeandintroducedpopulations[40].Thehighlydiversegenepoolpresentin
EuropehaspromotedarapidevolutionandadaptationofA.artemisiifoliatodifferentenvi-
ronments,e.g.field,roadside,river-sideandwasteland[41].Inaddition,thepresenceofa
north-southgradientinfloweringtimewithinintroducedEuropeanpopulationsfromlatitude
54˚Nto44˚Nwasrecentlyshowninacommongardenexperimentlocatedwithinthe
invadedarea[16].
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PhenologicalVariationinA.artemisiifolia
TheaimofthisstudywastoexplorevariationinphenologywithinA.artemisiifoliapopula-
tionsofdifferentoriginsandtestwhetherthereexistsalatitudinalclineinfloweringtime.
Commongardenexperimentshavealongtraditioninevolutionaryecology,andarewell
suitedforstudyingthegeneticdifferentiationamonggenotypes.Growingdifferentgenotypes
ofaspeciesinacommonenvironmentmakesitpossibletoattributephenotypicvariationto
eithergeneticorenvironmentalfactors[42].Areciprocalcommongardenexperimentwas
conductedatonesiteinthemiddleoftheinvadedarea(Osijek,Croatia;latitude45˚N),andat
onesitebeyondtherangelimit(Uppsala,Sweden;latitude59˚N).Theexperimentwascomple-
mentedwithphenologicalobservationsatseveralfieldsitesofpopulationslocatedinthe
northernpartoftheinvadedrange(Germany;latitude51–52˚N).Wehypothesizedthati)
thereexistsanorth-southgradientinfloweringtimeamongpopulations,withplantsfrom
northernpopulationsfloweringearlier,andii)thegradientshowsthesamepatternatboth
sites,butismoredistinctatthenorthernsiteduetonon-optimalphotoperiodic-thermalcon-
ditionsbeyondtherangelimit.Theabsenceofanorth-southgradientwouldindicatethat
thereisnolocaladaptationtophotoperiodic-thermalconditions,butratherthatthevariation
infloweringtimeisbasedonphenotypicplasticity.
MaterialsandMethods
Commongardenexperiment
AtotalofelevenpopulationsofAmbrosiaartemisiifoliawerecultivatedinareciprocalcom-
mongardenexperiment,withonegardensitelocatedbeyondtherangelimit,atahighlatitude
(Uppsala,Sweden:N59˚48´55”,E17˚38´47”)andtheotherwithintheinvadedEuropean
range(Osijek,Croatia:N45˚31´16”,E18˚40´54”).Commongardenexperimentswerecon-
ductedonlandownedbytheSwedishUniversityofAgriculturalSciencesandUniversityof
theJosipJurajStrossmayer,respectively,andpermissionsfortheexperimentsweregrantedby
theuniversities.SeedsofA.artemisiifoliawerecollectedin2010and2011fromnineintro-
ducedpopulationsinfiveEuropeancountriesandtwonativepopulationsfromNorthAmerica
(Fig1,Table1).Nospecialpermissionwasneededforseedcollectionandfieldstudiessince
theydidnotinvolveendangeredorprotectedspecies.Theelevenpopulationswereselectedto
representthelatitudinalvariationfromlatitude38˚Ntolatitude51˚N.Seedsfromatleastten
individualplantswerecollectedperpopulation.Apopulationwasdefinedasasetof
Table1. DetailsoftheoriginofAmbrosiaartemisiifoliapopulationsgrowninareciprocalcommongardenexperiment.
Population Country Latitude,Longitude Yearofcollection Habitat Seedcontributor
Drebkau Germany N51˚38´21",E14˚11´50" 2011 fallowfield U.Starfinger
Martonva´sa´r Hungary N47˚20´37",E18˚50´31" 2011 field P.Bonis
Baracska Hungary N47˚18´03",E18˚45´51" 2011 field P.Bonis
Pluvet France N47˚11´18",E05˚15´01" 2011 maizefield B.Chauvel
Kaposva´r Hungary N46˚22´12",E17˚51´17" 2011 maizefield G.Kazinczi
Besate Italy N45˚18´25",E08˚58´21" 2011 roadedge M.Bonini
StClothilde Canada N45˚10´03",E73˚40´50" 2011 fieldedge D.Benoit
Bassens France N44˚54´04",E00˚31´58" 2010 wasteland B.Laitung
Pribinic Bosnia&Herzegovina N44˚35´59",E17˚49´40" 2011 wasteland B.Vuckovic-Kelevic
Dions France N43˚56´03",E04˚18´22" 2010 wasteland B.Laitung
Lexington USA N38˚01´00",E84˚33´10" 2010 oldpasture C.&J.Baskin
TenindividualsfromeachpopulationweregrowninUppsala,Sweden(N59˚48´55”,E17˚38´47”)andinOsijek,Croatia(N45˚31´16”,E18˚40´54”).
Latitudeandlongituderefertoseedsamplingsite,andhabitatreferstovegetationtypeatseedsamplingsite.
doi:10.1371/journal.pone.0166510.t001
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PhenologicalVariationinA.artemisiifolia
individualsgrowinginonefieldwithatleast7.5kmbetweensampledfields.Seedweightwas
calculatedasthemeanweightof5x50seeds.Pretreatmentofseedsandhandlingofseedlings
wereconductedaccordingtothesameexperimentalprotocolinbothSwedenandCroatia,as
follows.Atotalofca.100seedsperpopulationwereplacedonmoistpaperinPetridishesat
5˚Cforstratification.Subsequently,thePetridisheswereplacedinagrowthchamberwitha
temperatureregimeof15˚Catnight(nolight)for8hoursand27˚Catday(lightintensity:
50μmolm-2s-1)for16hourstoinduceseedgermination.Whenaround50seedlingsperpop-
ulationhadbeenproduced,seedlingswereplantedinplastictrayswithindividualwells(5cm
diameter,6cmdepth).Eachtraywasassignedtoaspecificpopulation,andthesoilusedtofill
thetrayswasidenticaltothatusedforthegardenphase.Soilswithsimilarcompositionwere
usedatbothsites(fertilizedpeatsubstrate;HasselforsGardenS-jordinUppsalaandKlasmann
TS1inOsijek).Thepositionofthetrayswasrandomizedonceaday.Theseedlingswere
growninaclosedgreenhouse(20˚C)untilthefour-leafstage,whichoccurredca.3weeksafter
germination.Subsequently,tenfour-leafseedlingsofeachoftheelevenpopulations,assimilar
insizeaspossible,wereselectedandtransplantedinpots(5.5L,topØ19.5cm,height25.5cm;
Soparco,Conde´-sur-Huisne,France)filledwith5Lsoil.Thepotswereplacedoutdoorsinthe
gardeninacompletelyrandomizeddesignwith1mbetweeneachpot.Theplantswere
wateredthroughouttheexperimentalperiodtoavoiddroughtandfertilizedthreetimeswith
intotal0.1418gNperpot,correspondingto50kgNha-1.Thecommongardenexperimentin
Uppsalastartedwiththetransplantationofseedlingsoutdoorson15Juneandendedon16
October,shortlybeforethefirstfrost.InSweden,A.artemisiifoliaplantshavebeenobserved
alreadyinMay[36]andthesettingsofthegerminationforthecommongardenexperiment
(endofMay)werechosenbasedontheseobservations.Hence,earlyfloweringbytheGerman
populationattheUppsalasiteismorelikelytobeaconsequenceoflocaladaptationrather
thanwrongtimingofthegardenexperiment.TheexperimentatOsijekstartedon16Juneand
finishedon11September,whenallpopulationshadproducedbothmaleandfemaleflowers.
Allplantswerevisuallyinspectedatleasttwiceaweektorecordthedifferentphenological
stages:i)firstmaleflower(i.e.thefirstemergenceofanthersoutsideonesinglemaleflower,
usuallylocatedintheterminalmaleinflorescence),andii)firstfemaleflower(i.e.appearance
ofpistilsoutsideonesinglefemaleflower).Afterthefirstreleaseofpollengrainsfromanindi-
vidualplant,maleinflorescencesoftheplantsatthegardensiteatUppsalawerecutcontinu-
ouslyuntiltheendoftheexperiment.Thisprecautionaryprocedurewastakenfortwomain
reasons:i)topreventcontaminationoftheairbyhighlyallergenicpollenandii)toprevent
possibleseedproductionandputativesoilcontaminationofthegarden.Attheendofthesum-
merseason,theplantheightcorrespondingtothedistancebetweenthetopandthebaseofthe
plantwasmeasured.
Fieldstudy
Todetermineitsabilitytoflowerandtoproducematureseedsinthenorthernpartofthe
invadedEuropeanrange,atotalof17sitesinvadedbyA.artemisiifoliawereobservedduring
thegrowingseason2010.SiteswerelocatedinandaroundtwonorthernGermanlocalities;
Drebkau(N51˚39´19”,E14˚13´25”)andBerlin(N52˚30´59”,E13˚23´09”).Theformer
localitywaschosenbecausethepopulationisknowntobewellestablishedanddistributedin
thearea,whiletheBerlinsitesareprobablyinfestedwithpopulationsintroducedatdifferent
occasions.Duetoweedmanagementmeasuresduringthegrowingseasonthenumberofsites
wasrestricted,especiallyaroundBerlinwhereonlyfourfieldandroadsidesiteswerevisited,
withaminimumdistanceof19kmbetweensites.Toaccountforpossibledifferencesinselec-
tionpressureinthedifferenthabitats,duetoe.g.timeofharvestorcontrolmeasures,sixfield
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PhenologicalVariationinA.artemisiifolia
sitesandfiveroadsidesites,respectively,wereselectedatDrebkau,withaminimumdistance
of0.5kmbetweensites.Thenumberofplantspersitevariedinmostcasesbetween70and600
plants,butwithtwoextremesof3000and15000plants.Theselattertwositesweretransect-
likestretchedpatches(50mx1mand20mx2m,respectively,andthenumberofplants
therewasestimatedbycountingindividualsperm2andcalculatingforthearea.Everyweek,
200plantswererandomlyselectedateachofthesetwositesfordeterminationofthephenolog-
icalstage.Atallsitesthephenologicalstagesofindividualplantswererecordedweeklyfrom
theendofJuneuntilmid-October.Fivesuccessivestagesofthedevelopmentofthemaleinflo-
rescencewerepre-defined,recordedandestimatedatsitelevel:stage0=absenceofmale
floweratthesite,stage1=budsarevisible,stage2=maleflowers(startofpollenproduction),
stage3=80%ofindividualsatthesitehavematuremaleflowers(releasingpollen),stage
4=maleinflorescencesdying.Fourstagesofthedevelopmentofthefemaleflowerwerepre-
defined:stage5=absenceoffemaleflowers,stage6=femaleflowers(appearanceofstyles),
stage7=80%oftheindividualshaveopenfemaleflowers(visiblestyles),stage8=80%ofthe
individualshavepollinatedfemaleflowers(swollenovaries),whilethreestagesofthefruit
developmentwerecharacterized:stage9=80%oftheindividualshavematuringfemaleflow-
ers(ovariesatfinalseedsize),stage10=80%oftheindividualshaveseedswithcolorchanging
fromgreentodarkbrown,stage11=matureseedsdispersing.Hence,thelaststageofthe
femaleflowerdevelopmentcorrespondstothestagebeforethestartofseedmaturationinthe
caseofasuccessfulpollination.Thelengthoftwoimportantphenologicalstageswerequanti-
fied:the“pollenproduction”stage(numberofweeksbetweenstage2andstage4ofthedevel-
opmentofmaleflowers),andthe“totalreproduction”stage(numberofweeksbetweenstage1
ofthedevelopmentofmaleflowerandstage11ofthefruitdevelopment)(Table2).
Table2. Timingandlengthofphenologyinfield.
Phenologicalstage Locality HabitatatDrebkau
Drebkau Berlin S P Field Roadside S P
Mean±SE n Mean±SE n Mean±SE n Mean±SE n
1.Firstbud 186.4±1.6 13 200.2±1.8 4 60.5 0.0048 189.2±2.8 6 185.2±1.7 5 24.5 0.3335
Pollenproduction
2.Start 204.7±1.5 13 224.8±5.2 4 61.0 0.0037 206.7±3.0 6 202.0±0.0 5 22.5 0.1588
4.End 249.4±1.6 13 276.7±9.3 3 44.0 0.0101 247.5±1.6 6 251.0±3.8 5 33.0 0.6138
Femaleflowers
6.Start 192.3±2.0 13 231.8±3.4 4 62.0 0.0030 196.2±3.3 6 188.0±3.8 5 23.0 0.2133
7.End 210.1±2.7 13 251.0±4.9 4 62.0 0.0030 210.2±3.3 6 213.2±5.7 5 31.5 0.8500
Fruitdevelopment
8.Start 207.4±2.8 13 244.0±6.4 4 62.0 0.0036 210.2±3.3 6 209.0±4.9 5 29.0 0.9256
11.End 282.8±2.7 11 310.5±3.5 2 25.0 0.0320 277.6±3.4 5 286.0±4.9 4 25.5 0.1904
Lengthofstages
Pollenproduction 45.2±2.2 13 56.0±8.1 3 35.5 0.1800 40.8±2.2 6 49±3.8 5 27.0 0.0993
Totalreproduction 97.4±3.5 11 108.5±3.5 2 20.5 0.2245 89.6±3.4 5 101.5±6.1 4 26.0 0.1688
DifferencesintimingandlengthofthephenologicalstagesofA.artemisiifoliaplantsrecordedattwoGermanlocalities(DrebkauandBerlin),andseparately
fortwohabitatsinDrebkau(fieldsandroadsides).ThetimingofthephenologicalstagesisgivenasJuliandays,whilethelengthofthephenologicalstages
arepresentedasthenumberofdaysbetweenthetwostagesdefiningit.Thenumberforeachphenologicalstagecorrespondstothenumbergiveninthe
textforthedifferentstagesofmaleandfemaleflowering.“n”denotesthenumberofsites.ValuesofS(teststatisticassociatedwiththesmallersample)and
P(probability)werecalculatedusingtheWilcoxonranksumtest.
doi:10.1371/journal.pone.0166510.t002
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Statistics
Inter-populationvariationsofmaleandfemalefloweringtimeandplantheightwereanalyzed
bymeansofanalysisofcovariance,ANCOVA,usingprocGLMinthestatisticalsoftwareSAS
9.3(SASInstituteInc.Cary,NC)withgardensiteasfixedfactor,originandtheinteractionas
randomfactors.Sincebirdseed,whichisthemainvectorforentranceofAmbrosiaseedsto
Sweden,maybeimportedfromNorthAmerica,itwasconsideredimportanttoincludethese
populationsintheanalyses.ThenumberofJuliandaysbetweenthestartofthegardenexperi-
mentandthedatesoffirstmaleorfemalefloweringwerecalculatedasmeanforeachpopula-
tionateachgardensite.Whenanalyzingthestartofflowering,finalplantheightwasincluded
inthemodeltotestforcovariation,andwhenanalyzingfinalplantheight,seedweightwas
includedinthemodeltotestforcovariation.Forplantheight,thepartitioningofthevariation
betweengardensites(environment)andamongthedifferentoriginsoftheAmbrosiapopula-
tionswithinsite(genetic)wascalculatedusingprocGLM.
ToexplorepossibledifferencesinthefieldobservationsbetweenthetwoGermanlocalities,
andbetweenthedifferenthabitats(6sitesdefinedasfieldand5asroadside)withinthelocality
ofDrebkau,thenon-parametricWilcoxonsumranktestwasused.Thenon-parametrictest
wasusedsincemostvariablestested(firstbudappearance,endofmaleflowering,andstart
andendoffemalefloweringandfruitdevelopment)didnotfollowanormaldistribution.The
Juliandaysfora)appearanceofbuds,b)startandendofmaleflowering,c)startoffemale
floweringandsuccessfulpollination(appearanceofswollenovaries)andd)startandendof
seedmaturationateachsitewasregistered.Inaddition,thedifferenceinlengthoftheperiod
ofpollenproductionandthetotalreproductionperiod,calculatedasthedifferencebetween
theJuliandaysrecordedfortwophenologicalstages,wasanalyzed.
Results
Commongardenexperiment
Theresultsrevealedaclearnorth-southgradientforfloweringtimeofA.artemisiifolia(Figs2
and3).Significantdifferencesinthetimerequiredtotriggerthemalefloweringandthefemale
floweringwereobservedbetweenpopulationswithinbothgardensandalsobetweenthegar-
dens,eveniftherewerefewerpopulationsproducingfemaleflowersinUppsalathaninOsijek
(Table3,Figs2and3,S1Appendix).PlantsgrowninUppsalaneededsignificantlymoretime
toproducethefirstmaleandfemaleflowerthanplantsfromthesamepopulationsgrownin
Osijek(Figs2and3).Finalplantheightdidnothaveasignificanteffectoneitherfirstmaleor
femaleflower,hencethevariablewasexcludedintheANCOVAandonlysignificanteffects
werestatedinthefinalmodel.Early-floweringandlate-floweringpopulationswereidentified
ascorrespondingtothepopulationsfromtheextremenorthernandsouthernlatitudesofour
sampling(North:Germany51.4˚N;South:USA-Kt38.0˚N).InOsijek(Croatia)alltheindivid-
uals,grownundergoodphoto-thermicconditions(accordingtoDeenandcolleagues[34]),
producedmaleandfemaleflowersbeforetheendofthecommongardenexperiment.Incon-
trast,atthesitebeyondtherangelimit(Uppsala,Sweden),50%oftheindividualplantsfrom
themostsouthernpopulationofoursampling(Lexington,Kentucky)failedtoproducemale
flowersbeforeonsetoftheSwedishfrost.Intotal,approximatelyonefifthoftheplantsgrown
inUppsala,representingfourpopulationsfromthesouthernpartoftheinvadedEuropean
range(southernFrance,Italy,Bosnia&Herzegovina)andthetwoAmericanpopulations,did
notproducefemaleflowers(S1Appendix).
PlantsofdifferentpopulationsgrowninOsijekwereingeneralsignificantlytaller(differ-
ence27.6cm,SE±3.4)thanplantsofthesamepopulationsgrowninUppsala(Table3,Fig4).
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PhenologicalVariationinA.artemisiifolia
Fig2.FirstmalefloweringofelevenAmbrosiaartemisiifoliapopulationsinareciprocalcommon
gardenexperiment.GardenswerelocatedinUppsala,Sweden(N59˚48´55”,E17˚38´47”)andinOsijek,
Croatia(N45˚31´16”,E18˚40´54”).Meannumberofdaysfromstartofcommongardenexperimentuntil
firstmaleflowerappearanceofeachpopulation.Latitudesrefertositesfromwhichseedswerecollected(for
detailsofpopulationsandexactlatitudeinformationseeTable1).
doi:10.1371/journal.pone.0166510.g002
Theeffectoftheoriginofthepopulationwasnotsignificantandrepresentedonly14%ofthe
varianceofthefinalplantheight(geneticeffect“G”)whilethedifferencebetweenthetwogar-
densitesexplained75%.Seedweightdidnothaveasignificanteffectonfinalplantheight,
hencethevariablewasexcludedintheANCOVAandonlysignificanteffectswerestatedin
thefinalmodel.
Fig3.FirstfemalefloweringofelevenAmbrosiaartemisiifoliapopulationsinareciprocalcommon
gardenexperiment.GardenswerelocatedinUppsala,Sweden(N59˚48´55”,E17˚38´47”)andinOsijek,
Croatia(N45˚31´16”,E18˚40´54”).Meannumberofdaysfromstartofcommongardenexperimentuntil
firstfemaleflowerappearanceofeachpopulation.Latitudesrefertositesfromwhichseedswerecollected
(fordetailsofpopulationsandexactlatitudeinformationseeTable1).Onepopulation(Lexington)inUppsala
didnotproduceanyfemaleflowers.
doi:10.1371/journal.pone.0166510.g003
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PhenologicalVariationinA.artemisiifolia
Table3. Analysisofcovarianceoflatitudeandcommongardenlocationonfloweringtimeandplantheight.
Sourceofvariation Maleflower Femaleflowera Plantheight
Fb P Fb P Fb P
Commongarden 637.50 <0.0001 275.64 <0.0001 33.0 <0.0001
Latitude 101.20 <0.0001 65.16 <0.0001
CG*Latitude 23.16 0.0001 11.04 0.0040
Ambrosiaartemisiifoliaplantsfrom11populationsweregrowninreciprocalcommongardenexperimentsinUppsala,SwedenandinOsijek,Croatia.Inthe
analysisofcovariancefirstmaleflower,firstfemaleflowerandfinalplantheightweredependentvariableswithoriginallatitudeofsampledpopulationsas
theindependentcontinuousvariableandcommongardensiteastheindependentcategoricalvariable.
aTheLexingtonpopulationsfailedtoproducefemaleflowerswhengrowninUppsala.
bDegreesoffreedomfortheresidualis18,17and18formaleandfemaleflowerandplantheight,respectively.
doi:10.1371/journal.pone.0166510.t003
Fieldstudy
OneoftheGermanpopulationslocatedinthenorthernpartoftheinvadedEuropeanrange
(Drebkau)wasalsousedinthecommongardenexperiment.Phenologicalrecordsfromsev-
eralsitesconfirmeditsearly-floweringinfieldconditions(middleofJuly;Table2).Datesof
theonsetandendingofallphenologicalstagesstudiedoccurredsignificantlyearlieratthesites
in/aroundDrebkau(51˚39”N)thanatthesitesin/aroundBerlin(52˚30”N).However,nosig-
nificantdifferencewasobservedforthelengthofthe“pollenproduction”stageandofthe
“totalreproduction”stage(Table2)betweenthesetwoseparateGermanlocalities.Nosignifi-
canttemporaldifferencewasfoundamongsiteslocatedinfieldsandsitessituatedalongroads
withintheDrebkaulocalityforthestartofthereproductivestage,thestartandtheendofmale
andfemaleflowering,orthestartandtheendofthefruitdevelopment(Table2).
Fig4.PlantheightofelevenAmbrosiaartemisiifoliapopulationsinareciprocalcommongarden
experiment.GardenswerelocatedinUppsala,Sweden(N59˚48´55”,E17˚38´47”)andinOsijek,Croatia
(N45˚31´16”,E18˚40´54”).Latitudesrefertositesfromwhichseedswerecollected(fordetailsof
populationsandexactlatitudeinformationseeTable1).
doi:10.1371/journal.pone.0166510.g004
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PhenologicalVariationinA.artemisiifolia
Discussion
Photoperiodicresponsehasbeenproposedasoneofthemostimportantfactorslimitingthe
Europeandistributionofinvasiveplants[12].Attemptstopredictthenorthwardrangeshiftof
A.artemisiifoliahaveledtotheconclusionthattheextensionoftherangelimitistoalarge
extentconstrainedbythephotoperiodrequirementforinductionofflowering(e.g.[1,4,43]).
However,ourresultsindicatethatthephotoperiodic-thermalrequirementisatraitwitha
largepotentialforlocaladaptation.Indeed,theresultsofthecommongardenexperiment
showedthatA.artemisiifoliacanformmaleandfemaleflowersupto,atleast,latitude59˚N,
withaclearnorth-southgradientinfloweringtime.Thelargedifferencesobservedinthecom-
mongardenexperimentmakeitpossibletodifferentiateearlyandlatefloweringpopulations
and,thus,toestimatethepotentialthreatofcommonragweedpopulationstoFennoScandina-
vianpublichealthandagriculture(Figs2and3).
ThepopulationrepresentingthelargestpotentialforestablishmentinFennoScandinavia,if
introduced,isthemostearly-floweringpopulation,originatingfromGermany.Incontrast,
thepopulationfromthesouthernpartofthenativerange(Kentucky,USA)constitutesanegli-
gibleriskforinvasionatnorthEuropeanlatitudes.TheGermanpopulationisobviouslywell
adaptedtophotoperiodic-thermalconditionsatlatitude51˚N,andourcommongardenexper-
imentindicatesthatitisalreadypre-adaptedforreproductioninnorthernEuropeuptoat
leastlatitude59˚N.Thus,thenichelimitoftheGermanpopulationseemstobebeyondits
presentrangelimit.
IthasbeenshownthatthelargeecologicalamplitudeofA.artemisiifolia,togetherwith
unoccupiedniches,isafactorbehindsuccessfulinvasioninFrance[22].However,ifaspecies
hassimilarecologicalandclimaticnichesininvasiveandnativepopulations,ithasbeenseen
thatinvasionstypicallyfailwithoutpre-adaptation.Undersuchcircumstances,therateof
migrationofsuitableallelesalongtheselectivegradientbecomescrucialforadaptation[38].
Further,rangeexpansionofaspeciesisnotonlydependentonsuitableintraspecificecological
variation,availablenichespaceandpre-adaptedalleles,butalsoonsuccessfulestablishmentof
locallyadaptedalleles.Thelatterisinturninfluencedbygeneflow,selectionpressuresand
mutationrates[38].InFennoScandinavia,theselectionpressureonreproductionisrather
strong,asonlyindividualswithseedssetbeforethefirstfrostwillcontributetonextgeneration.
Suchselectionmightcounteractswampingofthelocallyadaptedgenepoolbypollenfromcen-
tralareasofthedistributionrange[38].SeveralpopulationgeneticstudiesindicatethatA.arte-
misiifoliainEuropehasahighgeneflow[27–29]andpollenfromthecentraldistribution
rangehasbeenfoundinSweden[44,45].Inaddition,seedsaredispersedbyimportationofA.
artemisiifoliainfectedbirdseed.Hence,fromthisperspective,thefutureestablishmentofviable
populationsofA.artemisiifoliainFennoScandinaviaisarealisticscenariowithstrongselection
pressureandtheprerequisitesformigrationofpre-adaptedalleles.Thisconclusionissup-
portedbyresultsfromtheGermanfieldstudy.Despitelocaladaptationatbothlocalities,flow-
eringoccurredsignificantlyearlierinDrebkauthaninBerlin,whichindicatesthatA.
artemisiifoliahasbeenintroducedinnorthernGermanyonseveraloccasions.
Toavoidexposingpeopleintheareatoallergenicpollen,individualmaleflowerheadswere
cutatthefirstsignofpollendistributionattheSwedishcommongardensite,andthusitwas
notpossibletorecordthedateofseedsetting.TheearlyfloweringoftheGermanpopulation
atthenorthernsiteis,however,aclearindicationthatthispopulationispre-adaptedtoset
seedsbeforefrostatnorthernlatitudes.Also,thedatesofmaleandfemalefloweringrecorded
atthenortherncommongardensitecoincidedwellwithfieldobservationsoftheDrebkau
populations.ThefieldobservationsatbothGermanlocalities(DrebkauandBerlin)indicate
thataperiodof10–12weeksafteronsetoffemalefloweringisneededforseedstomature
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