Evolution of the Selfing Syndrome in Arabis alpina

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Evolution of the Selfing Syndrome in Arabis alpina (Brassicaceae)
Tedder, Andrew ; Carleial, Samuel ; Gołębiewska, Martyna ; Kappel, Christian ; Shimizu, Kentaro K ;
Stift, Marc
DOI: https://doi.org/10.1371/journal.pone.0126618
Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-111113
Journal Article
Published Version

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Originally published at:
Tedder, Andrew; Carleial, Samuel; Gołębiewska, Martyna; Kappel, Christian; Shimizu, Kentaro K;
Stift, Marc (2015). Evolution of the Selfing Syndrome in Arabis alpina (Brassicaceae). PLoS ONE,
10(6):e0126618.
DOI: https://doi.org/10.1371/journal.pone.0126618
RESEARCHARTICLE
Evolution of the Selfing Syndrome in Arabis
alpina (Brassicaceae)
AndrewTedder1,SamuelCarleial2,MartynaGołębiewska1,ChristianKappel3,Kentaro
K.Shimizu1*,MarcStift2*
1 InstituteofEvolutionaryBiologyandEnvironmentalstudies,UniversityofZurich,Zurich,Switzerland,
2 Ecology,DepartmentofBiology,UniversityofKonstanz,Konstanz,Germany,3 InstitutfürBiochemieund
Biologie,UniversitätPotsdam,Potsdam-Golm,Germany
* [email protected](KKS); [email protected](MS)
Abstract
Introduction
Thetransitionfromcross-fertilisation(outcrossing)toself-fertilisation(selfing)frequentlyco-
OPENACCESS
incideswithchangestowardsafloralmorphologythatoptimisesself-pollination,theselfing
Citation:TedderA,CarleialS,GołębiewskaM,
syndrome.Populationgeneticstudieshavereportedtheexistenceofbothoutcrossingand
KappelC,ShimizuKK,StiftM(2015)Evolutionofthe
SelfingSyndromeinArabisalpina(Brassicaceae). selfingpopulationsinArabisalpina(Brassicaceae),whichisanemergingmodelspeciesfor
PLoSONE10(6):e0126618.doi:10.1371/journal. studyingthemolecularbasisofperennialityandlocaladaptation.Itisunknownwhetherits
pone.0126618
selfingpopulationshaveevolvedaselfingsyndrome.
AcademicEditor:HidenoriSassa,ChibaUniversity,
JAPAN Methods
Received:July26,2014
Usingmacro-photography,microscopyandautomatedcellcounting,wecomparedfloral
Accepted:April4,2015 syndromes(size,herkogamy,pollenandovulenumbers)betweenthreeoutcrossingpopu-
Published:June3,2015 lationsfromtheApuanAlpsandthreeselfingpopulationsfromtheWesternandCentral
Alps(MaritimeAlpsandDolomites).Inaddition,wegenotypedtheplantsfor12microsatel-
Copyright:©2015Tedderetal.Thisisanopen
accessarticledistributedunderthetermsofthe litelocitoconfirmpreviousmeasuresofdiversityandinbreedingcoefficientsbasedonallo-
CreativeCommonsAttributionLicense,whichpermits zymes,andperformedBayesianclustering.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
ResultsandDiscussion
credited.
Plantsfromthethreeselfingpopulationshadmarkedlysmallerflowers,lessherkogamy
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles. andlowerpollenproductionthanplantsfromthethreeoutcrossingpopulations,whereas
pistillengthandovulenumberhaveremainedconstant.Comparedtoallozymes,microsat-
Funding:ThisworkwassupportedbytheUniversity
ofKonstanz(ExcellenceInitiativeIndependent ellitevariationwashigher,butrevealedsimilarpatternsoflowdiversityandhighFisinself-
ResearchStartupGranttoMS,http://www.exzellenz. ingpopulations.Bayesianclusteringrevealedtwoclusters.Thefirstclustercontainedthe
uni-konstanz.de/en/);theSwissNationalScience
threeoutcrossingpopulationsfromtheApuanAlps,thesecondcontainedthethreeselfing
Foundation(grantnumbersCRSI33_127155and
populationsfromtheMaritimeAlpsandDolomites.
SinergiaAVE31003A_140917,http://www.snf.ch/en/
Pages/default.aspx);theUniversityofZurich
(UniversityResearchPriorityProgramEvolutionin Conclusion
Action,http://www.uzh.ch/research/priorityprograms/
Weconcludethatincomparisontothreeoutcrossingpopulations,threepopulationswith
university_en.html);andtheHumanFrontiers
ScienceProgram(YoungInvestigatorAwardtoKKS, highselfingratesarecharacterisedbyaflowermorphologythatisclosertotheselfing
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 1/17
EvolutionoftheSelfingSyndromeinArabisalpina
http://www.hfsp.org/).Thefundershadnorolein syndrome.Thepresenceofoutcrossingandselfingfloralsyndromeswithinasinglespecies
studydesign,datacollectionandanalysis,decisionto willfacilitateunravellingthegeneticbasisoftheselfingsyndrome,andaddressingwhich
publish,orpreparationofthemanuscript.
selectiveforcesdriveitsevolution.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Introduction
Mostangiospermsareoutcrossing,buttransitionstoself-fertilisation(selfing)arefrequent
[1,2].Basedonphylogeneticdata,thesetransitionsappeartobeunidirectionalandirreversible,
withselfingasthederivedstate[3–6].Therepeatedparalleltransitiontoselfinghasprovideda
uniqueopportunitytofindrecurrentpatternsinadaptiveevolutionamongdifferentspecies
[7,8].Selfingprovidestwodistinctadvantageswhencomparedtooutcrossing[1,2,9]:transmis-
sionadvantageandreproductiveassurance.Selfershaveatransmissionadvantagebecausethey
passtheircompletegenometothenextgenerationwithoutdilutionoftheirgeneticmaterial
fromamatingpartner.Atthesametime,theycanalsoactasoutcrosspollendonorsforseed
producedbyotherindividuals[10].Reproductiveassuranceisduetoselfersbeingabletore-
producewhenmatesarelimitedorwhenpollinatorsarescarce[11].Forselfingtoevolvefrom
anoutcrossingbackground,anybarrierstoself-fertilisation(e.g.,self-incompatibilitymecha-
nisms)mustbreakdown,andtransmissionadvantageandreproductiveassurancemustout-
weighinbreedingdepressionandanyothernegativeeffectsofinbreeding[12,13].Conditions
conducivetotheevolutionofselfingareforexamplefoundduringpostglacialgeographic
rangeexpansion,wheremateandpollinatorlimitationarelikely[14,15].
Transitionstoselfingoftenresultinspeciationinwhichchangesinthefloralsyndromeplay
animportantpart[16,17].Selfingspecieshaveatypicalfloralsyndrometermedtheselfingsyn-
drome[18].Comparedwithoutcrossingsyndromes,theselfingsyndromeconsistsofsmaller
flowersthatopenless,havereducedherkogamy(ashorterdistancebetweenstigmaandan-
thers)andatendencytowardsreducedpollen:ovuleratios,nectarproductionandscentemis-
sion[18].Severalselectiveforcesmaydrivetheevolutionoftheselfingsyndrome,forexample
selectionforrapidmaturationinmarginalhabitatsleadingtoareductioninoverallorgansize
[19],selectionforsmallerflowersthroughpreferentialpredationonlargerflowersbyflorivores
[20,21],orselectionforincreasedresourceallocationtoprogenyandreducedallocationtoat-
tractivetissuesthroughthereducedrequirementforpollinatorattraction[22].Theabundant
examplesofcloselyrelatedself-incompatibleandself-compatibletaxawithcontrastingfloral
syndromes(forexampleIpomoeacordatotrilobaandI.lacunosa[23];orCapsellagrandiflora
andC.rubella[16])suggestthattheevolutionofselfingisamajorforcedrivingspeciationin
angiosperms.Accordingly,theevolutionarytransitionfromoutcrossing,self-incompatiblean-
cestorstoself-compatible,selfinglineagesisofprimeinteresttoevolutionarybotanists[5].
Comparisonsbetweencloselyrelatedtaxawithcontrastingmatingsystemshavebeenused
tostartunravellingthegeneticbasisofthelossofself-incompatibilityandoffloralchangesto-
wardstheselfingsyndrome.Interspecificcrossesbetweenoutcrossingandselfingsistertaxa
havebeenusedforlinkagemappingoftheselfingsyndromeinalimitednumberofsystemsin-
cludingCapsella[24],Leptosiphon[25],Lycopersicon[26,27]andMimulus[28,29].Thecom-
monpatternemergingfromthesestudiesisthatalargenumberoflocicontributetothefloral
traitsthatdifferbetweentheoutcrossingandselfingsisterspecies.SpecificallyinC.rubellait
hasbeensuggestedthattheselfingsyndromehasevolvedfromthecommonancestorwithC.
grandiflorainastepwisemanner,involvingmultipleconsecutivemutationsatdifferentloci
[24].Furtherunravellingthetimelineofsuchstepwiseevolutionrequiressystemsinwhich
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 2/17
EvolutionoftheSelfingSyndromeinArabisalpina
outcrossingandselfinglineageshavedivergedlessthanthe30-50kythathasbeenestimated
forC.grandifloraandC.rubella[16,30].Therefore,itisofinteresttocharacterisesystemswith
intraspecificmatingsystemandfloralsyndromevariation,whichhastheextraadvantagethat
thesemaybecrossedmorereadily.Suchsystemswouldalsofacilitatemoredirectlyaddressing
theselectiveforcesthatdrivetheevolutionoftheselfingsyndrome.
ThenaturalvariationinmatingsystemthathasbeendiscoveredinArabisalpinamayprovide
apromisingmodelinthiscontext.AcrossitsEuropeanrange,A.alpinaoccursinalpineand
sub-alpineareasfromSpaintoScandinavia[31,32].Thiswidedistributionrangehasbeenattrib-
utedtorecentexpansionafterglacialperiods[31].Initsnorthernrange,A.alpinaisself-compat-
iblewithvariableF valuessuggestingamainlyselfingreproductivestrategy,withamaintained
IS
abilitytooutcross[33–35].Inthreepopulations,allozymegenotypingofprogenyarrayshadcon-
firmedthatplantsalmostexclusivelyreproducedbyselfing(outcrossingrates<0.15[34]).South
oftheAlps,F valuessuggestedapredominantlyoutcrossingstrategy[33]andinlinewiththis
IS
werecentlydiscoveredthreeself-incompatiblepopulationsintheApuanAlpswithoutcrossing
rates>0.78andhighpolymorphismintheself-incompatibilitygeneSRK[34].
Withitsdraftgenomeassemblypubliclyavailable[36],A.alpinaisanemergingmodelspe-
cies,amongothersforstudyingperenniality[37]andlocaladaptation[38,39].Usingtransgenic
techniquesandmutantscreenings,themolecularpathwaysresponsibleforperennialflowering
havebeenextensivelystudied[40–42].Inaddition,giventhepresenceofbothself-incompati-
bleoutcrossingpopulationsandpredominantlyselfingpopulations,A.alpinamaybeafeasible
modeltostudythemolecularandevolutionarybasisofthetransitiontoselfing,andthefloral
changesassociatedwiththistransition.However,itisstillunknownwhetherchangestowards
aselfingsyndromehaveoccurredinA.alpina.
Inthispaper,weusesixpopulations(Fig1)forwhichoutcrossingrateshadbeenestimated
previouslytotestwhetherplantsinselfingpopulations(MaritimeAlpsandDolomites)areassoci-
atedwithsmallerflowerscomparedtooutcrossingpopulations(ApuanAlps),andwhetherother
traits(herkogamy,pollenandovulenumber)havealsochangedtowardsstatestypicalfortheself-
ingsyndrome.Inaddition,weusedasetofmicrosatellitemarkers[35]toconfirmpreviouspopu-
lationgeneticestimations(allelicrichness,Ho,He,Fis,populationstructure)basedonallozymes,
becausetheseallozymeshadsufferedfromlackofvariationintheselfingpopulations[33,34].
MaterialsandMethods
Plantmaterial
Tocomparefloralmorphology(sizeandherkogamy)amongthreeoutcrossingandthreeselfing
populationsofArabisalpina(Fig1),wemadeuseofacollectionofrandomlyarrangedindividu-
allypottedplantsgrowinginacommongardenenvironment(BotanicalGarden,Universityof
Konstanz)sinceOctober2012.TheseindividualsstartedtoflowerinApril2013,andagainin
March2014.Theplantsoriginatedfromwild-collectedmaternalseedfamiliesfromthreeout-
crossingpopulationsintheApuanAlpsinItaly(Rotondo,14seedfamilies;Colli,14seedfami-
lies;PorteStrazzema,13seedfamilies)andthreeselfingpopulations,onefromtheDolomitic
AlpsinItaly(Presolana,13seedfamilies)andtwofromtheMaritimeAlpsinFrance(Valde
Roya1,14seedfamilies;ValdeRoya2,4seedfamilies)[34](Fig1).TheseweresowninOctober
2011foranotherexperiment.Eachseedfamilywasrepresentedbyasingleplant,sothetotalde-
signincluded41individualsfromoutcrossing,and31individualsfromselfingpopulations.
Flowermacrophotography
Todetermineflowersize(petalarea)andherkogamy,inspring2013,wetookstandardised
photographsofflowersusingaNikonD7100DSLRcamerawithaSigma150mmF2.8EXDG
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 3/17
EvolutionoftheSelfingSyndromeinArabisalpina
Fig1.SamplinglocationsofthreeoutcrossingpopulationsandthreeselfingpopulationsofArabis
alpina(detailsonoutcrossingrates,seeTable3).Piechartsindicatethepopulationmeanposterior
assignmentprobabilitybasedonBayesianclusteringanalysis(Fig5).Thepredominantlybluecluster
includesthethreeoutcrossingpopulations:COLL=Colli;POST=PorteStrazzema;ROTO=Rotondo.The
predominantlyredclusterincludesthethreeselfingpopulations:PRES=Presolana;VDR1and2=Valde
Roya1and2.
doi:10.1371/journal.pone.0126618.g001
OSHSMAPOMacrolensthatwasmountedonaheight-adjustablesupport.Foreachplant,
wephotographedtwotofiveflowerswiththecameraattheminimumfocaldistance.Thede-
velopmentalstateofflowerswasstandardisedbycollectingflowersbetween1000hand1600h,
whenmatureflowerswerefullyopenedandonlyusingflowersofwhichtheantherswerein
theprocessofdehiscing,andthatstillhadbrightwhitepetals(i.e.,petalsthatwereneitherlos-
ingbrightness,norhadalreadystartedwilting;petalstendtolosebrightness1–3daysafteran-
therdehiscence).Ifaninflorescencehadmultipleflowers,thesecriterianormallymeantthat
weusedtheyoungestfullyopenedflower.Immediatelyaftercollection,wephotographedflow-
ersagainstablackbackground(1%agarwith0.5%activatedcarbon),alwaysincludingasize
standardintheframe.First,torecordpetalarea,weplacedthepedicelofintactflowersina
capillarytube,andpositionedthetop-viewplaneoftheflowerparalleltothelensplane.Then,
torecordherkogamy,wecarefullyremovedthepetalsandtwoofthelongstamens,andposi-
tionedtheplanethroughthestyleandshortstamensparalleltothelensplane.
Flowersize,herkogamyandshapeanalysis
Flowersandsizestandardwereseparated(segmented)fromtheblackbackgroundusing
MatlabR2014a(Version8.3)anditsimageprocessingtoolboxbyapplyingOtsu'sthresholding
method.Areaandperimeterforbothobjects(flowerandstandard)wereextractedusingthe
regionpropsfunction,automaticallyidentifyingtheobjectsbasedontheirinherentlydifferent
shapes(standardswererectangularandthushadahighereccentricitythantheflowers).Flower
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 4/17
EvolutionoftheSelfingSyndromeinArabisalpina
Fig2.PlacementofeightlandmarksondissectedsideviewphotographsofArabisalpinaflowers.Photosarerepresentativeexamplesforeachofthe
threeselfingpopulations(toppanel)andthethreeoutcrossingpopulations(bottompanel).Numbersindicatelandmarks:1—baseofshortleftstamen;2—
baseofpistil;3—baseofshortrightstamen;4—anthertipofshortleftstamen;5—stigmatip;6—anthertipofshortrightstamen;7—anthertipoflongleftleft
stamen;8—anthertipoflongrightstamen.Withinoneofthepictures,linesindicatethemeasuretakenbasedonthelandmarks:averageherkogamylong
stamens(redlines)as(a+b)/2;averageherkogamyshortstamens(bluelines)as(c+d)/2;averagelengthlongstamens(orangelines)as(f+g)/2;average
lengthshortstamens(orangelines)as(h+i)/2;averageanglelongstamens(reddoubledashedline)as(α+β)/2;averageangleshortstamens(reddouble
dashedline)as(γ+δ)/2.
doi:10.1371/journal.pone.0126618.g002
parameterswerescaledusinginformationfromthesizestandard.Flowershapes(i.e.,the
shapesofthepetaloutlines)weredescribedbycalculatingadissectionindexasperiffimffiffieffitffiffier.Toquan-
parea
tifyherkogamyonphotographsofthedissectedflowers,weusedthesoftwareTPSdig2(ver-
sion2,http://life.bio.sunysb.edu/morph/)toplace8digitallandmarksonthedissectedflower
(Fig2).LandmarkcoordinateswereimportedinR(version2.15.3[43]),andusingbasictrigo-
nometricfunctionswecalculatedmeanherkogamyandangleforbothshortandlongstamens
andtheratiobetweentheshortandlongstamenlength(Fig1andTable1).
Table1. Overviewoflandmark-basedtraitsextractedfromdissectedsideviewphotographs(seeFig
2foranoverviewofthelandmarks).
Trait Formulaa
Meanherkogamylongstamens(mm) dL5 L7 dL5 L8
ð (cid:2) Þþ2 ð (cid:2) Þ
Meanherkogamyshortstamens(mm) dL5 L4 dL5 L6
ð (cid:2) Þþ2 ð (cid:2) Þ
Meananglelongstamens(degrees) L7L2L5 L8L2L5
ff þ2ff
Meanangleshortstamens(degrees) L4L2L5 L6L2L5
ff þ2ff
Pistillength(mm) d(L2–L5)
Meanlongstamenlength dL2 L7 dL2 L8
ð (cid:2) Þþ2 ð (cid:2) Þ
Meanshortstamenlength dL2 L4 dL2 L6
ð (cid:2) Þþ2 ð (cid:2) Þ
Traitsforwhichtheanalysisispresentedinthemainpaperareinbold,analysesfortheremainingtraitsare
presentedinS1TableandS1Fig.
ad=distance(inmm)
doi:10.1371/journal.pone.0126618.t001
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EvolutionoftheSelfingSyndromeinArabisalpina
Pollencounting
Toobtainpollencounts,inspring2014,wecollectedthreetosixflowerbudsforfourtoeight
plantsperpopulation(n=5forRotondo,n=7forPorteStrazzema,n=7forColli,n=6for
Presolana,n=8forValdeRoya1,n=4forValdeRoya2).Tostandardisethedevelopmental
stageofbuds,weonlycollectedbudsonwhichthepetalswerejuststartingtobecomevisible
(nomorethan1–2mm)between1000hand1600h.Budsweremanuallyopenedanddried
overnightat65°C.Toreleasepollenfromtheanthers,weadded100μlof5%Tween-20and
sonicated(BioruptorPlus,Diagenode,Liege,Belgium)for10cyclesof30seconds‘ON’and30
seconds‘OFF’.Finallyweadded10mLofCASYTonebuffer(RocheDiagnostics,Mannheim,
Germany)andthetotalvolumewasthenanalysedonaCASYModelTT—CellCounterand
Analyzer(RocheDiagnostics,Mannheim,Germany),usingthe150μmcapillary(whichcovers
asizerangeof3.2–120μm)withameasurementrangefrom12.5to25μm.Ifabortedpollen
wasdetected,themeasurementrangewasalteredto7.5–25μmtoensureaccuratecountingof
bothpollentypes.Pollencountswerewellbelow10000in18outof137cases(8fromoutcross-
ingpopulations,10fromselfingpopulations).Asweassumedthatthesewereduetotechnical
artefacts,weexcludedthemfromanalysis(excludedsampleshadpollencountsthatranged
from269to7612,includedsamplesrangedfrom10100–218100).
Ovulecountingandovarylengthmeasurement
Tocountovules,wecollectedthreetosixbudsinthesamestageandfromthesameplantsas
usedforpollencounting(forRotondo,samplesfortwoextraplantswereincluded),andfixed
theminethanol:aceticacid(9:1)foraminimumof12hours.Tosoftenthetissue,pistilswere
cutatthereceptacleandstigma,andsoakedin1NNaOHfor2hat60°C.Aftersoftening,sam-
pleswereplacedontoamicroscopicslideandovulenumberwascountedunderaLeica
DM5000BmicroscopewithaDICprism(Leica,Wetzlar,Germany).
Todetermineovarylengthonthesamesamplesforwhichovuleshadbeencounted,we
tookphotographswithaLeicaMonochromeDigitalCameraDFC345FX(Leica,Wetzlar,Ger-
many)connectedtothemicroscope(magnification5x)usingtheLeicaApplicationSuiteAF
version1.0.0build0(Leica,Wetzlar,Germany).Tofitthewholeovary,1–4photographswere
takenforeachsample,withsufficientoverlaptoallowlatermergingoftheimageswiththe
‘photomerge’functioninAdobePhotoshopCCversion14.2.1x64(AdobeSystems,California,
USA).Usingthemergedimages,wemanuallymeasuredovarylengthusingImageJ1.48v.
Statisticalanalysis
Totestwhetherfloraltraitsdifferedbetweenmatingsystems,weusedlinearmixedmodelsas
implementedinthelmefunctioninthenlmepackageinR(v2.15.3[43]).Thefixedpartof
themodelwasMatingSystem(outcrossingvs.selfing),andtherandompartofthemodelin-
cludedPopulation(nestedinmatingsystem,sincewecomparedthreeoutcrossingandthree
selfingpopulations)andPlant(nestedinPopulation,sincemultipleflowerswereanalysed
foreachplant).Weinspectedmodelresidualstructurefornormalityandvariancehomoge-
neity,andappliedtransformationstomeetmodelassumptionswhereneeded.Withoneex-
ception,insuchcaseseitherasquarerootorlog transformationwassufficient.Inthecaseof
e
pollennumbertherewasstrongvarianceheterogeneityamongmatingsystems,whichwead-
dressedbymodellingaseparatevarianceforthefactorlevelsoutcrossingandselfing(using
thesubcommandVarIdentwithinthelmefunction),sothatinterpretationofresultsisnotbi-
asedbyvarianceheterogeneity[44].
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 6/17
EvolutionoftheSelfingSyndromeinArabisalpina
DNAextractionandmicrosatellitegenotyping
Forgeneticanalysis,inOctober2012,wecollectedanddried(onsilica)leaftissuefromplants
fromthesamethreeoutcrossingandselfingpopulationsthatwehadusedtostudyflowermor-
phology.Thegeneticanalysisincludedall72individualsforwhichflowermorphologyhad
beendetermined,and23additionalsamplesforwhichtissuehadbeencollected,butthathad
eitherdiedbeforetheflowermeasurements(inwinter2012–2013)orthatdidnothaveflowers
duringtheflowerphotography(inspring2013).Samplesizesperpopulationwere:Rotondo
(n=23),Colli(n=17),PorteStrazzema(n=20),ValdeRoya1(n=15);ValdeRoya2
(n=4),Presolana,n=16).ToextractDNA,upto50mgofdesiccatedtissuewasgroundfor
two30-secondburstsusingaTissueLysermixer-milldisrupter(Qiagen,CA,USA)withthead-
ditionofasingle5mmstainlesssteelbeadpersample.TotalgenomicDNAwasthenextracted
usingaBiosprint96workstation(Qiagen,CA),followingthemanufacturers’defaultprotocols.
Todeterminewhetherpopulationsclusteredaccordingtomatingsystemandregionoforigin,
wegenotyped12microsatellitelocidevelopedforA.alpinaacrossall96individuals:DJ5E,
DEET,4MDH,A1T8T,5GTC,6U3S,A93Q,A4JW7,9VSH,7PJQ,BWF1and3Q19[35].In
eachcase,theforwardprimerwastaggedwithoneoffourfluorescentdyes,6-FAM,ATTO550,
ATTO565,orYakimayellow(MicroSynthAG,Balgach,St.Gallen,Switzerland).Productswere
amplifiedbysingleplexPCRusingthefollowingreagents:1μLof50–100ng/μLDNAtemplate,
4μL5xPCRbuffer,1μLof2mMMgCl ,1μLof10mMdNTPs,0.2μLof10μMforwardandre-
2
verseprimer,1UTaqpolymerase,andddH Otoareactionvolumeof20μL.Thermocyclingwas
2
performedonPTC-200(MJresearch,Watertown,MA,USA)machinesusingthefollowingpro-
gramme:initialdenaturationat94°Cfor15minfollowedby30cyclesof94°Cfor30s,57°Cfor
90s,72°Cfor60s,(rampto72°Cat0.7°C/s)andafinal72°Cextensionfor30min.PCRprod-
ucts(1:200dilutions)weregenotypedusinganABI3730sequencer.Genotypeswereanalyzed
usingGENEMAPPER4.0(AppliedBiosystems,FosterCity,CA,USA)andcorrectedmanually.
Toconfirmpreviousfindingsbasedonallozymes[33],foreachpopulationweusedourmi-
crosatellitegenotypestocalculatetheaveragenumberofallelesperlocus,expected(H )andob-
E
served(H )heterozygosity,andtheinbreedingcoefficient(F )usingGENEPOP4.0.10[45,46].
O IS
Bayesianinferenceofpopulationstructure
Toanalyseclusteringofpopulationsinrelationtomatingsystemandgeography,weusedthe
BayesianclusteringalgorithmimplementedinSTRUCTURE2.3.4[47].Thismethodusesa
multilocusgenotypetoprobabilisticallyassignindividualstooneormoreclusters.Usingthe
admixturemodelwithdefaultsettings(correlatedallelefrequencies),weran10simulationsper
priorK(K=1toK=6);foraburn-inperiodof200,000generationsand1,000,000MCMCrep-
licatesafterburn-in.WeplottedKvs.thelikelihoodsobtainedoverallsimulationsandinferred
theoptimalnumberofclustersKasrecommendedbytheSTRUCTUREmanual.Additionally,
weconfirmedKbasedontheΔKmethod[48],usingSTRUCTUREHARVESTERv0.46[49]
tocalculatethemodeofsecondorderderivativesofthelikelihooddistributiondividedbythe
standarddeviationsacrossreplicates(m|L (K)|/s[L(K)]),whichwasplottedagainstKforvisual
00
evaluation.CLUMPP1.1wasusedtocombinesimulationoutputforeachK[50].
Results
Floralchangestowardstheselfingsyndrome
Comparedtooutcrossingpopulations,plantsfromselfingpopulationshad2.5timessmaller
flowers(petalarea),a2.5foldreductioninthedegreeofherkogamyofthelongstamens,anda
1.3foldincreaseinherkogamyoftheshortstamens(Fig3andTable2).Moreover,selfing
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 7/17
EvolutionoftheSelfingSyndromeinArabisalpina
Fig3.BoxplotsformatureflowertraitsofthreeoutcrossingandthreeselfingpopulationsofArabisalpina.A)petalareaoflandingplatform,inlay
showstworepresentativeexamplesoftheoriginalphotographs;B)pistillength;C)absoluteherkogamyoflongstamens;D)absoluteherkogamyofshort
stamens.Theindividualpointsindicatethemeantraitvaluesforthereplicateflowersanalysedwithinindividualplantsonwhichtheboxplotswerebased.
doi:10.1371/journal.pone.0126618.g003
populationsproduced3.4timesfewerpollengrainswhilethenumberofovuleswasnotsignifi-
cantlydifferent(Fig4andTable2).Usingthemeanpollennumberandmeanovulenumberof
eachplant,thismeantthatpollen:ovuleratiosweremarkedlylowerintheselfingpopulations
(667,669and546,forPresolana,ValdeRoya1andValdeRoya2,respectively)thantheout-
crossingpopulations(2187,2244and2188forRotondo,PorteStrazzemaandColli,respective-
ly).Pollensizeswereslightlylargerfortheselfingpopulations(S1Table).
Thereducedherkogamyofthelongstamensinselfingpopulationscouldnotbeattributedto
anincreasedanglebetweenpistilandlongstamens.Infacttheanglewaslargerforselfingpopu-
lations(S1Table),whichwouldhavehadtheoppositeeffect(i.e.,increasingherkogamy).Pistil
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 8/17
EvolutionoftheSelfingSyndromeinArabisalpina
Table2. LinearmixedmodelanalysisoftheeffectofMatingSystemonfloraltraitsofArabisalpina.
Trait(unitof Transformationfor Modelestimate(transformed)ofdifferencebetweenoutcrossing t- p
measurement) analysis andselfingmeansa value (df=4)b
Flowerarea(mm2) log -0.94 -8.68 0.001
e
Pistillength(mm) squareroot -0.14 -2.15 0.0978
Herkogamyshortstamens - +0.58 6.54 0.0028
(mm)
Herkogamylongstamens squareroot -0.58 -5.94 0.004
(mm)
Pollennumber[x1000] - -61.6 -10.7 <0.001
(count)c
Ovulenumber(count) - -2.55 -1.20 0.298
Caseswheremodelestimatesforselfingpopulationsdiffersignificantlyfromthoseoftheoutcrossingpopulationsareindicatedinbold.
aModelFixedpart:Matingsystem;Randompart:Population(nestedinMatingSystem)andPlant_ID(nestedinPopulation),analysedwiththelme
functioninthenlmepackageinR[43]
bdf:degreesoffreedom.Fortestingdifferencesbetweenmatingsystemdf=4becausetherewerethreeoutcrossingandthreeselfingpopulations
cThismodelincludedaseparatevarianceforthetwomatingsystems(varIdentoptionoflme),astherewasalargevarianceheterogeneityamongmating
systemsthatcouldnotberemovedbytransformation.
doi:10.1371/journal.pone.0126618.t002
lengthdidnotdifferbetweenoutcrossingandselfingpopulations(Fig3andTable2),andhence
thereducedherkogamyofthelongstamens(Fig3andTable2)wasmainlyduetoareduced
longstamenlength(andanthersize)inselfingpopulations(S1Table).Theincreasedherkogamy
oftheshortstamens(Fig3andTable2)wasduetoacombinedeffectofanincreasedanglebe-
tweenpistilandshortstamens,andareducedshortstamenlengthinselfingpopulations(S1
Table).Therewerenoflowershapedifferencesbasedondissectionindex(S1Table).
Fig4.GameteproductioninthreeoutcrossingandthreeselfingpopulationsofArabisalpina.A)Pollennumbers;(B)andovulenumbers.Individual
pointsindicatethemeantraitvaluesforthereplicateflowersanalysedwithinindividualplantsonwhichtheboxplotswerebased.
doi:10.1371/journal.pone.0126618.g004
PLOSONE|DOI:10.1371/journal.pone.0126618 June3,2015 9/17