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Beatty&Provan—ClonaldiversityinH.monotropa
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hermaphroditeandplantscanreproducebothsexuallyandclonally.Hypopitysmonotropaisepiparasitic,persistingundergroundforthemajorityoftheyearandproducingaerialspikesfromlateJulyuntillateSeptember.Insectpolli-natorshavebeennotedvisitingthe owers(Wallace,1977),andwhenseedproductiondoesoccur,persistenceofseedlingsisdependentonthepresenceofmycorrhizalfungiofthegenusTricholoma,asisthecasewithmanymembersoftheEricaceae(BidartondoandBruns,2001;Leakeetal.,2004).ThemainaimofthepresentstudywastoassessthelevelsofgeneticdiversityintheremainingpopulationsofH.monotropainNorthernIreland.ApreviousstudyonanothermemberoftheMonotropoideae,Orthiliasecunda,whichalsoexistsasaseriesofsmall,highlyfragmentedpopulationsattheedgeofitsrangeinNorthernIreland,revealedextremelyhighlevelsofclonality(Beattyetal.,2008).Eachpopulationcomprisedasingleclone,withacompletelackofwithin-populationgeneticvariation.Consequently,wewereparticularlyinter-estedindeterminingwhethertheseperipheralpopulationsofH.monotropawerealsocharacterizedbyhighlevelsofclonalgrowth,sinceextantpopulationsofbothspeciesarerestrictedtothesametwoareasandsincesuchdataisimpor-tantfortheformationofrational,sustainableconservationmeasuresforthesethreatenedpopulations.
MATERIALSANDMETHODS
Surveysandstudypopulations
Surveyswerecarriedoutin2007and2008forCo.Fermanaghpopulations,and2009and2010forCo.Antrimpopulations.AllsiteswhereHypopitysmonotropahadpreviouslybeenrecordedwerevisitedandthenumbersofplantspresentwererecordedandtheirpositionsineachpopulationmapped(Fig.1andTable1).ErrigalBanks,Co.Londonderry,wasalsosurveyedin2007and2008,sincethespecieshadpre-viouslybeenrecordedthere,althoughithadnotbeenfoundinrecentyears.
SamplingandDNAextraction
HypopitysmonotropaisprotectedunderSchedule8oftheWildlife(NorthernIreland)Order(1985)and,assuch,itisanoffencetopick,uprootordestroytheplant.Consequently,twoscalesweretakenfromeachplantandstoredinsilicagelfortransportationtothelaboratory.DNAwasextractedfromonescaleperindividualusingtheQiagenDNeasyPlantMiniKit,afteraninitial3-mingrindingat30HzusingaRetschMM300mixermill.DNAwasquan-ti edvisuallyon1%agarosegelsstainedwithethidiumbromideanddilutedtoaconcentrationof50ngmL21forsub-sequentPCR.
Microsatellitegenotyping
Individualsweregenotypedfor veH.monotropamicrosa-tellitelocipreviouslydescribedinKloosteretal.(2009):Mono02,Mono15,Mono20,Mono21andMono22.ThreeadditionallocidevelopedforthisstudyusingtheISSR-cloningtechniqueoutlinedinProvanandWilson
(2007)werealsoused(Table2).Forwardprimersweremodi- edbytheadditionofa19-bpM13tail(5′-CACGACGTTGTAAAACGAC-3’)andreverseprimersweremodi edbytheadditionofa7-bptail(5’-GTGTCTT-3’).PCRwascarriedoutinatotalvolumeof10mLcontaining100nggenomicDNA,10pmolofdye-labelledM13primer(6-FAMorHEX),1pmoloftailedforwardprimer,10pmolreverseprimer,1×PCRreactionbuffer,200mMeachdNTP,2.5mMMgCl2and0.25UGoTaqFlexiDNApolymerase(Promega).PCRwascarriedoutonaMWGPrimusthermalcyclerusingtheconditionsdescribedinKloosteretal.(2009)andgenotypingwascarriedoutonanAB3730xlcapil-larygenotyping.system.AllelesizeswerescoredinGENEMAPPERV41(AppliedBiosystems)usingLIZ-500sizestandardsandwerecheckedbycomparisonwithpreviouslysizedcontrolsamples.
Dataanalysis
AsH.monotropapossessesthecapacityforclonalreproduc-tion,clonemateswereidenti edbycalculatingtheprobability(PGEN)ofeachmulti-locusgenotype(MLG)arisingthroughsexualasopposedtoclonalreproductionfollowingthemethodofParksandWerth(1993):
PGEN=[2hP(x1ix2i)]n 1
wherehisthenumberoflociatwhichthegenotypeishetero-zygous,xotypeatlocus1iistheallelefrequencyofthe rstalleleinthegen-i,xat2iistheallelefrequencyofthesecondalleleinthegenotypelocusi.Theobservationofheterozygosityallowsthedifferentiationbetweengeneticidentityduetoclonalpropagationandidentityduetoinbreedingorsel ng,whichwouldleadtoanincreaseinhomozygosity.SampleswhichsharedMLGswithPGEN,0.05wereconsideredclone-matesandidenti edassuchonthemaps,andduplicategeno-typeswereremovedfromsubsequentanalyses.PwerecalculatedusingtheGGENvaluesENCLONEsoftwarepackage(V2.0;Arnaud-HaondandBelkhir,2007).Levelsofexpectedhetero-zygosity(HE)basedonnuclearmicrosatelliteallelefrequen-cieswerecalculatedusingtheARLEQUINsoftwarepackage(V3.01;Excof eretal.,2005)forpopulationswithasamplesizeofN≥5.Thesigni canceofdifferencesinvaluesofHEbetweenyearswasestimatedusingtwo-tailedpairedt-tests,andaSpearman’srankcorrelationcoef cientwascal-culatedtotestforanyassociationbetweenpopulationsizeandgeneticdiversity.Levelsofclonaldiversitywereestimatedbycalculatinggenotypicrichness(R),de nedas(G–1)/(N–1),whereGisthenumberofMLGsandNisthenumberofplantsinthepopulation.Wheresampleswerelocatedinsuccessiveyears,anestimateofpopulationsizewasderivedusingasimplecapture–recaptureformula(numberofMLGsinyear1×numberofMLGsinyear24numberofMLGsidenti edinbothyears).Inbreedingcoef cients(FusingtheGpackage(V4.0.1.0;IS)wereestimatedENEPOPsoftwareRaymondandRousset1995).Totestforgeneticdifferencesinpopulationsbetweensuccessiveyears,analysesofmolecularvariation(AMOVA)werecarriedoutusingtheARLEQUINsoftwarepackage(V3.01;Excof eretal.,2005).AMOVAswerealso