Post on 22-Jun-2020
FinalreporttotheWestVirginiaDepartmentofNaturalResourcesProjectTitle:Ageneticassessmentofthepopulationhealthandconnectivityofakeystonespecies
inhighelevationAppalachianforestecosystems:redspruce(PicearubensSarg.)
PrincipalInvestigator:Dr.StephenR.Keller,AdjunctFaculty1
KeyPersonnel:ReginaTrott,FacultyResearchAssistant
SupportingInstitution:AppalachianLaboratory
UniversityofMarylandCenterforEnvironmentalScience
301BraddockRd.
Frostburg,MD21532
301-689-7203
email:skeller@umces.edu
AwardNumber:WDUProject#13-R10Datesubmitted:July20,20171CurrentAddress:AssistantProfessorofPlantBiology
DepartmentofPlantBiology
UniversityofVermont
111JeffordsHall,63CarriganDrive
Burlington,VT05465
Email:srkeller@uvm.edu
Phone:802-656-5121
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BackgroundandMotivation
Highelevationconiferousforestsdominatedbyredspruce(PicearubensSarg.)areabiodiversityhotspotintheCentralAppalachians,representativeofborealforest
ecosystemstypicallyrestrictedtomorenortherlyregionsofeasternCanadaand
NewEngland.Theseforestsarecriticalhabitatformanyspeciesofplantsand
wildlife,andassuchP.rubensrepresentsa“keystonespecies”whosepopulationviabilityandresiliencetoenvironmentalchangehasfarreachingconservation
impacts(Byersetal.2010).
TheCentralAppalachianredspruceecosystemattainsitsgreatest
concentrationinthehighelevations(>2500ft.)oftheAlleghenyMountainsinWest
Virginia,andwesternMaryland.Estimatesoftheoriginalexpanseofredspruce
forestinthisregionsuggest>400,000hectaresofthisimportantecosystem
blanketedWestVirginia.Today,<10%ofthisareaiscurrentlyoccupiedbynative
redspruce(anestimated30,000hectares;WVWCAP),andmuchofthisissecondor
thirdgenerationgrowth.Thislargehistoricalreductionindistribution,alongwith
itsecologicalsensitivitytoavarietyofsourcesofenvironmentalchange(e.g.,acid
rain,insectpathogens,deforestation,climate)hascausedredsprucedominated
foreststoberegardedasendangeredecosystemsatthestateandglobalscales
(Beane2010;Byers&Norris2011).
Itiswellknownthatdemographicbottlenecksandhabitatfragmentationcan
reducegeneticdiversitywithinpopulationsandaltergeneflowacrossthe
landscape.Whenpopulationsbecomesmallerandmorefragmented,diversityislost
throughinbreedingandgeneticdrift,anddifferentiationamongpopulationswith
limitedconnectivityincreasesasaresultofrestricteddispersalandgeneflow(i.e.,
“populationstructure”).Theroleofgeneticdiversityinconservationisnowwell
established,includingtheimportanceofheterozygositytoindividualphysiological
performanceandavoidanceofinbreedingdepression,andthenecessityofgenetic
diversityforpopulationstobeabletoadapttonewselectionpressures,suchas
changesinthetypeorabundanceofinsectherbivores,orshiftsintheabiotic
environment(ReedandFrankham2003).Thedominantviewamongconservation
biologistsandrestorationecologistsisthattheeffectivepopulationsize(Ne)isakeyvariableintomaintainingminimumviablepopulations(Schwartzetal.2007).Best
practicescallfortheminimumeffectivepopulationsizetobeatleast500inorderto
avoidthedetrimentaleffectsofinbreedingandgeneticerosion,andtomaintain
viablepopulationsthatareadaptableandresilientinthefaceofenvironmental
heterogeneityanddisturbance(Shaffer1981).
Itiscurrentlynotclearwhatimpactshistoricaldecimationhavehadonthe
reservoirofgeneticdiversitypresentinCentralAppalachianP.rubens,orifNeintheregionislargeenoughtomeetconservationneeds.Geneticdiversityscalesstrongly
andpositivelywithreproductivefitnessinP.rubensinotherpartsofitsrange,probablyreflectingthedegreeofinbreedingdepressionarisingamongclose
relativesinlowdiversitypopulations(Rajoraetal.2000;Mosseleretal.2003).
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TheecologicalimportanceofP.rubensanditsvulnerabilitytoecologicaldisturbancehasledtoalargescaleecologicalrestorationeffortbytheCentral
AppalachianRedSpruceInitiative(CASRI).Thegoalofthismulti-agencygroupisto
restore,viasupplementalplantingsandothersilviculturaltechniques,afunctioning
redspruceecosystemthroughoutitsformerrangeinintheCentralAppalachians
(www.restoreredspruce.org).Geneticdiversityplaysakeyroleinrestoration
(Mckayetal.2005),withrestoredpopulationsbenefittingfrominformedselectionandplantingofdiverse,endemicgenotypes.Thus,suchrestorationeffortscould
benefitfromknowledgeofthegeneticdiversityandNeofrestorationstock,andifcertainexistingstandssufferfromusuallylowdiversityorlackofgenetic
connectivitywithotherstands.
Giventheseconservationconcerns,thereisaneedtoassessthestabilityand
healthofexistingnaturalstandsofP.rubensacrosstheCentralAppalachianlandscape.Thisprojectsoughttofillsomeofthesegapsbyconductingapopulation
geneticstudytoestimatelevelsofgeneticdiversityanddegreeofpopulation
structureamongremnantstandsofredsprucewithinWVandwesternMD,withthe
goalofcharacterizingthegeneticdiversityofP.rubensintheregion,itseffectivepopulationsizeandevidenceforhistoricalbottlenecksinNe,andtheextenttowhichgeneflowmaintainsconnectivityamongremnantsprucestands.Specifically,we
soughttoaddressthefollowingthreesetsofquestions:
1. Whatarethestandinglevelsofgeneticdiversitywithinstands?Docertainstandscontainunusuallylowdiversity,orexhibitevidenceof
inbreeding,andassuchcouldbeprioritizedforgeneticrestoration?
2. WhatistheeffectivepopulationsizeofCentralAppalachianP.rubens?IsthereevidenceoftemporalchangesorbottlenecksinNe?HowdoestheNeofCentralAppalachianP.rubenscomparetopopulationsinotherpartsofitsrange?
3. HowmuchgeneticstructureexistsamongexistingstandsintheCentralAppalachians?Whataretheenvironmentalvariables(climate,geographic
distance)thataremostimportanttogeneticisolationamongremnantred
sprucestands?
MethodsFieldSampling
WorkinginclosecollaborationwithbiologistsattheWVDNRNaturalHeritage
Program,theNatureConservancy,andtheUSDAMonongahelaNationalForest,we
prioritizedoldergrowthorgeographicallyisolatedremnantstandsofredsprucefor
fieldsamplingofneedletissueforgeneticanalysis(Byersetal.2010).SamplinglocalityinformationissummarizedinTable1.
Table1.SamplinginformationforP.rubenspopulationscollectedduringthisstudy.SiteCode
State/Prov
LocationName NHPPlotCode1 N2 Latitude(dd.ddd)
Longitude(dd.ddd)
Elevation(m)
MinimumStandAge(yrs)3
BAT WV BarlowTop MONF.302 16 38.2292266 -80.2326555 1362 170BLM WV BlackMountain MONF.303 16 38.2951654 -80.2391810 1369 BNT WV Boar'sNestTrail,DollySods MONF.313 16 38.9391179 -79.3913178 1315 CBA WV CranberryGladesBotanicalArea MONF.205 16 38.1980991 -80.2713728 1027 144CMK WV CheatMountainKnob MONF.272 16 38.6307291 -79.8953463 1212 CNH WV CanaanHeights
16 39.0919726 -79.4498189 1155
CNS WV CranesvilleSwamp CRSW.9 16 39.5338726 -79.4817813 787 COW WV CowPasture MONF.198 16 38.2022533 -80.2570746 1047 103CPT WV CowPastureTrail MONF.194 16 38.1955116 -80.2619217 1038 210CSP WV CanaanStatePark CASP.30 16 39.0484004 -79.4739629 995 FSR WV ForestServiceRoad75
16 39.0111999 -79.3196768 1186
GDK WV GaudineerKnob
16 38.6141002 -79.8429144 GLR1 WV GladeRun-Shaver'sFork MONF.288 16 38.6420113 -79.8414660 1193 215GLR2 WV GladeRunsite2
16 38.6571443 -79.8407687
GRK WV GreenKnob MONF.318 16 38.8977829 -79.4411622 1406 HAK WV HaystackKnob MONF.320 16 38.9043187 -79.4406518 1336 140HKB WV HuckleberryTrail MONF.293 16 38.7347977 -79.5089717 1393 KSF WV KumbrabowStateForest KUMB.22 17 38.6294772 -80.1327991 1058 301LFW WV LaurelForkWilderness MONF.165 16 38.6905218 -79.6991719 1060 90OPR WV OldPineyRoad MONF.316 16 38.4729537 -79.7011336 1316 133PHK WV PharisKnob MONF.266/267 16 38.7437972 -79.6307336 1287 196PKB WV PantherKnob PEND.14 16 38.5706214 -79.4900388 1295 PRT WV PineyRidgeTrail KUMB.23 16 38.6383999 -80.1315325 1105 287RRN WV RedRun
16 38.6280000 -79.8995600
RSK WV RedSpruceKnob MONF.300 18 38.3346989 -80.1540319 1377 125
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SEN WV SenecaCreek MONF.321 16 38.7066954 -79.5473580 1201 135SHV WV Shaver'sMountain MONF.58/59 16 38.9896590 -79.6065495 1137 SKB WV Stuart'sKnob
16 38.9388145 -79.7135679 1206
SKL WV SpruceKnobLake
16 38.7180241 -79.6120161 1152 SMR WV SawMillRun PEND.17 16 38.6679848 -79.5704751 1165 90SPK WV SpruceKnob MONF.292 16 38.7166488 -79.5219644 1430 104SPT WV SouthProngTrail MONF.295 16 38.9560255 -79.3565877 1220 TKR WV Turkey(McGowan)Mountain MONF.277 16 39.0192477 -79.6750557 1131 TOA WV TopofAllegheny MONF.315 16 38.4742984 -79.7166843 1299 116WMT WV Whitmeadow MONF.291 16 38.6670538 -79.8530919 1171 WSW WV WhitmeadowSwamp MONF.212 16 38.6720019 -79.8866321 1132 117YCK WV YellowCreek MONF.103 16 38.9547280 -79.6637386 913 128NGR WV4 NewGermanySeedlings 100
FZL MD FinzelSwamp
16 39.7039308 -78.9384452 820 GLD MD TheGlades
16 39.5621173 -79.2781106 825
WLF MD WolfSwamp
16 39.6613345 -79.0917602 808 BMB PA BearMeadow'sBog
16 40.7303320 -77.7638050
LAC PA Lackawanna
16 41.2008810 -75.6193360 LKP NY LakePlacid
16 44.3312656 -73.9019769 494
BPT NH BlackPondTrail
16 44.1057363 -71.5813700 RAT NH Ripley-ArethusaTrail
16 44.1489594 -71.3884488
SMN VT Smuggler'sNotch
17 44.5734909 -72.7787015 595 GT ON GloucesterTownship
6 45.36199629 -75.53139755 90
1PlotcodesbasedontheWVDNR’sNaturalHeritageProgramforestinventoryplots2Numberofsampledindividualsforgeneticanalysis3Minimumstandage,basedontheoldestdatedtreecorereportedintheWVDNRNaturalHeritagePrograminventoryplots(WVDNR2017).4SourceofNGRseedlingswasfrompooledseedcollectedinWV.SeedlingsweresampledforanalysisatNewGermanyStatePark,MD,priortoplanting.
Treeswereselectedforsamplingacrossarangeofrepresentativediameterclasseswithineachsite,andbasedonfeasibilityofaccesstoneedletissue.Consequently,wewerenotalwaysabletosampletissuefromsomeverylargetreesduetotheheightofthecanopy.Foreachtreeselected,weclippedasmallamountoffresh,currentyearbranchgrowthwithhealthyneedles.WealsomeasuredDBHtothenearest0.1cm,andrecordedindividualtreeGPScoordinatesandelevation(ma.s.l.)usingaGarminGPSmap60Csx.Wealsonotedseveralcategoricaldescriptorsforeachsample,includingpositiononslope(riparian,flat,lowerslope,upperslope,crest),aspect(N,NE,E,SE,S,SW,W,NW),abundanceofP.rubens(dominant,abundant,common,uncommon,rare,solitary),lifehistorystageofthesampledindividual(juvenile,mature(conebearing),over-mature(crownsenescing)),andtookaphotographofthesampledtree.
Intotal,wesampledfortysitesacrossWV(N=37)andwesternMD(N=3),
withanaverageof16individualspersite,foratotalof643individuals(Figure1).Wealsoincludedadditionalsamplingfrom7sitesoutsideoftheCentralAppalachianregiontoprovideabroadergeographicandgeneticcontextandtoassessthedistinctivenessoftheCentralAppalachiandiversity.ThisconsistedofP.rubensfromNH(N=32),PA(N=32),NY(N=16),VT(N=16),andOntario(ON;N=6).Lastly,toassessthediversityencompassedbyseedlingsdistributedbyCASRI’srestorationseedlingprogram,wesampled100individualsfromasingleseedlinglotdeliveredtoNewGermanyStatePark,nearGrantsville,MD(NGR).Theproject-widesamplesizeforgeneticanalysiswasthusN=845individualsrepresenting47naturalpopulationsand1cohortofrestorationseedlings.AllsampledneedletissuewastransportedbacktotheUniversityofMaryland’sAppalachianLaboratory(Frostburg,MD)andstoredfrozenat-80°Cuntilfurtherprocessing.CollectiondetailsforindividualsamplesaregiveninAppendixA.
DNAIsolationandMicrosatelliteGenotypingWeextractedwholegenomicDNAfromneedletissueusingtheQiagenDNeasy96Plantkitandquantifieditfluorometrically(InvitrogenQUANT-IT).WeusedthispurifiedDNAtoPCRamplify18microsatellitelocidevelopedinotherPiceaspecies(mostlyP.glauca)thathavebeenshowntoreliablycross-amplifyinP.rubens(Pfeifferetal.1997;Hodgettsetal.2001;Rajoraetal.2001;Scottietal.2002;Rungisetal.2004).Theselociweretestedinpreliminaryanalysesandfoundtobepolymorphicandproducereliableamplificationproductsforscoringallelesizes.Weemployedamultiplexingstrategy(Blacketetal.2012)toamplifymultiplelocisimultaneously.Specifically,weusedacombinationofauniversalprimerfluorescentlylabeledwith1of4dyes(6-FAM,NED,PET,andVIC)andapairoflocus-specificprimers,withtheforwardlocus-specificprimermodifiedtohavea5’universalsequencetailcomplimentarytothelabeledprimer.Thisenabledahighlyefficientandflexiblesystemtodevelopmultiplexedsetsoflocilabeledwithdifferentfluorophores(Table2).
Figure1.Samplinglocalitiesforredspruce.Bluepointsindicatesamplinglocalitiesoftreesusedinthisstudy.Leftpanel:Mapof40samplinglocalitiesintheCentralAppalachianregion(WVandMD).Greenshadingindicatesestimateddensityofredsprucecover(high,medium,low)basedonWVDNRNHPmapping.Rightpanel:Regionalmap,showingadditionalsamplinglocalitiesinthenortheast.InsetshowstheCentralAppalachianregion.Shadingindicatestherange-widedistributionofP.rubens(Little1971).
Table2.Primerinformationfor18microsatellitelociamplifiedinP.rubens.LocusName Motif Size(bp) Dye Plex TA(°C) ReferenceGAT64 GAT 102-112 6-FAM A 57 Hodgettsetal.(2001)TG25 TG 94-106 PET A 57 Hodgettsetal.(2001)CT189B CT 114-116 NED A 57 Hodgettsetal.(2001)CA24 AC 184-212 6-FAM A 57 Hodgettsetal.(2001)PGL12 AG 222-250 PET A 57 Rajoraetal.(2001)PGL14 AG 136-180 VIC A 50 Rajoraetal.(2001)CT144 CT 145-154 NED A 50 Hodgettsetal.(2001) SPAGG3 GA 130-146 6-FAM B 57 Pfeifferetal.(1997)UAPgAG105 AG 175-179 NED B 57 Hodgettsetal.(2001)WS0082.E23 TA 248-266 VIC B 57 Rungisetal.(2004)PAAC23 GT 284-294 NED B 57 Scottietal(2000) WS0022.B15 AG 210-225 NED C 57 Rungisetal.(2004)WS0092.A19 AC 241-245 6-FAM C 57 Rungisetal.(2004)EAC6B03 AC 129-135 VIC C 57 Scottietal.(2002)SPL3AG1H4 GA 135-137 PET C 57 Pfeifferetal.(1997)WS00111.K13 AT 234-238 PET C 57 Rungisetal.(2004)WS0082.023 TA 226-240 VIC C 57 Rungisetal.(2004)
PCRamplificationsusedtheQiagenMultiplexPCRkitin10ulreactions,consistingof1ulddH2O,5ulMultiplexPCRMasterMix,1ulQ-solution,andamplifiedonanEppendorfMasterCyclerproPCRmachinewiththefollowingconditions:95°Cfor15min.,30cyclesof94°Cfor30s,TA(either50or57°C;seeTable2)for90s,72°Cfor90s,followedbyafinal72°Cextensionfor30min.andthena4°Chold.AsampleofamplificationproductswerescreenedusinggelelectrophoresisbeforecombiningwithLIZ500sizestandardandshippingtotheWestVirginiaUniversityGenomicsCoreLabforfragmentanalysisonanABI3130xlsequencer(AppliedBiosystems).RawdataweresizedusingthesoftwarePeakScanner(AppliedBiosystems)usingautomatedscoring,followedbymanualchecking.AllelefragmentsizeswerebinnedusingtheprogramTANDEM(Matschiner&Salzburger2009).StatisticalAnalysis1.GeneticDiversitywithinPopulationsandIndividualsWeestimatedpopulationgeneticdiversitystatisticsaveragedacrosslociforeachsiteusingthe‘diveRsity’package(Keenanetal.2013)inRv.3.3.2(RCoreTeam2014).Diversitymeasuresincludedtherawnumberallelesperlocusunadjustedforsamplesize(A),theproportionofallallelesobservedwithinsites(%A),allelicrichnessadjustedforsamplesizedbasedonrarefaction(Ar),observedheterozygosity(Ho),expectedheterozygosity(He),andthepopulationinbreedingcoefficient(FIS).SignificanceofFISwastestedwith1,000bootstrapresamples.
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Wewerealsointerestedinassessinggeneticdiversityattheindividuallevel,totestforanassociationbetweensizeclass(DBH)andheterozygosity.Suchanassociationmightbeexpectedifselectionactingacrosslifehistorystagesfavorsmoreheterozygous,lessinbredgenotypes,yieldinganenrichmentinheterozyogistyamongthelargestDBHtrees(aproxyforage)relativetomorerecentlyrecruitedsmallDBHtrees.Toestimatemulti-locusheterozygosityforeachindividualacrossthe18loci,weusedtheRpackage‘Rhh’(Alhoetal.,2010)toestimatethemulti-locusstandardizedheterozygositymeasure(SH;Coltmanetal.,1999).IncomparingSHtoDBH,werecognizethatDBHisgoingtobehighlyinfluencedbyenvironmentaldifferences,bothacrosssitesandwithinasite,duetovariationinreleaseratesofsubcanopytrees.Therefore,wetestedforheterozygosity~DBHassociationsbyrestrictingthesampletojustthelower(<9.60DBH)andupperquartileoftreeDBH(>34.40DBH),andincludedsiteasarandomeffectinamixedlinearmodelusingthe‘lme4’packageinR(Batesetal.2015).2.PopulationStructureandGeneticAncestryWeestimatedgeneticdifferentiationamongallpopulationsusingNei’sstandardizedestimateofallelefrequencydivergenceamongpopulations(GST),andHedrick’sre-scaledestimatethatdeterminesGSTrelativetoitsmaximumpossiblelevel(G’ST).Estimationwasdoneusingthe‘diveRsity’package,withsignificancedeterminedwith1,000bootstrapresamples.Toassessdifferencesingeneticancestrywithoutoursample,andtoassignindividualstotheirmostlikelygenepools,weusedtheBayesianclusteringprogramSTRUCTUREversion2.2(Pritchardetal.,2000;Falushetal.,2003),usingtheadmixturemodelwithcorrelatedallelefrequencies.Weperformed10independentrunsforeachK(1-10)with1,000,000MCMCiterationsafteraburn-inperiodof200,000iterations.Post-processingofSTRUCTURErunsandad-hocestimationofthenumberofclusters(K)basedonthedelta-Kmethod(Evannoetal.2005)wasimplementedinthesoftwareSTRUCTUREHARVESTER(Earl&vonHoldt,2012).AncestrycoefficientsacrossrunswerecombinedusingCLUMPP(Jakobsson&Rosenberg,2007). 3.GeneticConnectivityandIsolationbyEnvironmentToestimatethefactorscontributingtohistoricalconnectivityandgeneflowamongCentralAppalachianstands,weusedalandscapegeneticsapproachtorelatepopulationstructuretoenvironmentalfeaturesacrossthelandscape(Maneletal.2003).Specifically,weusedGeneralizedDissimilarityModeling(GDM)torelatepairwisegeneticdistancesamongsitestodifferencesintheirlocalclimateenvironment(Ferrieretal.2007;Fitzpatrick&Keller2015).TheGDMapproachisaformofmultivariatematrixregressionthatfitsnon-linearI-splinestomodeltherelationshipbetweenbiologicaldistanceandmultipleenvironmentalpredictors.Climatedataconsistedof19bioclimaticvariablesthatsummarizeseasonalaspectsoftemperatureandprecipitation(http://www.worldclim.org/bioclim),basedontheWorldClimdatasetdownscaledto30sresolution(Hijmansetal.2005).WealsoincludedpairwiseEuclideangeographicdistanceasanadditionalpredictorto
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accommodateeffectsofisolationbydistance.WeusedHedrick’sG’STasthegeneticdistancemetric,normalizedbetween0-1byscalingbasedonthelargestpairwisevalueobserved.GDMmodelswerefitusingtheRpackage‘gdm’(Manionetal.2016).Aftermodelfitting,weretainedallsignificantenvironmentalpredictorsandusedthemtotransformcontinuousbioclimaticspaceintheCentralAppalachianregionbasedonthemodeledassociationbetweengeneticandclimaticdistances.Theresultingtransformationwasthenmappedtovisualizethecontinuoussurfaceofgenetically-transformedenvironmentaldifferencesbetweensites.Suchananalysiscanbeusedtohighlightwhichregionsofthelandscapeareestimatedtobewellconnectedbygeneflowthroughclimaticspace,andwhicharelikelytobegeneticallyisolatedbyenvironmentaldifferences.
WealsoexploredtheextentofgeneticconnectivityamongCentral
AppalachianP.rubensusingDyer’spopulationgraphmethod(Dyer&Nason2004;Dyer2014).Populationgraphsemployagraph-theoreticframeworktomodelgeneticcovarianceinallelefrequenciesamongsitesusingnetworktheory,andestimateconditionalgeneticdistancesthatreflectthepatternofconnectivityandgeneflowamongsites.PopulationgraphsforP.rubensintheCentralAppalachianswereestimatedusingthe‘popgraph’Rpackage,withanalphathresholdtoleranceof0.025,andoverlaidontoGDMmodelpredictionstomaphowpopulationnetworkconnectivitycorrespondstogeneticisolationbyclimaticdistance.4.CurrentandHistoricalEffectivePopulationSize(Ne)Toestimatetheeffectivepopulationsize(Ne)intheCentralAppalachians,weemployedtheLDNemethodimplementedinNeEstimator(Doetal.2014).ThismethodestimatescurrentNebylookingattheextentoflinkagedisequilibriumpresentwithinapopulation,relativetowhatwouldbeexpectedundergeneticdriftinapopulationofsizeNe.WeinitiallyattemptedtoestimateNeseparatelyforeachsite,buttheaveragesamplesizeof16individuals(Table1)ledmostvaluestobeinestimable.Thus,weinsteadconsidered4differentdatasubsetsforestimatingNe:(1)asingleCentralAppalachian“population”consistingof643sampledindividualsfromWVandMD(excludingtherestorationseedlingsfromNGR);(2)the100NGRrestorationseedlings,toassesswhatfractionoftotaldiversitywasrepresentedbyrestorationstock;(3)the102northeasternsamplesfromPA,NY,VT,NH,andON,torelateCentralAppalachiandiversitytothatpresentfurthernorth;and(4)theentirepooledsampleof845individuals.
AsacomplimenttotheLDNemethod,wealsoestimatedcurrentandhistoricalNeusingacoalescentapproach.UnliketheLDNemethodbasedonpatternsoflinkagedisequilibrium,thecoalescentapproachmodelsthegenealogicalrelationshipsgivingrisetothedistributionofallelesizessampledinthepopulation.The“birth-death”processofallelesthenprovidesanestimateoftherateatwhichthepopulationwassubjecttogeneticdrift,whichisproportionaltoNe.UnliketheLDNemethodhowever,thecoalescentcanbeusedtoinferpasttemporalchangesinNethathaveshapedthegenealogyofalleles.Weusedthecoalescentmethod
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implementedintheRpackage‘VarEff’tomodelcurrentNeaswellasancestralNeusingasize-changemodel(Nikolic&Chevalet2014).ThetestedmodelallowedfortwohistoricalsizechangesleadinguptothecurrentNeintheCentralAppalachians.Thus,therewere3effectivepopulationsizesestimatedlookingbackwardsintime:currentNe,Neafterthesizechange(Ne-2),andtheancestralNepriortothesizechange(Ne-3).Asisstandardincoalescentapproaches,estimationwasforthecompoundparameterθ(=4Neµ,whereµisthemicrosatellitemutationrateperlocuspergeneration).Similarly,timingofthesizechangewasestimatedastheparameterτ(=gµ,wheregistimemeasuredingenerations).WefirstestimatedappropriaterangesforpriorsonNeandgfrompreliminaryrunsusingtheTheta()function.WethenperformedBayesianestimationofparameterswithMarkovChainMonteCarlo,withaburn-inof10,000iterationsfollowedby10,000samplingiterationsandathinningintervalof10.AsadvisedbyNikolicandChevalet(2014),wetookthemedianvaluefromtheposteriordistributionofeachparameterasourdemographicestimate,andintegratedacrossthe95%posteriorprobabilitytoobtainconfidenceintervals.Toconvertcompoundparameterstoanabsolutedemographicscale,wesubstitutedamutationrateof5x10-4forµ.Thisisvalueanover-simplificationthatdoesnotaccommodateuncertaintyinthemutationratenorvarianceamongloci,butisconsistentwithmicrosatellitemutationratesreportedintheliterature(Marriageetal.2009),andestimationofNeinthiswayprovidesapointofcomparisontotheLDNemethodwhichdoesnotrequireuseofamutationrate.Therefore,agreementbetweenthetwodifferentNeestimators,whichmakeuseofdifferentsignaturesinthegeneticdata,lendsconfidencetoourinferenceofeffectivepopulationsize.ResultsGeneticDiversityMicrosatellitediversitywithinsitesshowedlimitedvariabilityacrosssamplinglocations,withseveralnotableexceptions(Table3).Allelicrichness(Ar)withinCentralAppalachiansites(WVandMD)variedfrom2.8to3.86,withameanof3.12.SitesthatstoodoutashavingunusuallyhighdiversityofallelesincludedSKB(Stuart’sKnob),CNH(CanaanHeights)andYCK(YellowCreek),whileKSF(KumbrabowStateForest)andPKB(PantherKnob)possessedthelowestvaluesforthisregion,consistentwiththegeographicisolationoftheselocalities.Thiswasadditionallyreflectedbytheproportionoftotalallelicrichnesspresentwithinsites(%A),inwhichSKBstoodoutclearlywith>45%ofthetotaldiversityobservedwithinthisonesite.TheNGRrestorationseedlingspossessedthelargestabsolutenumberofalleles(111),likelyreflectingthelargesamplessize(N=100)andpoolingofseedsacrosscollectinglocalities.Afterrarefaction,allelicrichnessofNGRwasstillrelativelyhighbutlowerthanSKB,CNH,orYCK(Table3).OutsideoftheCentralAppalachians,LAC(Lackawanna)alsopossessedhighallelicdiversity(A,%A,andAr)whilepopulationsfurthernorthsuchasLKP(LakePlacid,NY),SMN(Smuggler’sNotch,VT)andGT(GloucesterTownship,ON)exhibitedsomeofthelowestvalues
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Table3.Populationgeneticdiversitywithinsites.BoldfacevaluesofFISaresignificantbasedon95%confidenceintervalsfrom1000bootstrapreplicates.SiteCode State N A %A Ar Ho He FISBAT WV 15.67 83 31.52 3.23 0.51 0.54 0.0607BLM WV 15.44 74 29.29 3.04 0.51 0.52 0.0172BNT WV 15.5 79 31.12 3.27 0.52 0.53 0.0254CBA WV 13.78 76 29.44 2.94 0.51 0.52 0.0264CMK WV 15.44 78 30.29 3.08 0.53 0.53 -0.01CNH WV 14.89 100 38.96 3.4 0.49 0.56 0.1344CNS WV 15.78 88 35.15 3.27 0.51 0.54 0.0582COW WV 15.78 77 30.18 3.25 0.57 0.54 -0.0676CPT WV 15.67 78 29.9 3.03 0.48 0.5 0.0394CSP WV 15.5 79 30.76 3.11 0.52 0.52 0.0138FSR WV 15.5 74 27.92 2.98 0.52 0.51 -0.0258GDK WV 15.17 81 31.4 3.14 0.54 0.53 -0.0068GLR1 WV 14.06 77 29.07 2.98 0.54 0.5 -0.0775GLR2 WV 15.61 79 31.07 3.13 0.53 0.53 0.016GRK WV 15.5 78 30.72 3.05 0.5 0.53 0.0667HAK WV 15.61 76 30.19 3.04 0.5 0.5 0.0098HKB WV 15.83 70 27.59 3 0.5 0.52 0.0327KSF WV 14.5 72 28.47 2.8 0.47 0.51 0.0672LFW WV 14.5 79 30.51 3.04 0.49 0.5 0.0266OPR WV 15.33 77 30.02 3.07 0.54 0.54 -0.013PHK WV 15.11 78 30.36 3.2 0.54 0.54 -0.0017PKB WV 15.28 67 26.09 2.8 0.48 0.47 -0.0246PRT WV 14.78 75 29.96 2.95 0.51 0.54 0.0419RRN WV 15.72 77 30.38 3.11 0.54 0.52 -0.0459RSK WV 16.17 80 31.13 3.02 0.53 0.54 0.024SEN WV 15.17 83 32.59 3.13 0.54 0.53 -0.0133SHV WV 15.67 83 31.98 3.25 0.55 0.54 -0.016SKB WV 14.94 112 45.19 3.86 0.52 0.64 0.1894SKL WV 15.33 80 31.13 3.12 0.48 0.52 0.0646SMR WV 15.5 84 33.24 3.14 0.5 0.53 0.042SPK WV 15.5 83 31.27 3.2 0.5 0.53 0.057SPT WV 14.94 74 28.37 3.03 0.55 0.53 -0.0394TKR WV 15.33 78 30.82 3.19 0.56 0.53 -0.0448TOA WV 15.67 75 29.01 3.02 0.56 0.52 -0.0789WMT WV 15.33 79 31.14 3.12 0.53 0.53 -0.0028WSW WV 15.28 77 29.29 3.17 0.55 0.53 -0.0415YCK WV 15.22 90 34.73 3.4 0.57 0.56 -0.0084FZL MD 14.44 81 33.56 3.09 0.47 0.55 0.1369GLD MD 15.39 82 33.07 3.17 0.5 0.55 0.0843WLF MD 14.72 71 28.97 2.94 0.47 0.52 0.0967NGR WV 96.5 134 49.71 3.31 0.51 0.56 0.086BMB PA 14.61 86 35.19 3.23 0.52 0.57 0.0918LAC PA 14.67 118 48.97 3.88 0.56 0.66 0.1466SMN VT 14.78 75 30.61 2.79 0.49 0.48 -0.0122LKP NY 15.22 67 27.49 2.71 0.43 0.49 0.1228BPT NH 15.61 77 31.87 3.05 0.53 0.51 -0.0387RAT NH 15.67 78 31.45 3.04 0.53 0.52 -0.0244GT ON 5.06 63 27.09 2.87 0.48 0.53 0.1005
13
observedacrosstheentiredataset,possiblyreflectingalossofdiversityalongthe
pathwayofpost-glacialrangeexpansion.
Heterozygosityshowedsimilartrendsamongsites,withSKBandCNHhaving
highvalues(Table3).However,levelsofobservedheterozygosityatthesesites
causedasignificantdeparturefromHardy-Weinbergequilibrium(significantly
positiveFISvalue),indicatingadeficitofheterozygotes.Whileselfingorbiparental
inbreedingisonepossibleexplanationforpositiveFISvalues,analternative
explanationconsistentwiththehighdiversityatthesesitesisarecentadmixtureof
multiplesourceswithdivergentallelefrequencies(e.g.,aWahlundeffect),causing
allelicdiversityandheterozygositytoincrease,butwithobservedheterozygosity
laggingduetoinsufficienttimeformatingtohomogenizeallelefrequenciesamong
offspring.Theexplanationofrecentadmixturebothraisingdiversitywhilealso
creatingadeficitofobservedheterozygotesisalsoconsistentwiththediversityof
theNGRseedlings,whichshowedsignificantFIS.Inthiscase,weknowthat
“admixture”occurredduringthepoolingofseedsfrommultipledifferentsource
localities.Incontrast,FZL(FinzelSwamp)andWLF(WolfSwamp)inwesternMD
bothshowedsignificantFISaccompaniedbyrelativelylowallelicrichness,
suggestingthatheterozygotedeficitwithinthesesitesatthenorthernedgeofthe
CentralAppalachiansmaybeattributabletoinbreeding.Attheotherendofthe
spectrum,siteTOA(TopofAllegheny)showedasignificantlynegativeFIS,indicatinganexcessofobservedheterozygotesrelativetoexpectationsunderrandommating.
OnepossibilityisselectionforheterozygousgenotypesatTOA,butgiventhatthisis
theonlypopulationtoshowsuchapattern,thesignificanceofFISheremaysimply
beattributabletomultipletestingacrossalargenumberofsites.
Asamorerigoroustestofthehypothesisthatselectionmayfavorthe
eventualdominanceofmoreheterozygousgenotypes,wetestedforanassociation
betweenindividualmulti-locusheterozygosityandDBHclass.Heterozygositywas
weaklybutnotsignificantlycorrelatedacrosslociwithinindividuals(r=0.042;95%CI:-0.016-0.099),providinglimitedsupportforselectionfavoringindividuals
thatareheterozygousacrosstheirgenomes.Consistentwiththis,weobservedno
supportforanassociationbetweenmulti-locusheterozygosityandDBHsizeclass
aftercontrollingforsiteeffects(β=0.019;t=0.729;P=0.467)(Figure2).Thus,unlikepreviousreportsfromP.rubenspopulationsinCanada(Rajoraetal.2000;Mosseleretal.2003),wefindlittleevidenceofincreasedheterozygositywithsizeclass.
14
Figure2.Associationbetweenstandardizedheterozygosity(SH)andthelowerandupperquartileoftreeDBH,usedhereasaroughproxyofage.
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PopulationStructureGeneticdifferentiationamongsiteswaslowoverall(GST=0.0109,95%C.I.0.0051-0.0179;G’ST=0.0245,95%CI:0.0122-0.0386),butinlinewithlowdifferentiationreportedforotherwind-pollinated,predominantlyoutcrossingforesttrees(Savolainenetal.2007).TheseGSTvaluesindicatethatroughly1-2%ofthetotalgeneticdiversitypresentintheCentralAppalachiansispartitionedamongdifferentsites,withthemajorityofvariabilityexistingwithinsites.Estimatesofpairwisedivergenceamongsitesshowedmorenuancedvariability,withmostsite-pairsshowinglittledivergence,andafewshowingrelativelylargegeneticdivergence,ofteninvolvingeitherFLZorWLFandotherCentralAppalachianpopulations(AppendixB). STRUCTUREanalysisofgeneticancestryreturnedK=3clustersbasedonthedelta-Kmethodofmodelselection(Evannoetal.2005).AtK=3,individualancestryassignmentsshowedmostsitesintheCentralAppalachianscontainedmixturesof2differentgeneticclusters,possiblyreflectingadmixturebetweentwodifferentrefugialregionsinthesoutheasternU.S.duringthePleistoceneglacialmaximum(AppendicesC1andC2).Interestingly,manyindividualsinSKBandCNHsharedancestryinageneticclusterthatispredominantlyfoundinnortheasternsites(redinK=3;AppendixC1).AtK=4,thisclusterseparatesintoafourthgroupathighestfrequencyintheLACsite,anditiswiththisgroupthatsomeSKBindividualssharegeneticancestry.AlongwithevidenceofelevatedallelediversityanddeparturesfromHardy-Weinbergequilibriuminthesesites,thisprovidesindicationthatSKBandCNHwereprobablysupplementedatsomepointinthepastwithseedlingmaterialoriginatingfromoutsidetheCentralAppalachianregion,likelyfromnortheasternP.rubens.AlsoofinterestistheancestryofNGRrestorationseedlings,whichhasapproximatelyequalancestryacrossallthreeclusters,includingthenortheasterncluster(AppendixC2).Thiscouldindicatethattheseedsourcesofrestorationplantingsmaythemselveshavebeensupplementedatsometimeinthepastwithgeneticancestryfromthenortheast,butthisremainsspeculativewithoutadditionaltesting.GeneticConnectivityandIsolationbyEnvironmentTheclimatezoneinhabitedbysampledP.rubensstandsinthisstudyshowtheCentralAppalachiansoccupyingadistinctclimatenichefromnortheasternredspruceforests(Figure3,top).Specifically,CentralAppalachiansiteswereseparatedfromothersamplesalongthefirstPrincipalComponentclimateaxis(54%ofvariance),withWVandMDsitescharacterizedbyhighertemperatures,higherprecipitation,andlowerseasonalityrelativetonorthernsites.WhentheCentralAppalachianregionwasanalyzedseparately,thethreewesternMDsites(WLF,FLZ,andGLD)separatedalongaprecipitationgradientrepresentedbythefirstPCaxis(51%ofvariance),indicatingthatthesesitesoccupyadriermicroclimatewithintheAlleghenyMountains(Figure3,bottom).
16
Figure3.PrincipalComponentAnalysis(PCA)ofclimatespaceoccupiedbyredsprucepopulationsinthisstudy,asdefinedby19bioclimaticvariables.Toppanelsshowrange-widePCA;bottompanelsshowPCAofCentralAppalachianredspruce.Bioclimvariabledefinitions:bio1(meanannualtemp.),bio2(meantemp.diurnalrange),bio3(isothermality),bio4(temp.seasonality),bio5(maxtemp.ofwarmestmonth),bio6(mintemp.warmestmonth),bio7(temp.annualrange),bio8(meantemp.wettestquarter),bio9(meantemp.driestquarter),bio10(meantemp.warmestquarter),bio11(meantemp.coldestquarter),bio12(annualprecip.),bio13(precip.wettestmonth),bio14(precip.driestmonth),bio15(precip.seasonality),bio16(precip.wettestquarter),bio17(precip.driestquarter),bio18(precip.warmestquarter),bio19(precipcoldestquarter).
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WethenaskedwhetherthesebioclimaticdifferencesinoccupiedclimatenichespacewithintheCentralAppalachianshaveshapedpatternsofgeneflowandpopulationconnectivityacrosstheregion.UsingGeneralizedDissimilarityModeling(GDM),werelatedpairwisepopulationgeneticdivergence(G’ST)todifferencesamongsitesforthe19bioclimaticvariables.Theoveralldevianceexplainedbyenvironmentaldifferenceswasonly17%ofthetotaldeviance,suggestingmostoftherelativelyweakgeneticdivergenceamongsitesisleftunaccountedfor,probablyduetootheraspectsoflandusehistoryaswellasstochasticdemographicandsamplingprocesses.Interestingly,onlyfourenvironmentalpredictorsweresignificantinthemodel,showingnon-zerodevianceinexplaininggeneticconnectivityamongsites(Figure4).Ofthese,themostimportantbyfarwasprecipitationduringthewarmestquarteroftheyear(bio18),withsteepchangesingeneticconnectivityacrossthedryportionoftheprecipitationgradientfrom32.0–36.0cm,followedbylittlechangeinconnectivityacrossthewetterportionofthegradientabove36.0cm(Figure4).Theotherimportantpredictorofgeneticconnectivitywasthemeantemperatureofthecoldestquarteroftheyear(bio11),whereconnectivitychangedthemostalongthecoldportionofthegradient(below-3.5°C).Devianceexplainedbytheremainingvariablesshowedrelativelyminoreffectsofgeographicdistanceandprecipitationseasonality(bio15). TheturnoverfunctionsproducedbyGDMallowtransformationofthecontinuousbioclimaticsurfaceintoageneticallyinformedmapofhowtheenvironmentisexpectedtomediategeneticconnectivityamongsites(Fitzpatrick&Keller2015).Thesetransformationsarebasedonthemodeledturnoverfunctionsthatassociatespecificportionsalongenvironmentalgradientswithhighorlowturnoveringeneticdiversity(Figure4).InGDMtransformedmaps,pixelswithsimilarcolorsarepredictedtoexperiencelowgeneticisolationbyenvironment,andhencesharehighconnectivitythroughthegene-environmentspace.GDMtransformationintheCentralAppalachiansshowsahighdegreeofconnectivityamongmuchofthecoreportionofP.rubensrangeintheregion(Figure5).ThegeneraltrendinconnectivityisalongaSWtoNEtrendingaxis,withabrupttransitionstowardstherainshadowoftheAlleghenyfront,inthevicinityofPKB(PantherKnob)neartheWV/VAborder.StrongchangesinconnectivityarealsoapparentnorthwardintoGarretCounty,MD,wheresitesGLD(Glades),WLF,andFZLshowlowpredictedconnectivitywiththerestoftherangeinWV.ThisdropinconnectivityislikelydrivenbyregionsofreducedsummerprecipitationnorthandeastoftheAlleghenyFront,althoughthesespecificstandsofredsprucehavefoundsuitablesitesmediatedbylocalhydrology.Nevertheless,theGDMunderscoresthegeneticandclimaticisolationofthewesternMDsprucesitesfromtherestoftheCentralAppalachianregion.Thisreducedconnectivityisalsoaccompaniedbyreducedwithin-sitediversityandevidenceforinbreeding(Table3). OverlayingthenetworktopologyofconditionalgeneticdistancesfromthepopulationgraphanalysisclearlyshowsthegeneticisolationofwesternMDP.rubenssites,includingCNS(CranesvilleSwamp)ontheWV/MDborder(Figure6).
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Figure4.GeneralizedDissimilarityModeling(GDM)resultsshowinginfluenceofbioclimaticvariablesonpopulationgeneticstructure.Toppanelsshowmodelperformanceatpredictingobservedgeneticdistanceamongpopulations.Bottomtworowsoffiguresshowgeneticturnoveralongenvironmentalgradientsofgeographicdistance,meantemperatureofcoldestquarter(bio11),precipitationseasonality(bio15),andprecipitationofwarmestquarter(bio18).
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Figure5.PredictedspatialvariationinpopulationgeneticdifferentiationbasedonGeneralizedDissimilarityModeling(GDM).AreaswithsimilarcolorsrepresentareasofpredictedgeneticsimilaritybasedonGDMtransformationofbioclimaticvariablesandtheirassociationwithgeneticdistance.Theleftpanelshowsthepredictedgradientacrosstheregion.TherightpanelshowsthesamegradientcroppedusingtheWVDNRRedSpruce(Picearubens)CoverinWestVirginia2013maplayerinordertobettershowthepredictedconnectivitywithinthecoredistributionalrangeofredspruceinWV.
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Figure6.PopulationgraphgeneticnetworkstructureamongCentralAppalachiansites.Leftpanelshowsheatmapandhierarchicalclusteringofsamplingsitesaccordingtotheirconditionalgeneticdistances.Warmercolorsindicatehigherconnectivity.Rightpanelspatiallymapsthepopulationgraphnetworktopologyofsites(nodes;blackcircles)linkedbyconditionalgeneticdistances(whiteedges).NetworktopologyisoverlaidontothegeneticallytransformedclimaticsurfacefromGDM.
OPR TOA
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Thepopulationgraphalsorevealsseveraladditionalinsightsintothegeneticconnectivityamongsitesthatwerenotapparentfromotheranalyses.ThemoststrikingfeaturebeingthealmostcompletelackofconnectivityofapairofpopulationsalongtheeasternedgeofP.rubensinWV,namelysitesTOA(TopofAllegheny)andOPR(OldPineyRoad),althoughbothsitesappearwellconnectedtoeachother(Figure6,leftpanel).OthersitesalsoshowsomewhatlimitedconnectivitywithinthenetworksuchasKSFandPRTintheKumbrabowStateForest,andPKBtotheeast.EffectivePopulationSizeandDemographicHistoryCurrentbestestimatesoftheeffectivepopulationsize(Ne)ofP.rubensintheCentralAppalachiansusingthelinkage-disequilibriummethodimplementedinNeEstimatorarelow:Ne=322,witha95%C.I.of287-364(Table4).ThisisslightlylowerthantheminimumNerecommendedforlong-termviablepopulations,andreflectstheknownlowgeneticdiversityreportedforP.rubensinothergeneticstudies(Hawley&DeHayes1994;Perronetal.2000).However,whenputintoabroaderregionalcontext,theCentralAppalachianNeislargerelativetotheNeestimatedfornortheasternP.rubens(ca.68individuals,CI:60-77),andrepresentsabout76%ofthetotalspecies-wideNeestimatedfromthepooledsampleofallindividuals(Table4).However,theseconclusionsregardingNeinthenortheastremainverytentativeuntilmuchmorethoroughsamplingcanbeconducted.TheNGRrestorationseedlingscapturedasurprisinglylargefractionoftheavailableNeintheCentralAppalachians,Ne=157(CI:123-212),roughlyhalfoftheregionalNe(Table4).Table4.Estimatesofcurrenteffectivepopulationsize(Ne).
SampleGroupSamplesize
(Nindividuals)Effectivepopulation
size(Ne)Lower95%
C.I.Upper95%
C.I.CentralAppalachians
(WV&MD) 643 322 287 364
Restorationseedlings
(NGR) 100 157 123 212
Northeast(PA,NY,NH,
VT,ON) 102 68 60 77
Pooled 845 539 473 619
EstimatesofcurrentNebasedonthecoalescentmethodinVarEffwerehighly
congruentwiththosefromNeEstimator.Assumingamutationrateof5x10-4,VarEffestimatesacurrentNefortheCentralAppalachianregionof535.7(95%CI:88.5–1765.3)(Figure7).ThisisslightlylargerthantheNefromNeEstimator,buttheconfidenceintervaloverlaps.Thus,giventwoalternatemethodsthatmakeuseofdifferentsignaturesofgeneticdriftinthedata,thereemergesarelativelyrobustpicturethatcurrentNeisinthehundreds,andveryclosetotheminimumrecommendedleveladvisedbyconservationbiologistsforlong-termviability.
Figure7.Coalescentmodelofeffectivepopulationsize(Ne)ofredspruceatdifferenttimeperiodsintheCentralAppalachians.NeisshownattimepointscorrespondingtotheancestralNebeforethesizechange(Ne-3;green),theNefollowingthesizechange(Ne-2;red),andthecurrentNe(blue).
0 2000 4000 6000 8000 10000
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Effective population size (Ne)
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23
Interestingly,Neintheregionhasnotalwaysbeenatthislowlevel.Thesize
changemodelinVarEffestimatesthattheancestralpopulationtoCentralAppalachianP.rubenshadamuchlargerNeinthepast(Figure8).ThesizeofthisancestralpopulationtotheCentralAppalachianregionisestimatedtobe7,209(95%CI:268.3-10,248.5)--anorderofmagnitudelargerthancurrentNeestimates,butwithawideconfidenceintervalreflectingtheuncertaintyinherentinestimatingchangesintheevolutionarypast.Thetimingofthesizechangeisestimatedat376generationsago(95%CI:13.3-1,229.0).Dependingonthevalueassumedforgenerationtime,thisplacesthetimingofthesizereductionsometimeinthemidtolateHolocene,probablyreflectinghistoricalreductionsinabundanceasthepost-glacialclimatewarmed.Thedominantsignatureleftinthegeneticdatareflectthismoreancientevent,andapparentlyoverwhelmanyeffectsonNefrommorerecentevents.ThissuggeststhatP.rubenswasalreadyquitebottleneckedintermsofitsgeneticdiversitypriortotheonsetoflandusechangeinthe19thand20thcenturies.
Figure8.2-Dposteriordensitydistributionsof(log10)effectivepopulationsizechangethroughtime.,measuredasgenerationsinthepast.Warmercolorsindicateareasofgreaterposteriorprobability.
200 400 600 800 1000
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N-T joint distribution (coverage = 99.2093 % )
/Volumes/kellrlab/STEVE/wvdnr_spruce/popgen_analysis/VarEff/job
24
ConclusionsTheresultsofthisresearchprovideafirst,detailedassessmentofthegeneticstatusofremnantpopulationsinP.rubens,akeystonespeciesinhighelevationconiferforests.Whilemanyquestionsremain,theresultshereshouldprovidesomeguidancetocurrentandfutureecosystemrestorationandmanagementeffortsintheCentralAppalachianeco-regionseekingtomaximizethedistributionofdiversityandgeneticconnectivityinordertomaintainapopulationthatpossesslong-termviability.Belowweprovidesomehighlightsandsynthesisofthemorestrikingresultstocomeoutofthisstudyandtheirpotentialrelevancetoredsprucerestoration:
• GeneticdiversityofP.rubensisquitelow,particularlyforanoutcrossingforesttree.Thisisreflectedbothinthelowlevelsofallelicrichnesswithinsites(Table3)andtheverysmallvaluesofitscurrenteffectivepopulationsize(Table4;Figure7).Lowlevelsofgeneticdiversityhavealsobeenfoundbyotherpopulationgeneticstudiesofredspruceindifferentportionsofitsrange,andappeartobeauniquefeatureofthistaxonthatstandsoutamongotherwind-pollinatedoutcrossingforesttrees.SomeresearchershavepositedthatP.rubensisactuallyarecentlyderived(andnotcompletelyreproductivelyisolated)speciesfromP.mariana(Perronetal.2000;Jaramillo-Correa&Bousquet2003;DeLafontaineetal.2015).
• ThelowcurrentNeisnearorslightlybelowwhatisgenerallyrecognizedasaminimumacceptableeffectivepopulationsizeforconservationofimpactedtaxa(Waples2002;Reed&Frankham2003;Frankham2005;Latta2008).ThesestudiessuggestthattheratioofNetocensuspopulationsizeisoften~0.1,butsucharatiogivesrisetounrealisticallysmallcensussizeestimatesofredspruceintheCentralAppalachians(ontheorderofafewthousand).Thus,itismorelikelythattheNe/NratioisthereforeevensmallerinP.rubens,possiblyreflectingitsdemographichistoryofrecentfoundereffectspeciationduringthePleistocenewithP.marianaaswellasthehistoricalbottleneckdetectedhereinthemid-lateHolocenethatreducedNebyanorderofmagnitude.ComplicationsduetoprolongedperiodsatlowNeincludereducedabilitytopurgedeleteriousmutationsandavoidinbreedingdepression,aswellaslimitedcapacityforresponsetonovelselectionpressures,suchasemergingpestsandpathogensorachangingclimate(Charlesworth2009).Thus,restorationpracticesshouldworktomaximizeNeofCentralAppalachianP.rubensbydrawingfromadiverseseedstockwithintheregion,andmaximizingtheallelicdiversityofrestorationseedlings.Thatsaid,theNepresentintheofCentralAppalachiansappearstobelargerelativetootherpartsofthespeciesrange,butconfidenceinthisassessmentwillhavetoawaitfurthersamplingofP.rubensinthenortheast.
25
• PresenceofgeneticancestryintheCentralAppalachianscharacteristicofnortheasternredsprucediversitysuggestshistoricanthropogenicmovementofgenesfromfurthernorth.Theeffectofthismovementrepresentsapotentialopportunitytoassesstheconsequencesoflong-distancegeneflowasamechanismofgeneticrescue,orifsuchgeneflowismaladaptivebydilutinglocaladaptation.Detailedcomparisonsoftreeswithvs.withoutnorthernancestrygrowinginthesamemicro-environmentwouldbepotentiallyrevealingonthispoint.
• GeneticconnectivityamongCentralAppalachianstandsishigh,butthereareisolatedpocketsofpopulationsseparatedfromthecoreoftheregionbymarginalclimateconditions.TheGDManalysisindicatessummerprecipitationisakeyfactorregulatingthisconnectivity.WesternMDsitesarenotablehere,beingclimaticallyandgeographicallyisolatedfromtherestoftheCentralAppalachianspruce,aswellasshowingreduceddiversityandevidenceforinbreeding.ItisimportanttonotethatthisstudypurposefullysampledtreesdeemedtobenativeandavoidedtherestorationplantingsconductedbytheNatureConservancyinthisregion.Thus,thepotentialthattheseplantingsmightsupplementtheexistinggeneticdiversityofthesestandswouldseemtobehigh,andcouldbeassessedastheplantingsmature.
AcknowledgementsIamgratefulfortheassistanceandadviceofmanypeoplewhocontributedtothisproject.Mostimportantly,myresearchassistantReginaTrottwhowashighlycommittedtothecollectioneffortandmolecularanalyses.Ialsoappreciatemanybiologistswithexpertiseontheredspruceecosystemwhoassistedmeinlocatingandaccessingsamples,includingKentKarriker,JimVanderhorst,BrianStreets,ElizabethByers,DaveSaville,MikePowell,andDeborahLandau.Finally,IacknowledgetheWVDNRandtheUniversityofMarylandCenterforEnvironmentalScienceforfundingsupport.
26
Appendices
AppendixA.Individualsamplinginformation.
AppendixB.Excelfilecontainingestimatesofpairwisegeneticdivergence(GST,
G’ST)amongpopulations.
AppendixC:STRUCTUREancestryassignmentsofindividualstogeneticclusters.
AppendixD.Microsatellitegenotypedataforallsampledindividualsat18loci.
27
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