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.

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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).

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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.

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/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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