Submitted August 31, 2015; Approved by USFWS September 4, 2015

GenomicAssessmentofTaxonomicStatusofCentralTexasEurycea

Salamanders

ThisprojectwasfundedbytheU.S.FishandWildlifeService,Section6grantTXE158-R-1,throughTexasParksandWildlifeDepartmentcontract#443022

PrincipalInvestigator: DavidM.Hillis DepartmentofIntegrativeBiology UniversityofTexas Austin,TX78712 Phone:512-471-5792 E-mail:dhillis@austin.utexas.eduCo-PrincipalInvestigator: DavidC.Cannatella DepartmentofIntegrativeBiology UniversityofTexas Austin,TX78712 Phone:512-232-4862 E-mail:catfish@utexas.eduAdditionalPersonnel: ThomasJ.Devitt DepartmentofIntegrativeBiology UniversityofTexas Austin,TX78712 currentaddress: EnvironmentalScientist WatershedProtectionDepartment CityofAustin Austin,Texas78704 AprilM.Wright DepartmentofIntegrativeBiology UniversityofTexas Austin,TX78712

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AbstractWeconductedphylogeneticanalysesofallknowndescribedandundescribedspeciesofcentralTexasneotenicEurycea(basedonanalysesof34,518loci),andpopulationSTRUCTUREanalysesofallknownpopulationsofE.tonkawae,E.naufragia,andE.chisholmensis(basedonanalysesof75,296loci).Phylogeneticanalysissupportedthemonophylyandgeneticdistinctivenessofalldescribedspeciesinthegroup;inparticular,E.tonkawae,E.naufragia,andE.chisholmensisfromnorthoftheColoradoRiverwereeachreciprocallymonophyletic,significantlysupported,andgeneticallydistinct.PopulationSTRUCTUREanalysesofthespeciesnorthoftheColoradoRiveralsosupportedthespeciesboundariesbetweenE.tonkawae,E.naufragia,andE.chisholmensis.AfewpopulationsthathavebeenprovisionallyassignedtoE.naufragiaweredeterminedheretobelongtoE.tonkawae(GeorgetownSprings)orE.chisholmensis(allknownpopulationsnorthofLakeGeorgetown).TheanalysesshowstrongsupportforthreeadditionalundescribedspeciesfromsouthoftheColoradoRiver.Oneofthesehasbeenreportedpreviouslyasanundescribedspecies(thePedernalesSalamander);weextendtherangeofthisundescribedspeciestoincludeacaveonthewestsideofthePedernalesRiver.Thetworemainingundescribedspeciesoccurinwest-centralandwesternportionsoftheEdwardsPlateau.WepresentgeoreferencedlocalitydataanddistributionmapsforallthespeciesofTexasneotenicEurycea.IntroductionThespringandcavesalamandersofcentralTexas(genusEurycea)representamajorradiationofdistinctandendemicTexasamphibians.Thespeciesinthisgrouphavehighlyrestrictedhabitatrequirements,andeachdescribedspeciesisrestrictedtospecificaquifersandriversystems.Manyoftheseaquifersareunderpressurefromdevelopment,whichcanresultindecreasedwaterqualityandquantityinsurfaceflows.Asaresultofthesepressures,severaloftheendemicTexasEuryceaarelistedasFederalThreatenedorEndangeredspeciesbytheU.S.FishandWildlifeService.AlthoughthetaxonomicstatusofmanyTexasEuryceaiswellestablished(Chippindaleetal.,2000;Hillisetal.,2001),thespeciesboundariesamongseveraloftheprotectedspecieshaverecentlybeenquestioned(Forstner,2012).Thesystematicrevisionsofthisgrouphavebeenbasedonacombinationofmorphological,allozyme,andDNAsequencingstudies.However,thesesalamandersaresubjecttorapidmorphologicalchangeinresponsetoenvironmentalfactors,especiallyrelatedtothedifferencesbetweensurfaceandcavehabitats(Wiensetal.,2003).Forexample,theenormousmorphologicaldifferencesbetweenthesubcladeTyphlomolge(BlindSalamanders)andepigeanspecies(thosecommonlyfoundatsurfacesprings)ledWake(1966)tosuggestthatthesetwogroupswerenotcloselyrelated,butrathertheresultoftwoindependentdispersaleventsintoTexas.However,molecularstudieshavemadeitclearthatTyphlomolgeisactuallymuch

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morecloselyrelatedtoepigeanspeciessouthoftheColoradoRiverthanitistotheepigeanspeciesnorthoftheriver(Hillisetal.,2001).ThethreedescribedspeciesnorthoftheColoradoRiverarehighlydistinctfromtheremainingspeciesinstudiesofallozymesandmitochondrialDNA,butafewpopulationshavebeenassignedtodescribedspeciesonlyprovisionally(Chippindaleetal.,2000).Inaddition,mitochondrialDNAsequencesrepresentonlyasinglegeneticlocus,andcaptureofmitochondrialDNAbetweenspeciescanmakeuseofthismarkerforspeciesassignmentproblematicattheindividualandpopulationlevel(Avise,2004).Thisisespeciallytrueingroupsinwhichhybridizationisknowntooccur,asitisinTexasEurycea(Sweet,1984).AlthoughthegreatgeneticdivergenceamongEuryceaspeciesnorthoftheColoradoRiver(inTravis,Williamson,andBellcounties)clearlyshowsthatatleastthreespeciesinhabitthisarea,additionaldataareneededtoclarifythestatusofsomeofthepopulations.Thisisespeciallytruefornumerouscavepopulationsinthisarea,whichareoftenmorphologicallydivergent.Whetherthismorphologicaldiverencerepresentslocaladaptationoradditionalspeciationiscurrentlyunresolved.TherehavebeenrapidadvancesingenomicsequencingtechnologiessincethetaxonomicrevisionofthisgroupbyChippindaleetal.(2000).Inaddition,manynewpopulations(andperhapsnewspecies)ofTexasEuryceahavebeencollectedinthepastdecade.HereweusethesenewtechnologiestoexaminethousandsofnuclearDNAlociacrosstherangeofTexasEurycea,andalsoconductdetailedpopulationanalysesofthefederallythreatenedspeciesofEuryceanorthoftheColoradoRiver(E.tonkawae,E.naufragia,andE.chisholmensis).Thisanalysisteststhecurrenttaxonomyofthegroup,andexaminesthestatusandspeciesboundariesofthethreedescribedspeciesthatoccurnorthoftheColoradoRiver.Wealsousetheseanalysestoexaminethestatusofseveralcavepopulationsthathavebeenprovisionallyassignedtothesespecies.ObjectiveToexamineandtestthetaxonomicstatusthedescribedspeciesofTexasEurycea,withparticularemphasisonpopulationsoftheproposedendangered[orthreatened]species,E.tonkawae,E.naufragia,andE.chisholmensis,usingabroadsurveyofnuclearDNAmarkersfromhundredsofindependentloci.[Note:theObjectivestatementaboveistakendirectlyformtheoriginalgrantproposal,asrequestedintheReportGuidelines.Weproposedtoexamineatleast“hundreds”ofloci.Inourfinalanalyses,wewereabletoexamine34,518lociforthephylogeneticanalyses,and75,296locifortheSTRUCTUREanalyses.]GeographicLocationSeeAppendixTable1forageoreferencedlistofallspecimensexamined.These

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includedsamplesfromallknownspecies,threeundescribedspecies(herecalledEuryceasp.1,sp.2,andsp.3),andsamplesfromallavailablelocalitiesofE.tonkawae,E.naufragia,andE.chisholmensis(thethreespeciesthatoccurnorthoftheColoradoRiver,whichwerethefocusofthestudy).WeexaminedsamplesfromthroughouttherangeoftheTexasneotenicEuryceaclade,fromBellCountyinthenortheasttoValVerdeCountyinthesouthwest.Weexcludedtwoindividualsfromthegenomicanalysesduetolowdatayield(PecanSpringsandKickapooSprings)andonebecauseofproblematiclabelingofthesample(PantherCanyon).MethodsOurfocuswasonthespeciesnorthoftheColoradoRiver:E.chisholmensis,E.naufragia,andE.tonkawae,althoughwecomparedallknownspeciesintheTexasneotenicEuryceacladetocompareinaphylogeneticanalysis.Alllocalitieshavebeengeoreferenced(AppendixTable1),andallassociateddata,specimens,andremainingtissuesamplesaredepositedintheGeneticDiversityCollectionoftheBiodiversityCollections,DepartmentofIntegrativeBiology,UniversityofTexas.

MolecularMethodsWeuseddouble-digestrestriction-siteassociatedDNAsequencing(ddRADseq)(Petersonetal.,2012).ThisapproachemployedtworestrictionenzymestocleaveDNAfromtheentiregenomeofoneindividualintofragments.Thesearethenseparatedbysizeintoalibrarythatisareducedrepresentationofthegenomecontainingfragmentsofaspecifiedlength(inthiscase,300–400nucleotides).TheseDNAfragmentswerethensequenced(Illuminaplatform)inmillionsofparallelreactions,producingmanycopiesofeachfragmentthatwerethenalignedandanalyzedusingasoftwarepipelinespecificallydesignedtohandleRADdata(Catchenetal.,2013).Thedataexaminedweresinglenucleotidepolymorphisms(SNPs)sampledfromthroughoutthegenomeofrespectivespecies.BioinformaticsPipelineWeexaminedarangeofvaluesforfivekeyparametersintheSTACKS1.30pipeline.TheseparametersarepresentedinAppendixTable2.Foreachparameter,wetestedatleastthreevalues,includingthedefaultvalueforvaluesthathaverecommendeddefault.Weselectedparametervaluesforuseinthefinalpipelineifthatvaluebothminimizedthetotalmissingdataandthenumberoftaxonpairsforwhichthereisnodataincommon.ThefinalstepintheSTACKSpipelineinvolvedexportingthematrixofretainedSNPs.Atthisstep,itwaspossibletoexcludeincompleteSNPs(SNPsforwhichoneormoreindividualsdonothavedata)fromthefinalphylogeneticdatamatrix.Ourfinaldatamatricesincluded34,518lociforthephylogeneticanalyses(allpopulations),and75,296lociforthepopulationStructureanalyses(populationsnorthoftheColoradoRiver).

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PhylogeneticAnalysisWefitamodelofDNAsequenceevolutiontoeachofthedatamatricesusingjModelTest(PosadaandBuckley,2004).Thebest-fitmodelwastheGeneralTime-Reversible(GTR)modelwithGamma-distributedratevariation.WeperformedphylogeneticestimationinfourdifferentsoftwarepackagesoneachofthefivefinalSNPmatrices.Thefirsttwoanalysesweremaximumlikelihoodmethods.WeusedGarli(Zwickl,2006)toobtainapointestimateoftopologyforeachdatamatrix.BecauseSNPdatasetsinherentlyexcludeinvariantsites,wealsousedRAxML(Stamatakis,2006)toestimateatree,asRAxMLhasrecentlyimplementedcorrectionsforthistypeofascertainmentbias(Leacheetal.,2015).WeestimatedasampleoftreesinMrBayes(Ronquistetal.,2012),andsummarizedthissamplewithamajority-ruleconsensustreetoproduceposteriorprobabilitysupportvalesforeachbranch.TheMCMCchainwasrunfor10milliongenerations,andcheckedforconvergenceusingTracer(Rambautetal.,2014).Finally,weusedPAUP*version4.0a146(Swofford,2003;updated2015)toconducttheSVDQuartetsmethodofChifman and Kubatko (2014) forconstructingspeciestreesfromgenetreesthatevolveunderthecoalescentprocess. PopulationSTRUCTUREAnalysisWeusedBayesianclusteringmodelsimplementedinSTRUCTURE2.3.4(Pritchardetal.,2000;Falush,2003;Falushetal.,2007)toinferthenumberofpopulations(K)andestimateindividualpopulationassignments.Forpopulationsnorthoftheriver,weexploredvaluesofKfrom1–5.Weassumedcorrelatedallelefrequenciesandadmixtureamongpopulationsusingthedefaultsettings.ThreereplicaterunswereconductedforeachvalueofKinSTRUCTURE,witheachrunconsistingof50,000sweepsafteraburn-inperiodof10,000sweeps.WeassessedMCMCconvergencebycomparingKacrossreplicateruns.WedeterminedthebestKbyexaminingboththelogprobabilityofthedata(logPr(X|K))andtheΔKstatisticfollowingEvannoetal.(2005).ResultsweresummarizedandvisualizedusingthewebprogramCLUMPAK(Kopelmanetal.,2015).ResultsDataMatrixAssemblyOurphylogeneticmatrixcontained34,518SNPs.Whenassemblingaphylogeneticmatrix,STACKSdistillsthedatasetdowntoasinglerepresentativefromeachpopulation.Therefore,somesiteswerenotutilizedinthephylogeneticanalysisiftheywerepolymorphicwithinpopulations.OutputforSTRUCTUREdidnotincludethisfiltering,soeachindividualinthedatamatrixwasequivalenttooneindividualorganism.Therefore,theSTRUCTUREmatricesaremuchlarger(75,296loci)than

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thephylogeneticmatrices.Alldatamatriceswillbereleasedinpublicdatarepositoriesuponfinalpublicationofthisstudy.PhylogeneticAnalyses

Allofourphylogeneticanalysesproducedverysimilarresults,anddifferedonlyintherelationshipsofpopulationswithinspecies.Hereweshowtworepresentativetrees:ThetreefromtheSVDQuartetanalysistoshowofsummaryspeciestree(Figure1),andatreefromtheBayesiananalysistoshowtheposteriorprobabilitysupportvaluesforeachbranch(Figure2).AllphylogeneticanalysesprovidedstrongsupportfortherecognizedspeciesofEurycea;inparticular,eachofthethreedescribedspeciesfromnorthoftheColoradoRiverwassignificantlysupported(Figure2).Asinpreviousstudies(e.g.,Chippindaleetal.,2000;Hillisetal.,2001),ourphylogenetictreesindicatedthedeepestdeepsplitintheTexasneotenicEuryceafromnorthandsouthoftheColoradoRiver.NorthoftheColoradoRiver,whereoursamplingwasthemostthorough,allanalysesshowedstrongandsignificantsupportforgroupsreferabletoE.tonkawae,E.naufragia,andE.chisholmensis(Figures1and2).

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Figure1.SpeciestreefromtheSVDQuartetsanalysis.Speciesareindicatedbycolors.Thescalebarindicates100nucleotidechanges.

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Figure2.PhylogenetictreefromtheBayesiananalysis.Posteriorprobabilitysupportvaluesforeachbranchareindicatedbythecolorscale;bluebranchesshowstatisticallysignificantsupport(PP>95%).PopulationStructureAnalysisTheoptimalvalueofK(numberofgeneticallydistinctgroups)forthepopulationsofEuryceanorthoftheColoradoRiverwas3,whichcorrespondstothethreedescribedspeciesE.tonkawae,E.naufragia,andE.chisholmensis(Figure3).Afewpopulationsshowedthepresenceofgenesthatwereotherwisefoundprimarilyinanotherspecies;thesegenesarelikelyancestralretainedpolymorphisms(polymorphismsretainedfromacommonancestorofthetworespectivespecies).However,thesegenescouldbealsobeindicativeoflowlevelsofgeneflowbetweensomeadjacentpopulations.Todistinguishbetweenthesetwopossibilities,itwouldbenecessarytoseeifthe“misplaced”genesinapopulationformlinkedblocksinthegenome.Wedidnotexaminelinkageamonganyoftheloci,soourdataareinsufficienttodistinguishthesetwopossibilities.

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Figure3.Structureanalysis(K=3)ofEuryceafromnorthoftheColoradoRiver,basedon75,296loci.Verticalbarsrepresentindividuals;blacklinesseparatepopulations.Thethreecolorsrepresentthethreemajorgeneticdivisions,andcorrespondtopopulationsofthethreedescribedspecies.Gray=E.tonkawae;Red=E.naufragia;Blue=E.chisholmensis.SpeciesStatusandGeographicDistributionFigure4presentsthedistributionofallpopulationsofcentralTexasneotenicEuryceaexaminedinthisstudy.Figure5presentsthedetailsofthedistributionofE.tonkawae,E.naufragia,andE.chisholmensisfromnorthoftheColoradoRiver.

Figure4.DistributionofalldescribedandknownbutundescribedspeciesofTexasneotenicEurycea.GeoreferenceddataforallsamplelocalitiesarepresentedinAppendixTable1.

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Figure5.DistributionofE.tonkawae,E.naufragia,andE.chisholmensislocalities(thespeciesthatoccurnorthoftheColoradoRiver).GeoreferenceddataforallsamplelocalitiesarepresentedinAppendixTable1.DiscussionOurresultssupportthetaxonomicstatusofthedescribedspecies,andoftheknownbutundescribedspeciesofTexasneotenicEurycea(seeAppendixTable1).EachofthecurrentlyrecognizedspeciesformsamonophyleticgroupintheanalysesshowninFigures1and2,andthelevelsofgeneticdivergenceareconsistentwiththerecognitionofthecurrentbreaksamongspecies.Inparticular,wefoundstrongsupportfromboththephylogeneticanalyses(Figures1and2)andthepopulationSTRUCTUREanalysis(Figure3)forthethreedescribedspeciesfromnorthoftheColoradoRiver:E.tonkawae,E.naufragia,andE.chisholmensis(seedistributionsin

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Figure5).TheboundariesamongE.tonkawae,E.naufragia,andE.chisholmensishavebeenclarifiedbyouranalyses.Chippindaleetal.(2000)provisionallyassignedthepopulationofEuryceaatGeorgetownSpringstoE.naufragia,buttheyonlyhadaccesstoasinglejuvenilespecimenanddidnothaveenoughtissuetoincludethispopulationintheirallozymeanalyses.Ouranalyses(Figures1-3)indicatethatthispopulationisactuallyE.tonkawae.AlthoughGeorgetownSpringsisgeographicallyproximatetotherangeofE.naufragia,theBalconesFaultextendsnorthwardandseparatesthepopulationsofE.naufragiatothewestfromE.tonkawaepopulationstotheeastofthefault(andInterstate35)atBrushyCreekSpringandGeorgetownSprings.Thus,therangeofE.tonkawaeextendsalongandeastoftheBalconesFaultnorthtoGeorgetown.GeorgetownSpringshavebeenseverelyimpactedbywaterintakefortheCityofGeorgetown,andwehavebeenunabletolocateanyadditionalspecimensofEuryceaatthislocalitysincecollectionofthesinglejuvenilein1991.ThesinglespecimenfromGeorgetownSpringsdoesshowsomeindicationofsharedalleleswithE.naufragiapopulations(seeFigure3),butnotmorethanseveralotherpopulationsofE.tonkawaethataregeographicallywellseparatedfromtherangeofE.naufragia(includingthetypelocalityofE.tonkawaeatStillhouseHollow,andseveralcavepopulationsintheButtercupCreekregion).Giventhesepatterns,thesesharedalleleslikelyrepresentretainedancestralpolymorphismsratherthanevidenceofongoinggeneflow.TheGeorgetownSpringspopulationclustersstronglyandsignificantlywiththeotherE.tonkawaepopulationsinthephylogeneticanalyses(Figure1-2).Chippindaleetal.(2000)alsoprovisionallyassignedacavepopulationofEuryceafromBatWellCaveinWilliamsonCountytoE.naufragia;thispopulationwaslocatedbetweenotherknownpopulationsofE.naufragiaandknownpopulationsofE.chisholmensis.Sincethen,severalspringpopulationshavebeendiscoveredinthisgeneralarea,andtheyusuallyhavebeentreatedaspopulationsofE.naufragiaaswell.Ouranalysesindicatethatthesepopulationsformthesister-grouptoknownpopulationsofE.chisholmensis(fromthespringsalongSaladoCreek),andshouldbereferredtothatspecies.AlthoughthesepopulationsaregeneticallydivergentanddistinctfromthepopulationsofE.chisholmensisintheSaladoCreekdrainage,werecommendthattheybeincludedinE.chisholmensis,ratherthandescribedasadistinctspecies.ThisrestrictstherangeofE.naufragiatospringssouthandeastofLakeGeorgetownintheSanGabrielCreekwatershed;allknownpopulationsofEuryceanorthofLakeGeorgetownarehereincludedwithinE.chisholmensis(Figures1-5).OthercavepopulationsfromnorthoftheColoradoRiverarecloselyrelatedtoadjacentsurfacepopulations.Althoughsomeindividualsfromthesecavepopulationsappeartobemorphologicallydistinctfromsurface-dwellingsalamanders,allcavepopulationsappeartobeassignabletooneofthedescribedspecies.Mostofthecavepopulations(i.e.,allofthepopulationsintheButtercupCreekarea)fallwithinknowngeneticvariationofE.tonkawae.Asnotedabove,the

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BatWellCavepopulationisassignabletoE.chisholmensis(asheredelimited),andthepopulationfromWaterTankCaveisassignabletoE.naufragia(seeFigure5andAppendixTable1).AcknowledgmentsThisprojectwasfundedbytheU.S.FishandWildlifeService,Section6grantTXE158-R-1,throughTexasParksandWildlifeDepartmentcontract#443022.WethankCraigFarquharandAndyGluesenkampofTexasParksandWildlifeDepartmentforassistance.TissuesampleswerekindlyprovidedbyNathanBendik,PaulChippindale,AndyGluesenkamp,JohnKarges,LisaO’Donnell,ChadNorris,ColinPeden,andAndyPrice.

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AppendixTable1.Samplesexamined:Fieldnumbers,localities,latitudeandlongitude,andidentification.Fieldandspecimennumberabbreviations:NathanF.Bendik(NFB),PaulChippindale(PC),ThomasJ.Devitt(TJD),AndyG.Gluesenkamp(AGG),DavidM.Hillis(DMH),JohnK.Karges(JKK),LisaO’Donnell(LOD),ColinE.Peden(CEP),AndyPrice(AHP),andTexasNaturalHistoryCollection(TNHC).SampleID latitude longitude Locality TaxonDMH_90.417 30.943616 -97.544174 SaladoSprings EuryceachisholmensisAGG_1893 30.943616 -97.544174 RobertsonSpring EuryceachisholmensisAGG_1920 30.943616 -97.544174 RobertsonSpring EuryceachisholmensisAGG_1919 30.943611 -97.544167 SaladoSprings EuryceachisholmensisDMH_90.1 30.943611 -97.544167 SaladoSprings EuryceachisholmensisDMH_90.2 30.943611 -97.544167 SaladoSprings EuryceachisholmensisDMH_90.23 30.943611 -97.544167 SaladoSprings EuryceachisholmensisDMH_90.3 30.943611 -97.544167 SaladoSprings EuryceachisholmensisDMH_90.322 30.943611 -97.544167 SaladoSprings EuryceachisholmensisPC_031 30.943611 -97.544167 SaladoSprings EuryceachisholmensisPC_032 30.943611 -97.544167 SaladoSprings EuryceachisholmensisAGG_1885 30.899673 -97.639036 SolanaRanchSpring EuryceachisholmensisAGG_1886 30.899673 -97.639036 SolanaRanchSpring EuryceachisholmensisAGG_1887 30.899673 -97.639036 SolanaRanchSpring EuryceachisholmensisAGG_1900 30.895873 -97.644216 SolanaRanchSpring EuryceachisholmensisAGG_1895 30.895577 -97.633658 SolanaRanchSpring EuryceachisholmensisAGG_1896 30.895577 -97.633658 SolanaRanchSpring EuryceachisholmensisAGG_1897 30.895577 -97.633658 SolanaRanchSpring EuryceachisholmensisAGG_1837 30.790375 -97.728266 CobbSpring EuryceachisholmensisAGG_1838 30.790375 -97.728266 CobbSpring EuryceachisholmensisAGG_1839 30.720469 -97.736801 CowanCreekSpring EuryceachisholmensisAGG_1840 30.720469 -97.736801 CowanCreekSpring EuryceachisholmensisAGG_1842 30.720469 -97.736801 CowanCreekSpring EuryceachisholmensisPCDMH_92.64 30.702778 -97.716389 BatWellCave EuryceachisholmensisAGG_1823 30.698386 -97.781728 TaylorRayHollowSpring EuryceachisholmensisAGG_1824 30.698386 -97.781728 TaylorRayHollowSpring EuryceachisholmensisAGG_1825 30.698386 -97.781728 TaylorRayHollowSpring EuryceachisholmensisAGG_1826 30.698386 -97.781728 TaylorRayHollowSpring EuryceachisholmensisAGG_1175 30.698386 -97.781728 TaylorRayHollowSpring EuryceachisholmensisAGG_1176 30.698386 -97.781728 TaylorRayHollowSpring EuryceachisholmensisAGG_1930 30.697268 -97.781929 TwinSprings EuryceachisholmensisDMH_91.4 30.663894 -97.751119 KnightSpring EuryceanaufragiaDMH_91.6 30.663894 -97.751119 KnightSpring EuryceanaufragiaDMH_91.8 30.663894 -97.751119 KnightSpring EuryceanaufragiaDMH_91.3 30.663894 -97.751119 KnightSpring Euryceanaufragia

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DMH_91.7 30.663889 -97.751111 KnightSpring EuryceanaufragiaAGG_1914 30.662200 -97.710500 SwinbankSpring EuryceanaufragiaAGG_1915 30.662200 -97.710500 SwinbankSpring EuryceanaufragiaAGG_1916 30.662200 -97.710500 SwinbankSpring EuryceanaufragiaAGG_1917 30.662200 -97.710500 SwinbankSpring EuryceanaufragiaAGG_1828 30.662087 -97.768887 CedarHollowSpring EuryceanaufragiaAGG_1829 30.662087 -97.768887 CedarHollowSpring EuryceanaufragiaAGG_1830 30.662087 -97.768887 CedarHollowSpring EuryceanaufragiaDMH_91.91 30.660839 -97.726674 BufordHollowSprings EuryceanaufragiaDMH_91.89 30.660833 -97.726667 BufordHollowSprings EuryceanaufragiaAHP_3255 30.660005 -97.750563 CedarBreaks EuryceanaufragiaAHP_3254 30.660005 -97.750563 CedarBreaks EuryceanaufragiaAHP_3246 30.660000 -97.750556 CedarBreaks EuryceanaufragiaAHP_3252 30.660000 -97.750556 CedarBreaks EuryceanaufragiaAHP_3253 30.660000 -97.750556 CedarBreaks EuryceanaufragiaAHP_3255 30.660000 -97.750556 CedarBreaks EuryceanaufragiaAGG_2020 30.653951 -97.793219 WaterTankCave EuryceanaufragiaAGG_2022 30.653951 -97.793219 WaterTankCave EuryceanaufragiaAGG_2024 30.653951 -97.793219 WaterTankCave EuryceanaufragiaAHP_3331 30.645561 -97.736397 AvantSpring EuryceanaufragiaAHP_3325 30.645561 -97.736397 AvantSpring EuryceanaufragiaAHP_3334 30.645561 -97.736397 AvantSpring EuryceanaufragiaAHP_3322 30.645561 -97.736397 AvantSpring EuryceanaufragiaAHP_3324 30.645561 -97.736397 AvantSpring EuryceanaufragiaDMH_91.96 30.650494 -97.668045 GeorgetownSprings EuryceatonkawaeAGG_1447 30.530746 -97.725400 KrienkeSpring EuryceatonkawaeAGG_1448 30.530746 -97.725400 KrienkeSpring EuryceatonkawaeAGG_1449 30.530746 -97.725400 KrienkeSpring EuryceatonkawaeAGG_1450 30.530746 -97.725400 KrienkeSpring EuryceatonkawaeDMH_90.24 30.516757 -97.661295 BrushyCreekSpring EuryceatonkawaeDMH_90.13 30.516757 -97.661295 BrushyCreekSpring EuryceatonkawaeDMH_90.20 30.516757 -97.661295 BrushyCreekSpring EuryceatonkawaeDMH_90.17 30.516757 -97.661295 BrushyCreekSpring EuryceatonkawaeNFB_4.28.10 30.507504 -97.749315 AveryDeerSpring EuryceatonkawaeDMH_89.3 30.497086 -97.846977 TwasaCave EuryceatonkawaeDMH_91.74 30.492730 -97.856637 TestudoTube EuryceatonkawaeDMH_89.4 30.492652 -97.845872 IlexCave EuryceatonkawaeDMH_91.69 30.489836 -97.845272 ButtercupCreekCave EuryceatonkawaeDMH_91.71 30.489836 -97.845272 ButtercupCreekCave EuryceatonkawaeAGG_1741 30.463037 -97.847873 HouseSpring EuryceatonkawaeAGG_1742 30.463037 -97.847873 HouseSpring EuryceatonkawaeAGG_1482 30.461708 -97.874325 WhelessSpring EuryceatonkawaeAGG_1483 30.461708 -97.874325 WhelessSpring EuryceatonkawaeAGG_1492 30.461708 -97.874325 WhelessSpring Euryceatonkawae

17

AGG_1511 30.461708 -97.874325 WhelessSpring EuryceatonkawaeAGG_1766 30.457373 -97.838669 KellyHollowSprings EuryceatonkawaeAGG_1767 30.457373 -97.838669 KellyHollowSprings EuryceatonkawaeAGG_1937 30.457106 -97.862539 BlizzardSpring EuryceatonkawaeAGG_1938 30.457106 -97.862539 BlizzardSpring EuryceatonkawaeAGG_1939 30.457106 -97.862539 BlizzardSpring EuryceatonkawaeNFBLOD31 30.449846 -97.853812 McDonaldWell EuryceatonkawaeNFBLOD32 30.449846 -97.853812 McDonaldWell EuryceatonkawaeNFBLOD33 30.449846 -97.853812 McDonaldWell EuryceatonkawaeAGG_1754 30.430984 -97.782260 TanglewoodSpring EuryceatonkawaeAGG_1756 30.430984 -97.782260 TanglewoodSpring EuryceatonkawaeAHP_3438 30.425985 -97.814481 CanyonCreek EuryceatonkawaeNFBLOD18 30.425222 -97.814706 Trib6BullCreek EuryceatonkawaeAGG_1776 30.421819 -97.850954 Kretschmarr EuryceatonkawaeAGG_1777 30.421819 -97.850954 Kretschmarr EuryceatonkawaeAGG_1778 30.421819 -97.850954 Kretschmarr EuryceatonkawaeAGG_1718 30.421454 -97.818068 CanyonCreek EuryceatonkawaeAGG_1719 30.421454 -97.818068 CanyonCreek EuryceatonkawaeAGG_1720 30.421454 -97.818068 CanyonCreek EuryceatonkawaeDMH_90.312 30.420847 -97.840243 SchlumbergerSprings EuryceatonkawaeNFBLOD21 30.419450 -97.851433 SASCanyon EuryceatonkawaeNFBLOD22 30.419450 -97.851433 SASCanyon EuryceatonkawaeNFBLOD23 30.419450 -97.851433 SASCanyon EuryceatonkawaeNFBLOD1 30.419009 -97.812697 FranklinTract EuryceatonkawaeNFBLOD2 30.419009 -97.812697 FranklinTract EuryceatonkawaeNFBLOD3 30.419009 -97.812697 FranklinTract EuryceatonkawaeNFBLOD4 30.419009 -97.812697 FranklinTract EuryceatonkawaeAGG_1134 30.418630 -97.814135 PitSpring EuryceatonkawaeAGG_1135 30.418630 -97.814135 PitSpring EuryceatonkawaeAGG_1797 30.414890 -97.757822 SierraSpring EuryceatonkawaeAGG_1534 30.413647 -97.822471 LanierSpring EuryceatonkawaeAGG_1549 30.413647 -97.822471 LanierSpring EuryceatonkawaeAGG_1454 30.412557 -97.825715 LanierRiffle EuryceatonkawaeAGG_1477 30.412557 -97.825715 LanierRiffle EuryceatonkawaeAGG_1737 30.409597 -97.752569 TrollSpring EuryceatonkawaeAGG_1738 30.409597 -97.752569 TrollSpring EuryceatonkawaeAGG_1739 30.409597 -97.752569 TrollSpring EuryceatonkawaeAGG_1734 30.408727 -97.754535 HearthSpring EuryceatonkawaeAGG_1735 30.408727 -97.754535 HearthSpring EuryceatonkawaeAGG_1444 30.408555 -97.838956 WTP4Site EuryceatonkawaeAGG_1446 30.408555 -97.838956 WTP4Site EuryceatonkawaeAGG_1621 30.404649 -97.826447 RibelinTract EuryceatonkawaeAGG_1630 30.404649 -97.826447 RibelinTract EuryceatonkawaeNFBLOD8 30.404644 -97.826440 LowerRibelin EuryceatonkawaeNFBLOD19 30.404644 -97.826440 LowerRibelin EuryceatonkawaeAHP_3288 30.375833 -97.767222 BarrowHollowSpring Euryceatonkawae

18

DMH_88.151 30.371796 -97.763522 StillhouseHollowSpring EuryceatonkawaeDMH_88.131 30.371796 -97.763522 StillhouseHollowSpring EuryceatonkawaeDMH_90.10 30.371796 -97.763522 StillhouseHollowSpring EuryceatonkawaeDMH_90.8 30.371796 -97.763522 StillhouseHollowSpring EuryceatonkawaeDMH_88.130 30.371796 -97.763522 StillhouseHollowSpring EuryceatonkawaeDMH_88.149 30.371796 -97.763522 StillhouseHollowSpring EuryceatonkawaeDMH_88.148 30.371796 -97.763522 StillhouseHollowSpring EuryceatonkawaeAGG_1850 30.362957 -97.748072 SpicewoodSpring EuryceatonkawaeAGG_1851 30.362957 -97.748072 SpicewoodSpring EuryceatonkawaeAGG_1852 30.362957 -97.748072 SpicewoodSpring EuryceatonkawaeAGG_1817 30.361560 -97.759886 IndianSprings EuryceatonkawaeAGG_2061 30.340256 -98.258956 MaplesCave Euryceasp1DMH_91.41 30.341506 -98.137589 MartinSpring Euryceasp1DMH_91.43 30.341506 -98.137589 MartinSpring Euryceasp1DMH_91.52 30.341506 -98.137589 MartinSpring Euryceasp1DMH_91.39 30.341506 -98.137589 MartinSpring Euryceasp1DMH_91.51 30.341506 -98.137589 MartinSpring Euryceasp1DMH_91.42 30.341506 -98.137589 MartinSpring Euryceasp1DMH_91.55 30.339727 -98.137508 HammettsCrossingSpring Euryceasp1DMH_91.54 30.339727 -98.137508 HammettsCrossingSpring Euryceasp1DMH_91.53 30.339727 -98.137508 HammettsCrossingSpring Euryceasp1DMH_90.6 30.339727 -98.137508 HammettsCrossingSpring Euryceasp1AGG_1993 30.263683 -97.770820 OldMillSpring EuryceawaterlooensisAGG_1994 30.263683 -97.770820 OldMillSpring EuryceawaterlooensisPCDMH_92.121 30.263688 -97.770827 BartonSprings EuryceasosorumDMH_88.18 30.263688 -97.770827 BartonSprings EuryceasosorumDMH_88.137 30.263688 -97.770827 BartonSprings EuryceasosorumDMH_88.135 30.263688 -97.770827 BartonSprings EuryceasosorumDMH_88.8 30.263688 -97.770827 BartonSprings EuryceasosorumDMH_88.138 30.263688 -97.770827 BartonSprings EuryceasosorumDMH_88.140 30.263688 -97.770827 BartonSprings EuryceasosorumDMH_88.142 30.263688 -97.770827 BartonSprings EuryceasosorumAGG_1924 30.279593 -97.780434 ColdSpring EuryceasosorumTailclip_76 30.279593 -97.780434 ColdSpring EuryceasosorumTJD_992 30.158211 -97.914991 SpillarRanchSpring EuryceasosorumTJD_993 30.158211 -97.914991 SpillarRanchSpring EuryceasosorumTJD_994 30.114376 -97.957963 TaylorSpring EuryceasosorumDMH_90.270 30.091672 -98.658341 PeaveysSpring EuryceapterophilaDMH_90.272 30.091672 -98.658341 PeaveysSpring EuryceapterophilaDMH_88.15 30.106180 -98.481959 ZercherSprings EuryceapterophilaPCDMH_94.4 29.928205 -98.451048 SattlersDeepPit EuryceapterophilaDMH_90.265 30.076672 -98.329452 TCave EuryceapterophilaDMH_90.210 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_90.211 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_90.205 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_90.209 30.111116 -98.301952 BoardhouseSprings Euryceapterophila

19

DMH_88.23 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_88.28 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_90.206 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_88.29 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_90.208 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_88.26 30.111116 -98.301952 BoardhouseSprings EuryceapterophilaDMH_90.124 29.802005 -98.252848 BearCreekSpring EuryceapterophilaDMH_90.119 29.802005 -98.252848 BearCreekSpring EuryceapterophilaDMH_90.118 29.802005 -98.252848 BearCreekSpring EuryceapterophilaDMH_90.120 29.802005 -98.252848 BearCreekSpring EuryceapterophilaDMH_90.176 29.713616 -98.136952 ComalSprings EuryceapterophilaDMH_90.174 29.713616 -98.136952 ComalSprings EuryceapterophilaAHP_3072 29.713616 -98.136952 ComalSprings EuryceapterophilaAHP_3071 29.713616 -98.136952 ComalSprings EuryceapterophilaJKK_2003.1 30.032789 -98.125214 JacobsWell EuryceapterophilaPC_016 29.983338 -98.013619 FernBankSprings EuryceapterophilaPC_015 29.983338 -98.013619 FernBankSprings EuryceapterophilaPC_013 29.983338 -98.013619 FernBankSprings EuryceapterophilaPC_012 29.983338 -98.013619 FernBankSprings EuryceapterophilaPC_010 29.983338 -98.013619 FernBankSprings EuryceapterophilaDMH_88.192 29.893061 -97.930563 SanMarcosSprings EuryceananaDMH_90.179 29.893061 -97.930563 SanMarcosSprings EuryceananaDMH_88.200 29.893061 -97.930563 SanMarcosSprings EuryceananaDMH_88.196 29.893061 -97.930563 SanMarcosSprings EuryceananaDMH_88.203 29.893061 -97.930563 SanMarcosSprings EuryceananaDMH_88.199 29.893061 -97.930563 SanMarcosSprings EuryceananaDMH_90.183 29.893061 -97.930563 SanMarcosSprings EuryceananaAHP_3077 29.893061 -97.930563 SanMarcosSprings EuryceananaAHP_3076 29.893061 -97.930563 SanMarcosSprings EuryceananaAHP_3074 29.893061 -97.930563 SanMarcosSprings EuryceananaAHP_3075 29.893061 -97.930563 SanMarcosSprings EuryceananaAGG_1816 29.713658 -98.138800 PantherCanyon EurycearathbuniAGG_1981 29.868294 -97.967597 JohnsonsWell EurycearathbuniDMH_91.12 29.901950 -97.921397 RattlesnakeCave EurycearathbuniDMH_91.13 29.901944 -97.921389 RattlesnakeCave EurycearathbuniDMH_90.115 29.755833 -98.620278 BadweatherPit EuryceatridentiferaDMH_91.57 29.847227 -98.491674 HoneyCreek EuryceatridentiferaAGG_2062 29.847227 -98.491674 HoneyCreek EuryceatridentiferaPCDMH_92.101 29.762222 -98.666389 PfeiffersWaterCave EurycealatitansDMH_92.5 29.662783 -98.637230 LeonSprings EuryceaneotenesDMH_90.139 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_90.138 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_90.147 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_90.149 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_90.150 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_159.162 29.637505 -98.694452 HelotesCreekSprings Euryceaneotenes

20

DMH_90.144 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_90.137 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_90.146 29.637505 -98.694452 HelotesCreekSprings EuryceaneotenesDMH_93.4 29.634170 -98.852702 PecanSpringsCave EuryceaneotenesAHP_3043 30.005838 -99.361952 EdmunsonCreek Euryceasp2AHP_3042 30.005838 -99.361952 EdmunsonCreek Euryceasp2AHP_3039 30.005838 -99.361952 EdmunsonCreek Euryceasp2AHP_3040 30.005838 -99.361952 EdmunsonCreek Euryceasp2AHP_2837 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_3050 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_2978 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_3052 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_3055 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_3065 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_3053 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_3054 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_2839 30.166672 -99.342230 FessendenSprings Euryceasp2AHP_3009 30.088338 -99.320564 Hwy176Spring Euryceasp2AHP_3007 30.088338 -99.320564 Hwy176Spring Euryceasp2AHP_3010 30.088338 -99.320564 Hwy176Spring Euryceasp2AHP_3317 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3013 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3319 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3320 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3016 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3022 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3020 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3023 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3318 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3017 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_3019 29.915283 -99.252230 RobinsonCreek Euryceasp2AHP_2989 30.143338 -99.077786 TroughSpring Euryceasp2AHP_2985 30.143338 -99.077786 TroughSpring Euryceasp2AHP_2982 30.143338 -99.077786 TroughSpring Euryceasp2AHP_2986 30.143338 -99.077786 TroughSpring Euryceasp2AHP_2979 30.143338 -99.077786 TroughSpring Euryceasp2AHP_3006 30.143338 -99.077786 TroughSpring Euryceasp2AHP_2988 30.143338 -99.077786 TroughSpring Euryceasp2AHP_2990 30.143338 -99.077786 TroughSpring Euryceasp2AHP_2991 30.143338 -99.077786 TroughSpring Euryceasp2TNHC_62775 29.628694 -99.177972 WestVerdeSprings EuryceatroglodytesTNHC_62774 29.629000 -99.178000 WestVerdeSprings EuryceatroglodytesDMH_90.75 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.80 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.74 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.77 29.749450 -99.426675 SutherlandHollowSpring Euryceatroglodytes

21

DMH_90.76 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.79 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.109 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.110 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.111 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.105 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesDMH_90.96 29.749450 -99.426675 SutherlandHollowSpring EuryceatroglodytesTNHC_62773 29.808000 -99.558000 WedgeworthCreekSprings EuryceatroglodytesTNHC_62772 29.807778 -99.558000 WedgeworthCreekSprings EuryceatroglodytesDMH_89.70 29.823894 -99.566953 SabinalCanyon EuryceatroglodytesDMH_89.69 29.823894 -99.566953 SabinalCanyon EuryceatroglodytesTNHC_62777 29.829722 -99.592806 WishingWellSprings EuryceatroglodytesTNHC_62778 29.829722 -99.592806 WishingWellSprings EuryceatroglodytesTNHC_62776 29.427500 -99.784611 BeeCaveSprings EuryceatroglodytesAHP_3467 29.989444 -99.953611 GreenwoodValley EuryceatroglodytesAHP_3465 29.989444 -99.953611 GreenwoodValley EuryceatroglodytesCEP_038 29.958636 -99.958675 MorrissSpring EuryceatroglodytesAHP_3449 29.986389 -99.964167 GreenwoodValley EuryceatroglodytesAHP_3453 29.986389 -99.964167 GreenwoodValley EuryceatroglodytesAHP_3441 29.955556 -99.971389 GreenwoodValley EuryceatroglodytesAGG_1989 29.901088 -100.998815 FineganSpring Euryceasp3AGG_1818 29.893833 -100.994528 BlueSpring Euryceasp3PCDMH_94.12 29.890350 -100.986140 DolanFallsPreserve Euryceasp3PCDMH_94.10 29.890350 -100.986140 DolanFallsPreserve Euryceasp3Dolan_Falls_JKK 29.890350 -100.986140 DolanFallsPreserve Euryceasp3TJD_988 29.890350 -100.986140 DolanFallsPreserve Euryceasp3TJD_989 29.890350 -100.986140 DolanFallsPreserve Euryceasp3TNHC_62781 29.758000 -100.397000 KickapooSprings Euryceasp3CEP_034 29.745000 -100.360000 PaisanoSpring Euryceasp3DMH_90.52 29.652783 -100.103342 SmithsSpring Euryceasp3DMH_90.31 29.652505 -100.103342 SmithsSpring Euryceasp3DMH_90.33 29.652505 -100.103342 SmithsSpring Euryceasp3DMH_90.29 29.652505 -100.103342 SmithsSpring Euryceasp3DMH_90.32 29.652505 -100.103342 SmithsSpring Euryceasp3DMH_90.30 29.652505 -100.103342 SmithsSpring Euryceasp3TNHC_62779 29.568500 -100.086306 MachineryHollowSprings Euryceasp3TNHC_62780 29.533000 -100.131000 BoilingSprings Euryceasp3DMH_90.56 29.480561 -100.078897 CarsonCave Euryceasp3JKK_2004.1 29.416802 -100.905223 FourMileCave Euryceasp3CEP_030 29.410000 -100.450000 LowerPintoSpring Euryceasp3CEP_039 29.410000 -100.450000 LowerPintoSpring Euryceasp3

22

AppendixTable2.ChoiceofAnalyticalParametersforSTACKS1.3Pipeline.Inthe‘ValuesTested’column,thedefaultvalueisindicatedinboldlettering.

Parameter PipelineStep ParameterFunction

ValuesTested ValueSelected

-M ustacks Minimumstackdepth(innumberofreads)torecordalocus

3,5,7 3

-m ustacks Maximummismatchesallowedbetweenreadsinastack

2,3,4 2

-n cstacks Maximummismatchesallowedbetweenaproposedlocusandalocusinthecatalog

0,1,2,3 2,3

-m populations Minimumstackdepthrequiredatalocusforindividualtobeaddedtopopulation

3,6,9 3

-r populations Percentofindividualsinapopulationthatmustpossessalocustoincludelocus

20,40,60,80,90

NotesonAppendixTable2:1.Weexaminedthreevaluesfortheminimumstackdepthparameter(-M):M=3,M=5andM=7.AstackdepthofM=3producedaconsistentamountofmissingdataacrossmostindividualsinthedataset.M=5andM=7producedmissingdatathat

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werebiased;populationsfromthesecondsequencingrunhadabouttwiceasmuchmissingdata,inproportiontothetotalnumberofsites,asthosefromthefirst.Therefore,weselectedM=3astheoptimalvalue.2.Weexaminedthreevaluesforthemaximumwithin-individualmismatchparameter(-m;ustacks):m=2,m=3andm=4.m=2isthedefaultandmaximizesthenumberoflociinthedatamatrix.m=2andm=3resultinthesameaverageamountofmissingdatapertaxon(35%),whereasm=4producesanaveragemissingdataof47%.Inthedistancematrix,m=2minimizesthenumberoftaxonpairsforwhichthereisnodataforacomparison(n=177).3.Weexaminedatfourvaluesofthemaximumcatalogmismatchparameter(-n):n=0,n=1,n=2,andn=3.Thedefaultvalueofthisparameterisn=0.Thedefaultvalueforourdataseverelylimitedthestacksdepthvaluesforthe-mand-rparametersofthepopulationsstep:Wedidnotretrieveanylocithathadastackdepthofmorethan2,orwerepresentinmorethan30%ofpopulations.Thissuggestsavalueofn=0isoverly-conservativeforourdata.Therewasnovaluethatalwaysminimizedmissingdataacrosssamples.Bothn=1andn=3maximizedandminimizedmissingdatafordifferentindividuals.n=2tendedtoperformintermediately,neverproducingeitherthemostorleastmissingdata.However,n=3producedthefewesttaxonpairsforwhichthereisnomissingdata.Therefore,bothn=2andn=3wereusedinthefittingofthefinaltwoparameters.Whenfitting-m,usingn=3retainedmoreloci,butthedistributionofmissingdataandnumberoftaxonpairsforwhichtherewerenodataincommonwasthesameforn=2andn=3.Whenthefinalparameter,r,wasincluded,n=2consistentlyproducedmoretaxonpairsforwhichnodatawereavailable.Wethereforeselectedn=3toproduceourfinaldatamatrix.4.Weanalyzedthedatausingthreevaluesforthe(-m,populations)parameter:m=3,m=6andm=9.Muchlikethe-nparameter,thisparametermadelittledifferencetotheoverallamountofmissingdata.However,increasingthisvalueincreasestheamountoftaxonpairsbetweenwhichnodataareshared(n=3gives174taxonpairsforwhichthereisnobasistocompare,n=9gives354).Increasingthisparameteralsohalvesthenumberoflociretainedinthephylogeneticmatrix.We,therefore,chosetomaximizedataexaminedbyusingavalueofm=3.5.Therparametergovernsthepercentofindividualsthatmustberepresentedatalocustousethatlocusinthefinalphylogeneticmatrix.Weexaminedfivevaluesofthisparameter:r=20,r=40,r=60,r=80,r=90.Asthevalueofrincreases,thenumberoftaxonpairsforwhichtherearenoshareddatapointsincreases.However,fortaxawheredataispresent,thecompletenessofthedataincreaseswiththevalueofr.We

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analyzedallfivematrices,andfoundthatphylogeneticresultsremainedconsistentacrossallruns,withgreaterresolutionamongpopulationsfromthelargestmatrix.Therefore,wepresentresultsfromthislargestmatrix(34,518lociforthephylogeneticanalysesofallpopulations;75,296locifortheStructureanalysisofindividualsnorthoftheColoradoRiver).