1

Phylogeographicstructureofthedunessagebrushlizard,anendemichabitat1

specialist2

3

Authors4

LaurenM.Chan,DepartmentofBiology,PacificUniversity,ForestGrove,Oregon,USA*5

CharlesW.Painter,EndangeredSpeciesProgram,NewMexicoDepartmentofGameand6

Fish,SantaFe,NewMexico,USA† 7

MichaelT.Hill,Albuquerque,NewMexico,USA.8

TobyJ.HibbittsBiodiversityResearchandTeachingCollections,DepartmentofWildlife9

andFisheriesSciences,andNaturalResourcesInstitute,TexasA&MUniversity,College10

Station,Texas,USA11

DanielJ.Leavitt,NaturalResourcesProgram,NavalFacilitiesEngineeringCommandSouth12

West,SanDiego,California,USA13

WadeA.Ryberg,NaturalResourcesInstitute,TexasA&MUniversity,CollegeStation,Texas,14

USA15

DanielleWalkup,NaturalResourcesInstitute,TexasA&MUniversity,CollegeStation,Texas,16

USA17

LeeA.Fitzgerald,BiodiversityResearchandTeachingCollections,DepartmentofEcology18

andConservationBiology,andEEBPhDProgram,TexasA&MUniversity,College19

Station,Texas,USA20

21

*Correspondingauthor:lchan@pacificu.edu;+1503-352-146922

†deceased23

2

24

Keywords:Sceloporusarenicolus,fragmentation,conservationgenetics,population25

genetics,shinneryoaksanddunes,MescaleroSands,MonahansSandhills.26

27

ShortTitle:PhylogeographyofSceloporusarenicolus 28

3

Abstract29

Phylogeographicdivergenceandpopulationgeneticdiversitywithinspeciesreflect30

theimpactsofhabitatconnectivity,demographics,andlandscapelevelprocessesinboth31

therecentanddistantpast.Characterizingpatternsofdifferentiationacrossthegeographic32

rangeofaspeciesprovidesinsightontherolesoforganismalandenvironmentaltraits,on33

evolutionarydivergence,andfuturepopulationpersistence.Thisisparticularlytrueof34

habitatspecialistswherehabitatavailabilityandresourcedependencemayresultin35

pronouncedgeneticstructureaswellasincreasedpopulationvulnerability.WeuseDNA36

sequencedataaswellasmicrosatellitegenotypestoestimaterange-widephylogeographic37

divergence,historicalpopulationconnectivity,andhistoricaldemographicsinanendemic38

habitatspecialist,thedunessagebrushlizard(Sceloporusarenicolus).Thisspeciesisfound39

exclusivelyinduneblowoutsandpatchesofopensandwithintheshinneryoak-sanddune40

ecosystemofsoutheasternNewMexicoandadjacentTexas.Wefindevidenceof41

phylogeographicstructureconsistentwithbreaksandconstrictionsinsuitablehabitatat42

therange-widescale.Inaddition,wefindsupportforadynamicandvariableevolutionary43

historyacrosstherangeofS.arenicolus.PopulationsintheMonahansSandhillshave44

deeplydivergentlineagesconsistentwithlong-termdemographicstability.Incontrast,45

populationsintheMescaleroSandsarenothighlydifferentiated,thoughwedofind46

evidenceofdemographicexpansioninsomeregionsandrelativedemographicstabilityin47

others.Phylogeographichistoryandpopulationgeneticdifferentiationinthisspecieshas48

beenshapedbytheconfigurationofhabitatpatcheswithinageologicallycomplexand49

historicallydynamiclandscape.Ourfindingsidentifyregionsasgeneticallydistinctive50

4

conservationunitsaswellasunderscorethegeneticanddemographichistoryofdifferent51

lineagesofS.arenicolus.52

53

Introduction54

Patternsofpopulationgeneticdiversitywithinspeciesareshapedbyboth55

evolutionaryandcontemporaryhistory(Rissler,2016).Thoughanthropogenicchangesto56

landscapesalterpatternsofconnectivitythatcanresultinthedivergenceorcoalescenceof57

populations,theseprocessestakeplaceonabackgroundofevolutionaryhistory58

determinedbychance,species’lifehistory,andalsogeologicandclimaticchanges.59

Characterizingthisevolutionaryhistory,andidentifyingtherolethatorganismaltraits,60

evolutionaryprocesses,andecologicalconditionshaveonpatternsofphylogeographic61

divergenceaddstoourunderstandingofevolution,andisalsofundamentaltoconserving62

evolutionarypotentialinthefaceofanthropogenicdisturbanceandclimatechange63

(Olivierietal.,2015).64

Thephylogeographichistoryofspeciescanreflecttherolesthathabitat65

connectivity,geneflow,andpopulationstabilityhaveplayedinaspecies’evolutionary66

persistence.Somespeciesmaybecharacterizedbydeeplydivergentlineages,suggestinga67

historyoflimiteddispersalandlowconnectivityamongsites(e.g.,Richmondetal.,2013,68

2014;Chanetal.,2013),especiallyinecosystemswithsteepenvironmentalgradientsand69

discontinuoushabitat(Vandergastetal.,2008).Plantandanimaltaxainnaturally70

fragmentedlandscapes,forexample,canexhibitstrongpatternsofgeneticpopulation71

structurewithselectionfavoringlimiteddispersal.Phylogeographicanalysesof72

Stenopelmatusspecies(Jerusalemcrickets)insouthwesternNorthAmerica,forexample,73

5

revealedlimiteddispersalamongpopulations,andidentifiedarecentresponseto74

anthropogenicchange(Vandergastetal.,2007).Ameta-analysisofgeneticdiversityamong75

21speciesofterrestrialanimalsidentifiedhotspotsofgeneticdiversitythatmayalsobe76

regionswithhighlevelsoftraitdivergenceduetonaturalselection(Vandergastetal.,77

2008).Alternatively,populationsmaybeonlyweaklydivergentacrossaspecies’range78

indicatinghighconnectivity(e.g.Lippéetal.,2006;ChanandZamudio,2009)eveninthe79

faceofstronglocaldynamics(e.g.Piersonetal.,2013).Identifyingevolutionaryscenarios80

andprocessesthathaveresultedinparticularphylogeographicpatternscanhelpus81

disentangleprocessesthatunderliepopulationgeneticdivergencefromthosethat82

maintaingeneticdiversity.Understandingthedriversofpopulationgeneticstructure83

acrosstherangeofaspeciescanalsohelpuspredicttheresponsetolossofhabitatandthe84

overallvulnerabilityofspeciestoanthropogeniclandscapechange.85

Ecologicalspecialistscanhavegreaterpopulationgeneticandphylogeographic86

structurethangeneralistsbecauseindividualsandpopulationsmayberestrictedto87

spatiallyisolatedpatchesofsuitablehabitat.(Rodericketal.,2012;Schäretal.,2018;Wort88

etal.,2019).Ecologicalspecialistsmayhavenarrowphysiologicaltolerances,specific89

habitatrequirements,andbelocallyabundantbutrareatregionalscales(Devictoretal.,90

2008).Habitatspecialistsusespecificlandscapefeaturesandvegetationassociations91

withintheirrange,andoftenpossesseco-morphologicalandbehavioraladaptations(Miles,92

1994a,1994b).Traitsthatmakehabitatspecialistswell-suitedforanarrowhabitatniche93

alsotendtomakethemrelativelypoordispersers(Clobertetal.,2012).Lowtolerancefor94

unsuitablelandscapesisexpectedtorestrictmovementsamongisolatedpatchesof95

preferredhabitat.Ecologicalstudiesfocusingonthedemographyanddistributionof96

6

habitatspecialistshavefoundtheyaresensitivetolandscapefragmentation(Leavittand97

Fitzgerald,2013;Walkupetal.,2017).98

Localprocessesareoftenlinkedtopatternsobservedacrosslong-term,99

evolutionarytimescalesandatbroaderspatialscales(seereviewsbyCutter,2013;Rissler,100

2016).Thus,inspecialistswithstricthabitatspecificityandlimiteddispersalamong101

populations,wemightexpectphylogeographicstructuretoreflecthistoricalpatternsof102

divergenceandlowpopulationconnectivityoverall(e.g.,Rodericketal.,2012).103

Alternatively,habitatspecialistsmayhavewell-connectedpopulationsthroughouttheir104

range,indicatingastrongrolefordispersalandmigrationthatcountersthedivergenceof105

potentiallyisolatedlocalpopulationsacrosslongertime-scales(Piersonetal.,2013).106

Characterizingevolutionarypatternsofdivergenceandhistoricaldemographicsinhabitat107

specialistscanhelpuspredicttherolethatshortandlong-termdynamicsplayinshaping108

populationgeneticstructure.Inaddition,describingspatialpatternsofdiversityand109

identifyingindependentevolutionaryunits,historicalbarrierstogeneflow,bottlenecks110

andfounderevents,andregionsofhighconnectivityallowstheeffectsofcontemporary111

pressurestobedisentangledfromhistoricaldriversandalsoprovidesimportant112

informationforthefuturemanagementandconservationofspecies.113

Thedunessagebrushlizard,Sceloporusarenicolus,isendemictotheMescaleroand114

MonahansSandhillsecosystemofsoutheasternNewMexicoandadjacentTexas(Fitzgerald115

andPainter,2009;LaurencioandFitzgerald,2010).ThisspeciesispartoftheSceloporus116

graciosusclade(Chanetal.,2013),butincontrasttoothermembersofthisgroupwhich117

tendtobegeographicallywidespreadgeneralists,S.arenicolusisahabitatspecialist.118

Withinthisecosystem,itonlyusesshinnery-oaksandduneformationswithinterconnected119

7

duneblowouts(sandydepressionscreatedbywind)andinsomecasesshinnery120

hummocksinduneswithsteepslopes(Fitzgeraldetal.,1997;LaurencioandFitzgerald,121

2010;Hibbittsetal.,2013).IntheMescalero-MonahansSandhillsEcosystem,dune122

blowoutsareemergentlandformsthataremaintainedbytheinteractionsamongwind,123

movingsand,andtheshinneryoak(Quercushavardii)whichstabilizesthedunes(Ryberg124

andFitzgerald,2016).IndividualS.arenicoluslizardsdemonstrateanestedhierarchyof125

habitatselection(Fitzgeraldetal.,1997),selectingforthermallysuitablemicrohabitatsand126

havingpreferenceforrelativelylargeduneblowouts.Asand-divingspecies,theydonot127

occurinareaswithrelativelyfinesand(Fitzgeraldetal.1997;RybergandFitzgerald128

2015).Atthehighestlevelofhabitatselection,theyareendemictothenarrowly129

distributedMescalero-MonahansSandhills(FitzgeraldandPainter,2009).130

Specialistscanreachhighpopulationdensitiesintheirpreferredhabitat,andcan131

outcompetegeneralistsinthesameareaeveninsomedegradedhabitats(Brown,1984;132

Attumetal.,2006).ThisistruetooforS.arenicolus,wherepopulationsofthisecological133

specialistthrivewheretheconfigurationofkeylandscapefeaturessupportslargergroups134

ofinteractingindividuals,definedasneighborhoods(sensuWright,1946;Rybergetal.,135

2013).Diffusiondispersalthroughoutinterconnectedareasofsuitablehabitatappearkey136

tomaintainingpopulationsincontiguoushabitatoverthelongterm(Rybergetal.,2013).137

Thequantityofhabitatispositivelycorrelatedwiththequalityofhabitat(Smolenskyand138

Fitzgerald,2011),andtheoccurrenceofS.arenicolusisassociatedwithrelativelylargecore139

areasofshinneryoakdunes.140

Sinceatleastthe1930s,anthropogenicdisturbancesfromherbicidespraying,oil141

andgasmining,andmorerecently,sand-mining,haveresultedinfragmentationand142

8

degradationoftheshinneryoakdunes.Long-termmonitoringandextensivefieldworkalso143

demonstratethatfragmentationoftheshinneryoakdunelandsleadsdirectlytopopulation144

collapsebecausequalityofhabitattendstodegradeinresponsetofragmentation,and145

dispersalisdisrupted(LeavittandFitzgerald,2013;Walkupetal.,2017).146

Toadequatelyinformconservationandmanagementactions,itisnecessaryto147

understandtheevolutionaryhistoryofthisspeciesatbothbroadandfine-scales148

throughouttherange.Previousgeneticworkconfirmedthatatbroadspatialscales,S.149

arenicolusiscomprisedofatleastthreedistinctgeneticgroups(Chanetal.,2009).150

However,itisunclearwherethesegeneticbreaksoccurgeographicallyandwhetherthey151

coincidewithputativenaturalorman-madebarrierstomovement.Thepurposeofthis152

studyistocharacterizetheevolutionaryhistoryofthedunessagebrushlizardusing153

completegeographicandgeneticsampling.Toidentifyevolutionarydistinctgeographic154

lineagesandtoreconstructthepopulationhistoryoftheselineages,weevaluate155

mitochondrialandnuclearsequencedataaswellasmultilocusmicrosatellitegenotypes.156

Samplingforindividualsoccurredevenlythroughouttheentireknownrangeofthis157

endemicandthreatenedlizard.158

159

MaterialsandMethods160

Sampling161

WesurveyedforSceloporusarenicolusthroughouttheirrange(Figure1).Liveror162

muscletissuewascollectedfromvoucheredspecimensdepositedintheBiodiversity163

ResearchandTeachingCollections(symboliccode:TCWC),orMuseumofSouthwestern164

Biology(MSB).Additionally,toeand/ortailtipswerecollectednon-destructivelyfrom165

9

animalscaughtinthefieldthatweresubsequentlyreleased.Alltissuesampleswerestored166

in95%EtOH.WholegenomicDNAwasextractedfromtissuesusingtheDNeasyBloodand167

Tissuekit(Qiagen).168

169

DNAsequencedata170

WetargetedtwomitochondrialandfournuclearlociforDNAsequencing.PCR171

amplificationofthemitochondriallociNADH-dehydrogenase1(ND1)andcytochrome-b172

(cyt-b)andtwoproteincodingnuclearlociprolactinreceptor(PRLR)andR35used173

previouslypublishedprimers(Irwinetal.,1991;LeachéandMcGuire,2006;Leaché,2010).174

Weusedtwoadditionalanonymousnuclearlocidesignedfromagenomiclibraryenriched175

formicrosatelliterepeats:sarANL298(scar298anl.F:5’-ATGGGAAGGCTTAAAATGAATC;176

scar298anl.R:5’-TGTGACTTAGGGAACTGGGTATGT)andsarANL875(scar875anl.F5’-177

CTTACCATTCAACCCTTCCTTG;scar875anl.R5’-CTAGAGCAGACCAGTTCAATGTAAT).All178

PCRwereconductedin10µltotalvolume.Annealingtemperatureforthenewnuclearloci179

was54°C.180

Weused0.4µlExoSAP-IT(USB/Affymetrix)and1.6µlwatertoclean5µlofPCR181

product.OneµlofcleanPCRtemplatewasusedineachcyclo-sequencingreactionusingthe182

samelocus-specificprimersusedinamplification.Sequencingreactionswerecleanedand183

runonanABI3730xlattheDukeSequencingFacilityortheBiotechnologyResources184

CenterofCornellUniversity.ChromatogramswereverifiedandcleanedinGeneiousR9185

(https://www.geneious.com).Heterozygoussitesinnuclearsequenceswerecalledwith186

theappropriateambiguitycode.SequencesateachlocuswerealignedusingtheMAFFT187

(Katoh,2005)plug-ininGeneious.AllsequencedatawillbesubmittedtoGenBank.188

10

Becausethemitochondrionisinheritedasasingleunitwithoutrecombination,we189

concatenatedthetwoloci(ND1andCyt-b)intoasinglealignment.Eachofthefournuclear190

lociweretreatedindependently.AllsequencesateachlocuswerealignedinGeneiousand191

allelesatnuclearlociweredeterminedusingtheprogramPHASE(Stephensetal.,2001)192

andthehelperprogramSeqPHASE(Flot,2010).193

194

Microsatellitegenotypedata195

Nuclearmicrosatellitelociweredevelopedfroma454-libraryenrichedfor196

microsatellitemotifsdevelopedatCornellUniversityEvolutionaryGeneticsCoreFacility.197

Afterinitialscreeningofloci,weusedtheQiagenType-ItmicrosatellitePCRkittogenotype198

individualsattheselociinfivemultiplexreactions(Supp.Mat.Table1).Forwardprimers199

foralllociweretaggedwithafluorescentdyeandsamplesweregenotypedonan200

ABI3730xlattheBiotechnologyResourceCenterofCornellUniversitywithGeneScan500201

LIZsizestandard(ThermoScientific).Alleleswerecalledandverifiedforallindividuals202

usingGeneMarker2.6.Priortosubsequentgeneticanalyses,allvariablelociweretested203

forthepresenceofnullallelesandselectionbytestingforHardy-WeinbergEquilibrium204

(HWE)andforevidenceoflinkagedisequilibriumusingGenePop(Rousset,2007).Thefinal205

datasetincludedgenotypesforallindividualsat27variableandneutrallyevolvingnuclear206

microsatelliteloci.207

208

Dataanalysis209

Summarystatistics210

11

WeusedPAUP(Swofford,2002)todeterminethenumberofparsimonyinformative211

sitesforeachsequencealignmentandDNAspv6(Rozasetal.,2017)tocalculatethe212

numberofuniquehaplotypes,thenumberofsegregatingsites(S),nucleotidediversity(π),213

andtheaveragenumberofnucleotidedifferences(k)foreachsequencealignment.214

215

Haplotypenetworks216

WeconstructedparsimonynetworksinTCS(Clementetal.,2000)forcomplete217

mtDNAhaplotypesforS.arenicolus.Becausetheresultsgeneratedbynetworkmethodscan218

bestronglyinfluencedbymissingdata(Jolyetal.,2007),wefirstomittedallindividuals219

withmissingsequencedataforoneofthetwomitochondrialloci.Weadditionallyomitted220

individualsforwhichwedidnothavelocalityinformation.Thefinalhaplotypenetworkfor221

mtDNAcontained195individuals.Weadditionallyconstructedparsimonynetworksfor222

thephasedallelesateachnuclearlocus.223

224

Phylogeneticanalysis225

ForthemitochondrialDNA,weestimatedthephylogeneticrelationshipsamongS.226

arenicolusunderbothmaximumlikelihoodandBayesianframeworks.Urosaurusornatus,227

Utastansburiana,Phrynosomacoronatum,Sceloporusjarrovii,S.merriami,S.occidentalis,228

andnineindividualsofS.graciosuswereusedasoutgroups(followingChanetal.,2013).229

ConcatenatedmtDNAalignmentswerefirstreducedtouniquesequencesusingaPython230

scriptfromBioPython(sequence_cleaner.py).Weestimatedthebest-fitmodelofsequence231

evolutionateachcodonpositionofeachgeneinDT-ModSel(Mininetal.,2003)and232

partitionedphylogeneticanalysesbygeneandcodonposition.ThebestfitmodelsbyDT-233

12

ModSelwereaSYM+Gforthefirstcodonpositionofeachgene,HKY+Iforthesecondcodon234

positionofeachgene,andTrN+I+GandTrN+GforthethirdcodonpositionofND1and235

Cyt-brespectively.WeestimatedthephylogenyunderaBayesianframeworkinMrBayes236

v3.2.6(RonquistandHuelsenbeck,2003;Ronquistetal.,2012)excludingindividualswith237

missingdata.TrNmodelswereexpandedtoGTRforBayesiananalysesandthefinal238

analysisconsistedoftwoindependentrunseachof50milliongenerationssampledevery239

5,000generations.Allparameterswerecheckedforadequatemixingandconvergence,and240

themaximumcladecredibilitytreewassummarizedinMrBayes.241

242

Populationgeneticanalysis243

WeestimatedwithinpopulationdiversityandamongpopulationpairwiseFSTfor244

mtDNAaswellasmicrosatellitedataassumingmembershiptothephylogroupsbasedon245

theBayesianphylogeny.EstimatesofFSTweredoneinArlequin(ExcoffierandLischer,246

2010)formtDNAandinFSTATformicrosatellitedata.Becausesamplesweredistributed247

evenlythroughouttherangeofS.arenicolus,weadditionallyconductedpopulationgenetic248

analyseswithoutanyassumptionofpopulationmembershipusingassignmentmethodsin249

Structure2.3.4(Pritchardetal.,2000).InStructure,wetestedassignmentofallindividuals250

toKpopulationsfromK=1to10.AteachKweconducted10replicaterunseachconsisting251

of1milliongenerationswiththefirst50%discardedasburn-in.Weused252

StructureHarvester(EarlandvonHoldt,2011)toexamineallrunsandCLUMPP(Jakobsson253

andRosenberg,2007)andDISTRUCT(Rosenberg,2004)tovisualizepopulation254

membership.StructurerunswithallindividualssupportedK=2,sosubsequentruns255

investigatedfurtherpartitioningwitheachmajorgroup.Foreachsubsetofdata,wetested256

13

K=1to5eachwith10replicaterunsateachKeachconsistingof2millionrunswiththe257

first50%discardedasburn-in.258

259

Demographicanalyses260

Weestimatedthehistoricaldemographicsforeachoffiveprimaryphylogeographic261

regions(A-E)identifiedinmtDNAanalyses.Weusedmultilocussequencedatatoconstruct262

extendedBayesianskylineplotsinBEAST2.5.0(Drummondetal.,2005,2012;Heledand263

Drummond,2008).EachdatasetincludedconcatenatedmtDNAalignmentsinadditionto264

phasedgenotypesforeachofthefournuclearloci.Substitutionmodelsforeachlocuswere265

setbasedonMrModeltest(Nylander,2004;Supp.Mat.Table2).Allrunsassumedarelaxed266

molecularclockwithalog-normaldistributionforthemtDNApartitionandstrict267

molecularclocksforthenuclearpartitions.TherateformtDNAwassetwithalognormal268

distributionwithmeanof1x10-8substitutions/site/yearandSDof0.27following(Chanet269

al.,2013).ParametertrendswereexaminedinTracertocheckforadequatemixingwithin270

runsandconvergenceacrossruns.Finalrunswere50millionstepssampledevery5,000271

stepsforregionsB,C,andE.ThefinalrunsforregionsAandDwere100and200million272

stepssampledevery10,000and20,000steps,respectively.ExtendedBayesianskyline273

plotswegeneratedafterdiscardingthefirst25%sampledstepsasburn-in.274

275

Hypothesistesting276

Basedontheresultsofphylogeneticanalysesandassignmenttests,wetestedthree277

alternativehypothesesofdivergenceandpopulationexpansionamongthreegeographic278

groups(NorthernMescaleroSands,SouthernMescaleroSands,andMonahansSandhills)279

14

assumingthattheMonahansSandhillspopulationswereancestralandofconstant280

populationsize(Chanetal.,2009;Supp.Mat.Figure1).WeusedapproximateBayesian281

computationtoevaluatesupportforthesemodelsandestimatedemographicparameters282

ofthebestsupportedmodelinDIYABC(Cornuetetal.,2008).AnalysesincludedmtDNA283

andphasednuclearsequences.Locusparameterswerespecifiedafterestimationof284

substitutionmodelsforeachlocusinDT-ModSel.ThepriorforthemtDNAmutationrate285

wassetasanormaldistributionwithameanof1x10-8substitutionspersiteperyearand286

nuclearsubstitutionratesweresetasuniformdistributions.Initialrunswereusedto287

determineadequatepriorsfordemographicparameters.Thefinalanalysisincluded2288

millionsamplesforeachdivergencemodel(6milliontotal)withalinearregressionstepto289

extracttheclosest1%ofsamplesanddeterminethebestsupportedmodelofthethree.For290

thebestsupportedmodel,weusedthesameselection/rejectionprocesstoestimate291

divergencetimesanddemographicparametersfromtheclosest1%ofthe2million292

samples.293

294

Results295

Summarystatistics296

Samplesizes,alignmentlengths,thenumberofuniquehaplotypes,numberof297

segregatingsites,averagenucleotidedifferences,andnucleotidediversityarereportedin298

Supp.Mat.Table3.Asexpected,nuclearlociwerelessvariablethanmtDNAthough299

nucleotidediversitywassimilarformtDNAandtwonuclearloci.Wealsorecovered300

multilocusgenotypesfor237individualsat27microsatellitelocithatconformedtoHWE301

15

expectationsanddidnotshowanyevidenceoflinkageornullalleles.Theaveragenumber302

ofallelesperlocuswas16.4witharangefrom3to35(Supp.Mat.Table4).303

304

Haplotypenetworksandphylogenetics305

Mitochondrialhaplotypenetworksrevealedgeographicallyassociatedhaplotype306

groupsformtDNAthatlargelycorrespondtoregionsofgrosslycontiguoushabitat(Figure307

2).IntheNorthernMescaleroSands,therearethreemainhaplotypegroupscorresponding308

largelywiththeAregions(Figure1;AAandAB),theBregions(BAandBB),andtheC309

region,thoughthegeneticdivergenceamongthesethreegroupsissmall.Common310

haplotypesaresharedacrossregions,butderivedhaplotypesareuniquetoeachregion.311

RegionsABandBBhavegeneticdiversitythatisprimarilyasubsetofthediversityfoundin312

AAandBA,respectively.TheSouthernMescaleroSands(RegionsDAandDB)are313

geneticallydivergentfromtheNorthernMescaleroSandspopulationswiththebarrier314

betweenthetwogroupsreflectingawest-eastconstrictioninthedistributionofpotentially315

suitablehabitat(referredhereafteras“theSkinnyZone”).AmongtheSouthernMescalero316

SandsindividualsinregionDA,wefindasinglewidespreadhaplotypeandmultiplederived317

haplotypes.Inaddition,regionDBatsouthernmosttipoftheSouthernMescaleroSands318

containsaclusterofderivedhaplotypes.319

PopulationsintheMonahansSandhillsaregeneticallydistinctfromallotherS.320

arenicoluspopulations,butdonotformasinglehaplotypegroup.Thereishighsequence321

divergenceamonghaplotypesfromtheMonahansSandhillsdespiteoccurringina322

relativelyrestrictedgeographicareaandtheyaredistantlyrelatedtoMescaleroSands323

haplotypes.TheEAandECareaseachhaveuniquehaplotypeswithoutasingle,most324

16

commonhaplotype.TheEBhaplotypesfalloutintotwomaingroups,onethatisequally325

distanttonorthernandsouthernMescaleroSandshaplotypesandonethatisdistantly326

relatedtoallotherrecoveredhaplotypes(Figure2).327

Ingeneral,nucleargeneregionshadmuchlowergeneticdiversitywithverylittle328

geneticstructure(Supp.Mat.Figure2).Acrossallfournuclearloci,wefoundasimilar329

patternwiththemostcommonhaplotypesoccurringinmost,orallregions.AtPRLRand330

scar875,severalderivedlociwereuniquetoMonahansSandhillspopulationsand331

MonahansSandhillsplusSouthernMescaleroSandspopulations.Withoneexception(AB332

locusR35)regionsAA,AB,andBBdidnothaveanyuniquenuclearalleles.333

Phylogeneticreconstructionslargelycorroboratedthegroupsfoundinthenetwork334

analyses(Figure3).WerecoverS.arenicolusasmonophyletic(PP=1).MonahansSandhills335

populationswereparaphyleticwithrespecttoMescaleroSandspopulationswiththe336

southern-mostMonahansSandhillsindividualsformingaweaklysupportedclade(PP=337

0.8657)sistertoallotherS.arenicolus.Amongtheremainingindividuals,thereisstrong338

supportforaNorthernMescaleroSandscladeincludingindividualsnorthoftheskinny339

zone(PP=0.9758)andmoderatesupportforaSouthernMescaleroSands–Monahans340

Sandhillscladethatincludesindividualssouthoftheskinnyzoneandthenorthernand341

centralMonahansSandhills(PP=0.9345).WithintheNorthernMescaleroSandsclade,we342

recoversupportforsomeclustersofindividuals,butdonotfindwell-supportedclades343

correspondingtodistinctgeographicregions.IndividualsfromregionA,atthenorthern344

endoftherange,formabasalpolytomyrelativetootherwisewell-supportedclades345

containingmostindividualsfromregionsBandC,andseveralfromregionAandonefrom346

D(TCWC94831).SupportforacladethatincludesmostBindividualsishigh(PP=0.9639)347

17

asissupportfortwodifferentcladesthateachprimarilyincludeindividualsfromC(PP=348

1).Wedonotrecoververymuchgeneticresolutionforindividualssouthoftheskinnyzone349

intheSouthernMescaleroSandsorthenorthernorcentralMonahansSandhills.Notably,350

individualsfromMonahansSandhillsareparaphyleticandtheirrelationshipslargely351

unresolved.Whilemostindividualsfromregionsclusterwithotherindividualsfromthe352

sameregion,asexpectedfromthehaplotypenetwork,thereareafewindividualsthatfall353

outwithindividualsfromdifferentregions.354

355

Demographicestimates356

ExtendedBayesianskylineplotsmatchtheinferencesmadefromthehaplotype357

networks(Supp.Mat.Figure3).Weseeevidenceofrecentpopulationexpansioninregion358

DanddemographicstabilityinregionE.RegionsA,B,andCshowsomeevidenceof359

populationexpansionthoughthecredibleintervalsaroundthemostrecentpopulation360

sizesislargeanddoesnotexcludethepossibilityofdemographicstability.361

362

PopulationGenetics363

PairwiseFSTamongpopulationswashighamongregionswithvaluessignificantly364

differentfromzerorangingfrom0.099to0.904formtDNAand0.026to0.236basedon365

microsatelliteloci(Table1).366

Assignmenttestsbasedonmicrosatellitedatarevealnestedstructureatmultiple367

spatialscales(Figure4).Acrossallsamples,ouranalysesrecovertwogroupswithsome368

admixture.ThegeographicbreakbetweenthesetwogroupscorrespondedtotheSkinny369

ZoneoftheMescaleroSandswithsomeindividualsinthisareabeingadmixed.Further370

18

assignmenttestsinStructurewithnestedsubsetsofthedataindicatethattheseadmixed371

individualsarealignedwithindividualsintheSouthernMescaleroSands.Werecover372

distinctivegroupsintheNorthernMescaleroSandswithsomeadmixtureaswell.RegionA373

individualsaredistinctfromregionB+Cindividualsalthough,assignmentplotssuggest374

someadmixturebetweenwesternApopulations(AA)andpopulationsinregionC,375

corroboratingresultsfromthemtDNAhaplotypenetworks.AnalysisoftheAA-ABgroups376

recoverABasgeneticallydistinctcorroboratingFSTestimates(Table1).AnalysisoftheBA-377

BB-CgroupsupportsBBandCasdistinctfromoneanotherwithBAhavinggenetic378

affinitiestoboth.TogethertheseresultsshowacleargeneticbreakbetweentheSouthern379

MescaleroSandsandMonahansSandhillspopulations.FortheSouthernMescaleroSands380

populationsthereisanadditionalgeneticbreakbetweenregionsDAandDBcoinciding381

withanotherconstrictioninsuitablehabitat.AmongMonahansSandhillssamples,EA382

individualsarelargelydistinctfromEB+EC.IndividualsfromEBandECaresomewhat383

distinctfromoneanotherthoughnotallindividualswithinaregionclusterunambiguously384

withothersinthegroup.385

386

Hypothesistesting387

Werecoverstrongestsupport(PP=0.9991)foradivergencescenariothatinvolved388

colonizationoftheNorthernMescaleroSandsfromMonahansSandhillspopulations389

around34.8Kya(CI17.7-108Kya)followedbycolonizationoftheSouthernMescalero390

SandsfromMonahansSandhillspopulationsmorerecently,around16.3Kya(CI7.9-41391

Kya;Figure5).ItispossiblethattheinitialcolonizationofNorthernMescaleroSands392

includedcolonizationoftheSouthernMescaleroSands,withsubsequentlocalextinction393

19

andrecolonization,orgeneticreplacement.Itisimportanttonotethatthe95%credible394

intervalsforallestimatesofdivergencetimeandpopulationsizearebroad.Infact,though395

thetimeofexpansionintheNorthernMescaleroSands(Texp1)wasconstrainedinindividual396

ABCsimulationstooccurafterthedivergencebetweentheNorthernMescaleroSandsand397

theotherregions(Tanc),themedianestimatefortheformerTexp1precedesthemedian398

divergencetime,Tanc(Figure5),thoughbothestimateshaveextremelybroadand399

overlappingcredibleintervals.400

401

Discussion402

SamplingofS.arenicolusthroughouttheentirerangeprovidesgreaterresolutionof403

theevolutionarypatternsofdivergenceofthisnarrowlydistributedhabitatspecialist.We404

findsupportformultiplegeneticgroupswithinS.arenicolussuggestinglimitedmigration405

inthishabitatspecialist.Inparticular,wefindgeneticstructurebeyondthethree406

mitochondrialgroupsdescribedinChanetal.(2009).Patternsofdivergencerecoveredby407

mtDNAcorroboratenuclearmicrosatellitedataanddemonstratetheimportanceofthe408

landscape-scaleconfigurationofareasofhabitatonthephylogeographicstructureofthis409

habitatspecialist.Withthoroughgeographicsamplingweareabletoidentifyregionsthat410

haveservedasbarrierstopopulationconnectivityandcharacterizehistorical411

demographicsacrossevolutionarytimescales.412

LineagesofS.arenicolusintheMescaleroSandsandMonahansSandhillshave413

independentanddistincthistoriesthatareassociatedwiththetimingofsanddeposition414

andduneformationinthesesub-regions.Indeed,theMescaleroSandsandMonahans415

Sandhillshaverelated,butdistinguishablegeologichistories(MuhsandHolliday,2001;416

20

RichandStokes,2011;Muhs,2017).BothMescaleroSandsandMonahansSandhillsare417

sandsheetsoftheSouthernHighPlainsdepositedoverolder,compacteoliandeposits418

comprisingtheBlackWaterDrawformation(204-43Kya;RichandStokes,2011).Sand419

accumulationandduneformationhasoccurredrepeatedlywithcurrentsandsheetage420

estimatesforMescaleroandMonahansas29.2and22.2Kyarespectivelyandwithamore421

recentdeposition~7.5Kya(RichandStokes,2011).ThoughS.arenicolussampledfrom422

theMonahansSandhillsdonotformamonophyleticgroup,itisclearthattheyaredistinct423

fromMescaleroSandspopulationswithestimatedinitialdivergencebetweenthesetwo424

regionsoccurringlongago(34.8Kya,CI108-17.7Kya;Figure5).Whiletheestimated425

divergenceisolderthantheestimatedageofthemostrecentsanddeposition,thisisa426

dynamiclandscapethathasundergonecyclesofsanddepositionduringperiodsofaridity427

(Holliday,1989;RichandStokes,2011)suchthatthisdivergenceismostlikelyassociated428

withpreviousepisodesofsedimentationandduneformation.TherearebroadCIaround429

estimatesofdivergenceandpopulationexpansion,buttheseestimatesgenerallycoincide430

withthesandageofNorthernMescaleroSands.Furthermore,theestimateofthemost431

recentdepositionfallswithintheCIforcolonizationandexpansiontimesfortheSouthern432

MescaleroSands.433

Thelocationandmovementofsandduneformationshaschangedovermillennia434

(MuhsandHolliday,1995,2001;Muhs,2017).Whilethepresenceofsanddunesalonedoes435

notindicatethepresenceofshinneryoak-sandduneecosystem,thedistributionofhabitat436

suitabletoS.arenicolushaslikelyshiftedinitsoccurrenceandconnectivityovergeologic437

time.GiventhedynamicnatureofthelandscapetowhichS.arenicolusisendemic,itstands438

toreasonthatdynamichistoriesalsocharacterizethephylogeographicandpopulation439

21

geneticstructureinthisspecies.ThoughtheageoftheMescaleroSandsandMonahans440

Sandhillsgeologicformationsareuncertain,ourdatasuggestthatMonahansSandhillswas441

thesourcepopulationfromwhichMescaleroSandsS.arenicoluspopulationswere442

colonized.MescaleroSandspopulations,whichlietothenorthoftheMonahansSandhills,443

arecomprisedofatleasttwodistinctlineages,butnuclearmicrosatellitedataandABC444

analysessuggestthattheSouthernMescaleroSandspopulationsaremorecloselyrelated445

totheMonahansSandhillspopulationsthantonorthernMescaleroSandspopulations.The446

twosandformationsarenotcurrentlyconnectedbysuitablehabitat(Figure1),but447

presumablywereconnectedinthepastfacilitatingthecolonizationofMescaleroSands448

fromMonahansSandhills.byS.arenicolus.Thecurrentrangemap(Figure1)isinformedby449

currentlyoccupiedhabitat,butgiventhedynamichistoryoftheshinneryoak-sanddune450

ecosystem,potentiallysuitablehabitatconnectingregionsmayhaveoccurredinthepast.451

Theconfigurationofavailablehabitatisvariesacrosstimewhichpresumablycauses452

concordantshiftsinspecies’distributions.453

Ourgeneticdatasuggestthatthecolonizationeventassociatedwiththecurrent454

SouthernMescaleroSandspopulationsoccurredseparatelyfromtheeventthatresultedin455

theNorthernMescaleroSandspopulations.ColonizationoftheNorthernMescaleroSands456

anddivergencefromtheMonahansSandhillsourcepopulationisestimatedtohave457

occurredapproximately34Kyafollowedbypopulationexpansion(Figure5;Supp.Mat.458

Figure3).Whilerecognizingthattherearebroadconfidenceintervalsaroundthe459

estimatedtimeofthisevent,itisplausiblethatthisdivergencewasassociatedwiththe460

depositionoflooseaeoliansandsovertheBlackwaterDrawFormation(RichandStokes,461

2011).TheseconddivergencewasbetweenthesouthernMescaleroSandsandMonahans462

22

Sandhillsoccurringlater,around16.3Kya.Thisissimilartotheageofmorerecentsand463

depositsintheMescaleroSands,andsubsequentpopulationexpansionwithamedian464

estimateof9.9Kyacoincidesroughlywiththeagesofthemostrecentaeoliandeposits.465

ThisresultsuggeststhatafterthecolonizationoftheMescaleroSands34KyabyS.466

arenicolus,habitatbetweentheMescaleroSandsandMonahansSandhillscontractedor467

thatSouthernMescaleroSandspopulationsbecameextirpatedandthisareawaslater468

recolonized.BothscenariosseemplausiblegivenwhatweknowaboutS.arenicolusecology469

andthedynamicnatureofthissystem.470

Sceloporusarenicolusrequiresinterconnectedshinnery-oakblowoutstosupport471

populations(Rybergetal.,2013;LeavittandFitzgerald,2013).Shinneryoakflatsor472

isolatedduneblowoutsimpedemovementsandisolatepopulations.Thedivergencesthat473

weseeacrosstheMescaleroSandsandMonahansSandhillscorrespondlargelywiththe474

geographicextentofpotentiallysuitablehabitatidentifiedinseveralstudiesofS.arenicolus475

(Fitzgeraldetal.,1997;LaurencioandFitzgerald,2010;Walkupetal.,2018).Wearealso476

abletoreconstructhistoricalpopulationdemographyandrecovervariable,andsometimes477

dynamic,historiesacrosspopulationsofS.arenicolus.Forinstance,wefindsupportfora478

majorgeneticbreakthatcoincideswiththeSkinnyZone,anarrowconstriction(~3km479

wide)inthecentralMescaleroSands.ThisnarrowzoneofhabitatforS.arenicolusis480

indicativeofalong-standingbarriertodispersalandisnowapointofsecondarycontact481

betweendivergentNorthernandSouthernMescaleroSandspopulations.482

Wefindshallowdivergence,butdistinctgeneticdiversityamongtheNorthern483

MescaleroSandsregionsindicatingthathabitatsuitabilityalsoimpactspopulationgenetic484

connectivityatthesefinerspatialscales.Populationsinsomeoftheseregions,likeABand485

23

BB,havedivergedinisolation,suggestingafoundereffectinlinewiththemajordirection486

ofsanddunemovement(Muhs,2017).Weadditionallyconfirmarecentcolonizationand487

subsequentrapidpopulationexpansioninthesouthernportionoftheMescaleroSands488

(RegionD).Finally,amongtheMonahansSandhillssampleswedocumentedhighly489

divergentalleles,deepdivergenceamongpopulations,andrelativepopulationstability.We490

recoveredatleastthreedivergentgroupsamongtheMonahansSandhillsindividuals491

indicatinglimitedmovementamongolderpopulationsretainingancestralgeneticdiversity.492

Thehistoricaldemographyandpatternsofdivergencearereflectedinthe493

microsatellitedataaswellasthemoreslowlyevolvingmtDNAsequencedataindicating494

thatpopulationstructureistheresultoflongstandinghabitatdynamicsandrestrictionsto495

geneflowatmultiplespatialscales,notjustmorerecentanthropogenicchange.496

DemographicstudiesofS.arenicolushaveemphasizedtheimportanceofanetworkof497

suitablehabitatatmultiplespatialscalestosupportmetapopulationdynamicsand498

populationpersistence(Rybergetal.,2014).Landscape-ecologicalanalysesofpresence499

andabsenceoflizardcommunitymembershipacrosstheMescaleroSandsdemonstrated500

thatlandscapeheterogeneity,notdispersal,explainedcommunityassemblyandmeta-501

communitystructure(RybergandFitzgerald,2015,2016).Theoccurrenceofthehabitat502

specialistS.arenicoluswasadriverofthispattern.Assuch,becausethefine-scale503

distributionofsuitablehabitatiscriticalforlocalpresenceofS.arenicolus,wesuggestthe504

compositionandconfigurationofthelandscapewithrespecttounsuitablehabitattypes505

determinespatternsofgeneticconnectivityacrosstherange.Thedivergenceswedetect506

reinforcethatextensivehabitatmaybenecessarytosupportgeneflowamongpopulations507

andthathabitatqualityandhabitatconfigurationatfinerscalesmaybeofcritical508

24

importancetoidentifyingpotentialcorridors.Importantly,itisclearthattheshinneryoak-509

sandduneecosystemisadynamiclandscapewheretheconfigurationofhabitatpatches510

canchangeoverdecadesandmillennia.Weknowfromphylogeographicstudiesthat511

specialistsinchangingenvironmentsundergorepeatedepisodesofisolationand512

divergence(Rodericketal.,2012).Whileitisimpossibletoreconstructthespecific,513

chronologicalhabitatconfigurationfortheMescaleroSandsandMonahansSandhills,itis514

likelythatnetworksofsuitablehabitathavedivergedandcoalescedrepeatedlyovertime515

(e.g.,Dzialaketal.,2013).Source-sinkdynamicsareimportantatlocalandcontemporary516

spatialandtemporalscales(Rybergetal.,2013;Walkupetal.,2019),andthismay517

translatetoevolutionarypatternsofpopulationgeneticstructureatbroaderspatialscales518

andlongertimescales.Underthismodel,habitatpatchesshiftintheirextentand519

distributionovertimeduetogeologicalprocesses.Thedivergenceandcoalescenceof520

habitatpatchesacrosstimeresultsinrepeatedlocalextinction,populationdivergence,and521

recolonization.Insupportofthisscenario,wefindpopulationgeneticanddemographic522

patternsthatreflectsuchdynamicprocessesandtheirvariabilityacrossthelandscape.For523

instance,theSouthernMescaleroSandsisamorerapidlyshiftingsandduneformation524

(MuhsandHolliday,1995,2001;Muhs,2017)incomparisontothesandsheetsofthe525

MonahansSandhillsformationwhicharemorestableandlessdynamic(Machenberg,526

1984).TheSouthernMescaleroSandsmaybecharacterizedbylocalextinctionand527

recolonizationwhereastheslowermovementoftheMonahansSandhillsmaymaintain528

demographicallystableandisolatedpopulationsoverlongertimeperiods.529

ThepatternsofdivergenceandgeneflowthatweseeinS.arenicolusarenot530

surprisingofahabitatspecialistinhabitingadynamiclandscape.Basedondemographic531

25

studies(LeavittandFitzgerald,2013;Walkupetal.,2019)andobservationsofS.arenicolus532

(Rybergetal.,2012;LeavittandAcre,2014;Walkupetal.,2018,p.2018;Youngetal.,533

2018),individualsdonotmovelargedistances.Theirstricthabitatrequirements,andthe534

naturallypatchyandtemporallydynamicqualitiesofthishabitat,suggeststhatpopulations535

shouldbesubdivided.ThenestednessofgeneticstructureinS.arenicolusmirrorsthe536

hierarchicalnatureoftheirhabitatpreference:individualsrequiresuitableblowouts537

withinamatrixofshinneryoak,andpopulationsaresupportedbyanetworkofconnected538

shinneryoak-sanddunecomplexes.Whilethegeneticconsequencesofmetapopulation539

dynamicshavetypicallybeenexploredatfinespatialandtemporalscales,our540

phylogeographicstudyshowsthatthesemetapopulationdynamicsmayalsoleavetheir541

signatureatbroaderspatialscales,inthiscase,acrosstherangeofthisendemiclizard.542

543

Conservation544

Theshinneryoak-sanddunehabitatsofMescaleroSandsandMonahansSandhills545

haveexperiencedseverehabitatdegradationandfragmentation,particularlyinthe546

southernportionsoftherangeofS.arenicolus(LeavittandFitzgerald,2013;Walkupetal.,547

2017).Recentongoingfragmentationduetohumanactivities(e.g.highwaysandcaliche548

roadsbuiltforoilfielddevelopment)isknowntodecreaseconnectivityamongpopulations549

andinterruptmetapopulationdynamicsleadingtoextinctionoflocalpopulations(Ryberg550

etal.,2013,2014;LeavittandFitzgerald,2013;Walkupetal.,2017).Fragmentationofthe551

shinneryoak-sandduneecosystemincreasesthelikelihoodthatancestraldiversityand552

uniqueevolutionarylineageswillbelost.Ourfindingshighlightregionstobeconsideredas553

26

geneticallydistinctiveconservationunitsaswellasunderscoretheuniquegeneticand554

demographichistoryofdifferentregionswithintherangeofS.arenicolus.555

556

Acknowledgments557

ThankyoutoJ.T.Giermakowski(MSB),R.Macey,C.Spencer(MVZ),andJ.Vindum(CAS)558

foraccesstotissuesamples.Wethankthemanyresearchassistantswhohelpedwithtissue559

collection,DNAsequencing,andgenotyping,includingJ.Moberg,E.Gibson,D.Cavero,and560

T.Caspi.SequencingwasconductedattheGenomeSequencing&AnalysisCoreResourceof561

theDukeInstituteforGenomeSciencesandPolicy.Genotypingofmicrosatellitelociwas562

completedattheBiotechnologyCoreFacilityofCornellUniversity.Portionsofthis563

researchwereprovidedbytheDunesSagebrushLizard/LesserPrairieChickenCandidate564

ConservationAgreement(CCA)ResearchFundadministeredbyCEHMM,Carlsbad,New565

Mexico,andbyUSABureauofLandManagement,andbytheStateofTexasComptrollerof566

PublicAccounts.ThisiscontributionnumberxxxxoftheBiodiversityResearchand567

TeachingCollections,TexasA&MUniversity.568

569

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756 757

31

Tables758

759

Table1.PairwiseFSTvaluesamongregionsformicrosatellitegenotypes(abovediagonal)760

andmitochondrialsequencedata(belowdiagonal).Valuessignificantlydifferentfromzero761

(atalpha<0.05)areindicatedinbold.Thesignificanceofsomevalueswasnotabletobe762

determinedbecauseoflowgeneticvariability,indicatedwithitalics.763

AA AB BA BB C DA DB EA EB ECAA 0.0807 0.0349 0.0689 0.0358 0.1443 0.1723 0.1488 0.1654 0.1457

AB 0.0279 0.1077 0.1911 0.0749 0.1988 0.2374 0.2080 0.2356 0.2010BA 0.3389 0.3151 0.0512 0.0264 0.1337 0.1594 0.1349 0.1388 0.1033

BB 0.6593 0.7667 0.2147 0.0807 0.1643 0.2116 0.1688 0.1830 0.1592

C 0.4102 0.3688 0.4605 0.6583 0.1271 0.1523 0.1213 0.1350 0.1071

DA 0.8466 0.8584 0.8083 0.8687 0.8503 0.0411 0.0882 0.1188 0.0990

DB 0.8381 0.8395 0.7483 0.8439 0.8173 0.3311 0.1213 0.1554 0.1302EA 0.7984 0.7723 0.7372 0.7842 0.7858 0.5767 0.4206 0.1188 0.1017

EB 0.5554 0.4000 0.5021 0.4513 0.5523 0.6668 0.4663 0.5302 0.0701EC 0.8406 0.8125 0.7540 0.8216 0.8099 0.8872 0.8424 0.8108 0.4119 764 765

32

FigureLegends766

Figure1.CollectionlocalitiesforsamplesofS.arenicolusfromNewMexicoincludedinthis767

studyandtheoutlineofthespecies’rangeandsuitableshinneryoak-sanddunehabitat768

(fromLaurencioandFitzgerald,2010).SpecificlocalitiesarenotshownforTexasdueto769

legalconfidentialityagreementswithlandowners.Coloredportionsofthespecies’range770

correspondtophylogroupsandgeographicregionsreferredtointext.Brownindicates771

potentialhabitatinTexaswhereS.arenicolushasnotbeenfound;onelocalityexistsinthis772

regionfrom1970.Presence/absencedataandhabitatsuitabilitymapscouldbeusedto773

morepreciselydelineategeographicboundariesofthephylogroupswithinareasofsuitable774

habitat.775

776

Figure2.HaplotypenetworksbasedonconcatenatedmtDNAsequences.Circlesrepresent777

uniquehaplotypeswiththesizeofthecirclecorrespondingtotherelativeabundanceand778

thecolorreferringtotheregionoforiginofindividualswiththathaplotype(seeboxesin779

upperleftrepresentinggeographicapproximationsofeachregion).Linesconnecting780

haplotypesrepresentonemutationalstep.Smallwhitecirclesrepresentunsampled781

haplotypes.[Alternateversionforindividualswithcolorvisiondeficienciesincluded].782

783

Figure3.Majority-rulesconsensustreefromBayesianphylogeneticanalysisof784

concatenatedmtDNAsequencedata.Posteriorprobabilityforallnodesis1unless785

otherwiseindicated.Tipsarelabeledwithasamplenamefollowedbythenumberof786

sampleswithanidenticalhaplotype.Regionsofcollectionareindicatedverticallywith787

severalexceptionslistedparentheticallyinterminalname.788

33

789

Figure4.IndividualassignmentplotsfromnestedBayesianassignmenttestsin790

STRUCTURE.ResultsatalternatevaluesofKareshownforsomesubsetsofindividuals.791

792

Figure5.Estimatesofdivergenceandexpansiontimesaswellascurrentandhistorical793

effectivepopulationsizesforthebestsupportedmodelfromABCanalysisofthecomplete794

geneticdataset.795

103°0'0"W104°0'0"W

33°0'0"N

32°0'0"N

0 20 40 60 8010

Kilometers

AB

AA

BA

BB

C

DA

DB

EA

EB

EC

Figure 1

New Mexico

Texas

29 31

41

22

AABBBA

C

DA

EAEBEC

AB

DB

Figure 2

AABBBA

C

DA

EAEBEC

AB

DB

Figure 2 (alternate version)

0.005

ESP9606

TCWC93505

SCAR10.2

ESP9325_ 3 (BA)DJL286_3

TJH2880

TJH2927_2

TJH2918

TJH2840

TCWC92573

TX2012.001

TX2011.241

ESP9057_2

ESP9451

ESP9070_9

TJH2939_2

TJH2846

DJL297_2

TX2011.286

SAR0229

MSB57694

SAR0023

SAR0361

ESP9485

MSB57705

SAR0226SAR0227

ESP9557

LAF10450_3

DED075

0.974

0.918

0.976

0.866

0.984

0.874

0.910

0.964

TJH2971_6CSA059

CSA043_8

TJH3000_2

TJH2984CSA061

TJH2966_3

TJH3033

0.977

0.943

0.945

0.560

MTH270

NM12.09

NM12.04NM12.07

NM12.08

ESP9196_8 (1 BA)

ESP9174

TCWC 94831 (D)

MTH468

ESP9417ESP9232

MTH275

MSB57684

ESP9186

ESP9208

0.989

0.526

MTH474

MTH489_4MTH470

SCAR31

ESP9179

SCAR09

ESP9261

SCAR35

ESP9273_2

MTH472

MTH478

SAR0003_2

MTH490

ESP9064

MTH475_5

SCAR33

SCAR08

SCAR34

ESP9264_7

ESP9480

SAR0200

MTH469

0.981

0.973

0.913

0.956

0.951

0.964

0.935

DL916

SAR0204

DJL868_36

SAR0252_2

DED046_2

ESP9340

DL914

SAR0351

DED051

ESP9351

SAR0264

DJL869_3

SAR0379

DJL878

DED049

ESP9258

ESP9357

ESP9147_2

ESP9245_2

ESP9092

DL902

SAR0262

ESP9621

ESP9202_2

SAR0259

TCWC91345

DJL870

LMC10.02_2

2010.7_3

SAR0383

SAR0261

DJL872

SAR0381

0.788

0.877 0.973

0.887

0.985

0.967

0.865

0.984

0.1

S. merriami

Phyrnosoma

S. occidentalis

Uta

S. jarrovii

Urosaurus

MVZ149956

S. graciosus

CAS223822

SGR3MVZ241596

JWA470CAS229140

SGR4

MVZ237413JWA338

0.976

0.7353

0.8084

0.9881

S. arenicolus (see below)

Region A

Region B

Region C

Region D

Regions

EA

, EB

, EC

Figure 3

AA

AB

BA

BB

C

A

B

C

Skinny

D

E

A

B

C

SkinnyD

Skinny

D

E EA

EB

EC

AA

AB

BA

BB

C

Skinny

DA

DB

K=2 K=3

K=2 K=3

K=4

K=2 K=3 K=4

K=2

K=2

K=2

K=2

K=2

Figure 4

N. Mescalero MonahansS. Mescalero

Texp136.7 Kya

(15.7-90.4 Kya)

Texp29.91 Kya

(1.51-32.4 Kya)

Tanc34.8 Kya

(17.7-108 Kya)

Tdiv16.3 Kya

(7.9-41.8 Kya)

Ne anc155k

(103-195k)

Ne N1129k

(28.5-195k)

Ne N2263k

(198-297k)

Ne S168.2k

(15.9-150k)

Ne S2166k

108-198k

Figure 5