Biogeography, phylogeny, and morphological evolution of central Texas cave and spring salamanders


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Biogeography, phylogeny, and morphological evolution of central Texas cave and spring salamanders

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Biogeography, phylogeny, and morphological evolution of central Texas cave and spring salamanders
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BMC Evolutionary Biology
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Bendik, Nathan F.
Meik, Jesse M.
Gluesenkamp, Andrew G.
Roelke, Corey E.
Chippindale, Paul T.
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Springer Nature
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Eurycea ( local )
Blepsimolge ( local )
Salamanders ( local )
Troglobites ( local )
Cave Adaptation ( local )
Morphological Evolution ( local )
Troglomorphism ( local )
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serial ( sobekcm )

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BMC Evolutionary Biology, Vol. 13, no. 201 (2013-09-17).

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Biogeography,phylogeny,andmorphological evolutionofcentralTexascaveandspring salamanders Bendik etal. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 http://www.biomedcentral.com/1471-2148/13/201

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RESEARCHARTICLEOpenAccess Biogeography,phylogeny,andmorphological evolutionofcentralTexascaveandspring salamanders NathanFBendik 1,2* ,JesseMMeik 3 ,AndrewGGluesenkamp 4 ,CoreyERoelke 1 andPaulTChippindale 1 Abstract Background: Subterraneanfaunalradiationscanresultincomplexpatternsofmorphologicaldivergenceinvolving bothconvergentorparallelphenotypicevolutionandcrypticspeciesdiversity.Salamandersofthegenus Eurycea in centralTexasprovideaparticularlychallengingexamplewithrespecttophylogenyreconstruction,biogeography andtaxonomy.Thesepredominantlyaquaticspeciesinhabitkarstlimestoneaquifersandspringoutflows,and exhibitawiderangeofmorphologicalandgeneticvariation.Weextensivelysampledspringandcavepopulations ofsix Eurycea specieswithinthisgroup(eastern Blepsimolge clade),toreconstructtheirphylogeneticand biogeographichistoryusingmtDNAandexaminepatternsandoriginsofcave-andsurface-associated morphologicalvariation. Results: Geneticdivergenceisgenerallylow,andmanypopulationsshareancestralhaplotypesand/orshow evidenceofintrogression.Thispatternlikelyindicatesarecentradiationcoupledwithacomplexhistoryof intermittentconnectionswithintheaquatickarstsystem.Cavepopulationsthatexhibitthemostextreme troglobiticmorphologiesshownoorverylowdivergencefromsurfacepopulationsandaregeographically interspersedamongthem,suggestingmultipleinstancesofrapid,parallelphenotypicevolution.Morphological variationisdiffuseamongcavepopulations;thisisincontrasttosurfacepopulations,whichformatightclusterin morphospace.Unexpectedly,ouranalysesrevealtwodistinctandpreviouslyunrecognizedmorphologicalgroups encompassingmultiplespeciesthatarenotcorrelatedwithspringorcavehabitat,phylogenyorgeography,and maybeduetodevelopmentalplasticity. Conclusions: Theevolutionaryhistoryofthisgroupofspring-andcave-dwellingsalamandersreflectspatternsof intermittentisolationandgeneflowinfluencedbycomplexhydrogeologicdynamicsthatarecharacteristicofkarst regions.Shallowgeneticdivergencesamongseveralspecies,evidenceofgeneticexchange,andnested relationshipsacrossmorphologicallydisparatecaveandspringformssuggeststhatcaveinvasionwasrecentand manytroglobiticmorphologiesaroseindependently.Thesepatternsareconsistentwithanadaptive-shift hypothesisofdivergence,whichhasbeenproposedtoexplaindiversificationinotherkarstfauna.Whilecaveand surfaceformsoftendonotappeartobegeneticallyisolated,morphologicaldiversitywithinandamong populationsmaybemaintainedbydevelopmentalplasticity,selection,oracombinationthereof. Keywords: Eurycea , Blepsimolge ,Salamanders,Troglobites,Caveadaptation,Morphologicalevolution, Troglomorphism *Correspondence: nathan.bendik@austintexas.gov 1 DepartmentofBiology,UniversityofTexasatArlington,Arlington,Texas 76019,USA 2 CityofAustin,WatershedProtectionDepartment,Austin,Texas78704,USA Fulllistofauthorinformationisavailableattheendofthearticle ©2013Bendiketal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 http://www.biomedcentral.com/1471-2148/13/201

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BackgroundRadiationsofkarstlimestonefaunaarecharacterizedby multipleinvasionsintocavesystemsthatmayresultin convergentorparallelphenotypicevolution[1-4],geneticallyadmixedpopulations[5],andalternatingperiods ofisolationandgeneflowbetweensurfaceandcave populations[6].Thesecomplexhistoriesposeproblems forphylogeneticandphylogeographicreconstruction,assessmentofbiodiversity[7],andinferenceofevolutionarytransitions[8].Becauseofthediscontinuousand sensitivehabitatsthatdefinekarstsystems,manyare “ hotspots ” ofthreatenedandendangeredspecies.The EdwardsPlateauofcentralTexasexemplifiesthispatternandharborsdiverse,endemicinvertebrateandvertebratespecies[9].ThePlateauisanupliftedCretaceous limestonethathaserodedtoformacomplex,highly subdividedaquifersystemwithnumerousspringsand water-filledcaves.Thesekarsthabitatshavebeenwidely colonizedbyagroupoflungless,primarilypaedomorphic(retainingaquaticlarvalformwhileattaining reproductivematurity)spelerpineplethodontidsalamandersofthegenus Eurycea [10-14],ofwhichthirteen speciesarerecognized.Giventhatmanypopulations of Eurycea inTexasarethreatenedbyeffectsof urbanization,suchasdecliningwaterqualityanddecreasedwaterlevelsfrompumpingoftheEdwardsand Trinityaquifers[15,16],adetailedunderstandingofgeneticstructureanddiversityinthegroupisessential(particularlywithregardtoidentificationofspeciesandtheir distributions).Inaddition,thesesalamandersexhibitextensivemorphologicalvariationassociatedwithboth cave(subterranean)andsurfacehabitats,makingthem wellsuitedforinvestigationofparallelevolutionofmorphologicaltraitsinsimilarenvironments(e.g.,[17]). ThecentralTexas Eurycea haveacomplicatedtaxonomic history[10,11,14,18-21],inpartbecauseconvergenceor parallelismincavepopulatio nshasconfoundedstudiesthat reliedsolelyonmorphologyormorphometrics(e.g.,[22], butsee[17]).Conversely,morph ologicalconservatism(primarilyamongsurface-dwellers )hasalsoledtounderestimationofspeciesdiversity[10,20].Wheremorphological datahavefailed,molecularphylogeneticstudieshaveclarifiedhigher-level,andinsomecasesspecies-levelrelationshipswithinthegroup[10,21].Althoughmembersofthis groupbelongtothegenus Eurycea underatraditional Linnaeanclassificationscheme,Hillisetal.[21]recognized additionalwell-supportedcladesunderanunrankedsystem (PhyloCode[23]).Thedeepestsplit(atleast15MaBP; [24])correspondstoacladeoccurringnorthofthe ColoradoRiver( Septentriomolge )andacladesouthof theColoradoRiver( Notiomolge ,consistingofclades Blepsimolge and Typhlomolge ).Thedistributionof Blepsimolge includescavesandspringsfromthevicinityof AustinandSanMarcosintheeastandextendingwestto ValVerdeCounty. Typhlomolge comprisesexclusivelysubterraneanspeciessistertoandessentiallysympatricwith Blepsimolge alongthesoutheasternperipheryofthe EdwardsPlateau[10,21]. Blepsimolge canfurtherbedivided intoeasternandwesterngroups,whichappeartobegeographicallydiscont inuousandarewelldifferentiatedgenetically[10,20].Thewesterngroupisoftentermedthe E.troglodytes complex[10,20].Herewefocusontheclade Blepsimolge fromtheeasternregion,whichcomprises populationsassignedtosixnominalspecies( E.latitans , E.pterophila , E.nana , E.neotenes , E.sosorum and E. tridentifera ).Hereafter,werefertothesepopulationsasthe “ eastern Blepsimolge ” .Relationshipsamongmanypopulationsremainuncertainandthevalidityofsomespeciesin thisgroupisquestionable[10,20].PopulationsofcentralTexas Eurycea exhibithabitatassociatedmorphologicalvar iation.Surfacepopulations, foundinsprings,springoutflowsandlow-orderstreams, aretypicallypigmented,withmuscularlimbs,elongated trunks,andwell-developedeyes.Subterraneanpopulations exhibitacontinuumofvariationrangingfromsurface-like tohighlytroglomorphic[22],withthemostextremeexamplesbeing E.tridentifera (eastern Blepsimolge )and,atthe furthestendofthe “ troglomorphicspectrum ” , E.rathbuni , E.robusta ,and E.waterlooensis ( Typhlomolge ).These “ extreme ” specieshavevestigialeyes,longspindlylimbs, shortenedtrunks,broadflattenedsnouts,andhighlyreducedskinpigmentation.Althoughmorphologicalconvergencebetween E.tridentifera and Typhlomolge hasbeen established[17],theextento fmorphologicaldivergence (andparallelism)amongothersurfaceandsubterranean populationsoftheeastern Blepsimolge hasnotbeenformallyevaluated.Patternsofmorphologicalvariationwithin andamongthesepopulationsarecomplex,astheremay havebeenmultipleindependentinvasionsintocavesystems,andbecauseofthestructuralandhydrogeological complexityofcaveandsurfacehabitats.Hereweprovidea phylogeneticanalysisoftheeastern Blepsimolge basedon mtDNAsequencedatafromext ensivesamplingofsurface andsubterraneansites,andwerelateourphylogenetichypothesistonewdataonmorphologicalvariationwithinthe group.Specifically,wecharacterizebroadpatternsofmorphometricvariationusingmultivariateanalysis,evaluate theextentandphylogeneticdistributionofcave-associated morphologies,anddiscussevolutionary,developmental, andtaxonomicimplications.MethodsTaxonsamplingformolecularanalysesSalamanderswerecollectedfromspringsandcaves acrossasevencountyareaofthesoutheasternEdwards Plateau.Ourdatasetincludesmorethantriplethenumberofsitessampledinpreviousstudies[10,21]including extensive,fine-scaleexaminationofcritical,andformerlyBendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page3of18 http://www.biomedcentral.com/1471-2148/13/201

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unsampledand/orpreviouslyinaccessibleregions.Data wereobtainedfor112specimenscollectedfrom45 springsand26cavesinthesoutheasternPlateauregion. Theseincluderepresentativesofthe E.latitans complex (sensu[10,20]) ,E.pterophila,E.nana,E.neotenes, E.tridentifera and E.sosorum (eastern Blepsimolge ),plus multiplenewlysampledpopulationsnotpreviously assignedtospeciesandoutgroupsamplesfrom E.rathbuni ( Typhlomolge )andthe E.troglodytes complex(western Blepsimolge ).WeadheredtoanimalwelfareprotocolsoutlinedbytheUniversityofTexas Arlington(IACUC#A.07.021).Specimendetailsare availableinAdditionalfile1.LaboratorymethodsDNAwasextractedfrommuscleorlivertissueusing severalmethods.Forallofthetissuesamplesobtained between2003and2007,DNAwasextractedusingthe DNeasykitfromQiagen.DNAforspecimenscollected priorto2003wasextractedprimarilyusingtheSTE methoddescribedbyHillisetal.[25]andamodification oftheChelexextractionmethod[26],describedby Chippindaleetal.[10].WefocusedontwomitochondrialDNA(mtNDA)sequenceregionsforourphylogeneticanalysis:a1110bpfragmentofcytochrome b (Cytb),anda619bpfragmentofNADHdehydrogenase subunit2plusadjacenttRNATRPandpartialtRNAALA(ND2).MostPCRproductswereamplifiedusinga standard Taq polymerase(NewEnglandBiolabsor Promega)orHotStart Ex-Taq (Takara-Mirus)onMJResearchPTC200gradientandPTC100thermalcyclers. AmplificationforPCRandsequencingwasperformed usingtheprimerslistedinTable1.PCRconditionsthat yieldedthemostconsistentresultswereasfollows: Reactionsconsistedof1 – 2 lofdiluteDNA(typically 10-50ngofDNA,butsometimesashighas300ng), 0.5 – 1.0 Mofeachprimer,0.75mMdNTPsmix,polymerasebuffer(1.5mMMgCL2),and1 – 2U Taq (or0.5 U ExTaq )polymeraseinatotalvolumeof20 l.Occasionally,2.5%DMSO(finalconcentration)wasusedin difficultPCRreactions.Thermalcyclingconditionsvariedgreatlydependingonthetemplateanddifficultyof amplification.Typicalconditionswereasfollows:Step1: 96°3min;Step2:Annealingtemp.50°30s;Step3:72° 1min/kb;Step4:96°20s;Step5:repeatsteps2 – 4 (×30);Step6:72°10min;Step7:4°hold.Variationsof thisprofileincludedastep-upannealingtemperature, wherebythefirst2or3replicationsincludeda3 – 5° lowerannealingtemperature,andthenwereraisedto thestandardannealingtemperaturefortheremaining cycles.SomePCRreactions(especiallyfordifficultsamples)wereperformedusingPhusionorPhusionIIDNA polymerase(NewEnglandBiolabs),generallyfollowing manufacturer ’ sinstructionsbutin5 – 10 LtotalvolumesandincludingBSAandDMSO( “ 1X ” and3.0mM finalconcentrations,respectively).Often,thesereactions wereperformed “ semi-nested ” withathirdprimeradded at0.1Xtheconcentrationofeachoftheothertwo (PGLU-TATforCytb,andcytochromeoxidaseIprimer MVZ202forND2;Table1).Typicalreactionprofilesinvolvedinitialdenaturationfor2minat98°,subsequent denaturationsat98°for10s,annealingtimesof5 – 10s, andextensiontimesof10 – 20sat72°withafinal 10minextensionstep.Generally “ touchdown ” methods wereusedinwhichannealingtemperaturesweresuccessivelydroppedinincrementsof2-3°,fromabout58 – 50° Table1ListofprimersusedtoamplifyandsequencegenefragmentsinthisstudyPrimernamePrimersequence(5 ’ -3 ’ )Amplifiedregion PGLUGAARAAYCANTRTTGTATTCAACCytb PGLU-TAT**GAARAAYCANTRTTGTATTCAACTATCytb MVZ-15[ 27 ]GAACTAATGGCCCACACWWTACGNAACytb HEM-CB1-5 ’ CCATCCAACATCTCAGCATGATGAAACytb CYTBTN5Fv2CATATTTAGGRGAAACACTTGTTCACytb CYTBTYPHmRGTCKGGGYTAGAATTAATTCCTGCytb EurTXCRThrGYCAATGTTTTTCTAAACTACAACAGCATCCytb METfL4437AAGCTTTTGGGCCCATACCND2 COIrH5934TGCCAATATCTTTGTGATTTGTTND2 ND2fL5002*AATCAACCACAAATCCGAAAAATND2 ASNrH5692*TTAGGTATTTAGCTGTTAAND2 MVZ202**[ 28 ]GCGTCWGGGTARTCTGAATATCGTCGND2ND2includesadjacentAlaandTrptRNAgenes. * indicatesprimerwasonlyusedforsequencing,andnotPCR. **indicatesexternalprimerfornestedPCR.Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page4of18 http://www.biomedcentral.com/1471-2148/13/201

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forCytband62 – 55°forND2,withincreasingnumbers ofcyclesateachlowertemperatureforatotalof35 – 40cycles. PCRproductswerepurifiedusingQiagengelextractionorPCRpurificationkitsfollowingthemanufacturer ’ sprotocols,orifnonspecificproductswereabsent, acombinationofexonucleaseIandshrimpalkaline phosphataseenzymes(USB)wereusedtodigestsinglestrandedDNAandphosphorylatedNTPs.Bothstrands ofeachampliconweresequencedforcompleteor nearly-completeoverlapformosttemplatesusingABI BigDyev3.1chemistry.Unincorporatedproductswere removedviaethanolprecipitationusing0.75Msodium acetateand125mMEDTA.AppliedBiosystems377and 3130xlmachineswereusedforsequencing.AlignmentandphylogeneticanalysisRawsequencechromatogramswereeditedwith Sequencherv4.2,v4.3andv4.5(GeneCodesCorp.,Ann Arbor,MI,USA).Multiplealignmentswereconducted withMEGAv5[29]usingMUSCLE[30].Forphylogeneticanalyses, Eurycearathbuni waschosenasthe outgroupbecauseitiswellsupportedassisterto Blepsimolge inpreviousmolecularstudies[10,17,21]. BayesianphylogeneticanalysisofthecombinedCytb andND2genesegmentswasrunusingMrBayesv3.1.2 [31]ontheCIPRESScienceGateway[32].Nucleotide modelsofevolutionweredeterminedusingjModeltest v0.1.1[33,34].InMrBayes,theparametersstatefreq, revmat,shapeandpinvarwereallowedtovarybygene segment.DefaultpriorssetbyMrBayeswereusedexcept forthebranch-lengthpriors;branch-lengthpriorswere setasexponentialwithmeansof1,0.1,0.01and0.001 infourseparaterunstotestpriorsensitivity,sincea largebranch-lengthpriorcanresultinunrealistically longtrees[35].Eachanalysiswasruntwicewith4 chains(onecold,threeheated),5millionMCMCgenerationsandasamplefrequencyof100.Burn-inwasdeterminedbyexaminingthelogfilesgeneratedby MrBayesusingprogramTracerv1.5.0[36];parameter traceswerevisuallyassessedforstationarity.Thepostburn-intreesfrombothrunswerecombinedtocalculate amajority-ruleconsensuswithacutoffof50%.MorphologicaldataandanalysesWemeasuredaseriesoftenstandardizeddistances basedonexternalmorphologyfrom255ethanolpreservedspecimenscataloguedatthefollowingcollections:TexasNaturalHistoryCollection,TheUniversity ofTexasatAustin;MuseumofVertebrateZoology,UniversityofCalifornia,Berkeley;andtheAmphibianand ReptileDiversityResearchCenterattheUniversityof TexasatArlington[Additionalfile2].Oursamplewas primarilyorganizedbycollectinglocalityandincluded representativesofeachspeciesofeastern Blepsimolge ,as wellascomparativematerialfromtheexclusivelysubterraneanspeciesof Typhlomolge ( E.rathbuni and E. waterlooensis ).Measurements 20mmweretakenwith adigitalcaliperandroundedtothenearest0.1mm; measurements<20mmweremadewithanocularmicrometermountedonadissectingmicroscopeand roundedtothenearest0.01mm.MorphometricvariableswereselectedfromChippindaleetal.[10]andincludedthefollowingmeasurements:AG(axilla-groin length);ALL(anteriorlimblength,frominsertiontotip ofthirdtoe);ED(eyediameter);HLA(headlengthA, distancefromtipofsnouttogularfold);HLB(head lengthB,distancefromposteriormarginofeyeto posterior-mostgillinsertion);HLC(headlengthC,distancefromtipofsnouttoposterior-mostgillinsertion); HLL(hindlimblength,frominsertiontotipofthird toe);HW(headwidthatrictusofmouth);IOD (interoculardistance);SL(standardlength,distancefrom tipofsnouttoposteriormarginofvent).Eyediameter wasdeterminedusingmaximumoculardiscdiameter, i.e.,portionofdarkpigmentincludingandsurrounding thefocusingportionoftheeye,under64Xmagnification withbacklighting.ResultswerehighlyconsistentandrepeatableamongspecimensexaminedbyPC,CR,and AG.Weusedprincipalcomponentsanalysis(PCA)to characterizebroadpatternsofmorphologicalvariation, andtoexplorecorrelationstructureamongvariables. PCAwascarriedoutwithSystat12.02(SystatSoftware, Inc.,Chicago,IL,USA),usingthecorrelationmatrixderivedfromlog10-transformedvariables.Althoughgreater separationofgroupsinordinationspacecouldbe achievedbyreducingtheinfluenceofsizethroughanalysisofmeasurementresiduals,weelectedagainstthis procedureforthefollowingreasons:(1)thejointinfluenceofsizeandshapeonphenotypeisbiologically relevant(e.g.,[37]);(2)inourdataset,individualmeasurementsscaledtoSLwithdifferentslopesandfunctions,makingresidualsproblematictocomparewith eachother[38];and(3)measurementsspannedalmost threeordersofmagnitude;thus,acommonscalingfactorwouldlikelymodelmorenoisethansignalfor shortermeasurements.Furthermore,varianceattributabletosizeandshapecannotbeseparatedwithonlylinearmeasurements[39].Someoftheseissuesalso influencerawortransformedmeasurementdatathatis inputintoPCA,butmaybecompoundedbyfurther processing.ResultsPhylogeneticsThelengthofthecombinedCytbandND2alignment was1729bp.Thirty-foursequenceswereslightlyshorter thantheothers;unsequencedregionsweretreatedasBendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page5of18 http://www.biomedcentral.com/1471-2148/13/201

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missingdata.Themaximumnumberofmissingbases (includingbothCytbandND2)was91foronespecimen;theaveragevaluewas7.Includingoutgrouptaxa, 380siteswerevariableand226ofthesewere parsimony-informative.Of107eastern Blepsimolge specimens,47haplotypeswererecoveredfrom71 differentspringsandcaves,forwhich155siteswere variableand72wereparsimony-informative.Genbank accessionnumbersareKC355860 – KC355971and KC355972 – KC356083forCytbandND2,respectively [seeAdditionalfile1].Themodelwiththehighest AICcscoreaccordingtojModeltestwasGTR+Gfor bothCytbandND2alignments.Branchlengthpriors ofexponentialmean1,0.1and0.01resultedinunrealisticallylongtreelengths;resultsfromthesmallest(exponentialmeanof0.001)branch-lengthprioranalysis arepresented(meantreelength=0.28,LCL=0.26, UCL=0.30),althoughtherewerenosignificantdifferencesintreetopologies.Samplesfromthefirst 500,000iterationsweredi scardedasburn-in. Phylogeneticresultsagreewiththoseofprevious studiesregardingthedeepestsplitswithin Eurycea from thesouthernEdwardsPlateau(exclusiveofclade Typhlomolge ,theoutgroup),andthedeepnodesare stronglysupported.Themostbasalsplitisbetween the E.troglodytes complex(western Blepsimolge )andthe eastern Blepsimolge [10,21]).Alsoconsistentisthe distinctivenessofapopulationthatmayrepresentan undescribedspecies,fromPedernalesSprings[10]. Eurycea sp.Pedernalesissistertotheremainingeastern Blepsimolge ,followedby E.nana fromthetypelocality, SanMarcosSprings(andindividualsthatappeartorepresent E.sosorum butpossess E.nana -likehaplotypes; collectivelycladeS/N)(Figure1). Euryceasosorum (primaryhaplotypeatBartonSprings,thetypelocality)is sistertotheremainingclade,whichincludespopulations representing E.latitans , E.neotenes , E.pterophila and E. tridentifera plusothersthathavenotbeenassignedto species(Figure1).Werefertothiscladeasthe Eurycea neotenes complex(afterthefirst-describedmemberof thegroup[40];seeDiscussion),whichisdistributed throughouttheCibolo,GuadalupeandBlancoriverwatersheds(TrinityAquifer),butalsoincludesseveralpopulationsthatoccuralongthesoutheasternedgeofthe EdwardsPlateau(EdwardsAquifer;Figure2). Althoughtherearesomehighlysupportedclades withinthe E.neotenes complex,primarilycomprising geographicallyproximatepopulations,thesedonot strictlycorrespondtocurrentlyrecognizedspecies boundaries(Figures1and2;[10]).Divergencesamong populationswithinthisregionarelow(averageuncorrectedp-distance=0.4%)andthereisextensiveinterandsometimesintrapopulationmorphologicalvariation (e.g.,Figure3).TheweaklysupportedcladeLTincludes representatives(includingtopotypes)ofboth E.latitans and E.tridentifera (Figure1).CladesBP1,BP2(Blanco Riverdrainagepopulationsof E.pterophila )andBGP (Blanco&GuadalupeRiver E.pterophila )eachcontain populationsassignedto E.pterophila [10],butforma polytomyinthe50%majority-ruletreewithcladeLT andseveralotherpopulations(Figure1).GroupBGC containsfourpopulationsdistributedacrosstheBlanco, GuadalupeandCibolodrainagesthatformapolytomy withBP1,BP2,BGPandLT.CollectivelyBP1,BP2,BGP, LTandBGCformahighlysupportedcladethatincludes mostpopulationspreviouslyassignedtoE.latitans , E. tridentifera and E.pterophila .CladeNincludespopulationsassignedto E.neotenes aswellasthepopulation fromComalSprings(Figure1),whichhasbeensuggestedtobeadistinctspecies[10].CladeFTincludes populationspreviouslyassignedto E.latitans [10]that occurinorneartheFortTerrettlimestoneformation. CladesN,FTandtherecentlydiscoveredpopulationat WhiteSpringsformapolytomy,whilethepreviously unsampledBullisSprings9 – 29and9 – 83areweakly supportedassistertotherestofthe E.neotenes complex.Thelatterthreepopulationsexhibitrelativelyhigh geneticdivergencescomparedtotherestofthe Eurycea neotenes complex[averageuncorrectedp-distances1.2% (WhiteSprings)and0.7%(BullisSprings9 – 29&9 – 83)]. Patternssuggestiveofmitochondrialintrogressionare alsoevidentforseveralpopulations(Figure1).HaplotypesfromJacob ’ sWell(BlancoRiverdrainage)occurin cladesBP1andBGP.Additionally,thereispotential introgressionand/orgeneflowbetweenadjacentpopulationsofcladesLTandN,ashaplotypesfrombothclades arefoundintheStealthCaveandBucketo ’ ToadsCave populations.Oursampleof E.sosorum (putativelyendemictoBartonSprings)alsocontainstwodistinct mitochondrialhaplotypes,oneuniquetoBartonSprings athighfrequency(approximately70%;unpublisheddata, [41])andonethatgroupswiththatof E.nana (SanMarcosSprings)andotherBartonSpringssegmentpopulations(BlowingSink,ColdSpringandTaylor/Upper TaylorSprings;Figures1and2).MorphologyThefirstthreeprincipalcomponents(PCs)accountfor 96.9%ofthetotalvarianceinthemorphologicaldataset. ThefirstPC(78.7%oftotalvariance)hashighpositive factorloadingsforallvariables,althoughsomewhat lowerforED,andreflectstheoverallpositivecorrelationsbetweenindividualmeasurementsandbodysize (Table2).ThesecondPC(9.5%oftotalvariance)is structuredprimarilybythebroadanddiffusevariation exhibitedbycavepopulations(Figure4a).Alongthis axis,surfacepopulationsformacohesiveclusterwith primarilynegativefactorscores.ThemostextremeBendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page6of18 http://www.biomedcentral.com/1471-2148/13/201

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troglomorphs(i.e., Typhlomolge )grouptogetherwith highfactorscoresonbothPC1andPC2.Subterranean populationsoftheeastern Blepsimolge ,particularlythose assignedto E.tridentifera ,overlappartiallywith Typhlomolge ,butonaveragehaveslightlylowerfactor scoresalongPC1andPC2.Thus,thecombinationofthe firstandsecondPCs,andparticularlyPC2,corresponds toagradientfromsurfacetocavemorphologies,with surfacespecimensoverlappingbroadlyinordination spacewithcavespecimensbutnotviceversa.PC2was Figure1 Fiftypercentmajority-ruleconsensusphylogramofeastern Blepsimolge basedonBayesiananalysis. Posteriorprobabilitiesof nodesupportgreaterthanorequalto95%areindicatedbyasterisks.Speciesdesignations(indicatedbycoloredblocks)followthosegivenby [10],andweincludeallofthesamepopulationsfromtheirmolecularanalysisplusnumerousnewsamples.Hollowsquaresindicate topotypicalspecimens. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page7of18 http://www.biomedcentral.com/1471-2148/13/201

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structuredprimarilybytheinverserelationshipbetween twosetsofvariables:AGandEDhadhighnegativefactorloadingswhilebothHLLandIODhadhighpositive factorloadings.Insummary,cavepopulationswere characterizedbysmalleyediametersandshortaxillagroinlengths,andbylonghindlimbsandinterocular distances. Morphologicalvariationwithrespecttocavepopulationsiscomplex(Figure4).Somecavepopulations(e.g., HoneyCreekCave,Pfeiffer ’ sCave)separateintoatleast twodiscretegroupsinordinationspace.ForHoney CreekCave(thetypelocalityof E.tridentifera ),one groupisextremelytroglomorphicwhiletheotherseems tobeintermediatebetweentroglomorphicandsurface forms.ForPfeiffer ’ sCave(nearthetypelocalityof E. latitans ),bothgroupsarewellseparatedfromeach other,butalsofromthemainclusterofsurfaceforms. Onlyfivecavepopulations(PreserveCave,HoneyCreek Cave,BadweatherPit,CampBullisCave#1,andCamp BullisCave#3)overlappedpartiallyinordinationspace withthe Typhlomolge specimens;thus,cavepopulations ofeastern Blepsimolge weredifferentfrom Typhlomolge Figure2 Geographicdistributionofeastern Blepsimolge mtDNAcladesinrelationtospeciesboundaries,habitatandmajor physiographicfeatures. Squaresandcirclesrepresentspringandcavelocalities,respectively.Cladeassociationsarelabeledbycolorforeach sampledpopulation;populationswithhaplotypesfromtwodistinctcladesarebicolored.Approximatedistributionsfor Euryceaneotenes complex species(coloredlines)aredrawnaccordingtodesignationsby[10]althoughthesedesignationsarenotentirelyconsistentwithourphylogenetic hypotheses.Similarly,physiographicboundariesalsoappeartobepoorpredictorsofmtDNAcladedistributions.B9=Bullissprings9 – 83and 9 – 29;CS=ComalSprings;PS=PedernalesSprings. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page8of18 http://www.biomedcentral.com/1471-2148/13/201

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inatleastsomeaspectsofmorphology.Someofthese populationsexhibitdistinctivetroglomorphicvariation, includingEbertCave,Pfeiffer Â’ sCave,andGrosser Â’ sSinkhole.Yetothercavepopulationsformrelativelyhomogeneousclustersthatwereintermediateinordination spacebetweensurfaceandextremelytroglomorphic populations(e.g.,StealthCave,SharonSpring,and Sattler Â’ sDeepPit).Finally,specimensfromT-Caveform acohesiveclusterindistinguishablefromthemaingroup ofsurfacespecimens. TheordinationofPC1andPC3(8.7%oftotalvariance) revealsanunexpectedandnovelpatternofmorphological variationincentralTexas Eurycea :theeastern Blepsimolge formtwodiscretegroups,withspecimens of Typhlomolge mostlyperipheraltoorseparatefrom thesegroups(Figure4b).Thesedistinctgroupswithin eastern Blepsimolge donotcorrespondtogeography, recognizedspecieslimits,phylogeographicstructure,or habitat(unlessatascalefinerthanthecave/surface dichotomyusedinthisstudy).Atthelevelofindividual localities,caveandsurfacesitesexhibitedparallelpatterns;somesurfaceandcavepopulationsincludespecimensfromonlyonemorphologicalgroup,whileother populationsarecomposedofspecimensfrombothgroups (Figure5).Ascatterplotoffactorscoresfromeachgroup indicatesanegativerelation shipbetweenPC3andPC1for eachgroup(Figure4b),whichsuggeststhatallometricdifferencesinfluencegroupordination.Althoughthesegroups Figure3 Diversityofheadmorphologyandpigmentationwithintheeastern Blepsimolge . Parallelpatternsofmorphologicalevolutionare evidentinthetroglomorphicspecimensfromcladesLT,NandBGP,althoughalllabeledcladescontainsurfaceforms(i.e.,havingfully-developed eyesanddarkpigmentation).Localitiesforindividualspicturedareasfollows: 1 HoneyCreekCave, 2 CascadeCaverns, 3 CampBullisCave#3, 4 CascadeCaverns, 5 BullisBatCave, 6 GoldenFawnCave, 7 PreserveCave, 8 CMCave, 9 PreserveCave, 10 HoffmanRanchEstavelle, 11 Fern BankSpring, 12 Jacob Â’ sWell, 13 HectorHole, 14 LewisValleyCave, 15 SharonSpring, 16 MoralesSpring, 17 TaylorSprings. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page9of18 http://www.biomedcentral.com/1471-2148/13/201

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arenotstructuredonthebasisofhabitatdesignation,the variablesthatmoststronglyinfluencePC3includemeasurementsthatwereimportantinthegradientofsurface-to -caveformsidentifiedfromPC2.Thehighestpositivefactor loadingisforEDandtheonlynegativeloadingsareforAG andSL(Table2).Incontrast,bothAGandEDweight negativelyonPC2.Thus,PC3representsprimarilyresidual, uncorrelatedvariationinEDa ndAG,variablesthatotherwiseshowastrongpositivecorrelationalongthegradient ofsurface-to-cavemorphologies.DiscussionPhylogeographyThemtDNA-basedphylogenyshowsacomplexpattern inwhichthreespecies, E. sp.Pedernales, E.nana and E. sosorum ,aresuccessivelysistertoaclade(the Eurycea neotenes complex)thatincludes E.latitans , E.neotenes , E.pterophila , E.tridentifera ,plusotherpreviouslyunassignedpopulations.Shortbranchlengthsandlackof reciprocallymonophyleticrelationshipstodistinguish specieswithinthe Euryceaneotenes complex(Figure1) indicaterecentdivergencescoupledwithincomplete lineagesortingandrecentorongoinggeneflow(thus, potentialconspecificity).Relationshipsamongpopulationsofthe Euryceaneotenes complexfollowapattern thatappearstobedeterminedmorebygeographicproximitythanbyhabitat(springvs.cave)ormorphology (Figure2),andthispatternisnotentirelyconsistentwith currentlyrecognizedspeciesboundaries(Figure1). Thiscomplexphylogeographicpatternlikelyreflects thedynamicnatureofkarstaquifersystemsinhabitedby centralTexas Eurycea .Thedissolutionoflimestone strataalterswaterflowroutesovertime,generatingnew connectionsbetweenformerlydisjunctpopulationsand severingothersintheprocess.Onashortertimescale, varyingclimaticconditions(e.g.,floodsordroughts)can influencehydrogeologicpathways[42]andtransiently facilitateorhindergeneflowacrossthekarsticlandscape.Althoughgeographicproximityisgenerallya goodpredictorofrelatednesswithintheeastern Blepsimolge ,therearemanyexceptionstothispattern thatreflectthecomplexityofgeneflowamongsalamanderswithinthesekarstaquifers.Somepopulationsof Eurycea appeartobelocallyisolatedwhileothershave maintainedgeneticandhydrogeologicalconnectivity withotherpopulations.Forexample,WhiteSpringsisa smalloutflowintheBlancoRiverdrainage,andbased onitslocation,wouldbeexpectedtohaveclosegenetic affinitytootherBlancoandGuadalupeRiverpopulations (e.g.,cladesBP1,BP2,BGP).However,salamandersfrom WhiteSpringsaresubstantiallydivergentinmtDNAsequenceandthispopulationformsapolytomywithFTand N,whichcollectivelyaresistertotheremainingmembers ofthe E.neotenes complex(Figure1).Inaddition,the WhiteSpringspopulationisdi stinguishedbyseveralunique nuclearsequencealleles(PC,unpublisheddata).Intwo otherinstanceswefoundthatpopulationsfromgeographicallyclosespringsappeareddi stantlyrelated.Bullissprings 9 – 83and9 – 29areadjacenttomanyNpopulationsbutare weaklysupportedassistertotherestofthe E.neotenes complex;FTpopulationsareaveryshortgeographicdistancefromLTpopulationsaswellbutdonotfallwithin thatgroup(Figure2). Incontrasttotheaboveexamplesofmoredistantlyrelated,butgeographicallyproximatepopulations,other populationswithinthe E.neotenes complexthathave sharedorsimilarhaplotypesoccuracrossarelatively widegeographicrangedespitepotentialbarrierstogene flow.Becausepopulationsofeastern Blepsimolge arerestrictedtokarst-associatedwaters(wetcaves,springs, spring-fedstreams),riverscanactasbarrierstogene flow[10,43].However,theBGPcladeisdistributed acrosstworiverdrainages(BlancoandGuadalupe), suggestingrecentgeneflowandhydrogeologicalconnectivitybetweentheseregionsdespitemodernriverine barriers.Forexample,thePreserveCavepopulation(in whichindividualsexhibittroglomorphismsimilartothat of E.tridentifera ;Figure3)issouthoftheGuadalupe RiverbutsharesanidenticalhaplotypewithHorsejump Spring,whichisnorthoftheBlancoRiver.Whilegene flowbetweenthesepopulationsmaynotcurrentlybe ongoing,theirsharedmitochondrialhaplotypesuggests recenthydrogeologicalconnectionsand/ordispersal acrosscontemporarybarriers. Temporaryhydrogeological connectionsmayalsoresult inintrogressionbetweendistinctspecies. Euryceasosorum and E.nanainhabitspringsthataredischargepointsfor Table2Factorloadingsforvariables,eigenvalues,and percentoftotalvarianceexplainedforprincipal componentsMeasurementPC1PC2PC3 AG0.741 0.315 0.583 ALL0.9650.1500.065 ED0.342 0.8570.384 HLA0.978 0.0060.128 HLB0.9700.1010.149 HLC0.9790.0320.157 HLL0.9600.1740.100 HW0.9630.0670.093 IOD0.9450.1580.076 SL0.821 0.150 0.538 Eigenvalue 7.8700.9490.868 %VarianceExplained 78.79.58.7Principalcomponentswerederivedfromthecorrelationmatrixoflog10transformedmorphometricdata.Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page10of18 http://www.biomedcentral.com/1471-2148/13/201

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largeregionsoftheEdwardsAquifer(theBartonSprings andSanAntoniosegments)andpopulationsofboth speciesfromthetypelocaliti esaremorphologicallyand geneticallydistinctive[10,41, 44,45].However,thepresence of E.nana -likehaplotypes(cladeS/N)throughoutBarton Springssegmentpopulationsincluding E.sosorum (atafrequencyof100%ingeographicallyintermediatespringsand caves,andapproximately30%atBartonSpringsitself;NB andPC,unpublisheddata[41])indicatesrecentmtDNA introgressionbetweenthesespecies.Thisisunexpected, sincethetraditionalviewofEdwardsAquiferhydrogeology suggestedagroundwaterdivid ebetweentheBartonSprings segment,whichdrainsnorthtoBartonSprings,andaSan Antoniosegment,whichdrainstoSanMarcosandother largespringstothesouth(Figure2;[46,47]).Recentdye tracingstudiesarebeginning tochallengethisview[47],as groundwateralmostanywherewithinHaysCountymay floweithernorthtoBartonSprings(TravisCounty)or southtoSanMarcosSpringsdependingonenvironmental conditions(e.g.,drought[42]).Thishydrogeologicpattern mayexplainthepresenceof E.nana -likehaplotypeswithin theBartonSpringssegment.Thisandseveralotherexamplesofsharedhaplotypesbetweengeneticallydistinct groups(betweenNandLT;BGPandBP1)suggestthat manyofthesespeciesarenotreproductivelyisolated.Althoughwecannotruleoutsharedancestralpolymorphism andincompletelineagesorting,thefactthatmostofthese casesoccuramongpopulationsinrelativelyclosegeographicproximity(withthepotentialforhydrologicconnection,pastorpresent)issuggestiveofatleastsporadic geneflow. Ecologicalsegregationhasalsofacilitateddiversificationoftheeastern Blepsimolge throughrepeated colonizationofsubterraneanhabitats.However,genetic divergencebetweenspringandcavepopulationsgenerallyislowdespitethemorphologicaldiversityofthe Figure4 Scatterplotsoffactorscoresfromprincipalcomponentsanalysis(PCA)oflog 10 -transformedmeasurements.4a – c :Ordination ofspecimensoftheeastern Blepsimolge (circles)and Typhlomolge (squares;includes E.rathbuni and E.waterlooensis )cladesareshown.Closed andopensymbolsrepresentspecimenscollectedfromcaveandsurfacelocalities,respectively. 4d :Ordinationofmorphologicalvariationwithin andamongcavepopulationsofcentralTexas Eurycea (PC1vs.PC2).Populationswith N >3areshownwithcoloredconvexhullpolygons (individualspecimensremovedexceptforoutliers).Lightgraycirclesindicatesurfacespecimens;darkgraycirclesindicatecavespecimensfrom localitieswith N 3. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page11of18 http://www.biomedcentral.com/1471-2148/13/201

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group.Mostcavepopulationswithinthe E.neotenes complexaredistributedthroughouttheLowerGlen Rose(LGR)limestoneformationandthemostextreme troglomorphicforms(e.g., E.tridentifera )occurwithin thesouthernLGR(Figure2).Themorphologicaldistinctivenessof E.tridentifera promptedSweet[22]to suggestthatthesecavesareamongtheoldestinthe plateauregion,whichwouldhaveallowedampletime fortheevolutionofcave-associatedfeatures.Caverndevelopmentinthisregioncoincidedwiththeerosionof theUpperGlenRose(UGR)limestoneformation, exposingtheLGRtoextensivekarstification.This processhasbeendatedtoapproximately1.3Mato990 kaBP[48],puttingatheoreticalboundontheearliest cavecolonizationbysurfacepopulations.WhilecavedwellingtroglomorphsmayhavealreadyinhabitedUGR cavesandlatercolonizednewlyavailablehabitatwhen theLGRwaskarstified,thisscenarioislesslikelygiven thatthemuchlowerextentofkarstificationexhibitedby theUGRinthisregion[48]suggeststhat(1)theextensivesubterraneanhabitatcreatedwithintheLGRwas novel,and(2)theextremelylowmtDNAdivergences betweensubterraneanandsurfacepopulationswithin the E.neotenes complexareconcordantwithapattern ofrecentratherthanoldcolonization.Thepatternsof karstaquiferevolutionandthecomplexevolutionary historyofeastern Blepsimolge poseachallenging phylogeographicpuzzlethatlikelywillonlybesolved withadditionalsamplingandincorporationofrapidly evolvingnuclearmarkersaswellasabetterunderstandingofregionalhydrogeology. Morphology Theremarkablearrayofmorphologicaldiversityineastern Blepsimolge (Figure3)isrelatedtotheextentto whichpopulationsexploitsurfaceversuscavehabitats. Ingeneral,subterraneanformsshow,tovaryingdegrees, lossofpigmentation,shorteningofthetrunk,flattening andbroadeningoftheskull,lengtheningofthelimbs, andreduction(andsometimeslossoffunction)ofeyes. PCAindicatesthatsurfacesalamandersoccupyarelativelytightclusterinmorphospace,whilesubterranean salamandersshowmorediffusevariationalongboth PC1andPC2(Figure4).Overall,theordinationsuggests thattherearevarious “ cave-type ” morphologiesincontrasttoamorecohesive “ surface-type ” morphology.This observationisconsistentwiththeresultsofprevious studiesthathaveattemptedtoassessspeciesdiversity withincentralTexas Eurycea usingprimarilymorphologicaldata(forreview,see[10,20]). Thepatternofreducedvariationinmorphologyof surface-dwellingsalamandersrelativetocave-dwelling populationsmayresultfromstrongerstabilizingselection,whichtendstoreducephenotypicvariation.Anobviousdifferenceinselectionpressurebetweencaveand surfacehabitatsispredation;salamanderpredatorssuch asfishes,aquaticinsectsandcrayfishesaremostlyabsent fromcavesincentralTexas,butcanbeabundantonthe surface(personalobservations).Consistentwiththis idea,thediffusevariationexhibitedbycave-dwellingsalamandersmayresultfromtherelaxationofselectionfor traitsthatareimportantonthesurface,andinparticular thosetraitscriticalforevadingpredation.Ourmorphologicaldataindicateasymmetricmigrationbetweenhabitatsbecausesurfaceformsaremorefrequentlyfoundin Figure5 Congruentpatternsofmorphologicalvariationfor selectedsurface(a)andcave(b)populations. Localitiesare shownwherespecimensclusterineitheroftwogroupsorwhere specimensclusterinbothgroups.Lightgraycirclesandsquares indicate Blepsimolge and Typhlomolge ,respectively. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page12of18 http://www.biomedcentral.com/1471-2148/13/201

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cavesthanviceversa.Thisobservationmayreflectlow survivorshipoftroglomorphicsalamandersthatare flushedtothesurfaceandtemporarymigrationofsurfacepopulationsduetoperiodicdryingofthesurface habitat[49].However,thefrequentparallel(sensu[17], giventhecloserelationships)evolutionoftroglomorphic traits(seebelow)suggeststhatdirectionalselectionmay beoperatingoncave-associatedmorphologies.Forexample,eyedevelopmentandmaintenancemaybemetabolicallycostly,andthereforeselectedagainstin perpetuallydarkenvironments[4,50].Similarly,eyedegenerationmayarisethroughpleiotropicenhancement ofothersensoryorgans[51].Moreover,mechanosensory detectionmaybeenhancedbychangesinheadandbody shape,forexampletoreduceswimmingnoiseorsupport largernumbersofsuperficialneuromasts(e.g.,in amblyopsidfishes[52]).Extensivevariationinmorphologyacrossdifferentcavesystemsmayresultfromdifferencesinthefollowing:(1)selectionregimes,(2)time sinceinvasionintovariouscavesystems,(3)spatialextentsandconnectivityofcaves(whichmayinfluencethe dependenceofsomepopulationsegmentsoncavehabitats),(4)habitatstability,and(5)frequenciesofgenetic admixturewithsurfacepopulationsandassociatedcave populations. Mostofthemajormitochondrialcladesrecoveredfromourphylogeographicanalysesincludeboth caveandsurfacepopulations(Figure1).Furthermore, mtDNA-baseddivergencesbetweencaveandsurface populationsintheeastern Blepsimolge areconsistently small.Thesepatterns,alongwiththeoveralllowgenetic diversitywithinthe E.neotenes complex,indicatethat troglomorphismaroserapidlyandindependentlyinthe variouscavepopulations( contra previousviews,e.g., [22]re: E.tridentifera ;populationsassignedtothisspeciesdonotformawell-differentiated,monophyletic groupbasedonmtDNAsequencedata).Whenconsidering mtDNAdataalone,wecannotr uleoutthepossibilitythat troglomorphictraitsaremaintaineddespiteon-goinggene flowfromsurfacepopulations .Overall,thisscenarioseems mostlikelygiventhatthephylogeographicstructurereflects dynamichydrogeologicalsyst emsthatconnectsubterraneanandsurfacehabitats,andthatdispersalofsurface formsislargelydependentonsubterraneancorridors. Perhapsthemostintriguingpatternrecoveredfromour analysisofmorphologicaldataistheexistenceoftwo discretegroupsseparatingprimarilyalongPC3,mostly inassociationwithPC1,buttoalesserextentwith PC2(Figure5).Thesegroupsdonotcorrespondto phylogeographicstructure,previouslyestablishedtaxonomy,geography,orwhetherpopulationsinhabitsubterraneanorsurfacehabitats.Infact,individuallocalities, whethertheyarecaveorsurfacesites,haveindividuals thatgroupwithoneortheothercluster,orinboth discreteclusters(Figure5).Overall,thesepatternsindicate thatthetwomorphologicalgroupsrecoveredarenot influencedbygeographicproximityorpopulation-level geneticdivergence.Thethreevariablesthatweighmost heavilyonPC3areAG,SL,andED(onlyAGandSLhave negativefactorloadingsforthiscomponent),traitsassociatedwithmorphologicaldifferencesbetweensurfaceand caveforms(Table2).However,becausePC2clearlyrepresentsasurface-to-cavemorphologygradient,thevariation alongPC3thatisseparatingspecimensmustberesidual uncorrelatedvariationinthesetraits.Thus,amajoraspect ofthevariationencompassedbyPC3seemstoincludereductionineyediameterwithoutconcomitantreductionin trunklength,orvice-versa.Themoredistinctseparation ofthesetwogroupsalongPC1ratherthanPC2indicates thatscalingrelationshipsarealsoinvolvedincreatingthe patternofgroupseparation. Atleasttwofactorsmayresultinsegregationofmorphologicalvariationintogroupsthatdonotcorrespondtogeographicvariationorgeneticdivergence.First,sexual dimorphismcouldgeneratethispattern,andinmostcases thesexesofspecimensmeasuredcouldnotreliablybedetermined.However,giventhatsexualdifferencesinmorphometricvariablesusuallydivergewithontogenyfroma commonstartingpoint[53,54],morphologicaldispersion basedonsuchdifferencesshou ldnotbediscontinuous.Anotherpossibilityisdevelopmentalplasticity,whichweconsidertobethemostplausibleexplanationforthese groupingsbasedonthelimited dataavailable.Developmentalplasticitymayoperatethroughtwomechanisms:(1)a thresholdresponsetoenvironmentalstimuliwherebyadevelopmentalswitchproducesalt ernativeforms(i.e.,adevelopmentalpolyphenism),or( 2)somaticordevelopmental selection,wherebylargenumbersofvariantsareproduced andsomevariantsareselectivelypreservedwhileothersare eliminated[55].Themostimpo rtanttraitstructuringvariationalongPC3istrunklength,asindicatedbysimilarcorrelationstructureofAGandSLvariables(althougheye diameterdoescontributesome whataswell).Trunklength iscorrelatedwithnumberofvertebralelements,whichis determinedbyperiodicsomiteformationinembryogenesis duetoamolecularoscillator(i.e.,the “ segmentationclock ” ) [56,57].Thus,plasticitycanmodifyvertebralnumbersby randomlyadjustingrateparametersofthesegmentation clock[58].However,whethervariationinthenumberof trunkelementshascontributedtogroupseparationremainsunknown. Plasticityitselfissubjecttoselection,andthedegree ofplasticityinatraitispredictedtocorrelatewiththe amountofenvironmentalvariationtowhichthetraitresponds[55].Thisfacetofplasticityisimportantbecause thedistinctionbetweencaveandsurfacehabitatsastheyrelatetosalamanderpopulationsincentralTexasis artificialinmanycases.SalamandersfromsurfaceBendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page13of18 http://www.biomedcentral.com/1471-2148/13/201

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populationsmayspendconsiderabletimeinsubterraneanrefugia,particularlywhenperiodsofdroughtcause surfacespringstogodry[49].Manypopulationsrelyon springsystemsthatarespatiallyheterogeneousintotal availablehabitatbothaboveandbelowground.Furthermore,surfacepopulationsmaydependmoreoninterstitialgroundwaterthantheopenwatercolumn,andease ofmovementthroughinterstitialcavitiesmaybe influencedbyfeaturessuchastrunklength.Overall,the extremeheterogeneityandstructuralcomplexityofkarst habitatsmayselectforplasticityintraitssuchas somitogenesisandeyedevelopment,andsomatic selectionofvariantsmayresultinthediscordantmorphologicalpatternsobservedatthelocalscale.Developmentalplasticityisalsoconsistentwithapparentlyrapid shiftsinmorphologybetweensurfaceandcavepopulations,andthemaintenanceofdifferentphenotypesdespitethehomogenizinginfluenceofperiodicgeneflow. Wecannotruleoutotherenvironmentalinfluences(e.g., clutcheffects[59]),butherethemorphologicalextremes appearhighlycorrelatedwithprimaryuseofcaveversus surfacehabitat.TaxonomicimplicationsWedonotwishtoprovideaformaltaxonomictreatmenthere(andthus,refrainfromproposingtaxonomic changes),butweoffercommentsoncurrentspeciesdesignationswithinthe E.neotenes complex. Eurycea tridentifera MitchellandReddell[14]exhibitsthemost extremecave-associatedmorphologicalfeatures,which havelongservedasthebasisforitstaxonomicrecognition[10,14,18,22,60,61].Theprevailingviewwasthat thisspeciesrepresentedasinglelineagethatindependentlyevolvedcave-associatedmorphologicaltraitssimilartothoseof E.rathbuni [17].Allozymefrequencydata alsoweaklysupporteditsdistinctiveness , althoughthere werenofixeddifferences(inthethreepopulationsexamined;[10]).Ouranalysisofmultiplepopulationswith variousdegreesoftroglomorphism(includingmany moreindividualsassignedto E.tridentifera andpreviouslyunassigned,nearbypopulationswhosemembers exhibit tridentifera -likemorphologies)challengesthis view.Populationsformallyassignedto E.tridentifera occurwithintheLTcladeandarenotdistinctfrom othercaveandsurfacepopulationsinthatgroup accordingtomtDNAsequencedata(Figure1).SalamandersfromPreserveCavearemorphologicallysimilarto thoseassignedto E.tridentifera (Figure5),yetfallwithin theBGPclade,whilethesingle tridentifera -likespecimenfromHectorHoleispartoftheNclade(Figures1 and3). Euryceatridentifera appearstobecomposedof populationscloselyrelatedtosurfaceformsthathave evolvedextremetroglomorphismindependently(Figures1, 3and4),andmaynotwarrantrecognitionasadistinct species.Chippindaleetal.[10]resurrected E.latitans Smith andPotter[62]fromsynonymyunder E.neotenes ,but regardeditasa “ catch-all ” groupofproblematictaxonomic status.Ourresultssupportthisview,showingextensive mitochondrialpolyphylyforp opulationsassignedtothis species(Figure1).Sweet[22] regardedoccurrenceofsalamanderswithmorphologies"intermediate"betweenthose ofsurfaceandcaveformsatCascadeCaverns(thetypelocalityfor E.latitans )asevidenceofhybridizationbetween E.tridentifera and E.neotenes ,butwefindnoindicationof thisbasedonmtDNA,norwasthissupportedbyallozyme data[10].Thus,thestatusof E.latitans asadistinctspecies alsoishighlyquestionable.C hippindaleetal.[10]tentativelyrecognized E.pterophila Burger,SmithandPotter [63](whichhadbeensynonymizedunder E.neotenes by Sweet[19])onthebasisofsimilarallozymefrequencies (butnodiagnosticalleles)andageographicdistributionexclusivetotheBlancoRiverdrainage[10].Whileourresults doshowgeneticaffinitiesamongsomepopulationswithin thebroaderBlancoRiverwater shed(e.g.,cladeBP2),other Blancopopulationsaremorecloselyrelatedtothosewithin theGuadalupewatershed(includingRebeccaCreekSpring, previouslyassignedto E.latitans [10]).Thisobservationis alsoconsistentwithapopulationgeneticstudythatdocumentedevidenceofgeneticisolationamongseveralpopulationsof E.pterophila [43].Cladescontainingpopulations assignedto E.pterophila formapolytomywiththeLTclade (Figure1)anddivergencebetweenthesegroupsislow.Finally,populationsassignedto E.neotenes BishopandWright[40],plusothercaveandsurfacemembersoftheN clade,formalargelycohesivegroupgeographically. The E.neotenes complexexhibitsdiscordancewith previouslydelimitedspeciesboundaries,containsa morphologically-basedspeciesunsupportedbymtDNA evidence( E.tridentifera )andincludespotentiallycryptic species(e.g.,WhiteSprings).Extremelylowgeneticdivergencebetweensubterraneanandsurfacepopulations isevident,andmorphologicalgroupsdonotcorrespond tocleargeographicorphylogeneticpatterns.Although themorphologicalvariabilityofthisgroupmayhave resultedinover-splittaxonomy,therehasalsobeenlack ofrecognitionofgeneticallydivergentbutmorphologicallysimilarspeciesofTexas Eurycea (mostnotablythose with “ surface ” morphologies,e.g., E.chisholmensis , E. naufragia , E.tonkawae [10]and E.sosorum [45]).We recognizethatconsistentmorphologicaldifferencesbetweenpopulationsmayalsobeindicativeofgenomicdivergenceandwecannotruleoutthepossibilitythatthis divergencemayhaveoccurredfasterthanmtDNA lineagesorting.Additionally,incongruencebetweenspeciestrees,mtDNAandnucleargenetreeshasbeendocumentedinnumerouscases[64-66],highlightingthe potentialpitfallsofrelyingsolelyonmitochondrialsequencedatafortaxonomicassessment.However,ourBendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page14of18 http://www.biomedcentral.com/1471-2148/13/201

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resultsarenotinconsistentwithnucleardatapresented inpreviousstudies[10,43]. Cavecolonization,adaptationandspeciation Speciationincavefaunashasoftenbeenexplainedby twocontrastingmodels:the ‘ climate-relict ’ andthe ‘ adaptiveshift ’ hypotheses.Thedistinctionbetweenthese twomodelsliesinwhethervicarianceordivergentselectionpressuresdrivespeciation[67-69].Underthe climate-relicthypothesis,speciationoccurswhensurface andcavepopulationsareseparatedafterclimatic changesresultinprolongedgeographicisolation[70].In thisscenario,surfacepopulationsareextirpated,gene flowtocavepopulationsiseliminated,andpopulations speciateinallopatry.The ‘ adaptive-shift ’ hypothesis explainsspeciationasaresultofecologicalniche separation[68,69].Inthiscase,ancestral “ surface ” populationsarenotisolatedgeographicallyfrompopulations exploitingthenovelcaveniche[68,71].Instead,natural selectiondrivesdifferentiationandeventuallysevers geneflowbetweentheincipientsurfaceandcavesibling species[69]. Theoccurrenceofphylogenetically-nestedtroglomorphic populationsofspringsalamanders(genus Gyrinophilus ) withinthegeographicrangesofmorewidespreadsurface formshasbeeninvokedasevidenceforspeciationwith geneflow,andthus,supportiveofanadaptive-shifthypothesis[72].Basedonourresults,th eadaptive-shifthypothesis isabetterexplanationofdiversificationandcaveinvasion (butnotnecessarilyspeciation)ineastern Blepsimolge than theclimate-relicthypothesisbecauseof(1)lowdivergence betweenspringandcavepopulationsand(2)genetically similar(orindistinct)caveandspringformsoccurringin sympatry.Buthowaredisparatesurfaceandcavemorphologiesmaintainedinspiteofecologicaloverlapandapparentgeneflow? SharedmtDNAhaplotypesbetweensurfaceandcave populationscanresultfromincompletelineagesorting, causingalagbetweentheprocessoflineagesplittingand ourabilitytodetectit[73].However,thereareseveralreasonswhygeneticadmixturebet weensurfaceandsubterraneanformswithinthe E.neotenes complexislikely.For example,populationsofspring-dwellingcentralTexas Eurycea aredependentuponsubterraneanhabitat[13],eitherforrefugefromdrought[49]orreproduction[74]. Additionally,severalcavepopulationsharborarangeof troglomorphicandsurfaceforms(Figures4dand6)that sharemitochondrialhaplotypes .Thus,thereispotentialfor extensiveoverlapbetweentheseniches,andourresults suggestthatsympatricsurf aceandsubterraneanforms withinthe E.neotenes complexdonotmaintainisolation (althoughSweetregardedthispatternasevidenceforassortativematingamongsim ilarforms[22]).Whether troglomorphismarisesduring briefperiodsofisolationor arisesinsympatry,thepersistenceofdivergentmorphologicalformsingeneticallyadmixedpopulationsmaybe duetostrongselectionforcavephenotypes[6],developmentalplasticity,orboth. Conclusions Themt-genephylogenyofeastern Blepsimolge reveals patternsofintermittentisolationandgeneflow,a Figure6 Cave(top)andsurface(bottom)morphsof Eurycea fromHoneyCreekCave(typelocalityfor Euryceatridentifera ). These individualswereobservedonlymetersapartwithinthesamecavestream:thesurfacemorphwasencountered5mintothecavewhilethecave morphwasobservedapproximately20mfromtheentrance. Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page15of18 http://www.biomedcentral.com/1471-2148/13/201

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reflectionofthedynamicnatureofkarstaquifers.Shallowgeneticdivergencesamongseveralrecognizedspeciessuggestthatthe E.neotenes complexmayhavebeen over-splitbyearlyworkersduetoanemphasison phenotypicdivergence,particularlybetweencaveand surfacepopulations.Thisisincontrasttoconsiderable crypticspeciesdiversityamongspring-dwellingpopulationsofcentralTexas Eurycea owingtomorphological conservatismamongspring-dwellers.Evidenceofgenetic exchangeandnestedrelationshipsacrossmorphologicallydisparatecaveandspringformswithinthe E. neotenes complexsuggeststhatcaveinvasioninthis groupwasrecentandmanytroglomorphicmorphologies (ofindividualstypicallyassignedto E.tridentifera )arose independently.Thesepatternsareconsistentwithan adaptive-shifthypothesisofdiversification.Inmany casescaveandsurfaceformsdonotappeartobe geneticallyisolated,andevenoccurinmicrosympatry (Figure6),suggestingthattroglomorphismismaintained bystrongselectionand/ordevelopmentalplasticity.AvailabilityofsupportingdataNucleotidesequencedatasupportingtheresultsofthisarticleareavailableinGenbank.Accessionnumbersare KC355860 – KC355971andKC355972 – KC356083forCytb andND2,respectively[seeAdditionalfile1].AdditionalfilesAdditionalfile1: MSExcelfileofcollectionandGenbank informationforspecimensusedingeneticanalysis. Additionalfile2: MSExcelfileofcollectioninformationfor specimensusedinmorphometricanalysis. Competinginterests Theauthorsstatethattheyhavenocompetinginterestsforthispublication. Authors ’ contributions NB,PC,andAGconceivedtheproject;NB,PC,andAGconductedfieldwork andobtainedtissuesamples;NBandPCgatheredmoleculardata;NBand JMperformedstatisticalanalyses;NB,JM,andPCwrotethemanuscript;NB andJMpreparedfigures;AGcollatedinformationonhydrogeologyand facilitatedpermits;CRobtainedmorphometricdata;PCprovidedsupport andlaboratoryfacilities;allauthorseditedandapprovedthefinal manuscript. Acknowledgements NewlyacquiredspecimensandsampleswereobtainedunderTexasParks andWildlifePermitSPR-0904-423andU.S.FishandWildlifePermitTE0818840.Thisworkwouldnothavebeenpossiblewithoutassistancefromthe followingindividualsandinstitutions:AndyPrice,JamesReddell,George Veni,DavidHillis,DavidCannatella,TravisLaDuc,BrianSullivan,CarlFranklin, JeanKrejca,LaurieDries,DeeAnnChamberlain,numerouslandownersand fieldcompanions,UniversityofTexasatAustinTexasNaturalHistory Collections,UniversityofTexasatArlingtonAmphibianandReptileDiversity ResearchCenter,andUniversityofCaliforniaBerkeleyMuseumofVertebrate Zoology.JeanKrejca(ZaraEnvironmental)providednumeroussalamander photographs.FundingwasprovidedinpartbytheUniversityofTexasat Arlington,PhiSigmaBiologicalHonorSociety,AustinCommunity Foundation,CityofAustin,TexasParksandWildlifeDepartment,U.S.Fish andWildlifeServiceandtheNationalScienceFoundation.BrianFontenot andDavidHillisprovidedhelpfulsuggestionsonanearlierdraftofthis manuscript.WealsothankDavidWakeandthreeanonymousreviewersfor theircomments. Authordetails1DepartmentofBiology,UniversityofTexasatArlington,Arlington,Texas 76019,USA.2CityofAustin,WatershedProtectionDepartment,Austin,Texas 78704,USA.3DepartmentofBiologicalSciences,TarletonStateUniversity, Stephenville,Texas76402,USA.4TexasParksandWildlifeDepartment,Austin, Texas78744,USA. Received:3April2013Accepted:12September2013 Published:17September2013 References1.WilkensH,StreckerU: Convergentevolutionofthecavefish Astyanax (Characidae,Teleostei):geneticevidencefromreducedeye sizeand pigmentation. BiolJLinnSocLond 2003, 80: 545 – 554. 2.XiaoH,ChenS,LiuZ,ZhangR,LiW,ZanR,ZhangY: Molecularphylogeny of Sinocyclocheilus (Cypriniformes:Cyprinidae)inferredfrom mitochondrialDNAsequences. MolPhylogenetEvol 2005, 36: 67 – 77. 3.DerkarabetianS,SteinmannDB,HedinM: Repeatedandtime-correlated morphologicalconvergenceincave-dwellingharvestmen(Opiliones, Laniatores)frommontanewesternNorthAmerica. PLoSONE 2010, 5: e10388. 4.CulverDC,KaneTC,FongDW: AdaptationandNaturalSelectioninCaves: TheEvolutionofGammarusminus. Cambridge:HarvardUniversityPress; 1995. 5.StreckerU,BernatchezL,WilkensH: Geneticdivergencebetweencave andsurfacepopulationsof Astyanax inMexico(Characidae,Teleostei). MolEcol 2003, 12: 699 – 710. 6.BradicM,BeerliP,LeónFJG,Esquivel-BobadillaS,BorowskyRL: Geneflow andpopulationstructureintheMexicanblindcavefishcomplex ( Astyanaxmexicanus ). BMCEvolBiol 2012, 12: 9. 7.PaquinP,HedinM: Thepowerandperilsof  moleculartaxonomy Ž :acase studyofeyelessandendangered Cicurina (Araneae:Dictynidae)from Texascaves. MolEcol 2004, 13: 3239 – 3255. 8.JuanC,GuzikMT,JaumeD,CooperSJB: Evolutionincaves:Darwin  s  wrecksofancientlife Ž inthemolecularera. MolEcol 2010, 19: 3865 – 3880. 9.ReddellJR: ThecavefaunaofTexaswithspecialreferencetothewestern EdwardsPlateau ,TheCavesandKarstofTexas.AGuidebookforthe1994 ConventionoftheNationalSpeleologicalSocietywithEmphasisonthe SouthwesternEdwardsPlateau.Huntsville:NationalSpeleologicalSociety; 1994:31 – 50. 10.ChippindalePT,PriceAH,WiensJJ,HillisDM: Phylogenetic relationshipsandsystematicrevisionofcentralTexashemidactyliineplethodontidsalamanders. HerpetolMonogr 2000, 14: 1 – 80. 11.PotterFE,SweetSS: GenericboundariesinTexascavesalamanders,anda redescriptionof Typhlomolgerobusta (Amphibia:Plethodontidae). Copeia 1981, 1981: 64 – 75. 12.SweetSS: Naturalmetamorphosisin Euryceaneotenes ,andthegeneric allocationoftheTexas Eurycea (Amphibia:Plethodontidae). Herpetologica 1977, 33: 364 – 375. 13.SweetSS: Adistributionalanalysisofepigeanpopulationsof Eurycea neotenes incentralTexas,withcommentsontheoriginoftroglobitic populations. Herpetologica 1982, 38: 430 – 444. 14.MitchellRW,ReddellJR: Euryceatridentifera ,anewspeciesoftroglobitic salamanderfromTexasandareclassificationof Typhlomolgerathbuni . TexJSci 1965, 17: 12 – 27. 15.BowlesBD,SandersMS,HansenRS: EcologyoftheJollyvillePlateau salamander( Euryceatonkawae :Plethodontidae)withanassessment ofthepotentialeffectsofurbanization. Hydrobiologia 2006, 553: 111 – 120. 16.ChippindalePT,PriceAH: ConservationofTexasspringandcave salamanders( Eurycea ) .In AmphibianDeclines:TheConservationStatusof UnitedStatesSpecies. EditedbyBerkeleyLM.LosAngeles:Universityof CaliforniaPress;2005:193 – 197. 17.WiensJJ,ChippindalePT,HillisDM: Whenarephylogeneticanalyses misledbyconvergence?AcasestudyinTexascavesalamanders. SystBiol 2003, 52: 501 – 514.Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page16of18 http://www.biomedcentral.com/1471-2148/13/201

PAGE 17

18.MitchellRW,SmithRE: Someaspectsoftheosteologyandevolutionof theneotenicspringandcavesalamanders( Eurycea ,Plethodontidae)of centralTexas. TexJSci 1972, 23: 343 – 362. 19.SweetSS: Onthestatusof Euryceapterophila (Amphibia: Plethodontidae). Herpetologica 1978, 34: 101 – 108. 20.ChippindalePT: Speciesboundariesandspecie sdiversityinthecentralTexas hemidactyliineplethodontidsalamanders,genus Eurycea. In TheBiologyof PlethodontidSalamanders. EditedbyBruceRC,JaegerR,HouckLD.NewYork: KluwerAcademic/PlenumPublishers;2000:149 – 165. 21.HillisDM,ChamberlainDA,WilcoxTP,ChippindalePT: Anewspeciesof subterraneanblindsalamander(Plethodontidae:Hemidactyliini: Eurycea: Typhlomolge )fromAustin,Texas,andasystematicrevisionofcentral Texaspaedomorphicsalamanders. Herpetologica 2001, 57: 266 – 280. 22.SweetSS: SecondarycontactandhybridizationintheTexascave salamanders Euryceaneotenes and E.tridentifera . Copeia 1984, 1984: 428 – 441. 23.CantinoPD,deQueirozK: Phylocode. http://www.ohio.edu/phylocode. 24.WiensJJ,EngstromTN,ChippindalePT: Rapiddiversification,incomplete isolation,andthe  speciationclock Ž inNorthAmericansalamanders(genus Plethodon ):testingthehybridswarmhypothesisofrapidradiation. Evolution 2006, 60: 2585 – 2603. 25.HillisDM,MableBK,LarsonA,DavisSK,ZimmerEA: NucleicacidsIV: sequencingandcloning. In MolecularSystematics. 2ndedition.Edited byHillisDM,MoritzC,MableBK.Sunderland:SinauerAssociates; 1996:321 – 381. 26.WalshPS,MetzgerDA,HiguchiR: Chelex100asamediumforsimple extractionofDNAforPCR-basedtypingfromforensicmaterial. Biotechniques 1991, 10: 506 – 513. 27.MoritzC,SchneiderCJ,WakeDB: Evolutionaryrelationshipswithinthe Ensatinaeschscholtziicomplexconfirmtheringspeciesinterpretation. SystBiol 1992, 41: 273 – 291. 28.VencesM,ThomasM,BonettRM,VieitesDR: Decipheringamphibian diversitythroughDNAbarcoding:chancesandchallenges. PhilosTransR SocLondBBiolSci 2005, 360: 1859 – 1868. 29.TamuraK,PetersonD,PetersonN,StecherG,NeiM,KumarS: MEGA5: molecularevolutionarygeneticsanalysisusingmaximumlikelihood, evolutionarydistance,andmaximumparsimonymethods. MolBiol Evol 2011, 28: 2731 – 2739. 30.EdgarRC: MUSCLE:multiplesequencealignmentwithhighaccuracyand highthroughput. NucleicAcidsRes 2004, 32: 1792 – 1797. 31.RonquistF,HuelsenbeckJP: MrBayes3:Bayesianphylogeneticinference undermixedmodels. Bioinformatics 2003, 19: 1572 – 1574. 32.MillerMA,PfeifferW,SchwartzT: CreatingtheCIPRESScienceGateway forinferenceoflargephylogenetictrees ,2010GatewayComputing EnvironmentsWorkshop:14November2010;NewOrleans.Washington DC,USA:IEEE;2010:1 – 8. 33.GuindonS,GascuelO: Asimple,fast,andaccuratealgorithmtoestimate largephylogeniesbymaximumlikelihood. SystBiol 2003, 52: 696 – 704. 34.PosadaD: jModelTest:phylogeneticmodelaveraging. MolBiolEvol 2008, 25: 1253 – 1256. 35.MarshallDC: CrypticfailureofpartitionedBayesianphylogenetic analyses:lostinthelandoflongtrees. SystBiol 2010, 59: 108 – 117. 36.RambautA,DrummondAJ: Tracerv1.5 2009.http://tree.bio.ed.ac.uk/ software/tracer/[AccessedMarch122013]. 37.MarroigG: Whensizemakesadifference: allometry,life-historyand morphologicalevolutionofcapuchins( Cebus )andsquirrels( Saimiri ) monkeys(Cebinae,Platyrrhini). BMCEvolBiol 2007, 7: 20. 38.McCoyMW,BolkerBM,OsenbergCW,MinerBG,VoneshJR: Sizecorrection: comparingmorphologicaltraitsamongpopulationsandenvironments. Oecologia 2006, 148:547 – 554. 39.AdamsDC,RohlfFJ,SliceDE: Geometricmorphometrics:tenyearsof progressfollowingthe  revolution. ItalianJournalofZoology 2004, 71: 5 – 16. 40.BishopSC,WrightM: AnewneotenicsalamanderfromTexas. ProcBiolSoc Wash 1937, 50: 141 – 144. 41.ChippindalePT: Statusofnewlydiscoveredcaveandspringsalamanders (Eurycea)insouthernTravisandnorthernHaysCounties. Austin,Texas:Texas ParksandWildlifeDepartment;2012:31. 42.JohnsonS,SchindelG,VeniG,HauwertN,HuntB,SmithB,GaryM: Tracing groundwaterflowpathsinthevicinityofSanMarcosSprings,Texas. San Antonio,Texas:EdwardsAquiferAuthority;2012:139. 43.LucasL,GompertZ,OttJ,NiceC: Geographicandgeneticisolationinspringassociated Eurycea salamandersendemictotheEdwardsPlateauregionof Texas. ConservGenet 2009, 10: 1309 – 1319. 44.ChippindalePT,PriceAH,HillisDM: SystematicstatusoftheSanMarcos salamander, Euryceanana (Caudata:Plethodontidae). Copeia 1998, 1998: 1046 – 1049. 45.ChippindalePT,PriceAH,HillisDM: Anewspeciesof perennibranchiatesalamander( Eurycea :Plethodontidae)fromAustin, Texas. Herpetologica 1993, 49: 248 – 259. 46.AndrewsF,SchertzT,SladeRJ,RawsonJ: Effectsofstorm-waterrunoffon waterqualityoftheEdwardsAquifernearAustin,Texas.Water-Resources InvestigationsReport. Austin,Texa:UnitedStatesGeologicalSurvey;1984:50. 47.HauwertNM: GroundwaterflowandrechargewithintheBartonSprings SegmentoftheEdwardsAquifer,southernTravisandnorthernHays Counties,Texas ,PhDthesis.TheUniversityofTexasatAustin: DepartmentofGeologicalSciences;2009. 48.VeniG: Geomorphology,hydrology,geochemistry,andevolutionofthekarsticlower GlenRoseaquifer,south-centralTexas ,PhDthesis.ThePennsylvaniaState University:DepartmentofGeosciences;1994. 49.BendikNF,GluesenkampAG: Bodylengthshrinkageinan endangeredamphibianisassociatedwithdrought. JZool 2013, 290: 35 – 41. 50.ProtasM,ConradM,GrossJB,TabinC,BorowskyR: Regressive evolutionintheMexicancavetetra, Astyanaxmexicanus . CurrBiol 2007, 17: 452– 454. 51.YamamotoY,ByerlyMS,JackmanWR,JefferyWR: Pleiotropicfunctionsof embryonicsonichedgehogexpressionlinkjawandtastebud amplificationwitheyelossduringcavefishevolution. DevBiol 2009, 330: 200 – 211. 52.NiemillerML,PoulsonTL: SubterraneanfishesofNorthAmerica: Amblyopsidae. In BiologyofSubterraneanFishes. EditedbyTrajanoE,Bichuette ME,KapoorBG.Enfield:SciencePublishers;2010:169 – 280. 53.BadyaevAV: Growingapart:anontogeneticperspectiveontheevolution ofsexualsizedimorphism. TrendsEcolEvol 2002, 17: 369 – 378. 54.GluesenkampAG,AcostaN: Sexualdimorphismin Osornophryneguacamayo withnotesonnaturalhistoryandreproductioninthespecies. JHerpetol 2001, 35: 148 – 151. 55.West-EberhardMJ: DevelopmentalPlasticityandEvolution. 1stedition.New York:OxfordUniversityPress;2003. 56.CookeJ,ZeemanEC: Aclockandwavefrontmodelforcontrolofthenumber ofrepeatedstructuresduri nganimalmorphogenesis. JTheorBiol 1976, 58: 455 – 476. 57.GomezC,ÖzbudakEM,WunderlichJ,BaumannD,LewisJ,PourquiéO: Controlofsegmentnumberinvertebrateembryos. Nature 2008, 454: 335 – 339. 58.MüllerJ,ScheyerTM,HeadJJ,BarrettPM,WerneburgI,EricsonPGP,PolD, Sánchez-VillagraMR: Homeoticeffects,somitogenesisandtheevolution ofvertebralnumbersinrecentandfossilamniotes. ProcNatlAcadSci USA 2010, 107: 2118 – 2123. 59.AdamsDC: Quantitativegeneticsandevolutionofheadshapein Plethodon salamanders. EvolBiol 2011, 38: 278 – 286. 60.WakeDB: Comparativeosteologyandevolutionofthelungless salamanders,familyPlethodontidae. MemoirsoftheSouthernCalifornia AcademyofSciences 1966, 4: 1 – 111. 61.SweetSS: Euryceatridentifera. CatalogueofAmericanAmphibiansand Reptiles 1977, 199: 1 – 199.62.SmithHM,PotterFEJr: Athirdneotenicsalamanderofthegenus Eurycea fromTexas. Herpetologica 1946, 3: 105 – 109. 63.BurgerW,SmithHM,FloydE: Potter:Anotherneotenic Eurycea from theEdwardsPlateau. ProceedingsoftheBiologicalSocietyof Washington,D.C. 1950, 63: 51 – 57. 64.ShawKL: Conflictbetweennuclearandmit ochondrialDNAphylogeniesofa recentspeciesradiation:whatmtDNA revealsandconcealsaboutmodesof speciationinHawaiiancrickets. ProcNatlAcadSciUSA 2002, 99: 16122 – 16127. 65.Fisher-ReidMC,WiensJJ: Whataretheconsequencesofcombining nuclearandmitochondrialdataforphylogeneticanalysis?Lessons from Plethodon salamandersand13othervertebrateclades. BMCEvolBiol 2011, 11: 300. 66.LeachéAD: Speciestreesforspinylizards(genus Sceloporus ): identifyingpointsofconcordanceandconflictbetweennuclearand mitochondrialdata. MolPhylogenetEvol 2010, 54: 162 – 171.Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page17of18 http://www.biomedcentral.com/1471-2148/13/201

PAGE 18

67.Desutter-GrandcolasL,GrandcolasP: Theevolutiontowardtroglobiticlife: aphylogeneticreappraisalofclimaticrelictandlocalhabitatshift hypotheses. MémoiresdeBiospléologie 1996, 23: 57 – 63. 68.HowarthFG: Theevolutionofnon-relictualtropicaltroglobites. IntJ Speleol 1987, 16: 1 – 16. 69.RiveraMAJ,HowarthFG,TaitiS,RoderickGK: EvolutioninHawaiiancaveadaptedisopods(Oniscidea:Philosciidae):vicariantspeciationor adaptiveshifts? MolPhylogenetEvol 2002, 25: 1 – 9. 70.Peck,FinstonTL: GalapagosIslandstroglobites:thequestionsoftropical troglobites,parapatricdistributionswitheyed-sister-species,andtheir originbyparapatricspeciation. MémoiresdeBiospléologie 1993, 20: 19 – 37. 71.HowarthF: High-stresssubterraneanhabitatsandevolutionarychangein cave-inhabitingarthropods. AmNat 1993, 142: S65 – S77. 72.NiemillerML,FitzpatrickBM,MillerBT: Recentdivergencewithgeneflow inTennesseecavesalamanders(Plethodontidae: Gyrinophilus )inferred fromgenegenealogies. MolEcol 2008, 17: 2258 – 2275. 73.WiensJJ,PenkrotTA: DelimitingspeciesusingDNAandmorphological variationanddiscordantspecieslimitsinspinylizards( Sceloporus ). SystBiol 2002, 51: 69 – 91. 74.CityofAustin: BiologicalAssessmentBartonSpringsFloodDebrisRemovaland BypassRepairs,Austin,Texas. Austin,Texas:U.S.ArmyCorpsofEngineers,Ft. WorthDistrictOffice;2010. doi:10.1186/1471-2148-13-201 Citethisarticleas: Bendik etal. : Biogeography,phylogeny,and morphologicalevolutionofcentralTexascaveandspringsalamanders. BMCEvolutionaryBiology 2013 13 :201. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Bendik etal.BMCEvolutionaryBiology 2013, 13 :201 Page18of18 http://www.biomedcentral.com/1471-2148/13/201


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