DiversificationintheNeotropics:InsightsfromDemographicandPhylogeneticPatterns

ofLanceheadPitvipers(Bothropsspp.)



DISSERTATION



PresentedinPartialFulfillmentoftheRequirementsfortheDegreeDoctorof PhilosophyintheGraduateSchoolofTheOhioStateUniversity 

By

ChristianDavidSalazarValenzuela,B.S.

GraduatePrograminEvolution,EcologyandOrganismalBiology



TheOhioStateUniversity

2016

DissertationCommittee:

Dr.H.LisleGibbs(Advisor) Dr.PaulA.Fuerst Dr.ThomasHetherington Dr.JohnFreudenstein  1                  Copyrightby

ChristianDavidSalazarValenzuela

2016



  

   





2  



 Abstract  



TheNeotropicsisthemostspeciesͲrichregionintheworld.Thecurrentdiversity anddistributionoflineagespresentinthisregionisinparttheresultofcomplex ecologicalandevolutionarytrendsdeterminedbyenvironmentalvariablesthathave operatedatdiversespatialandtemporalscales.Inaddition,demographicprocesses havealsoinfluencedthestructureofpresentͲdayphylogeographicpatterns.Several studieshaveusedNeotropicalpitvipersasmodelorganismstoexplorehistorical diversificationpatternsandecologicalprocessesthatproducediversityinthisregion.

However,fewofthosestudieshaveexploredpatternsofdiversificationforgroupsof pitviperslikelyinfluencedbyoneofthemostsalientfeaturesoftheSouthAmerican continent:TheAndes.Here,Iuseacombinationofmolecular,morphological,and geographicaldatatoexplorediversificationpatternsandtheevolutionarymechanisms implicatedinthedivergenceoftwodistinctmembersofthegenusBothrops.First,I examinecrypticdiversitypresentinthewidespreadandmedicallyimportantsnakesof theB.asperspeciescomplex(Chapter2).Usingagenomicandmorphologicaldataset collectedacrossthedistributionofthegroup,Iidentifiedextensivephylogeographic ii structure,suggestingtheinfluenceofgeographicbarriersand/ordifferencesin ecologicalnichesintherecentdiversificationinthegroup.Adeepdivergencebetweena

CentralandSouthAmericancladeisevident,butmorerecentlydivergedgroupsin

SouthAmericashowcomplicatedpatternssuggestiveofrecentdivergenceand/orgene flowamonglineages.Next,IusethisinformationtoperformmodelͲbasedanalysesto investigatethedemographicprocessesinvolvedintherecentoriginoftwoEcuadorian montanelineagesofthesepitvipers(Chapter3).Thisapproachallowedmetoresolve someofthediscrepanciesofevolutionaryrelationshipsfoundinChapter2.Ifound evidencefortheisolationofoneofthemontanelineagesindryinterͲAndeanvalleys, whichseemtobeimportantdriversforthediversificationofthisgroupinSouth

America.Finally,inChapter4Ideveloppredictivedistributionmapsforanendangered speciesofAndeanpitviper,B.lojanus,andbasedongeneticandmorphologicaldata identifytwodistinctgeneticgroupsinneedofconservationaction.Theevolutionary mechanismsandpatternsidentifiedinthesesnakesprovideinsightsintotheforces shapingtropicalbiodiversitywhilealsoofferingpracticalinformationforthe conservationandimprovedcoexistencewiththesefascinatingorganisms.







 

iii 





Dedication 



ToPaola,Ruth,andMario





























iv 

 

Acknowledgments 



Thisefforthascometofruitionthankstoanumberofindividualsandinstitutions thathavecollaboratedwithme.Iamdeeplygratefulforhavingmetandworkedwith suchamazingindividualswhomadethisexperienceenrichingandlifechanging.First,I owemygratitudetomyresearchadvisor,H.LisleGibbs,foracceptingmeintohislab andgivingmeboththeindependencetodelineateandworkonmyresearchquestions, aswellassettinghighstandardsthatmotivatedandinspiredme.Hispatience,support andmotivationhavebeenkeytomefinishingthisdissertationfeelingthatI accomplishedwhatIsetouttodowhenIfirstarrivedatOSU.Iwouldalsoliketo expressmysincerethankstomycommitteemembers,JohnFreudenstein,Tom

Hetherington,andPaulFuerstfortheiradviceandguidanceonhowtoimprovethis work.Myinteractionswiththemhavebeenthoughtprovokingandusefultoincrease myunderstandingofEvolutionaryBiology.Furthermore,Iwouldliketothankother

EEOBfacultywhoseseminarshaveenhancedmyknowledge:BryanCarstens,Laura

Kubatko,MegDaly,andAndreaWolfe.

v Severalpeoplehelpeddevelopthisresearchthroughstimulatingdiscussions, professionalcollaboration,andlogisticsassistance.IwouldliketothankUlrichKuch,

OmarTorresͲCarvajal,PabloAmaruLoaiza,EricSmith,WolfgangWüster,Mahmood

Sasa,MónicaSaldarriaga,JimmyGuerrero,FelipeLiévano,SergioCubides,Jorge

Valencia,SantiagoAyerbe,JoséMaríaGutierrez,BrunoLomonte,MarioGrijalva,Juan

ManuelDaza,GustavoSilva,LuisColoma,ChristopherParkinson,andJonathan

Campbell.

Also,myearnestthankstocurrentandformermembersoftheGibbslab.Their friendship,insightfulobservations,andconstructivecriticismshavebeeninvaluable.

ThankstoMikeSovic,TonyFries,JoséDiaz,MattHolding,RobDenton,JimmyChiucchi,

SarahSmiley,OleksanderZinenko,andKatieGreenwald.SpecialthankstoJoséand

Mikefortheirassistanceandinstructioninthelaboratory.Iwouldalsoliketothankmy fellowEEOBgraduateandpostͲdoctoralstudents:IsaacLigocki,EricMcCluskey,Erin

Lindstedt,JordanSatler,PaulBlischak,MichaelBroe,JasonMacrander,Orlando

Combita,JuanCarlosPenagos,SuiPhang,andBenTitusfortheirsupport,friendship, andconstantencouragement.

Iwouldequallyliketoacknowledgethecollaborationgivenbymycolleaguesand friendsattheMuseumofZoology,PontificalCatholicUniversityofEcuador:Santiago

Ron,AndreaRodríguez,FernandoAyala,andDiegoPaucar.Thankstoallthepeoplethat havecomewithmetoremotelocationsinthemiddleofthenighttolookforsnakes:

DarwinNuñez,WilliamandPaolaSantacruz,RuthValenzuelaandespeciallyPablo

vi AmaruLoaiza.TothefacultyandstudentsoftheMaster’sPrograminConservation

Biology,PontificalCatholicUniversityofEcuadorfortheirsupportandsolidarity.Also,I wanttorecognizethefinancialandlogisticalsupportfromtheinstitutionsthathave grantedresearchfundstopursuethisworkandextendmygratitudetotheCommission

FulbrightEcuadorandtheFulbrightͲSenescytcooperationforprovidingmewitha scholarshiptostartmygraduatestudies.

Finally,Iwouldliketoextendmydeepestgratitudetomyfamilyforsupporting methroughthisprocess,inspiringmetoworkhard,andkeepingmesane.Mymother’s strengthofcharacter,myfather’sdedication,andtheircommitmenttomedicine startedmeonthispath.Tomysiblingsandtheirfamiliesfortheirloveandsupport.I wouldespeciallyliketothankmywife,Paola,forthesacrificesshehasmadetobeable tosupportandencourageme.Abigpartofthisaccomplishmentbelongstoyou!

Finally,thisworkwouldnothavebeenpossiblewithoutthepeopleoftheruralareasof

Ecuadorwhoduringmyfieldtripswerealwayscooperative,curiousaboutmywork,and wagertosharetheiranecdotesandexperiences.Ihavegreatappreciationfortheirhard workandtheirconstantwillingnesstosharetheirknowledgeaboutnature.







 

vii 

  Vita 

2007...... B.S.Biology,PontificiaUniversidadCatólica

delEcuador,Quito,Ecuador.

2009Ͳ2011...... FulbrightScholar,DepartmentofEvolution,

EcologyandOrganismalBiology,TheOhio

StateUniversity

2011topresent...... GraduateTeachingandResearchAssociate,

DepartmentofEvolution,Ecologyand

OrganismalBiology,TheOhioState

University



Publications

SalazarͲValenzuela,D.,A.Martins,L.AmadorͲOyola,andO.TorresͲCarvajal.2015.Anew speciesandcountryrecordofthreadsnakes(Serpentes:Leptotyphlopidae)from northernEcuador.Amphibian&ReptileConservation8:107–120.

SalazarͲValenzuela,D.,D.MoraͲObando,M.L.Fernández,A.LoaizaͲLange,H.L.Gibbs, andB.Lomonte.2014.Proteomicandtoxicologicalprofilingofthevenomof Bothrocophiascampbelli,apitviperspeciesfromEcuadorandColombia.Toxicon 90:15–25.

viii SalazarͲValenzuela,D.,O.TorresͲCarvajal,andP.Passos.2014.Anewspeciesof Atractus(Serpentes:Dipsadidae)fromtheAndesofEcuador.Herpetologica70: 350–363.

SalazarͲValenzuela,D.,E.O.Carrillo,andS.Aldás.2010.Tricheilostomaanthracinum: geographicdistribution.HerpetologicalReview41:111–112.

Bravo,F.andD.SalazarͲValenzuela.2009.AnewspeciesofSycoraxCurtis(Diptera, Psychodidae)collectedonharlequinfrogs(Anura:Bufonidae,Atelopus)inthe EcuadorianAndes.Zootaxa2093:37–42.

Passos,P.,D.F.CisnerosͲHeredia,andD.SalazarͲValenzuela.2007.Rediscoveryand redescriptionoftherareAndeansnakeAtractusmodestus.Herpetological Journal17:1–6.

ProañoͲBolaños,C.,A.MerinoͲViteri,P.PeñaͲLoyola,andD.SalazarͲValenzuela.2007.A midaltitudereportofBatrachochytriumdendrobatidisinEcuador.Froglog82:3– 4.

Boada,C.,D.SalazarͲValenzuela,A.FreireLascano,andU.Kuch.2005.Thedietof Bothropsasper(Garman,1884)inthePacificlowlandsofEcuador.Herpetozoa 18:77–79. 



FieldsofStudy



MajorField:Evolution,EcologyandOrganismalBiology



    

 ix 

  TableofContents 





Abstract...... ii

Dedication...... iv

Acknowledgments...... v

Vita...... viii

TableofContents...... x

ListofTables...... xvi

ListofFigures...... xviii

Chapter1:Introduction...... 1

References...... 5

Chapter2:AnalysisofgenomicͲlevelvariationprovidesinsightintotherecent diversificationofawidespreadtropicalsnake,the“ultimatepitvipers”(Bothropsasper speciescomplex)...... 8

Abstract...... 8

x Introduction...... 10

MaterialsandMethods...... 14

Taxonsampling...... 14

Conceptualapproach...... 16

Geneticdata...... 16

Genomiclibrarypreparation,sequencing,andbioinformaticmethods...... 17

MitochondrialDNAdata...... 18

Speciesdiscoverymethods...... 20

CoalescentͲbasedspeciesdelimitation...... 23

Speciestreeinference...... 24

Morphologicalanalyses...... 25

Results...... 26

GenotypingofRADseqdata...... 26

Speciesdiscoverymethods...... 27

CoalescentͲbasedspeciesdelimitation...... 28

Speciestreeinference...... 29

Morphologicalanalyses...... 29

Discussion...... 30

xi Lineagediscoveryandspeciesdelimitationinyoungspeciescomplexes...... 31

CrypticdiversityintheBothropsasperspeciescomplex...... 32

EvolutionarybiogeographyoftheBothropsaspercomplex...... 35

Biomedicalimplications...... 39

Taxonomicrecommendations...... 40

Acknowledgements...... 41

References...... 42

Tables...... 55

Figures...... 58

Chapter3:Divergenceoftropicalpitviperspromotedbyindependentcolonization eventsofdrymontaneAndeanhabitats...... 65

Abstract...... 65

Introduction...... 66

MaterialsandMethods...... 70

Studysystem...... 70

Genomicmethods...... 72

Geneticstructureanalyses...... 73

Populationsplitsandmixtures...... 74

xii Demographicmodeling...... 74

Results...... 75

GenotypingofRADseqdata...... 75

Geneticclustering...... 76

Populationsplitsandmixtures...... 76

Historicaldemography...... 77

Discussion...... 77

EvolutionaryrelationshipsamongB.asperEcuadorianlineages...... 78

EvolutionarydynamicsoflowlandandhighlandB.asperEcuadorianlineages...... 79

RecenteventsofpitvipercolonizationanddiversificationinSouthAmerica...... 81

Implicationsandfuturedirections...... 83

Acknowledgements...... 84

References...... 85

Tables...... 94

Figures...... 96

Chapter4:Distribution,geneticstructureandmorphologicalvariationofanendangered

Andeanpitviper,theLojanlancehead(Bothropslojanus)...... 99

Abstract...... 99

xiii Introduction...... 100

MaterialsandMethods...... 102

Environmentalnichemodelingandsnakesurveys...... 102

Moleculardata...... 104

Phylogeneticanalysesandgeneticdiversity...... 105

Morphologicalanalyses...... 106

Results...... 108

Ecologicalnichemodeling...... 108

Phylogeneticreconstructionandgeneticstructure...... 109

Morphologicalvariation...... 111

Discussion...... 111

Acknowledgements...... 115

References...... 115

Tables...... 120

Figures...... 122

Appendices...... 127

AppendixA:MorphologicalVariables...... 127

AppendixB:MuseumSpecimensUsedinMorphologicalAnalysis...... 128

xiv MuseumAcronyms:...... 128

AppendixC:GeneticClusteringAnalyses...... 138

AppendixD:MorphologicalVariables...... 139

AppendixE:MuseumSpecimensUsedinMorphologicalAnalysis...... 140

AppendixF:ConcatenatedMitochondrialandNuclearTree...... 142

ReferencesforAppendices...... 144

Bibliography...... 145























 

xv   

ListofTables 





Table2.1.LocalitydataforsamplesusedwiththeRADseqprotocol.Countryabbreviationsareas follows:MX(Mexico),BZ(Belize),GT(Guatemala),NI(Nicaragua),CR(CostaRica),PA(Panama),

CO(Colombia),VE(Venezuela),EC(Ecuador),PE(Peru),TT(TrinidadandTobago),andBR(Brazil).

...... 55

Table2.2SpeciesdelimitationmodelstestedwiththeBFD*approach.Lineageabbreviationsare as follows: Bothrops asper (BAS), B. ayerbei (BAY), B. rhombeatus (BRH), Mexico and Nuclear

Central America (MNCA), Pacific Isthmian Central America (PICA), Darien Panama and Choco

(CHOCO), Magdalena Valley Colombia and Venezuela (MVV), Highlands Ecuador 1 (HEC1),

Highlands Ecuador 2 (HEC2), Pacific Ecuador (PEC). Other abbreviations include marginal

likelihoodestimator(MLE)andBayesfactors(BF)....... 57

Table3.1VoucherandlocalityinformationforEcuadorianspecimensusedinthisstudy...... 94

Table3.2AICmodelselectionresultsforfastsimcoalanalyses...... 95

Table 3.3 Maximum likelihood estimates for demographic parameters estimated in the fastsimcoalanalysisforthebestͲsupportedmodel:((PEC,HEC1)HEC2)....... 95

xvi Table4.1.VoucherandlocalityinformationforBothropslojanusspecimensusedinthisstudy.

...... 120

Table4.2GeneticdiversityindicesforcladeA,cladeB,andthecompletedatasetofBothrops lojanus.H=numberofhaplotypes;Hd=haplotypediversity;ʋ=nucleotidediversity;K=average numberofwithinͲpopulationpairwisedifferences....... 121

Table 4.3 Selected morphological characters that showed variation in specimens of Bothrops lojanussensustrictoandB.lojanusCladeB....... 121

TableB.1.MuseumSpecimensUsedinMorphologicalAnalysis....... 129

TableE.1:SpecimensusedinMorphologicalAnalysis....... 141

















   

xvii  



ListofFigures 





Figure2.1GeographicdistributionoftheBothropsasperspeciescomplex(asdefinedinthetext) inLatinAmerica:B.asper(gray)acrosstherange,andB.ayerbei(darkgray),andB.rhombeatus

(black)inColombia.SampledlocalitiesinthisstudyforRADseqdataonly(redcircles),RADseqand

morphologicaldata(reddiamonds),andmorphologicaldataonly(bluesquares).Asinglesymbol

maycoverseveralcloselysituatedlocalities.YellowtrianglesrepresentB.atroxspecimensfrom

EcuadorandBrazilusedasoutgroups.PictureofaB.asperfemalefromthePacificlowlandsof

EcuadorbyOmarTorresͲCarvajal....... 58

Figure2.2Maximumlikelihoodphylogram(left)andStructureplot(K=7)generatedfrom864

SNPs.Nodalsupportwasderivedfrom1000bootstrappseudoreplicatesperformedinRAxML.

Cladeswithstrongsupport(>70)arehighlightedinshadesofgray.Countrycodesinphylogram

arethoseindicatedinTable2.1andStructureabbreviationsareasfollows:CaribbeanIsthmian

CentralAmerica(CICA),MexicoandNuclearCentralAmerica(MNCA),PacificIsthmianCentral

America(PICA),MagdalenaValleyColombiaandVenezuela(MVV),DarienPanamaandChoco

(CHOCO),andPacificlowlandsandhighlandsofEcuador(EC)....... 59

xviii Figure 2.3 Results from the DAPC clustering analysis in adegenet with the outgroup samples included.PlotshowstheresultfortheoptimalKvalueinferredfromBICvaluesandcolorͲcoded

to their distribution in Latin America. Lineage abbreviations are as follows: Bothrops atrox

(ATROX),B.ayerbei(BAY),B.rhombeatus(BRH),MexicoandNuclearCentralAmerica(MNCA),

PacificIsthmianCentralAmerica(PICA),DarienPanamaandChoco(CHOCO),MagdalenaValley

ColombiaandVenezuela(MVV),HighlandsEcuador1(HEC1),HighlandsEcuador2(HEC2),and

PacificEcuador(PEC)...... 60

Figure2.4UltrametrictreeofuniquecytͲbandND4haplotypes.Asterisksabove/belownodesof

majorcladesrepresentBayesianposteriorprobabilityvalues>0.95.Verticalbarstotherightof

terminalbranchesrepresentcoalescentunitsrecoveredbythebGMYCalgorithm,colorͲcoded

accordingtotheirposteriorprobabilities:yellow=0.5<p<0.9,orange=0.9<p<0.95,red=0.95

<p<1.Namesindicategeneraldistributionareas....... 61

Figure 2.5 Results from the kͲmeans clustering analysis applied to the morphological data of females(resultsformalesweresimilarandarenotshown).Thefirsttwoprincipalcomponents wereextractedandcoloredbythekͲmeansresults(above).ColorͲcodeisasfollows:Mexicoand

NuclearCentralAmerica(black),CostaRica,PanamaandmostsamplesfromSouthAmerica(red), andBothropsayerbeispecimensandhighlandsofEcuador(green).PlotfortheoptimalKvalueof threegroups(below)...... 62

Figure2.6ConsensusspeciestreesobtainedinSNAPP(left)andSVDquartets(right)basedonthe models supported by BFD*. Bars indicate the divergence of lineages from Central America

(above), those present mainly in Colombia (middle), and those mainly from Ecuador (below).

Posteriorprobabilitiesandbootstrapsupportvaluesareshownonnodes.Abbreviationsareas

xix follows:MexicoandNuclearCentralAmerica(MNCA),PacificIsthmianCentralAmerica(PICA),

DarienPanamaandChoco(CHOCO),MagdalenaValleyColombiaandVenezuela(MVV),Highlands

Ecuador1(HEC1),HighlandsEcuador2(HEC2),andPacificEcuador(PEC)....... 63

Figure2.7Canonicalvariateanalysisplotsforfemales(A)andmales(B).Lineagescorrespondto thoseshowninFigure2.3....... 64

Figure 3.1 Topographic map of Ecuador (left) showing sampled populations from the Pacific lowlandsandthemontanelineagesdescribedinthetext:PEC(redtriangles),HEC1(blacksquare)

and HEC 2 (green circle). Two populations (north and south) were used for the PEC lineage.

Isolationmodels(A,B)andisolationwithmigrationmodels(C,D)testedinfastsimcoal....... 96

Figure3.2Structureplot(K=2)(above)andresultsfromtheDAPCclusteringanalysisinadegenet

(K=3)(belowright)generatedfrom1,241polymorphicloci.Inplot(belowleft)showstheresult fortheoptimalKvalueinferredfromBICvalues....... 97

Figure3.3MLpopulationtreeinferredwithTreeMix.Onlyonetreeisshownbecauseweobtained similar results when one to five migration events were allowed. Graph depicts splits among differentpopulationsandtheweightassociatedwithmigrationevents(redindicatesahigher weight).Numbersatnodesindicatebootstrapsupport....... 98

Figure 4.1 Geographic distribution of Bothrops lojanus in Ecuador; provinces are named and outlined.Localityrecordsfromtheliterature(reddots;bluedotrepresentsthetypelocalityfor thespecies:Loja,Lojaprovince),extremepopulationsregisteredduringthisstudy(triangles),and

materialfromPeruwithuncertaintaxonomicaffiliation(square)(CampbellandLamar,2004)are shown....... 122

xx Figure4.2PredictivedistributionmapsforBothropslojanusmodeledinMaxent3.3.2.Original predictivemap(above)usingsixlocalities(whitesquares)fromsouthernEcuador;areaswithhigh

probability(>85%)ofspeciesoccurrenceareshowninredandorange.Predictivedistribution mapswithninelocalitiesforB.lojanussensustricto(left)and12localities(right)forbothclades recoveredinthephylogeneticanalyses....... 123

Figure4.3ABayesianmitochondrialphylogramandhaplotypenetwork(left).Numbersabove branchesareBayesianposteriorprobabilities,whereasnumbersbelowaremaximumͲlikelihood bootstrappercentages.Branchsupportindicesarenotshownformostsubcladestopreserve

clarity.Thephysicalmapontherightshowsthemainareassampledduringthiswork;notethe presenceoftheTiolomaandCordoncillomountainrangeseparatingtheCladeBlocality(Gulag,

Azuayprovince)...... 124

Figure4.4DistributionofB.lojanusfemale(A)andmale(B)specimensalongthefirstandsecond principal component axes. Individuals representing B. lojanus sensu stricto (light blue) and B.

lojanusCladeB(red)areshowninpanelAandBleft.AcomparisonofB.lojanusmaleswith

specimensfromthePeruvianpopulation(pink)areshowninpanelBright....... 125

Figure 4.5 Map of southern Ecuador showing the known distribution of Bothrops lojanus.

Minimumconvexpolygonsareshownforbothlineagesidentifiedwithphylogeneticmethods

(bluedottedline),B.lojanussensustricto(orange)andCladeB(red)....... 126

FigureC.1OptimalKvaluesasidentifiedbythedeltaKmethodofEvannoetal.(2005)inStructure

Harvester(A)andlowestvaluesofBICscoresinadegenet(B)...... 138

xxi Figure F.1 A Bayesian mitochondrial and nuclear phylogram. Numbers above branches are

Bayesianposteriorprobabilities....... 143



xxii 



 Chapter1:Introduction 

TheNeotropics—thegeographicalregionextendingfromcentralMexicoto southernBrazil—harborsthelargestportionofthebiodiversityontheplanet(Antonelli andSanmartín,2011;Myersetal.,2000;Rull,2011).Theoriginandmaintenanceofthis diversitydependsonsynergisticenvironmentaldriversthathaveoperatedatdiverse spatialandtemporalscalesindifferentgroupsoforganisms(Rull,2008;TurchettoͲZolet etal.,2013).Itisnowbelievedthatthecurrentdiversityanddistributionofmodern lineagesintheregionisinlargeparttheresultofcomplexecologicalandevolutionary trendsdeterminedbytheinterplayofNeogeneorogeniceventsandPleistoceneclimatic oscillations.However,recentstudieshavealsostressedtheimportanceofassessingthe influenceofdemographicprocessesinthestructuringofpresentͲdayphylogeographic patterns(BrumfieldandEdwards,2007;HarveyandBrumfield,2015;Smithetal.,2014).

Therefore,tofullyunderstandthetimingandpotentialdriversofneotropical biodiversity,alargernumberofnaturalsystemsneedtobeexploredwithimproved datasetsandanalyticalapproachestobettercharacterizetheevolutionaryhistoryof organismsinthisregion(Rull,2013).

NeotropicalsnakesofthefamilyViperidae(i.e.,pitvipers)aremodelorganisms toexplorehistoricalpatternsandecologicalprocessesthatmolddiversityinthisregion. 1 Thesesnakespossessseveralcharacteristicsthatmakethemidealforsuchstudies:1)

PitvipershaveradiatedextensivelyintheNewWorldandcurrentlyoccupyawiderange ofenvironmentsandvegetationtypes(CampbellandLamar,2004);2)Theirlowvagility andstrongresponsetolocalenvironmentalfactorsmakethemwellsuitedforstudies assessingtheimpactofenvironmentandgeographyonlineageformation(Pyronand

Burbrink,2009);3)Duetotheirimpactonpublichealth,arobusthypothesisof phylogeneticrelationshipsexistsformostgroupsofpitvipers(QuijadaͲMascareñasand

Wüster,2010;Wüsteretal.,2008),and4)Distinctlineagesaredistributedwidelyacross

NorthandSouthAmericaorbroadlycoͲdistributedinspecificregionsmakingthemgood choicesfordiversephylogeographicquestions(Castoeetal.,2009;Wüsteretal.,2005).

Inaddition,theirvenomshaveevolvedasaresultoftheactionsofdifferent evolutionaryandecologicalforces(Gibbsetal.,2013;Mackessy,2010;Mebs,2001;

SalazarͲValenzuelaetal.,2014),addingawholenewlevelofphenotypicdiversitylikely shapedbysimilarprocesses.

Severalofthecharacteristicsmentionedabovearepresentinneotropical pitvipersofthegenusBothrops.ThisgroupismainlypresentinSouthAmericaand constitutesoneofthebestͲstudied,wideͲrangingneotropicalsnakeclades,dueto extensivestudiesonvenomcompositionandpathophysiologicalactionoftheirtoxins

(BeamanandHayes,2008;Fenkeretal.,2014).Nevertheless,somelineageswithinthe genusarepoorlyknownbecauseoftheirrestricteddistributionsintheAndesofSouth

2 Americaorbecausetheyconstitutespeciescomplexeswhosephylogeneticaffinities havebeendifficulttoestablishwithtraditionalmethodsanddatasets.

Here,Iuseacombinationofmolecular,morphological,andgeographicaldatato explorediversificationpatternsandtheevolutionarymechanismsimplicatedinthe divergenceoftwodistinctmembersofthegenus.First,Iconcentrateonthewidely distributedBothropsasperspeciescomplex,whichcomprisesagroupof morphologicallysimilarpopulationsofsnakespresentinMesoamericaand northwesternSouthAmericathatdivergedrecently(~3Mya).Theyarealsotheleading causeofsnakebiteaccidentsacrosstheirdistributionandamodelorganismin toxinologicalresearch.Incontrast,theLojanlancehead(B.lojanus)isanendangered pitviperwithanextremelylocalizeddistributioninthehighlandsofsouthernEcuador.

LittleinformationisavailableforthisspeciesandhereIdocumentdifferentaspectsof itsbiologytoassessitsconservationstatus.

InChapter2,IusehighdimensiongeneticdataandcoalescentͲbasedspecies delimitationmethodstodetectcrypticlineagesandidentifyspeciesboundariesintheB. asperspeciescomplex.Ialsoanalyzedmorphologicaldatacollectedacrossthe distributionofthegrouptoassessthedegreeofassociationbetweenphenotypicand genotypicdata.Myresultsindicatethatpitviperpopulationsbelongingtothisspecies complexshowextensivephylogeographicstructure,suggestingtheinfluenceof geographicbarriersand/ordifferencesinecologicalnichesintherecentdiversification ofthegroup.Ialsofoundthatthereisgeneticandmorphologicalevidenceforadeep

3 divergencebetweenaCentralandSouthAmericanclade,andthatmorerecently divergedgroupsinSouthAmericashowcomplicatedpatternssuggestiveofrecent divergenceand/orgeneflowamonglineages.

SomeofthosecomplicatedphylogeneticpatternsfoundintheB.asperspecies complexareexploredinChapter3withanalysesthatgobeyondtreeͲbasedmethods.I usegenomicdataandapopulationͲlevelsamplinginsouthwesternEcuadortoperform modelͲbasedanalysesandinvestigatetherecentoriginoftwoEcuadorianmontane lineagesofthesepitvipers.Mygoalwastodisentanglewhatrolesthecontrasting factorsofgeneflowandisolationbydriftplayduringtheprocessofdivergenceand resolvesomeofthediscrepanciesofevolutionaryrelationshipsfoundinChapter2.I foundstrongsupportfortheindependentoriginofmontanelineagesanddetected evidenceformigrationafterdivergenceonlybetweenalowlandlineageandoneofthe highlandlineages.Theothermontanelineagehasbeenisolatedforapproximately

200,000yearsanditsrecognitionatthespecieslevelispossiblywarranted.

Finally,inChapter4Idevelopspeciesdistributionmodelsbasedoninformation fromrecentfieldsurveysandmuseumspecimenstoassessthepotentialdistributionof

B.lojanusintheAndesofsouthernEcuador.Myresultsshowthattherangeofthe speciesislargerthanpreviouslysuggested.AnalysesofmitochondrialandnuclearDNA lociidentifytwodistinctgeneticgroupswithinthiscurrentlydescribedsinglespecies thatshouldeachhavestatusasseparateconservationunits.Morphologicalanalyses

4 showthattherearedifferencesincharactervariationthatmirrorstoalimitedextent thegeneticdifferences.

Overall,myexplorationoftheevolutionaryhistoryofthesepitvipersprovides insightsintotheevolutionaryforcesthatpromotetropicalbiodiversitybutalsopractical informationthatisusefulfortheirconservationandinaidofmanagingtheirbiomedical effectsthroughenvenomationofhumans.



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7      Chapter2:AnalysisofgenomicǦlevelvariationprovidesinsightintotherecent diversificationofawidespreadtropicalsnake,the“ultimatepitvipers” (Bothropsasperspeciescomplex) 

Abstract

GenomicͲlevelanalysesoftropicalvertebrateshavebeenincreasinglyusedto detectcrypticlineagesbuthaverarelybeenappliedtotropicalreptileswithbroad geographicdistributions.TheBothropsasperspeciescomplexcomprisesagroupof morphologicallysimilarpopulationsofsnakesdistributedinMesoamericaand northwesternSouthAmerica.Theyaretheleadingcauseofsnakebiteaccidentsinparts ofLatinAmericaandareamodelorganismfortoxinologicalresearchonvenom compositionandphysiologicalactionofsnaketoxins.Pastworkhassuggestedthe strongpossibilitythatthis“species”consistsofmultipledistinctlineagesyetanalyses thatusegenomicͲleveldatacombinedwithnewlydevelopedspeciesdelimitation analyseshaveyettobeappliedtothesesnakestoevaluatethispossibility.Here,weuse recentlydevelopedreducedͲrepresentationgenomesequencingmethodstoidentify

SNPsacross864polymorphiclocifor52B.aspersensulatoindividualsdistributed acrossthegeographicrangeofthegroup.Clusteringandphylogeneticmethodswere usedtoidentifygeneticstructureinthedataset.Wethencombinedthisinformation

8 withevidencederivedfrommitochondrialDNA,morphologicalvariation,andcurrent taxonomytotestspeciesdelimitationmodelsunderaBayesianframeworkandestimate speciestreesemployingthemultispeciescoalescentmodel.Ourresultsshowedthat pitviperpopulationsbelongingtotheB.aspercomplexshowextensivephylogeographic structure(7Ͳ10lineages),suggestingtheinfluenceofgeographicbarriersand/or differencesinecologicalnichesintherecentdiversificationinthegroup.Wealsofound thatthereisgeneticandmorphologicalevidenceforadeepdivergencebetweena

CentralandSouthAmericanclade,andthatmorerecentlydivergedgroupsinSouth

Americashowcomplicatedpatternssuggestiveofrecentdivergenceand/orgeneflow amonglineages.Finally,wedemonstratethatthecurrenttaxonomyforthegroupdoes notreflectitsevolutionaryhistory,andexpectthatthattheevolutionaryhypotheseswe providehelppromotetheestablishmentoffuturestudiesfocusedondifferentaspects ofthebiologyoftheseanimals,aswellascontributetoabetterunderstandingand managementoftheirbiomedicaleffects.

Keywords:Crypticspecies,morphology,multispeciescoalescent,Neotropics, phylogenomics,RADsequencing,speciesdelimitation.









9 Introduction

Identifyingcrypticdiversity–uniqueevolutionarylineagesthatarehiddenunder onespeciesname–isamajorchallengeinsystematicandevolutionarybiology(Adams etal.,2014;Mayden,1997;PfenningerandSchwenk,2007;Smithetal.,2011).Reasons fortheexistenceofcrypticlineagesincludeselectiveordevelopmentalconstraintsthat promotemorphologicalstasisandtheinabilityofresearcherstodifferentiaterelated taxaduetoabiastowardsvisuallydiscriminatingfeatures(Bickfordetal.,2007;

MalhotraandThorpe,2004).Failuretorecognizethesehiddenlineageshasimplications foraccurateassessmentsofbiodiversity,comparativeevolutionaryandecological studies,conservationefforts,biologicalcontrolapplications,bioprospectingventures, andpublichealthprograms(BeheregarayandCaccone,2007;Bickfordetal.,2007).

Crypticdiversityfrequentlyinvolvesrecentlydivergedlineagesforwhichmultiple sourcesofdataandanalyticalapproachesmaybecriticalforreliabledelimitation

(Carstensetal.,2013;RittmeyerandAustin,2015).Recently,thisareaofresearchhas seenmajorbreakthroughsduetotheincreasingavailabilityofhighdimensiongenetic datasetscombinedwithmethodsofanalysisthataccommodatesomeofthecausesof incongruencebetweendifferentgenegenealogies(Edwards,2009;Fujitaetal.,2012;

McCormacketal.,2013;Wiens,2007).

Asanexample,newlygeneratedgenomeͲscaledatasetsareimprovingthe inferencesresearcherscanmakeaboutthehistoricaldiversificationofnonͲmodel organisms(HarveyandBrumfield,2015).Theavailabilityofalargernumberofvariable 10 sitesfrommoreregionsofthegenomehasprovidedtobeespeciallyusefulfor phylogeography,populationgenetics,andspeciesdelimitationduetotheincreased accuracyandprecisionofparameterestimation(Leachéetal.,2015;McCormacketal.,

2013;Pyron,2015).However,methodsarestillbeingdevelopedandinthecaseof coalescentͲbasedspeciesdelimitationonlyafewapproachessofarareabletoutilize thewealthofgenomicdatafortheassessmentofcrypticlineagehypotheses(Fujitaet al.,2012;Leachéetal.,2014;YangandRannala,2014).Atshallowlevelsof diversificationsuchmethodsareworthexploringbecausetheymergeapproachesfrom phylogeneticsandpopulationgeneticsandcanprovideinsightintospeciesdelimitation

(Carstensetal.,2013;Satleretal.,2013).Nevertheless,otherlinesofevidence(e.g., morphological,behavioral,and/orecologicaldata)alsoremainrelevantand complementarytogeneticdatasets(Carstensetal.,2013;SchlickͲSteineretal.,2010;

Wiens,2007;butseeMeiketal.,2015).

Lineageidentificationisespeciallyproblematicinsystemswhere(1)rapidand recentdivergencesorinterͲlineagehybridizationhaveproducedgroupsthatarewell differentiatedmorphologicallybutnotgeneticallyand(2)systemsthatarecomposedof geneticallydifferentiatedgroups,whicharemorphologicallysimilar(Barleyetal.,2013).

Thelattercharacterizecrypticspeciescomplexesinhabitingwidespreadgeographic distributions,whichduetologisticalconstraintsarerarelyanalyzedacrosstheirentire distributionsorusingalargenumberofmolecularmarkers(Funketal.,2012;Geharaet al.,2014).Nevertheless,suchsystemsalsorepresentgreatopportunitiestobetter 11 understanddiversificationprocesses,especiallywhentheirrangesincludedifferent biomesindiverseregionsoftheearth.

Worldwide,onlyafewvenomoussnaketaxaareresponsibleformostcasesof humanmorbidityandmortality(Gutiérrezetal.,2006).IntheNeotropics,theBothrops asperspeciescomplexisonesuchtaxonandconstituteanimportantunittoexplore lineagediversificationandspeciesboundariesforthreereasons.First,thesesnakesare theonlymembersofthegenustoalsobepresentinCentralAmericaasfarnorthas centralMexico(CampbellandLamar,2004);thus,insightsintotheevolutionaryhistory ofthegroupcouldbeenlighteningfromabiogeographicalperspectiveoftheGreat

AmericanInterchange.Second,theyaretheleadingcauseofsnakebiteaccidentsacross theirwidespreaddistributioninLatinAmerica(OteroͲPatiño,2009;Warrell,2004), whichcoupledtotheirlargebodysizeandreadinesstodefendthemselveshavebeen arguedasreasonstocallthemthe“ultimatepitvipers”(Hardy,1994;Sasaetal.,2009).

Finally,theseanimalshavebecomeamodelorganismintoxinologicalresearchdueto numerousstudiesonvenomcompositionandpathophysiologicalactionthatdateback tothefirsthalfofthe20thcentury(Gutiérrez,2009).Unfortunately,mostofthis informationisbasedonpooledvenomsamplesfromrestrictedareasacrossthe distributionofthegroupandknowledgeabouttheevolutionaryhistoryofthecomplex haslaggedbehindthestudyoftheirtoxins.Previouslyundetectedgeneticdivergence andphylogeneticaffinitiesinthegroupshouldpromptareevaluationofpatternsand causesofvenomvariation,aswellasaidindefiningtheappropriatemixtureofvenoms 12 forimmunizationtoproducemoreeffectiveantivenomsandexpandourunderstanding ofclinicalmanifestationsofsnakebiteaccidentsproducedbytheseorganismsin humans(Fenwicketal.,2009;Gutiérrez,2014;Williamsetal.,2011;Wüsteretal.,

1997a).Therefore,aphylogeneticframeworkfortheB.asperspeciescomplexisneeded inordertopromotefuturecomparativetoxinologicalstudies,understandits biogeographichistory,andevaluatetaxonomicissuescurrentlypresentinthegroup.

TwopreviousstudieshaveexploredgeneticlineagedivergenceintheB.asper complex.Saldarriagaetal.(2009;in.prep.)estimatedphylogeneticrelationshipswithin thegroupbasedonmitochondrialDNA(mtDNA)data.Theseauthorsfoundeight phylogroupspresentinthecomplexandtimedtheirdiversificationtohaveoccurred duringthelast3Myr.However,thesestudiesdidnotincludesamplesfromlineages previouslyhypothesizedtobepartofthespeciescomplex(i.e.,B.ayerbeiandB. rhombeatusFollecoͲFernández,2010;seesection2.1)andtheyalsorecovered uncertainrelationshipsbetweenputativecladesthataredifficulttoreconcilein biogeographicalterms(e.g.,individualsfromEcuadorclusteredwithindividualsfrom

NuclearCentralAmerica).ThelattercouldprobablybearesultofthelongͲrecognized problemsassociatedwiththeexclusiveuseofthismarkerforsystematicinquiries(i.e., evolutionofthemitochondrialgenomemaynottrackphenotypicevolutionbecauseof lineagesortingandhybridizationandintrogressionevents)(ToewsandBrelsford,2012).

Here,weexploretherecentevolutionaryhistoryofthegroupusinghighdimension geneticdatageneratedthroughareducedͲrepresentationgenomesequencing 13 technique(RestrictionsiteͲassociatedDNAsequencing,RADseq)(Daveyetal.,2011;

Etteretal.,2011;Milleretal.,2007)andanalyzedwithnewlydevelopedcoalescentͲ basedspeciesdelimitationmethods.Thisapproachhasbeensuccessfullyappliedtoa rangeoforganismstoexplorerecentdiversificationeventsbydetectingcrypticlineages andresolvingtheirphylogeneticrelationships(Emersonetal.,2010;McCormacketal.,

2012;RittmeyerandAustin,2015;Wagneretal.,2013).Ourgoalsinthisstudyareto(1) identifyuniquegeneticgroupingspresentintheB.asperspeciescomplexusingsamples collectedfromacrossthespeciesrange,(2)establishphylogeneticrelationshipsamong thesegroups,(3)testdifferentspeciesdelimitationmodelsusingsinglelocusand genomicͲscaledatasets,and(4)assessthecorrespondenceofmorphologicaldivergence betweenidentifiedunitsinordertodelineatespeciesboundaries.



MaterialsandMethods

Taxonsampling

TheBothropsasperspeciescomplexiscomposedofmorphologicallysimilar populationsofpitvipersnakeswhosesystematicshavebeenhistoricallyconfusing

(CampbellandLamar,1989,1992;Wüsteretal.,1996).Severaltaxaweredescribedin thesecondhalfofthe19thcenturybasedonspecimensfromdifferentpopulations throughoutthedistributionofthegroup(McDiarmidetal.,1999).Untilrecently,allof thosenamesweresynonymizedwithB.asperalthoughsomeresearchersarguedforthe existenceofacomplexofspecies(CampbellandLamar,2004).FollecoͲFernández(2010) 14 analyzedmorphologicalvariationinalimitednumberofspecimensfromsouthwestern

ColombiaanddecidedtoresurrectthenameB.rhombeatusforpopulationsfrominterͲ

AndeanvalleysofcentralandnorthernColombiaanddescribeB.ayerbeifromaninter

AndeanͲvalleyofsouthernColombia.Thesethreetaxarepresentthecurrenttaxonomy ofthegroup(UetzandHosek,2015;Wallachetal.,2014).

Weobtainedbloodortissuesamplesfrom114B.aspersensulatoindividuals acrossthespeciescomplexrange,whichextendsfromcentralMexicoalltheway throughCentralAmericaandintoVenezuelaontheeastandPeruonthesouthin northwesternSouthAmerica(CampbellandLamar,2004)(Fig.1).Oursamplingtargeted snakesfromallthemitochondriallineagesidentifiedbySaldarriagaͲCórdobaetal.

(2009;in.prep.),aswellastaxaandgeographicregionsnotavailabletothem(e.g.,B. ayerbei,B.rhombeatus,westernVenezuela,southwesternColombia,northwestern

Ecuador).Specimensfromtypelocalitiesofmostoftheothertaxacurrently synonymizedwiththenameB.asperorfromgeographicareaswithincloserangeto themwerealsoincluded.BasedonFenwicketal.(2009),Parkinsonetal.(2002),

SaldarriagaͲCórdobaetal.(in.prep.)andWüsteretal.(2002),weusedtwoB.atrox samplesfromeasternEcuadorandAmazonianBrazilasoutgroupsforphylogenetic analysesbasedonRADseqdata,aswellassequencesofB.atrox,B.barnetti,B. caribbaeus,B.lanceolatus,B.osborneiandB.punctatusspecimensforthemtDNA dataset.

15 Additionally,werecordedmeristicandmorphometriccharacters(AppendixA) forB.aspersensulatoindividualsfrom82SouthAmericanlocalities.Datafromthese specimensincludedmostoftheindividualsgenotypedwiththeRADseqprotocoland werecombinedwithasubsetofthosereportedbySasa(2002)forCentralAmerican populationsoftheB.aspercomplex(Fig.2.1).Weexaminedpreservedandanesthetized livespecimenshousedininstitutionsfromtheUnitedStates,Colombia,andEcuador.A totalof600specimenswereexamined,comprising340femalesand260males.

Conceptualapproach

 Ourtheoreticalspeciesconceptisderivedfromtheevolutionaryspeciesconcept

(Simpson,1961;Wiley,1978)andthegenerallineageconceptofspecies(deQueiroz,

1998,2007).Weseektoidentifyuniquelyevolvingevolutionarylineagesasspecies usingoperationalcriteriainthecontextofcoalescenttheoryandmorphological distinctiveness(Fujitaetal.,2012).Therefore,themultiplesourcesofdataandmodels usedinthisstudyaretakenaslinesofevidencethatwhencongruentjustifythe conservativedecisionofrecognizingsuchlineagesasspecies(Carstensetal.,2013;

Padialetal.,2010;SchlickͲSteineretal.,2010).

Geneticdata

WeextractedgenomicDNAfromeachsampleusingeitheraQiagenDNAblood andtissuekit(Qiagen,Valencia,CA,USA)orliquidͲliquid(phenolͲchloroformand guanidiniumͲisothiocyanate)protocols.TheconcentrationofDNAisolateswas

16 examinedonaQubit2.0fluorometerusingadsDNABRassaykit(LifeTechnologies,

Carlsbad,CA,USA)oronaNanoDropNDͲ1000(NanoDropTechnologies,Wilmington,

DE,USA).

Genomiclibrarypreparation,sequencing,andbioinformaticmethods

 FiftyͲtwoB.aspersensulatoindividualsfrom48differentlocalitiesacrossthe speciescomplexrangewereincludedinourRADseqprotocol(Table2.1).Overall,2–10 individualsfromeachofSaldarriagaͲCórdobaetal.(in.prep.)mitochondriallineagesand taxainthecurrenttaxonomyofthegrouprepresentedourdataset.WefollowedSovic etal.(in.prep.)fortheconstructionofdoubleͲdigestRADseqlibraries(DaCostaand

Sorenson,2014;Petersonetal.,2012).Briefly,themethodconsistsofthefollowing steps:1)digestionofapproximately250ngofDNAforeachindividualusing15unitsof

EcoRIandSbfIrestrictionenzymes(NewEnglandBiolabs,Ipswich,MA,USA),2)ligation ofIlluminauniquebarcodedadapterstoeachDNAsample,3)sizeselectionof fragmentsrangingbetween300and450bpbyextractionfromagarosegels,4)qPCR quantification(KAPABiosystemskit,Wilmington,MA,USA)ofgelextractionproductsin ordertopreventhighlevelsofmissingdata;aminimumthresholdof150,000molecules waschosenandsamplesnotattainingthisnumberwerediscardedfromthelibraryand preparedagain,5)PCRamplificationofthelibrariesusingaPhusionpolymerasekit

(NewEnglandBiolabs),and6)purificationoftheproductswithAmPurebeads(Beckman

CoulterInc.,Pasadena,CA,USA)andasecondqPCRquantificationinordertopool equimolarconcentrationsofeachindividualintoasinglelibrary.Sequencingwas 17 performedin50Ͳbprunsusing10–20%ofalaneofanIlluminaHiSeq2000atthe

GenomicsSharedResourceoftheOhioStateUniversityComprehensiveCancerCenter.

 ThepipelineAftrRAD4.1(Sovicetal.,2015)wasusedtoassembleandgenotype theRADseqdata,aswellastoproduceinputfilesfordownstreamanalyses.Weused defaultsettings,exceptfortheparametersdescribedbelow.Onlylociscoredinatleast

95%oftheindividualswereretained;thislevelwaschosentobothreducetheeffectsof alleledropout(Arnoldetal.,2013;Gautieretal.,2013)andtokeepsomelociscoredin allingroupsamplesbutmissinginoutgroupindividualsduetopolymorphismat restrictionsites.Amaximumoffourindelswereallowedbetweenreadstoconsider themalternativeallelesfromthesamelocusandaminimumoffivereadswasrequired atagivenlocustocallagenotype.Finally,inordertoavoidspuriousSNPsthatformat theendofreadsduetolocusassemblymethods,onlySNPsoccurringinthefirst34 positionswereretainedafterremovalofbarcodesandrestrictionsites(Sovicetal.,

2015).

MitochondrialDNAdata

WeusedPCRtoamplifythecytochromeb(cytͲb)andNADHdehydrogenase subunit4(ND4)mtDNAfragmentsusingtheGludgandAtrCB3(Parkinsonetal.,2002) andND4andLEU(Arévaloetal.,1994)primerpairs,respectively.Sequenceswere obtainedfor109B.aspersensulatoindividuals,includingallanimalsusedintheRADseq protocol.TheywerecoupledwiththedatasetusedbySaldarriagaͲCórdobaetal.(in.

18 prep.),whichconsistedofsequencesforthesamemarkersfrom111B.asper individuals.ThefinalmtDNAdatasetconsistedofbothpublishedandnewsequences: cytͲb(116/246newsequences)andND4(113/231newsequences)fortheB.asper sensulatoingroupandsixspeciesoftheBothropsgenusthatwereusedasoutgroup taxa.

AmplificationreactionsusedeitherBioMixRedmastermix(BiolineInc.,

Springfield,NJ,USA)orindividualreagents(PlatinumTaqDNApolymerase,dNTPmix) fromLifeTechnologies.ThecytͲbfragmentswereamplifiedusinganinitial2.5min denaturationcycleat95°C,followedby30sdenaturingat95°C,1minannealingat45°C and1.5minextensionat68°Cfor2cycles,followedby30sdenaturingat95°C,30s annealingat48°Cand45sextensionat72°Cfor40cycles,followedbya15min extensionat72°C;ND4amplificationconditionsinvolvedaninitial5mindenaturation cycleat95°C,followedby30sdenaturingat94°C,45sannealingat52°Cand1min extensionat72°Cfor38cycles,followedbyafinal5minextensionat72°C.PCR purificationswereperformedusingExoSAPͲIT(Affymetrix,Cleveland,OH,USA)ora polyethyleneglycolprotocol.Sequencingreactionsforforwardandreversestrands wereconductedusingtheBigDyeterminatorcyclesequencingkit(LifeTechnologies) andproductsweresequencedbyMacrogenInc.(Seoul,SouthKorea)oranalyzedonan

ABI3100GeneticAnalyzer.Complementarysequenceswereassembledandeditedwith

CodonCodeAligner4andweusedMUSCLE(Edgar,2004)inGeneious7.0toalignthe sequencesusingdefaultsettings. 19 Speciesdiscoverymethods

InitialhypothesesaboutthenumberandidentityoflineagespresentintheB. asperspeciescomplexwereformulatedbyanalyzingourgenomicdatasetwithtreeͲ basedandclusteringmethods,ourmtDNAdatawithamodelͲbasedmethodknownas thegeneralmixedYuleͲcoalescent(Ponsetal.,2006),andourmorphologicaldataset usingastatisticaltransformationandclusteringmethod.Theseapproachesare collectivelyknownasspeciesdiscoverymethodssincetheydonotrequiredatatobe assignedaprioritoputativegroups.Forconsistencywithpublishedstudies(SaldarriagaͲ

Córdobaetal.,2009;in.prep.),wheneverpossibleweusednamesandabbreviationsof phylogroupspreviouslyusedinthisspeciescomplex.

First,weimplementedamaximumlikelihood(ML)approachtotheconcatenated matrixofSNPsderivedfromtheRADseqmethodinordertoestimatephylogenetic relationshipsamonglineagesofthesepitvipers.InRAxML8.0(Stamatakis,2014)we usedtheGTRGAMMAsubstitutionmodelandperformed1,000bootstrapreplicatesina rapidbootstrapanalysis.Twoclusteringmethodswereusedtoevaluatethegenetic structurepresentintheRADseqdataset.AmatrixcontainingbiͲallelicdatafromeach sampleandselectedfromthefirstSNPofeachlocuswasobtainedfromAftrRAD.We firstemployedtheBayesianalgorithmimplementedintheprogramStructure(Pritchard etal.,2000),whichclusterssamplesintopopulationsbyminimizingHardyͲWeinberg disequilibrium.Weusedanadmixturemodelanditerativelyconductedfive independentrunsofKvaluesrangingfrom1–20withaburnͲinof100,000generations 20 andeachanalysissamplingevery100iterationsfor1milliongenerations.Structure

Harvester(EarlandvonHoldt,2012)wasusedtoimplementtheȴKstatisticofEvannoet al.(2005)inordertoidentifyanappropriatenumberofclusters.Resultswere summarizedwithCLUMPP1.1.2(JakobssonandRosenberg,2007)usingtheFullSearch algorithmandvisualizedwiththeprogramdistruct1.1(Rosenberg,2004).Additionally, weusedthekͲmeansclusteringmethodavailableinadegenet1.4Ͳ2(Jombart,2008;

JombartandAhmed,2011).Thisprogramidentifiesthemostappropriateclustering solutionsbasedonBayesianinformationcriterion(BIC)scoresfromaxesderivedfroma

PrincipalComponentsAnalysis(PCA),andthereforedoesnotrelyontheHardyͲ

WeinbergassumptionsthatStructureuses.WeevaluatedKvaluesrangingfrom1–40 andperformedadiscriminatefunctionanalysisofPCAs(DAPC)basedontheoptimal clusteringsolutionsuggestedbyadegenet.Thesefunctionsareavailableintheade4 packageandwereconductedinR3.1.3(RCoreTeam,2015).

 Inaddition,weappliedasinglelocusdiscoverymethodtoourmtDNAdataset, whichconsistsofaBayesianimplementationofthegeneralmixedYulecoalescent

(bGMYC)model(Ponsetal.,2006;ReidandCarstens,2012).Thegoalofthisapproachis tomodelthetransitionpointbetweenallelecoalescenceandcladogenesisonan ultrametricphylogeny,incorporatinggenetreeuncertaintybysamplingoverthe posterioroftheoutputgenetrees.WefollowedCastoeandParkinson(2006)forthe partitionschemeofcytͲbandND4fragmentsandgeneratedultrametrictreesinBEAST

1.8.2(Drummondetal.,2012)usinguniquehaplotypespresentinthisdataset. 21 SubstitutionandclockmodelswereunlinkedforeachpartitionandweappliedthebestͲ fitsubstitutionmodelasidentifiedusingtheBICimplementedinjModeltest2.1.7

(Darribaetal.,2012)andusedanuncorrelatedlognormalrelaxedclock,respectively.

Treemodelsontheotherhandwerelinkedacrosspartitionsandacoalescentconstant ratemodelwasappliedassuggestedbyMonaghanetal.(2009).AMarkovchainMonte

Carlo(MCMC)simulationwasrunfor50milliongenerations,samplingtreesevery5000 generations.Weselectedarandomsampleof100ofthelast500genetreesestimated inBEASTandusedthemasinputforthebGMYCpackage1.0.2inR.Thelatterprogram wasusedwithdefaultsettingsspecifyingthestartingnumberofspeciestohalfthetotal numberoftipsanditwasrunfor50,000generations,withaburnͲinof50%,and sampledevery100thgeneration.

 Finally,weperformedanexploratoryanalysisofthemorphologicalvariation presentintheB.aspercomplexinordertoidentifystructurethatcouldpotentially correspondtospeciesgroups.WeconductedaPrincipalComponentAnalyisis(PCA)inR withthefunctionprcompinthestatspackage.Onlyindividualswithcompletecounts andmeasurementswereconsidered(AppendixB),anddataforfemalesandmaleswas consideredseparatelyduetosexualdimorphismpresentinpitvipersnakes.Wealso conductedakͲmeansclusteringanalysisinRacrossthefirsttwoPCscoreswiththe preferredsolutionofclustersbeingidentifiedbyplottingthewithingroupssumsof squaresagainstthenumberofextractedclusters.

22 CoalescentͲbasedspeciesdelimitation

 Basedonevidenceobtainedwiththeproceduresdescribedabove,wetested sevendifferentspeciesdelimitationhypothesesinaspeciescoalescentframework

(Table2.2).WeusedBFD*,whichisaBayesfactordelimitationapproachadaptedfor genomeͲwideSNPdata(Leachéetal.,2014).Thisapproachtestsdifferenthypotheses ofspeciesgroupingsbycomparingtheirmarginallikelihoodestimates(MLE)through pathsamplinganalysesandhasbeenimplementedinthespeciestreeestimation methodSNAPP(Bryantetal.,2012)executedwithintheprogramBEAST2.2.1

(Bouckaertetal.,2014).WeselectedthefirstbiallelicSNPfromeachlocusandused defaultsettingsforthepriordistributionsofeachparameter.Becauseofcomputational demandswelimitedoursamplingto2–5individuals(4–10chromosomes)perlineage foratotalsamplesizeof32individuals.Individualswerechosentomaximizethe geographicalrepresentationofsamples.

Thesevenmodelswererankedfromhighesttolowestbasedontheirmarginal likelihoodestimation(MLE)values.Thelatterwereestimatedviapathsampling

(LartillotandPhilippe,2006)witheachanalysisconsistingof48steps.Foreachstepwe ranachainlengthof100,000generationsand10%burnͲin,whichwassufficientto obtaineffectivesamplesizesabove200.Additionally,weevaluatedthestrengthof supportforthemodelsbyusingtheMLEresultstocalculateBayesfactors(BF).This modelselectiontoolwascomputedas2xlnBF,whereBFisthedifferenceinMLE valuesfortwocompetingmodels(Grummeretal.,2014;Leachéetal.,2014).Bayes 23 factorswerethenanalyzedusingtheframeworkofKassandRaftery(1995),which statesthatthestrengthofsupportisgivenasfollows:0<BF<2isnotconsidered support,2<BF<6ispositiveevidence,6<BF<10isstrongsupport,andBF>10 representsdecisiveevidence.

Speciestreeinference

 BasedonthespeciesdelimitationmodelchosenbytheBFD*approach,we subsequentlyestimatedthespeciestreeusingSNAPPtoinferrelationshipsamongthe groupsidentifiedfromtheBFD*analysis.Weperformedthreeindependentrunsinthis programfor2millionMCMCiterations,samplingevery1,000generations,thefirst10% ofwhichwerediscardedasburnͲin.Becauseofcomputationaldemandswelimitedour samplingto2–3differentindividuals(4–6chromosomes)peridentifiedgrouponeach run.AmaximumcladecredibilityconsensustreewasgeneratedwithTreeAnnotator v.2.2.0andoutputparameterswereexaminedinTracerv.1.5(Drummondand

Rambaut,2007).

Forthesamepurpose,wealsorantheprogramSVDquartets(Chifmanand

Kubatko,2014)asimplementedinPAUP4.0a143(Swofford,2002).Thisprogramuses thecoalescentmodeltoaccountforthedifferentgenealogicalhistoriesofindividualloci bysamplingamongallpossiblequartetsofdifferenttaxa.Weincludedallthespecimens

(n=54)presentintheoriginaldataset,evaluated500,000quartetsandperformed200 bootstrapreplicates.

24 Morphologicalanalyses

Specimenswereassignedtoeachofthegroupsidentifiedaboveinorderto assessthecorrespondenceofmorphologicaldivergencebetweenrecognizedunits.Due tolackofmaterial,wewerenotabletoincludespecimensbelongingtoB.rhombeatus ortooneofthelineagesidentifiedinthehighlandsofEcuador(seeResults).Weuseda combinationofourphylogeneticanalyses(mtDNA,nuDNA)andgeographicproximityto includespecimensintotheirrespectivegroups.Individualsthatwerenoteasily assignablewerenotfurtherused;intotal,197/340femalesand124/260wereassigned tothedifferentgroups.

Significantsexualdimorphismisprevalentinpitvipersnakesandseveralofour charactersshowedthesamedifferencewithineachlineagewhenanalyzedwithatwoͲ wayANOVA;therefore,maleandfemaledatasetswereevaluatedseparatelytoavoid anyconfoundingeffectofsexinouranalyses.WetestedforsignificantbetweenͲgroup variationinmeristiccharactersusingaoneͲwayANOVAortheequivalentBrownand

ForsythetestwhenLevene’stestofhomogeneityofvariancewassignificant.

MorphometriccharacterswereadjustedtoaccountforallometriceffectsusingoneͲway

ANCOVAappliedseparatelytoeachgroup.Snouttoventlength(SVL)wasusedasthe covariateforheadandtaillengths,andheadlength(HL)forallothercharacters.Only charactersthatshowedsignificantbetweenͲgroupvariationatthe5%levelwerefurther used.

25 Multivariateanalyseswereusedtodetermineifindividualscouldbeassignedto thecorrectspeciesgroupidentifiedbymoleculardataandalsotodeterminethe morphologicalcharactersthatvarysignificantlybetweenthem.Onlyindividualswith completecountsandmeasurementswereconsideredandourfinaldatasetconsistedof

136femalesand125males.Canonicalvariateanalysis(CVA)wereappliedtomeristic charactersaftertheywerestandardizedtozeromeanandunitstandarddeviation,as wellastomorphometriccharactersforwhichtheresidualsofthelinearregressions wereemployed.AnalyseswereperformedinR3.1.3andSPSSStatisticsversion22(IBM

Corp.).



Results

GenotypingofRADseqdata

 Werecoveredameanof659,047sequencereadsforindividualsincludedinour

RADseqdataset(range:77,505–1,959,382).Themeanreaddepthperlocuswas86.8 readswhilethemedianreaddepthwas52reads.Atotalof18,461nonͲparalogousloci wereidentifiedinthedatasetthatincludedBothropsatroxsamples.Oftheseloci,

14,195weremonomorphicandtheremaining4,266containedatleastonepolymorphic site.Ofthese,864werescoredinatleast95%ofthesamplesandwereusedin subsequentanalyses.



26 Speciesdiscoverymethods

TheMLapproachusedforthegenomicdatasetrecoveredaphylogramwiththe followingfeatures:1)strongbootstrapsupport(>70)fortheearlydivergenceofthe lineagecorrespondingtoB.ayerbei;2)statisticalsupportforthereciprocalmonophyly ofaCentralAmericanandaSouthAmericanclade;and3)weaksupportforevolutionary relationshipsamongcladespresentinSouthAmerica,althoughthereisstrongsupport fortheclusteringofsamplesbelongingtotheMagdalenaValleyinColombiaand

Venezuela,B.rhombeatus,andthehighlandsofEcuador(Fig.2.2).

GeneticclusteringinStructuresuggestedanoptimalKof7withthegroups closelymatchingthosefromRAxML(Fig.2.2;Fig.C.1A).SamplesfromCentralAmerica werepartitionedinthreegroups:CaribbeanIsthmianCentralAmerica(CICA),Mexico andNuclearCentralAmerica(MNCA),andPacificIsthmianCentralAmerica(PICA);B. rhombeatusspecimenswereclusteredwiththeChocoangroupandmostofthe

Ecuadoriansampleswereclusteredinonegroup.Individualsfromthehighlandsof

Ecuador(LojaandAzuayprovinces)andColombia(B.ayerbeiandB.rhombeatus),as wellasfromthelowlandsofEcuadorshowedapatternofadmixture(Fig.2.2).

 TheDAPCapproachinadegenetalsosuggestedahighlevelofstructuringinthe

B.aspercomplex(Fig.2.3;Fig.C.1BforBICplot).Eighttotengeneticgroupswere recognizedwiththehighernumberofclustersmainlyexplainedbytherecognitionof samplesfromSouthAmericanInterͲAndeanvalleys(i.e.,B.rhombeatusinColombiaand

27 oneortwogroupsfromthehighlandsofEcuador)intodistinctgroupsnotidentifiedby thepreviousanalysis.IndividualsfromNuclearCentralAmericanwereclusteredina singlegroup.

 FortheB.asperingroup,thebGMYCanalysisreturned8entities(“species’)with aposteriordistributiongreaterthan90%and12entitieswithaposteriordistribution greaterthan50%(Fig.2.4).Mostofthesegroupingsweresimilartothoseidentifiedby adegenet,althoughthegreaternumberofsuggestedentitieswasduetothe partitioningofsamplesfromtheMagdalenaValley,PacificEcuador,andMexicoand

NuclearCentralAmericaintosmallerunits.

 Finally,threegroupswereidentifiedbythekͲmeansprocedureappliedtothe morphologicaldatainfemalesandmales(Fig.2.5).Thefirstgroupwasprimarilymade upofanimalsfromMexicoandNuclearCentralAmerica,whilethesecondandthird clusterswereamixofsamplesfromCostaRica,Panamaandmostspecimensfrom

SouthAmerica.Interestingly,individualscorrespondingtoB.rhombeatusandthe highlandsofEcuadorwerelocatedattheextremeofoneofthesegroupsinthe multivariatespace.

CoalescentͲbasedspeciesdelimitation

 OutofthesevenmodelstestedwithBFD*,thosethatpartitionedthesamples intomoregroups(modelsAͲD)wererankedhigherbasedontheirMLEscores(Table

2.2).Theyweredifferentfromeachothermainlyinwhethertheylumpedorsplit1)the

28 ChocoangroupwithB.rhombeatusspecimensand2)thePacificEcuadoriangroupwith samplesoriginatinginthehighlandsofthiscountry.WhencomparingthehighestͲ rankinghypothesis(modelA)totherest,theBayesFactorswereover179infavourof themostpartitionedmodel.Therefore,theseresultsstronglysuggestthatmodelsthat partitionthesamplesintomoregroupsareagoodfittothedata.

Speciestreeinference

 ConsensusspeciestreesobtainedwithSNAPPandSVDquartetsshowedsimilar topologies,althoughtherewereinterestingdifferences(Fig.2.6).Ingeneral,both recoveredadeepdivergenceofCentralandSouthAmericanlineages.TheSNAPPtree showedabetterresolutionasindicatedbyhighposteriorprobabilitiesformostnodes.

Accordingtothistopology,theMagdalenaValleydivergedearlierinSouthAmericaand thereisgoodsupportforthecloserelationshipofEcuadorianlineages.However, incongruencebetweenSNAPPandSVDquartetstreessuggestsunresolvedrelationships betweenspecimensfromtheChoco,B.ayerbeiandB.rhombeatus,aswellasthe relationshipbetweenlineagesinthePacificlowlandsofEcuadorandthoseinthe highlands.

Morphologicalanalyses

 Allmeristicandmorphometriccharacters,exceptforthenumberofpreocular andlorealscales,weresignificantlydifferentbetweengroupsinfemales.Inmales,all meristicandmorphometriccharacters,exceptforthenumberofsupralabial,preocular,

29 internasalanddorsalscalesoneheadbeforethevent,weresignificantlydifferent betweengroups.

 ResultsfromtheCVAsweresimilarinbothsexesalthoughthepatternwas clearerinfemales.Infemales,individualsfromMexicoandNuclearCentralAmerica differentiatedalongthefirstcanonicalvariate(Fig.2.7A)andshowedalongerheadas wellasalargernumberofintersupraocularandventralscales.Femalespecimensfrom thehighlandsofEcuadorandthoserepresentingB.ayerbeidifferentiatedalongthe secondcanonicalvariate;theyshowedalowernumberofsubcaudalandcanthalscales.

Thepatternwasnotclearinmales,althoughtherewassomedifferentiationof individualsfromMexicoandNuclearCentralAmericaalongthefirstcanonicalvariate

(Fig.2.7B).



Discussion

 Themainresultsofourstudyarethat1)pitviperpopulationsbelongingtothe

Bothropsaspercomplexshowextensivephylogeographicstructuresuggestingthatthe recentlineageformationinthegrouphasbeenimportantlyinfluencedbygeographic barriersand/ordifferencesinecologicalniches,2)evolutionaryrelationshipsamongthe twodeeplydivergentCentralandSouthAmericancladesareresolvedthroughtheuse ofgenomicdata,3)morerecentlydivergedgroupsinSouthAmericashowcomplicated patternssuggestiveofrecentdivergenceand/orgeneflowamonglineages,and4)the

30 currenttaxonomyforthegroupdoesnotreflectitsevolutionaryhistory.Belowwe discussourfindingsinthecontextofthechallengesassociatedwithidentifyinglineages anddelimitingspeciesinrecentlyevolvedgroups,insightsforinterpretingthe biogeographichistoryoftheB.aspercomplex,andimplicationsfortoxinologicalstudies centeredontheseorganisms.

Lineagediscoveryandspeciesdelimitationinyoungspeciescomplexes

ThetimescaleatwhichtheRADSeqmethodismostusefulcorrespondstorecent diversificationevents,andassuchithasbeenappliedtophylogeographicand populationͲlevelstudies(Leachéetal.,2015).Atthislevelofdivergence,RADSeqisone ofthefewgenomicmethodsthatcurrentlyallowresearcherstoexplorepatternsof geneticstructureinnonͲmodelorganisms.However,someanalyticalchallengeshave beenrecognizedwhenusingthisapproachinyoungandcrypticspeciescomplexesand weoutlinethembelow.TheSNPdatasetderivedfromRADSeqhasbeenusedtoidentify putativedistinctlineagesandpatternsofadmixturebyconductingMLanalysesofthe concatenatedallelesandclusteringapproachesbasedondifferentalgorithms(Leachéet al.,2014;RittmeyerandAustin,2015;Streicheretal.,2014).Duetodisadvantages relatedwithapplyingthesameevolutionmodeltotheentiredatasetinconcatenated analysisorknownissuesintheprogramStructurewithȴKestimationwhensamplesizes aredifferent(Kalinowski,2011;Puechmaille,2016),someresearchershavesuggested thatforlargeandvariableSNPdatasetsthatincludevariationbeyondpopulationlevels

31 thealgorithmthatadegenetusesmaybeabetteranalyticaltooltoapproximatethe numberofnaturalgroups(Pyronetal.,2016;Streicheretal.,2014).

Potentiallineagesidentifiedusingclusteringapproachesareinferredwithout referencetothehistoryofpopulationdiversification.Exploringwhichofthedistinct populationclusterscorrespondtophylogeographiclineagesand/orspeciesͲlevel divergencesisamorecontentioussubject.AtendencyfordifferentspeciesͲdelimitation methodstooverͲdelimitgeographicclustersasindependentlineageshasbeen suggested;explanationsforthisphenomenonincludelocalfixationofSNPsdueto isolationbydistanceorextremepopulationstructuringacrosssmallgeographicalscales

(Carstensetal.,2013;Pyronetal.,2016).Inaddition,animportantcaveatofspecies delimitationmethodsisthattheycanbeaffectedbygeneflowbecauseofthe assumptionthatithasceaseduponspeciation(BurbrinkandGuiher,2015;

Gruenstaeudletal.,2016).

CrypticdiversityintheBothropsasperspeciescomplex

 Giventhelimitationsdescribedabove,weexploredcrypticdiversityintheB. asperspeciescomplexbyadoptingtherecommendedstrategyofusingmultiplesources ofmorphological/geneticdataandmodelsinordertoidentifygeographicallydefined geneticclusters,estimateevolutionaryrelationshipsbetweenthem,andevaluate whethertheyrepresentdistinctspecies(Carstensetal.,2013;butseeRannala,2015).

Asexpectedinagroupwherecrypticdiversityissuspected,ourexploratory

32 morphologicalanalysesonlyidentifiedalimitednumberofdifferentiatedgroups.

Interestingly,theseunitsareinagreementwithapreviousanalysisofmorphological variationthatidentifiedsignificantdifferencesbetweentwosetsofCentralAmerican populationsoftheB.aspercomplex:thoselocatedinMexicoandNuclearCentral

AmericaandthosefromIsthmianCentralAmerica(Sasa,2002).Wefoundadditional supportforthisdistinctionbecausesomespecimensfromthelowlandsofSouth

AmericaclusteredwiththosefromIsthmianCentralAmericaandsomeothersformeda thirdgroupthatincludedindividualsfromthehighlandsofColombiaandEcuador. 

Lineagediscoverymethodsappliedtotheindependentgeneticdatasets(mtDNA, nDNA)usedinthisstudysuggestthepresenceofextensivephylogeographicstructure

(between5Ͳ12lineages)intheB.asperspeciescomplex.OurmtDNAresultswere similartothoseofSaldarriagaetal.(inprep.)inthatsamplesfromthePacificlowlands ofEcuadorclusteredwithsequencesfromNuclearCentralAmericapopulations.

However,noneofournuDNAanalysesrecoveredthisrelationshipandtherefore stronglydemonstrateaninstanceofpervasivemismatchbetweenthesetwotypesof geneticdatathatobscuredpreviouseffortstoexplorethediversitypresentinthe group.BecauseourmtDNAsamplingwasmorecompletethanthatusedbySaldarriaga etal.(inprep.),webelieveapossibleexplanationforthisfindingismorerelatedtothe retentionofancestralpolymorphismsthantoasamplingissue(ToewsandBrelsford,

2012).TheMLanalysisandclusteringalgorithmsusedtoanalyzeourRADseqdataset convergedonsimilaranswersbetweenthem.Theyidentifiedalargernumberofgenetic 33 groupsthanpreviousstudies(SaldarriagaͲCórdobaetal.,2009;in.prep.),supportedthe inclusionofsamplesrepresentingB.ayerbeiandB.rhombeatusaspartofthisspecies complex,andrecognizedthepresenceofdistinctgeneticclustersinthehighlandsof southernEcuador.Overall,theseresultssuggestthatgeographicbarriersand/or differencesinecologicalnicheshaveimportantlyinfluencedtherecentlineage formationinthiswidespreadgroup(PyronandBurbrink,2009).

CoalescentͲbasedspeciesdelimitationmethodsarenovelapproachesthathelp improvingthestatisticalrigorandobjectivityoftaxonomy(Fujitaetal.,2012).Onlyone method(BFD*;Grummeretal.,2014;Leachéetal.,2014)iscurrentlyavailableto handleSNPgenomicdataandweuseditheretotestdifferenthypothesesof“species groups”basedontheevidenceprovidedbythediscoverymethodsmentionedabove.

Modelsthatpartitionedthedatasetintothehighestnumberofgroupswerehighly supported,whereasthecurrenttaxonomymodelwasdecisivelyrejected.Inagreement withthediscoveryapproaches,thespeciestreesestimatedinSNAPPandSVDquartets whichwerebasedonthebestͲsupportedhypothesissuggestedbyBFD*,alsoprovide evidencefortheearlydivergenceofCentralandSouthAmericanclades.Additionally, bothtreessupportthephylogeneticdistinctionbetweentwogroupsinSouthAmerica: onecladethatincludestheMagdalenan/Chocoan/B.rhombeatus/B.ayerbeigroupsand anothercladethatclusteredEcuadorianlineages.Withineachofthesetwoclades,both speciestreesshowedlowsupportvaluesandtopologicalinconsistencies.Asstated above,geneflowbetweenlineagescouldbeimportantwheninterpretingresultsfrom 34 speciesdelimitationmethodsandthisseemstobethecaseinSouthAmerican populationsbelongingtothesetwoclades,asevidencedbypatternsofadmixed individualsinStructureandpoorresolutionofrelationshipsbyestimatedspeciestrees.

Futurestudiesassessingtheamountofintrogression(e.g.,Patterson’sDͲstatistics) betweentheselineagescouldbefruitfultodeterminetheextentofgeneflowinthis youngspeciescomplex(Durandetal.,2011;Meiketal.,2015;Streicheretal.,2014).

Thesmallamountofmorphologicaldifferentiationbetweenmostofthelineages identifiedinthiscomplexprovidesindependentsupportfortheideaofsignificantgene flowoccurringbetweenpopulationsinSouthAmerica.Nevertheless,theidentification ofmorphologicaldifferencespresentinspecimensfromtheAndeanhighlandscouldbe relatedtoinstancesofvicarianceordifferentiationalongecologicalgradients(Brumfield andEdwards,2007).Finally,alltheevidencegatheredinthisstudypointstosignificant geneticandmorphologicaldifferentiationbetweentheCentralandSouthAmerican clades.

EvolutionarybiogeographyoftheBothropsaspercomplex

Foraround150years,theB.asperͲatroxcomplexhasbeenoneofthemost problematicgroupsofpitvipersoftheNewWorldintermsoftheirsystematicsandhas beenproposedtoincludecrypticspecies,discordantmorphologicalandmolecular variation,andseveralareasofcontact(CampbellandLamar,1992;Wüsteretal.,1996).

TheB.atroxgroupincludespopulationsdistributedeastoftheAndesacrossthetropical

35 lowlandsofSouthAmerica,exclusiveofParaguay,Uruguay,andArgentina(Campbell andLamar,2004),andtheirsystematicshavebeenstudiedseparately(Puortoetal.,

2001;Wüsteretal.,1996,1997,1999).Traditionally,B.atroxandB.asperhavebeen consideredtobesistertaxabutrecentworkhassuggestedadifferentrelationship.

Specifically,Saldarriagaetal.(inprep.)foundbasedonmtDNAͲbasedanalysesthat specimensbelongingtotaxausuallyassociatedwiththeB.atroxgroup(B.atrox,B. colombiensis,B.isabelae,B.marajoensis,B.moojeni,andB.leucurus)formeda monophyleticgroupnestedwithincladesrepresentingtheB.aspergroup.Thepresent studyrecoveredthesamemtDNArelationshipswithourmoreextensivesamplingofthe

B.asperͲatroxcomplex;however,althoughbasedonalimitednumberofB.atroxsensu latosamples,ourgenomicanalysesrecoveredacloserrelationshipbetweenindividuals fromTrinidadandtheAmazonianbasinofBrazil,Ecuador,Peru,andVenezuelathan withspecimensrepresentingtheB.aspercomplex.ThusourgenomicnDNAanalysis confirmsthetraditionalsisterrelationshipbetweenB.atroxandB.asper.

Althoughourstudywasnotaimedatreconstructingthebiogeographichistoryof theB.aspercomplex,ithasenabledustomakeinferencesthatsupplementprevious hypothesesabouttheevolutionaryhistoryofthegroup(Saldarriagaetal.,inprep.;

Werman,2005).BasedonmtDNAdata,Saldarriagaetal.(inprep.)estimatedthe divergenceoftheB.atroxgroupandB.asperlineagesatapproximately3.3Ma.Starting inthePaleogeneanduntilaround2.7Mawhencurrentelevationswereattained,the northernAndeshadalreadyachieved40%oftheirpresentelevationandthe 36 developmentofthetopographyoftheAndeswasoccurringatahighrate(Hoornetal.,

2010).Therefore,thedivergencebetweenthesetwogroupsislikelyexplainedbya vicarianteventthatconfinedancestralB.atroxpopulationstotheeastoftheAndesand toitspresentdistributionintheGuyanaShieldandAmazonianBasinandtheB.asper stocktonorthwesternSouthAmerica(Werman,2005).Thedeepdivergencebetween

CentralandSouthAmericanB.asperlineagesoccurredsoonafterthiseventaccording toSaldarriagaetal.(inprep.).Becausetheseauthorsdidnotrecoverasister relationshipontheirmtDNAphylogenybetweenbothCentralAmericanlineages

(MexicoandNuclearCentralAmericaandPacificIsthmianCentralAmerica),they suggestedthatdispersalacrosstheIsthmusofPanamaoccurredintwopulsesas suggestedbySavage(2002)toexplaintheunequaldistributionofamphibiansand reptilesofSouthAmericanorigininthisregion.However,ouranalysesdidrecovera sisterrelationshipbetweentheselineages.Therefore,basedonrecentevidenceofan earlierconnectionandmigrationpatternsacrosstheIsthmus(Baconetal.,2015),we proposethatthedivergencebetweenCentralAmericanlineagesisinagreementwith patternsfoundinotherlowlandpitvipersthatproposethefinalupliftoftheCordillera deTalamancaasamajorvicariantevent(Castoeetal.,2009;Dazaetal.,2010).

ThepatternofarecentandmoreextensivediversificationofB.asperlineagesin

SouthAmericaduringthePleistoceneisdifferentfromthefindingsofmostofthe phylogeographicstudiesconductedonherpetofaunaintheregion,inwhichintraspecific lineagesplitsoccurredmuchearlierduringthePlioceneand/orMiocene(TurchettoͲ 37 Zoletetal.,2013).AlthoughdiversificationinlowlandB.asperlineagesinthis subcontinentcouldberelatedtolargeͲscalelandscapechanges(e.g.,marine transgressions,Quaternaryclimatechanges)thatfragmentedpreviouslycontinuous distributions(Rull,2011),ourresultsshowtheimportanceofmontanehabitatsas driversofdiversification.Fouroftheidentifiedlineages(B.ayerbei,B.rhombeatus,

Ecuadorhighlands1,andEcuadorhighlands2)inSouthAmericaarepresentondry interͲAndeanvalleysinColombiaandEcuadorthatareisolatedfromthePacific lowlandsatvariousdegrees.TheseinterͲAndeanvalleysarelocatedataltitudes between1500Ͳ2500m.a.s.lintheCaucaandPatíariverbasinsinsouthwestern

Colombia,andtheJubonesandCatamayoriverbasinsinsouthwesternEcuador.Suchan effectofageographicallystructuredlandscapematrixpresentintheAndeanCordillera andvariabledispersalabilitiesfordifferentlineagesofbirdshavebeenrecently recognizedasimportantdriversofspeciationintheNeotropics(Smithetal.,2014).

SimilardiversificationprocessescouldhaveactedinlanceheadpitvipersoftheB.asper complex,astheseorganismsareprimarilydenizensoflowlandrainforestsacrosstheir distributioninLatinAmericabutattaintheirhighestverticaldistributionpreciselyin

ColombiaandEcuador.Thiscouldbetestedbyexaminingthelikelihoodofhistorical demographicmodelsthatevaluatetheevolutionarymechanismsinvolvedintheorigin ofthesemontanelineages(SousaandHey,2013).



38 Biomedicalimplications

Variationofvenomcompositioninsnakesisawidespreadphenomenon occurringatalltaxonomic(interfamilial,intergeneric,interandintraspecies)andsome biologicallevels(e.g.ontogeneticchanges)(Chippauxetal.,1991;Mebs,2001).

Althoughtherelationshipbetweenphylogeneticaffinities,venomcomposition,and antivenomcrossͲneutralizationishighlyvariable,arobustphylogeneticframework providesadefaulthypothesisforvenomvariationandantidoteresponse(Williamset al.,2011).DifferentresearchgroupsacrossLatinAmericahaveperformedanalysesof venomcompositionandtoxicologicalpropertiesinsnakesoftheBothropsasper complex(AlapeͲGirónetal.,2009;Kuchetal.,1996;Lainesetal.,2014;Saraviaetal.,

2001;Seguraetal.,2012);however,specimensusedinmostofthesestudieshavebeen sampledhaphazardlywithineachcountry.Interestingpatternsofvariationhavebeen showninstudiesthathaveactuallycomparedpopulationsrepresentingsomeofthe lineagesidentifiedbyus.Forexample,comparisonsbetweenpopulationsoriginatingin thePacificandCaribbeanversantsofCostaRica(AlapeͲGirónetal.,2008;Aragónand

Gubensek,1981;Gutiérrezetal.,1980;JiménezPorras,1964)andmorerecently betweenthePacificcoastofsouthernColombiaandthelineagecorrespondingtoB. ayerbei(MoraͲObandoetal.,2014)haverevealedstrikingdifferencesinvenom compositionandaction.Thephylogeneticrelationshipsprovidedinourstudyshould promotenewresearchfocusedonestablishingproteomicandfunctional characterizationsbetweendifferentcladesandlineages,especiallythosefrom

39 understudiedregionslikenorthwesternSouthAmerica,aswellasevolutionaryand immunologicaltrendsamongvenoms.

DespitethefactthatcrossͲneutralizationofantivenomsisextensiveamong membersoftheBothropsgenus,futurecomparativestudiesbasedonourresultscould improvetheefficacyofcurrentantidotesbypreͲclinicallyassessingtheirneutralization oflethalityandotherclinicallyͲrelevanteffectsalongwiththeidentificationofvenom componentsrecognizedbyantivenomantibodies(Gutiérrezetal.,2014;Gutiérrezetal.,

2010).Finally,thegeographicdistributionoflineagesandphylogeneticrelationships hypothesizedhereforthegroupcouldbeenlighteningwithrespecttothe characterizationofsnakebiteaccidentmanifestationsatclinicalsettings(Gutiérrez,

2014;OteroͲPatiño,2009).

Taxonomicrecommendations

Severaltaxonomicissueshavebeenclarifiedwithourresultsandherewe proposetaxonomicrevisionsthatwouldbetterreflectthediversityinthegroup.First,in agreementwithSaldarriagaetal.(inprep.),wedidnotfindanyevidencetoconsiderthe lanceheadpitviperpopulationinhabitingTrinidadaspartoftheB.aspercomplexand thereforeitshouldbebetterincludedintheB.atroxgroupuntilfuturestudiesare conductedinthatspeciescomplex.

Second,allofouranalysessupporttherecognitionoftheCentralandSouth

Americancladesasindependentlyevolutionarylineages(=species)andinorderfor

40 taxonomytorecognizethisevolutionaryhistoryweproposetoresurrectthenameB. septentrionalis(Müller1885)fortheCentralAmericanclade.B.asper(Garman1884)is thenamerecommendedfortheSouthAmericanlineage.

Finally,ataxonomicevaluationofthenamesB.ayerbeiandB.rhombeatusneeds tobeperformedinfutureanalyses.However,werecognizethatlineagespresentinthe highlandsoftheAndesshowinterestingpatternsofgeneticandmorphological differentiation.



Acknowledgements

WethankS.Ayerbe,J.M.Daza,J.A.Campbell,G.Rivas,W.E.Schargel,E.Tapia,

L.A.Coloma,M.Terán,J.Townsend,A.Batista,J.M.Ray,F.T.Burbrink,A.Guzman,D.

Amazonas,andL.Bustamanteforgenerouslyprovidingkeysamples.Wearegratefulto

M.Sovic,J.Díaz,andP.SantacruzͲOrtegaforadviceandhelpinthelab.Foradviceand commentsonthemanuscript,wethankmembersoftheGibbsLab,A.Leaché,J.

Freudenstein,B.Carstens,L.Kubatko,T.Hetherington,P.Fuerst,J.Satler,M.Broe,P.

Blischak,B.Titus,J.Streicher,O.Zinenko,C.SheehyIII,andE.Rice.WealsothankD.

Núñez,C.Tapia,P.SantacruzͲOrtega,M.Bolaños,S.Harris,C.Quito,W.Ventanas,L.

Pallatanga,A.FreireͲLascano,I.Tapia,R.Molina,M.PáezͲVacas,E.Arbeláez,J.Cuadros,

W.Santacruz,L.AmadorͲOyola,M.Salazar,andR.Valenzuelaforassistanceinthefield.

Wethankthefollowingcuratorsandstaffforallowingustoexaminespecimensunder

41 theircareandforfacilitatingourwork:J.M.Daza(MHUA),S.EstradaandA.M.Henao

(SUA),M.Rivera(CIBUC),C.Franklin(UTA),F.AyalaͲVarelaandD.PaucarͲGuerrero

(QCAZ),J.Valencia(FHGO),andM.YánezͲMuñoz(DHMECN).Wealsothankthehelp providedbythestaffofJamaͲCoaqueEcologicalReserveandBosqueProtectorCerro

Blanco.Specimenswerecollectedundercollectionpermit008Ͳ09ICͲFAUͲDNB/MAand weredepositedatMuseodeZoología(QCAZ),PontificiaUniversidadCatólicadel

Ecuador.Thisworkwasfundedinpartbythefollowingorganizations:GermanResearch

Foundation(DFG),SecretaríadeEducaciónSuperior,CienciayTecnologíadelEcuador

(SENESCYT),UniversidaddeAntioquia(projectSostenibilidad2014Ͳ2015),Tinker

Foundation,andTheOhioStateUniversity.



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54 Tables 

  e d

 u t i g  n  80.571 Ͳ 77.2813 76.2901 74.8208 78.51603 99.18152 78.31192 95.23417 79.67392 78.35956 78.75254 83.58174 77.32639 74.64338 89.40436 88.70096 76.63183 77.08369 83.58454 77.03397 84.46354 75.32146 89.03882 85.89949 84.31794 84.31718 83.57894 o Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ L

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 km  Lorenzo,  protocol. (Costa  2.0 

  San Volcano MX 735 18.591   Ha MX 263 18.548 Tobago), ca. Ͳ CR of   Cortés CR 18 9.01019 

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 N Xpujil   Lodge,  Branchi Hydroelectric Hydroelectric    ravine CO 15 5.60694 km Tuxlas: (Nicaragua),

    Marcos EC 1022 1.11435 El  (Trinidad   with   ICE ICE   Quesada CR 400 10.34587 los  4.0 NI     Matías CO 1639 6.55072 San Pérez CR 424 9.72218 Pérez CR 459 9.72175 TT   Cima MX 878 23.05201   Canal  between Bank BZ 13 17.60138   de   ca.  Rica CO 1100 2.54674    Sapo PA 977 7.97709 used   Tundaloma Chuchubí EC 739 0.88111 Urrao CO 965 6.40476 Don    Hill  Bajo Bajo Alta Ciudad      road  Livingston, Chical, Siquirres,  Siquirres, Melgar CO 345 4.20357   Victoria CO 370 5.32194 Sierra   Copé PA 300 8.62343 Acandi CO 28 8.51404 Nuqui, Yuto CO 60 5.53537  Huisitó Playa         Cerro El (Peru),  Gamboa PA 82 9.08822  El José: José:     

 Walk:  PE Izabal:  Limón: Limón:  Alajuela: San San Puntarenas: Matagalpa NI 975 12.95512 samples        Tolima: Choco: Choco: Caldas: Antioquia: Choco: Cauca: Cauca: Antioquia:            (Guatemala), Esmeraldas: Esmeraldas: Carchi: Coclé: Colón: Darién:        Tamaulipas: Veracruz: Campeche:   

 Orange for  Rica: Rica: Rica: Rica: Rica: Rica:        GT   Colombia: data

  (Ecuador),   r EC (Belize),   e 14932 Colombia: 14444 Colombia: 14437 Colombia: h     c BZ R R R 2672 Panama: Locality  51866 Mexico: Ͳ Ͳ Ͳ 3 71 Colombia: 40353 Guatemala: u   5760 Ecuador:  12585 Ecuador:      o R 4287 Colombia: 4102 Colombia: R 4318 Colombia:  1      24548 Mexico: 24348 Mexico: . 1421 Costa 3 Panama: 1562 Costa 1407 Costa 1377 Costa 1499 Costa 1106 Costa V          2   e l b  o 1JAC 2UTA 3JAC 8ICP W23Belize: 4WW263 5UTA 9ICP 7ICP 6N168 Nicaragua: a 24 QCAZ 23 QCAZ 14 ICP 15 MHCH 16 SUA 25 UTAT55E9 Colombia: 17 MHUA 19 SUA 26 MHUA 18 MHUA 20 CIBUC 21 CIBUC 27 SUA 10 ICP 22 SC2893 Ecuador: 11 ICP 12 ICP 13 TSP046 Panama: N (Mexico), (Venezuela),  T

55  e d u

 t i g 73.85 n  Ͳ 75.2729 75.3556 79.2255 79.2142 78.9705 79.15587 79.24583 80.65585 72.79024 79.21753 80.01972 72.71279 78.95705 61.24376 79.60593 76.74646 80.11595 76.74639 78.78519 76.64668 76.80572 76.69599 79.43453 80.17644 54.90247 79.15569 o Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ  L

 e d u t

 i 2.18 t Ͳ 3.307 4.2565 3.4158 Ͳ a 3.41749 1.81618 Ͳ 4.26699 0.42417 0.11889 3.89239 1.31172 3.84547 2.44721 3.47738 Ͳ 2.02584 Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ  L

  n o i )

t  a m v (

e  l E  y r t  n CO 100 7.97047 u

o  C u   Antioq   of    Achiote EC 137 0.1179  road EC 1875  road EC 1885 University    El Cumandá EC 1473    to Verde PE 700 to     Cuenca Cuenca Ͳ Ͳ road road    Campo Candelaria,  on Oña  Oña on     Reserve EC 580 Blanco EC 237   La

y   Pampas EC 1490 t old  old i   Park, l  Mangas EC 230  a Quito   Las c  Cerro the the    o  Dos de L   on on Pallatanga   Ecological  Hacienda Hondo CO 1600 2.52607  of   

Puerto  of National    Zumbi EC 824 S  Río NE  of     Protector bridge bridge E     Hills km  km  Coaque Francisco  Tapari BR 12  Ͳ   1 Barrancabermeja CO 91 7.06667 2 km  Caucasia,     Yunga, Yunga CO 1600 2.526 Tetilla CO 1750 2.51523 León León 2       San Valley TT 29 10.59265   José EC 1899  Pechiche EC 63  Jama  Bosque La La La Pomorroso CO 1300 2.38878 Corralejas CO 1550 2.27595  Chinchipe: Coloso CO 148 9.4967  Perijá VE 1149 10.04894 Perijá VE 394 10.04771          Gramalote EC 590 Río Río     Elena:  Amotape Vilcabamba EC 1541 Vilcabamba EC 1619 San     Ríos:  Santarém,  Zulia: Zulia:    Santander: Sucre: Antioquia: Cauca: Cauca: Cauca: Cauca: Cauca:         Guanapo Loja: Cotopaxi: Guayas: Loja: Azuay: Manabí: Zamora Los Loja: Pichincha: Chimborazo: Azuay: Azuay: Santa                 Pará:  Tumbes:       14604 Colombia: 14447 Colombia: Continued    r  R R 33608 Peru: e  Ͳ Ͳ 39 Colombia: 136 Colombia: 220 Colombia: 289 Colombia: 386 Colombia: 11622 Ecuador: 10580 Ecuador: 9126 Ecuador:  12765 Ecuador: 5873 Ecuador:   11397 Ecuador: 11620 Ecuador:   5785 Ecuador: 12615 Ecuador: 5013 Ecuador: 12460 Ecuador:          h   84768 Brazil: 4258 Colombia:   c  2.1:  u

o  V  o Table 30 MHUA 28 SUA 29 MHUA 50 QCAZ 39 QCAZ 44 QCAZ 31 WEST3014 Venezuela: 32 WEST3040 Venezuela: 33 CIBUC 51 WW740 Ecuador: 45 QCAZ 40 QCAZ 52 UWIZM20111915 Trinidad: 34 CIBUC 53 QCAZ 46 MUSM 35 CIBUC 41 QCAZ 36 CIBUC 54 IBSP 37 CIBUC 38 QCAZ 42 QCAZ 47 Bas020 Ecuador: 48 Bas024 Ecuador: 49 QCAZ 43 QCAZ N 

56       Central   ŽƚŚƌŽƉƐ likelihood Highlands      Isthmian   follows: (MVV),   6 marginal 6 8 4 4 . . .  8 2

as  6 F 8 7 5 Ͳ 4 8  B 0 7 8 8 4 4 2 1 3 9 Pacific  2  are  k  n include 2 3 1 5 4 6 7 a  R  Venezuela  2 8 9 6 9 9 2 ...... (MNCA),

 4 E  8 2 8 1 2 0 L 0 and 1 2 2 2 5 6 0  7 8 6 1 8 6  M 2 7 7 7 8 7 8 Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ

  abbreviations    E E Y  T T ) p d d R R e e e E E America d M e e u c c c   abbreviations U U e N N o n n  O n n n i i T T r d E E e e e N b C b C g u Colombia G G d d d t  l O i i U i U  E m m E c u v v v X R R o o D D n o E e e T i T A Lineage C C  ( A A Other  S S T Central    2 2 2 Valley C C C  E E  E H H H C (PEC).  E

 Nuclear H 1 1 C C  approach. C C E E  E E P  P S C H H and A E  B  P C C C Magdalena Ecuador BFD*  E E E   P P  P the V V V V V V

 Mexico   V V V V V V Pacific M M M M M M   with s (CHOCO),  e Y Y Y Y Y Y Y

  (BRH), g A A A A A A A  a B B B B B B B

 e n (HEC2), i  L  tested 2 H H H  H Choco   R R R R  B B B B O O  O C C C (BF).  O O O and O O O  H H H C C  C C C C models O O O  Ecuador H H H ͘ƌŚŽŵďĞĂƚƵƐ 

  C C C factors  S A A A A  A A C C C C C Panama I I I I I B  A P P P P P (BAY), C    Bayes N  A A A A A 2 M Highlands C C C C C   delimitation  , N N N N N  1 Darien and M M M M M    s e  . ͘ĂLJĞƌďĞŝ g 

 o a (HEC1), 8 8 6 5 8 9 3 (MLE)  e Species N  (PICA),  n 1 i   2  l . 2 l (BAS),    e e d l C B F E D A G o b a  M estimator ĂƐƉĞƌ Ecuador America  T  57 Figures 

    triangles   the  and America:  .  from 

 Yellow  Latin  Sampled  in   female diamonds),  

 text)  localities.  (red  the Colombia. asper 

   in in B.    data  a   situated  of   (black)  defined   closely (as   Picture   morphological 

 several  rhombeatus  complex  and  B.   outgroups.  cover  as and    species  RADseq may  

 used  gray),   asper   symbol circles), Brazil    (dark   Carvajal. and (red  Ͳ  single Bothrops 

  A  ayerbei  only  the

  Torres B.   of Ecuador   data

  and  Omar squares).   from   by  range, RADseq    (blue  distribution  for the    only  Ecuador specimens   of

 study   across data   atrox 

 this Geographic   B. .  in  (gray)   lowlands 2.1    asper  morphological Pacific represent B. localities  Figure

58      

 gray.  Central  Colombia derived of     was  Valley  Isthmian shades     in  support  (EC).   Magdalena Caribbean   Nodal  highlighted

  Ecuador  (PICA), of SNPs. are     follows:   as 70) 864    (>  are  America  from

 highlands   and  support 

 Central  generated  strong abbreviations  

 7) lowlands   = Isthmian   with  (K   Pacific  Structure plot  Pacific   Clades  and 

 and  1  (MNCA), Structure RAxML.     Table in   (CHOCO),  and in    America  (left) Choco   performed indicated and     Central  those   phylogram  Panama  are Nuclear    and  Darien  likelihood pseudoreplicates    phylogram  Mexico  (MVV), in    bootstrap Maximum   . (CICA), codes 2   . 2 

 1000 e  Venezuela r  u g i F America from and  Country 59      the    Lineage Ecuador  

 shows  Nuclear  Magdalena  Plot Pacific  and    America.  and   Latin  Mexico (CHOCO),  included.   in  (HEC2),

  2  (BRH), Choco   samples   and  distribution Ecuador    their outgroup rhombeatus    Panama  to B.    the  Highlands  Darien  with coded  (BAY),   Ͳ (HEC1),  color (PICA),  1    ayerbei  adegenet and  B.  

 in  Ecuador America   values   (ATROX), analysis  BIC   Central   Highlands  atrox from    clustering  Isthmian  (MVV),  inferred Bothrops 

  DAPC  Pacific  the value    K  Venezuela follows:   from as   and  (MNCA),   are  optimal 

 Results  the  America  2.3 Colombia  for      (PEC). Central Valley abbreviations  Figure result

60                                    Figure2.4.UltrametrictreeofuniquecytͲbandND4haplotypes.Asterisksabove/below nodesofmajorcladesrepresentBayesianposteriorprobabilityvalues>0.95.Vertical barstotherightofterminalbranchesrepresentcoalescentunitsrecoveredbythe bGMYCalgorithm,colorͲcodedaccordingtotheirposteriorprobabilities:yellow=0.5< p<0.9,orange=0.9<p<0.95,red=0.95<p<1.Namesindicategeneraldistribution areas.

61                                   Figure2.5.ResultsfromthekͲmeansclusteringanalysisappliedtothemorphological dataoffemales(resultsformalesweresimilarandarenotshown).Thefirsttwo principalcomponentswereextractedandcoloredbythekͲmeansresults(above).ColorͲ codeisasfollows:MexicoandNuclearCentralAmerica(black),CostaRica,Panamaand mostsamplesfromSouthAmerica(red),andBothropsayerbeispecimensandhighlands ofEcuador(green).PlotfortheoptimalKvalueofthreegroups(below) 

62        as  

 BFD*.  those and are    by   Choco and    (HEC2), and   2

  supported  (middle),

  Abbreviations  Panama   Ecuador  models 

 nodes.  Colombia Darien  the  on 

  in  on  Highlands

  (PICA),  shown   mainly  based  are   (HEC1),  1  America

  (right)  present  values   Ecuador  those  Central   support

  SVDquartets   (above),  Isthmian Highlands   and   bootstrap   (left) Pacific   (MVV), America  and     SNAPP  Central (MNCA), in     Venezuela   from probabilities   and 

 America  obtained   lineages  trees Posterior    Central of Colombia     species  Valley

  (below).   Nuclear   divergence  and  (PEC).   the  Ecuador  Consensus

  Magdalena  Mexico  from Ecuador 2.6.     indicate   Figure follows: (CHOCO), Bars Pacific  mainly 63 A                B                Figure2.7Canonicalvariateanalysisplotsforfemales(A)andmales(B).Lineages correspondtothoseshowninFigure2.3 

64 



 Chapter3:Divergenceoftropicalpitviperspromotedbyindependent colonizationeventsofdrymontaneAndeanhabitats 

Abstract

TheoriginandmaintenanceofNeotropicalbiodiversityhaslongbeenarguedto beinfluencedbyinteractionsbetweenevolutionaryprocessesandenvironmental factorsactingatdifferentspatialandtemporalscales.However,theimpactofspecific combinationsoftheseprocessesandfactorsisunclear.Onesuchcaseishowthe evolutionarydynamicsofcolonizationanddifferentiationinrelationtolowlandand highlandhabitatsoncetheAndesattainedtheircurrentaltitudes(~2.6Ma)has impactedlineageformation.Mostspeciationmodelsforthisregionhavefocusedon vicariantevents,buttheneedtoassesstheinfluenceofdemographicprocessesinthe structuringofpresentͲdayphylogeographicpatternshasbeenrecognizedonlyrecently.

ModelͲbasedanalysesofpopulationgenomicdatasetsfornonͲmodelorganismscanbe usedforthispurposeandareespeciallyusefulfordisentanglingwhatrolesthe contrastingfactorsofgeneflowandisolationbydriftplayduringtheprocessof divergence.Here,weusegenomicdataanddemographicmodelingtoinvestigaterecent diversificationeventsinSouthAmericausingavertebrategrouprarelyexploredin phylogeographicstudies:tropicalAndeansnakes.Specifically,theoriginoftwo 65 Ecuadorianmontanelineagesofterciopelopitvipers(Bothropsasperspeciescomplex) wasevaluatedgivenambiguousphylogeneticrelationshipswiththepresumably ancestralPacificlowlandlineage.Ourresultsindicatethatthesediscrepanciesof evolutionaryrelationshipspreviouslyobtainedwithtreeͲlikemethodsareresolved throughtheuseofmodelingapproaches.Wefoundstrongsupportfortheindependent originofmontanelineagesbasedonclusteringsolutionsandtopologiesinferredby maximumͲlikelihoodtreesandmodelingapproachesthattakeintoaccountpossible geneflow.Finally,evidenceofmigrationwasonlyidentifiedbetweenthelowlandand oneofthehighlandlineagesaftertheirdivergence.Theothermontanelineagehasbeen isolatedforapproximately200,000yearsanditsrecognitionatthespecieslevelis possiblywarranted.Thecurrentstudyillustratesthevalueofpopulationgenomicdata andmodelingapproachestobetterunderstandrecentdiversificationeventsinthe

Andes.

Keywords:Bothropsasperspeciescomplex,coalescentmodels,Neotropics,northern

Andes,phylogeography,RADsequencing,terciopelopitvipers



Introduction

TheNeotropicsharborsthelargestportionofthebiodiversitypresentinthe planet(Myersetal.,2000;Rull,2011;Smithetal.,2014).Fordecades,molecularstudies havecenteredonexploringdifferenthypothesestoexplaintheoriginofthisdiversity

(BagleyandJohnson,2014;LeiteandRogers,2013).Moststudieshaveusedgene 66 genealogiestoinferthetimingandgeographicallocationofdivergenceacrosstaxa,and havelinkedpatternsofevolutionaryrelationshipstospecificvicarianteventsgenerated byPliocene/Miocenelandscapechanges(e.g.,Andeanorogeny,emergenceofthe

PanamaIsthmus,drainageshifts,seatransgressions)orPleistoceneclimaticoscillations

(i.e.,glacial–interglacialcycles)(Rull,2011;TurchettoͲZoletetal.,2013).However, recentmetaͲanalyseshaveshownthatsuchgeneralizationsarenotwarranted,andthe originandmaintenanceofextantneotropicalbiodiversityisacomplexphenomenon thatdependsonsynergisticenvironmentaldriversthathaveoperatedatdifferent spatialandtemporalscales(Rull,2008;TurchettoͲZoletetal.,2013).Consequently,a thoroughunderstandingofthistopicrequiresalargernumberofsystemstobeexplored withdataandanalyticalapproachestoinferevolutionaryhistory(Rull,2013).

InSouthAmerica,theupliftoftheAndesmountainrangeundoubtedlyplayedan importantroleinthediversificationofseveraltaxonomicgroups(Antonellietal.,2010;

Hoornetal.,2010).However,oneaspectthatremainspoorlyunderstoodisthemore recentevolutionarydynamicsofcolonizationanddifferentiationinlowlandand highlandhabitatsoncetheAndesreachedtheircurrentaltitudes(~2.6Ma)(Brumfield andEdwards,2007).Forexample,recentstudiesindifferentlineagesofNeotropical birdshavesuggestedtheimportanceofdispersaleventsintoandoutofmontane habitatsasacauseofincreaseddiversificationratesduringthePleistocene(Brumfield andEdwards,2007;Smithetal.,2014;Weir,2006).Thesefindingsrepresentashift fromtheparadigmofmostspeciationeventsintheregionbeingrelatedonlyto

67 isolationduetolandscapechange(i.e.,vicariance)toaconsiderationofabroaderrange ofmechanisms.Theyalsodemonstratetheneedtoassesstheinfluenceofdemographic processesinthestructuringofpresentͲdayphylogeographicpatternsindiversetaxa

(HarveyandBrumfield,2015).

MovingbeyondasolefocusonvicarianceͲbasedhypothesestoabroaderrange ofscenariosthatincorporateotherhistoricaldemographicmechanismsisnowpossible duetotheincreasingavailabilityofgenomeͲscaledatasetsandmodelͲbasedapproaches inphylogeography(Garricketal.,2015;Hickersonetal.,2010).Includingalarger numberofvariablesitesfrommoreregionsofthegenomehasproventobeespecially usefulforthisdisciplineduetotheincreasedaccuracyandprecisionofparameter estimation(Leachéetal.,2015;McCormacketal.,2013;Pyron,2015).Such improvementshavealsobeenmatchedbytheimplementationofcoalescentͲbased modelingtechniquesthatincreasethestatisticalrigorofhypothesistestingby comparingalternativehistoricalscenarios(Beaumontetal.,2010;Knowlesand

Maddison,2002).Inrecentyears,computationallyefficientapproachesforanalyzing genomicdatasetsnowallowstheevaluationofmorecomplexandrealistichistorical processes(Excoffieretal.,2013;Gutenkunstetal.,2009;PickrellandPritchard,2012;

RittmeyerandAustin,2015).Specifically,modelingdivergenceinthisframeworkallows theincorporationofmultipledemographicprocesses(e.g.,populationsizechanges, migration)intoinferencesabouttruedivergencepatternsthatisnotpossiblewithtreeͲ basedmethods(SousaandHey,2013).

68 Inthisstudy,weusegenomicdataanddemographicmodelingtoinvestigate recentdiversificationeventsinSouthAmericausingavertebrategrouprarelyexplored inhistoricaldemographicstudies:tropicalAndeansnakes.Specifically,theoriginof montanelineagesofterciopelopitvipers(Bothropsasperspeciescomplex)inthe northernAndeswasevaluated.Thisgroupofvenomoussnakesmainlyoccursinlowland rainforestsfromcentralMexicoallthewaythroughCentralAmericaandintoVenezuela andPeruinnorthwesternSouthAmerica(CampbellandLamar,2004).Itisalsooneof themostcommonpitvipersinLatinAmericaandtheleadingcauseofsnakebite accidentsinhumansacrossitsdistribution(OteroͲPatino,2009;Warrell,2004).Recent analysesbasedonmitochondrialDNAandgenomicSNPdatasetshaveshownthatthis speciescomplexcomprisesbetween7Ͳ10lineages,withthedeepestdivergence betweenaCentralAmericanandaSouthAmericancladeestimatedtohaveoccurred around3Ma(SaldarriagaͲCórdobaetal.,in.prep.;SalazarͲValenzuelaetal.,in.prep.).

OnlythreeoftheselineagesaredistributedinCentralAmerica,whereB.asper populationshavebeenregisteredfromsealevelto1,200Ͳ1,300m,whiletheother geneticgroupsarepresentinSouthAmericawherepopulationscanbefoundashighas

2,640mintheAndes(CampbellandLamar,2004).Thismountainrangehadattainedits currentelevationsbythetimethesesnakescolonizedthenorthernAndesandlineages aroseinisolateddryinterͲAndeanvalleysinthehighlandsofColombiaandEcuador

(GutberletJrandHarvey,2004;Hoornetal.,2010;Werman,2005;SalazarͲValenzuelaet al.,inprep.).Assuch,anevaluationoftherecentevolutionarydynamicsinvolvedinthe

69 lowlandandhighlanddifferentiationsofthisecologicallydissimilargroupshouldaddto thelimitedknowledgeofhowrecentevolutionarydiversificationinvertebrateshas occurredinthishighlydiverseregion.

Specifically,weexaminethedistinctnessandevolutionaryhistoryofthree lineagesofthissnake:onelowlandPacificlineage(PEC)andtwohighlandlineages

(HEC1andHEC2)allͲpresentinthesouthernpartofEcuador.Thedegreetowhichthese lineagesaredistinctandwhattheirspecificevolutionaryhistoriesareisunresolved basedonphylogenetictreeͲbasedmethodsaloneduetohighlevelsofshared polymorphism(SalazarͲValenzuelaetal.,in.prep.).HereweuseapopulationͲlevel modelingapproachandagenomicSNPdatasettogenerateresultsfromgenetic clusteringanalyses,evaluationofhistoricalmigrationbetweengeneticclusters,and demographicmodelselection.Weusetheseresultstoevaluateiftheoriginofthe montanelineagesisexplainedbyeitheroftwohistoricalprocessesthatrepresent fundamentalphylogeographicmechanisms:1)Differentiationbyisolationwithinthe highlandsor2)Differentcolonizationeventsfromthelowlands.



MaterialsandMethods

Studysystem

Thehistoricaldemographyoflowlandandmontanelineagesofterciopelo pitvipers(Bothropsasper)inEcuadoridentifiedbySalazarͲValenzuelaetal.(inprep.) wasassessed.WetargetedEcuadorianpopulationsofthisvenomoussnakebecauseof 70 thepresenceoffourdifferentlineagesrecentlyidentifiedinthecountry(SalazarͲ

Valenzuelaetal.,in.prep.)andthehighlevelofhabitatheterogeneitypresentin westernEcuador(AndersonandMartínezͲMeyer,2004;LynchandDuellman,1997;

RidgelyandGreenfield,2001),whereB.asperispresentfromsealevelupto1,900m

(CampbellandLamar,2004;CisnerosͲHerediaandTouzet,2004;pers.obs.).Forthe demographicmodelselectiontests,wespecificallyevaluatedisolationandisolationwith migrationmodelsthatclusteredhighlandlineages(Fig.3.1A,3.1C)againstmodelsthat includedatopologythatclusteredthePacificlineageandtheHEC1lineage(Fig.3.1B,

3.1D).Theserepresentfundamentallydifferentmechanismsofphylogeographic differentiation(Avise,2000).Weusedrepresentativepopulationsoftheselineageswith thegoalofestablishingiftheoriginofthesemontanegroupscouldbeattributedto diversificationwithinthehighlandsofsouthernEcuador(i.e.,supportformodelsAorC) ortoindependentcolonizationsofmontanehabitatsfromPacificlowlandpopulations

(i.e.,supportformodelsBorD).

Weobtainedbloodortissuesamplesfor27Bothropsasperindividualsfromfour populationsdistributedinwesternEcuador(N=3–10perpopulation)(Fig.3.1,Table

3.1).Individualscollectedwereconsideredtobelongtoasinglepopulationiftheywere foundinlocalitieswithina5kmradius.TheselocalitiesweredistributedinthePacific lowlandsandwesternversantoftheAndes(300–1,000m),aswellasinthehighlands

(1,500–2,000m)ofsouthernEcuador.BasedonFenwicketal.(2009),Parkinsonetal.

(2002),SalazarͲValenzuelaetal.(in.prep.),SaldarriagaͲCórdobaetal.(in.prep.),and

71 Wüsteretal.(2002),weusedthreeB.atroxsamplesfromasinglepopulationin

AmazonianBrazilasanappropriateoutgroup.

Genomicmethods

WeextractedgenomicDNAfromeachsampleusingaQiagenDNAbloodand tissuekit(Qiagen,Valencia,CA,USA)andtheconcentrationofDNAisolateswas examinedonaQubit2.0fluorometerusingadsDNABRassaykit(LifeTechnologies,

Carlsbad,CA,USA).WefollowedSovicetal.(2016)fortheconstructionofdoubleͲdigest

RADseqlibraries(DaCostaandSorenson,2014;Petersonetal.,2012).Thedigestionof approximately250ngofDNAofeachindividualwasperformedusing15unitsofEcoRI andSbfIrestrictionenzymes(NewEnglandBiolabs,Ipswich,MA,USA).Sequencingwas performedin50Ͳbprunsusing10–20%ofalaneofanIlluminaHiSeq2000atthe

GenomicsSharedResourceoftheOhioStateUniversityComprehensiveCancerCenter.

WeusedAftrRAD4.1(Sovicetal.,2015)toassembleandgenotypetheRADseq data,aswellastoproduceinputfilesfordownstreamanalyses.Weuseddefault settings,exceptfortheparametersdescribedbelow.Onlylociscoredinatleast95%of theindividualswereretained;thislevelwaschosentoreducetheeffectsofalleledrop out(Arnoldetal.,2013;Gautieretal.,2013).Amaximumoffourindelswereallowed betweenreadstoconsiderthemalternativeallelesfromthesamelocusandaminimum offivereadswasrequiredatagivenlocustocallagenotype.Finally,inordertoavoid spuriousSNPsthatformattheendofreadsduetolocusassemblymethods,onlySNPs

72 occurringinthefirst34positionswereretainedafterremovalofbarcodesand restrictionsites(Sovicetal.,2015).

Geneticstructureanalyses

WeexaminedthestructureofthepopulationsusingtheBayesianclustering algorithmimplementedintheprogramStructure(Pritchardetal.,2000),whichclusters samplesintopopulationsbyminimizingHardyͲWeinbergdisequilibrium.Weusedan admixturemodelanditerativelyconductedfiveindependentrunsofKvaluesranging from1–4.AburnͲinof50,000generationswasusedandeachanalysissampledevery

100iterationsfor500,000generations.StructureHarvester(EarlandvonHoldt,2012) wasusedtoimplementtheȴKstatisticofEvannoetal.(2005)inordertoidentifyan appropriatenumberofclusters.ResultsweresummarizedwithCLUMPP1.1.2

(JakobssonandRosenberg,2007)usingtheFullSearchalgorithmandvisualizedwiththe programdistruct1.1(Rosenberg,2004).Additionally,weusedthekͲmeansclustering methodavailableinadegenet1.4Ͳ2(Jombart,2008;JombartandAhmed,2011).This programidentifiesthemostappropriateclusteringsolutionsbasedonBayesian informationcriterion(BIC)scoresfromaxesderivedfromaPrincipalComponents

Analysis(PCA),andthereforedoesnotrelyontheHardyͲWeinbergassumptionsthat

Structureconsiders.WeevaluatedKvaluesrangingfrom1–15andperformeda discriminatefunctionanalysisofPCAs(DAPC)basedontheoptimalclusteringsolution suggestedbyadegenet.Thesefunctionsareavailableintheade4packageandwere

73 conductedinR3.1.3(RCoreTeam,2015).Foranalysesranonbothprogramswe excludedtheB.atroxoutgroupsamples.

Populationsplitsandmixtures

TheprogramTreeMix1.12(PickrellandPritchard,2012)wasusedtoinferthe hypothesizedevolutionaryhistoryofthesampledpopulations.Themodelimplemented inthissoftwareallowsbothpopulationsplitsandadmixtureormigrationevents.The inputfileforTreeMixconsistedofthefirstSNPfromeachlocusandwasobtainedby convertingthediploidgenotypecallsforeachindividualintopopulationͲlevelallele countsusingAftrRAD.WeconstructedmaximumͲlikelihoodtreesallowing1–5migration eventsandperformed100bootstrapreplicateswhilesamplingblocksof10contiguous

SNPsinordertocontrolforstochasticsamplingerror.

Demographicmodeling

AlthoughTreeMixisausefulmethodtoinferrecentpopulationhistoriesthat consistofsplitsandinstancesofgeneflow,itonlymodelsmigrationasdiscreteevents anddoesnotconsidercontinuousgeneflow(PickrellandPritchard,2012;Sousaand

Hey,2013).Therefore,weusedthecoalescentͲbasedmodelingpackagefastsimcoal version2.5.2(Excoffieretal.,2013)tostatisticallycomparetherelativefitoffour historicaldemographicmodelsgivenourgenomicdata(Fig.3.1).Thesemodelsdiffered inthetopology:((PEC,HEC1)HEC2)inFig.3.1Avs((HEC1,HEC2)PEC)inFig.3.1B,and whethertheyallowedcurrentmigrationbetweenthedifferentlineages(Fig.3.1Cvs

3.1D).Duetocomputationaldemandsoftheprogramandbasedonourclustering

74 analyses,weonlyusedthesouthernpopulationofPECtorepresentthislineage.

Therefore,weselectedasubsetofouroriginaldatawhenbuildingtheunfoldedsite frequencyspectrum(SFS)andretainedonlylociscoredin100%ofthesamplesinorder todefinetheancestralallelefromB.atroxatallloci.Likelihoodsforeachmodelwere calculatedbasedontheSFS;weperformed100independentrunsoffastsimcoal

(200,000simulationsperrun),andtherunwiththehighestlikelihoodforeachmodel waschosentoperformmodelselectionwithAIC.Maximumlikelihoodestimatesfor parameterswereobtainedfromtheoptimalmodel/runandusedtogenerate confidenceintervalsbyaparametricbootstrappingapproachinwhich50independent

SFSweresimulatedfortheoptimalmodelandparameterestimates.Thesesimulated datasetsweretreatedasobserveddata,andparameterestimationwasperformedfor eachasabove.

Toconvertestimatesoftimefromgenerationstoyears,weuseddatafromSasa etal.(2009)andthemethodsuggestedbyGrazziotinetal.(2006)forBothropsjararaca.

Generationtimewasestimatedas8yearsandwasobtainedastheaverageofthe youngestreportedageatmaturity(3years)andtheshortestreportedlifespan(15 years)minus1yearasacompensationforsurvivalprobabilityuntiloldages.

Results

GenotypingofRADseqdata

Werecoveredameanof717,094sequencereadsforindividualsincludedinour

RADseqdataset(range:85,395–1,964,552)afterqualityfiltering.Themeanreaddepth 75 perlocuswas86.2readswhilethemedianreaddepthwas54reads.Atotalof15,393 nonͲparalogouslociwereidentified:12,442ofwhichweremonomorphicandthe remaining2,951containedatleastonepolymorphicsite.Ofthe2,951polymorphicloci,

1,241werescoredinatleast95%ofthesamples.

Geneticclustering

GeneticclusteringinStructuresuggestedanoptimalKoftwowithonegroup consistingofboththePEClineageandHEC1lineage,andanothergrouprepresentedby

HEC2.Thesegroupsaregeneticallyhomogeneousandformdistinctclusterswithalmost noevidenceofadmixture(Fig.3.2A).Incontrast,BICvaluesfortheDAPCapproachin adegenetsuggestedthreeindependentgroups;however,wenotethatbothPECand

HEC1samplesclusteredcloselyinthemultivariatespacerelativetoHEC2(Fig.3.2B).

Populationsplitsandmixtures

Similartotheclusteringsolutionssuggestedabove,MLpopulationtreesfrom

TreeMixinferredatopologythatclustersthePECandHEC1lineagestogether.This programidentifiedgeneflowbetweentheseB.asperlineageswhenallowedtochoose between1and5migrationevents.However,itneveridentifiedgeneflowbetweenthe

HEC2lineageandanyoftheothergroups.Finally,TreeMixalwaysallowedmigration eventsbetweentheoutgrouprepresentedbyB.atroxsamplesfromBrazilandthePEC lineagesuggestinghistoricalmigrationbetweenpopulationsoftheB.atroxcomplex locatedintheAmazonBasinandPacificpopulationsofB.asper(Fig.3.3).



76 Historicaldemography

Ourmodelingapproachsuggestedstrongsupportfora((PEC,HEC1)HEC2) relationship(model1B).Thismodelreceived98.1%oftherelativeweightbasedon evaluationusingAIC(Table3.2).Theseresultsindicatethatthereisaclear differentiationofoneofthehighlandlineages(HEC2),andthattheotherhighlandgroup

(HEC1)issistertothePEClineage.Thereisnoevidencethatsignificantlevelsofongoing migrationexistbetweenanyoftheselineages.

Maximumlikelihoodparameterestimatesandconfidenceintervalsunderthe bestͲsupportedmodel(Table3.3)indicatethateffectivepopulationsizesforthe highlandlineagepopulations(HEC1:11,915;HEC2:19,138)aresmallerthanthePEC population(85,918)consistentwithafoundingevent.DivergencetimesforthePacific andHEC1lineagesoccurredapproximately60,976yearsbeforepresent(CI33,376–

121,800ybp),whileestimatedtimesforthedivergencewiththeHEC2lineageoccurred approximately201,656ybp(CI116,008–298,000ybp).



Discussion

Themainresultsofourstudyarethat1)thediscrepancyofevolutionary relationshipsobtainedwithtreeͲlikemethodsisresolvedthroughtheuseofhistorical modelingapproaches,2)thereisstrongsupportforthepresenceandindependent originofmontanelineagesbasedonclusteringsolutionsandtopologiesinferredbyML treesandmodelingapproaches,and3)evidenceofmigrationwasonlyidentified 77 betweenthelowlandandoneofthehighlandlineagesaftertheirdivergence.We discusstheevolutionary,taxonomic,andbiomedicalimplicationsofourfindingsbelow.

EvolutionaryrelationshipsamongB.asperEcuadorianlineages

 Inadditiontopotentiallybeingaffectedbyincompletelineagesorting, coalescentͲbasedinferencesinsystemscharacterizedbyrapidspeciationeventscould alsobeaffectedbygeneflowbecausemethodsforlineageidentificationbasedon coalescentmethodsassumethatithasceaseduponspeciation(BurbrinkandGuiher,

2015).Thusgeneflowcouldreducetheaccuracyofspeciestreeinferences,resultingin underestimatesoflineagedivergencetimesand/orinferenceofanerroneoustopology

(Gruenstaeudletal.,2016).Newlyacquiredpopulationgenomicdatasetscanbeusedto disentangletheconflictingroleofgeneflowduringthedivergenceprocess(Sousaand

Hey,2013),andwehaveemployedthemheretoexplorepatternsofdiversification betweenlowlandandhighlandlineagesofterciopelopitviperspresentintheAndesof southernEcuador.

 Contrastingtopologiesbetweentheselineageswereinferredinapreviousstudy ofthiswidespreadspeciescomplex(SalazarͲValenzuelaetal.,in.prep.).Acloser relationshipbetweenhighland(HEC1andHEC2)lineageswasrecoveredwithanalysesof mtDNAdataandsomeapproachestoanalyzeRADSeqdata(estimationofamaximum likelihoodtreebasedontheconcatenateddatasetandspeciestreeanalyseswith

SVDquartets(ChifmanandKubatko,2014)).Incontrast,speciestreeanalysesofthese samedatawithSNAPP(Bryantetal.,2012)inferredatopologythatclusteredthePacific

78 Ecuadorianlineagewithoneofthehighland(HEC1)lineages.Allthemethodsweused heretoanalyzeourpopulationgenomicdatasetstronglysupportthelatterhypothesis

((PEC,HEC1)HEC2).Acloserrelationshipbetweenhighlandlineagescanthereforebe accountedforbythefactthatincompletelysortedancestralpolymorphismswerenot takenintoaccountwithsingleͲlocusanalysesortheevaluationofaconcatenated multilocusdataset.AlthoughSVDquartetsandSNAPPdealwiththisproblemusinga coalescentmodel,differencesbetweentheseprogramscouldbeduetodiscrete migrationeventsthatwereidentifiedafterthedivergenceofPECandHEC1lineages.

BurbrinkandGuiher(2015)recentlyusedmultilocusdataandalternative populationassignmentsofadmixedindividualstodemonstratetheimpactofgeneflow ontheidentificationanddelimitationoflineagesofNorthAmericanpitvipersofthe genusAgkistrodon.Theapproachimplementedinourstudygoesonestepfurtherby usingTreeMixandmodelͲbasedanalysesinfastsimcoalforourpopulationgenomicdata inordertodisentangletheroleofgeneflowduringtheoriginofmontanelineagesof terciopelopitvipersinEcuador.

EvolutionarydynamicsoflowlandandhighlandB.asperEcuadorianlineages

Vicarianceeventshavebeenemphasizedastheprimarymechanismexplaining thespeciationofAndeancolonistsfromlowlandancestors(Guarnizoetal.,2009;

Wingeretal.,2015).Supportforthisallopatricmodelhasbeenfoundinseveralstudies thathaveestablishedthatsisterlineagesindifferentgroupsofAndeanvertebrates occupyareasofsimilaraltitude(BrumfieldandEdwards,2007;PattonandSmith,1992;

79 Robertsetal.,2006).Alternatively,ecologicalgradients,availabilityofnewhabitats,and absenceofcompetitionaremechanismsofrapidlineagediversificationproposedto occuronrecentlyformedmountainrangessuchasthenorthernAndes(Brumfieldand

Edwards,2007;Caroetal.,2013;Chapman,1926;Endler,1977;Grahametal.,2004;

HughesandEastwood,2006).Parapatricspeciationalongamountainverticalaxis shouldfollowthedifferentialadaptationsassociatedwiththeseecologicalgradients

(Guarnizoetal.,2009).OurresultsforB.asperEcuadorianlineagesindirectlysupport thisalternatemodelofdiversificationsincewerejectedthehypothesisofhighland lineagesbeingeachother’sclosestrelatives.Divergentecologicalregimesinfluencing geneticdifferentiationpatternshavebeenproposedtobemoresignificantintaxawith morelimitedecologicalniches(e.g.,anurans,insects)(BrumfieldandEdwards,2007).

Althoughfewstudieshavebeenconductedwithsnakes,theseorganismscouldalsobe thoughtasheavilyinfluencedbyecotonesgiventheirlowvagilityandstrongresponse tolocalenvironmentalfactors(PyronandBurbrink,2009).

OurfindingssuggestthatB.asperpopulationsfromthePacificlowlandsof southernEcuadorindependentlycolonizedhighlandhabitatsandthenunderwent differentiationinisolation.TheHEC1populationanalyzedhereislocatedintheInterͲ

AndeanvalleyofVilcabamba,Lojaprovince.Isolationofthislineageagreeswith previousreportsofmorphologicaldifferencesforthesepopulationsrelativetootherB. asperpopulationsinEcuador(CampbellandLamar,2004)andwithpresentͲday distributionpatternsthatshownopublishedrecordsofcollectionlocalitiesbetween

80 populationsclosetothePacificcoastandthisvalley(BustamanteandArteaga,2013;

CisnerosͲHerediaandTouzet,2004).ThedistinctivenessoftheHEC2lineageismore intriguingasthereareatleastsomeinterveningpopulationspresentinthefoothillsof theAndes(SalazarͲValenzuelaetal.,in.prep.).Nevertheless,theInterͲAndeanvalley whereHEC2populationsarelocated(Jubonesvalley,AzuayandandLojaprovinces) seemstobeeffectivelyisolatedfromsurroundingareas,asitispartofa phylogeographicbarrierthathasbeenrecognizedasimportantforothermontane organisms(Weigend,2002).Thisbarrier,knownastheHuancabambaDepression,could alsoexplainourresultsofhistoricalmigrationbetweensnakesoftheB.atroxcomplex andPacificpopulationsofB.asper,asithasbeensuggestedthatthisregionoflow altitudeintheAndescouldhavefacilitatedtheexchangeoforganismslocatedonits easternandwesternversants(Duellman,1979;Tréneletal.,2008).

RecenteventsofpitvipercolonizationanddiversificationinSouthAmerica

RecentandextensivediversificationofB.asperlineagesinSouthAmericaduring thePleistocenecontrastwithpreviousphylogeographicstudiesconductedon herpetofaunaintheregionthathaveemphasizedolderdiversificationeventsduringthe

Plioceneand/orMiocene(TurchettoͲZoletetal.,2013).Althoughdivergenceinlowland

B.asperlineagesinthissubcontinentcouldstillberelatedtolargeͲscalelandscape changes(e.g.,marinetransgressions,Quaternaryclimatechanges)thatfragmented previouslycontinuousdistributions(Rull,2011),ourresultsshowtheimportanceof montanehabitatsfordiversificationinthegroup.

81 BesidestheB.aspercomplex,onlyafewadditionalgroupsofpitviperswere involvedinthegreatAmericanbioticinterchangeoftaxabetweenNorthandSouth

America(Baconetal.,2015;Werman,2005).Mostofthesesnakesdispersedfrom

CentralAmericaandcolonizedlowlandhabitatsontheeasternandwesternsidesofthe

Andes.Forexample,theNeotropicalrattlesnake(Crotalusdurissuscomplex)dispersed fromCentralAmericaduringthemiddlePleistoceneandsubsequentlydiversifiedin openanddryhabitatsinSouthAmericaeastoftheAndes(Wüsteretal.,2005).In contrast,theancestorsoftheB.asperspeciescomplexoriginatedinnorthwestern

SouthAmericaapproximately3.3MaandthenindependentlycolonizedCentralAmerica aswellasdispersingsouththroughanarrowstripoflowlandswestoftheAndes

(SaldarriagaͲCórdobaetal.,in.prep.;SalazarͲValenzuelaetal.,in.prep.).ThepresentͲ daydistributionofthegroupismainlyrestrictedtolowlandtropicalrainforestsinthese areas(CampbellandLamar,2004),butaswithothervertebrates(Milleretal.,2010)its southernlimitoccursinnorthwesternPeruwherehighlyaridconditionspresentduring thelast3Mamayhaverestrictedtheirdispersalfurthersouth(HartleyandChong,

2002).

Basedontheoverallassessmentoftreemethods(SalazarͲValenzuelaetal.,in. prep.)anddemographicmodelingpresentedhere,ourresultssuggestthatinterͲAndean valleyshavebeenapreviouslyunrecognizedimportantdriverofmontanelineage divergenceinterciopelopitvipers.Interestingly,environmentalconditionsinthese valleysonthehighlandsofEcuadorandColombiaaresimilarandareoccupiedby

82 seasonallydryforests,whichinthelast15yearshavebeenrecognizedasanimportant andthreatenedbiomerichinendemicspecies(Milesetal.,2006;Penningtonetal.,

2010;Penningtonetal.,2000;Wernecketal.,2011).Thereisevidencethatthesetypes offorestshavebeenpresentintheareasince10Ͳ15Ma(Särkinenetal.,2012),and thereforemayhaveprovidedecologicalopportunitiesforterciopelopitviper populationstocolonizeanddivergeinhighlandhabitats.Ourfindings,coupledwith recentdescriptionsofendemicsnakesandlizardsfromEcuadorandPeru(Kochetal.,

2015;Kochetal.,2013;TorresͲCarvajaletal.,2013;Venegasetal.,2008),suggestthat drymontanehabitatsmayplayapreviouslyunappreciatedroleasdriversof diversificationinAndeanreptiles.Thisfindingisnovelcomparedtodiversification eventsidentifiedinotherpitvipersfromtheNeotropics.Castoeetal.(2009)established coincidentMioceneandPliocenedivergencesforhighlandlineagesofpitvipersfrom

MiddleAmerica.However,diversificationinthoselineagesseemstobeassociatedwith vicarianteventsratherthanwiththemorerecentavailabilityofdrymontanehabitatsas isthecasewithterciopelopitviperscolonizingtheAndesofSouthAmerica.

Implicationsandfuturedirections

OurresultssupporttherecognitionoftheHEC2lineageasphylogenetically significant.Moreover,basedontheestimatedtimeofisolationassociatedwiththe

HEC2populationandonobserveddifferentiationinmorphologicaltraits(SalazarͲ

Valenzuelaetal.,in.prep.),werecommendrecognitionofthisgroupatthespecies level.Sinceterciopelosnakesaretheleadingcauseofsnakebiteaccidentsacrosstheir

83 distribution(Warrell,2004),futurestudiesestablishingproteomicandfunctional characterizationsofvenoms,aswellasantivenomneutralizationoftoxinspresentin membersofthislineagearerecommended.

Finally,approacheslikethoseusedinthisstudycouldbeappliedtoanalyze divergencebetweenotherlineagespreviouslyidentifiedintheB.aspercomplex

(SalazarͲValenzuelaetal.,in.prep.).Diversificationpatternsofterciopelopitvipers presentoninterͲAndeanvalleysofsouthernColombiaseemtobesimilartothose analyzedhereandcouldprovideanindependentassessmentoftheimportanceof montanedryhabitatsforthedivergenceprocessesthathaveactedinthisgroup.



Acknowledgements

WethankW.Wüster,E.N.Smith,M.Terán,andD.Amazonasforgenerously providingkeysamples.WearegratefultoM.Sovic,J.Díaz,andP.SantacruzͲOrtegafor adviceandhelpinthelab.Foradviceandcommentsonthemanuscript,wethank membersoftheGibbsLab,J.Freudenstein,B.Carstens,T.Hetherington,P.Fuerst,and

G.Silva.WealsothankA.LoaizaͲLange,D.Núñez,P.SantacruzͲOrtega,S.Harris,A.

FreireͲLascano,R.Molina,andE.Arbeláezforassistanceinthefield.Specimenswere collectedundercollectionpermit008Ͳ09ICͲFAUͲDNB/MAandweredepositedatMuseo deZoología(QCAZ),PontificiaUniversidadCatólicadelEcuador.Thisworkwasfundedin partbythefollowingorganizations:GermanResearchFoundation(DFG),Secretaríade

84 EducaciónSuperior,CienciayTecnologíadelEcuador(SENESCYT),theTinker

Foundation,andOhioStateUniversity.



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93 Tables

Table3.1VoucherandlocalityinformationforEcuadorianspecimensusedinthisstudy.   Population Altitude Voucher Lineage Latitude Longitude  (Locality,Province) (m)  INSPI2PEC(north) Cumandá,Chimborazo Ͳ2.20379 Ͳ79.13756 298 INSPI6PEC(north) Cumandá,Chimborazo Ͳ2.20379 Ͳ79.13756 298 INSPI18 PEC(north) Cumandá,Chimborazo Ͳ2.20379 Ͳ79.13756 298  INSPI34 PEC(north) Cumandá,Chimborazo Ͳ2.20379 Ͳ79.13756 298 INSPI138 PEC(north) Cumandá,Chimborazo Ͳ2.20379 Ͳ79.13756 298 QCAZ10065 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939  QCAZ10066 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 QCAZ10067 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 QCAZ11284 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 QCAZ11285 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 QCAZ12568 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 QCAZ12569 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 Bas002 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 Bas004 PEC(south) Alamor,Loja Ͳ4.02578 Ͳ80.10719 939 QCAZ11622 HEC1 Vilcabamba,Loja Ͳ4.25649 Ͳ79.21753 1,541 WW740 HEC1 Vilcabamba,Loja Ͳ4.25649 Ͳ79.21753 1,541 WW753 HEC1 Vilcabamba,Loja Ͳ4.25649 Ͳ79.21753 1,541 QCAZ4468 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875 QCAZ4474 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875 QCAZ4475 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875 QCAZ5013 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875 ENS12878 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875  ENS12881 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875 Vip001 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875  Bas020 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875 Bas023 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875  Bas024 HEC2 RíoLeón,Azuay Ͳ4.31995 Ͳ79.15371 1,875 



 94  Table3.2AICmodelselectionresultsforfastsimcoalanalyses.    No. Model LnLikelihood AIC Akaikeweight Parameters  IsolationHighlandlineages 10 Ͳ3862.055 17805.4205 0.00000  (1A) IsolationPacific+HEC1vs 10 Ͳ3854.965 17772.7699 0.98101  HEC2(1B) Isolation+Migration  14 Ͳ3856.737 17788.9302 0.00030 Highlandlineages(1C)  Isolation+MigrationPacific 14 Ͳ3854.948 17780.6916 0.01869 +HEC1vsHEC2(1D) 



Table3.3Maximumlikelihoodestimatesfordemographicparametersestimatedinthe fastsimcoalanalysisforthebestͲsupportedmodel:((PEC,HEC1)HEC2).    Parameter Pointestimate Confidenceinterval  Effectivepopulationsizes  HEC1(current) 11915 6220–25785  HEC2(current) 19138 11454–28236   PEC(current) 85918 53381–121541  B.atrox (current) 194993 143273–225409  PEC+HEC1(ancestral) 102705 30850–120622  B.asper (ancestral) 83367 66314–112991  B.asper +B.atrox 223371 182374–253718  Divergencetimes  PECͲHEC1 60,976ybp 33,376–121,800ybp  PEC,HEC1–HEC2 201,656ybp 116,008–298,000ybp  B.asper ͲB.atrox 929,968ybp 672,808–1160,736ybp  

95 

Figures

   the   with 

 and  circle).   isolation  lowlands (green   2

 and   B)  HEC Pacific  

 (A,  the and    from models   square)   Isolation (black 

  1  populations 

 HEC  lineage.  sampled PEC    the  triangles),   for   showing  (red   used  PEC (left)   were text: 

  fastsimcoal.  Ecuador in the     in of south)     tested and  map 

  D)   described (C,   (north

      Topographic models   lineages   3.1 

 populations   migration montane Two  Figure 96                     PEC + HEC1 HEC2                  Figure3.2Structureplot(K=2)(above)andresultsfromtheDAPCclusteringanalysisin ĂĚĞŐĞŶĞƚ(K=3)(belowright)generatedfrom1,241polymorphicloci.Theplot(below left)showstheresultfortheoptimalKvalueinferredfromBICvalues.

97 

































Figure3.3MLpopulationtreeinferredwithTreeMix.Onlyonetreeisshownbecausewe obtainedsimilarresultswhenonetofivemigrationeventswereallowed.Graphdepicts splitsamongdifferentpopulationsandtheweightassociatedwithmigrationevents(red indicatesahigherweight).Numbersatnodesindicatebootstrapsupport. 

98  



Chapter4:Distribution,geneticstructureandmorphologicalvariationofan endangeredAndeanpitviper,theLojanlancehead(Bothropslojanus) 

Abstract

Thegeographicdistributionandgeneticstructureofraretropicalsnakesis poorlyͲknownyetsuchinformationisusefulforconservationplanning.Here,weprovide suchinformationforararepitviper,theLojanlancehead(Bothropslojanus),whichhasa limiteddistributioninsouthernEcuador.Environmentalnichemodeling,whichused informationonsamplelocationsfromrecentlyͲcompletedfieldsurveysandfrom museumspecimens,demonstratesthattheprojectedrangeofthisspeciesislargerthan previouslysuggestedandtargetsspecificregionsinsouthernEcuadorforfuturesurvey work.GeneticanalysesofmitochondrialandnuclearDNAlociidentifytwodistinct geneticgroupswithinthiscurrentlydescribedsinglespeciesthatshouldeachhave statusasseparateconservationunits.Morphologicalanalysesshowthatthereare differencesincharactervariationthatmirrortoalimitedextentthegeneticdifferences, butatthispointtheyarenotconclusiveenoughtosupportthehypothesisthatthese groupsrepresentdistinctevolutionarylineages.Ourworkdemonstrateshowmultiple linesofdatacanbeusedtoclarifyecologicalandevolutionaryfeaturesofraresnakes thathaveconservationimplications. 99 Keywords:ecologicalnichemodeling,conservationgenetics,morphometrics,Bothrops, pitvipers,TropicalAndes



Introduction

 Snakeshavehistoricallyplayedsignificantrolesinhumanevolutionandculture

(GreeneandCampbell,1992;Isbell,2006).DuetocertainlifeͲhistorycharacteristics

(e.g.,latesexualmaturity,sitefidelity,unnaturallyhighmortality),venomoussnakesof thefamilyViperidae(i.e.,vipersandpitvipers)areespeciallyvulnerabletopopulation declines(BeaupreandDouglas,2009).Recently,ithasbeenshownthatvipersand pitvipersexhibitsignificantlyhigherproportionsofthreatenedspeciesthanrandomly expected(Böhmetal.,2013).Despitethefactthathumanenvenomationresultingfrom snakebitesisstillanimportantcauseofhumanmortalityandmorbidityintropicalareas oftheworld(Gutiérrezetal.,2006;Williamsetal.,2010),venomoussnakesalsooffer directandindirectbenefitstohumanssuchasecologicalservicesandbiomedical applications(SeigelandMullin,2009;TakacsandNathan,2014).Therefore,the protectionandmanagementofvenomoussnakesthat,inadditiontobeingrare,also poseapotentialthreattohumanhealthconstitutesaspecialchallengeforconservation biologists(SeigelandMullin,2009).

Amajorobstacleforestablishingeffectiveconservationactionsinsnakesisthat littlebiologicalknowledgeexistsformosttropicalspecies(SeigelandMullin,2009).An exceptionistheinformationthatexistsforvenomoussnakesduetotheirimpacton 100 publichealth(QuijadaͲMascareñasandWüster,2010).Asignificantamountof informationaboutsystematicsandvariousaspectsofnaturalhistoryhasbeengained fordifferentgroupsofvenomoussnakes(Cabrellietal.,2014;Fenkeretal.,2014;

Wüsteretal.,2008).Nevertheless,somespeciesinhabitingisolatedplacesstillremain poorlyknown(CampbellandLamar,2004).Formontanespeciesofvenomoussnakes, forexample,thereisalackofabasicunderstandingoftheirdistributions,systematics, andeffectsonpublichealthataregionallevel(Ferchaudetal.,2011;Holycrossand

Douglas,2007;Werman,2005).Alackofawarenessaboutthesefeaturescanaffectthe conservationofmontanevenomoussnakesbypreventinganinformedassessmentof theirfutureriskofextinction.

 OnesuchspeciesisBothropslojanusParker,1930,whosecommonnameisthe

Lojanlancehead(CampbellandLamar,2004).Unlikemostofthelowlandpitvipersof theNeotropics,thismediumͲsizedspeciesisahighlyendangeredsnakeduetoits restricteddistributioninhumanͲimpactedmontaneregionsoftheTropicalAndes biodiversityhotspot(CisnerosͲHeredia,2010;GreeneandCampbell,1992).Itsknown distributioninsouthernEcuador(Fig.4.1)overlapswithareasidentifiedaspriority placesforbiodiversityconservationgiventhehighconcentrationofendemictaxathey harbor(Stattersfieldetal.,1998;Terribileetal.,2009).Despitetheendangered conservationstatusofthissnake,littleisknownaboutkeyfeaturesofitsbiologysuchas itsdistribution,habitatrequirements,andpopulationgeneticstructure.Indeed,theonly informationavailableforthisspeciesisthatmostoftheknownpopulationsarelocated

101 inthevicinityofthetypelocality:Loja,Lojaprovince,atelevationsbetween2,100and

2,300mandthatapopulationrepresentingthesamespeciesoranearrelativeisfound innorthernPeru(CampbellandLamar,2004).

 Inthisstudy,weexpandourknowledgeforthisrarepitviperbyanalyzingnew andexistingdistributionaldata,mitochondrialandnucleargeneticdata,and morphologicaldataforB.lojanus.Ourgoalwasto1)generatepredictivedistribution mapsforthespecies,2)examinepatternsofgeneticstructureanddiversitypresentin thegroup,and3)evaluatethecorrespondenceofmorphologicaldivergencebetween geneticgroups.Additionally,weassessedmorphologicalaffinitiesofspecimensfromthe populationinnorthernPerutodetermineifitbelongstothisspecies.



MaterialsandMethods

Environmentalnichemodelingandsnakesurveys

SpecimensofB.lojanusarerareinnaturalhistorycollectionsandnogenetic materialwasavailableforthespeciesbeforeourstudy.Therefore,weidentified potentialareasofoccurrenceusinganecologicalnicheͲbasedmodelingapproachasa wayofassessingthepotentialrangeofthisspecies(Austin,2002;ElithandLeathwick,

2009;Grahametal.,2004).Occurrencedataforthespeciesweregatheredmainlyfrom collectedspecimenshousedinfournaturalhistorymuseumsinEcuador.Allofthe localitiesidentifiedwereincloseproximitytothecityofLoja,Ecuador.Aqueryin

VertNet(www.vertnet.org),apublicdatabaseofvertebratecollectionsdata(Constable 102 etal.,2010),identifiedjustoneanimalincollectionsoutsideEcuadorwithuseful geographicinformation.Thisanimal(KU135213)comesfromalocalityca.40kmNof thecityofLoja.

WeidentifiedsixrecordsandtheselocalitieswereusedinMaxent3.3.2(Phillips etal.,2006)toproducethepredictivemapfoundinFig.4.2A.Basedonthismap,we conductedsurveysinfouradditionallocalitiesinsouthernEcuador(GulagandGima,

Azuayprovince;Yangana,Lojaprovince;roadLojaͲZamora,LojaandZamoraͲChinchipe province)predictedtohaveahighdegreeofprobabilityfortheoccurrenceofthe species.Outof19environmentallayers(1kmresolution)availableinWORLDCLIM

(Hijmansetal.,2005),tenwereusedtoconstructthemodel:annualmeantemperature, meanmonthlytemperaturerange,isothermality,meantemperatureofwettestquarter, annualprecipitation,precipitationseasonality,precipitationofwettestquarter, precipitationofdriestquarter,precipitationofwarmestquarter,andprecipitationof coldestquarter.Thesetenvariableswerechosenbecausetheyhavepreviouslybeen foundtobelargelyuncorrelatedinEcuadorbyValenciaetal.(2010)hencerepresent independentmeasuresofenvironmentalvariation.ValuesusedfortheMaxentrun werethoserecommendedbydefaultforconvergencethreshold(10Ͳ5)andmaximum numberofiterations(500).Wealsocollectednewdistributionrecordsbylocating preservedsamplesheldbylocalpeopleandbycapturingindividualsinthefield.Most specimensofB.lojanusweredonatedbylocalpeoplethatkeptethanolͲpreserved

103 animalsassouvenirsortosellthemtohealers,whereasafewindividualswerefoundin understoryvegetationwhileconductingsurveysintheearlymorningorlateafternoon.

Moleculardata

Weobtainedbloodortissuesamplesfor21B.lojanusindividualsfromsix localitiesinsouthernEcuador(N=1–12perlocality)(Table4.1).BasedonCarrascoetal.

(2012),weusedonesampleeachofBothrocophiasmicrophthalmusandCerrophidion godmaniasoutgroups.WeextractedgenomicDNAfromeachsampleusingeithera

QiagenDNAbloodandtissuekit(Qiagen,Valencia,CA,USA)oraphenolͲchloroform protocol.TheconcentrationofDNAisolateswasexaminedonaQubit2.0fluorometer usingadsDNABRassaykit(LifeTechnologies,Carlsbad,CA,USA).Outofthe21 samples,wecouldonlyobtaingoodqualityDNAathighconcentrationfor17ofthem.

Theremainingsamplesbelongedtoindividualsthatmayhavebeenkepttoolongin inappropriatestorageconditionsresultinginDNAdegradation.Weamplifiedthree portionsofthemitochondrialgenome(mtDNA):cytochromeb(cytb),NADH dehydrogenasesubunit4(ND4),andATP6Ͳ8(ATP),aswellasthreenuclearDNA introns:Etsoncogene(ETS),GlyceraldehydeͲ3Ͳphosphatedehydrogenase(GAPD),and

EͲFibrinogen(FGB).PrimersandPCRconditionsarethosedescribedinCastoeand

Parkinson(2006)andGibbsandDiaz(2010).Sequencingreactionsforforwardand reversestrandswereconductedusingtheBigDyeterminatorcyclesequencingkit(Life

Technologies)andproductsweresequencedonanABI3100GeneticAnalyzer.



104 Phylogeneticanalysesandgeneticdiversity

ComplementarymtDNAsequenceswereassembledandeditedwithCodonCode

Aligner4andweusedMUSCLE(Edgar,2004)inGeneious7.0toalignthemusingdefault settings.Nucleotidesequenceswereexploredalongsidetranslatedaminoacidsto evaluatethereadingframeandensureabsenceofprematurestopcodonsornonsense mutations.WeusedbothmaximumͲlikelihood(ML)andBayesianinference(BI)analyses toinferintraspecificphylogeneticrelationships.TheMLapproachwasconductedin

RAxML8.0(Stamatakis,2014)usingaGTRGAMMAsubstitutionmodelandperforming

1,000bootstrapreplicatesinarapidbootstrapanalysis.ForBIanalyses,weused

PartitionFinderv1.1.1(Lanfearetal.,2012)toselectthepartitionschemeandbestͲfit nucleotidesubstitutionmodelundertheBayesianInformationCriterion.Three independentruns,eachwithfourMarkovchains(onecoldandthreeheatedchains), wererunfor20milliongenerationsandsampledevery1,000generationsinMrBayes v.3.2.3(Ronquistetal.,2012).Stationarityandeffectivesamplesizes(ESS)forall parameterswereassessedwithTracerv.1.5(DrummondandRambaut,2007).Ofthe

20,000treesresultingperrun,thefirst25%werediscardedasburnͲin.Theremaining treeswereusedtocalculateposteriorprobabilities(PP)foreachbipartitionina50% majorityͲruleconsensustree.Finally,uncorrectedpairwisedistanceswithinandamong mtDNAcladeswereestimatedusingMegav.5.03(Tamuraetal.,2011).

Becausenuclearmarkersshowedlowlevelsofvariation(seeResults),we followedGuoetal.(2011)andanalyzedrelationshipsbetweensampledindividualsat

105 theselociusingamedianͲjoiningnetwork(MJN)approach.Weusedtheprogram

PopART(LeighandBryant,2015)forthisgoalandadditionallyconstructedamedianͲ joiningnetworkforthemtDNAdataset.Finally,weconductedacombinedanalysisof nuDNAandmtDNAconcatenatingallthelociandpartitioningournucleardatasetin threeseparatepartitions.Methodsforthisconcatenatedanalysisfollowedthe proceduredescribedaboveforthemtDNAdata.

Thenumberofhaplotypes(H),haplotypediversity(Hd),nucleotidediversity(S),and averagenumberofpairwisedifferences(K)werecalculatedforthemtDNAsetusingthe programDnaSP5.10(LibradoandRozas,2009).

Morphologicalanalyses

WerecordedmeristicandmorphometriccharactersforB.lojanusindividuals fromsixEcuadorianlocalities(seeAppendixDforspecificcharactersmeasured).We alsoexaminedthreespecimensfromthepopulationinnorthernPerudescribedby

CampbellandLamar(2004)inordertodetermineifitbelongstothisspecies.Preserved specimensarehousedinthefollowinginstitutions:Ecuador—MuseodeHistoria

Natural,EscuelaPolitécnicaNacional(EPN),Quito;FundaciónHerpetológicaGustavo

Orcés(FHGO),Quito;MuseodeZoología,PontificiaUniversidadCatólicadelEcuador

(QCAZ),Quito.Peru—MuseodeHistoriaNaturaldelaUniversidadMayordeSanMarcos

(MUSM),Lima.UnitedStatesofAmerica—NaturalHistoryMuseum,UniversityofKansas

(KU),Lawrence;VertebrateCollection,TheUniversityofTexasatArlington(UTA),

106 Arlington.Atotalof51specimenswereexamined,comprising21females,19males,and

11withundeterminedsex(AppendixE).

Specimenswereassignedtogeneticgroupsidentifiedaboveinordertoassess thecorrespondenceofmorphologicaldivergencebetweenrecognizedunits.Weuseda combinationofourphylogeneticanalyses(basedonmtDNAandnuDNAdata)and geographicproximitytoassignspecimensintotheirrespectivegroups.Significantsexual dimorphismisprevalentinpitvipersnakesandseveralofourcharactersshowedthe samedifferencewithineachlineagewhenanalyzedwithatwoͲwayANOVA;therefore, maleandfemaledatasetswereevaluatedseparatelytoavoidanyconfoundingeffectof sexinouranalyses.WetestedforsignificantbetweenͲgroupvariationinmeristic charactersusingaoneͲwayANOVAortheequivalentBrownandForsythetestwhen

Levene’stestofhomogeneityofvariancewassignificant.Morphometriccharacters wereadjustedtoaccountforallometriceffectsusingoneͲwayANCOVAapplied separatelytoeachgroup.Snouttoventlength(SVL)wasusedasthecovariateforhead andtaillengths,andheadlength(HL)forallothercharacters.Onlycharactersthat showedsignificantbetweenͲgroupvariationatthe5%levelwereusedfurther.

Weperformedamultivariateanalysisofthemorphologicalvariationpresentin

B.lojanustoidentifystructurethatcouldpotentiallycorrespondtogeneticgroups.We conductedaPrincipalComponentAnalyisis(PCA)inRwiththefunctionprcompinthe statspackage.Onlyindividualswithcompletecountsandmeasurementswere considered.AnalyseswereperformedinR3.1.3andSPSSStatisticsversion22(IBM

107 Corp.).Becauseofoursmallsamplesizes,weusedMannͲWhitneyUͲteststoassessthe presenceofstatisticallysignificantdifferencesinmorphologicalcharactersbetween animalsfromdifferentgeneticgroups.SincethethreePeruvianspecimensareallmales, weincludedthemasitsowngroupinourcomparisonofthemaledatasetforB.lojanus.



Results

Ecologicalnichemodeling

Asaresultoffieldworkandadditionalspecimensavailableinnaturalhistory collections,weidentifiedatotalof12localitiesforthespecies.Asexplainedbelow, animalsfromthreeoftheselocalitiesrepresentadifferentlineagewithstrongsupport fromthegeneticanalyses.Therefore,wemodeledtheecologicalnicheofB.lojanus sensustrictobasedonninelocalitieswherethislineagehasbeenfound(Fig.4.2B),and alsodevelopedanothermodelbasedonallthelocalitiesforbothlineages(Fig.4.2C).

Wewerenotabletomodeltheecologicalnichefortheotherclade(henceforthB. lojanusCladeB)duetothelownumberoflocalities(N=3),ashasbeenshownthat reliableestimatesofnichemodelsintheprogramMaxentrequireatleast5to12data points(Pearsonetal.,2007).

Comparingmodelsdevelopedbefore(Fig.4.2A)andaftersnakesurveys,wecan seethattheuseofninelocalitiesforB.lojanussensustricto(Fig.4.2B)didnotimprove themodelmuchasthesamegeneralareaswerepredictedtobeoccupiedbythe species.However,whenlocalitiesforbothlineageswereused(Fig.4.2C),the 108 distributionwasrestrictedtoafewareasinsouthernEcuador.Thispredictionseemsto beinagreementwithcurrentknowledgeaboutthisspeciessincefieldworkconductedin centralEcuadorduringthepastdecadeshasnotyieldedanyrecordsforthissnake.

Twoofthefourlocalitieswhereweperformedfieldworkconsiderablyexpand theknowndistributionofthespeciesinEcuador(Fig.4.1).Thefirstone(Gulag,Azuay province)expandsthedistribution34kmNEofthelimitsofthepreviousdistribution andrepresentsanewrecordforAzuayprovince.Theotherlocality(Yangana,Loja province)expandstheknowndistribution50kmSWfillingagapbetweenEcuadorian localitiesandtherecordofuncertaintaxonomicaffiliationfromPeru.

Phylogeneticreconstructionandgeneticstructure

Atotalof702bpofsequencewerealignedforcytb(126variable,50parsimonyͲ informativesites),615bpforND4(89variable,28parsimonyͲinformativesites),and774 bpforATP(165variable,62parsimonyͲinformativesites).Wewereabletoamplifythe threenucleargenefragmentsinmostingroupsamplesbutonlyinoneoutgroupsample

(Bothrocophiasmicrophthalmus).SixteensequenceswereobtainedforETS(709bp,3 parsimonyͲinformativesitesfortheingroup),18forGAPD(226bp,3parsimonyͲ informativesitesfortheingroup),and17forFGB(866bp,7parsimonyͲinformativesites fortheingroup).

FortheBIanalysis,PartitionFinderidentifiedthefollowingsubstitutionmodels:

K80forcytbandND4codonposition2;HKYforallremainingpartitions;andGTR+Ifor thecombineddataset.ThemtDNAphylogeniesresultingfromboththeBIandML

109 methodswereverysimilar,showingtwodistinctandwellͲsupportedclades(posterior probabilityandbootstrapsupportof1.0and100%,respectively)withinB.lojanus(Fig.

4.3).CladeAincludesthesamplefromthetypelocalityofB.lojanusandpopulations nearby(SanLucasandYanganainLojaprovince,andEstaciónCientíficaSanFranciscoin

ZamoraChinchipeprovince).CladeBincludedanimalsfromGulaginAzuayprovince.

Theconcatenatedmitochondrialandnuclearanalysisproducedatreesimilartothe mtDNAͲbasedtree(AppendixF).Geneticdistancesbetweenthesetwocladesare relativelyhigh:2.58വ2.72%uncorrectedgeneticdivergencesuggestingthatpopulations fromthesetwoareasshowsignificantgeneticdifferentiationwithlittleornogeneflow betweengroups.

Therewereninetotalcombinedmitochondrialhaplotypesidentifiedamongthe individualsanalyzed.Nohaplotypewassharedbyindividualsbelongingtothetwo clades.Overallhaplotypediversitywasrelativelyhigh(Hd=0.824)andnucleotide diversitywaslow(S=0.017).GeneticdiversityindiceswerelowerforcladeB,an observationprobablyrelatedtothefactthatonlyonepopulationwasincludedinthis group(Table4.2).MedianͲjoiningnetworksfornucleargenesdidnotshowthedistinct cladessupportedbymtDNAdatapossiblyduetothelowlevelofvariationpresentin thesemarkers.Finally,geneticdistanceswithinB.lojanussensustrictowerelow(0വ

0.52%);fivehaplotypeswereidentifiedinprogramDNASP5basedonsevensequences.

WithinB.lojanusCladeB,geneticdistancesrangedfrom0to0.19%andfourhaplotypes weredetected.

110 Morphologicalvariation

PCAanalysesshowedsomesupportforourhypothesisthatmorphologicalvariation wouldmirrorthestructurefoundbetweenB.lojanusgeneticgroups.However,both cladesdidnotseparatesignificantlyalongtheaxesofvariationidentifiedinthePCA(Fig.

4.4A;Bleft).Significantdifferencesinscutellationfoundbetweengroupswerefoundfor thefollowingcharacters:B.lojanussensustrictomaleshadahighernumberofventral andsubcaudalscales(MannͲWhitneyUͲtest,p<0.05,N=19)andB.lojanussensustricto femalesshowedahighernumberofventralscales(MannͲWhitneyUͲtest,p<0.01,n=21)

(Table4.3).

Incontrast,malesfromthePeruvianpopulationanalyzedinthisstudyseparated significantlyfromB.lojanusmales(Fig.4.4Bright),andmayrepresentadifferent species.Regrettably,thesearetheonlyspecimensknownfromthatpopulationand futuresurveyswillhavetovisitthisareainordertosecureadditionalmaterial.



Discussion

Themainresultsofourstudyarethat1)thegeographicdistributionofthe endangeredB.lojanusislargerthanpreviouslyinferred,2)populationsofthisspecies arecomposedoftwolineagesthatshowsignificantgeneticstructure,3)thereissome evidenceofcoincidentlevelsofmorphologicaldivergencebetweenthetwogeneticallyͲ distinctgroups,and4)thepopulationfromnorthernPerupreviouslyassignedtothis

111 taxonseemstorepresentadifferentspeciesgiventhelimitedsampleavailabletous.

Belowwediscusstheevolutionaryandconservationimplicationsofourfindings.

TheoccurrencepointsgeneratedforB.lojanusduringthisstudyconstitutea significantimprovementintheexistingknowledgeaboutthegeographicaldistribution ofthisendangeredsnake.Previousrecordsindicatedthatthespeciesonlyoccurredin anextremelyrestrictedareaofsouthernEcuadorandpossiblyinnorthernPeru

(CampbellandLamar,2004).Thelargergeographicdistributioninferredfromour recordsencompassestheentireAndeanregionknownastheHuancabambaDepression.

Inthisarea,thenarrownessandlowaltitudeoftheAndeanchainhavebeen hypothesizedtodeterminetheoverlapofsouthernandnorthernspeciesleadingtohigh levelsofdiversityandendemismindifferentmontanetaxa(Weigend,2002).Ourresults demonstratinghighlevelsofintraspecificgeneticstructureofB.lojanusinEcuador providesupportforthispatternaswefoundevidenceforahighlevelofgenetic divergencebetweentwomaingroupsinthenorthernpartofthisregion.Inthoseareas, altitudesarestillhighandthisfactcouldhavecontributedtotheisolationand divergenceofCladeB.Thisobservationagreeswithpreviousstudiesofmontane amphibiansandreptilesthathaveproposedtheideathatisolationinmontanesky islandscommonlypromotesdiversityintaxafoundinsuchregions(Shepardand

Burbrink,2008,2009).Underthosecircumstances,highlevelsofinterͲpopulation geneticdivergenceamongmontanepopulationsexistbecauselowͲelevationareaswith distinctenvironmentalconditionsactasbarrierstogeneflowleadingtodifferentiation

112 betweenpopulationsfoundonthetopsofdifferentmountains(Guoetal.,2011).

Interestingly,B.lojanuspopulationslocatedfurthersouthintheHuancabamba

Depressionaremoreconnectedpossiblybecauseoftheloweraltitudespresentinthe

Andesinthatarea.FurtherconsiderationsaboutthebiogeographicalhistoryofB. lojanusareprecludedbythelackofarobusthypothesisaboutthephylogenetic affinitiesofthisspecies.NoneoftherecentphylogeneticstudiesonSouthAmerican pitvipershaveincludedgeneticdataforB.lojanusandanalysisbasedonthelimited morphologicalevidenceavailablesuggestedthatthisspeciesmightnotactuallybepart oftheBothropsclade(Carrascoetal.,2012;Fenwicketal.,2009).

Thegeneticandmorphologicalevidencegatheredinthisstudysupportsthe interpretationthatthespecimensanalyzedarepartofthesameinterbreeding population.Althoughtwocladeswererecoveredinourphylogeneticanalyses, morphologicaldifferentiationbetweenthesegeneticgroupswaslimited.Future analysestargetingadditionalpopulationsrepresentingCladeBandwithadifferentset ofgeneticmarkerscouldassessifourcurrentinterpretationiscorrect.Becauseofthe morphologicaldifferentiationfoundbetweenB.lojanusandthespecimensfrom northernPeru,additionalworkisneededinthatareainordertoobtaincomparative geneticmaterialandfemalesfromthatpopulation.

ConservationImplications:Basedonlocalitieswherethespeciesispresent,B. lojanusisknownfromtheprovincesofAzuay,LojaandZamoraChinchipe,its distributioncoversabout1350km2,anditsaltitudinalrangeisfrom2,191to2,634m.

113 ThedistributionareaforB.lojanussensustrictois487.52km2andforCladeBis37.89 km2(Fig.4.5).B.lojanusisconsideredanendangeredspecies[IUCNRedListcriteria

B1ab(iii,v)]basedonanestimateddistributionoflessthan5000km2,afragmented distributionintwogeneralareas,adeclineinthequalityandextentofitshabitat,and becausematureindividualsareusuallykilledbylocalpeople(CisnerosͲHeredia,2010).

Weconsiderthattheinformationpresentedhereagreeswiththeendangered conservationstatusgivenearlierforthespeciesandsupportsoursuggestionof consideringB.lojanusCladeBasademographicallyͲindependentmanagementunitin needofconservationactions.Finally,givenearlierrecommendationsofstressing distinctivebiologicalcharacteristicsandculturalrelevanceofpitviperspeciesforthe prioritizationoftaxaforconservation(GreeneandCampbell,1992;SeigelandMullin,

2009),wesuggestthatB.lojanusisaspeciesworthofconservationeffortsbecauseof thefollowing:1)ItisamontanetaxonendemictotheTropicalAndesfacingpossible futurethreatsduetoclimatechange,2)Currentstudiessuggestthatitsphylogenetic affinitiesarenotwiththewidespreadgenusBothropsandsothistaxonrepresentsa possiblecaseofaphylogeneticallyͲdistinctsnakeworthpreserving,and3)B.lojanus maywellserveasaflagshipconservationspeciesfortheHuancabambaregiongiven thatitscommonnamerepresentsoneofthelargestcitiesinEcuadorthatislocated withinthisimportantandthreatenedecosystem.





114 Acknowledgements

Wethankthefollowingcuratorsandtheirstaff(inalphabeticalorder)for allowingustheexaminationofspecimensundertheircare:C.Aguilar,C.Torres,andA.

Guzman(MUSM),A.Almendáriz(EPN),R.BrownandA.Campbell(KU),J.Streicher

(BMNH),O.TorresͲCarvajal(QCAZ),andJ.Valencia(FHGO).WearegratefultoE.

Arbeláez,D.Armijos,J.Benítez,G.Cabrera,Z.Coronel,J.Maldonado,L.Ochoa,andM.

Reyesforassistanceinthefieldorfordonatingspecimens.WethankD.Salazarforhelp withgeographicdataandanalyses.Selectedsampleswerecollectedundercollection permit008Ͳ09ICͲFAUͲDNB/MAandweredepositedatMuseodeZoología(QCAZ),

PontificiaUniversidadCatólicadelEcuador.Thisworkwasfundedbyacooperative grantfromtheColumbusZooandOhioStateUniversity.



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 119 Tables

Table4.1VoucherandlocalityinformationforŽƚŚƌŽƉƐůŽũĂŶƵƐspecimensusedinthis study.   Altitude Voucher Province,locality Latitude Longitude  (m)   QCAZ11398 Loja,Loja Ͳ3.95872 Ͳ79.24211 2,191  QCAZ11399 Loja,SanLucas Ͳ3.79400 Ͳ79.27144 2,552  QCAZ11286 Loja,Yangana Ͳ4.37680 Ͳ79.14570 2,302  QCAZ11287 Loja,Yangana Ͳ4.37680 Ͳ79.14570 2,302   QCAZ11289 Loja,Yangana Ͳ4.37680 Ͳ79.14570 2,302  Blo015 Loja,Yangana Ͳ4.37680 Ͳ79.14570 2,302  Blo002 Nabón,Azuay Ͳ3.32492 Ͳ79.07149 2,578  Blo001 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503   QCAZ11291 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ11292 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ11293 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ11294 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503   QCAZ11295 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ11296 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ11297 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ4459 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503   QCAZ4460 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ4461 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  QCAZ4476 Gulag,Azuay Ͳ3.32425 Ͳ79.09200 2,503  ZamoraChinchipe,   QCAZ5054 EstaciónSan Ͳ3.97222 Ͳ79.09278 2,634  Francisco  ZamoraChinchipe,  QCAZ5055 EstaciónSan Ͳ3.97222 Ͳ79.09278 2,634   Francisco 

120 Table 4.2 Genetic diversity indices for clade A, clade B, and the complete dataset of ŽƚŚƌŽƉƐ ůŽũĂŶƵƐ. H = number of haplotypes; Hd = haplotype diversity; S = nucleotide diversity;K=averagenumberofwithinͲpopulationpairwisedifferences. 

 CladeACladeBTotal Samplesize 10 7 17  Polymorphicsites 16 10 67  H549 Hd 0.857 0.533 0.824  S 0.0031 0.0012 0.0171  K 5.524 2.156 29.103     Table 4.3. Selected morphological characters that showed variation in specimens of ŽƚŚƌŽƉƐůŽũĂŶƵƐsensustrictoand͘ůŽũĂŶƵƐCladeB. 

 B.lojanussensu Character B.lojanusCladeB stricto  Numberofventral 143–152(n=11) 138–146(n=5)  scalesinmales

 Numberof subcaudalscalesin 39–45(n=11) 35–42(n=5)  males

 Numberofventral 148–156(n=14) 143–148(n=7) scalesinfemales 







121 Figures



































Figure4.1GeographicdistributionofŽƚŚƌŽƉƐůŽũĂŶƵƐinEcuador;provincesarenamed andoutlined.Localityrecordsfromtheliterature(reddots;bluedotrepresentsthetype localityforthespecies:Loja,Lojaprovince),extremepopulationsregisteredduringthis study(triangles),andmaterialfromPeruwithuncertaintaxonomicaffiliation(square) (CampbellandLamar,2004)areshown. 122     



 Figure4.2PredictivedistributionmapsforŽƚŚƌŽƉƐůŽũĂŶƵƐmodeledinMaxent3.3.2. Originalpredictivemap(above)usingsixlocalities(whitesquares)fromsouthern Ecuador;areaswithhighprobability(>85%)ofspeciesoccurrenceareshowninredand orange.Predictivedistributionmapswithninelocalitiesfor͘ůŽũĂŶƵƐsensustricto (bottomleft)and12localities(bottomright)forbothcladesrecoveredinthe phylogeneticanalyses. 123     B     Clade  areas Bayesian    support the   are 

 main  the

  Branch  branches separating    shows   above range   right   percentages.  the   on  Numbers  mountain

  map  bootstrap   (left).   physical  Cordoncillo   likelihood network Ͳ  The  and    clarity.  maximum haplotype Tioloma     are  the and    of  preserve  to  below    phylogram  presence   numbers   the subclades    note most    whereas  for  province). mitochondrial    work; 

 this  Azuay shown   Bayesian

  not  A  probabilities,  during 3  . (Gulag,

 are   4  e  r u g i locality sampled indices posterior  F

124  



A   2 

 0    í2

 var.) PC2 (12.3% explained  í20 2 4  PC1 (23.2% explained var.)     B C

4

2

2

0 0

í2 í2 PC2 (19.4% explained var.) explained PC2 (19.4% PC2 (15.6% explained var.) explained PC2 (15.6%

í4 í20 2 4 036 PC1 (25.5% explained var.) PC1 (36.1% explained var.) 

Figure4.4.Distributionof͘ůŽũĂŶƵƐfemale(A)andmale(B)specimensalongthefirstand secondprincipalcomponentaxes.Individualsrepresenting͘ůŽũĂŶƵƐsensustricto(light blue)and͘ůŽũĂŶƵƐCladeB(red)areshowninpanelAandBleft.Acomparisonof͘ ůŽũĂŶƵƐmaleswithspecimensfromthePeruvianpopulation(pink)areshowninpanelB right.  125 



































Figure4.5MapofsouthernEcuadorshowingtheknowndistributionofŽƚŚƌŽƉƐůŽũĂŶƵƐ. Minimumconvexpolygonsareshownforbothlineagesidentifiedwithphylogenetic methods(bluedottedline),͘ůŽũĂŶƵƐsensustricto(orange)andCladeB(red). 

126 





Appendices AppendixA:MorphologicalVariables

CharacterswereselectedbasedonSasa(2002)andSaldarriagaͲCórdobaetal.

(2009)andincludedscalecounts,colorationpattern,andmeasurements.Inorderto reduceinterͲobservererror,weselected17outofthe28meristic,morphometricand colorationpatterncharactersusedinthosestudiesbecausesuchvariablesare standardizedandcommonlyusedacrosssimilarpitviperstudies(Malhotraetal.,2011;

Puortoetal.,2001).

Includedcharacterswere:1)headlength;2)headwidth;3)snoutlength;4) supralabialscales;5)infralabialscales;6)preocularscales;7)interocularscales;8) canthalscales;9)internasals;10)ventrals(Dowling,1951);11)preventrals;12) subcaudals;13)dorsalscalerows;14)loreals;15)interrictals;16)degreeofventral mottling:from1(almostnopigmentation)to4(heavilypigmented);17)blotchnumber: countedfromthenecktothevent,ontheleftsideofthespecimen.

127 





AppendixB:MuseumSpecimensUsedinMorphologicalAnalysis

MuseumAcronyms:AMNH,AmericanMuseumofNaturalHistory,NewYork;CAS, CaliforniaAcademyofSciences,SanFrancisco,California;CIBUC,Centrode InvestigacionesBiomédicasdelaUniversidaddelCauca,Popayán,Colombia;DHMECN, MuseoEcuatorianodeCienciasNaturales,Quito,Ecuador;FHGO,Fundación HerpetológicaGustavoOrcés,Quito,Ecuador;FMNH,FieldMuseumofNaturalHistory, Chicago,Illinois;ICN,InstitutodeCienciasNaturales,MuseodeHistoriaNatural, UniversidadNacionaldeColombia,Bogotá;JDL,JohnD.Lynchpersonalcollection;KU, UniversityofKansasBiodiversityInstitute;LACM,NaturalHistoryMuseumofLos AngelesCounty,LosAngeles,California;MCZ,MuseumofComparativeZoology, HarvardUniversity,Cambridge,Massachusetts;MHNUC,MuseodeHistoriaNaturalde laUniversidaddelCauca,Popayán,Colombia;MHUAͲA,MuseodeHerpetología, UniversidaddeAntioquia,Medellín,Colombia;MVZ,MuseumofVertebrateZoology, UniversityofCalifornia,Berkeley;QCAZ,MuseodeZoología,PontificiaUniversidad CatólicadelEcuador,Quito,Ecuador;SUA,SerpentarioUniversidaddeAntioquia, Medellín,Colombia;TCWC,BiodiversityResearchandTeachingCollectionsTexasA&M University,CollegeStation,Texas;USNM,NationalMuseumofNaturalHistory, SmithsonianInstitutionWashingtonD.C;UTA,UniversityofTexasatArlington;UV, UniversidaddelValle,Cali.       128  TableB.1MuseumSpecimensUsedinMorphologicalAnalysis  Voucher Sex Country State Location UTAR2748 F Mexico Veracruz Sontecomapan  UTAR3010 F Mexico Veracruz Tuxpan UTAR2920 F Mexico Veracruz Sontecomapan  UTAR3021 F Mexico Veracruz Sontecomapan UTAR3063 F Mexico Veracruz Sontecomapan  UTAR11072 F Belize Toledo BlueCreekVillage UTAR12996 MCostaRica Limon LindaVistadeSiquirres UTAR14530 F Mexico Oaxaca SierraJuárez  KU23062 M Guatemala Izabal LasEscobas UTAR8834 F Guatemala Izabal ElEstor  UTAR17031 F Mexico Quintana Tulum UTAR21873 F Guatemala Izabal Mariscos  UTAR21877 F Guatemala Escuintla VolcándeAgua UTAR21878 M Guatemala Escuintla VolcándeAgua  UTAR21882 M Guatemala Escuintla VolcándeAgua UTAR21885 F Guatemala Escuintla VolcándeAgua  UTAR21886 M Guatemala Escuintla VolcándeAgua UTAR21887 M Guatemala Escuintla VolcándeAgua UTAR21888 M Guatemala Escuintla VolcándeAgua  UTAR21889 F Guatemala Escuintla VolcándeAgua UTAR21890 M Guatemala Escuintla VolcándeAgua  UTAR21891 M Guatemala Escuintla VolcándeAgua UTAR21893 M Guatemala Escuintla VolcándeAgua  UTAR21894 F Guatemala Escuintla VolcándeAgua UTAR21898 M Guatemala Escuintla VolcándeAgua  UTAR21899 F Guatemala Escuintla VolcándeAgua UTAR21900 M Guatemala Escuintla VolcándeAgua UTAR21901 F Guatemala Escuintla VolcándeAgua  UTAR21904 F Guatemala Quetzaltenango FincaElCarmen UTAR14531 M Guatemala Izabal PuertoSantoTomás  UTAR21906 F Guatemala Escuintla VolcándeAgua UTAR22226 M Guatemala Peten Tikal  UTAR9444 F Mexico Veracruz Sontecomapan KU191154 M Guatemala Izabal MontañasdelMico  KU191155 M Guatemala Izabal MontañasdelMico UTAR26636 M Guatemala AltaVerapaz SierradeLasMinas UTAR26637 M Guatemala AltaVerapaz FincaTinajas  UTAR26638 F Guatemala AltaVerapaz SierradeLasMinas  Continued 

129  TableB1:ŽŶƚŝŶƵĞĚ Voucher Sex Country State Location  UTAR26640 F Guatemala AltaVerapaz SierradeLasMinas UTAR28620 F Guatemala Izabal SierradelEspírituSanto  KU191151 M Guatemala Izabal LosAmates UTAR32494 M CostaRica Puntarenas RíoPeñasBlancas  UTAR14531 F Guatemala Izabal MontañasdelMico KU191157 F Guatemala Izabal MontañasdelMico  UTAR35017 F Guatemala Peten Tikal MCZ26882 F Panama Chiriqui Chiriqui  MCZ26883 F Panama Chiriqui Chiriqui MCZ26884 F Panama Chiriqui Chiriqui  MCZ26885 F Panama Chiriqui Chiriqui MCZ26886 F Panama Chiriqui Chiriqui  AMNH93435 F Mexico Veracruz Tuxpan MVZ78768 F Panama Chiriqui PanamericanHighway MVZ83439 M Panama Darien Yaviza  MVZ83440 F Panama Darien Yaviza MVZ160504 F Guatemala Izabal LasDantas  KU191152 F Guatemala Izabal LosAmates KU23915 F Mexico Veracruz JesusCarranza  KU27009 F Mexico Veracruz JesusCarranza KU24032 F Mexico SanLuis Ebano  KU24080 M Mexico SanLuis Xilitla KU25677 F CostaRica Limon ElDiamante  KU26473 M Mexico Veracruz PasodelMacho KU55704 F Guatemala ElPeten Chinajá  KU57138 F Guatemala ElPeten Sayaxché KU63916 M CostaRica Puntarenas BuenosAires  KU70908 F Mexico Quintana PuertoJuárez KU75003 F Mexico Quintana Caobas  KU107857 F Panama Darien ElReal KU94138 F Mexico Chiapas SabanadeSanQuintín KU97031 F Panama Darien RíoTuira  KU102537 M CostaRica Puntarenas RincóndeOsa KU107853 M Panama Darien RioTuira  KU107854 F Panama Darien RioTuira KU107855 M Panama Darien RioTuira  KU107856 F Panama Darien RioTuira KU107858 M Panama Darien ElReal 

 Continued 130  TableB1:ŽŶƚŝŶƵĞĚ Voucher Sex Country State Location  KU80603 F Panama Darien ElReal KU107859 F Panama Darien RíoChucunaque  KU107860 M Panama Darien RíoChucunaque KU107861 M Panama Darien RíoChucunaque  KU107862 M Panama Darien RíoChucunaque KU112571 M Panama Darien SantaFe  KU112957 F Nicaragua Zelaya ElRecreo KU112958 F Nicaragua Zelaya ElRecreo  KU157657 F Belize Cayo Belmopan KU157659 F Mexico Quintana FelipeCarillo  KU157661 F Mexico Quintana VicenteGuerrero KU171758 F Mexico Quintana KantunilKin  KU157662 F Mexico Tabasco Macultepec AMNH12707 F Nicaragua RACS Bluefields AMNH12711 F Nicaragua N/A CupitnaCamp  AMNH12712 M Nicaragua N/A CupitnaCamp AMNH17384 M CostaRica SanJose SanJose  AMNH36209 F Panama Darien RíoSubcutí AMNH58225 F Mexico Puebla SanDiego  AMNH58231 F Mexico Puebla VegasdeSuchi AMNH64448 F CostaRica Limon Guapiles  AMNH66455 F Mexico Chiapas LaEsperanza AMNH69972 M Guatemala Peten Sojio  AMNH76433 F Mexico Puebla Necaxa AMNH79034 F Mexico Veracruz Veracruz  AMNH89163 M CostaRica Limon RioTortuguero AMNH89164 F CostaRica Limon RioTortuguero  LACM131113 M CostaRica Limon RioTortuguero AMNH99681 F CostaRica Limon Penshurt  KU24033 F Mexico SanLuis ElSaltodeAgua AMNH122764 F Guatemala Quirigua Quirigua AMNH126449 M Belize Cayo PrivasonCreek  AMNH126450 M Belize Cayo MountainPineRidge CAS71738 M Panama Darida TuriaValley  CAS71739 F Panama Darida TuriaValley CAS71740 M Panama Darida TuriaValley  CAS71741 F Panama Darida TuriaValley CAS74396 F Mexico Veracruz Tezonapa 

 Continued 131  TableB1:ŽŶƚŝŶƵĞĚ Voucher Sex Country State Location  CAS150329 F Mexico Quintana Tulum TCWC6974 F Mexico SanLuis AntiguaMorelos  TCWC21394 M Mexico Veracruz LagoCatemaco TCWC21395 M Mexico Veracruz Sontecomapan  TCWC21397 M Mexico Veracruz LasChaspas TCWC21546 F Mexico Chiapas MalPaso  USNM47931 F Mexico Oaxaca SantoDomingo USNM25047 F Mexico Veracruz Mirador  KU94137 F Mexico Chiapas RuinasdePalenque USNM25048 F Mexico Veracruz Mirador  USNM30220 M Mexico Veracruz Orizaba USNM47932 F Mexico Oaxaca SantoDomingo  USNM32149 M Mexico Veracruz SanRafael USNM46406 F Mexico Tabasco Teapa FMNH3480 M Belize Belize ManateeRoad  FMNH4197 M Belize Belize Belize FMNH105314 F Mexico Campeche Encarnación  SUA1060 M Colombia Antioquia Caucasia SUA957 F Colombia Antioquia Caucasia  SUA1380 F Colombia Antioquia Caucasia SUA815 F Colombia Antioquia Caucasia  SUA2321 F Colombia Antioquia Caucasia SUA2322 M Colombia Antioquia Caucasia  SUA2256 M Colombia Antioquia Cáceres SUA899 M Colombia Antioquia GomezPlata  SUA897 F Colombia Antioquia GomezPlata SUA4326 F Colombia Antioquia DonMatías  SUA4330 F Colombia Antioquia DonMatías SUA772 F Colombia Antioquia SanFransisco SUA413 F Colombia Antioquia SanCarlos  SUA2296 M Colombia Antioquia SanCarlos SUA2237 M Colombia Antioquia SanCarlos  SUA2173 M Colombia Antioquia SanCarlos SUA2419 M Colombia Antioquia SanCarlos  SUA3605 F Colombia Antioquia SanRafael MHUA14604 M Colombia Sucre Coloso  MHUA14447 M Colombia Antioquia Caucasia MHUA14084 F Colombia Nariño Barbacoas 

 Continued 132  TableB1:ŽŶƚŝŶƵĞĚ Voucher Sex Country State Location  MHUA14437 M Colombia Choco Nuqui MHUA14081 M Colombia Nariño Barbacoas  MHUA14187 F Colombia Atlantico Usiacuri MHUA14665 M Colombia Antioquia GomezPlata  MHUA14806 F Colombia Antioquia PuertoBerrio MHUA14853 F Colombia Choco Acandi  MHUA14065 F Colombia Choco Acandi MHUA14232 M Colombia Antioquia Maceo  MHUA14034 M Colombia Choco Nuqui MHUA14438 M Colombia Choco Nuqui  MHUA14057 F Colombia Choco Nuqui MHUA14494 F Colombia Antioquia Amalfi  MHUA14116 F Colombia Antioquia Maceo MHUA14444 F Colombia Caldas Victoria MHUA14872 M Colombia Bolivar Norosi  SUA4102 F Colombia Choco Yuto SUA4258 M Colombia Santander Barrancabermeja  SUA4318 F Colombia Antioquia DonMatias CIBUC00003 F Colombia Cauca Huisito  MHNUCR000025 F Colombia Cauca Huisito,SantaRita MHNUCR000026 F Colombia Cauca Huisito,SantaRita  MHNUCR000440 F Colombia Cauca Huisito MHNUCR000461 F Colombia Cauca PlayaRica,CostaNueva  MHNUCR000463 F Colombia Cauca PlayaRica,CostaNueva MHNUCR000288 F Colombia Cauca Huisito  MHNUCR000459 F Colombia Cauca PlayaRica,CostaNueva CIBUC00267 F Colombia Cauca Huisito,SantaRita  MHNUCR000001 M Colombia Cauca Huisito,SantaRita MHNUCR000033 M Colombia Cauca Huisito,SantaRita MHNUCR000274 M Colombia Nariño N/A  CIBUC00259 M Colombia Cauca Huisito,SantaRita MHNUCR000559 F Colombia Cauca SanJoaquin,Pomarroso  MHNUCR000471 F Colombia Cauca SanJoaquin,Pomarroso MHNUCR000339 F Colombia Cauca LaPaz,Corralejas  CIBUC00384 F Colombia Cauca SanJoaquin,Pomarroso CIBUC00197 F Colombia Cauca SanJoaquin  CIBUC00392 F Colombia Cauca SanJoaquin CIBUC00147 F Colombia Cauca SanJoaquin,Pomarroso 

 Continued 133  TableB1:ŽŶƚŝŶƵĞĚ Voucher Sex Country State Location  CIBUC00383 F Colombia Cauca LaPaz,Corralejas CIBUC00397 F Colombia Cauca SanJoaquin,Pomarroso  CIBUC00404 F Colombia Cauca SanJoaquin,Pomarroso CIBUC00161 F Colombia Cauca SanJoaquin,Pomarroso  CIBUC00382 F Colombia Cauca LaPaz,Corralejas CIBUC00190 F Colombia Cauca SanJoaquin  CIBUC00210 F Colombia Cauca SanJoaquin,Pomarroso CIBUC00148 F Colombia Cauca SanJoaquin,Pomarroso  CIBUC00289 F Colombia Cauca SanJoaquin,Pomarroso MHNUCR000547 M Colombia Cauca SanJoaquin,Pomarroso  MHNUCR000237 M Colombia Cauca LaPaz,Corralejas CIBUC00396 M Colombia Cauca SanJoaquin  CIBUC00412 M Colombia Cauca SanJoaquin CIBUC00314 M Colombia Cauca SanJoaquin,Pomarroso CIBUC00027 M Colombia Cauca SanJoaquin  CIBUC00065 M Colombia Cauca SanJoaquin CIBUC00048 M Colombia Cauca SanJoaquin  CIBUC00325 M Colombia Cauca SanJoaquin,Pomarroso QCAZ4534 F Ecuador Esmeraldas Durango  QCAZ5053 F Ecuador Esmeraldas Chuchubí QCAZ4538 M Ecuador Esmeraldas PlayóndeSanFrancisco  QCAZ5850 M Ecuador Esmeraldas ElCristal QCAZ6033 M Ecuador Esmeraldas AltoTambo  QCAZ11772 M Ecuador Esmeraldas AltoTambo QCAZ12466 M Ecuador Esmeraldas Chuchubí  QCAZ4215 F Ecuador Esmeraldas Durango QCAZ5760 M Ecuador Esmeraldas Tundaloma  QCAZ6998 M Ecuador Esmeraldas Durango QCAZ5845 F Ecuador Esmeraldas Chuchubí QCAZ5849 F Ecuador Esmeraldas ElCristal  QCAZ5900 F Ecuador Esmeraldas Durango QCAZ11627 F Ecuador Esmeraldas Chuchubí  QCAZ12467 F Ecuador Esmeraldas Chuchubí QCAZ12468 F Ecuador Esmeraldas Chuchubí  QCAZ12585 F Ecuador Esmeraldas Chuchubí QCAZ12591 F Ecuador Esmeraldas }Chuchubí  QCAZ12464 M Ecuador Esmeraldas Chuchubí QCAZ5843 Ecuador Esmeraldas Chuchubí 

 Continued 134  TableB1:ŽŶƚŝŶƵĞĚ Voucher Sex Country State Location  QCAZ1083 F Ecuador Cotopaxi SanFranciscodelasPampas QCAZ5854 F Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ7863 F Ecuador Cotopaxi LasJuntas QCAZ10516 F Ecuador Cotopaxi SanFranciscodelasPampas  QCAZ12438 F Ecuador Cotopaxi LasPampas QCAZ12463 F Ecuador Manabí LaTabladadelTigre  QCAZ12588 F Ecuador Manabí LaTabladadelTigre QCAZ12590 F Ecuador Manabí LaTabladadelTigre  QCAZ1068 M Ecuador Cotopaxi SanFranciscodelasPampas QCAZ5856 M Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ10515 M Ecuador Cotopaxi SanFranciscodeLasPampas QCAZ10517 M Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ12461 M Ecuador Manabí LaTabladadelTigre QCAZ7926 F Ecuador Cotopaxi SanFranciscodeLasPampas QCAZ11626 F Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ1247 M Ecuador Cotopaxi SanFranciscodeLasPampas QCAZ12574 M Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ12577 M Ecuador Cotopaxi CampoAlegre QCAZ12589 M Ecuador Manabí LaTabladadelTigre  QCAZ7868 F Ecuador Cotopaxi LasJuntas QCAZ10580 F Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ12575 F Ecuador Cotopaxi SanFranciscodeLasPampas QCAZ12578 F Ecuador Cotopaxi SanPablodelosCamotes  QCAZ12586 F Ecuador Manabí LaTabladadelTigre QCAZ12447 M Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ12576 M Ecuador Cotopaxi CampoAlegre QCAZ12587 M Ecuador Manabí LaTabladadelTigre  QCAZ12448 Ecuador Cotopaxi SanFranciscodeLasPampas QCAZ7988 F Ecuador Cotopaxi SanFranciscodeLasPampas QCAZ12580 F Ecuador Cotopaxi SanFranciscodeLasPampas  QCAZ5638 Ecuador Cotopaxi SanFranciscodeLasPampas QCAZ6378 F Ecuador Loja Alamor  QCAZ10067 F Ecuador Loja Puyango QCAZ12613 F Ecuador Chimborazo Pallatanga  QCAZ13136 F Ecuador Guayas BosqueProtectorCerroBlanco QCAZ9468 M Ecuador Loja Balzones  QCAZ12567 M Ecuador Loja Guararas QCAZ4484 F Ecuador Loja Puyango 

 Continued 135  TableB1:ŽŶƚŝŶƵĞĚ  Voucher Sex Country State Location QCAZ9466 F Ecuador ElOro Arenillas Continued  QCAZ10066 F Ecuador Loja Puyango QCAZ11284 F Ecuador Loja Balzones QCAZ11285 F Ecuador Loja Balzones  QCAZ11863 F Ecuador ElOro ReservaEcológicaBuenaventur QCAZ12614 F Ecuador Chimborazo Pallatanga  QCAZ12615 F Ecuador Chimborazo Pallatanga QCAZ9126 M Ecuador Guayas BosqueProtectorCerroBlanco  QCAZ10372 M Ecuador ElOro Palmales QCAZ12568 M Ecuador Loja Guararas  QCAZ12460 F Ecuador SantaElena ElRincóndelTigre QCAZ13135 F Ecuador Guayas BosqueProtectorCerroBlanco  QCAZ12570 F Ecuador Loja Guararas QCAZ6379 M Ecuador Loja Alamor  QCAZ8761 M Ecuador ElOro ElCarmen QCAZ12449 M Ecuador ElOro ElRemolino QCAZ12569 M Ecuador Loja Guararas  QCAZ13137 M Ecuador Guayas BosqueProtectorCerroBlanco QCAZ164 M Ecuador Guayas Naranjal  QCAZ10065 M Ecuador Loja Puyango QCAZ4468 MEcuadorAzuay Oña  QCAZ5300 FEcuadorAzuayOña QCAZ6017 FEcuadorLoja FincaelAlumbro  QCAZ4474 FEcuadorLoja SanJosé QCAZ5013 MEcuadorAzuay Oña  QCAZ4475 MEcuadorLoja SanJosé QCAZ2262 M Ecuador Loja Vilcabamba  QCAZ11622 Ecuador Loja Vilcabamba QCAZ803 F Ecuador Loja Vilcabamba  QCAZ1656 FEcuadorManabíGuale QCAZ4111 F Ecuador Cañar MantaReal QCAZ4112 F Ecuador Cañar MantaReal  QCAZ11621 F Ecuador LosRíos RecintoPechiche QCAZ12455 F Ecuador Guayas RecintoCongo  QCAZ1657 MEcuadorManabíGuale QCAZ11610 M Ecuador LosRíos Ventanas  QCAZ12566 M Ecuador Bolívar SanJosédelTambo QCAZ5862 F Ecuador LosRíos RecintoLaMuralla 

 Continued 136  TableB1:ŽŶƚŝŶƵĞĚ Voucher Sex Country State Location  QCAZ12583 F Ecuador Guayas ElEmpalme QCAZ5838 M Ecuador LosRíos ReservaForestalCerroSamama  QCAZ11619 M Ecuador LosRíos RecintoPechiche QCAZ11620 M Ecuador LosRíos RecintoPechiche  QCAZ5859 F Ecuador LosRíos RecintoPechiche QCAZ11299 F Ecuador Loja Pindal  QCAZ11618 F Ecuador LosRíos RecintoPechiche QCAZ12562 F Ecuador Guayas ElEmpalme  QCAZ12565 F Ecuador Guayas ElEmpalme QCAZ12581 F Ecuador Guayas ElEmpalme  QCAZ12592 F Ecuador LosRíos RecintoLasTolas QCAZ1237 F Ecuador Manabí Machalilla  QCAZ832 MEcuadorLosRíos RíoPalenque QCAZ4055 M Ecuador ElOro ElGuayabo  QCAZ5860 M Ecuador LosRíos RecintoLaMuralla QCAZ12457 F Ecuador Bolívar RecintoElPijio QCAZ12582 F Ecuador Guayas ElEmpalme  QCAZ12593 F Ecuador LosRíos RecintoLaIsla QCAZ309 F Ecuador Bolivar SanLuisdePambil  QCAZ12454 M Ecuador Guayas RecintoBocaPucón DHMECN4595 F Ecuador Azuay Oña  UTAR55962 F Ecuador Azuay Oña

















 137 





AppendixC:GeneticClusteringAnalyses

A













B Value of BIC versus number of clusters  C I B 270 290 310

 0 10203040  Number of clusters   FigureC.1OptimalKvaluesasidentifiedbythedeltaKmethodofEvannoetal.(2005)in StructureHarvester(A)andlowestvaluesofBICscoresinadegenet(B).  138       AppendixD:MorphologicalVariables



Morphologicalcharactersincludedscalecounts,colorationpattern,and measurementscommonlyusedinsimilarpitviperstudies(Malhotraetal.,2011;Puorto etal.,2001).Includedcharacterswere:1)headlength;2)headwidth;3)snoutlength;4) supralabialscales;5)infralabialscales;6)preocularscales;7)intersupraocularscales;8) canthalscales;9)internasals;10)ventrals(Dowling,1951);11)preventrals;12) subcaudals;13)dorsalscalerows;14)loreals;15)interrictals;16)fovealscales;17) lacunalscales;18)degreeofventralmottling:from1(almostnopigmentation)to4

(heavilypigmented);19)postocularscales.















139 



















AppendixE:MuseumSpecimensUsedinMorphologicalAnalysis

















140  TableE.1SpecimensusedinMorphologicalAnalysis.  Province/ Species Museum Number Sex Country Locality  Department ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 2516 M Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3756 F Ecuador Loja Loja  ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3757 F Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3758 M Ecuador Loja Loja  ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3759 F Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3760 F Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3763 F Ecuador Loja Loja  ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3764 F Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ EPN 3767 M Ecuador Loja Loja  ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 235 M Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 479 F Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 817 M Ecuador ZamoraCh. Zamora  ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 830 M Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 840 F Ecuador Loja Loja  ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 855 F Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 883 M Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ FHGO 5527 M Ecuador Loja Loja  ŽƚŚƌŽƉƐůŽũĂŶƵƐ KU 135213 F Ecuador Loja Saraguro ŽƚŚƌŽƉƐůŽũĂŶƵƐ LOUNAZ n/a F Ecuador Loja Loja  ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 4459 F Ecuador Azuay Gulag ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 4460 M Ecuador Azuay Gulag ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 4461 M Ecuador Azuay Gulag  ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 5054 F Ecuador Zamora SanFrancisco ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 5055 F Ecuador Zamora SanFrancisco  ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11125 M Ecuador Azuay Gulag ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11286 M Ecuador Loja Yangana ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11287 F Ecuador Loja Yangana  ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11289 M Ecuador Loja Yangana ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11291 F Ecuador Azuay Gulag  ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11292 F Ecuador Azuay Gulag ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11293 M Ecuador Azuay Gulag ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11294 F Ecuador Azuay Gulag  ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11296 M Ecuador Azuay Gulag ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11297 F Ecuador Azuay Gulag  ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11398 F Ecuador Loja Loja ŽƚŚƌŽƉƐůŽũĂŶƵƐ QCAZ 11633 F Ecuador Azuay Gulag ŽƚŚƌŽƉƐůŽũĂŶƵƐ UTA 23529 M Ecuador ZamoraCh. Zamora  ŽƚŚƌŽƉƐƐƉ͘ MHNSM 13997 M Peru Cajamarca Yauyucán  ŽƚŚƌŽƉƐƐƉ͘ MHNSM 13998 M Peru Cajamarca Yauyucán ŽƚŚƌŽƉƐƐƉ͘ MHNSM 13999 M Peru Cajamarca Yauyucán 



141 





















AppendixF:ConcatenatedMitochondrialandNuclearTree

















 142    

  B_lojanus_QCAZ11289 B_lojanus_QCAZ11286

B_lojanus_Blo015  B_lojanus_QCAZ11287 0.4119 B_lojanus_QCAZ5055 B_lojanus_QCAZ11399 0.5747 B_lojanus_QCAZ11398 0.9869 B_lojanus_QCAZ4459 0.9443 0.997 1 Bayesian B_lojanus_QCAZ11296 0.4554 B_lojanus_QCAZ4460  B_lojanus_QCAZ11292 B_lojanus_QCAZ11295 B_lojanus_QCAZ11297 B_lojanus_QCAZ11293 B_lojanus_QCAZ4461 B_lojanus_QCAZ4476 0.0853 0.0837 B_lojanus_QCAZ11291 0.016 0.0531 0.0741 0.8127 0.7017 0.9999 are

1  

 B_microphthalmus_QCAZ7397 branches   above   Numbers

 

 phylogram.   0.03 1 C_godmani_UTA40002

 nuclear  and  

  mitochondrial  

 Bayesian  A probabilities.   1  . F  e r u g  i posterior F

 143 ReferencesforAppendices

Dowling,H.G.1951.Aproposedstandardsystemofcountingventralsinsnakes.British JournalofHerpetology1:97–99.

Evanno,G.,S.Regnaut,andJ.Goudet.2005.Detectingthenumberofclustersof individualsusingthesoftwareSTRUCTURE:asimulationstudy.Molecular Ecology14:2611–2620.

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