HerpetologicalMonographs, 37,2023,41–69 2023byTheHerpetologists’League,Inc.
MolecularandMorphologicalAssessmentofRainFrogsinthe Pristimantisorestes Species Group(Amphibia:Anura:Strabomantidae)withtheDescriptionofThreeNewCrypticSpecies fromSouthernEcuador
PAUL SZ ´ EKELY1,2,3,DIANA SZ ´ EKELY1,2,3,7,DIEGO ARMIJOS-OJEDA1,2,4,SANTIAGO
HUALPA-VEGA1,5, AND JUDIT VOROS6
1 MuseodeZoolog´ıa,UniversidadTecnicaParticulardeLoja,SanCayetanoAlto,callePar´ıss/n,110107,Loja,Ecuador
2 LaboratoriodeEcolog´ıaTropicalyServiciosEcosist ´ emicos(EcoSs-Lab),FacultaddeCienciasExactasyNaturales,DepartamentodeCienciasBiol ´ ogicasy Agropecuarias,UniversidadT ´ ecnicaParticulardeLoja,SanCayetanoAltos/n,110107,Loja,Ecuador
3 ResearchCenteroftheDepartmentofNaturalSciences,FacultyofNaturalandAgriculturalSciences,OvidiusUniversityConstantxa,Al.Universita ˘ t xiino.1, Constant xa900470,Romania
4 ProgramadeDoctoradoenConservaci ´ ondeRecursosNaturales,EscuelaInternacionaldeDoctorado,UniversidadReyJuanCarlos,CalleTulipa ´ ns/n, 28933Mostoles,Madrid,Spain
5 Fundaci ´ onGreenJewel,Av.P´ıoJaramilloyJohnKennedy,Loja,Ecuador
6 DepartmentofZoology,HungarianNaturalHistoryMuseum,Barossu.13,1088Budapest,Hungary
ABSTRACT:Wereviewthespeciescontentofthe Pristimantisorestes groupand,byintegratinggeneticandmorphologicaldata,wedistinguish anddescribethreenewcrypticspeciesfromthesubpa ´ ramosofsouthernEcuadorianAndes.Genetically,thesenewspeciesaremostrelatedto P. matildae andtheotherspeciesofthe P.colodactylus subgroup;however,inmorphology,theyarestrikinglydifferentfromthem,especiallyin termsoftheshapeofthebody,havingless-compressedheadsandbodies.Additionally,theydifferbytheirecology,havingarborealhabitsin contrastwiththeirbromeliadspecialistsisterspeciesofthesubgroup.Thenewspeciesarealsoeasilydistinguishedfromthespeciesofthe P. orestes and P.simonbolivari subgroups,mainlybythelackoftympanum,longersnouts,andlongerfingersandtoes,andcoloration. Pristimantis sagedunneae hasashagreendorsum,subacuminatetoroundedsnoutindorsalview,ToeVlongerthanToeIII,andabrownishgraycolorationof thedorsumwithblackishordarkbrownbarsandanintenseredcolorationonthedorsalsurfacesofthighs,groin,andconcealedlimbsurfaces; P. paladines hasatuberculatedorsum,acuminatetosubacuminatesnout,ToeVlongerthanToeIII,anddorsumofvariousshadesofbrown,reddish brown,greenishbrown,green,orreddishorangewithdark,blackishbarsandorangeorreddishbrowncolorationonthedorsalsurfacesofthighs, groin,andconcealedlimbsurfaces; P.numbala hasashagreendorsum,roundedtobroadlyroundedsnout,ToeVmuchlongerthanToeIII, reddishbrowncolorationofthedorsumwithdarkbrownbarsandreddishbrowncolorationonthedorsalsurfacesofthighs,groin,andconcealed limbsurfaces.Allthreespecieshaveverysmalldistributionrangesoflessthan20km2 locatedinthewesternlimitofParqueNacionalPodocarpus, ataltitudesbetween2,800–3,100m.Finally,webrieflydiscussthecurrentspeciescompositionofthegroup,therolethatnaturalhistorymight haveindeterminingtheshapeofthebodies,andthecrypticdiversityanddistributionpatternofsomeofthespeciesinsouthernEcuador.
Keywords: AbradeZamora;Amphibians;Direct-developingfrogs;DNA;ReservaNumbala;ReservaTapichalaca;TropicalAndes
THENEOTROPICAL genusofdirect-developingfrogs Pristimantis Jim ´ enezdelaEspada1870(Amphibia:Anura: Strabomantidae)isthelargestamongvertebrates,encompassingasofJuly2022592species(Frost2022),andwith manymorebeingstillundescribed.Thisgenusisdistributed inCentralandSouthAmerica;however,mostofthespecies areencounteredintheAndeanregionsofColombia, Ecuador,andPeru(KieswetterandSchneider2013).
Withthecontinuousspeciesdescriptions,alreadyinthe 1970sarosetheneedtoorganizeandclassifyinsomewaythe manyspeciesofthegenus.Thus,JohnD.Lynch(1976) proposedaseriesofspeciesgroupsforthemanyspeciesthen knownfromSouthAmerica(atthatpointknownas Eleutherodactylus)andattemptedtoorganizethespecies grouparrangementfortheentiregenus.Onesuch arrangement,proposedin1997,wasthe Pristimantisorestes speciesgroup(LynchandDuellman1997).
Atthattime,the P.orestes groupcontainedonlythree species(LynchandDuellman1997),distributedinpa ´ ramos andhigh-altitudecloudforestsinEcuador: P.orestes (Lynch 1979), P.simonbolivari (WiensandColoma1992),and P. vidua (Lynch1979).By2008,inthegroupwereincluded14 species;tothepreviousthreebeingadded11Peruvian species(seeHedgesetal.2008;DuellmanandLehr2009).
However,by2014onlyfourspeciesofthesewereincluded inthecomprehensiveTerraranaphylogeneticrevisions,and onlytwo(P.orestes and P.simonbolivari)clusteredtogether toformagroup(seeHedgesetal.2008;Padialetal.2014). Twootherspecieswerelateraddedtothe P.orestes species group: P.bambu Arteaga-NavarroandGuayasamin2011and P.mazar GuayasaminandArteaga2013.
Britoetal.(2017a)redefinedthegroupandaddedtoit P. andinognomus LehrandColoma2008,thenewlydescribed P.muranunka,andtwoadditionalspecies(whichcurrently remainundescribed).TheyremovedthePeruvianspecies P. melanogaster (DuellmanandPramuk1999)and P.simonsii (Boulenger1900),andnoticedthat P.orestes isincorrectly defined,beingactuallyrepresentedbythreedifferent speciesintheirtree. Pristimantistiktik Sz ´ ekelyetal.2018 wasaddedtothegroupandUrgil ´ esetal.(2019)clarifiedthe identityof P.orestes,usingforthefirsttimegeneticmaterial fromthetypelocality,andincluded P.saturninoi Britoetal. 2017bandtwonewspecies, P.cajanuma and P.quintanai, intothegroup.
Byusingsamplesfromthetypelocality,AbradeZamora, Sz ´ ekelyetal.(2020)redescribed P.vidua and P.colodactylus (Lynch1979)andconfirmedtheirbelongingtothegroup, andaddedtwonewspecies, P.samaniegoi and P.matildae. Also,inordertofacilitatetheanalysisofthegroup,the authorsrecognizedthreelargesubgroups(lineages)of species(Fig.1):the P.orestes subgroup(with P.andinog-
FIG.1.—Morphologicaldiversityinthe Pristimantisorestes speciesgroup. Pristimantisorestes subgroup:(A) P.vidua MUTPL156,SVL ¼ 26.8mm,adult femalefromBosqueProtectorWashapamba,LojaProvince,3,003m;(B) P.cajanuma MUTPL347,SVL ¼ 22.0mm,femaleparatypefromCajanuma entrancetotheParqueNacionalPodocarpus,LojaProvince,3,042m;(C) P.orestes MUTPL242,SVL ¼ 20.7mm,adultmalefromVıaUrdaneta-Tutupali, LojaProvince,2,960m. Pristimantissimonbolivari subgroup:(D) P.tiktik MUTPL247,SVL ¼ 20.2mm,femaleparatypefromV´ıaUrdaneta-Tutupali, LojaProvince,3,108m;(E) P.quintanai ZSFQ4130,adultmalefromRivera,CanarProvince,3,050m(photobyJuanC.Sa ´ nchez-Nivicela);(F) P. samaniegoi MUTPL357,SVL ¼ 20.1mm,femaleholotypefromCajanumaentrancetotheParqueNacionalPodocarpus,LojaProvince,3,258m. Pristimantiscolodactylus subgroup:(G) P.colodactylus MUTPL311,SVL ¼ 20.2mm,adultfemalefromAbradeZamora,LojaProvince,2,822m;(H) P. muranunka MUTPL652,SVL ¼ 19.9mm,adultmalefromCerroPlateado,ZamoraChinchipeProvince,2,409m;(I) P.matildae MUTPL366,SVL ¼ 20.8 mm,femaleparatypefromAbradeZamora,LojaProvince,2,817m;(J) P.sagedunneae MUTPL597,SVL ¼ 21.7mm,femaleparatypefromCajanuma entrancetotheParqueNacionalPodocarpus,LojaProvince,2,991m;(K) P.paladines MUTPL1151,SVL ¼ 19.5mm,femaleparatypefromParque NacionalPodocarpusinthevicinityofReservaTapichalaca,ZamoraChinchipeProvince,2,880m;(L) P.numbala MUTPL1178,SVL ¼ 23.2mm,female paratypefromReservaNumbala,ZamoraChinchipeProvince,2,861m.AllspecimensarefromEcuador.
nomus, P.orestes, P.vidua, P.cajanuma,andanundescribed species),thecloselyrelated P.simonbolivari subgroup(with P.tiktik, P.saturninoi, P.simonbolivari, P.bambu, P.mazar, P.quintanai, P.samaniegoi,andtwoundescribedspecies) andthemorphologicallydistinct,bromeliadspecialist, P. colodactylus subgroup(with P.matildae, P.colodactylus, P. muranunka,andanundescribedspecies).Thus,by2020,the P.orestes speciesgroupcontained14speciesand4 undescribedspecies,allfromtheAndesofcentraland southernEcuador.Hereinwedescribethreenewcryptic speciesofthe P.colodactylus subgroupfromsouthern Ecuador;thesenewspeciesaremorphologicallydifferent fromthecongenersoftheothertwosubgroups,butalso fromallsisterspeciesinthe P.colodactylus subgroup,from whichtheyadditionallydifferintheirarborealhabits.
MATERIALSAND METHODS
SpecimenCollectionandStudySite
Specimenswerecollectedduringseveralfieldexpeditions carriedoutbetweenMay2015andMay2022inAbrade Zamora(LojaProvince,so uthernEcuador;3.9922 8S, 79.14558W;datum ¼ WGS84inallcases;2,812ma.s.l.), theCajanumasectorofParqueNacionalPodocarpus(Loja Province;4.11288S,79.1767 8W,2,991m),theCerroToledo sectorofParqueNacionalPodocarpus(LojaandZamora Chinchipeprovinces;4.3796 8S,79.1124 8W,3,080m), ReservaNumbala(ZamoraChinchipeProvince;4.40658S, 79.08858W,2,875m),ParqueNacionalPodocarpusinthe vicinityofReservaTapichalaca(ZamoraChinchipeProvince; 4.44728S,79.14598W,2,880m),andReservaTapichalaca (ZamoraChinchipeProvince;4.48618S,79.15298W,2,915 m).Intensivevisualencountersurveysandauditorysurveys weremade,bothduringthedayandduringthenight(1200 to0200h)inallthesampledareas.
Thestudysites(Fig.2)arelocatedinsideorintheclose vicinityofParqueNacionalPodocarpus,southernEcuador (LojaandZamoraChinchipeprovinces).Theparkwas declaredin1982andcomprisesanareaof146,289ha;ithas averyirregulartopographycoveringaltitudesfrom900to 3,650m,withlargeareasofdiversenaturalhabitats(Rahbek etal.1995;Ord ´ onez-Delgadoetal.2019),anditispartof theTropicalAndeshotspots,therichestinbiodiversityon theplanet(Myersetal.2000).Theareasampledforour studyhasanaltitudinalrangebetween2,800and3,100m andconsistsofsubpa ´ ramoorevergreenelfinforest(Homeieretal.2008).
Weusedthefollowingtermsforrankingspeciesrelative abundance(aspresentedinSz ´ ekelyetal.2020):common— whenthespeciespresencewasdetected(seenorheard),in theproperhabitat,inlargeormoderatenumbers,onmore than50%ofthesamplingdays/nights;uncommon—when thespeciespresencewasdetectedinmoderateorsmall numbers,on25–50%ofthesamplingdays/nights;rare— whenthespeciespresencewasdetectedinsmallnumberson lessthan25%ofthesamplingdays/nights.
Allcollectedspecimenswerephotographedalive,euthanizedusing20%benzocaine,fixedin10%formalin,and storedin70%ethanol.Tissuesamplesforgeneticanalyses werepreservedin96%ethanol.Examinedspecimens(listed inthetype-seriesandAppendixI)arehousedatMuseode Zoolog´ıa,UniversidadT ´ ecnicaParticulardeLoja,Loja,
Ecuador(MUTPL),MuseodeZoolog´ıa,PontificiaUniversidadCat ´ olicadelEcuador,Quito,Ecuador(QCAZ),and MuseodeZoolog´ıa,UniversidadTecnol ´ ogicaIndoam ´ erica, Quito,Ecuador(MZUTI).
MolecularAnalyses
Genomicextraction,amplification,andsequencingwere asdescribedinSz ´ ekelyetal.(2020);thenewlygenerated DNAsequencesweredepositedinGenBank(AppendixII). Forthephylogeneticanalysisweusedsequencesoftwo mitochondrialribosomalgenes(12Sand16SrRNA)andone nucleargene(recombination-activatinggene1[RAG-1]) from43individualsof21speciescorrespondingto19 differentlocalitiesfromEcuador(AppendixII),representing allthecurrentlyconfirmedspeciesofthe Pristimantisorestes speciesgroup(GenBank-availablesequencesand25new sequencesgeneratedbythisstudy).Asoutgroups,weused Pristimantisceuthospilus, P.diadematus, P.imitatrix, P. rhodoplichus, P.unistrigatus,and P.wiensi.Thetreewas rootedwith P.galdi.
Weedited,assembled,andaligned(MAFFTalgorithm withtheG-INS-iiterativerefinementmethod;Katohand Standley2013)thesequencesusingtheprogramGeneious Prime(v2022.2.2,BiomattersLtd.,Auckland,NewZealand). Weinspectedvisually,inPhyDEv0.9971(M¨ulleretal. 2010),theeditedalignmentsof12S,16S,andRAG-1 sequencestocorrectpotentialalignmenterrors,concatenatedthemintoasinglematrix,andthenusedthemforthe phylogeneticanalyses.Thephylogeneticanalyseswerebased ona2,733-basepair(bp)dataset(901bpfor12S,1,193bp for16S,and639bpforRAG-1).Thealignedand concatenatedmatrixisavailableathttps://dx.doi.org/10. 5281/zenodo.7262263.WeconductedBasicLocalAlignment SearchTool(BLAST)searchestoidentifysimilarsequences of12S,16S,andRAG-1inGenBankandusedFastTreev2.1 (Priceetal.2010)tobuildanexploratorytreewithmostof theavailableEcuadorian Pristimantis sequencesinorderto determinetherelationshipsofthenewspecies.Boththe BLASTsearchesandtheFastTreeindicatedthatthenew speciesarepartofthe P.orestes speciesgroup.
Phylogeneticrelationshipswereinferredusingboth MaximumLikelihoodandBayesianInference.Weused PartitionFinderv2.1.1(Lanfearetal.2017)toselectthebest partitionschemebasedonthecorrectedAkaikeInformation Criterion(AICc)formodels election.PartitionFinder identifiedthreepartitionschemes(bestmodelinparentheses):12Sand16S(GTRþIþG),RAG-11stand2ndposition (F81þG),andRAG-13rdposition(HKYþG).Maximum LikelihoodanalyseswereconductedinGARLIv2.1(Zwickl 2006),performing1,000treesearches(fourindependent searches,twowiththe ‘‘streefname’’ settorandomandtwo settostepwise,with250replicateseach)andnodesupport assessedwith1,000bootstrapreplicates.BayesianInference analysiswasimplementedinMrBayesv3.2.6(Ronquistetal. 2012),theMarkovChainMonteCarlorunsbeingperformed twice,independently,for50milliongenerations,withtrees sampledevery1,000generationsuntilconvergence(P , 0.001),andconsensustreesweresummarizedafterdiscardingtheinitial25%asburn-in.Moredetailsabouthowtree searcheswereperformedarepresentedinSz ´ ekelyetal. (2020).
FIG.2.—Distributionofthethreenewspecies.RecordsarebasedonspecimensdepositedattheMuseodeZoolog´ıa,UniversidadT ´ ecnicaParticularde Loja,Loja,Ecuador(MUTPL)andfielddata.Thenumbersrepresentimportantsamplingareas:(1)AbradeZamora;(2)CajanumasectorofParque NacionalPodocarpus;(3)CerroToledosectorofParqueNacionalPodocarpus;(4)ReservaNumbala;(5)ReservaTapichalaca.
Weaprioriregardedthatatreenodehadstrongsupport whenitsbootstrapvaluewas .75anditsBayesianposterior probabilitywas .0.95,moderatesupportfor50–75and 0.90–0.95,andweaksupportornonresolvedforvalueslower than50and0.90,respectively.Uncorrectedgeneticpdistanceswerecalculatedfor16SwithMEGA6(Tamuraet al.2013)andarepresentedinSupplementalFileS1, availableonline.
MorphologicalAnalyses
Forthedescriptionofqualitativeandquantitative morphologicalcharacters,aswellastheformatofthe description,welargelyfollowDuellmanandLehr(2009). Sexwasdeterminedbygonadalinspection.Juvenilesand subadultsweredistinguishedfromtheadultsbythesmaller size,slightlymoretuberculateskin,andconditionofoviducts and/orovarianeggs(DuellmanandLehr2009).Assuggested byDuellmanandLehr(2009),wedesignatedthelarge tubercleatthebaseofFingerI(thumb)asthethenar tubercle,andtheusuallymuchlargerandcommonly bifurcate(partiallydivideddistally)tubercleatthebaseof FingersIIIandIVasthepalmartubercle.BasedonLynch andDuellman(1997),weclassifiedtherelativelengthsof toesVandIIIasfollows:ToeVisslightlylongerthanToeIII whenthetipofToeVdoesnotreachtheproximaledgeof thedistalsubarticulartubercleonToeIV,whentheyare adpressed;ToeVislongerthanToeIIIwhenitstipreaches theproximaledgeofthedistalsubarticulartubercleinToe
IVbutdoesnotextendtoitsdistaledge;ToeVismuch longerthanToeIIIifthetipofToeVreaches,orextends beyond,thedistaledgeofthedistalsubarticulartubercleon ToeIV.Colorationoflivespecimenswasbasedonfieldnotes anddigitalphotographs.Werefertocolorationinlifeunless otherwisestated.
Forcomparisonofseveralimportantfeaturesweused predeterminedcategories,duetothefactthattheavailable morphometricdataislimitedtoarelativelysmallnumberof specimensormissingaltogether.Thus,thebodysize (relativesizeinsidethe P.orestes speciesgroup)wasdefined as:minute(whenthelargestrecordedfemalesnout–vent length[SVL]issmallerthan21mm);small(femaleSVL smallerthan23mm);medium(femaleSVLsmallerthan25 mm);andlarge(femaleSVLlargerthan25mm).Snout lengthwasdefinedas:veryshort(whentheaverageeye–nostrildistance[END]islessthan8%oftheSVL);short (averageENDislessthan9%oftheSVL);andlong(average ENDismorethan9%oftheSVL).FingerIIIlength(or handlength)wasdefinedas:veryshort(whentheaverage lengthofFingerIIIislessthan22%oftheSVL);short (averagelengthofFingerIIIislessthan24%oftheSVL); medium(averagelengthofFingerIIIislessthan26%ofthe SVL);long(averagelengthofFingerIIIislessthan28%of theSVL);andverylong(averagelengthofFingerIIIis largerthan28%oftheSVL).ToeIVlength(orfootlength) wasdefinedas:veryshort(whentheaveragelengthofToe IVislessthan38%oftheSVL);short(averagelengthofToe
IVislessthan40%oftheSVL);medium(averagelengthof ToeIVislessthan46%oftheSVL);long(averagelengthof ToeIVislessthan48%oftheSVL);andverylong(average lengthofToeIVislargerthan48%oftheSVL).Tympanum wasdefinedas:present(tympanicmembraneabsentbut tympanicannulusevidentorbothtympanicmembraneand annuluspresent)orabsent(bothtympanicannulusand tympanicmembraneabsent).
Alladultspecimenswereweighted(bodymass)before euthanasiausingaMyWeighTritonT3portablescalewith 0.01gprecision.Measurementsofthepreservedspecimens weretakenunderastereomicroscope,withaVernier caliper,androundedtothenearest0.1mm.Specimenswere measuredforthefollowingmorphometricvariables:(1)SVL, distancefromthetipofsnouttoposteriormarginofvent;(2) headwidth,widestportionofthehead,measuredatlevelof jawarticulation;(3)headlength,distancefromthetipof snouttoposteriorangleofjawarticulation;(4)interorbital distance,distancebetweentheinnermarginsoftheorbits;
(5)internarialdistance,distancebetweentheinneredgesof thenarialopenings;(6)uppereyelidwidth,theperpendiculardistancetotheouteredgeoftheeyelid;(7)eye diameter,distancebetweenanteriorandposteriorbordersof eye;(8)END,distancefromposteriormarginofnostrilto anteriormarginofeye;(9)thighlength,lengthofthighfrom venttoknee;(10)tibialength,lengthofflexedlegfromknee toheel;(11)footlength(lengthofToeIV),distancefrom proximalmarginofinnermetatarsaltubercletotipofToe IV;(12)handlength(lengthofFingerIII),distancefrom proximaledgeofpalmartubercletothetipofFingerIII (Sz ´ ekelyetal.2020).Additionally,wedefinedandmeasured the(13)headheight,asthehighestportionofthehead, measuredatlevelofjawarticulation,and(14)bodyheight, thehighestportionofthebody,measuredatleveloftheiliosacralarticulation.Measurementsaregivenasmean 6 SD. AllrawmeasurementsarepresentedinFileS2.
Toevaluatethedifferencesintheshapeofanimalsin relationtotheirpreferredmicrohabitat,weclassifiedthe speciesincategoriesaccordingtotheirphylogenetic subgroup,i.e., P.simonbolivari (includes P.tiktik and P. samaniegoi), P.orestes (P.andinognomus, P.cajanuma, P. orestes,and P.vidua),butdividingthe P.colodactylus subgroupintotwoseparatecategoriesbasedontheir microhabitat:bromeliad P.colodactylus (P.colodactylus, P. matildae,and P.muranunka)andarboreal P.colodactylus (P. numbala, P.paladines,and P.sagedunnae).Asproxiesforthe shapeofindividuals,wecalculatedtheheadratio(head height/SVL)andbodyratio(bodyheight/SVL).Comparison oftheseparametersbetweenthefourcategoriesofspecies wasdonethroughanalysisofvariance(ANOVA)tests,after checkingthatthenormality(Q–Qplotinspection)and homogeneityofvariance(Levene’stest)assumptionswere met;ifANOVAtestsweresignificant,wefollowedwith Tukey’shonestlysignificantdifference(HSD)posthoc.To checkfordifferencesinmorphologicaltraitsbetweenpairof species,weusedMann–Whitney U-tests(duetolowsample sizes),usingonlythemeasurementsfromfemales.All statisticalanalysiswasperformedinIBMSPSSStatistics v25(IBMSPSSStatistics,Armonk,NY),withasignificance levelof a ¼ 0.05.
RESULTS
Phylogeny
TheBayesianandMaximumlikelihoodphylogenetictrees showedalmostthesametopology,withminordifferencesin thepositionofsomeoftheunresolvedbranches,with strongerBayesianInferencesupportforsomeofthetree nodes(Fig.3).SimilartoBritoetal.(2017a),Urgil ´ esetal. (2019),andSz ´ ekelyetal.(2020),werecoveredthe Pristimantisorestes speciesgroupasmonophyletic,with strongsupport(bootstrapvalues ¼ 100%;posteriorprobabilities ¼ 1)inbothanalyses.Also,thethreesubgroups(P. orestes, P.simonbolivari,and P.colodactylus,sensuSz ´ ekely etal.2020)havestrongsupportinbothanalyses.Additionally,thenewtreehasasimilartopologywiththeone presentedinSz ´ ekelyetal.(2020),withsomesmall differencesinthepositionoftheunresolvedbranchesof the P.orestes and P.simonbolivari subgroups.
Thethreenewspeciesarepartofthe P.colodactylus subgroupandform,togetherwiththeirsisterspecies P. matildae,astronglysupportedclade(bootstrapvalues ¼ 99.6%;posteriorprobabilities ¼ 1);theexactrelationship betweenthenewspeciesremainsnonresolved(Fig.3). Uncorrectedgeneticp-distancesforthegene16Softhenew speciesandtheirsisterspecies, P.matildae,rangesbetween 2.4%and4.9%;thedistancesfromtheotherspeciesofthe P. colodactylus subgrouprangesbetween6.0%and10.5%,and fromthespeciesofthe P.orestes and P.simonbolivari subgroupsbetween7.5%and13.0%(FileS1).
Uncorrectedgeneticp-distancesbetweenthenewspecies areasfollows:between P.sagedunneae and P.paladines rangefrom2.0%to2.8%;between P.sagedunneae and P. numbala rangefrom4.3%to4.9%;andbetween P.paladines and P.numbala rangefrom3.4%to4.4%.Asforthe intraspecificuncorrectedgeneticp-distances,thesedidnot surpass0.2%for P.sagedunneae,0.7%for P.paladines,and 0.1%for P.numbala (FileS1).
Morphology
Ourmorphometricanalysissuggeststhatthebromeliad specialistspeciesfromthe P.colodactylus subgrouphavea differentbodyshapebyhavingbothflatterheads(ANOVA F3,122 ¼ 48.86, P , 0.001;Fig.4;Table1)andslender (more-compressed)bodies(ANOVA, F3,122 ¼ 14.19, P , 0.001;Fig.5;Table1)comparedtoboththespeciesinthe othertwosubgroupsandtothenewlydescribedarboreal sisterspecies.Thethreenewspecieshaveabodyshapemore similartothespeciesofthe P.orestes and P.simonbolivari subgroupsthantotheirbromeliadsisterspeciesfromthe P. colodactylus subgroup(Figs.1,4,5).
Taxonomy
Pristimantis sagedunneae sp.nov. (Figs.6–9)
Holotype.—MUTPL500(Fieldno.SC230;Figs.6–8), anadultfemalefromEcuador,LojaProvince,Abrade Zamora(3.99228S,79.14558W),2,812m,collectedbyP. Sz ´ ekelyandF.Sta ˘ nescuon13November2018.
Paratypes(4females,1juvenile).—MUTPL1094 (Fieldno.SC1206),MUTPL1095(Fieldno.SC1207; Figs.9B,E,H),twoadultfemalesandMUTPL1096(Field no.SC1208;Fig.9C,F,I),ajuvenile,collectedfromthe
typelocalitybyP.Sz ´ ekely,D.Sz ´ ekely,D.Armijos-Ojedaand A.Jara-Guerreroon4June2021;MUTPL597(Fieldno.SC 252;Fig.9A,D,G),adultfemale,fromLojaProvince, CajanumaentrancetotheParqueNacionalPodocarpus,on LosMiradorestrail(4.1128 8S,79.1767 8W),2,991m, collectedbyP.Sz ´ ekelyon12January2019;MUTPL911 (Fieldno.SC421),adultfemale,fromLojaProvince, CajanumaentrancetotheParqueNacionalPodocarpus,on LosMiradorestrail(4.1167 8S,79.1697 8W),2,861m, collectedbyS.Hualpa-VegaandD.Hualpa-Vegaon8 December2019.
Diagnosis.—Weassignthisspeciesto Pristimantis based onphylogeneticevidence(Fig.3)andonthegeneral morphologicalsimilaritytoothermembersofthegenus.
Pristimantissagedunneae isamedium-sizedspecies(among
the P.orestes group;Table2),distinguishedbythefollowing combinationoftraits:(1)skinondorsumshagreenwithsome scatteredtubercles(featuremoreevidentinlife);skinon venterareolate;discoidalfoldpresent;dorsolateralfolds absent;flanksusuallywithlongitudinallateralfoldson anteriorhalf;lowmiddorsalfoldpresent;(2)tympanic annulusandtympanicmembraneabsent;supratympanicfold present;(3)snoutsubacuminatetoroundedindorsalview, roundedinprofile;canthusrostralisconcaveindorsalview, roundedinprofile;(4)uppereyelidbearingonelarger tubercleandseveralsmalltubercles(featuremoreevidentin life),it’swidthabout68%ofinterorbitaldistance;cranial crestsabsent;(5)dentigerousprocessesofvomersconcealed inbuccalmucosa;eachprocessbearingthreetofiveteeth; (6)conditionofvocalsacs,vocalslits,andnuptialpads
unknown;(7)FingerIshorterthanFingerII;discson fingersbroadlyexpanded,truncate;circumferentialgrooves present;(8)fingersbearinglateralfringes;subarticular tuberclesprominent;hyperdistalsubarticulartubercles present;supernumerarypalmartuberclespresent;palmar tubercledividedintoalarger(inner)andasmaller(outer) tubercle(s);thenartubercleelliptical,samesizewiththe innerpalmartubercle;(9)ulnartuberclespresent(feature moreevidentinlife);(10)heelwithonetothreelarge tuberclesandseveralsmalltubercles(featuremoreevident inlife);outeredgeoftarsuswithrowofsmalltubercles (featuremoreevidentinlife);innertarsalfoldpresent;(11) innermetatarsaltuberclebroadlyovoid,about33 to43 the sizeofsubconical(inprofile)outermetatarsaltubercle; subarticulartuberclesprominent;hyperdistalsubarticular tuberclespresent;supernumeraryplantartuberclespresent; (12)toesbearingbroadlateralfringes;webbingbasal;ToeV
TABLE
longerthanToeIII;discsontoesbroadlyexpanded, truncate,aboutsamesizeasthoseonfingers;circumferential groovespresent;(13)inlife,dorsumbrownishgray,with blackishordarkbrownbarsthatonthemiddleoftheback conjoinin .X or XXX-likeshapes;flanks,dorsalsurfacesof arms,andofhindlimbswithdarktransversebars;largeareas ofdorsalsurfacesofthighs,groin,andconcealedlimb surfacesredorintensered;headwithawide,blackborderedlightinterorbitalbar,darklabialbars,anddark supratympanicstripes;venterandventralsurfacesof hindlimbsandarmspinkishwhiteorpinkishgray,withor withoutdarkmarkingsonthethroat;irisbronzewithblack reticulationsandamedian,wide,horizontaldarkredstreak; (14)SVL21.4–24.3mminadultfemales(22.5 6 1.41, n ¼ 5);malesunknown.
Comparisonswithsimilarspecies. Pristimantissagedunneae ismorphologicallyverydifferentfromthebrome-
diagonal)andbodyheight/SVL(shownunderthediagonal)ratios.Boldedtextindicatessignificantdifferenceswithan a ¼ 0.05.Thespeciesincludedinthe analysisare: Pristimantiscolodactylus subgroupbromeliadspecies(P.colodactylus, P.matildae,and P.muranunka), P.colodactylus subgrouparboreal species(P.numbala, P.paladines,and P.sagedunnae), P.simonbolivari subgroup(P.tiktik and P.samaniegoi),and P.orestes subgroup(P.andinognomus, P. cajanuma, P.orestes,and P.vidua).
FIG.4.—RelationshipbetweenheadheightandSVLinthethreesubgroupsofthe Pristimantisorestes speciesgroup.Amongthe P.colodactylus subgroup, P.colodactylus, P.matildae,and P.muranunka arebromeliadspecialists(opensymbols),while P.numbala, P.paladines,and P.sagedunneae arearboreal species.
liadspecialistspeciesofthe P.colodactylus subgroup,being distinguishedmainlybythegeneralhabitus,withnot compressedbodyandhead,muchlongerfingersandtoes andthegeneralcoloration,andfromtheotherspeciesofthe P.orestes group,mainlybythemuchlongerfingersandtoes andthegeneralcoloration(Figs.1,4,5;Table2).Itismost similartotheothertwonewspecies, P.paladines and P. numbala
Fromthe17speciesthatarecurrentlyincludedinthe P. orestes group,only6specieslacktympanicannuliand membranes: P.samaniegoi, P.colodactylus, P.matildae, P. paladines, P.numbala,and P.sagedunneae (Table2). However, P.sagedunneae differsfrom P.samaniegoi byits largersize(SVL . 21mmvs.SVL , 20.7mmin P. samaniegoi),longersnout(END . 2.1mmvs.END , 1.8 mm),longerfingers(FingerIIIlength . 6.3mmvs.Finger IIIlength , 5.1mm),longertoes(ToeIVlength . 10.1mm vs.ToeIVlength , 8.3mm),discsonfingersandtoes broadlyexpanded(vs.slightlyexpanded),andbydifferent colorationpattern,withblackishordarkbrownbarsthaton themiddleofthebackconjoinin .X or XXX-likeshapesand withlargeareasofthedorsalsurfacesofthighs,groin,and concealedlimbsurfacesbeingredorintensered(vs. dorsum,flanks,dorsalsurfacesofhindlimbsandarmsdark brownwithvariouswhite,irregularspotsanddarkgray venterinfemales).Withinthe P.colodactylus subgroup, P. sagedunneae canbedistinguishedfrom P.muranunka bythe lackoftympanicannulusandtympanicmembrane(vs.
presentin P.muranunka),andfrom P.colodactylus and P. matildae byhavinganoncompressedbodyandhead(vs. compressed;Figs.4and5;Table1),longerfingers(Finger IIIlength . 6.3mmvs.FingerIIIlength , 4.4mmin P. colodactylus and , 6.1mmin P.matildae),longertoes(Toe IVlength . 10.1mmvs.ToeIVlength , 7.5mmin P. colodactylus and , 9.8mmin P.matildae),andbythe typicalredcolorationpresentonthedorsalsurfacesof thighs,groin,andconcealedlimbsurfaces(vs.concealed colorationmissingin P.colodactylus and P.matildae;Table 2).
Thethreenewspeciesaremorphologicallysimilarand difficulttodistinguish.However,thereareseveralimportant featuresthatcanbeusedtoidentifyeachofthem(Table3). Thus, P.sagedunneae canbedistinguishedfrom P.paladines byitslargersize(Mann–Whitney U ¼ 1, n1 ¼ n2 ¼ 5, P ¼ 0.02),shagreendorsum(vs.tuberculatein P.paladines),low middorsalfold(vs.elevated,ortallmiddorsalfoldthathas usuallyseverallargertuberclesscatteredalongitslength), theintenseredcolorationonthedorsalsurfacesofthighs, groin,andconcealedlimbsurfaces(vs.orangeorreddish browncoloration),andthelackofgreeninthedorsal coloration(vs.frequentlygreenpresentinthedorsal coloration).From P.numbala, P.sagedunneae canbe distinguishedbythesubacuminatetoroundedsnout(vs. roundedtobroadlyroundedsnoutin P.numbala),the relativelengthofthetoes,withToeVlongerthanToeIII (vs.ToeVmuchlongerthanToeIII),theintensered
colorationonthedorsalsurfacesofthighs,groin,and concealedlimbsurfaces(vs.reddishbrowncoloration),and thelackofgreeninthedorsalcoloration(vs.frequently greenpresentinthedorsalcoloration).
Descriptionofholotype.—Adultfemale(MUTPL500; Figs.6–8),withsmallwhitisheggs,headwiderthanbody, widerthanlong,headlength82%ofheadwidth,headwidth
38%ofSVL;headlength31%ofSVL;snoutlong(END10% ofSVL),roundedindorsalviewandinprofile,withsmall rostralpapillaattipofsnout;canthusrostralisconcavein dorsalview,roundedinprofile;lorealregionslightlyconvex; eyediameterlargerthanEND;nostrilsslightlyprotuberant, orientedposteriorly;lipsnotflared;cranialcrestsabsent; uppereyelidbearingonelargertubercleandseveralsmall
tubercles(featuremoreevidentinlife);widthofupper eyelid69%ofinterorbitaldistance;tympanicannulusand tympanicmembraneabsent;supratympanicfoldpresent; twolarge,roundedpostrictaltubercles,separatedbyalarge spacebetweenthem;choanaelarge,round,partially concealedbypalatalshelfofmaxillaryarch;dentigerous processesofvomersconcealedinbuccalmucosabutwith processfromtherightsidebearingthreeteeth;tongue longerthanwider,slightlynotchedposteriorly,posteriorhalf notadherenttofloorofmouth.
Skinondorsumshagreenwithsomescatteredtubercles (featuremoreevidentinlife);thin,lowmiddorsalfold startingattipofsnoutandendingatcloaca;dorsolateral foldsabsent;flankswithlongitudinallateralfoldsonanterior half;skinonchest,belly,ventralsurfacesofthighsareolate; thoracicfoldabsent,discoidalfoldweak;cloacalregion borderedventrallybyseveralsmalltubercles.
Ulnartuberclespresent(traitmorevisibleinlife);outer palmartubercleprominent,dividedintoalarger(inner)and asmaller(outer)tubercle(s);thenartubercleelliptical,same sizewithinnerpalmartubercle;subarticulartubercles
prominent,roundandroundedinsection;hyperdistal subarticulartuberclespresentonallfingers;supernumerary palmartuberclesrounded,large,justslightlysmallerthan subarticulartubercles;fingersbearinglateralfringes;relative lengthoffingersI , II , IV , III;discsonfingersbroadly expanded,truncate;allfingersbearingpadswelldefinedby circumferentialgrooves.
Hindlimbslong,slender;tibialength52%ofSVL;foot length50%ofSVL;heelwithtwolargeandseveralsmall tubercles(traitmorevisibleinlife);outeredgeoftarsuswith rowofsmalltubercles(traitmorevisibleinlife);inneredge oftarsusbearingshortfold;innermetatarsaltubercle broadlyovoid,about33 subconical(inprofile)outer metatarsaltubercle;subarticulartuberclesprominent,round androundedinsection;hyperdistalsubarticulartubercles presentinalltoes;supernumeraryplantartuberclesrounded oroval,smallerthansubarticulartubercles;toesbearing lateralfringes;webbingba sal;discsontoesbroadly expanded,truncate,smallerthanthoseonfingers;toeswith ventralpadswelldefinedbycircumferentialgrooves;relative lengthoftoesI ,II , III , V , IV;ToeVlongerthanToe
III(tipofToeIIIextendsbeyondproximaledgeof penultimatesubarticulartubercleonToeIV,tipofToeV barelyextendsbeyondproximaledgeandnotreachesdistal edgeofdistalsubarticulartubercleonToeIV).
Colorationofholotype. —Inlife(Fig.6):dorsum pinkish/brownishgray,withdark,blackishbarsthatonthe middleofthebackconjoinina .X-likeshape;flankswith
wide,darktransversebars;dorsalsurfacesofarmsand hindlimbswithdarktransversebars;largeareasofdorsal surfacesofthighs,groin,andconcealedlimbsurfacesare intensered;headwithwide,blackborderedlight(whitish yellow),butterflyshapedinterorbitalbar,darklabialbars anddarksupratympanicstripes;venterandventralsurfaces ofhindlimbsandarmspinkishwhiteorpinkishgray;throat white;irisbronzewithblackreticulationsandamedian, wide,horizontaldarkredstreak.
Inpreservative(Fig.7):dorsumbrownishgraywithdark brownbars;flanks,dorsalsurfacesofarmsandhindlimbs brownishgraywithdarktransversebars;headwithblackborderedwhitishyellowinterorbitalbar,darklabialbarsand darksupratympanicstripes;venter,ventralsurfacesofarms, andthroatpredominantlybrownordarkbrown;ventral surfacesofhindlimbswhitishyellow.
Measurementsofholotype(inmm).—SVL21.4;head width8.1;headlength6.6;interorbitaldistance2.6; internarialdistance2.1;uppereyelidwidth1.8;eyediameter 2.4;END2.1;thighlength10.2;tibialength11.2;footlength 10.6;handlength6.3.
Bodymassofholotype.—0.91g.
Variation.—MorphometricvariationisshowninTable4. Thedark,blackishbarsthatconjoinonthemiddleofthe backhavevariousforms,suchas .X, XX, XXX,or W-like shapes,andwereuniqueforeachoftheencountered individuals.Thesecharacteristicpatternsformedbythebars probablycanbeusedfortheindividualphotographic identification.Ofallencounteredanimals,paratypeMUTPL 597(Fig.9A,D,G)hadthemost-intenseredcolorationof thedorsalsurfacesofthigh,groin,andconcealedlimb surfaces.Thisspecimenhadalsothelargestareascolored withred,havingeventhetoesandfingertipsred(Fig.9A). TheirisoftheparatypeMUTPL1095(Fig.9B)hadavery lightcoloration,beingalmostwhite,exceptforthehorizontal darkredstreak.Thesoleencounteredjuvenile(paratype MUTPL1096)hadjustalittlebitofthedorsalsurfacesof thighsandgroinscoloredwithred(Fig.9C).Wesuspect that,astheindividualsmature,theintensityofthecolorand thesuperficiescoveredwithredincreasesinthisspecies.
Advertisementcall.—Unknown.
Etymology.—Thespecificname sagedunneae isanounin thegenitivecaseandhonorsSageDunne,inrecognitionof herpassionforAndeanwildlifeandherfamily’sinvaluable supportofconservationworkinEcuador.Ofparticular importanceistheircontributiontotheamphibianconservationintheSangay-Podocarpusconnectivitycorridor,Ecuador’sfirstecologicalcorridor,whichprotects567,067haof high-elevationpa ´ ramograsslandsandcloudforestecosystems,aswellaschainsoflakesandwetlands,withunique biologicaldiversityandendemism.
CommonEnglishname.—SageDunne’sRainFrog.
CommonSpanishname.—Cut´ındeSageDunne.
Distribution. Pristimantissagedunneae isknownonly fromAbradeZamoraandabout13kmtothesouth,from theCajanumasectorofParqueNacionalPodocarpus(Fig. 2).AllthespecimensfromAbradeZamorawereencounteredinonlyoneverysmallarea(lessthan50m2)andin CajanumaonlyontheLosMiradorestrail.Wewerenotable toencounterthisspeciesinadjacentareas,withsimilar ecosystems,despiteintensivefieldworkcarriedoutsince 2016.Thespecimenswereencounteredatanaltitudinal
¼ 21.7mm,adultfemaleparatypefromtheCajanumaentrancetotheParqueNacionalPodocarpus,LojaProvince,2,991m;(B,E,H)MUTPL1095,SVL ¼ 23.8mm,adultfemaleparatypefromAbradeZamora,LojaProvince,2,812m;(C,F,I)MUTPL1096,SVL ¼ 14.7mm,juvenileparatypefromAbrade Zamora,2,812m.
rangebetween2,800and3,000minsubpa ´ ramoecosystems (Homeieretal.2008).
Naturalhistory.—Thisisararespecies,oratleastwitha verylowdetectability.Between2016and2022weencounteredonlysevenspecimensof P.sagedunneae,andduring only4doutofmorethan50doffieldworkinthearea.All animalswereencounteredduringthenight,perchingon moss-coveredbranchesorleaves,from40cmupto2mhigh, inhabitatswithshrubbyvegetation.Noindividualwas encounteredinsidebromeliads.Unfortunately,despite intensivesearches,wewerenotabletohearorencounter anymales.Sympatricfrogspeciesinclude Pristimantis aff. andinognomus, P.cajanuma, P.matildae, P.samaniegoi, P. versicolor (Lynch1979),and P.vidua. Conservationstatus. Pristimantissagedunneae is knownfromonlytwocloselocalities,fromanestimated areaoflessthan10km2.Althoughbothlocalitiesareinsidea nationallyprotectedarea,werecommendthatthisspeciesbe categorizedasEndangeredfollowingtheB1ab(iii,iv,v)þ 2ab(iii,iv,v)InternationalUnionforConservationofNature (IUCN)criteria(IUCN2001)because:(1)itsExtentof
Occurrence(EOO)andAreaofOccupancy(AOO)are estimatedtobelessthan10km2;(2)itisknownfromonlya fewindividualsandonlyfromtwolocalities;and(3)its habitatscouldbeaffectedinthenearfuture,astheyare situatedinthevicinityofdenselypopulatedareas(Ord ´ onezDelgadoetal.2020).
Pristimantis paladines sp.nov. (Figs.10–14)
Holotype.—MUTPL1159(Fieldno.SC1260;Figs.10–12),anadultfemalefromEcuador,ZamoraChinchipe Province,ParqueNacionalPodocarpusinthevicinityof ReservaTapichalaca(4.4472 8S,79.1459 8W),2,880m, collectedbyP.Sz ´ ekely,D.Sz ´ ekely,D.Armijos-Ojeda,and L.Carri ´ onon27August2021.
Paratypes(4females,5malesand2subadults). MUTPL1151(Fieldno.SC1252;Fig.13A,D,G),MUTPL 1155(Fieldno.SC1256),MUTPL1160(Fieldno.SC1261; Fig.13B,E,H),andMUTPL1161(Fieldno.SC1262;Fig. 13C,F,I),fourfemalescollectedwiththeholotype;MUTPL 122,MUTPL123,MUTPL125(Fig.14A,D,G),threeadult
group.ForthedefinitionofthecategoriesseeMorphologicalAnalysesintheMaterialand
2.—Comparisonofseveralimportantfeaturesofthecurrentlyconfirmedspeciesofthe Pristimantisorestes
kingsinaxilla,groin,oronconcealedlimbsurfaces.?
Methodssection.Specialcoloration:referstosomekindofdistinctivecoloration(infemalesorbothsexes),eitherventralordorsalspots,ormar
* IncorrectlydefinedasslightlyexpandedinSz
Indicates thatwelackinformationorthattheavailabledataareuncertainduetoincompletemeasurementsand/orpossibleidentificationerrors.
Special colorationSource
Vocal slits
Discsonfingers andtoesTympanum
Slightlyexpanded, rounded
Slightlyexpanded, rounded
Slightlyexpanded, rounded
Relativelengths oftoes
ToeVslightlylonger thanToeIII
ToeVslightlylonger thanToeIII
ToeVmuchlonger thanToeIII
ToeVslightlylonger thanToeIII
snout–ventlength;END ¼ eye–nostrildistance.
BodysizeSnoutlengthFingerIIIlengthToeIVlength
Short(lengthofToe IVabout39.6%of SVL)
Medium(lengthof FingerIIIabout 24.5%ofSVL)
Veryshort(END about7.8%ofSVL)
Large(femalesSVL upto26.8mm)
Pristimantisorestes subgroup P.vidua
Long(lengthofToe IVabout46.7%of SVL)
Long(lengthof FingerIIIabout 27.3%ofSVL)
Short(ENDabout 8.5%ofSVL)
Small(femalesSVL upto22.1mm)
P.cajanuma
Medium(lengthof ToeIVabout41.8% ofSVL)
Short(lengthof FingerIIIabout 23.4%ofSVL)
Veryshort(END about7.7%ofSVL)
Minute(femalesSVL upto17.9mm)
P.andinognomus
Short(lengthofToe IVabout39.9%of SVL)
Short(lengthof FingerIIIabout 23.0%ofSVL)
Short(ENDabout 8.4%ofSVL)
Large(femalesSVL upto27.2mm)
P.orestes
Coloma1992
PresentPresentYesArteaga-Navarroand Guayasamin2011
Arteaga2013
PresentAbsentNoBritoetal.2017a andthisstudy
AbsentAbsentNoThisstudy
Absent?YesThisstudy
Slightlyexpanded, rounded
ToeVslightlylonger thanToeIII
Short(lengthofToe IVabout39.0%of SVL)
Short(lengthof FingerIIIabout 22.9%ofSVL)
Short(ENDabout 8.0%ofSVL)
Pristimantissimonbolivari subgroup
???Slightlyexpanded, rounded
Expanded,roundedto truncate
ToeVslightlylonger thanToeIII
Medium?(lengthof ToeIVabout44.7% ofSVL)
Medium?(lengthof FingerIIIabout 24.9%ofSVL)
Veryshort?(END about7.6%ofSVL)
Short?(ENDabout 9.0%ofSVL)
Slightlyexpanded, rounded
ToeVslightlylonger thanToeIII
Short?(lengthofToe IVabout39.0%of SVL)
?Short?(lengthof FingerIIIabout 23.9%ofSVL)
Slightlyexpanded, rounded
ToeVslightlylonger thanToeIII
Medium?(lengthof ToeIVabout41.6% ofSVL)
Long?(lengthof FingerIIIabout 26.0%ofSVL)
Short?(ENDabout 8.4%ofSVL)
ToeVlongerthan ToeIII
Medium(lengthof ToeIVabout41.1% ofSVL)
Medium(lengthof FingerIIIabout 24.3%ofSVL)
Veryshort(END about7.5%ofSVL)
Slightlyexpanded, rounded
ToeVslightlylonger thanToeIII
Short(lengthofToe IVabout38.6%of SVL)
Short(lengthof FingerIIIabout 23.5%ofSVL)
Short(ENDabout 8.7%ofSVL)
Slightlyexpanded, rounded
ToeVlongerthan ToeIII
Veryshort(lengthof ToeIVabout36.1% ofSVL)
Veryshort(lengthof FingerIIIabout 21.6%ofSVL)
Long(ENDabout 9.2%ofSVL)
ToeVmuchlonger thanToeIII
Short(lengthofToe IVabout38.5%of SVL)
Short(lengthof FingerIIIabout 22.7%ofSVL)
Long(ENDabout 9.2%ofSVL)
ToeVlongerthan ToeIII
Medium(lengthof ToeIVabout40.2% ofSVL)
Medium(lengthof FingerIIIabout 25.2%ofSVL)
Long(ENDabout 9.1%ofSVL)
ToeVlongerthan ToeIII AbsentAbsentYesThisstudy
Verylong(lengthof ToeIVabout48.4% ofSVL)
Verylong(lengthof FingerIIIabout 28.4%ofSVL)
Long(ENDabout 10.1%ofSVL)
ToeVmuchlonger thanToeIII Absent?YesThisstudy
Verylong(lengthof ToeIVabout48.7% ofSVL)
Verylong(lengthof FingerIIIabout 28.6%ofSVL)
Long(ENDabout 9.6%ofSVL)
Verylong(lengthof ToeIVabout48.3% ofSVL)
Verylong(lengthof FingerIIIabout 28.9%ofSVL)
Broadlyexpanded, truncate 53 SZ ´ EKELYETAL.—THREE NEW CRYPTIC PRISTIMANTIS SPECIES Downloaded from http://meridian.allenpress.com/herpetological-monographs/article-pdf/37/1/41/3220628/i1938-5137-37-1-41.pdf by Ecuador user on 24 May 2023
Minute(femalesSVL upto20.4mm)
Small(femalesSVL upto22.0mm)
Large(femalesSVL upto26.4mm)
Medium(femalesSVL upto23.7mm)
Small(femalesSVL upto21.5mm)
Small(femalesSVL upto21.8mm)
Minute(femalesSVL upto20.7mm)
P.tiktik
P.simonbolivari
P.bambu
P.mazar
P.saturninoi
P.quintanai
P.samaniegoi
Pristimantiscolodactylus subgroup
Minute(femalesSVL upto20.7mm)
P.colodactylus
Medium(femalesSVL upto23.3mm)
P.muranunka
Medium(femalesSVL upto23.3mm)
Medium(femalesSVL upto24.3mm)
Long(ENDabout 9.6%ofSVL)
P.matildae
P.sagedunneae
Small(femalesSVL upto21.4mm)
P.paladines
Medium(femalesSVL upto23.2mm)
P.numbala
PristimantissagedunneaePristimantispaladinesPristimantisnumbala RelativebodysizeMediumSmallMedium Snoutshape(indorsalview)SubacuminatetoroundedAcuminatetosubacuminateRoundedtobroadlyrounded RelativeheadwidthHeadwidthabout37%ofSVLHeadwidthabout38%ofSVLHeadwidthabout40%ofSVL Canthusrostralis(indorsalview)ConcaveWeaklyconcavetostraightConcave LargetuberclesonuppereyelidOneTwotothreeOne
RelativewidthofuppereyelidWidthofuppereyelidabout68% ofinterorbitaldistance
Widthofuppereyelidabout72% ofinterorbitaldistance
Widthofuppereyelidabout83% ofinterorbitaldistance
LorealregionSlightlyconvexSlightlyconvexFlat ChoanaeLarge,round,partiallyconcealed bypalatalshelfofmaxillaryarch
Large,round,partiallyconcealed bypalatalshelfofmaxillaryarch
DentigerousprocessesofvomersConcealedinbuccalmucosaInconspicuous,oblique,ovoidor triangular,separatedmediallyby distancelowerthanthewidthof processes
SkintextureondorsumShagreenwithsomescattered tubercles
Tuberculate,withnumerous scatteredlargetubercles
MiddorsalfoldLowEvident(tall),usuallywithseveral largertuberclesscatteredalongits length
ThenartuberclesizeThesamesizewiththeinner palmartubercle
SupernumerarypalmartuberclesRounded,large,justslightly smallerthansubarticulartubercles
Largerthantheinnerpalmar tubercle
Rounded,small,muchsmallerthan subarticulartubercles
Large,oval,notconcealedby palatalshelfofmaxillaryarch
Evident,oblique,ovoid,separated mediallybydistanceequalor slightlylowertothewidthof processes
Shagreenwithsomescattered tubercles
Low
Largerthantheinnerpalmar tubercle
Oval,large,smallerthan subarticulartubercles
LargetuberclesontheheelOnetothreeTwotothreeOne
OuteredgeoftarsusWitharowofsmalltuberclesWitharowoflarge,conical tubercles
Withorwithoutarowofsmall tubercles
RelativelengthsoftoesToeVlongerthanToeIIIToeVlongerthanToeIIIToeVmuchlongerthanToeIII
males,andMUTPL124,MUTPL126twosubadultsfrom LojaProvince,theCerroToledosectorofParqueNacional Podocarpus(4.37968S,79.11248W),3,080mcollectedbyP. Sz ´ ekelyandD.Sz ´ ekelyon10May2016;MUTPL1107 (Fieldno.SC1210;Fig.14B,E,H)andMUTPL1109(Field no.SC1212;Fig.14C,F,I),twoadultmalesfromZamora ChinchipeProvince,theCerroToledosectorofParque NacionalPodocarpus(4.3857 8S,79.1066 8W),3,104m collectedbyP.Sz ´ ekelyandD.Sz ´ ekelyon26June2021.
Diagnosis.—Weassignthisspeciesto Pristimantis based onphylogeneticevidence(Fig.3)andonthegeneral morphologicalsimilaritytoothermembersofthegenus. Pristimantispaladines isasmallspecies(amongthe P. orestes group;Table2),distinguishedbythefollowing combinationoftraits:(1)skinondorsumtuberculatewith numerousscatteredlargetubercles(inlifethetuberculated
textureoftheskinismoreevident);skinonventerareolate; discoidalfoldpresent;dorsolateralfoldsabsent;flanks usuallywithlongitudinallateralfoldsonanteriorhalf; middorsalfoldevident,tall,usuallywithseverallarger tuberclesscatteredalongitslength(featuremoreevidentin life);(2)tympanicannulusandtympanicmembraneabsent; supratympanicfoldpresent;(3)snoutacuminatetosubacuminateindorsalview,roundedinprofile;canthus rostralisweaklyconcavetostraightindorsalview,rounded inprofile;(4)uppereyelidbearingtwotothreelarge tuberclesandseveralsmalltubercles(featuremoreevident inlife),itswidthabout72%ofinterorbitaldistancein femalesand81%ofinterorbitaldistanceinmales;cranial crestsabsent;(5)dentigerousprocessesofvomersinconspicuous,oblique,ovoidortriangular,separatedmediallyby adistancesmallerthanthewidthofprocesses;eachprocess
TABLE 4.—Bodymeasurementsofadultsofthethreenew Pristimantis species.Bodymass(ingrams),snout–ventlength(SVL)(inmm),and morphologicalproportions(inpercentages).Valuesaregivenasmean 6 SD(range).RawmeasurementsarepresentedinFileS2.Femalebodymass includeseggs. *n ¼ 4.END ¼ eye–nostrildistance;—indicatesmissingdata.
TABLE 3.—Morphologicaltraitcomparisonamongthethreenewspecies.SVL ¼ snout–ventlength.bearingtwotofiveteeth;(6)maleswithoutvocalsacs,vocal slits,andnuptialpads;(7)FingerIshorterthanFingerII; discsonfingersbroadlyexpanded,truncate;circumferential groovespresent;(8)fingersbearinglateralfringes;subarticulartuberclesprominent;hyperdistalsubarticulartu-
berclespresent;supernumerarypalmartuberclespresent; palmartubercleusuallynotdividedoronlypartiallydivided intoalarger(inner)andasmaller(outer)tubercle(s);thenar tubercleelliptical,largerthaninnerpalmartubercle;(9) ulnartuberclespresent(featuremoreevidentinlife);(10)
heelwithtwotothreelargetuberclesandseveralsmall tubercles(featuremoreevidentinlife);outeredgeoftarsus withrowoflarge,conicaltubercles(featuremoreevidentin life);innertarsalfoldpresent;(11)innermetatarsaltubercle broadlyovoid,about23 to33 thesizeofsubconical(in profile)outermetatarsaltubercle;subarticulartubercles prominent;hyperdistalsubarticulartuberclespresent;supernumeraryplantartuberclespresent;(12)toesbearing broadlateralfringes;webbingbasal;ToeVlongerthanToe III;discsontoesbroadlyexpanded,truncate,slightlysmaller thanthoseonfingers;circumferentialgroovespresent;(13) inlife,dorsumofvariousshadesofbrown,reddishbrown, greenishbrown,greenorreddishorangewith,dark,blackish bars,withorwithout X or W markingsontheback;flanks, dorsalsurfacesofarmsandofhindlimbswithdarktransverse bars;largeareasofdorsalsurfacesofthighs,groin,concealed limbsurfacesandsometimesofdorsumorangeorreddish brown;headusuallywithdark,blackishinterorbitalmarkings,darklabialbarsanddarksupratympanicstripes;venter andventralsurfacesofhindlimbsandarmspinkishwhiteor pinkishgray,withorwithoutdarkmarkingsonthethroat;iris
bronzewithfineblackreticulationsandamedian,wide, horizontaldarkredstreak;(14)SVL19.5–21.4mminadult females(20.5 6 0.82, n ¼ 5)and16.7–18.3mminadult males(17.4 6 0.73, n ¼ 5).
Comparisonwithsimilarspecies. Pristimantispaladines ismorphologicallyverydifferentfromthebromeliad specialistspeciesofthe P.colodactylus subgroup,being distinguishedmainlybythegeneralhabitus,withnot compressedbodyandhead,longerfingersandtoes,and thegeneralcoloration,andfromtheotherspeciesofthe P. orestes speciesgroup,mainlybythemuchlongerfingersand toesandthegeneralcoloration(Figs.1,4,5;Table2).Itis mostsimilartotheothertwonewlydescribedspecies, P. sagedunneae and P.numbala
Pristimantispaladines differsfrom P.samaniegoi byits longersnout(END . 1.9mmvs.END , 1.8mm),longer fingers(FingerIIIlength . 5.4mmvs.FingerIIIlength , 5.1mm),longertoes(ToeIVlength . 9.7mmvs.ToeIV length , 8.3mm),discsonfingersandtoesbroadly expanded(vs.slightlyexpanded),andbydifferentcoloration patternwithdark,blackishbars,withorwithout X or W
markingsonthebackandwithlargeareasofthedorsal surfacesofthighs,groin,concealedlimbsurfaces,and sometimesofthedorsumorangeorreddishbrown(vs. dorsum,flanks,dorsalsurfacesofhindlimbs,andarmsdark brownwithvariouswhite,irregularspotsanddarkgray venterinfemales).Withinthe P.colodactylus subgroup, P. paladines canbedistinguishedfrom P.muranunka bythe
lackoftympanicannulusandtympanicmembrane(vs. presentin P.muranunka),andfrom P.colodactylus and P. matildae byhavinganoncompressedbodyandhead(vs. compressed;Table1;Figs.4,5),longerfingers(FingerIII length . 5.4mmvs.FingerIIIlength , 4.4mmin P. colodactylus),longertoes(ToeIVlength . 9.7mmvs.Toe IVlength , 7.5mmin P.colodactylus),andbythetypical orangeorreddishbrowncolorationpresentonthedorsal surfacesofthighs,groin,andconcealedlimbsurfaces(vs. concealedcolorationmissingin P.colodactylus and P. matildae;Table2).
Thereareseveralimportantfeaturesthatcanbeusedto distinguish P.paladines fromitssisterspecies(Table3). Thus, P.paladines differsfrom P.sagedunneae byitssmaller size(Mann–Whitney U ¼ 1, n1 ¼ n2 ¼ 5, P ¼ 0.02), tuberculatedorsum(vs.shagreenin P.sagedunneae),tall middorsalfoldthathasusuallyseverallargertubercles scatteredalongitslength(vs.lowmiddorsalfold,without tubercles),orangeorreddishbrowncolorationonthedorsal surfacesofthighs,groin,andconcealedlimbsurfaces(vs. intenseredcoloration),andfrequentlygreenpresentinthe dorsalcoloration(vs.lackofgreeninthedorsalcoloration). Pristimantispaladines differsfrom P.numbala bythe acuminatetosubacuminatesnout(vs.roundedtobroadly roundedsnoutin P.numbala),thetuberculatedorsum(vs. shagreendorsum),tallmiddorsalfoldthatusuallyhasseveral largertuberclesscatteredalongitslength(vs.lowmiddorsal fold,withouttubercles),andshorterrelativelengthofthe toes,withToeVlongerthanToeIII(vs.ToeVmuchlonger thanToeIII).
Thedorsumskintexturecanbeusedonlyforidentificationinlife,asallthreenewspecieshavetheabilitytochange theirskintexturesimilarlyto P.mutabilis (Guayasaminetal. 2015).Furthermore,inpreservedspecimensthetextureof theskin,andparticularlytheshapeofthetubercles,is significantlymodifiedbythepreservationprocess(the tuberclesbecomelessconspicuous).Nevertheless,the tuberculatedorsumisanevidentdistinctionbetween P. paladines anditssisterspecies.
Descriptionofholotype.—Adultfemale(MUTPL1159; Figs.10–12),withlargewhite-yelloweggs,headnarrower thanbody,widerthanlong,headlength87%ofheadwidth, headwidth36%ofSVL;headlength32%ofSVL;snoutlong (END10%ofSVL),subacuminateindorsalviewand roundedinprofile,withsmallrostralpapillaattipofsnout; canthusrostralisweaklyconcaveindorsalview,roundedin profile;lorealregionslightlyconvex;eyediameterlargerthan END;nostrilsslightlyprotuberant,orientedposteriorly;lips notflared;cranialcrestsabsent;uppereyelidbearingtwo largertuberclesandseveralsmalltubercles(traitmore visibleinlife);widthofuppereyelid70%ofinterorbital distance;tympanicannulusandtympanicmembraneabsent; supratympanicfoldpresent;onelarge,roundedpostrictal tubercle;choanaelarge,round,partiallyconcealedbypalatal shelfofmaxillaryarch;dentigerousprocessesofvomers inconspicuous,slightlysmallerthanthechoanae,oblique, situatedposteriorandmediantochoanae,triangularin outline,separatedmediallybydistanceequaltohalfofwidth ofprocesses,eachprocessbearingthreetofourteeth; tonguejustslightlylongeraswide,notnotchedposteriorly, posteriorhalfnotadherenttofloorofmouth.
Skinondorsumtuberculatewithnumerousscattered largetubercles(traitmorevisibleinlife);evident,tall, middorsalfoldstartingattipofsnoutandendingatcloaca (traitmorevisibleinlife);dorsolateralfoldsabsent;flanks withlongitudinallateralfoldsonanteriorhalf;skinonchest, belly,ventralsurfacesofthighsareolate;thoracicand discoidalfoldspresent,weak;cloacalregionbordered laterallybyonetubercleoneachsideandventrallyby severalsmalltubercles.
Ulnartuberclespresent(traitmorevisibleinlife);outer palmartubercleprominent,partiallydividedintoalarger (inner)andasmaller(outer)tubercle(s);thenartubercle elliptical,largerthaninnerpalmartubercle;subarticular tuberclesprominent,roundandroundedinsection;hyperdistalsubarticulartuberclespresentinallfingers;supernumerarypalmartuberclesrounded,small,muchsmallerthan subarticulartubercles;fingersbearinglateralfringes;relative lengthoffingersI , II , IV , III;discsonfingersbroadly expanded,truncate;allfingersbearingpadswelldefinedby circumferentialgrooves.
Hindlimbslong,slender;tibialength51%ofSVL;foot length49%ofSVL;heelwithtwolargeandseveralsmall tubercles(traitmorevisibleinlife);outeredgeoftarsuswith rowoflarge,conicaltubercles(traitmoreevidentinlife); inneredgeoftarsusbearingshortfold;innermetatarsal tuberclebroadlyovoid,about23 subconical(inprofile)outer metatarsaltubercle;subarticulartuberclesprominent,round androundedinsection;hyperdistalsubarticulartubercles presentinalltoes;supernum eraryplantartubercles inconspicuous;toesbearingbroadlateralfringes;webbing basal;discsontoesbroadlyexpanded,truncate,slightly smallerthanthoseonfingers;toeswithventralpadswell definedbycircumferentialgrooves;relativelengthoftoesI ,II , III , V , IV;ToeVlongerthanToeIII(tipofToe IIIextendsbeyondproximaledgeofpenultimatesubarticulartubercleonToeIV,tipofToeVbarelyextendsbeyond proximaledgeanddoesnotreachdistaledgeofdistal subarticulartubercleonToeIV).
Colorationofholotype.—Inlife(Fig.10):dorsum greenishbrownwithdarkbarsand X and W darkmarkings onmiddleoftheback;flankswithwide,darktransversebars;
dorsalsurfacesofarmsandhindlimbswithdarktransverse bars;largeareasofdorsalsurfacesofthighsandofgroinare orange;headwithwide,whitishyellowinterorbitalbar, whichisborderedposteriorlybydark,blackishmarkings, darklabialbars,anddarksupratympanicstripes;venterand ventralsurfacesofhindlimbsandarmspinkishgray;theskin onrightsideofabdomenalmosttranslucent,revealingthe whitishyellowcolorationofthelargeeggs;throatpinkish gray;irisbronzewithfineblackreticulationsandamedian, wide,horizontaldarkredstreak.
Inpreservative(Fig.11):dorsumbrownishgraywithdark brownbars;flanks,dorsalsurfacesofarmsandhindlimbs brownishgraywithdarktransversebars;headwithdark interorbitalmarkings,darklabialbarsanddarksupratympanicstripes;venter,ventralsurfacesofarms,hindlimbs, andthroatpredominantlywhitishyellow,withsomesmall brownishmarkings.
Measurementsofholotype(inmm).—SVL20.6;head width7.5;headlength6.5;interorbitaldistance2.7; internarialdistance1.8;uppereyelidwidth1.9;eyediameter
2.2;END2.0;thighlength9.9;tibialength10.4;footlength 10.1;handlength5.7.
Bodymassofholotype.—0.96g.
Variation.—MorphometricvariationisshowninTable4. Besidestheevidentsizedifferencebetweenthefemalesand males(femalesbeingsignificantlylarger),thereareno noticeabledistinctionbetweenthesexes.Someindividuals, likethefemaleparatypeMUTPL1160(Figs.13B,E),had theentirebackreddishorange,orothers,likethefemale paratypeMUTPL1161(Figs.13C,F),hadapredominantly greendorsumwithadditionalreddishorangecoloration. Someindividuals(liketheMUTPL123,MUTPL1155or MUTPL1160paratypes)hadseveralsmallerorlargerwhite spotsscatteredonthedorsum,head,ordorsalsurfacesof armsandlegs(Figs.13B,E).ThemaleparatypeMUTPL 125hadaveryparticularcolorationwithawide,whitish yellowmiddorsalstripe(Figs.14A,D).
Advertisementcall.—Unknown.
Etymology.—Thespecificname paladines isanounin appositionandhonorsthePaladinesfamilyfromthecityof Loja,inparticularF
´ elixHumbertoPaladinesPaladines(1938–2022),forhisvaluablecontributiontotheacademic andculturalfieldsandforsafeguardingthehistoryand identityofsouthernEcuadorianpeople.Inaddition,it constitutesrecognitionoftheremarkableworkcarriedout byhischildren,Renzo,Bruno,Pedro,andMariaGabriela, whocreatedthenongovernmentalorganizationNaturalezay CulturaInternacional(NCI),aconsolidatedorganization dedicatedtotheprotectionofwildlifeinLatinAmerica whereithasmanagedtoprotect,todate,morethan8.5 millionhectaresoftropicalforestinvariouscountries.
CommonEnglishname.—PaladinesRainFrog.
CommonSpanishname.—Cut´ındePaladines.
Distribution. Pristimantispaladines isknownfrom threenearbylocalities:theCerroToledosectorofthe ParqueNacionalPodocarpus,thenfromabout10kmtothe south,insideParqueNacionalPodocarpusinthevicinityof ReservaTapichalaca,andfromanother5kmfurtherinthe southintheReservaTapichalaca(Fig.2).Thespecimens wereencounteredatanaltitudinalrangebetween2,800and 3,100minsubpa ´ ramoecosystems(Homeieretal.2008).
Naturalhistory.—Thisisacommonandlocallyabundant species.Individualswereencounteredduringthenight, perchingonmoss-coveredbranchesorleavesoronmosscoveredwallsneartheroad,sometimesnexttosmall streams,atheightsrangingfromthesoillevelupto1.5m high,inhabitatswithshrubbyvegetation.Noindividualwas encounteredinsidebromeliads.Also,nocallingmaleswere heard.Sympatricfrogspeciesinclude Pristimantisandinognomus, P.atratus (Lynch1979), P.versicolor,andthree undescribedspeciesof Pristimantis Conservationstatus. Pristimantispaladines isacommonandlocallyabundantspeciesknowncurrentlyfromonly threecloselocalities,fromanestimatedareaoflessthan20 km2.Allknownlocalitiesareinsidenationalorprivate protectedareasanditshabitatcurrentlydoesnotfaceany majorthreats.Nonetheless,werecommendthatthisspecies iscategorizedasNearThreatenedfollowingtheIUCN criteria,duetoitsverysmallExtentofOccurrence(EOO).
Pristimantis numbala sp.nov. (Figs.15–18)
Holotype.—MUTPL1190(Fieldno.SC1284;Figs.15–17),anadultfemalefromEcuador,ZamoraChinchipe Province,ReservaNumbala(4.40658S,79.08858W),2,875m, collectedbyS.Hualpa-Vega,P.Sz ´ ekely,andD.Sz ´ ekelyon3 September2021.
Paratypes(2femalesand1juvenile). —MUTPL1191 (Fieldno.SC1285;Figs.18C,F,I),ajuvenilecollectedwith theholotype;MUTPL1178(Fieldno.SC1271;Figs.18A, D,G),MUTPL1179(Fieldno.SC1272;Figs.16B,E,H), twofemales,fromReservaNumbala(4.40058S,79.0854 8W), 2,861m,collectedbyP.Sz ´ ekely,D.Sz ´ ekely,andS.HualpaVegaon2September2021.
Diagnosis.—Weassignthisspeciesto Pristimantis based onphylogeneticevidence(Fig.3)andonthegeneral morphologicalsimilaritytoothermembersofthegenus.
Pristimantisnumbala isamedium-sizedspecies(amongthe P.orestes group;Table2),distinguishedbythefollowing combinationoftraits:(1)skinondorsumshagreenwithsome scatteredtubercles(featuremoreevidentinlife);skinon venterareolate;discoidalfoldpresent;dorsolateralfolds
absent;flanksusuallywithlongitudinallateralfoldson anteriorhalf;lowmiddorsalfoldpresent(featuremore evidentinlife);(2)tymp anicannulusandtympanic membraneabsent;supratympanicfoldpresent;(3)snout roundedtobroadlyroundedindorsalview,roundedin profile;canthusrostralisconcaveindorsalview,roundedin profile;(4)uppereyelidbearingonelargertubercleand severalsmalltubercles(featuremoreevidentinlife),it’s widthabout83%ofinterorbitaldistance;cranialcrests absent;(5)dentigerousprocessesofvomersevident,oblique, ovoid,separatedmediallybydistanceequalorslightly smallerthanthewidthofprocesses;eachprocessbearing fourtosixteeth;(6)conditionofvocalsacs,vocalslits,and nuptialpadsunknown;(7)FingerIshorterthanFingerII; discsonfingersbroadlyexpanded,truncate;circumferential groovespresent;(8)fingersbearinglateralfringes;subarticulartuberclesprominent;hyperdistalsubarticulartuberclespresent;supernumerarypalmartuberclespresent; palmartubercleusuallynotdividedoronlypartiallydivided intoalarger(inner)andasmaller(outer)tubercle(s);thenar tubercleelliptical,largerthaninnerpalmartubercle;(9) ulnartuberclespresent(featuremoreevidentinlife);(10) heelwithoutorwithonelargertubercleandseveralsmall tubercles(featuremoreevidentinlife);outeredgeoftarsus withorwithoutrowofsmalltubercles(featuremoreevident inlife);innertarsalfoldabsentorpresent;(11)inner metatarsaltuberclebroadlyovoid,about23 to33 thesizeof subconical(inprofile)outermetatarsaltubercle;subarticular tuberclesprominent;hyperdistalsubarticulartubercles present;supernumeraryplantartuberclespresent;(12)toes bearinglateralfringes;webbingbasal;ToeVmuchlonger thanToeIII;discsontoesbroadlyexpanded,truncate,about samesizeorslightlysmallerasthoseonfingers;circumferentialgroovespresent;(13)inlife,dorsumreddishbrown, withdarkbrownbarsand X markingsontheback;flanks grey,pinkishgrey,orgreen,withwide,darktransversebars; dorsalsurfacesofarmsandofhindlimbswithdarktransverse bars;largeareasofdorsalsurfacesofthighs,groin,and concealedlimbsurfacesreddishbrown;headwithblack interorbitalmarkings,darklabialbarsandblacksupratympanicstripes;venter,throat,andventralsurfacesof hindlimbsandarmspinkishwhiteorpinkishgraywith blackishmarkings;irisbronzewithfineblackreticulations andamedian,wide,horizontaldarkredstreak;(14)SVL 20.4–23.2mminadultfemales(21.9 6 1.43, n ¼ 3);males unknown.
Comparisonwithsimilarspecies. Pristimantisnumbala ismorphologicallyverydifferentfromthebromeliad specialistspeciesofthe P.colodactylus subgroup,being distinguishedmainlybythegeneralhabitus,withnot compressedbodyandhead,muchlongerfingersandtoes, andthegeneralcoloration,andfromtheotherspeciesofthe P.orestes groupmainlybythemuchlongerfingersandtoes andthegeneralcoloration(Figs.1,4,5;Table2).Itismost similartotheothertwonewspecies, P.sagedunneae and P. paladines
Pristimantisnumbala differsfrom P.samaniegoi byits longersnout(END . 2.0mmvs.END , 1.8mm),longer fingers(FingerIIIlength . 6.1mmvs.FingerIIIlength , 5.1mm),longertoes(ToeIVlength . 10.0mmvs.ToeIV length , 8.3mm),discsonfingersandtoesbroadly expanded(vs.slightlyexpanded),andbydifferentcoloration
patternwithdarkbrownbarsand X markingsontheback andwithlargeareasofthedorsalsurfacesofthighs,groin, andconcealedlimbsurfacesreddishbrown(vs.dorsum, flanks,dorsalsurfacesofhindlimbsandarmsdarkbrown withvariouswhite,irregularspotsanddarkgrayventerin
females).Withinthe P.colodactylus subgroup, P.numbala canbedistinguishedfrom P.muranunka bythelackof tympanicannulusandtympanicmembrane(vs.presentin P. muranunka)andfrom P.colodactylus and P.matildae by havinganoncompressedbodyandhead(vs.compressed;
Figs.4,5;Table1),longerfingers(FingerIIIlength . 6.1 mmvs.FingerIIIlength , 4.4mmin P.colodactylus), longertoes(ToeIVlength . 10.0mmvs.ToeIVlength , 7.5mmin P.colodactylus and , 9.8mmin P.matildae),and bythetypicalreddishbrowncolorationpresentonthedorsal surfacesofthighs,groin,andconcealedlimbsurfaces(vs. concealedcolorationmissingin P.colodactylus and P. matildae;Table2).
Thereareseveralimportantfeaturesthatcanbeusedto distinguish P.numbala fromitssisterspecies(Table3). Thus, P.numbala differsfrom P.sagedunneae bythe roundedtobroadlyroundedsnout(vs.subacuminateto roundedsnoutin P.sagedunneae),therelativelengthofthe toes,withToeVmuchlongerthanToeIII(vs.ToeVlonger thanToeIII),thereddishbrowncolorationonthedorsal surfacesofthighs,groin,andconcealedlimbsurfaces(vs. intenseredcoloration),andfrequentlygreenpresentinthe dorsalcoloration(vs.lackofgreeninthedorsalcoloration).
Pristimantisnumbala differsfrom P.paladines bythe roundedtobroadlyroundedsnout(vs.acuminateto
subacuminatesnoutin P.paladines),theshagreendorsum (vs.tuberculatedorsum),lowmiddorsalfold(vs.tall middorsalfold,thathasusuallyseverallargertubercles scatteredalongitslength),andtherelativelengthofthetoes, withToeVmuchlongerthanToeIII(vs.ToeVlongerthan ToeIII).
Descriptionofholotype.—Adultfemale(MUTPL1190; Figs.15–17),withlargewhiteeggs,headslightlywiderthan body,widerthanlong,headlength81%ofheadwidth,head width40%ofSVL;headlength33%ofSVL;snoutlong (END9%ofSVL),roundedindorsalviewandinprofile, withsmallrostralpapillaattipofsnout;canthusrostralis concaveindorsalview,roundedinprofile;lorealregionflat; eyediameterlargerthanEND;nostrilsslightlyprotuberant, orientedposteriorly;lipsnotflared;cranialcrestsabsent; uppereyelidbearingonelargertubercleandseveralsmall tubercles(traitmorevisibleinlife),widthofuppereyelid 80%ofinterorbitaldistance;tympanicannulusandtympanic membraneabsent;supratympanicfoldpresent;two(onthe rightside)andone(ontheleftside)larger,rounded
postrictaltubercles;choanaelarge,oval,notconcealedby palatalshelfofmaxillaryarch;dentigerousprocessesof vomersevident,slightlysmallerthanthechoanae,oblique, situatedposteriorandmediantochoanae,ovoidinoutline, separatedmediallybydistanceequaltowidthofprocesses, eachprocessbearingfourtofiveteeth;tonguejustslightly
longerthanwide,slightlynotchedposteriorly,posteriorhalf notadherenttofloorofmouth.
Skinondorsumshagreenwithsomescatteredtubercles (featuremoreevidentinlife);thin,lowmiddorsalfold startingattipofsnoutandendingatcloaca(featuremore evidentinlife);dorsolateralfoldsabsent;flankswith longitudinallateralfoldsonanteriorhalf;skinonchest, belly,ventralsurfacesofthighsareolate;thoracicand discoidalfoldpresent;cloacalregionborderedventrallyby twolargertubercles.
Ulnartuberclespresent(featuremoreevidentinlife); outerpalmartubercleprominent,notdivided;thenar tubercleelliptical,largerthantheinnerpartofouter tubercle;subarticulartuberclesprominent,roundand roundedinsection;hyperdistalsubarticulartubercles presentinallfingers;supernumerarypalmartubercles inconspicuous;fingersbearinglateralfringes;relativelength offingersI , II , IV , III;discsonfingersbroadly expanded,truncate;allfingersbearingpadswelldefinedby circumferentialgrooves.
Hindlimbslong,slender;tibialength51%ofSVL;foot length49%ofSVL;heelwithonelargeandseveralsmall tubercles(traitmorevisibleinlife);outeredgeoftarsuswith rowofsmalltubercles(traitmorevisibleinlife);inneredge oftarsuswithoutfold;innermetatarsaltuberclebroadly ovoid,about23 subconical(inprofile)outermetatarsal tubercle;subarticulartuberclesprominent,roundand roundedinsection;hyperdistalsubarticulartubercles presentinalltoes;supernumeraryplantartuberclesoval, smallerthansubarticulartubercles;toesbearinglateral fringes;webbingbasal;discsontoesbroadlyexpanded, truncate,slightlysmallerthanthoseonfingers;toeswith ventralpadswelldefinedbycircumferentialgrooves;relative lengthoftoesI ,II , III , V , IV;ToeVmuchlonger thanToeIII(tipofToeIIIextendsbeyondproximaledgeof penultimatesubarticulartubercleonToeIV,tipofToeV extendsbeyonddistaledgeofdistalsubarticulartubercleon ToeIV).
Colorationofholotype.—Inlife(Fig.15):dorsum reddishbrownwithsomedarkmarkingsontheback;flanks pinkishgreyandgreenandwithwide,darktransversebars; dorsalsurfacesofarmsandhindlimbswithdarktransverse bars;largeareasofdorsalsurfacesofthighs,groin,and concealedlimbsurfacesreddishbrown;headwithblack interorbitalmarking,whitishyellowsnout,darklabialbars, andblacksupratympanicstripes;venter,throat,andventral surfacesofhindlimbsandarmspinkishgraywithblackish markings;irisbronzewithfineblackreticulationsanda median,wide,horizontaldarkredstreak.
Inpreservative(Fig.16):dorsumbrownishgraywithdark brownbars;flanks,dorsalsurfacesofarms,andhindlimbs brownishgraywithdarktransversebars;groinanddorsal surfacesofthighsyellowishorange;headwithdark interorbitalmarkings,whitishyellowsnout,darklabialbars anddarksupratympanicstripes;venter,ventralsurfacesof arms,hindlimbs,andthroatwhitishyellow,withdark brownishmarkings.
Measurementsofholotype(inmm).—SVL22.3;head width9.0;headlength7.3;interorbitaldistance2.5; internarialdistance2.1;uppereyelidwidth2.0;eyediameter 2.6;END2.1;thighlength10.8;tibialength11.4;footlength 10.8;handlength6.4.
Bodymassofholotype.—1.06g.
Variation.—MorphometricvariationisshowninTable4. Noneoftheparatypeshadsomuchoftheirdorsumcolored inreddishbrownasinthecaseoftheholotype.Also,inthe caseoftheparatypes,thedarkbrownmarkingsonthe dorsum,flanks,anddorsalsurfacesofarmsandofhindlimbs weremoreevident(contrasting)thanintheholotype.The paratypeMUTPL1178wastheonlyonethatdidnothavea whitishyellowsnout(Figs.18A,D).
Advertisementcall.—Unknown.
Etymology.—Thespecificname numbala isanounin appositionandreferstoReservaNumbala,animportant privateprotectedareamanagedbytheNGONaturalezay CulturaInternacional.Thereservewasestablishedin2006, withthemainaimofconservingthelastremnantsof romerillo( Retrophyllumrospigliosii and Prumnopitys harmsiana,Podocarpaceae)forests,whicharerepresented herebysomeofthelast,gianttreesofthesespecies.The reserveprotects1,800haofsubpa ´ ramoandmontanecloud forestandishometoanimportantdiversityofbirds, amphibians,mammals,andplants.Itislocatedbetweenthe
twoisolatedextensionsofthesouthernpartofParque NacionalPodocarpus,guaranteeingtheconnectivityneeded forthepreservationofthebiologicaldiversityofthenational parkanditsareaofinfluence.
CommonEnglishname.—NumbalaRainFrog.
CommonSpanishname.—Cut´ındeNumbala.
Distribution. Pristimantisnumbala isknownonlyfrom ReservaNumbalaanditsimmediateproximity(Fig.2). Specimenswereencounteredatanaltitudinalrangebetween 2,860and2,880minasubpa ´ ramoecosystem(Homeieretal. 2008).
Naturalhistory.—Thisisanuncommonspecies.However,thisisanincompleteassessmentbecausewewereable tocarryoutonlyfourcollectingexpeditionsinthereserve andweencounteredthespeciesonlyduringoneofthefield trips.Wefoundmerelyacoupleofindividuals,allfemales. Theanimalswereencounteredduringthenight,perchingon moss-coveredbranchesorleaves,fromthesoilupto1m high,inhabitatswithshrubbyvegetation.Noindividualwas encounteredinsidebromelia ds.Sympatricfrogspecies include Pristimantisandinognomus, P.atratus, P.cryptome-
las (Lynch1979), P.versicolor,andtwoundescribedspecies of Pristimantis Conservationstatus. Pristimantisnumbala iscurrently knownonlyfromonelocalitywithaverysmallestimated areaofabout300m2.Becausewelacksufficientdatato makeanassessment,weconsiderthisspeciestobeData DeficientfollowingtheIUCNcriteria.
DISCUSSION
Overviewofthe Pristimantisorestes Speciesgroup
Thedescriptionofthesethreenewspeciesbringsthe totalnumberofspeciesinthe P.orestes groupto17,withan additionalfourspeciesthatarecurrentlyundescribed(Fig. 3).Additionally,Britoetal.(2017a)identifiedsome morphologicalsimilaritiesbetween P.muranunka and P. proserpens (Lynch1979), P.paquishae Britoetal.2014,and P.tinajillas Urgil ´ esetal.2014.Basedontheirmorphology, thesespeciescouldbelongtothisgroup;however,because noreliablemorphologicalsynapomorphiesareknownforthe P.orestes group,futureworkisneededtoclarifytheir phylogeneticrelationshipsanddeterminetheirassociation withthegroup.Nonetheless,thereremainmanymore speciesfromthisgroupthatarecurrentlylackingformal description(atleast15species;PS,personalobservation). Thankstotheworkpublishedduringthelast6yr,wehavea clearerimageregardingthespeciescompositionofthe P. orestes groupincentralandsouthernEcuador;allthe currentlyknownspeciesbeingdistributedinAndean montaneforests,subpa ´ ramos,andpa ´ ramos.
Unfortunately,thesituationofthePeruvianspeciesis unknown,asthereisnotevenonespeciesmolecularly confirmedtobelongtothegroup.Thus,fromthe11 Peruvianspeciesincludedoriginally(Hedgesetal.2008; DuellmanandLehr2009), P.atrabracus (Duellmanand Pramuk1999), P.chimu Lehr2007, P.cordovae (Lehrand Duellman2007), P.corrugatus (Duellmanetal.2006), P. melanogaster, P.pataikos (DuellmanandPramuk1999), P. pinguis (DuellmanandPramuk1999), P.seorsus Lehr2007, P.simonsii, P.stictoboubonus (Duellmanetal.2006),and P. ventriguttatus LehrandKohler2007,DNAsequenceswere availableforonlytwo(P.melanogaster and P.simonsii);their analysisdeterminedtheremovalofthesespeciesfromthe group.In2012,twomorespeciesweretentativelyaddedto thegroup(P.mariaelenae VenegasandDuellman2012and P.stipa VenegasandDuellman2012),buttheyalsolack moleculardata.Thegrouppresenceiscertain,atleastfor northernPeru,closetotheEcuadorianborder.Specimens identifiedas P.colodactylus fromthere(Lynch1979;Lehr andDuellman2007)areprobablycloselyrelated,undescribedspecies,because P.colodactylus isendemictoAbra deZamora,muchfurtherinthenorth(Sz ´ ekelyetal.2020). Also, P.muranunka mightbepresentbecausetheknown populationsofthisspeciesareveryclosetothePeruvian border.
Microhabitat–BodyShapeRelationship
Ourresultssuggestthatthearborealspeciesofthe P. orestes and P.simonbolivari subgroupsshareacharacteristic bodyshape,withshortsnouts,robust(chubby)bodies, relativelynarrowheads,proportionatelyshortlimbs,and narrowandroundeddigitaldiscs,asobservedbyLynchand
Duellman(1997)intheiroriginaldiagnosisofthe P.orestes group(Fig.1).Incontrast,thebromeliadspecialistspecies fromthe P.colodactylus subgroup—P.colodactylus, P. muranunka,and P.matildae (Figs.1G–I)—haveastrikingly distinctbodyshape(Sz ´ ekelyetal.2020).Thesespeciesare characterizedbyadifferenthabitus,withlongersnouts, slender(compressed)bodies,andflatterheads(Figs.1,4,5; Tables1and2).Hereinweinferthatthedivergencein generalshapeofthe colodactylus lineagesfromthetypical orestes shapeisduetolifeindifferentmicrohabitats,and thatthesebodyfeaturesareprobablytheresultofthe adaptationtothelifeinsidetheoverlappingrosetteofleaves ofthebromeliads.Ontheotherhand,therobust(chubby), short-leggedbodyshapethatcharacterizesthe orestes and simonbolivari subgroupsisconsideredanadaptationtothe lifeinmosslayerandherbaceousvegetationbysomeauthors (Lehretal.2017).
Itisimportanttomentionthatthemeasurementsthatwe usedfortheheightofheadandbodyarenotideal,asthe shapeofthebodycansufferimportantchangesduetothe preservationprocess.Alternative,more-precisemethods, suchaslandmark-basedgeometricmorphometricsor osteologicalmeasurementsonhighresolutionmicrocomputedtomographyscans,shouldbeemployedinorderto confirmourinitialfindings.Also,specimensfromallthe speciesofthegroup(wehaddatafor12ofthe17species) shouldbeincludedinfutu reanalyses,andprobably measurementsonlivesspecimens(orscaled,goodquality photographsoflivespecimens),inordertocorrectlydefine andmeasuretheshapeoftheheadandbody.Nevertheless, ourpreliminaryanalysisconfirmsthatthebromeliad specialistspecieshaveflatter(compressed)bodyplanes comparedwiththearborealspeciesofthegroup.
Althoughphylogeneticallycloselyrelatedwiththem,the threenewarborealspeciesdescribedherearemorphologicallydissimilarfromthebromeliadspecialistsisterspecies, havingless-compressedheadsandbodies,andtheydisplay evidentmarkingsonthighs,groin,andconcealedlimb surfaces(Fig.1;Table2).Ontheotherhand,thenew speciesseemtobedifferentalsofromallthespeciesofthe P. orestes and P.simonbolivari subgroups,mainlybythelonger snouts(acharacterthatissharedwiththeirsisterbromeliad species)andlongerfingersandtoes(Table2),having somewhatintermediarybodyshapes(Fig.1).
CrypticDiversityinSouthernEcuador
Theresultsofthe2021EcuadorianamphibianRedList assessment(Ortega-Andradeetal.2021)showedthat57%of thespecies(363speciesoutofthe635assessed)are threatened,themaincausesbeinghabitatloss,theexpansion oftheagricultural/cattleraisingfrontier,andotheranthropogenicthreats(roads,humansettlements,andmining/oil activities),aswellaspotentialsynergiceffectswithclimate changeandemergentdiseases.Also,mostthreatenedspecies werefoundtobeinAndeanmontaneforestandpa ´ ramo ecosystems,withnearly10%ofthemlocatedoutside protectedareas.Theauthorsoftheassessmenthighlighted thattheincompletetaxonomicdelimitation,especiallyin megadiversecountrieslikeEcuador,hasthepotentialto seriouslyimpactamphibianconservation.Widelydistributed speciescomplexes,whichareoftenassessedasLeast Concern,sometimesincludecryptictaxathatmightbe
facingparticularconservationthreats(Ortega-Andradeetal. 2021).Inthiscontext,itisespeciallyimportanttoincrease theresearcheffortstowardthedescriptionofnewspecies, andparticularlydelimitcrypticspecies,inordertocorrectly evaluateextinctionrisksandimplementadequateconservationactions.
Thehigh-altitudeAndesarecharacterizedbyhighlevels ofendemismandspeciationforanuranfauna,despitea relativelylowspeciesrichness(BernalandLynch2008; Garcia-Retal.2014;Guarnizoetal.2015;Mendozaetal. 2015).Proposedmaindriversforthishighdiversification rateinthe Pristimantis arerelatedtolowdispersionabilities, ahighdegreeofspecializationtomicrohabitats,and ecologicalopportunitiesduetoabsenceofotherlineages (Mendozaetal.2015;Hutteretal.2017).The P.orestes speciesgroupevolutioninourstudyareaissuchanexample: atleastninespeciesandfourpotentialnewspeciesare presenthere,takingintoaccountonlythesamplestaken fromafewlocalities,fromamountainrangethathasless than50kminastraightline(fromAbradeZamorato ReservaTapichalaca)andataltitudesbetween2,700–3,300 m.
Theamphibianspeciesthatinhabitthemountainrange fromAbradeZamoratoReservaTapichalacaexhibita particularpatternintheirdistribution(Fig.2).This mountainrangerepresentsthewesternlimitsofParque NacionalPodocarpusandconsistsofevergreenupper montaneforest(onaltitudesusuallybetween2,100to 2,700m)andsubpa ´ ramoorevergreenelfinforest(onthe crest,between2,700and3,700m;Homeieretal.2008).The crestheightsfluctuatebetween2,700to3,700m,withan averageofaround3,200m,withitshighestaltitudesbeing encounteredtowardthesouthernhalfofthedistance, nearbythefamouscomplexofaround50glaciallakesknown asLagunasdelCompadre.
Thus,someofthespeciesencounteredintheAbrade Zamora/Cajanumaareaarereplacedbymorphologicallyand ecologicallysimilar,butgeneticallydifferent,speciesinthe CerroToledosectorofthenationalpark.Thisisthecaseof Pristimantisandinogigas Ya ´ nez-Mu nozetal.2019, P. sagedunneae,and Lynchiusflavomaculatus (Parker1938) fromAbra/Cajanumaand P.chomskyi Pa ´ ezandRon2019, P.paladines,andanundescribedspeciesof Lynchius (Sz ´ ekelyetal.2020)fromCerroToledo,respectively. However,wehaveadditionalevidencethatthesame situationappliestoseveralundescribedspeciesof Pristimantis (threeofthemfromthe P.orestes speciesgroup),and probableisthecaseof P.andinognomus and P.versicolor, whichneedcarefulrevisions(Sz ´ ekelyetal.2020).Inthese cases,thegeneticdistancevariesbetween2%and3%,which islowerthantheconservative3%geneticdistancethreshold proposedtoidentifycandidatespeciesinanurans(Fouquet etal.2007;Vieitesetal.2009).However,ampleevidence producedinrecentyearsconfirmsthatthisisanoverly prudentlimit(CaminerandRon2014;Guayasaminetal. 2019),especiallyinyoungevolutionaryradiationssuchas Pristimantis (Garcia-Retal.2014;Ortega-Andradeetal. 2015;Pa ´ ezandRon2019).Moreover,theAndean Pristimantis speciesareknowntohavesmalldistributionranges, andthefewexceptionsmightbeartefactsofincorrect speciesdelimitation,asevidencedbyrecentstudies(Pa ´ ez
andRon2019;Urgil ´ esetal.2019;Sz ´ ekelyetal.2020;Zumel etal.2022).Notallspeciesfromourstudyareafollowthisdistribution pattern.Speciessuchas P.vidua reachtheirsouthernlimit inAbradeZamora/Cajanuma,whileotherssuchas P. balionotus, P.cajanuma, P.colodactylus,or P.percultus are known,fornow,onlyfromthisrelativelysmallarea(Sz ´ ekely etal.2020).Additionally,thedistributionof P.numbala is intriguing,asthisspeciesisgeneticallymoredistantfromthe nearby P.paladines inCerroToledothan P.paladines is fromitssisterspeciesfromfurthernorth, P.sagedunneae (Fig.2).Probablythepopulationof P.numbala fromReserva Numbalarepresentsthespecies’north-westernlimitsand almostreaches P.paladines distributionflanks.Nonetheless, aswelackanydatafrominsidetheParqueNacional Podocarpus(mostlyduetotheinaccessibilityandremotenessofthearea),wedonothavetheinformationtobackup thishypothesis.Itisimperativetocollectadditional information,especiallyfromtheunexploredareassituated betweentheCajanumaandCerroToledosectors,aswellas fromtheremoteareasinsidethenationalpark,inorderto understandthedistributionoftheamphibiansinthis biodiversityhotspot.
Acknowledgments.—Wearegratefulforthehelpandsupportoffered byMinisteriodelAmbiente,AguayTransici ´ onEcol ´ ogica,Zona7,andfor theissuedresearchpermits(MAE-DNB-CM-2015-0016,MAAE-ARSFC2020-0727,andMAATE-DBI-CM-2021-0181).Fieldandlaboratorywork wasfundedby:CriticalEcosystemPartnershipFund(CEPF;Project: AmphibianConservationintheAbradeZamoraKeyBiodiversityAreaof Ecuador),NaturalezayCulturaInternacional(Projects:ECMCSPAnne Dunne/Anfibios,andSistematizaci ´ ondeunal´ıneadebasebiol ´ ogicaque caractericealaReservaNumbala,enelmarcodelProyectoExpansionof theNumbalaCloudForestReserve),andUniversidadT ´ ecnicaParticularde Loja(Project:Descripci ´ ondenuevasespeciesdeanfibiosyreptilesdelsur deEcuadorapartirdelascoleccionesdelMuseodeZoolog´ıa,UTPL).We thankallinstitutionsfortheirtrustandinvaluablehelp.Projectno. KH130360hasbeenimplementedwiththesupportprovidedfromthe NationalResearch,DevelopmentandInnovationFundofHungary, financedunderthe(KH_18)fundingschemetoJV.Weareindebtedto allpersonsandinstitutionsthathavehelpedwiththiswork:O.Peralta,J.C. Ortega,E.Armijos-Cano,andtheotherrangersofParqueNacional Podocarpus(MAATE),P.Paladines,F.Rodas,F.Serrano(NCI),Jocotoco Foundation,andA.Dunne.ThankstoA.Gordillo ‘‘DonPolo’’ andA. GordilloJr.fortheirimportanthelpduringfieldworkinReservaNumbala. WethankJ.C.Sa ´ nchez-NivicelaandV.L.Urgilesforsharingtheirdatawith us.SpecialthankstoS.G ´ omez-Eras,A.Ochoa-Carrillo,M.AngamarcaGuartan,D.Hualpa-Vega,M.C ´ ordova-D´ıaz,andL.Carri ´ onforhelpduring fieldwork.Finally,wewanttothankthetwoanonymousreviewersfortheir usefulcommentsandsuggestionsthatimprovedourmanuscript.
SUPPLEMENTAL MATERIAL
Supplementalmaterialassociatedwiththisarticlecanbe foundonlineathttps://doi.org/10.1655/ HERPMONOGRAPHS-D-22-00002.1.S1andhttps://doi. org/10.1655/HERPMONOGRAPHS-D-22-00002.1.S2.
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Acceptedon 17December2022
ZooBank.orgregistrationLSID: 8708D1C7-8AA7-4F0A-B32F-
48993C149D3B
Publishedon 10April2023
APPENDIX I
AdditionalSpecimensExamined
Pristimantisandinognomus (25).—ECUADOR:ZAMORA CHINCHIPE : ReservaTapichalaca(QCAZ26964,26965,27033,29248,35374,35375, 35377,35378,45614,45621,45622,45623,45656,45659,45661,45662; MUTPL338–340,743,744);ParqueNacionalPodocarpusnearbyReserva Tapichalaca(MUTPL1153,1157);ReservaNumbala(MUTPL1170,1173).
Pristimantis aff. andinognomus (24).—ECUADOR:LOJA:Abrade Zamora(MUTPL206–209);BosqueProtectorWashapamba(MUTPL 157–162,167);Huacapamba(MUTPL384,386,387);ParqueNacional Podocarpus-Cajanuma(MUTPL348–351;1097);ParqueNacional Podocarpus-ElPalto(MUTPL989);ReservaMadrigaldelPodocarpus (MUTPL193–195);SanLucas,Acacana(MUTPL1123).
Pristimantisbambu (29).—ECUADOR:CANAR:LaLibertad,Reserva Mazar(MZUTI3373,3378,3420;QCAZ68126,68127,68131,68133–68143,68145,68147,68151,68154,68161,68329,68335,68342,68374, 68375,68402);ZAMORA CHINCHIPE:Yacuambi,Imbana(QCAZ54356).
Pristimantiscajanuma (15).—ECUADOR:LOJA:ParqueNacionalPodocarpus-Cajanuma(MUTPL343–347,352,353,355,573,583,584,591–594).
Pristimantiscolodactylus (4).—ECUADOR:LOJA:AbradeZamora (MUTPL311,388,675,732).
Pristimantis aff. colodactylus (1).—ECUADOR:ZAMORA CHINCHIPE: ReservaTapichalaca(MUTPL742).
Pristimantismatildae (12).—ECUADOR:LOJA:AbradeZamora(MUTPL 366,394,731,733);ParqueNacionalPodocarpus-Cajanuma(MUTPL360–362,813);ParqueNacionalPodocarpus-ElPalto(MUTPL990,992–994).
Pristimantis aff. matildae (17).—ECUADOR:LOJA:ParqueNacional Podocarpus-CerroToledo(MUTPL527,682–689,817);ZAMORA CHINCHIPE:ParqueNacionalPodocarpus,nearbyReservaTapichalaca (MUTPL1152,1154,1156,1158);ReservaNumbala(MUTPL1192–1194).
Pristimantismazar (37).—ECUADOR:CANAR:ElTambo(QCAZ2569); LaLibertad,ReservaMazar(MZUTI3418,3488;QCAZ27493,27503–27505,27507,27508,27511,27514,27519,27553–27555,27560,27563, 27565,32619,49750,49764,68334,68344,68353,68368,68383,68386, 68387,68391,68392,68394–68397,68405,68409,68413).
Pristimantismuranunka (4).—ECUADOR:ZAMORA CHINCHIPE:Cerro Plateado(MUTPL652);ReservaCerroPlateado(MUTPL605,606,643).
Pristimantis aff. muranunka (1).—ECUADOR:ZAMORA CHINCHIPE: Loyola(MUTPL939).
Pristimantisorestes (8).—ECUADOR:AZUAY:V´ıaSigsig-Gualaquiza (QCAZ40783);BosqueProtectorShincata(MUTPL787,799,800);LOJA: VıaUrdaneta-Tutupali(MUTPL242,248,249,1060).
Pristimantis aff. orestes (50).—ECUADOR:AZUAY:CooperativaBellarica (QCAZ52584);ElGuillan,HaciendadelaUniversidaddelAzuay(QCAZ 59019–59021,61872–61874,61877);EnelcaminoentreGualaceoyPlande Milagro(QCAZ27056);Matanga(QCAZ29134);V´ıaSusudel-Cuenca,a aproximadamente1kmdeSusudel(MZUTI706);Yumate,Shoupshe(QCAZ 46991);8kmsurdeLaPazenPanamericana(QCAZ45935);CANAR:La Libertad,ReservaMazar(QCAZ27482,27498,27513,27559,27563,27606–27608,28136,32611,32613–32615,49753,68129,68358);CHIMBORAZO:Rıo Atillo(QCAZ3341);EL ORO:Chillacocha(QCAZ45093,45096);LOJA: BosqueProtectorWashapamba(QCAZ40804;MUTPL136–142,172); ParqueNacionalPodocarpus(QCAZ4974);Saraguro(QCAZ58567–58570); MORONA SANTIAGO:L´ımiteProvincialentreAzuayyMoronaSantiago,v´ıa Sigsig-Cutchil-Oriente,25kmestedeSigsig(QCAZ3443–3446).
Pristimantisproserpens (1).—ECUADOR:ZAMORA CHINCHIPE:Rio Blanco(MUTPL403).
Pristimantissamaniegoi (9).—ECUADOR:LOJA:AbradeZamora (MUTPL365,660,674,676,777);ParqueNacionalPodocarpus-Cajanuma (MUTPL356–358);ReservaMadrigaldelPodocarpus(MUTPL1075).
Pristimantis aff. samaniegoi (9).—ECUADOR:ZAMORA CHINCHIPE: ReservaNumbala(MUTPL1176,1177,1180–1183,1185–1187).
Pristimantissimonbolivari (40).—ECUADOR:AZUAY:Cuenca,Ba ˜ nos (QCAZ37665–37670);BOLIVAR:BosqueProtectorCashcaTotoras(QCAZ 932,939,942,12932,13755,13759,13763,16819,16823,16830,16832, 16925,25107,25108,25113,25120,25121,25123,30893,35747–35749, 36669,37665–37670,42591,42594,42598,49361);12kmestedeGuaranda, carreteraGuaranda-Riobamba(QCAZ1497).
Pristimantistiktik (9).—ECUADOR:LOJA:V´ıaUrdaneta-Tutupali (MUTPL239,240,245–247,251,252,276,277).
Pristimantisvidua (21).—ECUADOR:LOJA:AbradeZamora(MUTPL 295,296,312,496,501,805);BosqueProtectorWashapamba(MUTPL 143–150,164,173);ParqueNacionalPodocarpus-Cajanuma(MUTPL492, 581,586,590);ReservaMadrigaldelPodocarpus(MUTPL488).
APPENDIX II.—Voucher,GenBankaccessionnumbers,andlocalityforthe Pristimantis specimensusedinthephylogeneticanalysis.Allspecimensare fromEcuador. *IncorrectlylabeledasDHMECN3112inTable1ofBritoetal.(2017a)andinGenBank.Boldlettersmarkthesequencesgeneratedbythe presentstudy;—indicatesmissingdata.
SpeciesVouchernumber
GenBankaccessionno. Locality 12S16SRAG1
P.andinognomus QCAZ45534—KY967669KY967688LOJA,ParqueNacionalPodocarpus,guardian´ıaCajanuma
P.andinognomus QCAZ45661—KY967671KY967690ZAMORA CHINCHIPE,ReservaTapichalaca
P.bambu QCAZ46743—KY967674KY967692CANAR,ReservaMazar
P.bambu QCAZ46744—KY967659KY967693CANAR,ReservaMazar
P.cajanuma MUTPL344MK993331MK604535—LOJA,ParqueNacionalPodocarpus,Cajanuma
P.cajanuma MUTPL345MK993331MK604535MK602184LOJA,ParqueNacionalPodocarpus,Cajanuma
P.colodactylus MUTPL311MT764340MT764804MT810309LOJA,AbradeZamora
P.colodactylus MUTPL388MT778072MT762200MT810310LOJA,AbradeZamora
P.matildae MUTPL360MT778081MT762201MT810319LOJA,ParqueNacionalPodocarpus,Cajanuma
P.matildae MUTPL394MT778084MT762202MT810322LOJA,AbradeZamora
P.mazar QCAZ27559—KY967664KY967683CANAR,ReservaMazar,LaLibertad
P.mazar QCAZ27572JF906315KY967666KY967685CANAR,ReservaMazar,LaLibertad
P.muranunka MEPN14737—KY967661KY967680ZAMORA CHINCHIPE,ReservaCerroPlateado
P.muranunka MUTPL605MT778085MT764807MT810323ZAMORA CHINCHIPE,ReservaCerroPlateado
P.numbala MUTPL1178
OP290789OP290796OP302834 ZAMORA CHINCHIPE,ReservaNumbala
P.numbala MUTPL1190— OP290797OP302835 ZAMORA CHINCHIPE,ReservaNumbala
P.orestes KU218257EF493388EF493388—AZUAY,7kmESigsig
P.orestes MUTPL242MT778087MK604538MK602185LOJA,11kmNEUrdaneta
P.orestes MUTPL248MK993330MK604539MK602186LOJA,11kmNEUrdaneta
P.paladines MUTPL126
P.paladines MUTPL1107
P.paladines MUTPL1161
OP290790OP290798 —LOJA,ParqueNacionalPodocarpus,CerroToledo
OP290791OP290799OP302836 ZAMORA CHINCHIPE,ParqueNacionalPodocarpus,CerroToledo
OP290792OP290800OP302837 ZAMORA CHINCHIPE,ParqueNacionalPodocarpusnearby ReservaTapichalaca
P.quintanai MZUA.AN.1881MK993335MK604541MK602188CANAR,ComunidadGuangras
P.quintanai MZUA.AN.1900MK993336MK604544MK602189CANAR,Llavircay
P.sagedunneae MUTPL500
P.sagedunneae MUTPL597
P.sagedunneae MUTPL1096
OP290793OP290801OP302838 LOJA,AbradeZamora
OP290794OP290802OP302839 LOJA,ParqueNacionalPodocarpus,Cajanuma
OP290795OP290803OP302840 LOJA,AbradeZamora
P.samaniegoi MUTPL357MT778091MT764810MT810328LOJA,ParqueNacionalPodocarpus,Cajanuma
P.samaniegoi MUTPL365MT778092MT764811MT810329LOJA,AbradeZamora
P.saturninoi DHMECN12214MK993328MK604532—MORONA SANTIAGO,ParqueNacionalSangay
P.saturninoi DHMECN12232MK993327MK604533—MORONA SANTIAGO,ParqueNacionalSangay
P.simonbolivari KU218254EF493671EF493671—BOLIVAR,BosqueProtectorCashcaTotoras
P.simonbolivari QCAZ56567—KY967676KY967695BOLIVAR,BosqueProtectorCashcaTotoras
P.tiktik MUTPL239MH668274MH668275MH708575LOJA,21kmEUrdaneta
P.tiktik MUTPL247MH668161MH668276MH708576LOJA,14kmEUrdaneta
P.vidua MUTPL148MT778096MT764812MT810333LOJA,BosqueProtectorWashapamba
P.vidua MUTPL312MT778098MT764815MT810336LOJA,AbradeZamora
P.vidua MUTPL586MT778106MT764823MT810344LOJA,ParqueNacionalPodocarpus,Cajanuma
Pristimantis sp.DHMECN12237MK993329MK604534—MORONA SANTIAGO,ParqueNacionalSangay
Pristimantis sp.DHMECN9656* —KY967658KY967677ZAMORA CHINCHIPE,ReservaTapichalaca
Pristimantis sp.MUTPL939 MZ702105MZ702090MZ706290 ZAMORA CHINCHIPE,Loyola
Pristimantis sp.QCAZ45556—KY967670KY967689LOJA,ParqueNacionalPodocarpus,LagunasdelCompadre
Pristimantis sp.QCAZ56535—KY967675KY967694AZUAY,LagunaPatococha