Integrative Taxonomic Approach for Describing a New Cryptic Species of Bush Frog (Raorchestes

21 Pages·2016·6.37 MB·English

RESEARCHARTICLE
Integrative Taxonomic Approach for
Describing a New Cryptic Species of Bush
Raorchestes
Frog ( : Anura: Rhacophoridae)
from the Western Ghats, India
H.Priti1,2☯,RekhaSarmaRoshmi1,2,BadrinathRamya3,H.S.Sudhira3,4,G.Ravikanth1,
NeelavaraAnantharamAravind1,KotambyluVasudevaGururaja3,4☯*
1 SuriSehgalCentreforBiodiversityandConservation,RoyalEnclave,AshokaTrustforResearchin
EcologyandtheEnvironment,Sriramapura,JakkurPostOffice,Bengaluru,India,2 ManipalUniversity,
Manipal,India,3 ResearchandDevelopmentCenter,GubbiLabsLLP,IICrossExtension,Gubbi,India,
4 ScienceMediaCenter,GubbiLabsLLP,WS-5,IFloor,EntrepreneurshipCenter,SocietyforInnovation
andDevelopment,IndianInstituteofScienceCampus,Bengaluru,India
☯Theseauthorscontributedequallytothiswork.
*[email protected](KVG)
OPENACCESS
Abstract
Citation:PritiH,RoshmiRS,RamyaB,SudhiraHS,
RavikanthG,AravindNA,etal.(2016)Integrative AnewcrypticspeciesofbushfrogRaorchesteshonnamettisp.nov.isdescribedfromthe
TaxonomicApproachforDescribingaNewCryptic
south-easternpartoftheWesternGhats,India.Thisnewlydescribedspeciesbelongsto
SpeciesofBushFrog(Raorchestes:Anura:
Rhacophoridae)fromtheWesternGhats,India.PLoS theChariuscladeandismorphologicallysimilartootherclademembers—R.chariusandR.
ONE11(3):e0149382.doi:10.1371/journal. griet.Therefore,anintegrativetaxonomicapproachbasedonmolecularandbioacoustic
pone.0149382
analysisalongwithmorphologywasusedtodelimitthenewspecies.Raorchesteshonna-
Editor:UlrichJoger,StateNaturalHistoryMuseum, mettisp.nov.,iscurrentlyknownonlyfromBiligiriRangaswamyTempleTigerReserve,a
GERMANY
partofBiligiriRangaswamyhorstmountainrange(amountainformedduemovementoftwo
Received:June24,2015 faults)formedduringtheLateQuaternaryperiod(1.8–2.58Ma).Discoveryofcrypticspe-
Accepted:February1,2016 ciesfromahighlyspecioseandwell-studiedgenusRaorchesteshintsatthepossibleexis-
tenceofseveralmorecrypticspeciesinthisgenus.Wediscussthepossiblereasonsfor
Published:March2,2016
crypsisandemphasizetheneedforcontinuedsystematicsurveysofamphibiansacross
Copyright:©2016Pritietal.Thisisanopenaccess
theWesternGhats.
articledistributedunderthetermsoftheCreative
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
Introduction
DataAvailabilityStatement:Allrelevantdataare
Crypticspeciesaremorphologicallysimilaranddifficulttodistinguishfromtheirclosestcon-
withinthepaperanditsSupportingInformationfiles.
Allnucleotidesequencefilesareavailablefromthe geners[1].Studieshaveshownthatcrypticspeciesarecommonandmanyofthemgounde-
GenBankdatabase(accessionnumbersKT151650, tectedwhenstudiedsolelyonthebasisofmorphologicalevidence.Inrecentyears,molecular
KT151651,KT151652,KT151653). genetictechniquesandbioacousticshavehelpedinthediscoveryofcrypticanurans[2,3].
Funding:Theauthorsreceivednospecificfunding Identifyingcrypticspeciesisofparamountimportancenotonlyforbiodiversityconservation
forthiswork. butalsotounderstandprocessesofevolutionandbiogeography[1,4].
TheWesternGhatsofIndiaisknownforhighdiversityofamphibianswith220species
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. recorded.Alongwithhighdiversity,manystudieshavealsosuggestedpresenceofhighnumber
PLOSONE|DOI:10.1371/journal.pone.0149382 March2,2016 1/21
NewCrypticBushFrogfromtheWesternGhats
ofcrypticspeciesintheWesternGhats.Usingmoleculartechniques,Nairetal[5]carriedouta
studyonendemicgenusIndirana,anddemonstratedhighcrypticdiversityamongthem.Simi-
larly,twonewgenerainthefamilyRhacophoridaewerediscovered,inacrypticgenusPolype-
dates[6].Usingbioacousticsandmoleculartechniques,crypticspecieswerediscoveredfrom
widelydistributedgeneralikeEuphlyctis(2species,[7])andFejervarya(4species,[8]).
ThegenusRaorchestesbelongstofamilyRhacophoridae.Itincludesbushfrogswithadult
sizerangingfrom10mmto50.5mm.Theyaredistinguishedbythepresenceoftransparent/
translucentvocalsacandtheabsenceofvomerineteeth.Adultsarenocturnalandlackfree
swimminglarvae[9].Thereare59validspeciesinthegenusRaorchestes[10],ofwhich50spe-
ciesarefromtheWesternGhats,4fromEasternHimalaya,1fromEasternGhatsand4species
fromChina,MyanmarandVietnam.Beeetal[11]indicatedthepossibleexistenceofseveral
undescribedcrypticspeciesinthegenusRaorchestes.However,tilldate,thereisnopublished
literatureaboutcrypticspeciesfromthisgenus.Herewedescribeanewcrypticbushfrogspe-
ciesfromsoutheasternregionoftheWesternGhats.Thedescriptionisbasedonanintegrative
taxonomicapproachinvolvingmorphology,moleculartechniquesandbioacoustics.Wedis-
cussthepossiblereasonforcrypticspeciationandtheneedformoredetailedstudiesoncryptic
speciesofWesternGhats.
MaterialsandMethods
Ethicsstatement
FieldworkandsamplingwascarriedoutintheBiligiriRangaswamyTempleTigerReserve
(BRTTR)ofKarnataka,withduepermissionfromtheDirector,BRTTRdated10October
2012.Tissuesamplingwascarriedoutunderthestrictsupervisionoflocalforestersandused
solelyforscientificresearch.Minimumsampleswerecollected(sixindividuals)asrequiredfor
thestudyandthesamplingdonotinanywayaffecteffectthepopulationofthisspecies.Field
PermitissuedbyPrincipalChiefConservatorofForestsandChiefWildlifeWarden,Karnataka
StateForestDepartment.
Nomenclaturalacts
TheelectroniceditionofthisarticleconformstotherequirementsoftheamendedInterna-
tionalCodeofZoologicalNomenclature,andhencethenewnamescontainedhereinareavail-
ableunderthatCodefromtheelectroniceditionofthisarticle.Thispublishedworkandthe
nomenclaturalactsitcontainshavebeenregisteredinZooBank,theonlineregistrationsystem
fortheICZN.TheZooBankLSIDs(LifeScienceIdentifiers)canberesolvedandtheassociated
informationviewedthroughanystandardwebbrowserbyappendingtheLSIDtotheprefix
“http://zoobank.org/”.TheLSIDforthispublicationis:urn:lsid:zoobank.org:pub:93347FEF-
6BF8-40A2-9EEB-2F2C743E70FC.Theelectroniceditionofthisworkwaspublishedinajour-
nalwithanISSN,andhasbeenarchivedandisavailablefromthefollowingdigitalrepositories:
PubMedCentral,LOCKSS.
Studyarea
ThestudywascarriedoutinBRTTR(BiligiriRangaswamyhillrange;latitude:11°–12.15°N,
longitude:77°–77.2667°E,altitude:600–1800mamsl).Specimenswerecollectedfromshola
forests(sholaforestsareatypeofforestinthevalleysofamountainseparatedbymontane
grasslands)inHonnametti(11.8987°N,77.1741°E,1659mamsl)andDoddaSampige
(11.9473°N,77.1836°E,1142mamsl)withinBRTTR(Figs1and2).Thereservegotitsname
“Biligiri”fromthewhitecliffonwhichthetempleofLordRangaswamyissituated.Thistiger
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NewCrypticBushFrogfromtheWesternGhats
Fig1.BiligiriRangaswamyTempleTigerReserveandsamplingsitesofRaorchesteshonnamettisp.nov.(bluecircles).Mapsweregeneratedusing
QGIS1PisaVer.2.10.Datawassourcedfromwww.gadm.orgforadministrativeboundaryofIndiaandShuttleRadarTopographyMission(SRTM)90m
database(http://srtm.csi.cgiar.org)forelevation.
doi:10.1371/journal.pone.0149382.g001
reserveislocatedtotheeastoftheWesternGhatsinKarnatakaState.Thetotalareaofthetiger
reserveis540km2.Thelandscapeisveryheterogeneousandundulating.Theannualrainfall,
receivedmainlyduringthesouthwestmonsoon,rangesfrom600mmatthebaseto3000mm
atthehilltop.Thevariationintemperaturerangesfrom8°Cto25°C.Thewiderangeofcli-
maticconditionsaswellasaltitudinalvariationswithinthesmallareahasresultedinvaried
foresttypeslikescrubforest,drydeciduous,moistdeciduous,riparian,evergreen,sholasand
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NewCrypticBushFrogfromtheWesternGhats
Fig2.TypelocalityofRaorchesteshonnamettisp.nov.–ahighaltitudesholaforestatHonnamettiinBRTTR.
doi:10.1371/journal.pone.0149382.g002
grasslands[12].Theextentofthemajorforesttypesrepresentedaspercentoftotalareaisas
follows:deciduous(moistanddry)=61.1%,scrub=28.2%,grassland=3.4%,evergreen=6.5%
andhighaltitudesholas=0.8%[13].
Specimencollection
SixadultcallingmaleswerecollectedinOctober2012.Specimenscollectedwereeuthanized
andfixedin5%formalinfor24handpreservedin70%alcohol.Thighmuscletissuesamples
forgeneticanalysiswerecollectedbeforefixingthespecimen.Coloroflivespecimensandnat-
uralhistorynoteswererecordedatthetypelocalityduringmultiplevisits.Specimenswere
depositedintheBombayNaturalHistorySociety(BNHS)museum,Mumbai.
Geneticanalysis
ForDNAextraction,wefollowedthemethoddescribedinVencesetal[14].Asmallamountof
thighmuscletissuewasexcisedfromtheholotypeforextraction.ThePCRamplificationand
sequencingof16SrRNAandND1genes(subunitofNADHdehydrogenase)weredonefollow-
ingPalumbietal[15]andBossuytetal[16]respectively.Theamplifiedproductsweresentto
AmnionSequencingservices,Bangalore,India.Thesequenceswerecheckedmanuallyusing
programChromaslite2.01(http://www.technelysium.com.au/chromas_lite.html).The
sequenceswerealignedusingMAFFTalgorithm[17]andmanuallycorrectedinMEGA5.10
software[18]anddepositedinGenBank(Accessionnumbers,KT151650–KT151653).The
finaldatasetconsistedof1032basepairs.16SandND1genesof49validspeciesofthegenus
RaorchestesendemictotheWesternGhats[19,20,21]wereusedinphylogeneticanalysis.
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NewCrypticBushFrogfromtheWesternGhats
GenBankaccessionnumbersfor16SrRNAandND1genesof49validspeciesusedinthe
molecularanalysisaregiveninS1andS2Tables,respectively.Thephylogeneticanalyseswere
performedusingMaximumlikelihood(ML)algorithmandBayesianinferencemethods.The
MLanalysiswasexecutedinraxmlGUIv1.3[22]withGTR+Gmodelselectedasthebest-fit
nucleotidesubstitutionmodelinjModeltest[23]for1000bootstrapreplicates.TheBayesian
analysiswasperformedinMrBayes3.2.4[24].Thecombineddatasetof16SandND1parti-
tionedasseparategenefragmentswereusedfortheanalysiswithTIM2+I+GandTPM3uf+I
+Gselectedasbestfitmodelsfor16SandND1genesrespectivelyinjModeltest.TheMarkov
chainMonteCarloanalysisforthedatasetwasrunfor50milliongenerationsandtreeswere
sampledevery500cycles.Theconvergenceoftherunswasanalyzedbyassessingthesplitfre-
quencystandarddeviations(<0.001)andpotentialscalereductionfactor(PSRF~1.0).The
first10%ofthesampledtreeswerediscardedasburn-inandremainingsampleswereusedto
generatemajorityruleconsensustree.Forestimatingthegeneticdivergence,uncorrectedpair-
wisegeneticdistancebetweenthespecieswascalculatedinMEGA5.10.
Morphology
ThenewspecieswascomparedwithallvalidspeciesofRaorchestesbothfrompublishedlitera-
tureandthroughexaminationoftypespecimensdepositedinIndia(S3Table).Afterthephylo-
geneticanalysis,initialmorphologicalcomparisonsweremadewiththemembersofthe
Chariusclade.Raorchestesthodai[25]fromWesternGhatslackedgeneticdataandwasmor-
phologicallycomparedwiththenewspecies.Similarly,othernon-WesternGhatsspeciesof
Raorchestes,namely,R.terebransfromEasternGhats[26],R.annandalii[27],R.manipurensis
[28],R.sahai[29]andR.shillongensis[30]fromEasternHimalayas;R.gryllus[31],R.long-
chuanensis[32,33,34],R.menglaensis[32,35]andR.parvulus[36]fromMyanmar,Vietnam
andSouth-Chinawerealsocomparedmorphologicallywiththenewspecies.Morphological
comparisonsweremadewithsynonymisedspeciesbasedontheiroriginaldescription,namely,
R.emeraldi[21],R.montanus[37,38,39]andR.neelanethrus[40]toverifytheavailabilityof
1
thosenamestothenewspecies.MorphologicalmeasurementsweretakenusingaMitutoyo
digitalslidecallipertothenearest0.1mm.MeasurementandterminologyfollowsBijuand
Bossuyt[20]andGururajaetal[41].Abbreviationsusedareasfollows:snout–ventlength
(SVL);headwidth,attheangleofthejaws(HW);headlength,fromtherearofthemandibleto
thetipofthesnout(HL);interuppereyelidwidth,i.e.theshortestdistancebetweentheupper
eyelids(IUE);maximumuppereyelidwidth(UEW);snoutlength,measuredfromthetipof
thesnouttotheanteriororbitalborderoftheeye(SL);eyelength,i.e.thehorizontaldistance
betweenthebonyorbitalbordersoftheeye(EL);internarialdistance,i.e.leastdistancebetween
theinnermarginsofnares(IN);nostril–snoutdistance,i.e.distancebetweenmiddleofnostril
andtipofsnout(NS);eyetonostrildistance,i.e.distancebetweenanterior-mostpointofeye
andmiddleofnostril(EN);tympanum–eyedistance,i.e.anteriorrimoftympanumtoposte-
riorofeye(TYE);distancefromtherearofthemandibletothenostril(MN);distancefromthe
rearofthemandibletotheanteriororbitalborderoftheeye(MFE);distancefromtherearof
themandibletotheposteriororbitalborderoftheeye(MBE);distancebetweenanteriorcorner
ofeyes,i.e.theshortestdistancebetweentheanteriororbitalbordersoftheeyes(IFE);distance
betweenposteriorcornerofeyes,i.e.theshortestdistancebetweentheposteriororbitalborders
oftheeyes(IBE);largesttympanumdiameter(TYD);forelimblength,measuredfromthe
elbowtothebaseoftheouterpalmartubercle(FLL);handlength,measuredfromthebaseof
theouterpalmartubercletothetipofthethirdfinger(HAL);thighlength(TL);shanklength
(ShL);footlength,measuredfromthebaseoftheinnermetatarsaltubercletothetipofthe
fourthtoe(FOL);distancefromtheheeltothetipofthefourthtoe(TFOL);discwidthon
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NewCrypticBushFrogfromtheWesternGhats
fingersI,II,IIIandIV(FDI,II,IIIandIV);widthoffingerI,II,IIIandIVmeasuredatthe
baseofthedisc(FWI,II,IIIandIV);lengthsoffingersI,II,IIIandIVmeasuredfrombaseof
proximalsubarticulartubercletofingertip(FLI,II,IIIandIV);tibiawidth,i.e.widthoftibiaat
itswidestregion(TW);discwidthontoesI,II,III,IVandV(TDI,II,III,IVandV);widthof
toesI,II,III,IVandV(ToWI,II,III,IVandV)measuredatthebaseofdisc;lengthoftoesI,
II,III,IVandVmeasuredfrombaseofproximalsubarticulartubercletotipoftoe(ToLI,II,
III,IVandV);lengthofinnermetatarsaltubercle(IMT);distancefromdistaledgeofmetatar-
saltubercletomaximumincurvatureofwebbetweenfourthandfifthtoe(MTFF);distance
fromdistaledgeofmetatarsaltubercletomaximumincurvatureofwebbetweenthirdand
fourthtoe(MTTF);distancefrommaximumincurvatureofwebbetweenfourthandfifthtoe
totipoffourthtoe(FFTF);distancefrommaximumincurvatureofwebbetweenthirdand
fourthtoetotipoffourthtoe(TFTF).Intercalaryossification,whichisthecartilaginousstruc-
turebetweendistalandpenultimatephalangesinfingersandtoeswasobserved.Thepresence
ofanintercalaryossificationcanbenoticedwithoutanatomicalsectioningasaglandularpro-
jectionbetweenphalanges[42].Wecarriedoutlineardiscriminantanalysis(LDA)usingRv.
3.1.3[43]andRStudiov.0.98.1102(http://www.rstudio.com/)onmorphologicaldata.The
datawasconvertedasratioofSVLandtestedfornormality.Eightmorphologicalcharacters
viz.,HW,HL,UEW,SL,HAL,TL,ShLandFOLweresubjectedtoLDAtogetalinearcombi-
nationofthesecharacterstodiscriminateR.charius,R.grietandR.honnametti.Toidentify
multivariatedifferencesamongspeciesMANOVAwasperformedandTukeyHSDpost-hoc
testwasusedforpairwisecomparisonofcharacters.
Advertisementcallrecordingandanalysis
CallrecordingsweremadeusingOlympusstereodigitalvoicerecorder(LS-11)forR.charius
andR.honnametti.CallsofR.grietwererecordedusingCanonEOS600D.Callswithlessback-
groundnoiseweremanuallyselectedfromdifferentindividualcallrecordsandwereanalysed
usingAudacityVer.1.3(Beta)andRavenPro1.5.Nineteencallsfrom3individualsofR.hon-
nametti;20callsfrom4individualsofR.chariusand8callsfrom1individualofR.grietwere
selectedforfurtheranalysis.Duration,inter-callintervalduration,dominantfrequencyand
numberofpulsesofeachcallwasrecorded.CallterminologywasbasedonKokandKalamand-
een[44].AirtemperatureandrelativehumiditywererecordedusingTFAdigitalThermo-
Hygrometer.WeusedWelcht-testinRv.3.1.3tocomparethemeansofcallcharacteristics
betweentwospecies,assumingnormaldistributionofcallsampleswithunequalvarianceand
samplesize.
Results
Geneticanalysis
ThemoleculardataanalysissuggestedthatRaorchesteshonnamettisp.nov.belongedtothe
‘Chariusclade’[21]comprisingofR.chariusandR.grietTheuncorrectedpairwisegeneticdis-
tance(Mean±SD)betweenR.honnamettiandR.chariusis4.14±0.14%andbetweenR.hon-
namettiandR.grietis6.47±0.17%.Fig3showstheresultofMLtreewithastrongbootstrap
support(100%)fortheR.honnamettiandR.charius.Hence,themorphologicalcomparisons
aremadeonlywiththeclosestrelativesofthespecies,i.e.R.chariusandR.griet.Theoverall
geneticdivergenceof50species,includingR.honnamettiforthecombineddatasetof16S
rRNAandND1genesrangesfrom2to32%(S4Table).
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NewCrypticBushFrogfromtheWesternGhats
Fig3.MaximumLikelihoodtreefor50RaorchestesspeciesfromtheWesternGhatsbiodiversityhotspotandanoutgroupRhacophorus
malabaricus.NumbersaboveandbelowthenodesindicateBayesianPosteriorProbabilitiesandMaximumLikelihoodBootstrapvalues>50,respectively.
Asterisk(*)indicatesvalues<50.
doi:10.1371/journal.pone.0149382.g003
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NewCrypticBushFrogfromtheWesternGhats
Raorchesteshonnamettisp.nov.Gururaja,Priti,RoshmiandAravind
urn:lsid:zoobank.org:act:071B913C-BB18-4E6A-97C6-6BA2621F8D6E
Suggestedcommonname. Honnamettibushfrog.
Holotype:BNHS5941,anadultmalecollectedbyauthorsfromStrobilanthusshrubsat
0.48mabovegroundatHonnametti,on13thOctober2012at20:20hfromBiligiriRangaswamy
hills(11.8987°N,77.1741°E,1659mamsl).
Paratypes:BNHS5942,BNHS5943,BNHS5945andBNHS5946,maleindividualscol-
lectedbyauthorsinHonnametti,collectiondateandplacesameasholotype.BNHS5944,a
malecollectedbyauthorson14thOctober2012at19:45hDoddaSampige(11.9473°Nand
77.1836°E,1142mamsl).
Diagnosis:RaorchesteshonnamettibelongstothegenusRaorchestesastheyarerelatively
smallsizedfrogs(15–45mm),activeinnight,vomerineteethabsent,transparent/translucent
vocalsacwhilecallinganddirectdevelopmentwithoutfreeswimmingtadpoles.Itisasmall
sizedadult(male:21.7–24.8mm,n=6);snoutlongerthanthehorizontaldiameterofeye;
groinuniformlightbrownwith3–4yellowblotches;bothanteriorandposteriorpartofthigh
uniformlightbrownwithsmallroundtoovalshapedyellowblotchesandrelativelyshorthind
limbsShL/SVLratio<0.5.ItbelongstotheChariuscladeandmorphologicallysimilartoR.
chariusandR.griet.
Descriptionofholotype:Asmallsizedfrog(SVL=24,Fig4A–4H,Table1,allmeasurements
inmm),headwiderthanlong(HW=8.0;HL=7.4),snoutovalindorsalviewandinprofile
rounded.Snoutlongerthanorequaltoeye(EL=3.2;SL=3.6).Canthusrostralisrounded,
lorealregionslightlyconcave.Interorbitalspaceflat,almostequaltouppereyelidwidthand
internarialdistance(IUE=2.5;UEW=2.1;IN=2.2).Internarialdistancebetweenposterior
marginsofeyes1.7timesthatofanteriormargins(IFE=4.3;IBE=7.2).Nostrilsoval,without
flap,closertotipofsnoutthantoeye(NS=1.4;EN=2.0).Weaksymphysialknob.Pinealocel-
lusabsent.Tympanumindistinct,oval,closertoeye(TYD=1.4),2.3timesineyelength.
Supratympanicfolddistinctfrombackofeyetoshoulder.Mediansub-gularvocalsacwitha
pairofopeningsatthebaseoflowerjaw.Tonguebifid,chordate,sparselygranular.Lingual
papillaabsent.Eyesmoderatelylarge(EL=3.2),protruding,pupilhorizontal.
Forelimblengthshorterthanhandlength(FLL=5.1;HAL=7.1).Relativelengthsoffin-
gersI<II<IV<III(FLI=1.9;FLII=2.4;FLIII=3.2;FLIV=2.5).Fingertipswithwell-devel-
opeddiscs(FDI=0.7,FDII=0.9,FDIII=1.1,FDIV=1.2;FWI=0.6,FWII=0.4,FW
III=0.6,FWIV=0.6)withcircummarginalgrooves,groovesfoldupwards,givingbifid
appearanceindorsalview,intercalaryossificationdistinctbetweenpenultimateanddistalpha-
langes.Dermalfringeweak,onbothsidesofthefingers.Webbingbetweenfingersabsent.Sub-
articulartuberclesdistinct(finger:i=1,ii=1,iii=2,iv=1)roundedandpre–pollextubercle
oval,distinct.Supernumerarytuberclespresent.Nuptialpadabsent.
Hindlimbslong,donotoverlaportouchwhenfoldedatrightanglestobody.Shank4.2
timeslongerthanwide(ShL=10.6;TW=2.5),shorterthanthighlength(TL=11.3)andlon-
gerthanfootlength(FOL=10.0).Heeltotipoffourthtoe(TFOL=16.4)3.1timeslongerthan
fourthtoelength(ToLIV=5.3).RelativetoelengthI<II<III(cid:1)V<IV(ToLI=1.9;ToL
II=2.0,ToLIII=3.2;ToLIV=5.3;ToLV=3.8).Toeswithwelldevelopdiscsattip(TD
I=0.8,TDII=0.8,TDIII=0.8,TDIV=1.0,TDV=1.0;ToWI=0.5,ToWII=0.5,ToW
III=0.6,ToWIV=0.7,ToWV=0.6).Webbingmoderate(MTTF=5.1,MTFF=6.4,
TFTF=5.7,FFTF=4.7).Firsttoe(ToLI=1.9)1.9timesthelengthofinnermetatarsaltubercle
(IMT=1.0).Outermetatarsaltubercleabsent,supernumerarytuberclesandtarsaltubercle
present(toe:i=1,ii=1,iii=2,iv=3,v=2).
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Fig4. Holotype(BNHS5941)ofRaorchesteshonnamettisp.nov.a-b—Livespecimen;c—ventralview;d—dorsalview;e—ventralviewofhindlimb;f—
ventralviewofforelimb;g—lateralprofileofhead;h—Schematicviewofwebbinginhindlimb.
doi:10.1371/journal.pone.0149382.g004
Skin:Entiredorsum,snoutanduppereyelidwithverysmallhornyspines,mid-dorsum
densewithsmallhornyspines;dorsalpartofforelimbs,thighsandshankssmooth;throat,
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NewCrypticBushFrogfromtheWesternGhats
Table1. MorphometricdataofRaorchesteshonnamettisp.nov.(Holotypeandparatypes). Measurementsinmm.
Characters BNHS5941 BNHS5942 BNHS5943 BNHS5944 BNHS5945 BNHS5946 Mean±SD
Holotype Paratype Paratype Paratype Paratype Paratype
Sex Male Male Male Male Male Male
SVL 24.0 24.8 23.9 23.4 25.0 21.7 23.8±1.2
HW 8.00 8.2 8.5 8.4 8.3 8.7 8.4±0.2
HL 7.4 7.2 7.1 7.1 7.1 7.3 7.2±0.1
IUE 2.5 3.2 3.2 3.5 3.2 3.6 3.2±0.4
UEW 2.1 1.9 2.0 2.0 1.8 2.1 2.0±0.1
SL 3.6 3.6 3.7 3.1 3.3 3.9 3.5±0.3
EL 3.2 3.3 3.1 3.1 3.3 2.8 3.1±0.2
MN 5.6 5.9 5.8 5.7 5.9 6.1 5.8±0.2
MFE 6.0 4.6 4.5 4.5 4.3 4.4 4.7±0.6
MBE 2.5 2.1 2.2 2.1 2.2 2.1 2.2±0.2
IN 2.2 2.4 2.4 2.0 2.5 2.4 2.3±0.2
IFE 4.3 4.2 4.0 4.1 4.3 5.3 4.4±0.5
IBE 7.2 7.0 7.4 7.5 7.6 7.9 7.4±0.3
NS 1.4 1.6 1.5 1.3 1.5 1.4 1.5±0.1
EN 2.0 2.2 2.3 1.7 1.9 2.1 2.0±0.2
TYD 1.4 1.7 1.2 1.3 1.6 1.1 1.4±0.2
FLL 5.1 5.1 5.4 5.0 6.1 4.7 5.2±0.5
HAL 7.1 7.6 7.5 6.8 7.1 6.8 7.2±0.3
FD1 0.7 0.7 0.7 0.7 0.7 0.6 0.7±0.0
FD2 0.9 0.9 0.7 0.9 0.9 0.8 0.9±0.1
FD3 1.1 1.3 1.1 1.2 1.0 1.2 1.2±0.1
FD4 1.2 1.1 1.0 1.2 1.1 1.0 1.1±0.1
FW1 0.6 0.5 0.5 0.5 0.4 0.5 0.5±0.1
FW2 0.4 0.5 0.5 0.6 0.6 0.5 0.5±0.1
FW3 0.6 0.5 0.6 0.7 0.7 0.7 0.6±0.1
FW4 0.6 0.6 0.5 0.6 0.7 0.6 0.6±0.1
FIL 1.9 1.9 1.9 1.7 1.7 1.7 1.8±0.1
FIIL 2.4 2.3 2.4 2.3 2.2 2.1 2.3±0.1
FIIIL 3.2 4.4 3.8 3.6 4.5 3.9 3.9±0.5
FIVL 2.3 3.1 3.6 3.1 3.9 3.2 3.2±0.5
FL 11.3 11.5 11.4 10.3 11.9 11.9 11.4±0.6
ShL 10.6 10.9 11.3 10.3 10.9 10.7 10.8±0.3
TW 2.5 1.9 2.6 2.3 2.5 3.1 2.5±0.4
FOL 10.2 10.6 10.9 8.8 10.3 9.8 10.1±0.7
TW1 0.5 0.5 0.5 0.5 0.6 0.6 0.5±0.1
TW2 0.5 0.5 0.5 0.6 0.5 0.6 0.5±0.1
TW3 0.6 0.5 0.6 0.6 0.6 0.6 0.6±0.0
TW4 0.7 0.6 0.6 0.6 0.8 0.7 0.7±0.1
TW5 0.6 0.6 0.7 0.6 0.8 0.8 0.7±0.1
TD1 0.8 0.7 0.7 0.8 0.8 0.6 0.7±0.1
TD2 0.8 0.9 0.8 0.7 0.8 0.7 0.8±0.1
TD3 0.8 0.7 0.9 0.9 0.7 0.7 0.8±0.1
TD4 1.0 1.0 1.0 1.1 0.8 0.9 1.0±0.1
TD5 1.0 1.1 1.0 1.1 0.8 1.0 1.0±0.1
IMT 1.0 1.1 0.9 1. 0.9 0.8 1.0±0.1
TFOL 16.4 11.1 17.5 15.1 16.9 16.2 15.5±2.3
(Continued)
PLOSONE|DOI:10.1371/journal.pone.0149382 March2,2016 10/21