Using DNA barcoding and GenBank data for west Central African amphibian conservation

38 Pages·2017·8.4 MB·English

RESEARCHARTICLE
How many species and under what names?
Using DNA barcoding and GenBank
data for west Central African amphibian
conservation
JessicaL.Deichmann1*,DanielG.Mulcahy2,HadrienVanthomme1,ElieTobi1,Addison
H.Wynn3,BredaM.Zimkus4,RoyW.McDiarmid5
a1111111111 1 CenterforConservationandSustainability,SmithsonianConservationBiologyInstitute,NationalZoological
Park,Washington,DC,UnitedStatesofAmerica,2 GlobalGenomeInitiative,NationalMuseumofNatural
a1111111111
History,SmithsonianInstitution,Washington,DC,UnitedStatesofAmerica,3 DepartmentofVertebrate
a1111111111
Zoology,DivisionofAmphibiansandReptiles,NationalMuseumofNaturalHistory,SmithsonianInstitution,
a1111111111
Washington,DC,UnitedStatesofAmerica,4 MuseumofComparativeZoology,HarvardUniversity,
a1111111111 Cambridge,MA,UnitedStatesofAmerica,5 USGS,PatuxentWildlifeResearchCenter,NationalMuseumof
NaturalHistory,WashingtonDC,UnitedStatesofAmerica
*[email protected]
OPENACCESS
Abstract
Citation:DeichmannJL,MulcahyDG,Vanthomme
H,TobiE,WynnAH,ZimkusBM,etal.(2017)How
DevelopmentprojectsinwestCentralAfricaareproceedingatanunprecedentedrate,often
manyspeciesandunderwhatnames?UsingDNA
barcodingandGenBankdataforwestCentral withlittleconcernfortheireffectsonbiodiversity.Inanattempttobetterunderstandpotential
Africanamphibianconservation.PLoSONE12(11): impactsofaroaddevelopmentprojectontheanuranamphibiancommunity,weconducted
e0187283.https://doi.org/10.1371/journal.
abiodiversityassessmentemployingmultiplemethodologies(visualencountertransects,
pone.0187283
auditorysurveys,leaflitterplotsandpitfalltraps)toinventoryspeciespriortoconstructionof
Editor:StefanLo¨tters,UniversitatTrier,GERMANY
anewroadwithinthebufferzoneofMoukalaba-DoudouNationalPark,Gabon.Becauseof
Received:November15,2016 difficultiesinmorphologicalidentificationandtaxonomicuncertaintyofamphibianspecies
Accepted:September6,2017 observedinthearea,weintegratedaDNAbarcodinganalysisintotheprojecttoimprove
theoverallqualityandaccuracyofthespeciesinventory.Basedonmorphologyalone,48
Published:November13,2017
specieswererecognizedinthefieldandvoucherspecimensofeachwerecollected.We
Copyright:Thisisanopenaccessarticle,freeofall
usedtissuesamplesfromspecimenscollectedatourfieldsite,materialavailablefrom
copyright,andmaybefreelyreproduced,
distributed,transmitted,modified,builtupon,or amphibianscollectedinotherpartsofGabonandtheRepublicofCongotoinitiateaDNA
otherwiseusedbyanyoneforanylawfulpurpose. barcodelibraryforwestCentralAfricanamphibians.Wethencomparedoursequenceswith
TheworkismadeavailableundertheCreative
materialinGenBankforthegenerarecordedatthestudysitetoassistinidentifications.The
CommonsCC0publicdomaindedication.
resultingCOIand16Sbarcodelibraryallowedustoupdatethenumberofspeciesdocu-
DataAvailabilityStatement:16SandCOI
mentedatthestudysiteto28,therebyprovidingamoreaccurateassessmentofdiversity
sequenceshavebeendepositedinGenBankunder
anddistributions.WecautionthatbecausesequencedatamaintainedinGenBankareoften
theaccessionnumbersKY079387-KY080438.All
COIand16Srecordshavealsobeendepositedin poorlycuratedbytheoriginalsubmittersandcannotbeamendedbythird-parties,these
BOLD(ProcessIDs:WAFRA001-14toWAFRA542- datahavelimitedutilityforidentificationpurposes.Nevertheless,theuseofDNAbarcoding
14;WestCentralAfricanamphibianbarcodelibrary
islikelytobenefitbiodiversityinventoriesandlong-termmonitoring,particularlyfortaxathat
forbiodiversity).
canbedifficulttoidentifybasedonmorphologyalone;likewise,inventoryandmonitoring
Funding:ShellGabonfundedthefieldworkinthe
programscancontributeinvaluabledatatotheDNAbarcodelibraryandthetaxonomy
LMRstudyarea.TheSmithsonianInstitution’s
ofcomplexgroups.Ourmethodsprovideanexampleofhownon-taxonomistsand
DNABarcodeNetworkprovidedfundingforall
laboratorywork.Thefundershadnoroleinstudy
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DNAbarcodingforAfricanamphibianconservation
design,datacollectionandanalysis,decisionto parataxonomistsworkinginunderstudiedpartsoftheworldwithlimitedgeographicsam-
publish,orpreparationofthemanuscript.The plingandcomparativemorphologicalmaterialcanuseDNAbarcodingandpubliclyavailable
SmithsonianConservationBiologyInstituteandthe
sequencedata(GenBank)torapidlyidentifythenumberofspeciesandassigntentative
NationalMuseumofNaturalHistory’sLaboratories
ofAnalyticalBiologyprovidedin-kindsupport. namestoaidinurgentconservationmanagementactionsandcontributetotaxonomic
resolution.
Competinginterests:Whilethefieldwork
presentedinthispaperwasfundedbyShellGabon
(listedinthefinancialdisclosuresection),
representingprivatesectorfunds,theSmithsonian
Institutionmaintainsallintellectualpropertyand
rightsindata,andwedeclarenocompeting
interestsexist. Introduction
Specieslistsarenecessaryforconservationplanningandmanagement[1].Assuch,conserva-
tionbiologistsandecologistsmustprovidetaxonomicinformationtodecision-makersfor
conservationeffortstomoveforward.Unfortunately,taxonomyhasnotkeptpacewithhabitat
lossinhighbiodiversityareas[2],andacommoncriticismofstudiesinconservationbiology
andecologyarethattheylacktaxonomicrigor[2,3].Putsimply,thereisanurgentneedfor
taxonomicnamesandcountsofspecies,butassigningdefinitivenamesanddistinguishing
closelyrelatedspeciesisproblematic,moresoforsomegroupsthanothers.
InCentralAfrica,asinmanyotherpartsoftheworld,economicdevelopmentisputting
increasedpressureonbiodiversity[4].Expansionoflogging[5],roaddevelopment[6],agri-
culture[7],oilandgasdevelopment[8],andotherformsofenergydevelopmentarecausing
changesinlandusepatternsandconsequentlyaffectingbioticcommunities.Spendingon
infrastructureinAfricabetween1998and2007increasedannuallyby17percentandthat
growthisexpectedtoaccelerate[9].Itisestimatedthatupto$450billionwillbeinvestedin
energyinfrastructuredevelopmentandproductioninsub-SaharanAfricaoverthenextfew
decades[10].Consequently,EnvironmentalImpactAssessments(EIAs)areroutinelybeing
carriedoutinmanyAfricannationstoevaluatepotentialeffectsofvariousproposeddevelop-
mentprojectsontheenvironment.
Expectedchangesinlandusecombinedwithprojectedclimatechangearelikelytohave
particularlynegativeconsequencesfortropicalAfricanamphibianbiodiversity[11].Amphibi-
ansplayacrucialroleinproperlyfunctioningecosystems,contributingtonutrientcycling,
bioturbation,energyflow,foodwebs,andotherecosystemdynamics[12,13].Theseanimals
provideadditionalecosystemservicesvaluabletohumans,suchasregulatingpests,actingasa
foodsource,functioningasmodelsformedicalresearchandservingassubjectsofrecreational
andspiritualimportancethatvaryacrosscultures[13,14].Giventheircriticalimportancein
functionalecosystems,maintenanceofamphibiandiversityinthefaceofanthropogenic
changeisessential.However,inordertobeabletomaintainamphibiandiversity,wemust
firstaccuratelymeasureitsothatchangescanbedocumentedandrestorationmeasuresimple-
mentedindisturbedsystems.
Unfortunately,theissueofdeterminingthetotalnumberofspeciesofamphibiansina
givenareaisnotasimpletask.Inspiteofthefactthatamphibiansarethemostthreatened
groupofvertebratestobeassessedtodate[15,16],speciesofthisclassarealsoamongthe
mostpoorlyknownvertebrategroupsinmanygeographicareas(e.g.[17]).IntheAfrotropics
inparticular,thelackofbasicknowledgeregardingamphibianspeciestaxonomyandrichness
andhighnumbersofundescribedspeciesmakethetaskofsurveyingandidentifyingamphibi-
ansmoredifficult[18].Althoughrecenttaxonomicfieldguideshaveimproved(e.g.[19]),
anuransincludegroupsrifewithcrypticspecies[20],andotherswithhighlevelsofintraspe-
cificphenotypicpolymorphismexist[21,22].Thesefactorscomplicatenotonlyspecimen
identification,butalsosimpledocumentationofthenumberofspeciesatagivensite.
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DNAbarcodingforAfricanamphibianconservation
Confoundingmattersevenfurther,expertiseinamphibiantaxonomyislimitedgloballyand
tendstoberestrictedtocountrieswithhigheconomicincome[23].Inmanycases,basicbiodi-
versityassessmentsindevelopingcountriesarecarriedoutbyparataxonomists,invaluable
fieldpersonnelchargedwithsortingspecimensintotaxonomicunits,whovaryintrainingand
abilitytoevaluatediagnosticmorphologicaltraits[24–27].Makingmattersworse,published
keysmeanttoaideinidentificationcanbeunclear,confusing,orinneedofimprovement
becausemanyproblematicgroupshavenotbeenworkedouttaxonomically.
Integratingadditionaltoolsintotraditionalmorphologicalspeciesinventoriescancontrib-
uteagreatdealtotheaccuracyandprecisionofspeciesrichnessestimatesinanarea[28–30].
DNAbarcodingisonesuchtoolthatcanbeaddedatrelativelylimitedcostsandpresentsnon-
taxonomistswithawaytoverifyandimproveidentificationsandspeciesdiversityestimates
[31–33].DNAbarcodingislikelytobeaveryusefultoolinspeciesmonitoring,particularlyfor
taxathatcanbedifficulttodistinguishbasedonmorphologyalone(i.e.crypticspecies).In
addition,DNAbarcodingcanalsoreduceerrorsinidentifyingspecieswithhighphenotypic
variabilityandpolymorphisms,whichislessfrequentlydiscussedintheliteraturewhencom-
paredtoitspotentialtoaidincrypticspeciesidentification.
TheadventofDNAbarcodinghasopenedupnewpotentialsolutionstotheissueofcharac-
terizingdiversity;however,thismethodrequirescarefulinterpretationwhenusedtoassign
namestogroupsofsimilarspecimens(i.e.clustersoroperationaltaxonomicunits[OTU])
[34],particularlywithamphibians[35].WhenaDNAbarcodelibraryexistsforcomparisons
ofspecimensfromthefield,itcanbeusedtoidentifyspecimenstoaspecies[36].Infact,the
existenceofDNAbarcodelibrarieshasprovenusefulforavarietyofbiodiversityassessments
(e.g.[32,33,37,38])andecologicalstudies[39,40]representingdifferenttaxonomicgroups.
However,giventhattheintentofDNAbarcodingisnottoreconstructtheevolutionaryhistory
ofalineage[36],theusefulnessofaDNAbarcodelibraryforassigningnamestoOTUsis
heavilydependentontheaccuracyofreferencesequences.Misidentifiedandconsequently
mislabeledspecimensandtheirassociatedsequencedatacanhindertheabilitytoidentify
specimensaccurately.
Herewereporttheresultsofanintegratedamphibiansurveyinanareainsouthwestern
Gabonpriortotheconstructionofaroad.Ourprimarygoalwastodocumentamphibianspe-
ciesrichnesswithinthestudyareasothatappropriateconservationmanagementmeasures
couldbetakenduringandafterdevelopment.WeusetheresultsofourDNAbarcodinganaly-
sestoidentifyspecimens,therebyrefiningourinitialfieldidentificationsbasedonmorphol-
ogy,followedbysystematiccomparisonsofourgeneticmaterialtosequencesinGenBank,
morphologicalcomparisonstoadditionalmaterialsavailableintheNationalMuseumofNatu-
ralHistory(NMNH),andtheprimaryliterature,includingrecentworkandoriginalspecies
descriptions.Bydoingso,wecontributemorewidelytoaDNAbarcodelibraryforamphibians
ofthisregionandsummarizethecurrentknowledgeofwestCentralAfricananurantaxonomy
byincludinggeneticmaterialofspecimensavailabletousattheNMNH.
Wepresentthisasamodelstudyforusebyparataxonomists,ecologistsandconservation
biologistsneedingtorapidlydefinetaxonomyofproblematicgroupsusingDNAbarcoding
andlimitedcomparativematerial.Ourstudydemonstrateshowadditionaltaxonomicexper-
tiseandcomparativematerialcanbeusedforproblematicgroups.Inparallel,weofferadvice
totaxonomistswhohavemorecomparativematerialandadditionalresourcesavailableto
undertakefuturecomprehensivetaxonomictreatmentsofthesegroups.Throughthiswest
CentralAfricancasestudy,weprovidemethodologicalinsightsthat,ifimplemented,will
betterintegratebiodiversityinventoriesandtaxonomyforthebenefitofbiodiversity
conservation.
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DNAbarcodingforAfricanamphibianconservation
Materialsandmethods
Studyarea
ThestudysiteliesalongtheAtlanticcoastofGabonintheNyangaProvinceandincludesa
mosaicofgrassland,secondaryforests,andwetlandscoveringapproximately175km2(Fig1).
Annualrainfallaveragesroughly2,000mm.AshortdryseasonoccursinJanuarywithalonger
dryseasonfromlateMaytoSeptember.Theresearchareaisa52km-longbandcenteredon
theprojectedpathoftheLoubomo-MougagaraRoad(LMR)andextending2–3kmoneither
side.Moukalaba-DoudouNationalParkbordersmostoftheresearchareatothenortheast,
andtheeasternendoftheLMRpathterminatesattheN6road.Theareaisborderedtothe
southandwestbytheAtlanticOcean.In2014(afterbiodiversitysurveys),constructionofthe
two-laneLMRbeganandthelateritephaseoftheroadwascompletedin2016.
Fieldsampling
WesampledamphibiansbetweenMay20–June19,2012(earlydryseason)andMarch13–
April23,2013(shortwetseason2013).Weusedavarietyofsamplingmethods,includingnoc-
turnalanddiurnalvisualandacousticsurveysalong22pre-determinedandrandomlyselected
500mtransectsinallhabitattypes(forests,savannas,swampmargins),pitfalltrapsnearhabi-
tattransitionzones,andopportunisticvisualencounters.
Inthedryseason,theresearchteamconsistedofaparataxonomist(ET)previouslytrained
inamphibiantaxonomy,astudent,andafieldassistant.Inthewetseason,theteamwasjoined
byalabassistantandamoreexperiencedamphibianbiologist(JLD),albeitanoviceinAfrican
amphibiantaxonomy.Weusedpublishedassessmentsandspecieslistsofamphibiansof
Gabon[41–43]andafieldguide[19]toidentifyspecimensinthefield.Voucherswerecol-
lectedforallmorphologicallyrecognizedspecies(morphospecies),andsomewerealsophoto-
graphed.Specimenswereeuthanizedinthefieldusing20%benzocainegel(Oragel)appliedto
theventralsurfaceofthebody[44].Aftereuthanasia,asampleofthighmusclewastakenand
Fig1.Loubomo-MougagaraRoad(LMR)studyareaforamphibianbiodiversityinventoryinNyanga
province,Gabon.
https://doi.org/10.1371/journal.pone.0187283.g001
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DNAbarcodingforAfricanamphibianconservation
thetissuestoredin95%ethanol.Voucherspecimensweresubsequentlypreservedin10%for-
malinandstoredin70%ethanol.SpecimensweredepositedinthecollectionoftheUnited
StatesNationalMuseum(USNM)housedattheNMNH,SmithsonianInstitution,Washing-
ton,DC.Inordertoconfirmspeciesidentificationsmadeinthefield,webarcoded80speci-
mensfromthefirst(dry)seasonand97specimensfromthesecond(wet)samplingseasonfor
atotalof177specimensfromtheLMRstudyarea.
Ethicsstatement
LMRspecimenswerecollectedunderresearchpermitsAR0016/12,AR0004/13and
00170MEFfromtheCentreNationaldelaRechercheScientifiqueetTechnologique,the
AgenceNationaledesParcsNationaux,andtheDirectiondel’Ame´nagementdesAiresProte´-
ge´esofGabon.TheLMRfieldstudywascarriedoutinstrictaccordancewiththeguidelines
setforthbytheAmericanSocietyofIchthyologistsandHerpetologists(ASIH),theHerpetolo-
gists’League(HL),andSocietyfortheStudyofAmphibiansandReptiles(SSAR).TheLMR
specimeneuthanasiamethodhasbeenapprovedbytheaboveinstitutionsaswellastheAmeri-
canVeterinaryMedicalAssociation[44],andalleffortsweremadetominimizesuffering.The
protocolwasapprovedbytheSmithsonianNationalZoologicalPark’sInstitutionalAnimal
CareandUseCommittee(NZP-IACUCpermit#12–13).
DNAbarcodeprotocol
ExtractionsofgenomicDNAwereperformedonanAutoGenprep965(2011AutoGen,Inc.),
usingthemanufacturer’sstandardphenolprotocol.GenomicDNAwaselutedin100μlof
AutoGenR9re–suspensionbuffer.Polymerasechainreactions(PCR)wereconductedforthe
mtDNAlargeribosomalsubunit(rrnL:16S)andcytochromeoxidasesubunitI(COI)using
theprimers:16Sar5' CGCCTGTTTATCAAAAACAT 3'and16Sbr5' CCGGTCTGAACTCA
GATCACGT 3'[45]andCOI–fishCO1F5' TCAACYAATCAYAAAGATATYGGCAC 3'and
fishCO1R5' ACTTCYGGGTGRCCRAARAATCA 3'[46]orChmf45' TYTCWACWAAYCAY
AAAGAYATCGG 3'andChmr45' ACYTCRGGRTGRCCRAARAATCA 3'[47].PCRcondi-
tionswereperformedin10μlreactionsfollowingthe3.6PCRMethods:Amplificationprotocol
ofWeigtetal.[48]withannealingtemperaturesof54˚Cfor16Sand48˚CforCOI.Sequence
reactionswereperformedinbothdirectionswiththePCRprimersusingBigDye1Terminator
v3.1CycleSequencingKit’sin0.25x10μlreactionsandrunonanAutomatedABI3730Se-
quencer(2011LifeTechnologies).RawchromatogramswereeditedinSequencher1v5.1
(2012GeneCodesCorp.),complementarystrandswerealigned,andCOIwasinspectedfor
propertranslation,usingtheDNAbarcodetrimcriteriadescribedinWeigtetal.[48].Align-
mentsforbothgeneswereconductedusingtheMAFFTplug-ininGeneiousv8.1.7with
defaultsettings.Wechosetocharacterizeaportionofthe16Sgeneinadditiontoproducing
COIbarcodesforourspecimensbecauseweanticipatedneedingtocompareourdatatothe
largeamountof16SsequencedataavailableinGenBankforwestCentralAfricananurans.
Additionalreferencematerial
InordertoaidinidentificationofspeciesfromtheLMRstudysite,webarcodedadditionalref-
erencespecimensfromtheUSNMcollection.Weattemptedtosampleallspeciesknownto
occurinGabon,aswellassomemuseumspecimenscollectedinneighboringRepublicof
Congo(RC),forbarcodeanalyses(Fig2).InadditiontothematerialcollectedfromtheLMR
studysite,webarcoded90specimenscollectedpreviouslyfromsitesinEstuaireandOgooue-
MaritimeprovincesinGabon(approximately400and25kmfromtheLMRsite,respectively),
and275amphibianspecimenscollectedfromfoursitesintheRC(Fig2).Allmaterialis
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DNAbarcodingforAfricanamphibianconservation
Fig2.Referencemapshowinglocalitiesofallbarcodedmaterial.Whitecircle:Estuaireprovince(Gabon),
whitesquares:Ogooue-Maritimeprovince(Gabon),whitetriangles:Nyangaprovince(Gabon;LMRstudysite),
blackcircles:Lekoumoudepartment(RepublicofCongo,RC),blacksquares:Likoualadepartment(RC),and
blacktriangles:Pooldepartment(RC).
https://doi.org/10.1371/journal.pone.0187283.g002
catalogedandhousedintheUSNMcollection.Tissuesfromthesespecimenswereinitially
placedin95%ethanolandsubsequentlystoredat-80˚CafterdepositionintheUSNMcollec-
tion.Somevoucherspecimens,includingthosefromtheLMRsite(N=2),otherGabonese
sites(N=3),andCongolesesites(N=7),havebeenlostordestroyed.Forsomeofthosespeci-
mens,photographsserveasthevoucherandarereferredtoasUSNMHerpImageNumber
(N=3);theothertissuesampleswithoutphotographicvouchersarecataloguedasUSNM
HerpTissueNumber(N=9).
Inadditiontousingavailablemuseumspecimens,wealsoqueriedtheBarcodeofLifeData-
base(BOLD)andGenBankforsequencesfromallamphibianspeciesknowntooccurin
region(asof11November2016)andforallgeneraforwhichwegeneratedsequencedatafor
comparativepurposes.
Dataanalysis
ResearchersuseavarietyofmethodsforanalyzingDNAbarcodedata,includingpercent
sequencedivergence[49],neighbor-joining(NJ)trees[36],theBarcodeIndexNumber(BIN)
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DNAbarcodingforAfricanamphibianconservation
System[50],andhaplotypenetworks[51]amongothermethods.Givenourleveloftaxonomic
andgeographicsampling,wechoseavarietyofmethodstoanalyzeourdataincludingpercent
sequencedivergence,neighbor-joiningtrees,BINs,andtheAutomaticBarcodeGapDiscovery
(ABGD)method[52].
ForCOIdata,wefirstusedtheBarcodeIndexNumber(BIN)System[50]generatedin
BOLDforspecimenidentification.However,becausetheavailableDNAbarcodelibraryfor
anuransislimited,wecouldnotrelysolelyonthismethod.Forexample,asearchinGenBank
(Dec.2015)for‘Anura’and‘Africa’reveals170COIrecords(120ofwhichwerefromfrogsof
thegenusXenopus).InadditiontotheBINsmethod,weincorporatedanalternativeassess-
mentofspecimenidentificationusingtheAutomaticBarcodeGapDiscovery(ABGD)method
[52]withourCOIdata.TheABGDmethodusesadjustableparametersforgroupsequences
(weusedPmin-0.001,Pmax=0.1,10steps,X=1.5,andNb=20),ratherthanafixeddiffer-
ence(e.g.>2.2%;BIN).Wedirectlycomparedtheresultsofthesetwomethods,whereasothers
discusstheirdifferencesinmoredetail[52].WealsogeneratedNeighbor–joining(NJ)treesin
PAUP(cid:3)v4.0b10[53]forthe16SandCOIdataseparately,andthetwolociwereconcatenated
forallthesequenceswegeneratedinacombinedMaximumLikelihoodanalysisusingRAxML
[54]andtheGTRGAMMAmodel(partitionedbygene)withsimultaneousfastbootstrap
method(100replicates)andbestMLtreesearch.Wethenconductedneighbor-joiningtree
searchesinPAUP(cid:3)withtheGenBank16Smaterialtoassesssequencesimilarity(seebelow).
WeacknowledgethatNJtreesarenotidealforreconstructingevolutionaryhistory,particu-
larlywiththesmallsizedDNAsequencesofCOIor16S.However,weusethemasausefultool
forclusteringsimilarsequencesofputativemembersofthesamespecies,notforestimating
evolutionaryrelationships.Wedidnotusehaplotypenetworksbecausetheyworkbestwhen
taxonomicandgeographicsamplingaremostcomplete,whichisnotthecaseinourstudy.
Mostofourspecimensperspeciesarefromeffectivelyonlyoneortwolocalitieswhereasthe
specieslikelyoccurallthroughouttheregion.
Taxonomicassignments
Weattemptedtoassigneachspecimentoaputativespeciesgiventheresultsofvariouslinesof
evidence(seebelow).Ifspeciesassignmentwasnotpossible,weassignedatemporaryspecies
name(e.g.sp.A)toagivenspecimenorgroupsofspecimensthatwethinkrepresentthesame
undescribedspecies,i.e.“knownunknowns”[36].Weconsiderthisaprocessof“speciesdis-
covery”[36],wereconservativeinourassignmentsofspecimenstothesecategories,andwefol-
lowedthegenerallineageconceptofspecies[55]usingthecriteriadefinedbelow.
AsaresultofapaucityofexistingrecordsofanuranCOIbarcodesinBOLD,wewere
unabletorelysolelyontheBINsmethodtoverifyidentificationsofourspecimens;therefore,
wedeterminedthenumberofspeciesrepresentedbyourspecimensandtheirtaxonomybased
onmultiplelinesofevidence:1)COIBINscalculatedinBOLDandtheABGDmethod;2)16S
sequencescomparedwithGenBankmaterial;3)traditionalmorphologicalspeciesdescrip-
tions,includingcomparisonstotypedescriptions,assessmentsofproximitytotypelocalities,
andknowngeographicdistributionsoftheputativespeciesinquestion(forfurtherinforma-
tion,seebelow).
COIBINs. IfspecimenswereplacedinthesameCOIBINinBOLDandinasingle
ABGDgroup,theywereconsideredthesamespecies.Ifspecimensinitiallyidentifiedasthe
samespecieswereplacedinseparateBINsorABGDgroups,theywerefurtherevaluated.We
directlycomparedtheresultsoftheABGDmethodwiththeBINs.IftheseparateBINsor
ABGDgroupsincludedspecimensfromdifferentgeographiclocations,andthe16Sdata
placedtheminthesameclade,theyweretreatedasthesamespecieswithgeneticvariation
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DNAbarcodingforAfricanamphibianconservation
assumedtobebasedongeography.Ifspecimensfromthesamelocalitywereplacedinseparate
BINsorABGDgroupsandthe16Sdataplacedtheminthesameclade,theyweretreatedas
thesamespecieswithpopulation-levelgeneticvariation.Ifspecimensfromthesamelocality
wereplacedinseparateBINsorABGDgroupsandthe16Sdataplacedthemindifferent
clades,theyweretreatedasseparatespecies.
16Ssequences. ForthesesamplesweusedcriteriasimilartoourCOIBINsandABGD
groupsmethoddescribedabove,andtreated16ScladessimilartoCOIBINsandABGD
groups.Wealignedallofthe16SsequencesfromGenBankwithoursequencesusingMAFFT
inGeneious(defaultsettings),andgeneratedNJtreesinPAUP(cid:3).Specimensidentifiedasthe
samespeciesandinthesameclade,includingoursequencesandthosefromGenBank,were
treatedasthesamespecies.Specimensidentifiedasthesamespeciesbutindifferentclades
anddifferentlocalitiesbutmonophyletic(i.e.sistertoeachother)weretreatedasgeographic
variationofthesamespeciesiftheyfitthemorphologicaldescriptionofthatspecies(see
below).Iftheydidnotfitthemorphologicaldescriptionofthatspeciesorshowedsubstantial
geneticvariation,theyweretreatedasdifferentspecies.Specimensmorphologicallyidentified
asthesamespeciesbutindifferentclades,sistertootherspeciesweretreatedasdifferent
species.
Morphologicalspeciesdescriptions. Giventhetaxonomicscopeofourproject,thetime-
linessoftheprojectinconjunctionwithspecimenscurrentlyonloantootherresearchers,and
thefactthatmanyGenBankvoucherspecimensaredifficulttotrackdown,wereunableto
examinethemorphologyofallGenBankvouchers.Itislikelythatparataxonomistsworkingin
developingcountrieswouldalsohavedifficultyinsuchanundertaking.Therefore,weoffer
thismorerapidcursurymethodasanintermittentsolutiontothetaskofproperamphibian
taxonomy.Additionally,wecouldnotrelyentirelyonspeciesidentificationsinGenBank,we
realizedinconsistentandinaccurateidentificationscouldaffectallspecimens(fromtheLMR
studysite,referencematerial,andotherGenBanksequences).Accordingly,weattemptedto
verifytheidentityoftheLMRsitespecimensbycomparingthemtomorphologicaldescrip-
tionsofspecies,includingoriginaldescriptionsandsubsequentworks,andtakingintoaccount
thegeographicdistancebetweenourspecimensandtheGenBankrecords,aswellasthe
geneticdistancesbetweensequences.Unfortunately,nomethodoralgorithmtakesallofthese
factorsintoaccounttodeterminespeciesdesignationsautomatically.Therefore,wereliedon
ourtaxonomicexpertiseregardinganurandiversityinthisregion.Insodoing,weattempted
tobeconservative,recognizingwidespread,geneticallyvariablespecies(ratherthanidentifying
eachgeographiccladeasadistinctspecies),andpointoutcaseswhereadditionalsampling,
datacollection,andanalysesareneededtoinvestigatefurtherwhatmayrepresentgreater
diversitythanpreviouslyrecognizedinsuchgroups.
Weacknowledgethatthismethodisoverlysimplistic,andtruespeciesboundariesmaybe
maskedbyfactorssuchashybridization,incompletelineagesorting,andgeographicstructure
inconsistentwithtaxonomy.However,weproposethisasamethodforparataxonomistswork-
ingindevelopingcountriestoobtainaccuratespeciesinventories,whichmayalsobridgecon-
servationecologistsandtaxonomistsinfurthersortingoutwestCentralAfricanamphibian
taxonomythroughsupplementedgeographicandtaxonomiccoverage(demonstratedon
amphibianshere,butmayalsobeappliedtoothertaxonomicgroups).
Communityspeciesrichnessanalysis
Usingtheresultsoffield(in-situandmorphologically-based)identificationsandofDNAbar-
codeidentificationsofamphibianspecimenscollectedintheLMRstudyareaduringeachsam-
plingperiod(dryandwetseasons)andoverall,weemployedEstimateS[Version9.1,56]to
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DNAbarcodingforAfricanamphibianconservation
calculatesample-basedrarefactioncurvesforspeciesrichnessandtocalculatethenon-
parametricincidence-basedspeciesrichnessestimator,Chao2[57]foreachidentification
methodandineachseason.Weusedsample-basedratherthanindividual-basedrarefaction
becausereferencematerialwasnotcollectedforallindividualsencounteredinthefield;there-
fore,anyabundance-orindividual-basedrichnessestimateswouldbeinaccurate.Richness
estimatesfortheLMRstudysitepresentedherearebasedsolelyondatafromcollectedmate-
rial.Todeterminestatisticalsignificanceofrarefiedspeciesrichness,wecomparedoverlapof
84%(P=0.05)confidenceintervalsoftheaccumulationcurvesforeachseason[58,59].
Results
Moleculardata
WecharacterizedDNAsequencedatafrom540specimens,including524COIDNAbarcode
sequencesrangingfrom498–654base-pairs(bp)obtainedandplacedin94BINsinBOLDand
85ABGDgroups,and52816Ssequencesrangingfrom493–579bp(Table1).Ourmaterial
includedonecaecilianspecimen(Gymnophiona)and539specimensfrom12families,21gen-
era,and72speciesofanurans.OurconcatenatedalignmentofCOIsequenceswas654bp,and
the16Salignmentwas655bp.Our16SandCOIsequenceshavebeendepositedinGenBank
undertheaccessionnumbersKY079387–KY080438,andweincludedtracefilestoobtainthe
Keyword"Barcode"inGenBankforallCOIsequences.AllCOIand16Srecordshavealso
beendepositedinBOLD(ProcessIDs:WAFRA001-14toWAFRA542-14;WestCentralAfri-
canamphibianbarcodelibraryforbiodiversity).Ourconcatenated(COIand16S)maximum-
likelihoodphylogenyoftheDNAbarcodedspecimensshowsfamiliesobservedatourstudy
siteandadditionalfamiliessampledinourreferencematerialfromelsewhereinGabonand
theRC(Fig3).AlistofspeciesidentifiedattheLMRstudysite,elsewhereinGabon,andfrom
theRCisshowninTable2alongwithadirectcomparisonofBINsandABGDgroups.Alldis-
crepanciesbetweentheBINsandABGDgroupsarerestrictedtoinstanceswhereasinglespe-
ciesconsistedofmultipleBINs.Specieswereplacedinto2–3BINs,butonlyinoneABGD
groupinsevencases;twooftheseoccurredwheretherewere3BINsperspecies(Hyperolius
olivaceusandH.adspersus).Inonecase,PhyrnobatrachusaurituswasplacedinthreeBINsand
twoABGDgroups.ThesecasesresolvethedifferencebetweenthenumberofBINsandABGD
groups.Interestingly,eightcasesoccurredwherespeciesputintomultipleBINswereequally
dividedamongABGDgroups(Table2).Inallothercases,thetwomethodsagreed.Belowwe
presentspeciesaccountsforspecimensidentifiedinourstudy,organizedtaxonomicallyby
orderandfamily.TheassociatedNJtreescanbefoundinSupportingInformation(S1–S9
Figs).WediscussrelevantGenBanksequencesthatarepotentiallymisidentified.
Table1. NumberofsampledspecimensandCOIand16Ssequencessuccessfullyamplifiedfromspecimensateachcollectionsite.
CollectionLocality TotalNumberofSpecimens NumberofCOISequences Numberof16SSequences
LMRstudysite(Season1) 79 79 79
LMRstudysite(Season2) 97 93 95
Gabon(Estuaire) 33 31 33
Gabon(Ogooue-Maritime) 57 56 57
RepublicofCongo(Kouilou) 7 7 7
RepublicofCongo(Lekoumou) 123 117 116
RepublicofCongo(Likouala) 73 72 70
RepublicofCongo(Pool) 71 69 71
TOTAL 540 524 528
https://doi.org/10.1371/journal.pone.0187283.t001
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DNAbarcodingforAfricanamphibianconservation
Dermophiidae
Geotrypetesseraphini(Dume´ril,1859). Wesequencedonespecimen(USNM576262)
fromMayongongo,RC.The16Ssequenceisa99%matchtoaG.seraphinispecimencollected
inGabon(FMNH256782),usedinbothFrostetal.(DQ283337[60])andRoelantsandBos-
suyt(AY523754[61]).Our16Ssequenceis97%similartoaG.seraphinispecimen(BMNH
2005.2)fromCameroon[62].ThoughourCOIsequencewas94%similartotheBMNHspeci-
men,itwasplacedinaseparateCOIBINandABDGgroup.
Arthroleptidae(S1Fig)
Arthroleptis. SixspeciesofArthroleptisareknowntooccurinGabon[42,63–65],
although10differentmorphospecieswereidentifiedduringfieldworkattheLMRsite.Seven
Fig3.Maximum-likelihoodphylogenyofthecombined16SandCOIDNAbarcodedataforallspecimenssequencedforthis
study.Familycladesobservedatourstudysitearecolor-codedandshowarepresentativeimage.Thefourfamiliesshowningrey
(includingthecaecilian)werenotobservedattheLoubomo-MougagaraRoadstudysitebutwereincludedasreferencematerialfrom
othersitesinGabonortheRepublicofCongo.
https://doi.org/10.1371/journal.pone.0187283.g003
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