Vast underestimation of Madagascar's biodiversity evidenced by an integrative amphibian inventory

49 Pages·2009·3.35 MB·English

Vast underestimation of Madagascar’s biodiversity
evidenced by an integrative amphibian inventory
DavidR.Vieitesa,1,KatharinaC.Wollenbergb,FrancoAndreonec,Jo¨rnKo¨hlerd,FrankGlawe,andMiguelVencesb
aMuseoNacionaldeCienciasNaturales,ConsejoSuperiordeInvestigacionesCientíficas,c/Jose´GutierrezAbascal2,28006Madrid,Spain;bZoological
Institute,TechnicalUniversityofBraunschweig,Spielmannstrasse8,38106Braunschweig,Germany;cMuseoRegionalediScienzeNaturali,ViaGiolitti36,
10123Turin,Italy;dHessischesLandesmuseumDarmstadt,Friedensplatz1,64283Darmstadt,Germany;andeZoologischeStaatssammlungMu¨nchen,
Mu¨nchhausenstrasse21,81247Munich,Germany
EditedbyDavidB.Wake,UniversityofCalifornia,Berkeley,CA,andapprovedMarch27,2009(receivedforreviewOctober26,2008)
Amphibiansareindeclineworldwide.However,theirpatternsof Amongterrestrialvertebrates,amphibiansarecharacterizedbya
diversity,especiallyinthetropics,arenotwellunderstood,mainly rapidrateofspeciesdiscovery(8,9),withanoverallincreaseinthe
becauseofincompleteinformationontaxonomyanddistribution. number of amphibian species globally of 19.4% during the last
We assess morphological, bioacoustic, and genetic variation of decade, reaching 6,449 currently recognized species (10). An im-
Madagascar’s amphibians, one of the first near-complete taxon portant acceleration in the rate of new discoveries, mainly from
samplingsfromabiodiversityhotspot.BasedonDNAsequencesof tropicalareas,isobviousfrommanyrecentstudies(11–16).These
2,850 specimens sampled from over 170 localities, our analyses discoveriesarenottheresultoftaxonomicinflation(9,14,17),but
revealanextremeproportionofamphibiandiversity,projectingan correspond to real divergent species (18, 19). Although high
almost 2-fold increase in species numbers from the currently numbersofundescribedamphibianshavebeenestimatedtoexistin
described 244 species to a minimum of 373 and up to 465. This poorly studied tropical regions (11, 15), these results remain un-
diversity is widespread geographically and across most major verifiedforcomplete,highlydiverseamphibianfaunas.Inparallel,
phylogenetic lineages except in a few previously well-studied an increase of threatened amphibian species has been reported ON
genera,andisnotrestrictedtomorphologicallycrypticclades.We worldwide(8,20,21).Amphibiansareofhighconservationcon- UTI
classify the genealogical lineages in confirmed and unconfirmed cern,with43%ofspeciesbeinggloballythreatened(20),mostof VOL
candidatespeciesordeeplydivergentconspecificlineagesbased themintropicalregionswithhighamphibiandiversity. E
Madagascar is one of the top priority global hotspots for
onconcordanceofgeneticdivergenceswithothercharacters.This
biodiversityconservation(1),affectedbyahighrateofhabitat
integrative approach may be widely applicable to improve esti-
destruction(22).Itsfaunaandfloraevolvedlargelyinisolation
matesoforganismaldiversity.OurresultssuggestthatinMada-
(23), and many taxa are characterized by a high degree of
gascar the spatial pattern of amphibian richness and endemism
microendemismwithinMadagascar(24–27).Thenativeamphib-
mustberevisited,andcurrenthabitatdestructionmaybeaffecting
ian fauna is constituted by 5 endemic evolutionary lineages of
morespeciesthanpreviouslythought,inamphibiansaswellasin
frogs with 100% species-level endemism, 2 of which (the man-
other animal groups. This case study suggests that worldwide
tellidsandthecophylineandscaphiophryninemicrohylids)are
tropical amphibian diversity is probably underestimated at an
veryspecies-rich.Large-scaletaxonomicinventoriesconducted
unprecedented level and stresses the need for integrated taxo-
since 1991 have led to an increase from 133 to 244 described
nomicsurveysasabasisforprioritizingconservationeffortswithin
species, largely due to the exploration of new areas and the
biodiversityhotspots.
applicationofmoreefficienttechniques.About46specieswere
identified during the 1990s mainly based on bioacoustics; the
biodiversityestimates (cid:1) newcandidatespecies (cid:1) phylogeny (cid:1)
applicationofcombinedmethods,includingmoleculargenetics,
DNAbarcoding
wascrucialinthediscoveryof51newspeciessince2000aswell
asintheresurrectionofspeciesformerlyconsideredtorepresent
The current biodiversity crisis demands the study of broad- synonyms.
scale spatial variation in species richness and endemism to Undescribeddiversitymayhaveanimportantimpactonunder-
identifyareasthatmeritspecialconservationattention.Global standingthespatialpatternsofendemicradiationsontheisland,
efforts to minimize biodiversity loss have led to prioritizing butobjectiveestimatesofspeciesnumbersarenotavailablesofar.
biodiversity hotspots (1) which are defined as areas with high We report a comprehensive assessment of morphological, bio-
concentrations of endemic species and that are undergoing acoustic,andgeneticvariationoftheanuranfaunaofMadagascar.
exceptional habitat loss (2–4). A second step is the efficient Ourgoalsare(i)toprovideareliableestimateoftheproportionof
implementationofconservationmeasuresatalocalscalewhich yet-undescribed amphibian species from Madagascar and their
requires an understanding of spatial patterns of richness and phylogeneticandgeographicdistribution,(ii)todiscusstheimpact
endemism within these hotspots (5). Assessments of such re- ofourfindingsforglobalestimatesofamphibiandiversity,and(iii)
gional priority areas are often hampered by incomplete distri- to propose a novel terminology to be better able to assess the
butionalandtaxonomicinformation.Theoperationalunitsused increasing number of identified but taxonomically undescribed
to assess conservation priority areas are described species, candidatespeciesofanimals.
whereasestimatesofundiscoveredandundescribedspeciesare
usuallyignored.Recently,manypotentialnewspecieshavebeen
Authorcontributions:D.R.V.andM.V.designedresearch;D.R.V.,K.C.W.,F.A.,J.K.,F.G.,and
identified by DNA barcoding, but a taxonomic validation of
M.V.performedresearch;D.R.V.,K.C.W.,J.K.,andM.V.analyzeddata;andD.R.V.andM.V.
these species will outdate the short time span left for efficient wrotethepaper.
biodiversityconservationprioritization,andsofaritisnotclear
Theauthorsdeclarenoconflictofinterest.
how this undescribed diversity can nonetheless be considered.
ThisarticleisaPNASDirectSubmission.
Thisundescribeddiversityalsobearsrelevanceforunderstand-
Datadeposition:ThesequencesreportedinthispaperhavebeendepositedintheGenbank
ingthetreeoflife:Thecompletenessoftaxonsamplingisone database(accessionnos.FJ559069–FJ559372andFJ217329–FJ217345)
of the major prerequisites for reliable phylogenetic analysis, 1Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected].
reconstruction of character evolution, and inference of macro-
This article contains supporting information online at www.pnas.org/cgi/content/full/
evolutionary processes (6, 7). 0810821106/DCSupplemental.
www.pnas.org(cid:2)cgi(cid:2)doi(cid:2)10.1073(cid:2)pnas.0810821106 PNAS (cid:1) May19,2009 (cid:1) vol.106 (cid:1) no.20 (cid:1) 8267–8272
Fig.1. Phylogenetic,geographic,andhistoricalpat-
ternsofundescribedamphibiandiversityinMadagas-
car.(A)Bayesianphylogenetictreeof236(outof244)
describedspeciesand258deeplydivergentgenealog-
icallineagesofMalagasyfrogs(amongthem129CCS
and92UCSand37DCLwith(cid:1)3%geneticdivergence
tonearestdescribedneighbor)basedonafragmentof
the mitochondrial 16S rRNA gene. Circles represent
CCS(brown),UCS(orange),andDCL(lightorange).
Insetphotosshowsomeoftheseforms(seeSIAppen-
dix).He,Heterixalus(Hyperoliidae);Dy,Dyscophus;Sc,
Scaphiophryninae;Co,Cophylinae(Microhylidae);Sp,
Spinomantis; Md, Mantidactylus; Mt, Mantella; Gu,
Guibemantis; Ge, Gephyromantis; Bo, Boophis; Bl,
Blommersia;Ag,Aglyptodactylus(Mantellidae).(Band
C)Mapsofcollectinglocalitiesofdescribedspecies(B)
andofCCSandUCS(C)ofMalagasyfrogs,andremain-
ingprimaryvegetation(evergreenforests,blue;non-
evergreenforests,gray).Currentprotectedareanet-
workisshowninred.(D)Cumulativenumberofspecies
currently considered as valid per 10-year intervals
(2001–2008forthecurrentdecade),andtheCCS,UCS,
andDCLidentifiedinthepresentpaper.(E)Totalnum-
bers of described species and CCS, UCS, and DCL in
majorcladesofMalagasyfrogs.
Results frogs,indicatingthattheyarenottheresultoftaxonomicinflation
Fromourintegrativeanalysesofmorphological,bioacoustic,and (e.g.,elevatingsubspeciestospecificstatus).
geneticdata,weidentifiedmanymorespeciesofMalagasyfrogs Newdiscoveriesarealsogeographicallyubiquitous.Theyoccur
thantherearenamesavailable.Thenumberofdescribedspecies throughout Madagascar, both in poorly explored and in better-
ofMalagasyfrogshasslowlyincreasedduringthe20thcentury, studied areas. Described species are known from 451 sites and
reaching100describedspeciesaround1940,and133atthetime 87.3% are found in protected areas, whereas CCS and UCS are
knownfrom168sitesandonly66.4%arefoundinprotectedareas
of the last monographic account in 1991 (28) (Fig. 1). The
(Fig.1BandC).Evenin2ofthebest-studiedsitesforamphibians
currentnumberof244validnativespeciesindicatesanincrease
in Madagascar, Mantadia/Analamazaotra and Ranomafana Na-
of 83.5% since 1991.
tionalParks,harboringatotalof94and112speciesoffrogs,we
We classify frog lineages from Madagascar in 3 categories
found10and31CCSandUCS(seeSIAppendix).Mostdescribed
(Table 1): (i) confirmed candidate species (CCS) are those
speciesareknownfromoneorafewlocalitiesandhavenotbeen
differing clearly by morphological and bioacoustic characters
foundelsewhere.Thispatternisconsistentamongtheundescribed
andusuallyshowinghighgeneticdifferentiationthatwehypoth-
species;only6ofthe219CCSandUCSarewidespread,whereas
esize are distinct, undescribed species; (ii) unconfirmed candi-
63 are currently known from small ranges and 154 from single
date species (UCS) are deep genealogical lineages (15)—
localities.
bioacoustically and morphologically unstudied and usually
derived from geographically distant populations—for which Discussion
generalindicationsexistthattheyaredistinct,undescribedspecies;
The Concept of Candidate Species and Their Delimitation. By inte-
and (iii) deep conspecific lineages (DCL) are deeply divergent
grating molecular, morphological, and bioacoustic data, we have
genealogicallineages,studiedbutnothavingclearmorphologicalor
identifiedalargenumberofundescribedspeciesofamphibiansin
bioacousticdifferenceswithdescribedspecies.Ourdataprovided
Madagascar. The remarkable increase in estimated species num-
anadditional129CCS,92UCS,and37DCL.IfCCSplusUCSare
bers is independent of the species concept applied. Almost all
assumedtorepresentdistinctundescribedspecies,a90.6%increase
describedspeciesarewelldefinedasreciprocallymonophyleticand
fromthecurrent244to465frogspeciesisprojected.Thisprojection
stronglydifferentiatedunitsbymoleculardatasetsandbymorpho-
wouldconstituteanoverallincreaseof250%since1991.
logicaland/orbioacousticevidence.TheonlyexceptionsareMan-
UndescribeddiversityisphylogeneticallywidespreadinMalagasy tella milotympanum, Mantella nigricans, Dyscophus antongilii, He-
frogs (Fig. 1A and SI Appendix). We found candidate species in terixalus andrakata, Heterixalus variabilis, and Heterixalus tricolor,
most clades except in a few monospecific or species-poor genera which are not clearly diagnosable by molecular data and require
(e.g.,Wakea,Dyscophus).GenerasuchasthecolorfulMantellaor furtherstudy(29).Thesamecombinationofcharactersetsdefines
Heterixalusareratherwellstudiedandconsequentlycontainonlya allCCS(e.g.,Fig.2A),and,basedonourongoingstudies,weexpect
fewcandidatespecies,whereasinthe3mostspecioseanddiverse thesameformostUCS.Manyofthesetaxaoccurinsyntopywith
clades (Mantidactylus, Boophis, and Cophylinae), the number of theirnearestrelativeswithoutanysignalofadmixture,corroborat-
candidate species is close to or even exceeds the number of ingtheirspeciesstatusunderbiological,evolutionary,andphylo-
describedspecies(Fig.1E).Ingeneral,inmostgeneratheaverage genetic species concepts. In contrast, many DCL correspond to
differentiation in the 16S rRNA gene of CCS and UCS is (cid:1)4%. geneticallydifferentiatedallopatricpopulationsofwidespreadspe-
MostdescribedspeciesofMalagasyfrogsshowgeneticdistancesof cies for which the application of a biological species concept is
6–8%totheirclosestrelativesdependingontheperiodofdiscovery inherentlydifficult.IfcriteriasuchasthoseforMalagasyprimates
(9),butthesevaluesrefertodistancescorrectedbytheKimura-2- (30)wereappliedtotheseDCL,manyofthemwouldbeconsidered
parameter-model (K2P) which are higher than the uncorrected evolutionaryspeciesaswell.Becausetheprocessofspeciesforma-
distanceswereport.ThemoleculardifferentiationofCCSandUCS tioninamphibiansisanactiveandcontroversialareaofresearch
isthusatsimilarlevelsasbetweendescribedspeciesofMalagasy (31),wecurrentlydiscouragesuchconclusions.
8268 (cid:1) www.pnas.org(cid:2)cgi(cid:2)doi(cid:2)10.1073(cid:2)pnas.0810821106 Vieitesetal.
Table1.Definitionsofproposedcategoriesofcandidatespecies
Category Generaldefinition DefinitionusedinMalagasyfrogs
UnconfirmedCandidateSpecies(UCS) Defaultcategoryfordeepgenealogicallineagesof Uncorrectedpairwisegeneticdivergencesin16SrRNA
unknownstatus.Thegeneticdifferentiationmustbe gene(cid:1)3%toallotherdescribedspecies.Nodataon
aboveathresholdvaluetypicalforcomparisonsamong morphologyandbioacousticsduetounavailabilityof
closelyrelatedspeciesinthegroupofanimalsunderstudy. voucherspecimensorimmaturestateofvouchers.
Datadeficientformorphology,ecology,anddistribution.
ConfirmedCandidateSpecies(CCS) Specimensorpopulationscharacterizedbyadetectable Uncorrectedpairwisegeneticdivergencesin16SrRNA
geneticdifferentiationtoalldescribedspecies,not genetoallotherdescribedspeciesinmostcases(cid:1)3%,
necessarilyaboveanythreshold,butinconcordancewith sometimesonly1–2%.Concordanceofthismolecular
atleastoneofthefollowingcriteria: differentiationwithoneofthefollowing:
(i)adistinctdifferentiationinacharacterthatmediatesa (i)aqualitativedifferenceinadvertisementcalls,
prematingreproductivebarrier, (ii)diagnosticdifferenceinatleastonemorphological
(ii)adiagnosticmorphologicaldifferenceinacharacter characterknowntobegenerallyspecies-specificin
thatintherespectivegroupofanimalsisknowntobeof Malagasyfrogs:e.g.,presenceandextentofdermal
lowintraspecificvariabilityandofhighvalueto spines,ridgesandtubercles,extensionofterminalfinger
discriminatespecies, tips,morphologyoffemoralglands,relativetympanum
(iii)sympatricoccurrencewithoutadmixture,andwithat size,iriscolor,ortadpolemouthparts.
leastonephenotypiccharacterstatedifference,evenif
subtle,strictlycorrelatedtothegenealogyinferredfroma
neutralmolecularmarker.
DeepConspecificLineage(DCL) Deepgenealogicallineagesaboveathresholdvalue Uncorrectedpairwisegeneticdivergencesin16SrRNA
typicalforcomparisonsamongcloselyrelatedspeciesin gene(cid:1)3%toallotherdescribedspeciesincombination
thegroupofanimalsunderstudy.Oneorseveralofthe withoneorseveralofthefollowing:
followingmustapply: (i)nomorphologicaldifferences,ordifferencesonlyin ON
(i)morphologicalandchromaticdatarevealnodifferences charactersknowntoshowintraspecificvariability:e.g., UTI
totopotypicpopulationsofadescribedspecies,oronly size,relativehindlimblength,bodycolorandpattern, OL
V
subtledifferencesincharactersthatareknowntoshow (ii)nodifferenceinadvertisementcalls,oronlydifferences E
intraspecificvariability,oronlyquantitativedifferences(in invariablesknowntoshowintraspecificvariability,
characterssuchasbodysize), (iii)co-occurrencewithotherdeeplydivergenthaplotypes
(ii)ifcharactersrelevantforprematingisolationare withinthesamepopulations,notconcordantwithany
known,thentheseshownooronlyquantitative distinctorsubtlemorphologicaldifferenceamong
differencestotopotypicpopulationsofdescribedspecies, individuals.
(iii)phylogeographicstudiesareavailableandshow
indicationsforgeneticadmixturewithothergenealogical
lineageswithinaspecies.
Recent progress in molecular techniques leads to acquiring fixed time span needed for speciation, there should be a con-
newlydeterminedDNAsequencesatafasterpacethantaxon- tinuumofpairwisegeneticdivergencesofsisterspecies,downto
omistsareabletofollowupwithresults.Inmanycases,divergent 0% in cases of rapid adaptive speciation. Any threshold will
DNA sequences are observed that probably correspond to therefore miss a proportion of very young species (false nega-
distinct, undescribed species, but a taxonomic analysis of these tives).Thesecondkindoferroristowronglyidentifyintraspe-
dataareimpracticableintheshortterm,especiallyinmorpho- cificgenealogicallineagesasspecies(falsepositives).Theaccu-
logically cryptic and highly diverse groups (32, 33). The term racy of the method depends on the so-called barcoding gap
‘‘candidate species’’ is rarely used by zoologists, but has betweenintraspecificandinterspecificdivergences,butprevious
recently been proposed for newly discovered units that prob- work on amphibians has shown a wide overlap of these values
ablycorrespondtoundescribedspecies(31),andweadvocate and absence of a distinct barcoding gap (40). Because DNA
using it in a less formal way than the Candidatus status in barcoding can only be a preliminary tool for a first and rough
microbiology (34–36). identification of candidate species, we favor a conservative
Delimiting species is a resurgence issue in biology for which approachthatminimizestheerrorprobabilityoffalsepositives.
variousexplicitprocedureshavebeenproposed(37,38).Mostof Thisapproachwillmissspeciesofrecentorigin,butitwillmore
these procedures require a relatively good state of taxonomic efficiently help taxonomists to focus on those genealogical
knowledge.Fortree-basedmethods,organismsusuallyneedto lineages likely to be undescribed species. The categories CCS,
be sampled from several populations and to have their phylo- UCS,andDCLconstituteanadvanceoveruncriticalapproaches
genetic relationships well resolved. Many nontree methods re- toDNAbarcodingbecausetheyincorporatethisusefulmolec-
quire extensive datasets as well. These datasets are usually not ular tool but emphasize the need of complementary data to
availableforthosegeneticallydivergentindividualsforwhichwe understand biological reality.
propose the category of candidate species. Also, species are Following our definitions, assignment of a genealogical lin-
knowntobeoftenparaphyleticintheirmitochondrialhaplotypes eage to the CCS category has the highest reliability of the 3
(39).Evenmoreimportantly,phylogeniesbasedonsingleDNA categories proposed here, and the number of 129 CCS thus
fragments,suchasusedinDNAbarcodingapproaches,areoften provides a minimum estimate of undescribed species in Mada-
not sufficiently resolved. We therefore do not recommend gascar.ManyDCLarelargelybasedonthelackofevidencefor
explicit tree-based delimitations of candidate species, although CCS status. Some of these genealogical lineages may thus be
suchmethodsarecertainlyagreatimprovementfortheeventual upgraded to CCS when more extensive phylogeographic data
taxonomic description of species. and integration of nuclear genetic markers are available (e.g.,
Distance-basedDNAbarcodingmethodsbasedondivergence Boophisluteus,Fig.2B),althoughinmanycasestheDCLstatus
thresholds are prone to 2 kinds of errors. Because there is no iswellassessedbycomprehensivedatasets(e.g.,inGuibemantis
Vieitesetal. PNAS (cid:1) May19,2009 (cid:1) vol.106 (cid:1) no.20 (cid:1) 8269
Molecular Data in Integrative Taxonomy. Integrative taxonomic
analyses (46) are boosting the discovery and description of am-
phibians worldwide (17). The global biodiversity crisis requires
thesekindsoftaxonomicstudies(21)becausetheyaretheprereq-
uisiteforunderstandingdiversitypatternsandthusfortheidenti-
fication of priority areas for conservation. Despite the fact that
recenteffortsinamphibiansystematicssignificantlyincreasedthe
numberofspecies,inventories—especiallyfromtropicalareas—are
farfromcomplete.Theseinventoriesarealsoneededtocomplete
ourknowledgeofthetreeoflifeofamphibians.
Amphibianstendtohaveconservativemorphologicalevolution
(47–49),whichmakestheirspecies-levelidentificationdifficult.We
heredemonstratetheefficiencyofanapproachthatfirstidentifies
genealogical lineages with high genetic divergences (40) and in a
second step classifies these as CCS, UCS, or DCL by integrating
otherdatasets.Thisapproachdoesnotprecludedetailedmorpho-
logical and bioacoustic studies in cases where biological species
showlowgeneticdifferentiation.Infact,ourapproachiscloseto
currenttaxonomicpracticeinherpetology:Weproposediagnostic
character-state differences in morphology or bioacoustics as a
centralprerequisiteforCCSstatus,agreeingwiththefactthatmost
descriptionsofnewamphibianspeciesaretodaystillbasedonlyon
morphology,despitetheavailabilityofmoresophisticatedmethods
(37,38).Toextendcurrentstandardsinamphibiantaxonomy,we
strongly recommend the routine inclusion of DNA sequences in
descriptions of new species. Such a practice would simplify the
subsequent identification of divergent genealogical lineages and
their integrative taxonomical study (40), even if this sequence
informationwouldnotbesufficienttoassessphylogeneticrelation-
shipsofthenewspecies.
AmphibianDiversityPatternsinMadagascarandConservationPrior-
ities. Although Madagascar’s fauna has been studied extensively
duringthelastcentury,theincreaseinspeciesdiscoveriesismaking
diversity estimates tentative in most cases. Our data suggest that
currentdiversityfigureslargelyunderestimatetherealdiversityof
frogs, and probably of other terrestrial vertebrates as well. This
underestimationhasdirectconsequencesonourunderstandingof
Fig.2. Examplesofcombinedphylogeographic,bioacoustic,andmorpho- the pattern of biodiversity in Madagascar. Most of the Malagasy
logicalevidenceusedtoclassifydivergentmitochondriallineagesasCCSor amphibianspeciesareknownfrom(cid:2)10localities,andmanyfrom
DCL.(A)BoophismajoriandB.aff.majoriaresympatricCCS;despitetheironly
singlelocalitiesonly(50),makingspatialdiversityanalysesdifficult.
lowgeneticdivergence,theyhavedistinctandconstantdifferencesintadpole
In many cases, this analysis certainly does not represent species
morphologyandqualitativedifferencesinadvertisementcalls,withoutsignal
ofgeneticadmixture.(BandC)B.luteus(B)andGuibemantisliber(C)consist actualdistributionranges,butindicatesareallackofinformation
ofdeeplydivergentgenealogicallineagesclassifiedasDCL,becausethecall on species spatial distribution patterns in Madagascar whereas a
differencesonlyaffectquantitativeparameterssuchasnote-repetitionratein considerablenumberofspeciesareprobablyindeedrestrictedto
B.luteus,andtherearenomorphologicalorecologicaldifferencesbetween verysmallandisolatedranges.
the populations. Furthermore, genetic admixture of the lineages was de- Important increases in species numbers have also character-
tectedinG.liberatonelocality. ized other groups of Malagasy vertebrates during the last 2
decades (30, 51). Numerous undescribed species of reptiles,
mammals,andespeciallyfreshwaterfishesareknowntoexistbut
liber,Fig.2C).Finally,theUCSstatusis,bydefinition,unreliable
are usually not included in conservation assessments. Probably
and is used to accommodate those genealogical lineages with
byfarthelargestproportionofspeciesofinvertebratesremains
insufficient data.
undescribedasisusualformosttropicalregions.Foramphibians
Thesecategorydefinitionsarenotrestrictedtoamphibians.In
aswellasmanyothergroupsofanimals,theongoingdiscovery
Madagascar and elsewhere, molecular data have been used to
of undescribed diversity is being triggered by several factors,
detect unexpected levels of cryptic diversity in other animal
includingtheincreaseoftaxonomicworkinMadagascar,incor-
groups(32,33,41–43),andinmanycasesthebiologicalidentity
porating new molecular tools with traditional methods, and an
of the previously undescribed genealogical lineages remains
increase in field exploration efforts. Some larger patches of
largely unassessed. Our definition of CCS (Table 1) relies on rainforest in Madagascar have not been properly explored yet
geneticdivergencecombinedwithadistinctdifferenceineither andprobablyharbornumerousadditionalnewcandidatespecies.
morphologyorinacharacterthatmediatesprematingisolation However, many of the amphibians we have identified here are
(advertisementcallsinfrogs).Charactersandlevelsofmorpho- foundinsomeofthebest-studiedareasofMalagasyrainforests,
logicaldivergencewillbedifferentineachanimalgroup,butin such as Ranomafana and Analamazaotra/Mantadia National
general we expect a wide applicability of this approach. More Parks (Fig. 1 B and C; SI Appendix). Taxonomic exploration
work must focus on understanding conditions under which the should thus not be limited to unexplored areas but should
concordanceofmitochondrialandnucleargenegenealogiescan includeputativelywell-knownplotsaswell.Thecombinationof
become a defining standard for CCS (15, 44, 45). phylogeneticdatasetswithspatialmodelingofspeciesorclades
8270 (cid:1) www.pnas.org(cid:2)cgi(cid:2)doi(cid:2)10.1073(cid:2)pnas.0810821106 Vieitesetal.
will help to define their potential distribution in space and technique, are urgently needed, especially for poorly explored,
thereby help to identify target areas for further exploration. highly diverse regions.
Ourfindingsconfirmthatspatialpatternsofendemism,rarity,
MaterialsandMethods
andspeciesrichnesswithintheMadagascarbiodiversityhotspot
are poorly known. The protected area network of Madagascar DNASequencingandAnalysis.Afragmentofthemitochondrial16SrRNAgene
hasincreasedsignificantlyinrecentyears,andproposalsfornew wasamplifiedandsequencedforca.2,850specimens.BasedonNeighbor-
Joiningtrees,weidentifiedsequencesshowinghighdivergencesfromrefer-
protected areas based on multitaxon analyses (52) will cover
encespecimensofdescribedspecies.Selectedsequenceswereusedtocom-
most of the key biodiversity areas in Madagascar. Almost a
puteatreebyusingBayesianinferenceafterdeterminingtheappropriate
quarter(23.3%)ofthenewlydiscoveredfrogcandidatespecies substitution model (see SI Appendix). We used the software TaxI (56) to
arenotfoundwithinthecurrentlyexistingprotectedareas;many calculatepairwisedistancesbetweensequences,whichavoidspossiblealign-
of these are range-restricted and are more likely to disappear mentartifactsinthedistributionofindelsfromtheglobalalignmentthatmay
given the pace of habitat destruction observed in Madagascar affectthiscomputation.ExtendedmethodsareavailableinSIAppendix.
(22).Thispatternimpliesthatevenalargenetworkofprotected
AssessmentofBioacousticDifferentiation.Thecallsofmanycandidatespecies
areasmaynotfullyprotectthecurrentdiversity.Aconservation
aredocumentedinarecentlycompiledsoundguide(57).Advertisementcallsof
strategy should consider protecting additional small rainforest
anuransareexcellenttaxonomicindicators,butintraspecificcalldifferencesare
fragments,becausetheycancontainarelativelylargeproportion known(58):Temporalvariablesand(toafarlesserextent)alsofrequency(spec-
of amphibian species (53, 54). The apparent absence from tralcharacters)dependonenvironmentaltemperatureandstateofsexualmo-
Madagascarofemergingdiseasessuchastheamphibianchytrid tivation,whereasfrequencyismainlyinfluencedbybodysize.However,these
fungus, in concert with a strong commitment of national insti- differencesareinallcasesquantitativeandcontinuous,i.e.,noteornoteinterval
tutionstoconserveitsbiodiversity,characterizeMadagascaras duration,orfundamentalanddominantfrequency,becomecontinuouslylarger
orsmallerwithincreasingordecreasingtemperatureorbodysize.Similarcon-
auniqueopportunitytoproactivelyprotectanamphibianfauna
tinuousdifferencesarealsoknownamongpopulationsofaspecies,oftenbe-
so far untouched from catastrophic declines other than those
causeofcharacterdisplacementoradaptationtoenvironmentalfactors(59).
caused by ongoing habitat destruction (55). Mostwell-documentedcasesofgeographiccallvariationinanuransrefertosuch
variationincontinuouscharacters(60–62).Incontrast,qualitativedifferences N
ImplicationsforGlobalBiodiversityEstimates.This study analyzes suchasthepresence/absenceofdifferentcalltypesoramelodiousvs.unmelo- TIO
the complete amphibian diversity of a tropical biodiversity diouscallstructurearerarewithinspeciesandthefewdocumentedcasesshow LU
O
hotspot by using comprehensive genetic, morphological, and thesignatureofincipientspeciation(63).Weconsideredcallvariationin(i) EV
dominantorfundamentalfrequency,(ii)noteduration,(iii)noteintervaldura-
bioacousticdatasets.Previousstudiesofotherhotspotsreported
tion,and(iv)pulserateasquantitativecalldifferences,insufficienttodefineaCCS
highnumbersofundescribedamphibianspeciesaswell(11,15), exceptinsituationsofsympatry.Differencesin(i)numberofnotetypes,(ii)
but were more limited in taxonomic and geographic coverage. generalarrangementofnotetypes,and(iii)melodiousvs.noisyorpulsedstruc-
The estimated 250% increase since 1991 up to possibly 465 tureofnoteswereconsideredtobequalitativecalldifferences,definingaCCS
speciesisunparalleledandwouldmakeMadagascaroneofthe status.
top 5 most diverse countries on the planet for amphibians,
Geographic Analysis. A georeferenced database was compiled containing
togetherwithBrazil,Colombia,Ecuador,andPeru.Theglobal
amphibianrecordsfromliteratureandourownfieldinventories.Records
importance of Madagascar’s amphibian fauna is paramount,
weretakenintoaccountonlyifidentificationwasconsideredreliable,i.e.,
especially because of its extreme degree of endemism (100%
typelocalities,recordsofmorphologicallydistinctspecies,orrecordswith
among the native species). However, it should be taken into bioacousticormoleculardata(50).Thecurrentprotectedareanetworkwas
account that very few other tropical countries with diverse providedbyConservationInternational.Distributionmapsofalldescribed
amphibian faunas have been surveyed as intensively as Mada- speciesandCCS(29)largelyformthebasisforthegeographicanalysis.Table
gascar with comparable approaches. The unexpectedly great S1 in the SI Appendix lists the working names applied herein and in the
recentlypublishedfieldguide(29)forthecandidatespecies.The9described
increaseinspeciesnumbersthatweestimatemaythereforenot
speciesforwhichnogeneticdataareavailable(seeSIAppendix)aremorpho-
characterizeMadagascarasbeingmuchmorespecies-richthan
logicallydistinctandhavenarrowdistributionranges(29),andtheirconfusion
other tropical regions. Rather, it exemplifies the power of withanyCCSorUCSisunlikely.
integrative taxonomic assessments and predicts that applying
thesetootherregionsorothergroupsoforganismsmayleadto ACKNOWLEDGMENTS.WethankParfaitBora,NeilD’Cruze,LouisduPreez,
comparably high proportions of novel discoveries. LilianeRaharivololoniaina,Roger-DanielRandrianiaina,JasminE.Randriani-
rina,andCheWeldonfortheirhelpinthefield.WealsoaregratefultoSandra
As a primary conclusion from our study, the number of
NietoRoma´n,LouisBoumans,AngelicaCrottini,SusanneHauswaldt,Meike
tropical amphibian species is probably underestimated at an Kondermann,GabrieleKeunecke,EvaSaxinger,AxelStrauß,MeikeTeschke,
unprecedentedlevelataglobalscale.Extrapolatingourdatato and Jens Weste who helped to obtain the DNA sequences. David Knox
(ConservationInternational)providedthelatestversionofthemapsofMada-
other less-studied tropical regions predicts that the number of
gascar’sprotectedareanetwork.Thestudywasmadepossiblebycollabora-
amphibian species worldwide could double or possibly even tion agreements of the authors’ institutions with the Universite´
quadruplebeforesaturationinnewdiscoveriescanbeexpected. d’Antananarivo,De´partementdeBiologieAnimale,theAssociationNation-
Amphibiansarethevertebrategroupwiththehighestproportion alepourlaGestiondesAiresProtege´es,andtheParcBotaniqueetZoologique
deTsimbazaza,Madagascar.WearegratefultotheMalagasyauthoritiesfor
ofthreatenedspecies(20),andcurrentdeclinesmaybeaffecting researchandexportpermits.ThisworkwassupportedbyNationalScience
more diversity than previously thought. Paradoxically, we are FoundationAssemblingtheTreeofLifeProgramGrantEF-0334939,Consejo
living in an era of simultaneous mass extinction and mass SuperiordeInvestigacionesCientíficasIntramuralGrant200830I100,grants
fromtheVolkswagenFoundation,DeutscheForschungsgemeinschaft(VE247/
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Vieites et al. PNAS. 2009 1
Supplementary information S2: Amphibian species list for two major centers of
amphibian diversity, (1) Ranomafana National Park and (2)
Analamazaotra/Mantadia National Park with surrounding forests

(1) Ranomafana National Park (112 species of which 31 CCS and UCS)

This list contains records from various sites within and directly adjacent to Ranomafana
National Park (with maximum distances of less than 20 km between sites), and from
three localities that are at ca. 10, 20 and 40 km east of Ranomafana village (Mahakajy,
Ifanadiana, Ambohitsara). Several species that so far have only been recorded from
these three sites (marked with asterisks) can be expected to occur in poorly explored
low-altitude sites within the Park as well. CCS and UCS are marked reddish-brown.

Sites are coded as follows:
(1) Ranomafana village , 21º15'41"S,47º27'34"E.
(2) Ambatolahy forest, 21º14'37"S,47º25'34"E.
(3) Ranomafana National Park entrance, 21º9'10"S,47º31'39"E.
(4) Talatakely, 21º15'50"S,47º25'"E.
(5) Vohiparara, 21º15'30"S,47º24'"E.
(6) Ranomafanakely, 21º14'55"S,47º22'18"E.
(7) Maharira, 21º19'32"S,47º24'8"E.
(8) Mangevo, 21º23'13"S,47º27'18"E.
(9) Ambohitsara, 21º21'25"S,47º48'56"E.
(10) Mahakajy Private Reserve, 21º16'45"S,47º31'49"E.
(11) road Ifanadiana - Tolongoina, 21º21'12"S,47º36'28"E.

Family Ptychadenidae
Ptychadena mascareniensis (1, 2, 9)

Family Hyperoliidae
Heterixalus alboguttatus (1, 9)
Heterixalus betsileo (1, 2)

Family Microhylidae
Anodonthyla boulengeri (1, 10)
Anodonthyla moramora (5)
Anodonthyla sp. 1 (7)
Anodonthyla sp. 4 (7)

Platypelis pollicaris (4, 7)
Platypelis grandis (5, 4)
Platypelis tuberifera (4)
Platypelis cf. cowani (9)*

Plethodontohyla brevipes (2)
Plethodontohyla cf. bipunctata (7, 4)
Plethodontohyla inguinalis (5)
Vieites et al. PNAS. 2009 2
Plethodontohyla mihanika (4)
Plethodontohyla ocellata (8)*
Plethodontohyla notosticta (8)

Stumpffia sp. 9 (1, 10)
Stumpffia cf. tridactyla (7)
Stumpffia cf. tetradactyla (9)*

Paradoxophyla palmata (1, 6)
Scaphiophryne spinosa (1, 4)

Family Mantellidae
Aglyptodactylus madagascariensis (5)
Aglyptodactylus sp. 3 (1)
Boophis albilabris (2, 4)
Boophis albipunctatus (8)*
Boophis andohahela (2)
Boophis bottae (5, 6)
Boophis elenae (5, 7)
Boophis guibei (3, 7)
Boophis lilianae (11)*
Boophis madagascariensis (3, 6)
Boophis majori (5, 7)
Boophis cf. mandraka (2)
Boophis marojezensis (2, 7)
Boophis microtympanum (7)
Boophis opisthodon (11)*
Boophis pauliani (7)
Boophis periegetes (7)
Boophis picturatus (5, 7, 9)
Boophis pyrrhus (11)
Boophis rappiodes (1, 6)
Boophis reticulatus (2, 5)
Boophis rhodoscelis (6)
Boophis schuboeae (2, 5)
Boophis tasymena (5, 6)
Boophis tephraeomystax (1)
Boophis viridis (9)*
Boophis sp. 8 (6)
Boophis sp. 13 (6)
Boophis sp. 15 (2)
Boophis sp. 16 (2, 5, 9)
Boophis sp. 17 (2)
Boophis sp. 22 (1, 2, 6)
Boophis sp. 29 (10)*
Boophis sp. 30 (6)
Boophis sp. 35 (4, 6)
Boophis sp. 37
Vieites et al. PNAS. 2009 3
Boophis sp. 38
Boophis sp. 42

Blommersia blommersae (1, 5)
Blommersia domerguei (7)
Blommersia grandisonae (11)*
Blommersia sp. 9 (5)

Gephyromantis blanci (5, 6, 7)
Gephyromantis cf. boulengeri (2, 9, 11)
Gephyromantis enki (4, 5, 6, 7)
Gephyromantis runewsweeki (5, 7)
Gephyromantis cf. asper (6)
Gephyromantis plicifer (4)
Gephyromantis redimitus (8)
Gephyromantis sculpturatus (4, 9, 11)
Gephyromantis tschenki (2, 3, 4)
Gephyromantis malagasius (9)
Gephyromantis ventrimaculatus (5, 6)
Gephyromantis sp. 8 (9)*
Gephyromantis sp. 9 (3, 5)
Gephyromantis sp. 13 (5)*

Guibemantis liber (3, 5)
Guibemantis pulcher (5, 7)
Guibemantis depressiceps (3)
Guibemantis timidus (11)*
Guibemantis tornieri (2, 3)
Guibemantis sp. 7 (5, 7)
Guibemantis sp. 12 (3)

Mantella baroni (5, 6, 7, 8)
Mantella bernhardi (9)*
Mantella madagascariensis (5, 6)

Mantidactylus argenteus (8)
Mantidactylus alutus (6)
Mantidactylus betsileanus (3)
Mantidactylus femoralis (6)
Mantidactylus majori (2, 4, 5, 7)
Mantidactylus melanopleura (5, 7)
Mantidactylus delormei (7)
Mantidactylus opiparis (5)
Mantidactylus grandidieri (4, 5, 8, 9)
Mantidactylus cf. lugubris (1, 2, 4, 7, 10, 11)
Mantidactylus sp. 1 (4)
Mantidactylus sp. 13 (10)
Mantidactylus sp. 24 (2)
Mantidactylus sp. 28 (7)
Mantidactylus sp. 44 (9)
Vieites et al. PNAS. 2009 4
Mantidactylus sp. 47 (2)
Mantidactylus sp. 58
Mantidactylus sp. 59

Spinomantis aglavei (5)
Spinomantis elegans (7)
Spinomantis peraccae (7)
Spinomantis fimbriatus (2, 5)
Spinomantis sp. 7 (7)
Spinomantis sp. 8 (7)