Extant diversity and estimated number of Gracillariidae (Lepidoptera) species yet to be discovered in the Neotropical region

REVISTA

BRASILEIRA

DE

Entomologia

w w w . r b e n t o m o l o g i a . c o m

Review

Extant

diversity

and

estimated

number

of

Gracillariidae

(Lepidoptera)

species

yet

to

be

discovered

in

the

Neotropical

region

Rosângela

Brito

_,

_Jurate

_De

_Prins

_,

_Willy

_De

_Prins

_,

_Olaf

_H.H.

_Mielke

_,

_Gislene

_L.

_Gonc¸

_alves

_,

Gilson

R.P.

Moreira

a_{UniversidadeFederaldoParaná,ProgramadePós-Graduac¸ãoemEntomologia,Curitiba,PR,Brazil} b_{RoyalBelgianInstituteofNaturalSciences,Brussels,Belgium}

c_{UniversidadeFederaldoParaná,DepartamentodeZoologia,Curitiba,PR,Brazil}

d_{UniversidadeFederaldoRioGrandedoSul,InstitutodeBiociências,DepartamentodeGenética,PortoAlegre,RS,Brazil} e_{UniversidaddeTarapacá,InstitutodeAltaInvestigación,Antofagasta,Arica,Chile}

f_{UniversidadeFederaldoRioGrandedoSul,DepartamentodeZoologia,PortoAlegre,RS,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received28April2016 Accepted9June2016 Availableonline28June2016 AssociateEditor:RodrigoFeitosa Keywords:

Leafminermoths Microlepidoptera Speciesrichness

Phylogeneticdiversification

a

b

s

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a

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Gracillariidae(Lepidoptera)arecommonlyknownbytheleafminerhabitfoundinthelarvalstageof mostspecies.Byusingworldwide,publicdatabasesonspeciesdiversityandDNAsequencesavailablefor extantgracillariidspecies,wedeterminedchangesintherateoftaxonomicspeciesdescriptionsthrough time,mappedtheirspatialdistributions,examinedtheirphylogeneticdiversification,andestimatedthe numberofspeciesyettobedescribedforthefamilyintheNeotropics.Werecovered185species,a numberthatissmallerthanthatfoundinanyotherbiogeographicregion.However,itwasestimatedthat atleast3875additionalspeciesremaintobedescribedintheregion.Phylogeneticdiversificationshowed apatternofexpandingdiversity.Afewentomologistshavebeeninvolvedwithgracillariidtaxonomyin theNeotropics,having39%ofthespeciesbeendescribedbyasingletaxonomist.Inmostofsuchcases, descriptionswerebasedontheadultsonly.Afewspecieshavebeendescribedfrombiomesknownto havesomeofthegreatestdiversityonearth,suchastheAtlanticForest.Thus,suchascenarioresultsfrom lowsamplingandscarcetaxonomicactivitythathasprevailedforthisfamilyofmothsintheNeotropics. Itmayalsobeassociatedwiththeirsmallbodysizeandtothefactthatgracillariidsdonotseemtobe attractedtolighttrapsasmuchasothermoths,whichmaketheircollectionandidentificationbynon expertsdifficult.Wealsosuggestedscientificandpoliticalactionsthatcouldbeadoptedtoovercome suchanunfavorablescenario.

©2016SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Sincethepioneer,comprehensiveinventoriesthathavebeen conductedonthediversityofterrestrialarthropodsingeneral,it becameevidentthattheLepidopteraareamongthemostdiverse group of insects existing in tropical rainforests, including the Neotropicalregion(Erwin,1982;Lawtonetal.,1998;Bassetetal., 2012).Thesestudiesalsoledtothepredictionthatthediversityof Lepidopterainsuchareasismuchgreaterinnumbersthanwhat wereally know,andthus manyeitherremaintobediscovered and/orneedtobetaxonomicallydescribed.Infact,severalsurveys carriedoutaposterioriinsuchregionsfocusingonLepidoptera havegivensupporttosuchaprediction(e.g.Brehmetal.,2005;

∗ Correspondingauthor.

E-mail:gilson.moreira@ufrgs.br(G.R.P.Moreira).

Janzenetal.,2005;Linaresetal.,2009;Leesetal.,2014;Zenker

etal.,2016).Thesestudies,however,withtheexceptionofLees

etal.(2014),havebeenconductedwithprimaryattentionon

samp-lingofthemacrolepidoptera.Thisartificialgroupthatincludesthe Papilionoideabutterfliesandtheothermorederivedmoth super-families,accountsforca.107,475species(68.27%),andthusca.2/3 ofthetotal(=154,424species)extantLepidoptera(vanNieukerken etal.,2011).However,itincludesonly34familiesintheOrder;that is,25.56%ofthetotal(=133families).Fromaspeciesrank perspec-tive,thiswouldsuggestphylogeneticdiversification(sensuMorlon, 2014)ofyoungerlineageshasbeenproportionallygreaterin Lep-idoptera,whichissupportedbytheexistenceofasimilartrendat thegenericlevel;thatis,fromthetotalof14,655generalistedby

vanNieukerkenetal.(2011),9713(=66.28%)aremacrolepidoptera,

andtheremaining4942(=33.72%)microlepidoptera.However,one cannot rejecta priori thealternativehypothesis thatthis trend resultsatleastin partfrombias,associatedtolower collection http://dx.doi.org/10.1016/j.rbe.2016.06.002

0085-5626/©2016SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

effortsoftheolder,microlepidopteralineages,whichisexplored hereinfortheGracillariidae,asacasestudy.

Gracillariidae(Figs. 1–8)is consideredone themostdiverse microlepidopterafamilieshavinganendophagousfeedinghabitat, accountingfor105generaandmorethan1950speciesthatare distributedworldwide,exceptAntarctica(DePrinsandDePrins,

2016a).Althoughlarvaeofmostspeciesareleaf-miners,somemine

eitherstemsorfruits,andothersareflower/fruitborers,leafrollers, orgallinducers(e.g.Davis,1987;Guillénetal.,2001;Vargasand

Landry,2005;Huetal.,2011;Hansonetal.,2014;Kawakitaand

Kato,2016).ThefamilywasproposedbyStainton(1854)toascribe

atthat timethegenera GracilariaZeller,1839,CorisciumZeller,

1839,andOrnixKollar,1832.Theformerisnowasynonymof Gracil-lariaHaworth,1828,thetypegenusofthefamily.Thelattertwo aresynonymsofCaloptiliaHübner,1825,atthepresenttimealso withinGracillariidae.CaloptiliidaeFletcher,EucestidaeHampson, LithocolletidaeStaintonandOrnichidaeStaintonarenow consid-eredsynonyms ofGracillariidae(DePrinsand DePrins,2016b). Threesubfamiliesarecommonlyacceptedforthefamily: Gracil-lariinae,LithocolletinaeandPhyllocnistinae(DavisandRobinson, 1998).However,othershavebeenproposed,suchas Oecophyllem-biinae(RéalandBalachowsky,1966;Kumata,1998),Ornichinae

(Kuznetzov and Stekolnikov,1987)and Ornixolinae (Kuznetzov

andBaryshnikova,2001).Recently,themonophylyofGracillariidae

Figs.1–8.Leafmines(left)andadults(right)fromputativespeciesofNeotropicalgracillariids:(1,2)SpinivalvagauchaMoreira&Vargas(Gracillariinae)onPassifloraactinia Hook(Passifloraceae);(3,4)PorphyroselaminutaClarke(Lithocolletinae)onTrifoliumrepensLinnaeus(Fabaceae);(5,6)AngelabellatecomaeVargas&Parra (Oecophyllem-biinae)onTecomafulva(Cav.)G.Don(Bignoniaceae);(7,8)Phyllocnistissp.(Phyllocnistinae)onBaccharisanomalaDC.(Asteraceae).Scalebars=10,1,10,1,5,1,5,1mm, respectively.

wassupported by analysesbased onmolecular data,including foursubfamilies,whichareadoptedinthepresentstudy: Gracil-lariinae, Lithocolletinae, Phyllocnistinae and Oecophyllembiinae

(Kawaharaetal.,2011).Hypermetamorphosis,adramaticchangein

morphologyduringlarvalonthogeny,fromsap-drinkingto tissue-feedingand/orspinning(seeKumata,1978),hasbeenproposedas anapomorphyforGracillariidae(Davis,1987).However,recently

Britoetal.(2013)demonstratedthatthegracillariineSpinivalva

gauchaMoreira&Vargas,describedfromSouthernBrazildoesnot havesap-feedinglarvalinstars.Ontheotherhand,Kuznetzovand

Stekolnikov(1987)suggestedGracillariidaeareuniquewithin

Lep-idopterabyhavingmalegenitaliawithonlyfourpairsofmuscles andwithoutagnathos,andfemalegenitalia,withashort,laterally flattenedovipositor,whichshouldbefurtherexplored.Although ithasbeenacceptedthattherearemanyspeciesofGracillariidae stilltobedescribedintheNeotropicalregion(DavisandWagner,

2011;Britoetal.,2012;Leesetal.,2014), asfarasweare

con-cernednodataavailablesupportingsuchapredictionhasbeenever comparativelyexplored.

Byusing dataexistingin theliteratureandpublicdatabases availableaselectronicpublicationsintheWorldWideWeb,we hereinretrievedtaxonomicdataonNeotropicalgracillariids, list-ingthevalidspeciesbyauthorandyearofdescription,andmap thedistributionoftypesaccordingtocorrespondingbiogeographic regionsandpoliticalborders.Then,weadjustedtheannual vari-ationofthedescribedspeciestoalogisticmodel,predictingthe numberofgracillariidspeciesstilltobedescribedinthe Neotrop-ics,aswellasthetimeneededtoachievethatgoalgiventhepresent timerateofspeciesdescriptionsintheregion.Also,byusingDNA sequencesavailableinpublicdatabasesweestimatedthe phyloge-neticdiversificationofGracillariidaeintheNeotropicsthroughout time.Theseandadditionalfindingssuchasonhost–plantuseare discussedincomparisontootherbiogeographicregions.By review-ingthestatusoftheknowndiversity,evaluatingtheirphylogenetic diversificationandpredictingthenumberofgracillariidspeciesyet tobedescribedintheNeotropics,weaimtoestablisha correspond-ingbasis forfuture taxonomicstudiesintheregion,suggesting researchprioritiesandscientificactions,aswellastheexistenceof potentialdifficultiestobesurpassedregardingtaxonomic impedi-ment,ifany.Wehopeourfindingswillstimulatefurtherscientific interestinNeotropicalgracillariids,notonlyfromataxonomic per-spectivebutalsotheiruseasmodelsonevolutionaryecologybased studies,particularlythosefocusingonhost-plantinteraction,and alsoonconservationbiology.

Materialandmethods

Dataonspeciesdiversitywerecompiledfromthecatalogon NeotropicalGracillariidaeprovidedbyDavisandMiller(1984)and thepublicdatabaseonWorldGracillariidaemaintainedbyDePrins

andDePrins(2016a).TheglobaldatabaseofGracillariidae

(Lepi-doptera)waslaunchedin2006asanonlinesearchablecatalogof allknownGracillariidaespecies.Theemphasisisfocusedonthe followingaspects:i)completeness(taxonomicinformationonall 1956species,413synonymsofspeciesnames,8subspeciesand 309unavailable species-group names, 105 generaand 42 syn-onyms of genus-group names and 17 family-group names are foundin asearchableformat), ii)regularupdates,i.e. twiceper year,iii)presentationofcross-referencedverifieddata(dataon gracillariidtaxonomy,9054distributionrecords,7641hostplants recordsand4199parasitoidrecordsarepresentedonlinelinked withthepublicationreferenceandcitationpage),iv)generation ofdatawithuser-targetedmulti-searchablefunction(e.g.regional or country catalogs, detailed species checklists in association withtheirhostplantspecies/genera/families;v)documentationof

tri-trophic interactions of host(s)–gracillariidmoth species and theirparasitoid(s);vi)arobustgalleryof2188imageswiththeaim todocumentvisuallyeverygracillariidspeciesincludinginternal diagnosticcharactersofthelastabdominalsegments,morphology ofpre-imaginalstages,speciesbiotopes,aswellasbiologicaland ecological characteristics; vii)automatically generated country-linkedspeciesdistributionmaps;viii)typespecimens andtheir depository;ix)robustreferencelistof4806publications;x) fos-silspeciesandspeciesoriginallydescribedasgracillariidsbutlater transferredtootherfamilies.

To map the gracillariid species diversity, we adopted the regionalizationoftheNeotropicalregionasproposedbyMorrone

(2014),usingthecorrespondingshapefilethatwasprovidedby

Löwenberg-Neto(2014)toplotthedata.Theseauthorsadopteda

restrictivedefinitionoftheNeotropicalregion,excludingthe south-ernportionoftheAndeanareaandalsonorthernMexico.Toconceal thiswiththedatabaseprovidedbyDePrinsandDePrins(2016a), whoadoptedabroaderview,theseareaswerehereinincludedin theanalyses,andthustreatedinconjunctionwiththeremaining subregions(sensuCox,2001).Theideawastohaverepresentative subdivisionsfromabiogeographicperspective,withoutnecessarily takingintoaccounttheranksproposedbyMorrone(2014).

Weestimatethepotentialnumberofgracillariidspeciesstillto bedescribedintheNeotropicalregionbyadjustingthecumulative numberofdescribedspeciesthroughtime toathree-parameter logisticmodel(Ratkowsky,1990),byusingthesoftwareSigmaPlot 11.0(SystatSoftware,Inc.).Thus,weassumedthereisafinite num-berofspeciesexistingonearth.Also,thatasthenumberofrecorded speciesnearsthetotalnumberofspeciesinexistenceforagiven areaitbecomes increasinglydifficulttodiscovernewspeciesin

thatarea(Diamond,1985;Cabrero-Sa ˜nudoandLobo,2003).This

modelshowedasatisfactoryfitwhenusedrecentlybyCardosoetal.

(2015)topredictthenumberofstillundescribedmayflyspecies

existinginBrazil.Astheseauthors,thefunctionwasadjustedherein byusingtheQuasi-Newtonmethodandcriterionofconvergence 0.0001.Also,thesamedefaultvaluesadoptedbythemwereused: thatis,startingvalueof0.1andsept-widthof0.5,forallparameters. Theadjustmentwasbasedon200interactions,usingthenumber of173speciesconsideredasvalidatpresenttime,1867astheyear ofthefirstspeciesdescription,and2013,asthelastone.

We calculate the expectation for the number of lineages of Neotropicalgracillariidsinareconstructedphylogenyusing171 extantoperationaltaxonomicunits(OTUs),byconditioningonthe ageofthetreeaswellaswithassumingauniformpriorfortheage ofthetree.Weusedthebarcodeindexnumber(BIN),asystemthat clustersbarcodesequencesalgorithmically,asaproxyoflineages, sinceclustersshowhighconcordancewithspecies.Weemployed thisapproachtodocumentthediversityintheabsenceof taxo-nomicinformationformostoftheavailabledata.Sequencesofthe CytochromeoxidaseI(COI)gene(i.e.,barcodes)wereusedto recon-structthetaxonomicpositionofspeciesandgenera,mainlyfrom theBOLDand GenBank databases,but alsoincorporating novel OTUssequencedbyourresearchgroup(Tab.S1).Bucculatrixwas usedtorootthetree(basedonKawaharaetal.,2011;Wahlberg etal.,2013).SequenceswerealignedusingClustalXinMEGAv6

(Tamuraetal.,2013),accordingtodefaultparameters.Thebestfit

modelofsequenceevolution (GTR)wasimplementedaccording totheAkaikeInformationCriterionscoresforsubstitution mod-elsevaluatedusingjModeltestv.0.1.1(Darribaetal.,2012).BEAST v.2.1.3(Bouckaertet al.,2014)wasusedtoestimatethe diver-gencetimeunderaBayesianapproach(Drummondetal.,2006).An uncorrelatedlognormalmodelofratechangewasimplemented.To testwhetherthediversificationrateshavechangedovertime(i.e. increasedlineagespeciation)wecontrastedthelikelihoodvalues ofthedata(branchingtimesderivedfromthetrees)undertwo modelsofconstantspeciationrate().Twoindependentanalyses

usingastreepriorapurebirthmodelwitha=0andextinction rate=0(theYuleprocess),andabirth-deathmodelwith>0and >0,wererunusing100milliongenerationssampledevery10,000 generations.Convergencewasconfirmedbyeffectivesamplesizes over200forallparametersinTracer1.6(Rambautetal.,2014).The resultingtreesanddivergencetimesestimatedunderdifferenttree priorsweresimilar.BayesFactoranalysiswasperformedunderthe smoothedmarginallikelihoodestimateandwith1000bootstrap replicatescomputedinTracerv.1.6,butitdidnotprovidestrong supportforselectinganyofthepriors(BF<2).Wechosetousethe treesreconstructedunderthebirth–deathprocesstreepriorasit accountsforbothspeciationandextinction(Gernhard,2008).The timecalibration(inmillionsofyears[Ma])wascalculatedusing asecondorderdateforthegroupGracillariidae+Bucculatricidae, proposedbyWahlbergetal.(2013).Weappliedanormal distribu-tionforthepriorwithameanof115.34Ma(108.34–122.34;99% CI).Toexamine(visually)thetemporalpatternoflineage diversi-fication,meansemi-logarithmiclineagethroughtime(LTT)plots wereconstructedusingTracerv1.6with95%confidenceintervals generatedonthefinalBEASTtrees.

Results

Atotalof185GracillariidaespeciesfortheNeotropicalregion (Fig. S1)were recovered,belonging tofour subfamiliesand 26 genera(Tab. S2).The Gracillariinae werethe mostrepresented (145species),followedbytheLithocolletinae(21), Phyllocnisti-nae(17)andOecophyllembiinae(02)(Fig.S2).Correspondingtype

Fig.10.Annualvariationinnumberofgracillariidspeciesdescribedforthe Neotrop-icalregion.

localitiesarescattereddistributedinspace,varyinginnumberfrom zerospeciesintheSouth-easternAmazoniandominionto42inthe Pacificdominion(Fig.9).MostNeotropicalcountriesholdatleast onetypelocality;Brazilaccountedforthegreatestnumberoftypes, followedbyEcuadorandPeru(Fig.S3).

Speciesdescription-throughtimeplotshowedanerratic pat-tern(Fig.10), starting in 1867withthe firstdescribed species, and reachinga peak between1911 and 1920when 69 species weredescribed. A total of 24 authorsaccounted for the corre-sponding descriptions. The British taxonomist Edward Meyrick producedthegreatestnumberofcontributionsinthisregard(78 species;Fig.11).Typesweredesignatedintheoriginal descrip-tionformostcases.Theyweredepositedamong16museumsthat arelocatedmostlyabroad,mainlyintheNaturalHistoryMuseum,

Fig.9. Diversityofextantgracillariidspecies(Arabicnumbers)intheNeotropics,accordingtobiogeographicalregionalizationproposedbyMorrone(2014).Asterisksindicate areasnotcontemplatedinhisrestricteddefinitionoftheNeotropicalregion(seetextforfurtherdescription).

Fig.11.NumericalcontributionsbyauthorondescriptionofgracillaridspeciesfortheNeotropicalregion.Numbersabovebarsrepresentpercentagesinrelationtothetotal numberofspecies(n=175).

Fig.12.Relativerepresentationoftypespecimensforgracillariidspeciesinthe Neotropicalregion.Numbersabovebarsrepresentpercentagesinrelationtototal numberofextantspecies(n=175).

London(UK)andtheNationalMuseumofNaturalHistory– Smith-sonianInstitution (USA)(Figs. 12 and 13).Originaldescriptions werebasedprimarilyontheadultstage,particularlyonwing vena-tion and color pattern. Data onthe genitaliaof either male or

Fig.13.Variationinnumberoftypespecimensavailableamongmuseumcollections forNeotropicalgracillarids.Numbersabovebarsrepresentpercentagesinrelation tototalnumberofspecies(n=175).SeeTabS3fordescriptionofmuseumacronyms.

femalewereprovidedforonly46(25%)intheoriginaldescriptions ofspecies.Descriptionoftheimmaturestages (eitheregg,larva orpupa)arepresented in11%ofthearticlescontainingspecies descriptionsand, regarding moleculardata(DNAsequences), in only4%(Fig.14).Host–plantspecieswererecordedin29familiesof angiosperms,Fabaceaebeingthemostcommonlyused(TableS3,

Fig.15).

Cumulativespeciesdescription-throughtimedataadjustedwell (p<0.05)totheLogisticequation(Fig.16).Annualrateofspecies description was 1.18per year. The model predicted the exist-ence of 4048 species in the Neotropicalregion. Corresponding standarderrorwas91,004(t=44.487,p<0.001).Descriptionofthe remaining3875 speciesstill to bedescribed inthe Neotropical regionwouldbecompletedintheyear2174.

Phylogeneticdiversification revealedby theLTT plotfor the GracillariidaeintheNeotropicalregionsuggestsapatternofeven speciation rates, withexpanding diversity throughout the evo-lutionary time, with progressive, exponential accumulation of lineagesuntilthelast25Mya,and anearlyconstant,linearrate fromthattothepresenttime(Fig.17).

Fig.14.Variationinnumberofarticlesregardingoriginaldescriptionson Neotropi-calgracillariidsthatwerebasedonadultsbutthatincludedalsodataeitherongross morphologyofimmaturestagesorDNAsequences.

Fig.15.VariationinnumberofrecordsforangiospermfamiliesusedashostsforgracillariidspeciesintheNeotropicalregion.Numbersabovebarsrepresentpercentagesin relationtototalnumberofplantfamilies.

Fig.16. PredictivefuturecumulativespeciesdescriptioncurvefortheGracillariidae intheNeotropicalregion,basedontheLogisticmodel.

Fig.17.Lineage-Through-Time(LTT)plotofNeotropicalGracillariidae.LTTwas plottedusing1000treesfromtheCOIdatasetanalyses.Thenumberoflineages (yaxis)isplottedagainsttime(xaxis)insuchwaythateachincreaseonthenumber oflineagesrepresentsacladogenesis(anode)inoneofthe1000phylogenetictrees. 2.Dashedredandbluelinesrepresent95%-confidenceintervalsfortimeofstarting diversificationandLLTplot,respectively.

Discussionandconclusions

Findingsinthepresentstudyprovidedstrongevidencethatthe smallnumberofknownGracillariidaefortheNeotropicalregion resultsfromundersampling,associatedwithlowtaxonomic activ-ityonthefamilythroughouthistoryintheregion.Itcallsattention tothefact thatsucha numberis thesmallest(8%ofthetotal)

foundforanyotherbiogeographicregionintheworld(Fig.S1). Thisisnotexpectedgiventhesizeanddiversityofplantsknown toexistintheNeotropicalregion.Inotherwords,thistrenddoes notfindsupportonwhatisobservedforotherLepidoptera fam-ilieswhoseworldwidediversityisbetterknown.Forexample,if weconsiderGeometridae,PyralidaeandNymphalidae,this rela-tioninspeciesdiversitydoesnotsustain;tothecontrary,forthese families diversity is greater in theNeotropics compared tothe PalaearcticandNearctic(FigS5),thetwogreatestbiogeographic regionsintermsofareaonearth.Itisimportanttomentionthatthe larvalstageofmanymacrolepidopteranfamiliesareexternal feed-ers,andmanyareeitheroligophagousorpolyphagous.Asalready mentioned,larvaeofgracillariidsingeneralareinternalfeeders, havinga specializedfeedingapparatusand thusarehighly spe-cificintermsofhost–plantuse,manybeingmonophagous(Davis, 1987).Thus,foragivenarea,comparativelytosuchlepidopteran families, one would expectthe diversity of Gracillariidaetobe moreinfluencedbythatofplants,andinconsequenceshouldbe greater.Thistrendwouldnotbeexpectedonlyincaseof differ-encesinageandvariation ofhistorical diversificationrates due toanyreason,whichremains tobetestedfor these macrolepi-dopteranfamiliesin theNeotropics.However,Gracillariidaeare knowntobemucholderthanthesefamilies(DavisandRobinson, 1998),andweprovidedevidencehereinthatitsdiversificationin theNeotropicshasprogressivelyexpandedatanexponentialrate until25Mya,andataconstantratethereafteruntilthepresent. Thispatternofspeciationrateovertimehasoftenbeeninterpreted asadiversity-dependenteffectresultingfromasaturationofniche spacefollowingadaptiveradiations(PhillimoreandPrice,2008;

RaboskyandLovette,2008;EtienneandHaegeman,2012).In

addi-tion,LTTplotresultsaresensitivetoincompletetaxonsampling

(Neeetal.,1994;Ricklefs,2007),whichlikelyaffectedtheestimates

inthecaseofgracillariids.Inspiteofthis,asafirstphylogenetic diversificationanalysisongracillariids,we wereabletorecover evenratesofspeciationthroughouttheevolution,indicatingthat Neotropicallineageshave been thetargetof thediversification processfor alongtime inevolutionaryhistory. Thus,we found noindicationandhencerefutethehypothesisforoccurrenceofa greaterrateofdiversificationearlyinthegracillariidphylogeny.In otherwords,thereisnoindicationfortheexistenceofanegative balancebetweenspeciationandextinctionratesthatwouldlead totheexistenceoflowdiversityofgracillariidsintheNeotropical region.

Infactourdatashowsthatthenumberofgracillariidspecies describeduptonowfromtheNeotropicalregionisfarfrom real-istic.Forexample,theParanádominion,withinwhichtheAtlantic forestislocated,accountedonlyfor7species.TheAtlanticforest isknownasoneoftheareaswiththegreatestdiversityofplants andanimalsonearth,beingextremely richinendemics(Myers

etal., 2000;Carnavalet al.,2009;Freitas etal.,2011).

Approx-imately50%ofalmostseventhousandspeciesofplantsexisting intheareaareendemics(Stemannetal.,2009).Asalready men-tioned,leaf-minermothsarehighlyhost–plantspecific,andbased onthis,Britoetal.(2012,2013)predictedthathundredsof unde-scribed,endemicgracillariidspeciesshouldexistin theAtlantic forest.Indeed,severalgracillariidspeciesrearedfromleaves col-lectedinthesouthernpartoftheAtlanticforest,mostbelonging toangiospermfamiliesnotrecoveredashostplantofgracillariids inthepresentsurveyfortheNeotropics,awaitfordescriptionin ourlaboratory.OtherareasinBrazil,suchasthecentraland north-easternportionsoftheChacoandominion(CerradoandCaatinga biomes),andtheoccidentalAmazonareahavebeentraditionally misrepresentedinLepidopteracollectionsingeneral,duetolow samplingeffortsappliedfortheseinsectsinsuchareas(Santosetal.,

2008a,b).Recently,inasurveycarriedoutinsmallareasofFrench

GuianaandEcuador,Leesetal.(2014)estimatethecorresponding gracillariidrichnesstobe240species,fromwhichapproximately 85%arebelievedtorepresentnewspeciestoscience.While sur-veyinggracillariidsassignedtothegenusPhyllocnistisZellerina localizedareainCostaRica,DavisandWagner(2011)cametothe conclusionthattherichnessforthisgenusaloneshouldaccountfor hundredsofspeciesintheNeotropicalregion.Thistrendmaywell beextendedtothetemperateportionsoftheNeotropicalregion. ThegracillariidfaunaoftheSouthernConeof SouthAmericain particularis largelyunexplored,andthis regionaccountsforat least18,139speciesofplants,amongwhichca.43%areendemics

(ZuloagaandBelgrano,2015).

Thus,itisnotsurprisingthatthepredictionmadeinthisstudy suggeststhatthenumberofgracillariidspeciesinthe Neotrop-icalregioniscirca20timesgreater.Itisalsoimportanttonote thatthetaxonomicdescriptionsofsuchunknownspecieswould becompletedonlyabouttwocenturiesfromnow.Thisestimate, however,wasbasedonthelowcollectionintensityandscarce tax-onomyactivityongracillariidsthathavehistoricallyprevailedin theregion,asalreadymentioned.Thus,suchtimemaybealtered substantiallyinthefuture,dependinguponchangesinpriorities andtheinvestmentinfieldandlaboratoryresearch.Intensive sur-veysforgracillariidspecieswhencarriedoutinrichbiomes(e.g.

Leesetal.,2014)wouldsubstantiallyspeedupthecurve,reducing thetimetoreachsuchaplateau.Thiswouldbeparticularlytrue ifmodernmolecularbiologytechniques,suchasDNAbarcoding (sensuHebertetal.,2003)areusedinconjunctionwithtraditional alphataxonomicmethods.UseofDNAsequencesinthiscasewould facilitatenotonlytheidentificationofadultspecimensthatmaybe collectedbyusinganykindoftrap,butparticularly,inthe associa-tionwiththeirimmaturestagescollecteddirectlyfromhost–plants, andviceversa.

Ourdatashowedclearlythattheunderlyingcausesforpaucity intheknowledgeofgracillariiddiversityintheNeotropicsfitinto thetypicalissuesinvolvedinwhattaxonomistscalledlatelythe “taxonomicimpediment”(fordiscussion,seeLipscombetal.,2003;

Scotlandetal.,2003;Wheeler,2004;Carvalhoetal.,2007).Inother

words,whathasobstructedprogressofgracillariidtaxonomyin theNeotropics is primarily thescarce activityof specialists on suchafaunathroughouthistory.Correspondingspecies descrip-tionshavebeenlowanderraticintime,andwereprovidedina superiornumberbyforeigntaxonomiststhateventuallyworked withNeotropicalgracillariids,mostlyin thefirsthalfofthelast century.Consequently,correspondingtypesweredepositedmostly

abroad,andmoreimportantly,nomajor,wellcuratedcollection ofgracillariidshaseverbeencompiledfortheregion.Therewas no indication in this study that such a situation varies much amongNeotropicalcountries.Justtogiveoneexample,theSerra Bonitacollection(Camacãn,Bahia,Brazil),oneofthegreatest col-lectionsonmicrolepidoptera existingin theNeotropicalregion, hascirca1200pinned-driedpreservedadultsofgracillariidsthat are placed into one drawer (V.O. Becker, pers.comm.). In our opinion,however,additionalissues inherenttotheseleafminer mothsinparticularhavecontributedtothemaintenanceofapoor knowledge about theirtaxonomy in theregion, and should be takenintoaccountwhenestablishingpoliciesneededtoovercome such an undesirable scenario.First, as typical microlepidopter-ans, gracillariids are not easy to workwith due to theirsmall size,requiringextraabilityfromtaxonomiststomountthem,and especiallyduringdissectionsneededfortaxonomicdescriptions, asforexampletheirgenitalia.Thus,theexistenceofappropriate equipment,suchashighresolutionlightstereomicroscopesand scanningelectronmicroscopefacilities,andcorresponding train-ing,areimportant inthiscase. Second,theydonot seemtobe much attracted to light (Vári, 1961), and thus theuse of light trapsmaybelessefficienttocapturethemasadultscomparedto nocturnalmacrolepidopterans.Thismeansthatothermethodsof capture,includingrearingtheadultsfromhost–plantmines,are desirable,leading tobetterresultsinmany cases.Searchingfor mines,however,isatimeconsumingactivity,andsometimesthe larvaeoccurunderlowdensitiesandaredifficulttoaccess,asfor exampleinthecanopy.Third,recentstudieshaveindicatedthat forsomegracillariidlineages,asforexampleinthegenus Phyl-locnistisZeller,specificvariationisgreaterintheimmaturestages comparedtotheadults(DavisandWagner,2011;Britoetal.,2012). Andthus,locationofhost–plantsandrearingofthesestagesarenot onlydesirablebutrequiredforanaccurateidentificationinsuch cases.

Correspondingknowledgeaboutgracillariidbiologyingeneral isalsoattheinfancylevel intheNeotropics,exceptforspecies ofeconomicimportance,asforexamplethosewiththepotential foruseasbiologicalcontrolagentsofinvasiveplants(e.g.Davis

etal.,1991;McKayetal.,2012).PhyllocnistiscitrellaStainton,a

cos-mopolitangracillariidspeciesrecentlyintroducedintotheregion, becominganimportantpestofCitrusorchardsinmanyNeotropical countries,hasbeenthefocusofseveralstudies(e.g.Vargasetal.,

1998;ChagasandParra,2000;Jahnkeetal.,2005;Santosetal.,

2008a,b).ThebiologyoftheleafminerPorphyroselaminutaClarke,

whichcausesdamageonTrifoliumrepensinUruguaywasexplored

byBentancourtandScatoni(2007).Additionalstudiesthatdonot

focusontaxonomicdescriptionsareuncommonfor Neotropical gracillariidspecieswithoutanyapparenteconomicinterest(but

seeStorey-Palmaetal.,2012,2014;Maita-Maitaetal.,2015).On

theotherhand,theseleafminermothshavebeenstudiedabroad fromseveralbiologicalperspectives,andtheyhavebeensuggested tobeusedasmodelsinstudiesinvolvingtheevolutionofinsect host–plantinteractions. Thisisparticularlyduetothedramatic changesinlarvamorphology(hypermetamorphosis)foundinmost lineages(seeDavis,1987)thatareassociatedwithveryspecialized endophagousfeedinghabits(Wagneretal.,2000;Bodyetal.,2015). Theexistenceofhostshiftshasbeenreported,withstrongevidence fortheexistenceofco-speciationwithhost–plantsinsomecases

(OhshimaandYoshizawa,2006;Ohshima,2008).Also,some

gracil-lariidspecieshaveamutualisticassociationwithplantsbyacting aspollinatorsintheadultstageandfeedingaslarvaeonthe repro-ductivestructuresofsuchplants(Kawakitaetal.,2004;Kawakita

andKato,2006;Zhangetal.,2012).Allthesepervasiveavenues

inresearch,whichmustbeprecededbyasolidtaxonomic back-ground,remaintobeexploredfortheNeotropicalgracillariidfauna ingeneral.

In summary, it was herein clearly demonstrated that the diversityand biology of Neotropicalgracillariids remainlargely unknown,whichisattributedparticularlytotheexistenceofa tax-onomicimpediment,prevailingthroughouttheregion.Thus,the establishmentofscientificpoliciesbygovernmentsofallcountries isurgentinordertospeedupgracillariidtaxonomyandthusto overcomesuchanundesirablescenariointheNeotropics(foran exampleofsuchpolicies,seeAguiaretal.,2009).Finally,itisalso importanttomentionthatasfarasweareconcerned,no Neotropi-calgracillariidspecieshasbeentakenintoaccountuptonowfrom aconservationbiologyperspective.Asalreadymentioned, there aremany speciesinthisfamilythroughoutdifferentgeographic dominionsintheNeotropicalregionthatarespecialistsonrare and/orendemicplants(e.g.Davis,1994;VargasandLandry,2005;

VargasandParra,2005;Mundacaetal.,2013;Britoetal.,2013).By

beingdependentonendemichostplantsataregionalscale,these speciesinparticularareundercomparativelygreaterthreat,since suchplantsareusuallylocalizedindistribution(Lewinsohnetal., 2005).Touncoverfurthersuchkindsofhiddendiversityof gracil-lariidspecies,andthustosetthebasisfortheirconservationbefore extinction,wesuggestendemicplantsinparticularshouldbe prior-itizedonhost-focusedinventoriesfortheirassociatedleafminers intheNeotropics.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WearegratefultoCarlosLopezVaamonde(INRA)forallowingus toaccessbarcodedataintheBOLDSystemGracillariidaeprojects. Thanksarealsoduetwoanonymousreviewersforimportant sug-gestionsmadetothefinalversionofthemanuscript.R.Britoand G.L.Gonc¸alvesweresupported,respectivelybyaCNPqDoctoral ScholarshipandaPostdoctoralCAPESFellowship(PNPD),andO. H.H.MielkeandG.R.P.MoreirabygrantsfromCNPq.Thisarticleis dedicatedtoHéctorA.Vargas(UTA)whoearlyinthisdecadekindly introducedG.R.P.MoreiraandR.BritotothestudyofNeotropical gracillariids.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.rbe.2016.06.002.

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