A new species of Leurocephala Davis & Mc Kay (Lepidoptera, Gracillariidae) from the Azapa Valley, northern Chilean Atacama Desert, with notes on life-history

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REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution

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

Systematics,

Morphology

and

Biogeography

A

new

species

of

Leurocephala

Davis

&

Mc

Kay

(Lepidoptera,

Gracillariidae)

from

the

Azapa

Valley,

northern

Chilean

Atacama

Desert,

with

notes

on

life-history

Cristiano

M. Pereira

a

_,

_Denis

_S.

_Silva

b

_,

_Gislene

_L.

_Gonc¸

_alves

b,c

_,

_Héctor

_A.

_Vargas

c

_,

_Gilson

_R.P.

_Moreira

b,∗

a_Universidade_Federal_do_Rio_Grande_do_Sul,_Instituto_de_{Biociências,}_Programa_de_{Pós-graduac¸}_ão_em_Biologia_Animal,_Porto_Alegre,_RS,_Brazil b_Universidade_Federal_do_Rio_Grande_do_Sul,_Instituto_de_{Biociências,}_Departamento_de_Zoologia,_Porto_Alegre,_RS,_Brazil

c_Universidad_de_Tarapacá,_Facultad_de_Ciencias_{Agronómicas,}_Departamento_de_Recursos_Ambientales,_Arica,_Chile

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23August2016 Accepted2November2016 Availableonline19November2016 AssociateEditor:RodrigoGonc¸alves Keywords: Anacardiaceae Gracillariidmoths Leafminers Microlepidoptera Schinus

a

b

s

t

r

a

c

t

TheNeotropicalmicromothgenusLeurocephalaDavis&McKay,2011(Lepidoptera,Gracillariidae)was originallydescribedtoincludeonlythetypespecies,L.schinusaeDavis&McKay,2011,whoseleaf minerlarvaeareassociatedwithAnacardiaceaeinArgentina,BrazilandParaguay.Anintegrative analy-sisincludingmorphology,lifehistoryandDNAbarcodesequencesrevealedthatspecimenscollectedon SchinusmolleL.(Anacardiaceae)inthecoastalvalleysoftheAtacamaDesertofnorthernChilebelongto asecondspeciesofthisformerlymonotypicgenus.AdultsofLeurocephalachilensisVargas&Moreirasp. nov.arehereindescribedandillustratedinassociationwiththeimmaturestagesandlifehistory,and correspondingphylogeneticrelationshipsareassessedbasedonDNAbarcodesequences.Thisﬁnding providestheﬁrstrecordofLeurocephalafromwestoftheAndesRange,expandingremarkablyits geo-graphicrange.ItissuggestedthattheextentofdiversitywithinLeurocephalaismuchgreaterandthat variationingeographicfactorsandhostplantusemayhavemodeledit,anevolutionaryhypothesisthat shouldbeassessedinfurtherstudies.

Introduction

Gracillariidaeis ahighly diverselineage ofleaf-mining Lepi-doptera,with105recognizedgeneraand1952speciesdistributed worldwide;over180taxahavebeenrecordedintheNeotropical region(DePrinsandDePrins,2016;DePrinsetal.,2016).Recent studieshavesuggestedthatthecomparativelylowdiversityofthe Neotropicalfaunaof Gracillariidaeisanartifact due tothelow samplingeffortsinthisgeographicarea;accordingly,further sur-veysshouldrendermanyadditionalspecies(Leesetal.,2014;Brito etal.,2016).

Theuseinconjunctionofdistinctcharactersandmethodologies tostudy taxonomical problems, since 2005deﬁned as integra-tivetaxonomy,isthemodernbasisofdelimitationanddiscovery of species (Dayrat, 2005). The usefulness of this approach has beenwidelyrecognized,especiallyincasesinvolvingspecieswith closelysimilarmorphology(e.g.,Schlick-Steineretal.,2010;Barão etal.,2014;Kergoatetal.,2015;Kirichenkoetal.,2015),apattern

∗ Correspondingauthor.

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

probablywidespreadamonggeneraofNeotropicalGracillariidae (DavisandWagner,2011).

TheNeotropicalmicromothgenusLeurocephalaDavis&McKay, 2011(Lepidoptera,Gracillariidae)originallyincludedonlythetype species,L.schinusae,whoseleafminerlarvaeareassociatedwith AnacardiaceaeinArgentina,BrazilandParaguay(Davis&McKay, 2011).Molecularphylogeneticanalysesbasedonsequencesof21 nuclearprotein-codinggenesplacedLeurocephalawithinthe Parec-topagroupofGracillariinae(Kawaharaetal.,2011).Thisgroupof lineagesischaracterizedbytheplacementoftheostiumbursaeon theVIIsternumofthefemale,whichissupposedlyahighlydistinct morphologicalapomorphy(Kumataetal.,1988;Kawakitaetal., 2010).

Leurocephalaremainedasamonotypicgenusuntilnow. How-ever,aspartofastudyoftheLepidopteraassociatedwithnative plantsinthecoastalvalleysoftheAtacamaDesertofnorthernChile, adultsofLeurocephalawererecentlyrearedfromleafmines occur-ringonSchinusmolleL.(Anacardiaceae).Thus,representsanovel recordintermsofbothgeographicdistributionandhostplantuse forsuchamicromothgenus.Furthermore,apreliminaryanalysis ofthemorphologyofthemaleandfemalegenitaliaenabledusto hypothesizethatthesespecimenswerenotconspeciﬁcwiththe http://dx.doi.org/10.1016/j.rbe.2016.11.003

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typespecies.Thishypothesiswassubsequentlysupportedbyan integrativeanalysisofmorphology,lifehistory,andDNAbarcode sequences.

Accordingly,theaimofthisarticleis toprovidedescriptions ofallthelifestagesandthelifehistoryofanewspeciesof Leuro-cephalafromtheAtacamaDesert.Inaddition,theﬁrstassessmentof thephylogeneticrelationshipsforthetwospeciesofthisformerly monotypicgenusisprovidedbasedonsequencesofmitochondrial DNA.

Materialandmethods

Specimensusedfordescriptioninthisstudywereeither diss-ected orreared fromleaf minescollectedon S.molle plants in theAzapavalley,AtacamaDesert,northernChile,between2008 and 2016.They were broughtto theentomology laboratoryof theFacultad deCienciasAgronómicas,UniversidaddeTarapacá, Arica,wheretheywereeitherdissectedorrearedinsmallplastic vials.Theseweremaintainedatroomtemperatureandperiodically inspectedforemergedadults,whichwerepinnedanddried.

Morphologicalanalysis

ImmaturestageswereﬁxedinDietrich’sﬂuidandpreservedin 75%ethanol.Fordescriptionsofthegrossmorphology,the speci-menswereclearedina10%potassiumhydroxide(KOH)solution andslide-mountedineitherglycerinjellyorCanadabalsam.

Observationswereperformedwiththeaidofa Leica® M125 stereomicroscope, and measurements were performed using anattachedocularmicrometer(precision=0.01mm).Structures selectedtobedrawnwerepreviouslyphotographedwithaSony®. Cyber-shotDSC-H10digitalcameraattachedtothe stereomi-croscope. Vectorized line drawings were then made with the softwareCorelPhoto-Paint® X7,usingthecorresponding digital-izedimagesasaguide.Atleastﬁvespecimenswereusedforthe descriptionsofeachlifestageorinstar.

For scanningelectron microscopeanalyses, additional speci-mensweredehydratedinaBal-tec®CPD030critical-pointdryer, mountedwithdouble-sidedtapeonmetalstubsandcoatedwith goldinaBal-tec®SCD050sputtercoater.Theywereexaminedand photographedinaJEOL® JSM6060scanningelectronmicroscope attheCentrodeMicroscopiaEletrônicaofUniversidadeFederaldo RioGrandedoSul(UFRGS).

Forplantanatomicaldescriptions,ﬁeld-collectedleafportions ofS.mollecontainingminesofLeurocephalachilensiswereﬁxed in FAA(37%formaldehyde, glacialacetic acidand 50% ethanol, 1:1:18,v/v),andpreservedin70%ethanol.Leafportions contain-ingthedifferentlarvalinstarmorphotypeswereselectedunder a stereomicroscope in thelaboratory. Theywere then progres-sivelyhydrated,immersedin10%potassiumhydroxidefor20min, stainedfor24hwithtoluidineblue(aqueoussolution:200mg/L) andthenmountedwholeinglycerineonslides.Semi-permanent slideswerealsopreparedwithfreehandcrosssectionscutwitha razorblade,usingadditionalminescontaininglarvaeofdifferent agesandpreparedsimilarly.Head-capsuleexuviawerelocatedby transparencyintheslide-mountedminesandmeasuredunderthe stereomicroscopewithanattachedocularmicrometer.

Museumcollections

Abbreviationsoftheinstitutionsfromwhichspecimenswere examinedareasfollows:IDEA,ColecciónEntomológicadela Uni-versidaddeTarapacá,Arica,Chile;LMCI,LaboratóriodeMorfologia eComportamentodeInsetos,UniversidadeFederaldoRioGrande

do Sul,PortoAlegre,Brazil; MNNC,Museo NacionaldeHistoria NaturaldeSantiago,Santiago,Chile.

Molecularanalysis

TotalgenomicDNAwasextractedfromfreshlarvaltissueusing thePureLinkkit(Life,Invitrogen,USA)followingmanufacturer’s instructions.Specimensfromthetypelocalityofthenewtaxon L.chilensis(n=2;Azapa)andoftheonlyspeciesrecognizedinthe genus,L.schinusae(n=4;ParanáandRioGrandedoSul,Brazil)were surveyedtogenerateoriginaldata(Table1).Wealsoincorporated fromtheBOLDSystemandGenbankdatabasesthreeindividuals ofL.schinusaefromMisiones,Argentina.Thisdatasetwasusedto assessthemonophyleticstatusofL.chilensis.Gracillaridspeciesof theSpinivalvaMoreira&Vargas,likelythesisterlineageof Leuro-cephala,andalsoofParectopaClemensandEpicephalaMeyrick,all belongingtothe‘Parectopagroup’(Britoetal.,2013),wereused asoutgroup,thecorrespondingsequencesbeingdownloadedfrom GenBank(Table1).WeampliﬁedtheDNAbarcoderegion(partof themitochondrialcytochromeoxidaseI)including658basepairs, usingprimersandconditions describedbyFolmeretal. (1994). PCRproductswerepuriﬁedusingexonuclease(GEHealthcareInc.) andShrimpAlkalinePhosphatase(SAP),sequencedwithBigDye chemistry and analyzed in an ABI3730XL (Applied Biosystems Inc.).Chromatogramsobtainedfromtheautomaticsequencerwere read andsequenceswereassembled usingthesoftware Codon-Code Aligner(CodonCodeCorporation). Sequencesgenerated in this studyweredeposited in thedatabasesGenBank and BOLD System(Table1).

Correspondingtreewasconstructedusingmaximumlikelihood (ML)methodinthesoftwarePHYML3.0(Guindonetal.,2010). TheprogramjModelTest2(Darribaetal.,2012)wasusedto esti-matethesubstitutionmodel(GeneralTime-Reversible;Rodriguez etal., 1990), following theAkaikeInformationCriterion. Mono-phylyconﬁdencelimitswereassessedwiththebootstrapmethod (Felsenstein,1985)at50%cut-offafter1000bootstrapiterations. WealsoanalyzedthepairwisegeneticdistanceusingtheKimura 2-parametermodel(Kimura,1980)procedure,with1000bootstrap replications,betweenclustersdeﬁnedinthephylogenyand out-groups.

Results

LeurocephalachilensisVargas&Moreirasp.nov.

Typematerial.Maleholotype:Azapa,Arica,Chile,August2015, exleafminerlarvaonS.molle,July2015,H.A.Vargascoll.(MNNC). Paratypes: Two males, two females, same data as holotype (MNNC); two males and three females, same data as holotype (IDEA);onemaleandonefemaleAzapa,Arica,Chile,December 2010,exleafminerlarvaonS.molle,July2010,H.A.Vargascoll. (IDEA);threemalesAzapa,Arica,Chile,November2011,ex leaf minerlarvaonS.molle,October2011,H.A.Vargascoll(IDEA).

Genitaliadissected by H.A. Vargas(HAV) were deposited in IDEA,underaccessionnumbersasfollows,allfromAzapa,Arica, Chile,exleafminerlarvaonS.molle:HAV276,onemale, Decem-ber2010; HAV358,359and 402,three males,November2011; HAV108, onefemale,December 2010;HAV1020 and 1024,two males,August2015;HAV1021and1023,twofemales,August2015. H.A.Vargascoll.ImmaturespecimensofL.chilensisweredeposited inLMCI,dissectedfromleafminesonS.mollefromAzapa,Arica, Chile,August2012,H.A.VargasandG.R.P.Moreiracoll.asfollows: preservedin100%alcoholbelow−10◦_C,_used_for_DNA_extraction

(LMCI191-3);preservedin75%alcohol,usedformicroscopy stud-ies,seveneggs(LMCI191-37),sixﬁrstinstarlarvae(LMCI191-41), twelvelastinstarlarvae(LMCI43),sevenpupae(LMCI191-44).

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Table1

GracillaridspecimensusedinthisstudytoreconstructthephylogeneticstatusandevolutionaryrelationshipsofLeurocephalachilensisbasedon658basepairsofDNAbarcode (cytochromeoxidasesubunitIgene)sequences.

Group Genus Species Voucher Locality Accessionnumber

Genbank BOLDsystem Ingroup

Leurocephala

chilensis LMCI191-3-1 Chile,Azapa KY006921 MISA007-16.COI-5 LMCI191-3-5 Chile,Azapa KY006922 MISA008-16.COI-5 schinusae

DDAV-D546 Argentina,Misiones HM382092 RDOPO384-10.COI-5 DDAV-D547 Argentina,Misiones HM382093 RDOPO385-10.COI-5 DDAV-D576 Argentina,Misiones HM382112 RDOPO414-10.COI-5 LMCI295-19B Brazil,RioGrandedoSul KY006923 MISA009-16.COI-5 LMCI295-19C Brazil,RioGrandedoSul KY006924 MISA010-16.COI-5 LMCI309-01-10A Brazil,Paraná KY006925 MISA011-16.COI-5 LMCI309-01-10B Brazil,Paraná KY006926 MISA012-16.COI-5 Outgroup

Spinivalva

gaucha LMCI164-15 – KC512112 GBGL13506-14.COI-5P

sp. LMCI169-A1 – KC512114 GBGL13508-14.COI-5P

Parectopa ononidis CLV2269 – KP845416 GRSLO654-11.COI-5P

Epicephala sp. E312AK – FJ235388 –

SpecimensofL.schinusaeusedforcomparisonwereeither

diss-ected(immatures)orreared(adults)fromleafminescollectedon

Schinusterebinthifolius byC. M.Pereira (CMP)in Laranjeirasdo

Sul,PR,Brazil,as follows:LCMI 309-1,ﬁve larvaepreserved in

100%alcoholbelow−10◦_C,_used_for_DNA_extraction,_25.VII.2015;

LMCI309-3,onefemale,preservedin70%alcohol,withgenitalia

inslidepreparation(CMP001-16F),07.XI.2015;LMCI309-4,one

male,pinned,withgenitaliainslide(CMP001-18M),07.XI.2015;

LMCI309-5,onemale,in75%alcohol,withgenitaliainslide (CMP-22M),16.VI.2016;LMCI309-6,onefemale,pinned,withgenitaliain slide(CMP-31F),16.VI.2016.Also,ﬁvelastinstarlarvae,preserved

in100%alcoholbelow−10◦_C,_used_for_DNA_extraction,_dissected

fromleafminesonSchinusaff.polygamus, Coxilhadas Lombas,

SantoAntoniodaPatrulha,RioGrandedoSul,Brazil,April2015,

G.R.P.Moreira&S.L.Bordignoncoll.(LMCI295-19).

Diagnosis

Despitetheirmorphologicalandlifehistoryresemblance,the

twospeciesofLeurocephalacanbedifferentiatedbasedon

mor-phologyof theadultand larvalstages, andbytheshape ofthe

mine.TheapexofthesacculusofmalegenitaliainL.chilensisis

providedwithashort,spine-likeprocessthatprojectsupwards,

whichisabsentinL.schinusae;thedorsalsurfaceoftheaedeagus ofL.chilensis issculpturedwithseveralsmalltooth-like projec-tionsontheconcavearea,whichareabsentinL.schinusae.Inthe

femalegenitalia,twosignaarefoundonthecorpusbursaeofL.

chilensis,whileonlyonesignumisfoundinL.schinusae;

further-more,thehorn-likelateralextensionsoftheantrumarelaterally

projectedinL.chilensis, whilethesestructuresareapically pro-jectedinL.schinusaeAtthelarvalstage,theventralplateofthe prothoraxofthelastinstarofL.schinusaeisuniformlysculptured byagreatnumberofgranularprojectionsofsimilarsize,about12

ofwhich areattheposteriormarginoftheplate,almost

touch-inglaterally,whileinL.chilensisthegreatestgranularprojections, almost1.5timesthelengthofthesmallestones,arerestrictedtothe posteriorthirdoftheventralplate,withonlyfourattheposterior

margin,clearlyseparatedfromeachanotherbyadistancesimilar

tothediameteroftherespectiveprojection.Theserpentinemine

constructedbythesecondinstarofL.schinusaehasalittle

blotch-likebroadening ashortdistancefromtheemptychorion,while

broadeningisabsentintheserpentinemineofthesecondinstarof L.chilensis.

Description Adult(Fig.1)

Male.Head.Frontmostlywhitishgraywithbrownishgrayspots closetothecompoundeyes;vertexwhitishgray;maxillarypalpus whitishgray;labialpalpusmostlywhitishgray,secondsegment withbrownish-grayspotsdistally;proboscisshort,naked;antenna filiform,slightlyshorterthanforewing;scapeelongated,brownish graydorsally,whitishgrayventrally;withtwonarrowlongitudinal stripesonthemedialsurface,onebrownishgrayincontactwiththe ventralarea,theotherwhitishgrayincontactwiththedorsalarea; pedicelandtwofirstflagellomereswithcolorationsimilartothe scape,remainingflagellomeresbrownishgray.

Thorax.Mostlybrownishgraydorsallywithafewwhitishgray scales;teguladorsallybrownish,ventrallywhitishgraywithatuft oflongscalesattip;lateralandventralsurfaceswhitishgray. Fore-legmostlywhitishgray,medialsurfaceoffemurandtibiabrownish gray,tibialepiphysiswhitishgray,abrownish grayringatbase ofeachtarsomere.Middlelegmostlywhitishgray,tibiawithtwo brownishgrayrings,tibialspurswhitishgray,tarsomeressimilarto thoseoftheforeleg.Hindlegmostlywhitishgray,abrownishgray ringatbaseofthefemur,twobrownishgrayringsonthetibia, prox-imaltibialspursbrownishgray,distaltibialspursmostlywhitish graywithabrownishgrayringatmiddle,longwhitishgrayhair-like scalesontheanteriorandposteriorsurfaceofthetibia,tarsomeres whitishgray.

Forewing.Length:4.0–4.5mm(n=10).Mostlybrownishgray;a distinctivewhitetransversestripearisesinthemiddleofthecoastal andhindmargins,slightlyprojectedapicallyatthelongitudinalaxis ofthewing,sometimesinterruptedbygroundcolorscales;ashort oblique,apicallyprojectedstripearisesfrom3/4ofthehindmargin reachingthelongitudinalaxisofthewing;adistinctiveblackish graydotsubapically;fringearoundapexshort,concolorwiththe wing,asmallapicaltuftofplainscales;fringeonhindmarginwith longhair-likebrownishgrayscales.VenationasdescribedbyDavis etal.(2011)forL.schinusae.

Hindwing. Length: 3.2–3.4mm (n=10). Uniformly brownish graywithconcolorousfringeoflonghair-likescales.Venationas describedbyDavisetal.(2011)forL.schinusae.

Abdomen.Mostlybrownishgraydorsally,withoblique segmen-talstripesofwhitishgrayventrallyuntilsegmentVI,completely whitishgrayventrallyatapex.SegmentVIIwithtergumand ster-numreducedtoﬁnetransversalstripes.SegmentVIIIwithsternum asahood-likeslightlysclerotizedplate;sternumVIIIasaslightly

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Figs.1–7.Adultandlife-historyofLeurocephalachilensisontheabaxialsurfaceofSchinusmolleleaves:(1)pinned-driedmale,dorsalview;(2)egg;(3)freshlyhatchedﬁrst instarlarva,seenbytransparencewithinthemine(emptychoriumisindicatedbyarrowaside);(4)middleagemine(arrowindicatestheegg-choriumatthebeginningof themine;lettersandassociateddashed-linescorrespondtothelocationsoftissuesections,presentedintransversalviewinFigs.33–35);(5)old,emptymine(setaindicates exitoflastinstarlarva);(6)pupalcocoon,ornamentedwithbubbles(arrow);(7)pupalexuvium,partiallyprotrudingfromthecocoon.Scalebars=1,0.5,0.5,5,5,3and 1mm,respectively.

sclerotizedﬁnetransversalstripe.Membranousareabetween seg-mentVIIandVIIIlaterallywithapairofhair-likecoremataatthe apexofaﬁnger-likemostlymembranoselobeprovidedwitha rod-likesclerite.SegmentVIIIwithasecondpairofhair-likecoremata onthelateralapexofthesternum.

Male genitalia(Figs. 8–11). Uncus absent. Tegumen as two ﬁnestripestouchingdorsally.SaccusU-shapedinposteriorview, ventralareawiththeanteriormarginslightlyprojectedforward, posteriormargin slightlyconvex.Gnathos (Fig.8)astwoshort, slightlysclerotizedﬁnger-likelobes.Valva(Fig.8)broadlyjoined basallytotheposteriormarginofthelateralarmsofthesaccus; costalmarginstraight;cucullusmostlymembranous,ventral mar-ginparalleltothecosta;sacculuswellsclerotized,basalpartbroad, triangle-like,delimitedbyabroadconcavityontheventralmargin, withdistalpartstraight,down-curved,slightlydilatedsubapically, bearingashortspine-likeprojectionatapex(Fig.9).Transtillaasa

slightlysclerotizedtransversalbandjoiningthebaseofthecostal marginoftherightandleftvalvae.Juxtaabsent.Aedeagus(Fig.10) abitshorterthanvalva,withinsertionoftheductusejaculatorius dorsally,closetothemiddle;basalhalfforwarddirected,tipblunt, diameterincreasingtowardthemiddle;distalhalfupcurved,with lateralsidesslightlyasymmetricalatapex,dorsalsurface sculp-turedbyseveralsmalltooth-likeprojectionsontheconcaveportion (Fig.11).Asmallscleritejoineddorsallyonthemiddleofthe aedea-gus.Cornutiabsent.

Female.Similartomaleinsizeandcolor.

Femalegenitalia(Figs.12–14).Anteriorandposterior apophy-seswellsclerotized,withlengthsimilartothesternumVII.Ostium bursaebroad,coveringcompletelytheposteriormarginofthe ster-numVII.Antrumbroad,sclerotized,trapezoid-likeinventralview, lengthabouthalfofthesternumVII,cephalicsideabouthalfof theposteriorside,withtwolaterallydirectedhorn-likeextensions

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Figs.8–14.GenitaliamorphologyofLeurocephalachilensisunderlightmicroscopy:(8)male,posteriorview;(9)distalendofvalva,indetail(enlarged,rectangulararea markedinFig.8);(10)aedeagus,lateralview;(11)dorsalspinesofaedeagus,indetail(enlargedareaindicatedbysetainFig.10);(12)female,ventralview;(13,14)signa, indetail(indicatedbyopenandclosedarrowsinFig.12).Scalebars=200,50,100,20,200,50and50␮m,respectively.

onthemiddleof thelateralsides.Ductusbursae membranous, withsimilarlengthtotheantrum.Corpusbursaeelliptical, elon-gated,mostly membranous,withtwo signa(Fig.12);thelarger (Fig.13)oneontheventralsurface,triangle-like,withanarrow sclerotizedlongitudinalstripewiththecephalictipprojected for-wardasashortspineintothecorpusbursae;thesmaller(Fig.14) oneonthedorsalsurface,elliptical,elongated,lengthabout3/4of thelarger,withalongitudinalsclerotizedstripe.Ductusseminalis basallyinsertedontheventralsurfaceofthecorpusbursae(Fig.12).

Egg(Fig.2).

Roundandﬂat,withatranslucentchorion,allowingby trans-parencevisualizationoftheembryounderdevelopmentwithin.

Larva(Figs.15,16,19–26).

Therearethreemorphotypesandfiveintars,whicharesimilar inmorphologytothosedescribedbyDavisetal.(2011)forthetype species,exceptforthelastinstardescribedbelow.Thefirstinstar isofa“sap-feeding”type,havingmandiblesmodifiedforslicing theepidermiscells,differingfromtheremainingfourinstarsthat aretissue-feeders,whichhavemandiblesusedforchewingtheleaf parenchyma.Thesecanbeidentifiedbymeasuringtheirhead cap-sulewidth,whichdonotoverlapinsize(Table2).Corresponding exponentialgrowthcurveforthefourtissue-feedinginstarsofL. chilenisrearedonL.mollewas:y=0.0494e0.4787x_;_n₌_50;_r₌_0.98;

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Figs.15–18.Lastinstar(15,16)andpupal(17–18)morphologiesofLeurocephalachilensisunderlightmicroscopy,indorsalandventralviews,respectively.Scalebars=0.5mm. Lastinstar(Figs.15,16,19–26).Maximumbodylength=5.5mm

(n=10).Head(Figs.15,16,19,20).Brown,semicircularindorsal view,vertexpartiallycoveredbytheprothorax,slightlydepressed dorsoventrally,mostlysmoothwitharhomboid-likeareacovered byshortspine-likemicrotrichiabetweenthefrontoclypeousand thestemmata;epicranialnotchU-shaped,broadanddeep; fron-toclypeousrectangle-like,abouttwotimeslongerthanwide;six

circularstemmata,withstemmata1and2closetosetaA3, stem-mata3–5inadiagonallineventraltosetaA1,stemma5slightly displacedtotheventralsurfaceofthehead,stemma6isolatedon theventralsurfaceofthehead,almostequidistanttosetaeS2and SS2.Antennathree-segmented;ﬁrstsegmentannular,second seg-mentcylindrical,abouttwotimeslongerthantheﬁrstsegment, withsensillae distally,and third segmentcylindrical, similarin

Figs.19–26. LastlarvalinstarofLeurocephalachilensisunderscanningelectronmicroscopy:(19–20)head,underdorsalandlateralviews,respectively;(21)antenna,dorsal; (22)callumofﬁrstabdominalsegment,ventral;(23)sternalprothoracicplate,ventral;(24)mesothoracicleg,lateral;(25)pseudopodiumofﬁfthabdominalsegment,ventral; (26)spiracleofeighthabdominalsegment,lateral.Scalebars=100,50,20,50,25,50,50and10␮m,respectively.

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Table2

VariationinsizeofheadcapsulewidthamonginstarsofLeurocephalachilensisrearedonSchinusmolle(n=10perinstar).

Instar Headcapsulewidth(mm)

Mean±standarderror Range Growthrate

I 0.127±0.026 0.117–0.143 –

II 0.127±0.003 0.117–0.143 –

III 0.211±0.009 0.182–0.260 1.661

IV 0.340±0.007 0.312–0.377 1.611

V 0.534±0.005 0.507–0.559 1.571

lengthtosecondsegment,aboutahalfthediameterthesecond

segment,withsensillaeatapex.Mouthpartsofthechewingtype;

labrumbilobed,fourshorthair-likesetaeontheexternalsurface;

epipharyngealspinesclosetothedistalmarginofthelabrum,one

pairofplain epipharyngealsclerites closetothe each groupof

epipharingealspines;mandible well-developed,withﬁvedistal

cusps;maxillawithwell-differentiatedgaleaandpalpus;labium

withawell-developedcylindricalspinneretatapexandapairof

bi-segmentedpalpilaterallytothespinneret;hypopharynxprovided

withlonghair-likeprojections.Chaetotaxy.AFgroupbisetose,AF1

andAF2asmicrosetaeclosetothedorso-medianapexofthepatch

ofmicrotrichiae.Agroupbisetose,A1closetoantenna,A3dorsal

tostemma,abouttwotimesthelengthofA1.CDgroupof

microse-taetrisetose.Cgroupunisetose,C1asamicroseta.Fgroupofsetae absent,Faporepresent.Lgroupunisetose,L1asashorthair-like setaposteroventraltoA3.MGgroupofmicrosetaebisetose.Pgroup bisetose,P1atmiddleofthepatchofmicrotrichiae,sizesimilarto A1,P2greatlyreduced,slightlygreatestCDsetae.Sgroupbisetose,

S1abouthalfwaybetweenstemmata1and3,S2abouthalfway

betweenstemmata1and6.SSgrouptrisetose,SS1ventromedial

tostemma5,SS2abouthalfwaybetweenstemmata4and6,SS3

posteromedialtostemma6.Thoraxandabdomensculpturedby

shortspine-likemicrotrichiae.

Thorax(Figs.15,16,22–24).Prothorax.Dorsalshieldgrayish

brown,smooth,intheformoftwosubtriangularplatesseparated mediallybyanarrowmembranousstripe;eachplatewiththe ante-riormarginslightlyconvex,medialmarginstraight,lateralmargin widelyconcaveclosetotheanteriormarginandalmostparallel tothemedialmarginonthedistal 2/3,posteriorvertex widely rounded.Anellipsoidshieldpostero-ventraltoSVgroup.Ventral shieldsquare-like,lateralsidesslightlyconcaveclosetotheanterior margin,posteriormarginslightlyconvex;sculpturedbygranular projectionsvariableinsize,thelargestrestrictedtotheposterior thirdoftheplate,clearlyseparatedfromeachotherbyadistance similartothediameteroftherespectiveprojection.Acircular spira-clewithslightlyelevatedperitremalaterallyclosetotheposterior marginofthesegment.Alongitudinallyorientedcallus-like struc-turebetweenthelateralmarginofthedorsalshieldand theSV group;another callus-likestructure postero-lateralto thecoxa. Chaetotaxy:Dgroupbisetose,D1greatlyreduced,onthe poste-riorhalfofthedorsalshieldclosetothelateralmargin,D2about threetimesthelengthofD1betweenthedorsal shieldandthe callus-likestructure.XDgroupbisetose,XD1anteriortoD2,XD2 ventraltoXD1.SDgroupbisetose,lateroventraltothecallus-like structure,SD1similarinsizetoD2,SD2about4–5timeslonger SD1.Lgroupbisetose,similartoSD2insize,clearlyanteriortothe spiracle.SVgroupbisetose,antero-dorsaltotheellipsoidshield.V groupunisetose,V1betweenthecoxaandtheventralshield.

Mesoandmetathoraxwithoutdorsal,lateralorventralshields. A transversally oriented callus-like structure anterior to D2; anothercallus-likestructurepostero-lateraltothecoxa. Chaeto-taxy: D group bisetose, D1 anterior and D2 posterior to the callus-likestructure.SDgroupbisetose,ventro-lateraltothe callus-likestructure.Lgroupbisetose.SVandVgroupsunisetose.Legs moderatelywelldeveloped,bearinglargetarsalclaws.

Abdomen(Figs.15,16,25,26).Allsegmentsbearingdorsaland ventralsmoothshieldsvaryinginshape.DorsalshieldofA1–6in theformofasmallirregularplate,thoseofA7andA8little devel-oped,intheformofa smalldot; dorsalshieldofA9ellipsoidal, well-developed,transversallyarranged;dorsalshieldofA10inthe formoftwowidelyseparatedplatesclosetotheposteriormargin ofthesegment.VentralshieldofA1–2andA8circle-like,little evi-dentonA3–5,betweentherespectiveprolegs;thoseofA6–7similar insizeand shapetothose ofA3–5;ellipsoidalonA9, transver-sallyarranged,smallerthanthedorsalshieldofthesamesegment; absentonA10.Chaetotaxy:A1–2,6–7:Dgroupbisetose,D1 ante-riorandD2posteriortothecallus-likestructure.SDgroupbisetose, SD1latero-ventraltothecallus-likestructure,SD2greatlyreduced, dorsal tothe spiracle. L group unisetose,L1 postero-ventral to thespiracle.SVbisetose,dorso-lateraltothecalluslikestructure. Vgroupunisetose,V1betweenthecallus-likestructureandthe ventralshield.A3–5:similartotheprecedingsegments;SV dorso-lateraltotheproleg.A8similartopreceedingsegment,exceptthat SVgroupunisetose.A9similartopreceedingsegment,exceptthat SDgroupunisetose.A10withDandSDgroupsbisetose;D1 ante-riorandD2posteriortothedorsalshield,SD1,SD2onthemargin ofthelateralpartofthedorsalshield;L,SVandVgroups unise-tose.Spiraclesround,withmoderatelyelevatedperitreme.Prolegs presentonA3–5andA10;crochetsarrangedinastaggeredcaudal varyingfrom10to16hooksonA3–5(Fig.25);A10withcrochets reducedto4hooks.

Pupa(Figs.17,18,27–32).

Maximumbodylength:4mm(n=10).Similarincolorand gen-eralappearancetoL.schinusae,asdescribedbyDavisetal.(2011) forL.schinusae.Minutelyanddenselyspinose,particularlythe dor-salabdomen.Cocooncuttersubtriangular,withouterridgehaving numerousminuteteeth,thecentralthreeteeththelargest. Anten-naelong,surpassingtheabdomeninlength.Labialpalpica.1/3the lengthofproleg.Proboscisaslongastheproleg.Forewings nar-row,wellseparated,extendingtoabdominalsegmentA7.Hindlegs extendingto abdominal segment 9+10. SetaeD1, SD1, and L1 present onA1–7;only SD1presentonA8. Abdominalspiracles round, with elevatedperitreme (Fig.31). Cremaster formedby threepairsof slightlycurvedspines,two lateraland onedorsal (Fig.32).

Etymology.Thespeciﬁcepithetisderivedfromthecountryof thetypelocality.

Distribution.L.chilensisisknownonlyfromAzapa(type local-ity)ValleyintheAtacamaDesertofnorthernChile.

Lifehistory(Figs.2–7).Eggsarelaidindividually(Fig.2)onthe adaxialsurfaceoftheleaflet,mostlyclosetothemainleafletvein. Afterhatching,thefirst,sap-feederinstarintroducesitselfintothe leaflet,constructingasmall,superficial,blotch-likemineashort distancefromtheemptychorion(Fig.3).Thefecesaredeposited intothelumenofthechorionduringthetimethattheanalapexof thefirstinstarremainsthere.Thesecondinstarconstructsa nar-rowserpentinemine(Fig.4)ontheadaxialsurfaceoftheleaflet. Subsequentinstarsconstructaconspicuousblotchedmineonthe adaxialsurfacewhosediameterincreaseswiththesequenceofthe instars,generallycoveringmorethan50%oftheleafletwhenfully

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Figs.27–32. PupaofLeurocephalachilensisunderscanningelectronmicroscopy:(27)head,ventralview;(28)“cocoon-cutter”indetail,ventral;(29)labrumindetail,ventral; (30)sixthandseventhabdominalsegments,dorsal,(31)spiracleofeighthabdominalsegment,lateral;(32)lastabdominalsegments,dorso-posterior.Scalebars=100,25, 100,100,5and100␮m,respectively.

Figs.33–35. VariationintransversalhistologicalsectionsofLeurocephalachilensisonSchinusmolleleaf,accordingtolarvalontogeny;(33)ﬁrst,sapfeedinginstar(position indicatedbyletter“a”inFig.4);(34)earlytissue-feedinginstar(letter“b”inFig.4);(35)lasttissue-feedinginstar(letter“c”inFig.4).Ab,abaxialsurfaceofepidermis;Ad, adaxialsurfaceofepidermis;Lm,leafmine;Ep,epidermis;Pp,palisadeparenchyma;Sp,spongyparenchyma.Scalebars=0.2mm.

developed(Fig.5).Alargenumberoffecesaregluedontheinternal sideofthe“epidermal”surfaceoftheblotchmine.Thefully devel-opedfifthinstarmakesashortslitonthemarginoftheminetoexit fromittosearchforasiteforpupation.Pupationmostlyoccurs ontheabaxialsurfaceofa leafletofthesameplant;previously thefifthinstarconstructsanellipsoidalsmoothcocoon,generally withoneofthelateralmarginstouchingthelateralmarginofthe leaflet,andexternallydepositssilkbubbles(Fig.6)secretedbythe anus.Whenmetamorphosisiscompleted,thepupamakesa slit onthecocoonusingthecephaliccocoon-cuttertoenabletheadult toemerge,afterwhichthepupalexuviumtypicallyappears pro-trudedwiththeposteriorbodyportionremaininginthecocoon (Fig.7).Thefirst,sap-feedinginstarfeedsonlyontheepidermis cells(Fig.33),whiletheotherfourtissue-feedinginstarsfeedon thepalisadeparenchyma,leavingthespongyparenchymaintact (Figs.34,35).

Molecularanalyses.Tworeciprocallymonophyleticlineages werefoundwithinthegenusLeurocephala: thecurrently recog-nizedL.schinusaeandthenewspeciesL.chilensis(Fig.36).Genetic divergenceestimatedbetweentheselineageswas12%(±1%)andof bothspeciestogetherversustheoutgroups(Spinivalva,Parectopa andEpicephala),variedfrom16to18%(±1%,foranycomparison). Discussion

Morphologicalresemblancewiththetype speciesenablesus toincludethisnewgracillariidspeciesintheformerlymonotypic NeotropicalgenusLeurocephala.Nodifferenceswerefoundinbody colororwingvenationintheadultstageforthetwospecies.In addition,wewereunabletoseparateL.chilensisfromL.schinusae basedontheexternalmorphologyofthepupa;also,nodifferences werefoundbetweentheirlastlarvalinstarchaetotaxy.However,

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Fig.36.MaximumlikelihoodphylogenetictreeofLeurocephalainferredbasedon658bpofDNAbarcodesequences(cytochromeoxidasesubunitIgene).Asteriskabove branchindicatebootstrapsupportlowerthan50%.SpeciesofSpinivalva,ParectopaandEpicephalawereusedasoutgroup;seeTable1andtextforfurtherdescription.

asalreadymentioned,highlyconstantdifferenceswerefoundto separatethetwo speciesin theadultgenitalia,and inthe ven-tralprothoracicplateoflarvae.Inaddition,subtledifferenceswere foundintheshapeoftheminebuiltbythesecond,tissue feed-inginstar.Thesemorphologicaldifferenceswerereinforcedbythe molecularphylogeneticanalysis,asthetwospeciesweregrouped ascloselyrelated,butreciprocallymonophyletictaxathatdiverge fromeachotherbyca.12%(±1%)inDNAbarcodesequences.

The known distribution of Leurocephala was previously restrictedtothesouthernpartoftheAtlanticForest,including por-tionsofArgentina,BrazilandParaguay(Davisetal.,2011).Thus thediscoveryofL.chilensisintheAtacamaDesertprovidesthefirst recordofLeurocephalafromwestoftheAndesRange,expanding remarkablythegeographicrangeofthegenus.Additionalsurveys willbeneeded tocharacterize adequatelythe host(s)and geo-graphicrangeofL.chilensis.Itisverylikelytofindthisspeciesin othercoastalvalleysoftheAtacamaDesert,eitherinnorthernChile orsouthernPeru,inthesamewaythatotherGracillariidaewere foundinthishyperaridarea(Maita-Maitaetal.,2015),sincethe hostplantS.molleiswidespreadintheregion.ThetypespeciesL. schinusaeisabletobreedonseveralspeciesofSchinus,andalsoon speciesoftwoothergenerainAnacardiaceae(McKayetal.,2012). AdultsofLeurocephalaspecimenswererecentlyrearedfrom leaf-minescollectedonS.polygamus(Cav.)Cabrera,andminestypical ofLeurocephalawerealsofoundonleavesofSchinuslatifolius(Gill. exLindl.)EnglerintwolocalitiesofCentralChilenotincludedin thepresentstudy.Thesenewfindingssuggestthatallthespecies ofSchinusdistributedinsouth-centralChile(seeRodríguezetal., 1983)shouldbesurveyedtoknowtheeffectiverangeanddiversity ofLeurocephalawestoftheAndes.Inouropinion,furthertaxonomic decisionsonthisregardshouldwaituntilcomparative,finescale analyseswithanintegrativetaxonomicapproachareperformed, andafterabroadersurvey forthismicromoth genusbeen con-ductedthroughoutitsrangeinSouthAmerica.AccordingtoDavis etal.(2011),thetypematerialusedintheoriginaldescriptionof L.schinusaewasrearedfromminescollectedonS.terebinthifolius Raddi,butsimilarmineswerealsofoundbythemonother Schi-nusspecies,andalsoonanadditionalanarcadeacean,Astronium balansaeEngl.

Additionalmonospeciﬁc genera of Neotropical Gracillariidae havebeen described duringthe lastdecades (e.g.,Davis, 1994; VargasandLandry,2005;VargasandParra,2005;Mundacaetal., 2013a,b),whosemonotypicstatusmightbeassociatedwithlow samplingeffort.ThediscoveryofasecondspeciesofLeurocephala withmorphologyveryclosetothetypespeciesbutwithrelative highlevelofgeneticdivergence,andwithlarvaefeedingonaplant

ofthesamefamilyasthetypespeciessuggeststhatanalogous stud-iesshouldbecarriedoutalsofortheseotherapparentlymonotypic genera.Inotherwords,host-plantshiftsbetweencloselyrelated plantsandtheexistenceofcrypticspeciesinassociationshouldbe furtherexploredinsuchNeotropicalgracillariidgenera,asalready suggestedbyBritoetal.(2013).

Finally, ourresultsillustrate theexistence of wide diversity offeedinghabits atfinescale throughoutontogenyand among gracillariidlineages,whichshouldbebetterexplored.Asfarwe areaware,feedingconfinedtosingle-celledepidermis,as demon-stratedhereforthefirstsap-feedinginstarofL.chilensishasnot beendescribedforanyspecieswithintheParectopa group.This veryspecializedfeedingbehaviorwasdemonstratedforthe sap-feedinglarvaeofPhyllocnistiscitrellaStainton(Achoretal.,1997) andMarmaraarbutiellaBusck(Wagneretal.,2000).Sap-feeding instarsofothergracillariidsaresupposedlyassociatedprimarily withtheouterlayersofparenchyma(e.g.Britoetal.,2012). Feed-ingonpalisadeparenchymaisrestrictedtotissue-feedinginstars inL.chilensis,whichisalsothecaseforalltissue-feedinginstars ofSpinivalvagauchaMoreira&Vargas,asdescribedbyBritoetal. (2013).Ourdatagivefurthersupportinthesensethatthethree larvalmorphs(sap-feeder,apodalandlegged)Leurocephalafound (seeDavisetal.,2011)mightbeassociatedwiththethreemining typesdescribedhere(theepidermic,serpentineandblotchtypes, respectively).

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest. Acknowledgements

WearegratefultoThalesR.O.Freitas(UFRGS)andtheCentro deMicroscopiaEletrônica,UFRGS,forlaboratoryfacilitiesusedin themolecularanalysesandscanningelectronmicroscopy, respec-tively.WethankDavidLees(NHMUK)andDonaldDavis(USNM)for allowingustoaccessbarcodedatainBOLDSystemGracillariidae projects,andLafayetteEatonforeditingthetext.Thanksarealso dueLuizA.CamposandLucasA.Kaminski(UFRGS),GermánSan Blas(CONICET)andtwoanonymousrefereesforsuggestionsthat improvedtheﬁnalversionofthemanuscript.M.PereiraandG.L. Gonc¸alvesweresupported,respectivelybyaCNPqDoctoral Schol-arshipandaPostdoctoralCAPESFellowship(PNPD),G.R.P.Moreira bygrantsfromCNPq,andH.A.VargasbygrantDGI9711-12from theUniversidaddeTarapacá.

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