Two new species of Mycodrosophila (Diptera, Drosophilidae) proposed by molecular and morphological approaches, with a key to American species

(1)

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

REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution

Systematics,

Morphology

and

Biogeography

Two

new

species

of

Mycodrosophila

(Diptera,

Drosophilidae)

proposed

by

molecular

and

morphological

approaches,

with

a

key

to

American

species

João

Junges

a

_,

_Marco

_Silva

_Gottschalk

b

_,

_Elgion

_Lucio

_da

_Silva

_Loreto

a

_,

_Lizandra

_Jaqueline

_Robe

a,c,∗

a_Programa_de_Pós_Graduac¸_ão_em_{Biodiversidade}_Animal,_Universidade_Federal_de_Santa_Maria,_Santa_Maria,_RS,_Brazil b_Departamento_de_Ecologia,_Zoologia_e_Genética,_Instituto_de_Biologia,_Universidade_Federal_de_Pelotas,_Pelotas,_RS,_Brazil

c_Programa_de_{Pós-Graduac¸ão}_em_Biologia_de_Ambientes_Aquáticos_{Continentais,}_Instituto_de_Ciências_Biológicas,_Universidade_Federal_do_Rio_Grande,_Rio_Grande,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29August2015 Accepted12November2015 Availableonline14December2015 AssociateEditor:GustavoGraciolli Keywords:

Amazonianforest

CytochromeoxidasesubunitI DNAbarcoding

Mycophagousdrosophilids Pampabiome

a

b

s

t

r

a

c

t

Thereareapproximately130speciesofMycodrosophilaOldenberg,1914worldwide,althoughonlynine specieswererecordedinAmericancountriessofar,threeofwhichareexclusivelyNearctic,ﬁve exclu-sivelyNeotropicalandonefoundinbothbiogeographicregions(Mycodrosophilaprojectans).Suchasmall numberofAmericanspeciesislikelyaconsequenceofcollectingbias,whichfavorsthecaptureof fru-givorousdrosophilids,andtothegeneralabsenceofNeotropicalMycodrosophilastudiesinthelast50 years.Here,wedescribetwocommonlysampledspeciesofMycodrosophilafromtheAmazonianand PampaBrazilianbiomes,whichsharemorphologicalsimilaritieswithMycodrosophilaneoprojectansand M.projectans,respectively.Wecomparedsequencesofthemitochondrialgenecytochromeoxidase sub-unitI(COI),externalmorphologycharacteristicsandmaleterminaliaamongthesespecies.Basedona DNAbarcodingapproachcoupledtomorphologicaldifferences,weproposedthedelimitationoftwo newspecies,Mycodrosophilahofmannisp.nov.andMycodrosophilavalentaesp.nov.Anupdatedkeyto identifyingNeotropicalandNearcticMycodrosophilaspeciesisalsoprovided.

Introduction

MycodrosophilaOldenberg,1914isusuallyknownforits associ-ationwithfungalfructiﬁcationbodiesofthegenusPolyporusandis embeddedwithintheZygothricagenusgroup(WheelerandTakada,

1963;Bock,1980;Grimaldi,1990).Therearealmost130described

Mycodrosophila species (Bächli, 2015) and the genus is dis-tributedworldwide,althoughmostofthedescribedMycodrosophila species occurinAfrica, Asiaand Australia (Burla, 1954; Okada,

1956,1965,1968,1986;Bock,1980).Sixspecies(Mycodrosophila

brunnescens,Mycodrosophilaelegans,Mycodrosophilaneoprojectans, Mycodrosophilanigropleura,Mycodrosophilaprojectansand Myco-drosophilapseudoprojectans)havebeenrecordedfortheNeotropics, mostlyinCentralAmericaandBrazil,andallofthemweredescribed orredescribedinWheelerand Takada(1963).Infact,according

toGrimaldi(2010),Wheelerand Takada’sstudycan be

consid-eredthemostimportantworkforidentifyingAmericanspeciesof

∗ Correspondingauthor.

E-mail:lizbiogen@gmail.com(L.J.Robe).

Mycodrosophilabecauseitcharacterizesallspeciesthathavebeen reportedfromthiscontinent,andtherehasbeennootherstudy encompassingthesystematicsortaxonomyofMycodrosophilain thisregionsincethen.Suchadelay,associatedwiththe straight-forwardfruitbaitbiasindrosophilidsampling,canberesponsible forthescarceknowledgeregardingtheNeotropicalMycodrosophila fauna.

Here,wereportthecollectionoftwoMycodrosophila morpho-types,putativelycommontotheBrazilianAmazoniaandPampa biomes, which could not be correctly matched to any species inthekey providedbyWheelerand Takada(1963).After com-paring molecular sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene and re-evaluating the morpho-logical patterns among these and other previously described species, we were able to validate these as two new species. Thus,in thismanuscript,we providethevalidation oftwonew Mycodrosophila in a DNA barcoding like analysis and achieve their description using external morphology and male termi-nalia illustrations. Moreover, we also provide an update to

Wheeler and Takada (1963) key to American Mycodrosophila

species.

http://dx.doi.org/10.1016/j.rbe.2015.11.008

(2)

Fig.1.FliesrestingoverfungalfructiﬁcationbodiesofMycenasp.(Pers.)Roussel 1806(Agaricales,Mycenaceae).

Materialandmethods

DNAmanipulation

Mycodrosophilaspecimenswerecollectedflyingoverorresting onfungifructificationbodies(Fig.1)usingamanualentomological aspirator(Machadoetal.,2014)inforestedareasoffive munici-palitiesdistributedalongAmazoniaandPampabiomes(Table1). Theidentificationwasperformedbyanalysisofexternal morphol-ogyandmaleterminaliapatterns,followingWheelerandTakada

(1963).

TotalDNAwasextractedfromeachof14individuals(Table1) usingNucleoSpinTissueXSkit(Macherey-Nagel,Düren,Germany) andpolymerasechainreactions(PCR)wereperformedusingthe TYJ1460andC1N2329primersdescribedbySimonetal.(1994), for theampliﬁcation of approximately900bpof theCOI mito-chondrial gene. PCRproducts werepuriﬁed using a solutionof polyethyleneglycol(PEG)13%and NaCl1.6Mandthendirectly sequencedinaMegaBace500automaticsequencer,usingthe DYE-namicET®SequencingKit(Amersham,GEHealthcare,ChalfontSt.

Giles,UK)accordingtothemanufacturer’sprotocol.Both,forward andreversesequencesweredetermined,sothateachbasewasread atleasttwice.

Moleculardatamatrixassembly

Thesequencessoobtainedwereassembled,visualizedand cor-rectedin theGAP4 programfromtheStaden Package(Staden, 1996),andalignedusingtheClustalWalgorithmasimplemented

inMega5.0(Tamuraetal.,2011).Table1presentsGenBank

acces-sionnumbersforthe14newCOIMycodrosophilasequences,which wereaddedtotheorthologoussequencesfromtheNearctic Myco-drosophila dimidiataand Mycodrosophila claytonae.Additionally, COIsequenceswerealsodownloadedfromGenBankfordifferent species ofAcletoxenus, Amiota,Leucophenga and Ceratitis,which wereusedasoutgroupsbecausetheybelongtodifferent Drosophil-idaesubfamiliesoreventodifferentDipteranfamilies.

Molecularphylogeneticanalyses

The phylogenetic analyses were performed using different methods/algorithms and softwares: Bayesian analysis (BA), as performedinMrBayes3.2.1(Ronquistetal.,2012);maximum like-lihood(ML)analysis,asimplementedinPhyML3.0(Guindonand

Gascuel,2003);maximumparsimony(MP)analysis,asexecutedin

Paup4.0b10(Swofford,2003);andNeighbor-Joining(NJ)analysis, asachievedinMega5.0.BAanalysiswasperformedusing1,000,000 generationsofMarkovChainMonteCarlo(MCMC)search,adopting theGTR+G+ImodelselectedbytheAkaikeinformation(AIC) evalu-ationcriterionimplementedinMrModelTest2.3(Nylander,2004), withtreesamplingevery100generationsanddiscarding25%of theinitialresultsasburn-in.TheMLanalysiswascarriedoutwith theGTR+G+ImodelselectedbytheAICcriteriainModelTest3.7

(PosadaandCrandall,1998),usinganoptimizedNJstartingtree.

TheMPtreeswereinferredbyheuristicsearchwithTree Bisec-tionandReconnectionbranchswappingoptimizationappliedto agroupof100randomstepwiseadditionstartingtrees.Finally, theNJalgorithmwasimplementedusingtheTamura–Nei substi-tutionmodel(TamuraandNei,1993)(themostsimilartoGeneral TimeReversible(GTR)amongMega 5.0optionsavailableforNJ

Table1

Taxonomicposition,geographicalsourceandCOIsequencesGenBankaccessionnumbersforMycodrosophilaspeciesandoutgroupsusedforthemolecularphylogenetic reconstruction.Mycodrosophilasp.1,M.hofmannisp.nov.;Mycodrosophilasp.2,M.valentaesp.nov.

Family Subfamily Species Origin Accessionnumbera

Drosophilidae Drosophilinae Mycodrosophilahofmannisp.nov.1 ColoradodoOeste,RO,Brazil KC987477 Mycodrosophilahofmannisp.nov.2 ColoradodoOeste,RO,Brazil KC987478 Mycodrosophilavalentaesp.nov.1 SantaMaria,RS,Brazil KC987479 Mycodrosophilavalentaesp.nov.2 RioGrande,RS,Brazil KC987481 Mycodrosophilavalentaesp.nov.3 SantaMaria,RS,Brazil KC987480 Mycodrosophilavalentaesp.nov.4 RioGrande,RS,Brazil KC987482 Mycodrosophilavalentaesp.nov.5 RioGrande,RS,Brazil KC987483 M.elegans1WheelerandTakada,1963 DiamantedoNorte,PR,Brazil JX497750 M.elegans2 TeodoroSampaio,SP,Brazil KC987476 M.projectans1WheelerandTakada,1963 SantaMaria,RS,Brazil JX497751 M.projectans2 SantaMaria,RS,Brazil KC987484 M.projectans3 SantaMaria,RS,Brazil KC987485 M.projectans4 SantaMaria,RS,Brazil KC987486 M.projectans5 SantaMaria,RS,Brazil KC987487 M.claytonae LeonCo.,Florida,USA GU597470

M.dimidiata – EU493682

Steganinae AcletoxenusindicusLoew,1864 Hainan,Qionghai,China HQ701131 AmiotasetigeraMalloch,1924 – EU493568 LeucophengaalbfasciataMacquart,1851 – EU493569 L.angustaOkada,1956 Hachioji,Tokyo,Japan HQ842780 L.quadripunctatadeMeijere,1908 Hachioji,Tokyo,Japan HQ842781 L.variaWalker,1849 Florida,LeonCo.,USA GU597446 Tephritidae Dacinae CeratitiscapitataWiedmann,1824 – NC000857

(3)

Table2

Morphologicalcharactersusedforthemorphologicalphylogeneticreconstruction andtheirrespectiveclassandstateintheanalysis.

Morphologicalcharacter Class/states

Cindex Continuous

4vindex Continuous

5xindex Continuous

Acindex Continuous

LengthOr1/LengthOr2 Continuous Setulaeonparaphysis Continuous Outerbranchesonsurstyles Continuous Setaeongonopod Continuous Rowsofacrostichalsetae Categorical–6/8/10

Tergite2–palearea Categorical–presence/absence Tergite3–palearea Categorical–presence/absence Tergite4–palearea Categorical–presence/absence Tergite5–palearea Categorical–presence/absence Tergite6–palearea Categorical–presence/absence CloudbandonapicalCII Categorical–presence/absence Cloudbandon1stcostalbreak Categorical–presence/absence Cloudbandon2ndcostalbreak Categorical–presence/absence Shortpileonparaphysis Categorical–presence/absence

searches)withaGammacorrection,whosealphavaluewassetto 0.17,followingModelTestresults.Cladesupportwasinferredwith abootstraptestwith1000replicationsforML,MPandNJ analy-ses,whileforBA,theposteriorprobability(PP)ofeachcladewas estimated.Moreover,intra-andinterspeciﬁcCOIdistanceswere calculatedwiththeKimura2-Parameters(K2P)evolutionarymodel

(Kimura,1980),withstandarderrorestimatedthrough1000

boot-strapreplicates.

Morphologicalphylogeneticanalysis

A morphological phylogenetic analysis was performed with 18 categorical and continuous characters (14 based on exter-nal morphology and four based on male terminalia patterns)

(Table2)obtainedfromthedescriptionsofMycodrosophilaspecies

ofSturtevant(1916),WheelerandTakada(1963)andofthenew

speciespresentedbelow.Weusedtheaverageofeach measure forcontinuouscharacters,becausethis wastheonlydata avail-ableintheliterature.Additionally,continuouscharacterswerefirst categorizedinanascendingorderandlatterweightedininverse proportiontotheirminimumnumberofsteps,usingascaledMP search,asperformedinPaup4.0b10.Otherheuristicsearch prop-ertiesweresetaccordingtothefeaturesdescribedaboveforthe molecularanalysis.Giventhedifficultiesinestablishing homolo-giesforthesecharacterstootherspecies,theNearcticspecieswere usedasoutgroup,whichseemedjustifiablegiventheresultsofour molecularphylogeneticanalysis(seebelow).

Newspeciesdescriptions

Aftermorphological and molecular validation regarding the samplingoftwonewspecies,descriptionswereperformedfrom

individuals preservedinethanol 70◦ accordingtothe measure-mentsandnomenclatureofstructuressuggestedbyBächlietal.

(2004). The measurements were performed on a stereoscopic

microscopecoupledwithmicrometricreticulum.Theaverage val-ues for each characteristic are presented accompanied by the maximumandminimumobservedvalues(inbrackets).The post-abdomenof allthespecimens was disarticulatedand themale terminaliaweremountedinpermanentslides withCanada bal-sam.Illustrationsofmaleterminaliaoftheholotypesweremade usingacamaralucidaattachedtoanopticalmicroscope(Olympus BX40)witha40×objectivelensanda10×ocularlens.Weobtained photomicrographsoftheexternalmorphologystructuresandmale terminaliaofﬂieswithaZeissDiscoveryV.20stereomicroscope.

Wheneverpossible,wephotographedthehostfungiandsent thephotographstoDr.EduardoBernardi(UniversidadeFederalde Pelotas)foridentiﬁcation.Thetypesseriesofthenewspecieswill bedepositedintheMuseudeCiênciasNaturaisoftheFundac¸ão Zoobotânicado Rio Grandedo Sul(MCNZ,PortoAlegre,Brazil). OneparatypeofeachnewspecieswillbedepositedintheMuseu NacionaldoRiodeJaneiroofUniversidadeFederaldoRiodeJaneiro (MNRJ,RiodeJaneiro,Brazil).

Results

Speciesvalidationandphylogeneticanalyses

The sampledMycodrosophila specimens were determinedto fourmorphotypes:M.projectans,M.elegansandtwoinitially puta-tivenewspecies.Themultiplealignmentsofthe16Mycodrosophila sequences(14new+twoavailableinGenBank)spanned748base pairsfromthe5regionofCOIandcontained170variablesites,of which156wereparsimoniouslyinformative.Thegeneral frequen-ciesforeachnucleotidewereA=29.9,C=16.0,G=17.0andT=37.1, andtheaveragetransition/transversionratiowas0.98.Minimum, maximumandaverageofintraspecificandinterspecificK2PCOI distanceswithstandard errors arepresentedin Table3.In this case,whereasthetwosequencesobtainedforthefirstputativenew species(hereafterM.hofmannisp.nov.,seebelow)exhibited0.4% divergence,theintraspecificdistancesforthesecondputativenew species(hereafterM.valentaesp.nov.,seebelow)sequencesranged from 0.0 to 0.3%. The interspecific distances between thenew speciesandeachoftheothertwosampledcongenericNeotropical speciesweremuchhigher,andalwaysgreaterthan12%.TheK2P distancesbetweenthetwoputativenewspecieswerealsohigh, presentingvaluesoftheorderof13%.

TheCOIsupportedphylogramisdepictedinFig.2,whereitcan beseenthatallthesampledmorphotypesarereciprocally mono-phyleticinregardtoeachother,aresultpresentinghighsupport forallmethods.Concerningtheinterspeciﬁcrelationships,the phy-logenydisplaysMycodrosophilaasmonophyleticinregardtothe includedSteganinaespecies(PP=0.97),clusteringthetwoNearctic species(PP=1.00),andthepairM.hofmannisp.nov.andM.valentae

Table3

Intra-andinterspeciﬁcK2PCOIdistancesandtheirrespectivestandarderrorsinregardtothecomparisonsinvolvingMycodrosophilahofmannisp.nov.andM.valentaesp. nov.plustheotherevaluatedNeotropicalMycodrosophilaspecies.

Species Distancesvalues

Minimal Average Maximal

Intraspeciﬁcdistances M.hofmannisp.nov. – 0.004±0.002 –

M.valentaesp.nov. 0.000±0.000 0.001±0.001 0.003±0.002 Interspeciﬁcdistances M.hofmannisp.nov.×M.projectans 0.143±0.017 0.145±0.017 0.149±0.017 M.hofmannisp.nov.×M.elegans 0.163±0.018 0.164±0.018 0.165±0.018 M.valentaesp.nov.×M.projectans 0.127±0.014 0.134±0.015 0.144±0.016 M.valentaesp.nov.×M.elegans 0.146±0.014 0.148±0.015 0.150±0.016 M.hofmannisp.nov.×M.valentaesp.nov. 0.133±0.016 0.135±0.016 0.137±0.016

(4)

Fig.2.COImajority-ruleconsensusphylogenyinferredbyBayesiananalysisusingtheGTR+G+Imodelforthe23nucleotidesequences(Table1).Thevaluesabovethenodes aretheposteriorprobabilitiesinBayesianAnalysisandthevaluesbelowarethebootstrapvaluesforthemaximumlikelihood,maximumparsimonyandNeighbor-Joining trees,respectively.The“-”signalindicatesabsenceofsupportfortheconsideredrelationship.ThistreewasrootedwithCeratitiscapitatasequences.Branchlengthsare proportionaltothescale,giveninsubstitutionspernucleotide.

sp.nov.(PP=0.95)assisterspecies.Althoughwithlowsupport,the treealsorecoveredtheearlyseparationbetweenNeotropicaland Nearcticspecies(PP=0.51),andpresentedM.elegansasanearly Neotropicaloffshoot(PP=0.69).

Themorphologicalweightedparsimonyanalysisrecoveredsix treeswiththesamenumberofsteps(280),allofwhichpresenteda ciof0.85andariof0.68.Withoutconsideringrelationshipsamong

Fig.3. Majority-ruleconsensustreerecoveredthroughmaximumparsimony analy-sisofthemorphologicalcharacterslistedinTable2showingtheexternalsimilarities amongtheeightknownNeotropicalMycodrosophilaspecies.Thistreewasrooted withthethreeNearctic’sspecies(M.claytonae,M.dimidiataandM.stalkeri).The valuesabovethenodesrepresentthepercentageofmaximumparsimonioustrees recoveringtheclade,sothatconﬂictingrelationshipsarehighlightedby intermedi-atesupportvalues(i.e.50%).

theNearcticspecies,thissetoftreeswasfurtherreducedtotwo, oneofwhichispresentedinFig.3.InsteadofclusteringM.hofmanni sp.nov.andM.valentaesp.nov.,thetwomostparsimonioustrees recoveredM.hofmannisp.nov.asmorphologicallysimilartoM. neoprojectans.Moreover,althoughthepositioningofM.valentae sp.nov.differedbetweenthetwotreeswiththesamenumberof steps,oneofthetopologiespresentedthisspeciesassistertoM. projectans(Fig.3),whosemorphologicalresemblancewasalready recognizedbysomeoftheauthors(MSGandJJ)ataﬁrstsight.

Descriptions

Basedonthemolecularanalysespresentedaboveandonthe analysisofbodymorphologyandmaleterminaliapatterns,we pro-posetwonewspeciesofMycodrosophila,asdescribedbelow:

Mycodrosophilahofmannisp.nov.

(Figs.4A–C,5A,6A,7A–E)

Typematerial.HOLOTYPE:♂,labeled“Brasil,RO.Coloradodo Oeste13◦0037.7S;60◦3524.9W,Junges,J.col.5.I.2012/M. hof-mannisp.nov.Junges,Gottschalk,Loretoand Robe♂Holótipo”, post-abdomendisarticulatedandtheterminaliamountedonslide with Canada balsam. PARATYPES: ♂, labeled “Brasil, RO. Col-oradodoOeste.Zonarural,13◦0037.7S;60◦3524.9W,Junges, J.col.5.I.2012/M.hofmannisp.nov.Junges,Gottschalk,Loretoand Robe♂Parátipo”,post-abdomendisarticulatedandtheterminalia

(5)

Fig.4.Mycodrosophilahofmannisp.nov.,paratypemale,terminaliaremoved.(A)Lateralview;(B)dorsalview;(C)head,frontalview.Mycodrosophilavalentaesp.nov., paratypemale,terminaliaremoved.(D)Lateralview;(E)dorsalview;(F)head,frontalview.Bars:AandB=0.5mm;DandE=1.0mm;CandF=0.2mm.

mountedonslidewithCanadabalsam;male,slidewithterminalia labeled“Brasil,RO.ColoradodoOeste.Zonarural,13◦0037.7S; 60◦3524.9W,Junges,J.col.5.I.2012/M.hofmannisp.nov.Junges, Gottschalk,LoretoandRobe♂Parátipo”(GenBankaccession num-berKC987477).

Fig.5.IllustrationoftheabdominalpatternsforMycodrosophilaspecies.(A)M. hof-mannisp.nov.;(B)M.valentaesp.nov.DrawingoutlinesbasedonWheelerand Takada(1963).

Typelocality: Amazonianforestfragment,ColoradodoOeste city,RondoniaState,Brazil(13◦0037.7S;60◦3524.9W).

Diagnosis:Maincolordarkbrown.Thoraxwithdorsalregion darkbrownandpleurapaleyellowish(Fig.4AandB).Abdomen

(6)

Fig.7.MaleterminaliaofM.hofmannisp.nov.holotype.(A)Epandrium,cerci,surstylianddecasternum,posteriorview;(B)hypandriumandgonopods,posteriorview; (C–E)aedeagus,aedeagalapodemeandparaphyses,respectivelyinventral,dorsalandlateralviews.InA,arrowindicatesdecasternumposition.Bar=0.1mm.

withdark brownbands covering tergites 2–5, tergite2 with a palecentralareabackwardprojectedcentrally,tergite5withthe darkbandcoveringonlytheposteriormargin,withaslightcentral andanteriorprojection,andtergite6withacentrallongitudinal stripe(Fig.5A).Wingbearingtwocloudedbandsextending dorso-ventrallybelowtoeachcostalveinbreaks(Fig.6A),similartoM. projectans.Aedeagusprojectedanteriorlywithapicalregion bifur-catedandjagged(Fig.7C–E).

Maledescription

Head (Fig. 4C): Frons dark brown. Frontal length=0.24mm (0.21–0.25mm);frontalindex=0.81(0.86–0.91);frontaltapering ratio=1.18(1.13–1.22),lengthratiooforbitalsetae:or2/or1=0.16, or2/or3=0.33, or1/or3=1.00; vertical setae broken; ocellar tri-angle yellowish about 32% (30–33%) of frontal length. Facial carinaproeminentand noselike.Antennaebrown, aristae with 4 dorsal and 1 ventral branches plus terminal fork, ﬂagel-lomere I width to length ratio=0.90 (0.87–0.93). Palpi dark brown.Redeyeswithoutpile.Genaebrown,vibrissalindex=0.60 (0.45–0.75); cheek index=5.47 (4.43–7.00); eye index=1.11 (1.07–1.15).

Thorax(Fig.4A–B):Maincolordarkbrown.Length:0.67mm (0.62–0.70mm),width:0.54mm(0.50–0.57mm).Eightirregular rowsofacrostichals,anteriorpairofdorsocentralsetaeabsent, dis-tancebetweenposteriordorsocentral=0.26mm(0.22–0.30mm); no prescutellar setae. Scutellum dark brown. Basal scutellar setae convergent. Scut index=0.54 (0.45–0.64); scut position index=0.61 (0.50–0.70). Two prominent katepisternal setae.

Sternoindex=0.54 (0.45–0.63).Pleuraebrownishyellow. Halter darkbrown.Legsyellowish.

Wing (Fig. 6A): Yellowish with dark yellow veins, bear-ing two clouded bands extending dorso-ventrally below each costal vein breaks. Length=1.53mm (1.50–1.57mm). Indices: wing index=2.27 (2.17–2.33); C=0.90 (0.88–0.92); ac=5.95 (5.40–6.25);4v=2.04(1.52–2.38);5x=1.85(1.55–2.00);4C=1.79 (1.31–2.15); M=0.51 (0.42–0.70); hb=0.63 (0.60–0.66); prox. x=0.56(0.42–0.69).

Abdomen(Fig.5A):Tergites2–5withdarkbrownbands.Tergite 2withthepaleareaoccupyingalmosthalfoftheanterior mar-gin,withabackwardcentralprojection.Tergite5withtheband coveringonlytheposteriormarginandcontainingaslightcentral projection.Tergite6bearingonecentrallongitudinalstrip.

Bodylength:1.51mm(1.50–1.55mm).

Terminalia(Fig.7):Epandriummicrotrichosewith5upper bris-tles, ventral lobe not microtrichose with 7 large bristles. Cerci microtrichose bearing large bristles, not fused to epandrium. DecasternumasinFig.7A.Surstyliwith12–13prensisetae, 2–3 innerand3–4outersetae.Hypandrium(Fig.7B)inanarc.Gonopods large,fusedtohypandriumandcontainingshortpilesandoneseta. Aedeagus(Fig.7C–E)projectedanteriorly,withtheapicalregion bifurcatedandjagged,internalmarginoftheaedeaguswithrows oftinysetae.Aedeagalapodemeﬂattenedlaterallyandshorterthan aedeagus.Ventralrodprojectedanteriorlyandfusedwiththe pos-teromedianmarginofthehypandrium.Paraphysesmicrotrichose, fusedtothegonopodsandcontainingthreeinnersetulae.

(7)

Fig.8. Brazilianbiomes,accordingtoIBGE(2013),andtheoccurrencerecordsfromthetwonewMycodrosophilaspecies.StarsindicateM.hofmannisp.nov.,trianglesindicate M.valentaesp.nov.Theblacksymbolsindicatethetypelocalityofeachspecies.

Femaleunknown.

Etymology:Theepithet isa genitive patronymtohonorthe BrazilianDrosophilistPauloRobertoPetersenHofmann,from Uni-versidadeFederaldeSantaCatarina.

Geographicdistribution:M.hofmannisp.nov.wassampledin twofragmentedareasfromBrazilianAmazonForestinColoradodo Oestecity,Rondônia(RO)(13◦0037.7S;60◦3524.9W)(Fig.8).

Ecologicalnotes:Therecordsarerestrictedtothetypelocality, andthetypeseriesspecimenswerecollectedﬂyingover mush-roomsofMycenasp.(Agaricales,Mycenaceae)(Fig.1).

Mycodrosophilavalentaesp.nov.

(Figs.4D–F,5B,6B,9A–E)

Typematerial. HOLOTYPE: ♂, labeled: “Brasil, RS. Capão do Leão.HortoBotânicoIrmãoTeodoroLuis–UFPel.31◦4602.05S, 52◦2655.34W,Blauth,M.L.andValer,F.col.29.04.2011/M. valen-tae sp. nov. Junges, Gottschalk, Loreto and Robe, ♂ Holótipo”. PARATYPES:3♂,labeled:“Brasil,RS.CapãodoLeão.HortoBotânico Irmão Teodoro Luis – UFPel. 31◦4602.05S, 52◦2655.34W, Blauth,M.L.andValer,F.col.29.04.2011/M.valentaesp.nov.Junges, Gottschalk,LoretoandRobe♂Parátipo”.Allthespecimens (holo-typeandparatypes)hadtheirpost-abdomendisarticulatedandthe terminaliamountedonslideswithCanadabalsam.

Typelocality:HortoBotânicoIrmãoTeodoroLuis,Campusof UniversidadeFederaldePelotas,CapãodoLeãocity,RioGrandedo SulState,Brazil(31◦4602.05S,52◦2655.34W).

Diagnosis:Maincolordarkbrown,notumdarkbrownand pleu-raebrownishyellow(Fig.4DandE).Abdomenwithdarkbrown bandscoveringtergites2–6,tergite4withapalecentralareaasin M.projectans(Fig.5B).Wingbearingtwocloudedbandsbeloweach costalveinbreaks(Fig.6B).Theaedeagushasatubularformwith tinyscalesaroundit,beingapicallybifurcatedandslightlyserrated (Fig.9C–E).

Maledescription

Head(Fig.4F):Fronsdarkbrownwithamedianarealightbrown. Frontal length=0.29mm (0.29–0.30mm); frontal index=0.97 (0.96–1.00), frontal tapering ratio=1.35 (1.30–1.48), length ratiosof orbital setae: or2/or1=0.20 (0.12–0.29), or2/or3=0.21 (0.12–0.38),or1/or3=1.05(0.82–1.3),vtindex=0.99(0.77–1.15), ocellartriangledarkbrownabout35%(29–40%)offrontallength. Facedarkbrown,facialcarinaproeminentandnoselike.Antennae dark brown, aristae with4 or 5 dorsal, 1 ventral and 4 inter-nalbranches,plusterminalfork;ﬂagellomereIwidthtolength ratio=0.44(0.41–0.47).Palpidarkbrown.Redeyeswithoutpile. Genaebrown,vibrissalindex=0.27(0.16–0.33);cheekindex=9.00 (5.00–13.00);eyeindex=1.12(1.05–1.26).

Thorax(Fig.4DandE):Maincolordarkbrown.Length=0.95mm (0.92–1.00mm), width=0.72mm (0.69–0.77mm). Ten irregular rowsofacrostichals,anteriorpairofdorsocentralsetaeabsent, dis-tancebetweenposteriordorsocentral=0.34mm(0.28–0.36mm), noprescutellarsetae.Scutellumdarkbrown.Basalscutellarsetae convergent.Scutindex=0.41(0.36–0.51),scutpositionindex=0.63 (0.41–0.82).Twoprominentkatepisternalsetae,sternoindex=0.54 (0.47–0.59). Pleurae brownish yellow. Halterdark brown. Legs brownishyellow.

Wing (Fig. 6B): Yellow with veins dark yellowish, bearing clouded bands below the two costal vein breaks, extend-ing dorso-ventrally. Length=2.26mm (2.17–2.33mm). Indices: wing index=2.39 (2.17–2.81); C=0.84 (0.80–0.87); ac=5.40 (5.06–5.86);4v=1.90(1.81–1.97);5x=1.42(1.25–1.61);4C=1.78 (1.64–1.86); M=0.48 (0.45–0.50); hb=0.76 (0.72–0.79); prox. x=0.52(0.47–0.54).

Abdomen(Fig.5B):Tergites2,6darkbrown,onlytergite4with ananteriorandmedialpalearea.

Bodylength:2.36mm(2.12–2.61mm).

Terminalia(Fig.9):Epandrium(Fig.9A)highlymicrotrichose, with11 upperbristles.Ventral lobenotmicrotrichose, with10 largebristles.Cercimicrotrichose,bearinglargebristles,notfused

(8)

Fig.9.MaleterminaliaofM.valentaesp.nov.holotype.(A)Epandrium,cerci,surstylianddecasternum,posteriorview;(B)hypandriumandgonopods,posteriorview;(C–E) aedeagus,aedeagalapodemeandparaphyses,respectivelyinventral,dorsalandlateralviews.InA,arrowindicatesdecasternumposition.Bar=0.1mm.

toepandrium. Decasternum as in Fig.9A. Surstyli with6 peg-likeprensisetae,4innerand3outersetae.Hypandrium(Fig.9B) longerthanwide,v-shaped.Gonopodsfusedtohypandrium,longer thanwider,withonelongseta.Aedeagustubularwithtinyscales in themedial half,projected anteriorly, withthetip turnedto the dorsal region. Apex of aedeagus bifurcated, with marginal smallspinesandajaggedaspect.Aedeagalapodemeshorterthan aedeagus.Ventralrodprojectedand fusedwiththe posterome-dianmarginofthehypandrium.Paraphyseslinkedtotheaedeagal apodemebymembranoustissueandcontainingthreetinysetulae internally.

Femaleunknown.

Etymology:Theepithet isa genitive patronymtohonorthe BraziliandrosophilistVeraLúciadaSilvaValente,from Universi-dadeFederaldoRioGrandedoSul.

Geographicdistribution:M.valentaesp.nov.seemstobewidely distributedintheBrazilianstateofRioGrandedoSul(Fig.8).Until now,thisspecieswascollectedinthemunicipalitiesofCapãodo Leão(31◦4602.05S;52◦2655.34W),SantaMaria(29◦4302S; 53◦4334W), Rio Grande (32◦3218.28S; 52◦326.74W) and Viamão,RS,Brazil(30◦0517S;51◦0607W).

Ecologicalnotes:Thisspecies iscommonin Southern Brazil, wherespecimensofthetypeseriesweresampledﬂyingover mush-roomsofanunidentiﬁedPolyporaceae.

Discussion

Phylogeneticanalysis

Ourmolecularresultssupportthepresenceoftwonew Neotrop-icalMycodrosophilaspeciesintheBrazilianAmazonianandPampa biomes. In fact, M. hofmanni sp. nov. and M. valentae sp. nov. are reciprocally monophyletic species, which showed a broad barcoding gapbetweenintra-and interspecific distances. Since these are two premises for successful application of DNA bar-codingapproaches(MeyerandPaulay,2005),ourresultssupport thenotionthatthis methodologymayaidinspecies identifica-tion/discoveryissueswithinthisgroup,aspreviouslyshownfor othertaxons(Hebertetal.,2003;vanNieukerkenetal.,2012). Nev-ertheless,weneedtobeawarethattheseresultsmaysufferfrom samplingshortages,relatedtotheunderestimationofintraspecific variationand/ortotheoverestimationofinterspecificdivergence

(Moritzand Cicero,2004;Linares etal.,2009).In fact,whereas

thecompletedistributionrangeofeachof thetwonewspecies isnotyetunderstood,wewerenotabletosamplefourofthe cur-rentlyknownNeotropicalMycodrosophilaspecies(M.brunnescens, M.neoprojectans,M.nigropleuraandM.pseudoprojectans).Evenso, morphologicaldistinction fromthese (seebelow)attests tothe validityofM.hofmannisp.nov.andM.valentaesp.nov.

(9)

Concerningtherecoveredphylogeneticpatterns,interspecific relationshipsaredifficulttoinferbecauseofthelowsupportvalues presentedbyourphylogenies.Moreover,comparisonsinvolvinga singlegenemaynotaccuratelyreflectspeciesphylogeny(Nixon

andCarpenter,1993;vanNieukerkenetal.,2012).Nevertheless,as

somesimilaritieswererecoveredinbothmolecularand morpho-logicalanalyses,wemayusethemtomakeaﬁrstapproximation tothegroup’sphylogeny. Thisis thecase, forexample, forthe clusteringofM.projectans,M.hofmannisp.nov.andM.valentae sp.nov.,whichformedamonophyleticcladesistertoM.elegans inboth,molecularandmorphologicalphylogenies.Theformation ofaNeotropicalMycodrosophilaspeciesgroupindependentofthe NearcticspecieswasinferredthroughCOIphylogeneticanalysis and—despiteitslowsupportvalues—isconcordantwiththe propo-sitionofWheelerandTakada(1963).Inthissense,itisimportantto emphasizethatnomolecularstudy,toourknowledge,hasexplored thephylogenetic relationshipsamong AmericanMycodrosophila species.

TwonewspeciesofNeotropicalMycodrosophila

Thetwonewspeciesdescribedhereareincludedinthe Myco-drosophilagenusbythepresenceofthearistawithasingleventral branch,onlyonepairofdorsocentralsetae,acostallappetandthe entirelycoloredandshiningscutum,withoutspotsatthebasesof thesetae(WheelerandTakada,1963;Grimaldi,1990).M.hofmanni sp.nov.andM.valentaesp.nov.arealsoembeddedwithinthe sub-genusMycodrosophila,withadevelopedcostallappetandacloud bandbelowthesecondcostalvein.

The patterns of the tergites are remarkable in distinguish-ingthetwonewspeciesbetweeneach otherandinrelationto otherMycodrosophilaspecies.Thisdistinctioniscommonlyrelated tothe presence of pale areas between black bands in the ter-gites, which seems to be widespread in Mycodrosophila (Bock,

1980;ChassagnardandLachaise,2000;Okada,1956;Tsacasand

Chassagnard, 1991).In this case, whereas the Nearctic species

presentpaleareasinallthelastsixtergites(WheelerandTakada, 1963),inthespeciesdescribedherethesearerestrictedtosome ofthetergites. Thepresenceofthesepale areasalsoallowsthe distinctionofM.hofmannisp.nov.andM.valentaesp.novfrom M.brunnescens,M.nigropleuraandM.elegans,whichhavetheir abdomencompletelydark(WheelerandTakada,1963).

Althoughthedifferentiationofthetwonewspeciesinregardto theremainingNeotropicalMycodrosophilaspeciesisnotso remark-able,thecolorpatternofthetergitesisalsoveryinformativeinthis task.Inthissense,M.hofmannisp.nov.differsfromM.projectans bytheabsenceofpaleareasintergites3and4,andfromM. pseudo-projectansbyhavingadarkspotontergite6.Infact,assuggestedby ourmorphologicalphenogram,M.hofmannisp.nov.ismostsimilar toM.neoprojectans,althoughbothspeciesdifferbythecolor pat-ternoftergite2,whereapaleareaisencounteredonlyinthenew species.InthecaseofM.valentaesp.nov.,itdiffersfromM. neopro-jectans,M.projectansandM.pseudoprojectansbyhavingapalearea onlyintergite4,whereastheotherthreespecieshaveadditional paleareasintheirabdomens.

Themaleterminaliaofthetwonewspeciesalsopresentsthe regularformfoundinMycodrosophilaﬂies,withatubular, elon-gatedandapicallybifurcatedaedeagus(Bock,1980;Chassagnard

andLachaise,2000;McEveyandPolak,2005;WheelerandTakada,

1963).Infact,theaedeaguswithabifurcationorbifurcatedand jaggedintheapicalregionis a characteristicsharedby alarge numberofMycodrosophilaspecies,beingfoundineightofthenine previouslyknownAmericanMycodrosophilaspecies(themale gen-italiaofM.brunnescensisunknown),inadditiontothetwonew speciesdescribedhere.TheventrallycurvedaedeagusfoundinM. hofmannisp.nov.is alsoencounteredin Mycodrosophiladiversa

Bock,1980and Mycodrosophila erectaOkada,1968,two species

inhabitingotherZoogeographicalRegions,althoughM.diversahasa paleareaontergite6andathinnerapicalaedeagusregion,whereas M.erectahastheaedeagusmorestraightandwithsomenotched bifurcations. Otherwise, theaedeagus of M.valentaesp. nov. is similartothatfoundinM.claytonae,withastraightformatand tinyspinesintheapicalregion,althoughtheabdominalpatternis largelydifferentamongthesetwospecies.

KeytoAmericanspeciesofMycodrosophila(updatedfrom

WheelerandTakada,1963)

1. Darkbandsoftergites2and3broadlyinterruptedmediallyandall tergiteswithpaleareas;Nearcticspecies...2

1 _Dark_bands_of_tergites₂_and₃_without_broad_median_{interruptions;}

Neotropicalspecies...4

2. Darkbandoftergite4broadlyinterruptedmedially,thusappearing liketheprecedingones;terminaliaasinWheelerandTakada,1963: 396,ﬁgs.3–6...M.dimidiataLoew,1862

2_. _Dark_band_of_tergite₄_not_interrupted,_medially_shaped_like_an

invertedV;terminalianotasabove...3

3. Costalindexabout2.0,tergite6darkorwithsmallpaleareasinthe lateralregion.AbdominalpatternasshowninWheelerandTakada, 1963:394,ﬁg.2;terminaliaasinWheelerandTakada,1963:396,ﬁgs. 7–10...MycodrosophilastalkeriWheelerandTakada,1963

3_. _Costal_index_about_1.7,_tergite₆_with_large_pale_areas_in_the_lateral.

AbdominalpatternasshowninWheeler&Takada,1963:394,ﬁg.2; terminaliaasinWheelerandTakada,1963:396,ﬁgs.11–14)...M. claytonaeWheelerandTakada,1963

4. Wingwithﬁveprominentblackclouds,includingoneoverdm-Cu crossvein;R2+3bentabruptlytoCosta;terminaliaasinWheelerand

Takada,1963:397,ﬁgs.31–34)...M.elegansWheelerandTakada, 1963

4_. _Wing_less_clouded,_posterior_crossvein_(dm-Cu)_never_in_a_cloud;_R₂₊₃

veinapproachingCostagradually;terminalianotasabove...5 5. Pleuralargelybrown,withawhitishareaextendingobliquelyfromthe

axillararea(wingbase)tothepronotum(prosternum);legsbrown. Male:unknown...M.brunnescensWheelerandTakada,1963

5_. _Pleura_pale_yellowish,_at_most_the_anepimeron_{(pteropleura)}_dark;_legs

mostlypale...6

6. Anepimeron(pteropleura)dark,formingashortobliquebandfromthe baseofthehalteretothekatepisternum(sternopleural)corner; abdomenblack;terminaliaasinWheelerandTakada,1963:396,ﬁgs. 27–30...M.nigropleuraWheelerandTakada,1963

6_. _Anepimeron_{(pteropleura)}_yellowish_pale_or_brownish_pale;_some

tergiteswithpaleareas...7 7. Tergite6wholeshiningblack...8

7_. _Tergite₆_yellow_with_and_elongate,_narrow,_median_black_stripe;

tergite5withanapicaldarkbandexpandedbasallyinmidline...9 8. Tergites3,4and6shiningblack,tergite5withparamedianbasalpale

areas;humeralbreak(costalincision)weak,lappetscarcely developed;terminaliaasinWheelerandTakada,1963:396,ﬁgs. 23–26...M.pseudoprojectansWheelerandTakada,1963

8_. _Tergites₃_and₄_or_only_tergite₄_with_some_pale_areas;_tergites₅_and₆

shiningblack;costallappetlargeandblack;terminalianotasabove... 10

9. Tergite2withoutpaleareas;darkbandintergite5nottouchingthe lateralmargin;terminaliaasinWheelerandTakada,1963:397,ﬁgs. 19–20)...M.neoprojectansWheelerandTakada,1963

9_. _Tergite₂_with_a_pale_area_in_the_anterior_margin_and_a_dark_band_with

bilobedaspect(Fig.5A;terminaliaasinﬁg.7A–E...M.hofmannisp. nov.

10. Bothtergites3and4withpaleareasintheanteriormargin;tergite3 withapaleandroundedcentralareaandtergite4withanexpanded palearea;terminaliaasinWheelerandTakada,1963:396,ﬁgs.15–18 ...M.projectansSturtevant,1916

10_. _Only_tergite₄_with_a_pale_area_expanded_in_the_anterior_margin,_tergite

3darkbrown(Fig.5B);terminaliaasﬁg.9A–E...M.valentaesp.nov.

Conﬂictsofinterest

(10)

Acknowledgements

The authors are grateful to Ronaldo Golombiesky, Pedro Mesquita Fonseca and Francine Cenzi de Ré for helping with themolecularprocedures;to Monica LanerBlauth, Felipe Berti ValerandStelaMachado,forkindlyproviding somespecimens; toEduardoBernardiforfungalidentifications;andtothe Brazil-ianFundingAgenciesCoordenaçãodeAperfeiçoamentodePessoal deNívelSuperior(CAPES)andConselhoNacionalde Desenvolvi-mentoCientíficoeTecnológico(CNPq)forfinancialsupport.The collections here performedwere authorized by theSistema de Autorização e Informação em Biodiversidade (SISBIO), and we thank themto theircommitmentwithethicalguides and con-servation efforts as concerns Brazilian scientific activities.This studywassupportedbyUniversal-CNPq14/2013,processnumbers 471174/2013-0and472973/2013-4.

References

Bächli,G.,2015.TaxoDros:TheDatabaseonTaxonomyofDrosophilidae,Available fromhttp://www.taxodros.unizh.ch(accessed23.08.15).

Bächli,G.,Vilela,C.R.,Escher,S.A.,Saura,A.,2004.TheDrosophilidae(Diptera)of FennoscandiaandDenmark.FaunaEntomol.Scand.39,1–362.

Bock,I.,1980.DrosophilidaeofAustralia.IV.Mycodrosophila(Insecta:Diptera).Aust. J.Zool.28,261–269.

Burla,H.,1954.ZurKentnnisDerDrosophilidenderElfenbeinküste(Französisch, West-Afrika).Rev.SuisseZool.61,1–218.

Chassagnard,M.T.,Lachaise,D.,2000.Mycodrosophilamatilei,nouvelleespèce mycophageafrotropicaledécouverteparLoïcMatile(Diptera,Drosophilidae). Rev.Fran.Entomol.22,177–182.

Grimaldi,D.A.,1990.Aphylogeneticrevisedclassiﬁcationofgenerainthe Drosophil-idae(Diptera).Bull.Am.Mus.Nat.Hist.197,1–139.

Grimaldi,D.A.,2010.Drosophilidae(smallfruitflies,pomaceflies,vinegarflies).In: Brown,B.V.,Borkent,A.,Cumming,J.M.,Wood,D.M.,Woodley,N.E.,Zumbado, M.A.(Eds.),ManualofCentralAmericanDiptera.NRCResearchPress,Ottawa, pp.1197–1206.

Guindon,S.,Gascuel,O.,2003.Asimple,fast,andaccuratealgorithmtoestimate largephylogeniesbymaximumlikelihood.Syst.Biol.52,696–704.

Hebert,P.D.N.,Cywinska,A.,Ball,S.L.,deWaard,J.R.,2003.Biologicalidentiﬁcations throughDNAbarcodes.Proc.R.Soc.B:Biol.Sci.270,313–332.

IBGE–InstitutoBrasileirodeGeograﬁaeEstatística,2013.MapasMurais:Biomas, Availablefrom ftp://ftp.ibge.gov.br/CartaseMapas/MapasMurais/ (accessed 20.02.13).

Kimura, M.,1980. Asimplemethod forestimating evolutionaryrate of base substitutionsthroughcomparativestudiesofnucleotidesequences.J.Mol.Evol. 16,111–120.

Linares,M.C.,Soto-Calderon,I.D.,Lees,D.C.,Anthony,N.M.,2009.Highmitochondrial diversityingeographicallywidespreadbutterﬂiesofMadagascar:atestofthe DNAbarcodingapproach.Mol.Phylogenet.Evol.50,485–495.

Machado,S.,dosSantos,J.P.J.,Robe,L.J.,Loreto,E.L.S.,2014.Anefﬁcientandcheap entomologicalaspiratortocollectmycophylicandanthophilicadult Drosophil-idaeﬂies.Drosoph.Inf.Serv.97,169–171.

McEvey,S.F.,Polak,M.,2005.Mycodrosophila(Diptera:Drosophilidae)ofFijiand Vanuatuwithdescriptionofninenewspecies.Occas.Pap.BerniceP.BishopMus. 84,35–67.

Meyer,C.P.,Paulay,G.,2005.DNAbarcoding:errorratesbasedoncomprehensive sampling.PLoSBiol.3,2229–2238.

Moritz,C.,Cicero,C.,2004.DNAbarcoding:promiseandpitfalls.PLoSBiol.2,e354.

Nixon,K.C.,Carpenter,J.M.,1993.Onoutgroups.Cladistics9,413–426.

Nylander, J.A.A., 2004. MrModeltest v2. Program Distributed by the Author. Evolutionary Biology Center, Uppsala University, Available from

https://github.com/nylander/MrModeltest2(accessed10.10.12).

Okada,T.,1956.SystematicStudyofDrosophilidaeandAlliedFamiliesofJapan. Gihodo,pp.183.

Okada,T.,1965.DrosophilidaeoftheOkinawaIslands.Kontyû33,327–350.

Okada,T.,1968.AdditiontothefaunaofthefamilyDrosophilidaeofJapanand adjacentcountries(Diptera).II.GeneraParamycodrosophila,Mycodrosophila, Lio-drosophilaandDrosophila,includinganewsubgenusPsilodorha.Kontyû26, 324–340.

Okada,T.,1986.ThegenusMycodrosophilaOldenberg(Diptera,Drosophilidae)of SoutheastAsiaandNewGuineaII.Atypicalspecies.Kontyû54,291–302.

Oldenberg,L.,1914.BeitragzurKenntnisdereuropäischenDrosophiliden(Dipt.). Arch.Nat.80,1–42.

Posada,D.,Crandall,K.A.,1998.Modeltest:testingthemodelofDNAsubstitution. Bioinformatics14,817–818.

Ronquist,F.,Teslenko,M.,vanderMark,P.,Ayres,D.L.,Darling,A.,Höhna,S.,Larget, B.,Liu,L.,Suchard,M.A.,Huelsenbeck,J.P.,2012.MrBayes3.2:efﬁcientBayesian phylogeneticinferenceandmodelchoiceacrossalargemodelspace.Syst.Biol. 61,538–542.

Simon,C.,Frati,F.,Beckenbach,A.,Crespi,B.,Liu,H.,Flook,P.,1994.Evolution, weighting,andphylogeneticutilityofmitochondrialgenesequencesanda com-pilationofconservedpolymerasechainreactionprimers.Ann.Entomol.Soc.Am. 87,651–701.

Staden,R.,1996.TheStadensequenceanalysispackage.Mol.Biotechnol.5,233–241.

Sturtevant,A.H.,1916.NotesonNorthAmericanDrosophilidaewithdescriptionsof twenty-threenewspecies.Ann.Entomol.Soc.Am.9,323–343.

Swofford,D.L.,2003.PAUP: PhylogeneticAnalysisUsingParsimony(andOther Methods).Version4.SinauerAssociates,Massachusetts,Availablefromhttp:// www.sinauer.com/paup-phylogenetic-analysis-using-parsimony-and-other-methods-4-0-beta.html(accessed10.10.12).

Tamura,K.,Nei,M.,1993.Estimationofthenumberofbasenucleotidesubstitution inthecontrolregionofmitochondrialDNAinhumansandchimpanzees.Mol. Biol.Evol.1,512–526.

Tamura, K., Peterson, D., Peterson, N., Stecher, G.,Nei, M., Kumar, S., 2011.

Molecularevolutionarygeneticsanalysisusingmaximumlikelihood, evolu-tionarydistance,andmaximumparsimonymethods.Mol.Biol.Evol.28,2731– 2739.

Tsacas,L.,Chassagnard,M.T.,1991.DipteraDrosophilidaedeNouvelle-Calédonie. 2. Leucophenga, Mycodrosophila et Paramycodrosophila, avec une note sur les genitália de Leucophenga. Mem. Mus. Natl. d’hist. Nat. 149a, 302–328.

vanNieukerken,E.J.,Doorenweerd,C.,Stokvis,F.R.,Groenenberg,D.S.J.,2012.DNA barcodingoftheleaf-miningmothsubgenusEctoedemias.str.(Lepidoptera: Nepticulidae)withCOIandEF1-␣:twoarebetterthanoneinrecognisingcryptic species.Contrib.Zool.81,1–24.

Wheeler,M.R.,Takada,H.,1963.ArevisionoftheAmericanspeciesofMycodrosophila (Diptera,Drosophilidae).Ann.Entomol.Soc.Am.56,392–399.