REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolutionw w w . r b e n t o m o l o g i a . c o m
Systematics,
Morphology
and
Biogeography
New
systematic
position
of
Itatingamyia
Albuquerque
(Diptera,
Muscidae)
based
on
molecular
evidence,
and
description
of
the
female
of
I.
couriae
Kirstern
Lica
F.
Haseyama
a,∗,
Claudio
J.B.
de
Carvalho
b,
Ândrio
Zafalon-Silva
b,
Frederico
D.
Kirst
aaUniversidadeFederaldeMinasGerais,LaboratóriodeSistemáticadeInsetos,DepartamentodeZoologia,BeloHorizonte,MG,Brazil bUniversidadeFederaldoParaná,DepartamentodeZoologia,Curitiba,PR,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received7August2018 Accepted18October2018 Availableonline3November2018 AssociateEditor:AndrzejGrzywacz Keywords:
Atlanticforestbiome Cyrtoneurininae Neotropicalregion Newrecords Phylogeny
a
b
s
t
r
a
c
t
ItatingamyiaAlbuquerque,1979israreincollections,withonlyninespecimenspreservedinmuseums. Twospeciesareknown,andtheirplacementwithinMuscidaewasnevertestedusingmoleculardata. Here,weestimatethepositionofItatingamyiawithinMuscidaewithmitochondrial(COI)andnuclear (AATS,CAD,andEF1-␣)markersusingMaximumlikelihoodandBayesianposteriorprobabilitiesas opti-malitycriteria.Accordingtoourresults,weproposetoclassifyItatingamyiaasaCyrtoneurininae.We alsodescribethepreviouslyunknownfemaleandeggofItatingamyiacouriaeHaseyamaanddeCarvalho, 2011andexpandtheknowndistributionofthisspeciestothestateofMinasGerais,Brazil,throughthe discoveryof13newspecimensthere.WealsoarguethatItatingamyiadistributionisrestrictedtoAtlantic Forestareas.
©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
ItatingamyiaAlbuquerque,1979isaNeotropicalgenusof Musci-daewithtwodescribedspecies,I.bivittataAlbuquerque,1979andI. couriaeHaseyamaanddeCarvalho,2011.Thegenuswasincluded inMydaeinaebyAlbuquerque(1979),basedontheshapeofthe ovipositorandfemalecercus.However,theauthorhighlightedthat Itatingamyiahasasinglepost-suturalintra-alarseta,a character-isticfoundinmanyCyrtoneurininaegenera(sensuSnyder,1954). Later,deCarvalhoandCouri(1993)placeditinReinwardtiini,a tribeof Azeliinae(sensu deCarvalho, 1989), basedonthelong femalecercusprojectingbeyondthehypoproct,thedeveloped ster-niteeight,andtheanchor-shapedtergitesix.
Accordingtoaphylogeneticanalysisbasedonmorphological data,ItatingamyiaisthesistergroupofCariocamyiaSnyder,1951 and Charadrella (Haseyama and de Carvalho, 2012). Thisclade, formedbythreesmallNeotropicalgenera,wasplacedwithinan assembly ofPaleotropicalgenera(Alluaudinella Giglio-Tos,1895 andOchromuscaMalloch,1927),andDichaetomyiaMalloch,1921, agenuswidespreadfromAfricatoAustralasia.Duetotheclose relationshipwithDichaetomyia,Charadrellaandalliedgenerawere placedinDichaetomyiinae(HaseyamaanddeCarvalho,2012;see
∗ Correspondingauthor.
E-mail:[email protected](K.L.Haseyama).
alsoCourianddeCarvalho,2003).Ontheotherhand,someauthors
classified Charadrella and Cariocamyia within the
Cyrtoneurini-nae(Hennig,1965; deCarvalhoetal., 2005).Thiswasthefirst
time a species of Itatingamyia was included in a phylogenetic study,buttheresultsofthatanalysiscannotbeconsidered con-clusivebecauseitemployedabiasedtaxonsampling:ratherthan includingrepresentativesoftheentirefamily,itmostlyincluded generathathadpreviouslybeenconsideredasclosely-relatedto Charadrella.
Itatingamyiaisrareincollections.Itatingamyiabivittataisknown only from thetype-seriesstudied by Albuquerque (1979)(two malesand six females) fromPedra Azul,stateof MinasGerais, Brazil.Accordingtotheauthor,thelocalityis560metersabove sea level and is predominantly covered with Cerrado vegeta-tion(Braziliansavanna;Albuquerque,1979).Itatingamyiacouriae is known only by its male holotype (Santa Teresa, state of EspíritoSanto, Brazil;HaseyamaanddeCarvalho, 2011).It was collectedusingaMalaisetrapintheSantaLúciaBiological Sta-tion, at 867 meters above the sea level, in the Atlantic Forest biome.
Here,weusemoleculardatatoestimatethephylogenetic posi-tion of Itatingamyia. We also describethe previously unknown femaleand eggofI.couriae.Finally, wedescribemorphological variationsfoundamongthemalesofthisspecies,providenew geo-graphicrecordsforit,anddiscussthegeographicdistributionofthe genus.
https://doi.org/10.1016/j.rbe.2018.10.003
0085-5626/©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Materialandmethods
Sampledesign,DNAextraction,amplification,andsequencing Taxonsamplingincludedgenerafromthethreesubfamiliesof Muscidae(Muscinae,Cyrtoneurininae,andMydaeinae,according
toHaseyamaetal.,2015).Weaimedforadensersamplingwithin
theCyrtoneurininaeandMydaeinae,subfamiliesthatincludedthe groupspreviouslyhypothesizedtocontainItatingamyia(see sec-tion‘Introduction’).Weusedpreviouslypublisheddataavailableat GenBank,andnewCOIandnuclearprotein-codinggenes(AATSand EF1-␣)sequencesobtainedforthisstudy(SupplementaryTableS1). SincemostofthespecieswiththosegenesalsohadCADsequences, thisnuclearprotein-codinggenewasalsousedintheanalysis,even thoughwewerenotabletoamplifyitforItatingamyia.
DNAwasextractedusingthePurelinkGenomicDNAextraction kit (Invitrogen) following the protocol provided by the manu-facturer,withthefollowing modifications:(i)atthetissue lysis step,thespecimenwasnot destroyed,allowingittobepinned anddepositedatthebiologicalcollection‘UniversidadeFederalde MinasGerais,CentrodeColec¸õesTaxonômicas’(CCT-UFMG;see alsoexaminedmaterialinTaxonomysection).Thewholeflywas placedintissuepaperallowingfortheevaporationof cryopreser-vationliquid(alcohol),andthenincubatedinproteinaseKandlysis buffer;(ii)sincetheflywasnotmacerated,itwasincubatedfora longerperiodat56◦C(approximately90h);(iii)aimingtoobtaina concentratedgenomicDNA,thefinalDNAelutionusedonly50L ofbuffer.
Polymerase chain reactions (PCR) were carried out using primers previously indicated by Haseyama et al. (2015) (Sup-plementaryTable S2). Primerswereused ata concentration of 10pmol/Lforatotalvolumeof50Lperreaction.GoTaq® G2 DNAPolymerase(Promega,Madison,USA)wasusedfollowingthe protocolprovidedbythemanufacturer,exceptfortheadditionof 3LofMgCl2at25mMforallreactions.Moreover,1-2Lofeach
primerand1-3LofgenomicDNAwereusedforamplification. Fornucleargenes,touchdownPCRwasthechosenstrategy.This strategyminimizestheamplificationofspuriousproductsthrough thesuccessivedecreasingof theannealing temperature(Donet
al.,1991; KorbieandMattick,2008).PCRcyclesarespecifiedin
SupplementaryTablesS3andS4.
PCRproductsweresubjectedtoelectrophoresisinagarosegel at2.5% todiscard possibleunwanted amplifications. The prod-uctswerestainedwithGelRedStain(BiotiumInc.,Hayward,USA) and the target bands were cut and purified using Wizard® SV GelandPCRClean-UpSystemkit(Promega,Madison,USA).The cleanedproductsweresentforsequencinginbothdirectionsatthe BiochemistryDepartmentattheUniversidadeFederaldoParaná, throughWEMSeqCompany(Curitiba,Brazil).
ChromatogramswereanalyzedusingChromasLite2.6.2 (Tech-nelysiumPty,2016)andsequenceseditedwithBioEdit7.2.5(Hall,
1999,2013).The quality index, which represents thebase call
probability,was analyzedfor each nucleotide of thesequences andforwardandreversesequenceswerematched.Aconservative approachwasusedfornucleotidepositionswithaqualityindex below20oneachsequence,whichwerereplacedwithan‘N’on theconsensussequence.Theconsensussequenceswereobtained throughtheBayesianmethodimplementedatSeqTrace0.9(Stucky,
2012).
Phylogenyandtopologytests
SequenceswerealignedusingMAFFTonlineserver(Katohetal., 2017)byimplementinganautoselectionforaligningalgorithm, whileotheroptionswereusedasdefault.Asafirstapproach,each genewasanalyzedseparatelyusingmostavailablesequenceson
GenBank.Bestmodelsofnucleotideevolutionwerechosenby Mod-elFinderAUTO feature,simultaneouslywithtreereconstruction
(Kalyaanamoorthyetal.,2017)withdefaultoptions.Phylogenetic
relationshipswereestimatedusingtheMaximumlikelihood(ML) softwareIQ-Tree(Trifinopoulosetal., 2016).Then,RogueNaRok
(Aberer etal., 2013)web server(http://rnr.h-its.org/about) was
usedtoidentifyroguetaxaineachanalysisunderdefaultoptions. Wechosethis strategytoindividuallyidentifyroguesequences, insteadofroguetaxa.Using thisapproach,ifonlyasinglegene sequence,or asubgroup ofsequences,froma terminalis caus-inginstabilityintheanalysis,wecanstillpreservethatterminal byusingonlythesequenceswithphylogeneticsignal.After prun-ingroguesfromindividualmatrices,alignmentswereconcatenated usingSequenceMatrixv.1.7.8(Vaidyaetal.,2010).
ModelFinder(Kalyaanamoorthyet al.,2017)wasusedunder default options with partition merging activated both for esti-matingthepartitioningschemeandchoosingthebestnucleotide evolution models (Supplementary Table 5). Maximum likeli-hood(ML)wasused asanoptimalitycriterion throughIQ-Tree
(Trifinopoulosetal.,2016)with20independentsearchesusingthe
standardoptions.MLbranchsupportmetricswereSH-aLRT,aBayes andUltrafastBootstrap(AnisimovaandGascuel, 2006;Guindon
etal.,2010;Hoangetal.,2018),allcalculatedwithIQ-Tree.
Sup-portwasconsideredsignificantwhenover80%forSH-aLRT,0.95 foraBayes,and95%forUltrafastBootstrap.Datawasalsoanalyzed underBayesianInference(BI)usingMrBayes3.2.2(Ronquistetal., 2012)throughtheCipreswebserver(Milleretal.,2010).The anal-ysisusedtwosimultaneousrunswithfourchainseachfor2×108
generationswithasampleevery1000generations.The conver-genceofrunswascheckedusingthestandard deviationofsplit sequences.Bayesianposteriorprobabilitieswereusedasbranch supportontheconsensustree,andconsidersignificantwhenover 95%.BasedonthecurrentknowledgeoftheMuscidaephylogeny
(Kutty et al., 2014; Haseyama et al.,2015)we rootedall trees
betweenMuscinaeandthecladeweconsideredasthein-group, Cyrtoneurininae+Mydaeinae.Treefigures weregeneratedusing FigTree1.4(RambaudtandDrummond,2012).
Theclassificationof Itatingamyia atsubfamilylevelwasalso examinedusingtopologicaltests.Thetestswerecarriedoutusing allmetricsofstatisticalsignificanceavailableonIQ-Tree(Kishino
andHasegawa,1989;ShimodairaandHasegawa,1999;Strimmer
and Rambaut, 2002;Shimodaira, 2002).Threeconstraintswere
createdimposingalternativepositionsforItatingamyia:(i)within Cyrtoneurininae;(ii)withinMydaeinae;(iii)withinMuscinae.All testswereperformedwith10,000bootstrapresamplingusingthe resamplingestimatedlog-likelihood(RELL)method(Kishinoetal.,
1990).
Taxonomyanddistributionmap
Thestudiedmaterialbelongsto‘UniversidadeFederaldeMinas Gerais, Centro de Colec¸ões Taxonômicas’ (CCT-UFMG), ‘Univer-sidade Federal do Paraná,Colec¸ão Padre Jesus Santiago Moure’ (DZUP)and‘UniversidadedeSãoPaulo,MuseudeZoologiada Uni-versidadedeSãoPaulo’(MZUSP).Thenewspecimens,includingthe oneusedforDNAextraction,werecapturedusingMalaisetraps between1999and2015.Theywerepreservedin70%ethanolat roomtemperaturesincetheywerecollected.During thisstudy, theywerepinnedandaddedtoCCT-UFMG,andDZUPcollections (seeExaminedmaterialfordetails).ThecollectingsitesareinBelo Horizontemunicipality(MinasGeraisState,Brazil)orsurrounding regions(withina100kmray).ThecollectingsiteatBeloHorizonte isadisturbedforestfragmentinahighlyurbanizedareainsidethe campusofUniversidadeFederaldeMinasGerais.Thetrapsplaced atNovaLimaandPetiecologicalstationsiteswereinAtlanticforest fragmentswithnearbywatercourses.
The morphological terminology of the adult follows
Stuckenberg(1999),NiheianddeCarvalho(2007)andCumming
and Wood (2009). The following abbreviations are usedin the
description:A1+CuA2=anal1+anteriorcubital2vein,A2=anal2
vein,AD=anterodorsal,AV=anteroventral,D=dorsal,M=medial vein, P=posterior, PD=posterodorsal, PV=posteroventral, R=radialvein,andV=ventral.Thefemaleterminaliawasprepared
for observationusingcold KOHand wasdrawn usingacamera lucida.
The distribution map was built using data from the
litera-ture(Albuquerque,1979)andmuseumspecimens(seeExamined
material). The raster representing the continental surface was the ‘Natural Earth I with shaded relief, water, and drainages’
(Kelsoand Patterson,2010; https://www.naturalearthdata.com);
Fig.1.MolecularphylogenetichypothesisusingMaximumLikelihoodofthecombinedmitochondrial(COI)andnuclear(AATS,CAD,andEF1-a)protein-codinggenesfor68 speciesofMuscidaehighlightingthepositionofItatingamyiawithintheCyrtoneurininae.Subfamily-levelclassificationfollowsHaseyamaetal.(2015).Numbersonnodes areSH-aLRT,aBayes,andUltrafastBootstrapsupportvalues.
biomes boundaries were delimited using the shapefile from Ministério do Meio Ambiente (Brasil, 2018; http://mapas.
mma.gov.br/i3geo/datadownload.htm).
Results
Phylogeny
AtotalofthreenewsequencesweregeneratedforItatingamyia couriae.OnlytheforwardstrandoftheAATSsequencewasused, duetosequencingproblems.Since thequalityindexwasabove thehighprobabilitythreshold(>30),wefeltconfidentenoughto usethesequencebasedonasinglestrand.Thefinalconcatenated matrixhad4029basepairsand68terminals.
The topology of the trees using both the ML and BI opti-mality criteria was similar, estimating the three subfamilies of Muscidae, Muscinae, Cyrtoneurininae and Mydaeinae (sensu
Haseyamaetal.,2015).Itatingamyiawasnestedinbothcaseswithin
Cyrtoneurininae,withsignificantsupport(Figs.1and2).The inter-nalrelationshipsofthissubfamily,however,differedaccordingto theoptimalitycriteriaused.WithML,I.couriaeisthesister-groupof acladecomprisingChaetagenia,Cyrtoneurina,Cyrtoneuropsis, Mus-cina, Philornis, Pseudoptilolepis, Scutellomusca and Synthesiomyia (Fig.1).IntheBIanalysis,theresultissimilar,butwithlower res-olution.In this case, Itatingamyiais placed ina polytomy, with Chaetagenia,Cyrtoneurina, someCyrtoneuropsis species,Muscina, Philornis,Pseudoptilolepis,Scutellomusca,andSynthesiomyia(Fig.2). AlltopologicaltestssupportItatingamyiawithinthe Cyrtoneurini-naeandrejectitsplacementwithinalternativesubfamilies(Table S6).
Taxonomy
ItatingamyiacouriaeHaseyamaanddeCarvalho,2011
(Figs.3and4)
Fig.2. MolecularphylogenetichypothesisusingBayesianinferenceofthecombinedmitochondrial(COI)andnuclear(AATS,CAD,andEF1-a)protein-codinggenesfor68 speciesofMuscidaehighlightingthepositionofItatingamyiawithintheCyrtoneurininae.Subfamily-levelclassificationfollowsHaseyamaetal.(2015).NumbersareBayesian posteriorprobabilitiesvalues.
A
B
C
D
Fig.3. ItatingamyiacouriaeHaseyamaanddeCarvalho,2011,female UFMG-IDI-1600467.(A)lateralhabitusview.Scale:3mm;(B)dorsalhabitusview.Scale:3mm; (C)head,frontalview.Scale:0.75mm;(D)egg,dorsalview.Scale:0.3mm.
Fig.4.Itatingamyiacouriae HaseyamaanddeCarvalho,2011,femalegenitalia UFMG-IDI-1600467.(A)Ovipositor,dorsalview.Scale:1mm;(B)Ovipositor,ventral view.Scale:1mm;(C)Spermathecae.Scale:0.2mm.
Descriptionoffemale(similartomale).Measurements(2females): 5.4–6.6mm(lengthfromtheheadtotheposteriortipofthe scutel-lum),9.0–9.3mm(winglength).
Color.Head.Parafacialyellowtolightbrownwithsilver pruinos-ity(Fig.3C); gena,proboscis,andpalpusyellowtolightbrown; postpedicelyellowishwithsilverpruinosity;aristayellowtolight brownon basal fourthand reddish brown totheapex; frontal vittareddishbrownwithminutesilversetulae;ocellartriangleand fronto-orbitalplatereddishbrown,thelatterwithsilverpruinosity anteriorly(Fig.3C).Thoraxlaterally,postpronotallobe,and noto-pleuronyellowtolightbrown(Fig.3A);scutumdarkbrownwith three silver pruinosity stripes; lower calypteryellowish;upper calypterhyalinewithdarkenedborders(Fig.3A).Winghyaline yel-lowish,slightlydarkenedintheupperthird.Legsyellowtolight brown;clawsbrownwithblackapex;pulvilliyellowish.
Abdomen.Tergites1–4yellowishtolightbrownwithamedian brown stripe extending to the apical borders of the tergites (Fig.3A,B);tergite5brownwithyellowishapex(Fig.3A).
Head.Dichoptic;eyeswithshortsparsesetulae;frontalvittabare; fronto-orbitalplatewithafewsetulae;postpedicelendingbefore insertionofvibrissa;aristasetulose,itslongestsetulaefar exceed-ing width of base of arista; ventral setulae in arista present; secondarysetulaeoninner-dorsalsurfaceofaristaabsent; parafa-cialsetuloseonlowerthird;facialridgeprojected,partiallyhiding post-pedicelinlateralview;palpuscylindrical;proboscis retrac-tile; vibrissa inserted under ventral level of the eye and fully developed,2–3setaelongerthansubvibrissalsetae;inner verti-calsetaconvergent;outerverticalsetadivergent;postocularseta convergent,shorterthaninnerverticalseta;upwardcurvedsetae
ingenaabsent;ocellartriangleshort,notreachinghalfthelength offrontalvitta.
Thorax.Acrostichalsetae:0:1;dorsocentralsetae:2:3; postprono-talsetae2;presuturalintra-alarsetapresent;presuturalsupra-alar setapresent,twicelongerthanpresuturalintra-alarseta; notopleu-ron:2setaeandwithsparsecoveragesetulaearoundposteriorseta; prealarsetaabsent;supra-alarpostsuturalsetae:2;intra-alar post-suturalseta:1;basalscutellarsetae:1weakpair(about 80%of thelenghtofscutellum);lateralscutellarsetae:1pair3–4times longerthanbasalone;preapicalscutellarsetae:1pair,thesame sizeasbasalscutellarsetae;apicalscutellarsetae:1pair,thesame sizeaslateralscutellarsetae;anepimeron(lowerportion), anater-gite,katatergite,katepimeron,meron,greaterampulla,post-alar wall,supra-squamalridgeand membrane betweenanepimeron and lowercalypterand bare;proepisternum: 2 upwardcurved setaeandwithafewcoveragesetulae;proepimeronwithafew coveragesetulae;anepimeron(upperportion),katepisternumand basal lateralportion of scutellumsetulose; katepisternals: 1:2; anepisternumwitharowof8setae,withsetulaeposteriorlyand atanteriordorsalcorner;metathoracicspiraclelargerthanhalter’s knobandsetuloseatposteriorportion;metakatepisternumwitha fewweaksetulae;lateralbasalmembraneconnectingupperand lowercalypterabsent;lowercalypterglossiform.
Wing.Subcostalsinuous;A1+CuA2long,almostreachingthe
mem-braneborder;A2curvesmooth;subcostalscleritebare;theapical
portionofMstraight,subparalleltoR4+5;microtrichiapresentin
allthemembrane;allveinsbareexceptforcostalvein.
Legs.ForefemurwithacompleterowofsetaeinPD,D,andPV surfaces;foretibiawithdifferentiatedgoldencoveragesetulaeon AVsurface,2–3mediantosubmediansetaeontheADsurfaceand amediansetaonPDsurface.Midfemurwith2PDsubapicalsetae, 1strongmedianventralandaseriesofdistalslightlydifferentiated PVsetae;midtibiawith2–3Pmediansetae.Hindcoxabareon posteriorface;hindfemuronAV surfacewitharowofsetaein apicalhalfandastrongersubmedianseta,analmostcompleterow ofsetaeonADsurface,asubapicalsetaonPDsurface,anirregular rowofdistalsetaeonPVsurfaceandastrongersubmedianseta; hindtibiawith1–2submediansetaeonAVsurface,asubmedian,a strongermedianandastrongapicalsetaonADsurface;1–2median setaeonPDsurface;calcarabsent.
Abdomen.Firststernitesetuloseonthewholesurface.
Terminalia.Ovipositorlong;longsetaepresentonthetransition betweenovipositorsegments.Sixthsterniterectangularandpoorly sclerotized,almosttransparentandbarelyvisible;seventh stern-itenotsclerotized(notvisible);eighthsternitedividedandclearly visible;hipoproctpentagonal;cercidigitiform;sixthtergitedivided andanchor-shaped;seventhandeightsternitesnotsclerotized(not visible);epiprocttriangularandpoorlysclerotized,almost trans-parent(Fig.4A,B);threespermathecae(Fig.4C).
Egg(Fig.3D).Colordarkbrown.TheeggisPhaonia-type(Skidmore, 1985),elongated,roundedatbothpolesandwithone longitudi-nallycorrugatedmarkonventralsurfacethatextendsalmostfrom poletopole.Asimilarmarkisalsofoundoneachlateroventraland laterodorsalsidesoftheegg.Dorsalsurfaceflatwithabroadmedian area,andventralsurfaceconvex.Hatchingpleatsfoliaceous, paral-lel,andfoldinginwards.Theoutermarginsareserrated,eachpeak withadelicatesetulaonthetip.
Malemeasurements(9males):4.6–6.1mm(lengthfromthehead totheposteriortipofscutellum),6.7–10mm(winglength). Morphologicalvariationsbetweennewmalespecimensandthe holotype.Scutellumvaryingfromwhollyyellowtoyellowwitha brownspotatthebase.Parafacialsetulosefromthelowerthirdto thelowerhalf.Postocularsetaeconvergent.Foretibiawith1–2A setae,theproximalonlyslightlydifferentiatedfromground setu-lae;withorwithoutaPmedianseta.Midfemurwithaseriesof distalsetaethatareslightlydifferentiatedfromcoveragesetulae
onPV.Hindtibiawith1–2submedianAVsetae;2–3submedian ADsetae;1–2submedianPDsetae.Theoriginaldescriptionofthe holotypereportsasetaslightlydifferentiatedfromthecoverage setulaeastheparahumeral.Asnoneofthenewlyobservedmaterial hasthisseta,webelievetheparahumeralisactuallynotdeveloped inthisspecies.Thesameobservationisvalidfortheintrapostalar seta.
Remarks. The original description of the holotype states that “parafacialsetuloseonventralhalf(...)parafacialbare”(Haseyama
anddeCarvalho,2011,p.62).Theparafacialisactuallysetulose
inthelowerhalf.Othercharactersnot previouslydescribedare prosternumbareandnotopleuronsetuloseonlynearthePseta. Wehadonlytwofemalesavailableforthisstudy,andbothwere dissected.Wecouldnotvisualizethesixthtergiteandscleriteand theepiproctinthefirstone.Inthesecondfemale,thesestructures werepoorlysclerotizedandbarelyvisible.Thefemaleissimilarto male,regardingsize,color,andchaetotaxy.
Examinedmaterial
Type-material.Holotype,male.Labels:“BIOTA-FAPESP/BRASIL:ES: SantaTeresa/Est.BiológicaSantaLúcia–867m/1958373S4032 225W/Arm.Malaise–Ponto1–Trilha/9-12IV2001/C.O.Azevedo &eq.Col.”(MZUSP).
Additional material examined (n=13). All specimens belong to CCT-UFMG, except where noted. Brazil, Minas Gerais. 1♂ Belo Horizonte, Campus UFMG, Estac¸ão Ecológica, 19◦52’30”S, 43◦58’20”W, 842m, 20.i.1999, Malaise trap, A.F. Kumagai col. (UFMG-IDI-1700220); 1♀, same data,17–23.xi.1991 (UFMG-IDI-1700221);1♀ BeloHorizonte,CampusUFMG,Estac¸ãoEcológica, 10–16.xii.1991,A.F.Kumagaicol.(UFMG-IDI-1600467);1♂Nova Lima,19◦58’7.4”S,43◦51’22.7”W,27.x.2015,Malaisetrap,A.R.Lima col.(UFMG-IDI-1701192;voucherofthesequencessubmittedto GenBank);1♂samedata,27.xii.2015(UFMG-IDI-1700226);1♂São Gonc¸alodoRioAbaixo,EPDA/Peti,PO-44,19◦52’49”S,43◦22’06”W, 14.ii.2003,Malaisetrap,A.F.Kumagaicol.(DZUP245599);1♂,same data,14.xi.2013(UFMG-IDI-1701194);1♂,samedata,27.xii.2002, PO–37 (UFMG-IDI-1700225); 1♂, same data, 27.ii.2003, PO-47 (UFMG-IDI-1700224);2♂,samedata,17.i.2003,PO-40 (UFMG-IDI-1700228,UFMG-IDI-1700229);1♂,samedata,13.xii.2002,PO-35 (UFMG-IDI-1700223);1♂,samedata,07.ii.2003,PO-43 (UFMG-IDI-1700222).
Geographical distribution (Fig. 5): Brazil: Espírito Santo, Minas Gerais(newrecord).
Discussion
ItatingamyiaishighlysupportedasaCyrtoneurininaeby topo-logicaltestsandphylogeneticestimation.Nevertheless,wewere notabletoconsistently establishthesister-groupofthisgenus, sinceCyrtoneurininaeinternalrelationshipsarenotthesameunder MLandBI,andhavelowresolutiononthelattercriterion.More inclusivephylogenies,likethosepublishedbyKuttyetal.(2014)
and Haseyamaet al. (2015)alsohave a low resolution for this
clade,suggestingthatadensersamplingoftaxaorgenes,orboth,is neededtoconsistentlysolvetherelationshipswithinthesegenera. OurresultsagreewithdeCarvalhoandCouri(1993),who clas-sifiedItatingamyiawithintheReinwardtiini, whichin thesense usedhereincludestheCyrtoneurininaesensuSnyder(1954),the Reinwarditiini,theEginiini,andpartiallythePhaoniini(Haseyama
etal.,2015).Albuquerque(1979)classifiedthegenuswithinthe
Mydaeinae,basedontheovipositorofI.bivittata.However,his illus-trationofthisstructure(Fig.1,p.325)isverydifferentfromthe
onebydeCarvalhoandCouri(1993)forthesamespecies(Figs.8
Fig.5.KnowncollectionsitesforItatingamyiaspecies.Bluesquares:I.bivittata.Redcircles:I.couriae.Darkergreenarea:Atlanticforestbiome.Yellowishgreenarea:Cerrado biome.Yellowishgrayarea:Caatingabiome.
ovipositorandcerciareshort,likethosefoundinmanyMydaeinae, whileindeCarvalhoandCouri’sillustrationtheovipositorandcerci arelong.Thissecondinterpretationissimilartoourown(Fig.4). Thelongovipositorandthedigitiformelongatedcerciofthefemale arefoundinotherCyrtoneurininaegenera(e.g.,Chaetagenia, Neo-muscina,Philornis,amongothers)(Couri,1983;CouriandLamas,
1993;Pereira-ColaviteanddeCarvalho,2012).Furthermore,short
cerciarenotexclusiveoftheMydaeinae,andtherefore,this charac-terisnotappropriatetodeterminethesystematicpositionwithin Muscidae.Also,ouranalysesdonotcorroborateacloserelationship betweenItatingamyiaandCariocamyia(HaseyamaanddeCarvalho, 2012),whichwasestimatedasthesistergroupofProhardyia(ML,
Fig.1),orinapolytomywithCyrtoneuropsisspecies(BI,Fig.2). The egg of Itatingamyia couriae is in general terms similar to those found in other Muscidae species with broadly folia-ceoushatchingplates(i.e.,Phaonia-type),suchasMydaealateritia (Rondani,1866)(GrzywaczandPape,2010),Neodexiopsisrufipes (Macquart,1851)(PatitucciandCouri,2018)andPhilornistorquans (Nielsen,1913) (Patitucciet al.,2017).Sincethesestudiesused electronmicroscopyand oursonlylight microscopy,wecannot comparethestructuresindetail.However,toourknowledge,it isthefirsttimethatsetulaeareobservedinthehatchingplateof Muscidaeeggs.
ItatingamyiacouriaeisprimarilydistributedinAtlanticForest areas(Fig.5).Ontheotherhand,accordingtoAlbuquerque(1979),I. bivittatawascollectedinaCerradoareainthemunicipalityofPedra Azul,stateofMinasGerais.Interestingly,PedraAzuliscoveredwith dryforest,whichisakindofdeciduousvegetation(Golfari,1975). Whilesomeauthorsconsiderthiskindofvegetationasapartof Cerradobiome(e.g.,RibeiroandWalter,1998),theBrazilian gov-ernmentplacesPedraAzulwithintheboundariesoftheAtlantic forestfollowinglaw11428/06(Brasil,2006).Therefore,thegenus canbeconsideredrestrictedtotheAtlanticforestbiome.
Thediscoveryofthenewindividualsdescribedhere,especially thefemale,willfacilitatefuturemorphologicalandmolecular sys-tematicstudiessincetheknownspecimensofI.bivittataareinpoor condition(e.g.,Fig.1fromHaseyamaanddeCarvalho,2011).Even
thoughwewereabletoconsistentlyproposeasubfamily classifi-cationforthegenus,furtherinvestigationisneededtounderstand therelationshipswithinthiscladeandtouncoverthesister-group ofItatingamyia.
Authorscontribution
KLFHconceivedtheresearchprojectandanalyzedthedata.CJBC describedthefemale.AZFproducedthenewDNAsequencesand analyzedthedata.FDK sortedthespecimens andproduced the images.Allauthorscontributedtotheanalysisdesign,manuscript writingandapprovedthefinalmanuscript.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
Theauthorsthankthecollectorsofthestudiedmaterial;Carlos J.E.LamasforgrantingpermissiontousetheDipteracollectionof theMZUSP.WealsothankAdalbertoSantos(UFMG)forallowing ustousephotographicequipmenttoproducefiguresandBárbara Faleiro(UFMG) andPedro Henrique Martins(UFMG)for assist-ingusintheprocess.ThisworkwassupportedbytheConselho NacionaldeDesenvolvimentoCientíficoeTecnológico,CNPq[CJBC –processnumber309873/2016-9];CNPq[AZS–processnumber 161907/2015-6];UFMG/PRPQ[KLFH–projectnumber23853/344]; andFAPEMIG.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.rbe.2018.10.003.
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