A new species of Neotropical Drosophila (Diptera, Drosophilidae) belonging to the guarani group

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A

new

species

of

Neotropical

Drosophila

(Diptera,

Drosophilidae)

belonging

to

the

guarani

group

Vilma

Ratcov

a

_,

_Carlos

_R.

_Vilela

a,∗

_,

_Beatriz

_{Go ˜}

_ni

b

a_Universidade_de_São_Paulo,_Instituto_de_{Biociências,}_Departamento_de_Genética_e_Biologia_Evolutiva,_São_Paulo,_SP,_Brazil b_Universidad_de_la_República,_Facultad_de_Ciencias,_Instituto_de_Biología,_Sección_Genética_Evolutiva,_Montevideo,_Uruguay

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2March2017 Accepted8June2017 Availableonline22June2017 AssociateEditor:SarahOliveira Keywords: Atlanticforest Brazil Femaleterminalia Karyotypes Maleterminalia

a

b

s

t

r

a

c

t

Drosophilabutantansp.nov.,aspeciesbelongingtotheguaranigroupandcloselyrelatedtoDrosophila nigrifemurfromBolivia,isdescribedbasedonafemale,andsomeofitsoffspring,collectedattheforest reserveoftheInstitutodeBiociênciasdaUniversidadedeSãoPaulo,CidadeUniversitária“ArmandodeSalles Oliveira”,SãoPauloCity,stateofSãoPaulo,Brazil.Althoughexternallysimilar,thetwoapparently forest-dwellingspeciescanbetoldapartbyhavingdistinctoviscaptvalvesandspermathecalintrovertsand tips.Accordingly,aproposalismadetoalsoincludeD.nigrifemur,apreviouslyunassignedspecies,inthe guaranigroup.ThetwospeciesseemtobealsorelatedtoDrosophilaalexandreiandDrosophilaguaraja asindicatedbytheirexternalmorphology,theirelongatespermathecaeandthenotsosharplypointed oviscaptvalves.ThekaryotypesofthenewspeciesdifferfromthosedescribedforD.alexandreiandD. guaraja,whilethoseofD.nigrifemurremainstillunknown.Photomicrographsofthemaleandfemale imagines,inadditiontodrawingsandphotosoftheirterminalia,arealsoincluded.

Introduction

MostspeciesofDrosophilabelongingtotheNeotropicalguarani grouparereadilyrecognized bytheirdarkbrownbody and by havingwingswithcloudedmaincrossveins.Theguaranispecies groupwasproposedby Dobzhanskyand Pavan (1943:66) and is currently (Bächli, 2017)composed by 16 species: Drosophila alexandreiCordeiro,1951,D.araucana Brncic,1957,D. cuscungu Vela and Rafael, 2005, D. ecuatoriana Vela and Rafael, 2004, D. griseolineata Duda, 1927, D. guaraja King, 1947, D. guaru DobzhanskyandPavan,1943,D.huillicheBrncic,1957,D. limbin-ervisDuda,1925,D.maculifronsDuda,1927,D.ornatifronsDuda, 1927,D.pichinchanaVelaandRafael,2004,D.quitensisVelaand Rafael,2004, D. subbadia Patterson and Mainland in Patterson, 1943, D. tucumana Vilela and Pereira, 1985, and D. urubamba VilelaandPereira,1993.

Duringa two-yearDrosophilid faunasurveyconductedfrom September, 1995 through August, 1997 (Ratcov, 2002 unpub-lished thesis) in an urban forest reserve in São PauloCity, we collectedsomesmallunknowndarkbrownmaleandfemale ima-ginesbelongingtotheguaranigroup.Althoughwewereunableto

∗ Correspondingauthor.

E-mail:crvilela@ib.usp.br(C.R.Vilela).

identifythemtothespecieslevel,wesuspectedtheywere conspe-cific.Afterhavingestablishedanisofemaleline(codedI48F71)we couldconfirmthepreviousmale/femaleassociationandanalyze theirmaleterminalia,especiallytheaedeagusandtheinner sper-mathecalcapsules.Atfirstsight,basedontheshapeofthelatter structures,wesuspectedtheycouldbelongtoDrosophila alexan-drei,aspeciescollectedinRioGrandedoSulstate(Cordeiro,1951), onlyknownfromitsoriginaldescriptionandwhosetypespecimens arethoughttobelost.Theanalysisofthemaleterminaliawasof nohelpastheywereneitherdepictednorillustratedintheoriginal descriptionofthecandidatespecies.

Tenyearslater,inDecember2006,10specimens(4males:6 females)ofthisundescribedspecies wereidentiﬁedamong 495 drosophilids aspirated from living inﬂorescences of Goeppertia monophylla(Vell.)Borchs.&S.Suárez(Borchseniusetal.,2012) (Marantaceae),inthesameforestreserve(Vazetal.,2014).

More recently, analyses of the mitotic metaphase chromo-somes obtained from the larval brain sampled from isofemale lineI48F71 and described below suggest theunknown species wehave collectedand Drosophila alexandreibelongtodifferent species.

Thepurposeofthepresentpaperistodescribethisunknown forest-dwellingNeotropicalspeciesoftheDrosophilaguaranigroup, whichis attractedtobutapparently doesnot breedon inﬂore-scencesofGoeppertiamonophylla,a Marantaceaespecies locally knownascaeté.

http://dx.doi.org/10.1016/j.rbe.2017.06.002

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Figs.1–6.Drosophilabutantansp.nov.,paratypesandpupafromisofemalelineI48F71:(1,2)maleparatype,and(3,4)femaleparatype,indorsalandrightlateralviews, respectively;(5,6)ordinarypuparium+pupa,dorsalandobliquedorsalviews.Scalebar=1mm.

Materialandmethods

Onefemalecollectedwithanentomologicalnetovera banana-baited trap in late August 1996, including her first-generation offspring (5 males, 8 females), plus two specimens (1 male, 1 female) of a much later generation of the same isofemale line (I48F71) yielded in the laboratory in a modified banana-agar medium(Go ñiandVilela,2016)wereusedinthedescriptionof thenew species. The trap wasset in theforest reserve of the InstitutodeBiociências daUniversidadedeSãoPaulo, Cidade Uni-versitária“ArmandodeSallesOliveira”,SãoPaulocity,stateofSão Paulo,anurbanfragmentofthemontaneAtlanticforestof south-easternBrazil.Labeldataaccompanyingeachtypespecimenare recordedasgiven;acommaindicatesalinechange,andaslash, alabelchange.Ourownnotesorinterpretationsareincludedin brackets. Allspecimens aredeposited in theMuseu de Zoologia daUniversidadedeSãoPaulo, SãoPaulo,Brazil(MZUSP). Dissec-tionsofreproductivestructuresfollowWheelerandKambysellis (1966),asmodifiedbyKaneshiro(1969)andBächlietal.(2004). Formorphologicalterminology,measurements,indices, prepara-tionsofmicroscopeslidesaswellasillustrationsrefertoVilelaand Bächli(1990,2000)andBächlietal.(2004).Wheneverinthesame plate,alllinedrawingsweredrawntothesamescale,andall pho-tomicrographsweretakenandenlargedtothesamemagnification. Photomicrographs ofimagoesand pupa(4× objective)were takenwithanOlympuscamera(PM2)loadedwithananalog35mm FujichromeProfessional64TfilmandattachedtoanOlympus ste-reomicroscope(SZ11)witharingilluminator.Theanalogimages werelaterdigitizedwithanEpsonscanner(Perfection4180photo). Photomicrographsofwing(4×objective,optovar1×)and termi-naliastructures(10×objective,optovar1×)weretakenwithan OlympusQ-Color5digitalcameraattachedtoanOlympusBX60 microscope.Asetof20–50pictureswastakenbymanually focus-ingeachstructureatdifferentdepths.Thissetofphotomicrographs wasthendigitallystackedtocreateanall-in-focuscompositeusing

the CombineZP software (open source softwareCombineZP at http://combinezp.software.informer.com).

Initially,theisofemalelineI48F71waskeptinashort-lasting powderedmilk-agarmedium(Bächlietal.,2000)forabout10years. However,duringthepast11years,thecitedstrainhasbeenalso successfully maintainedin a long-lastingmodified banana-agar culturemedium(Go ñiandVilela,2016).Theimaginesarekeptin conventionalcylindricalglasstubes(20×100mm),containingca. 3mLofmodified banana-agarmediumfor3 weeks,at18±1◦C and13:11h(L:D)photoperiod.Duringthisperiod,theyare trans-ferredonceaweektonewvialstoreachsexualmaturation.Atiny ball(ca.1mmdiameter)ofsolidandfreshbaker’syeastisadded aftertransferringtheflies.Thefliesarediscardedthreeweeksafter emergenceand,oneweeklater,twoV-shapedfilterpaperstrips areinsertedintothemediumandthevialsareplacedinsideone ¼lflaskcontainingwetsandatthebottom.Emergedfliesofthe newgenerationarethenaspiratedoncea weekandtransferred againtoamodifiedbanana-agarmedium,restartingthecycle.This methodhasalsobeensuccessfullyusedinourlaboratoryinSão Paulocitytokeepstrainsofsomespeciesbelongingtothecardini andtripunctatagroups.SinceearlyFebruary2017thewetsandhas successfullybeenreplacedbyexfoliatedvermiculite.Itisworth mentioningthecalmbehavioroftheimaginesofthenewspecies insidethecultureflaskscontainingsandorvermiculite,facilitating theaspirationandtransferofemergedfliestonewvials.

Male and female mitotic chromosomes were obtained from cytologicalpreparationsofcerebralgangliaofthirdinstarlarvae of both sexes of the new species sampled from the isofemale lineI48F71,accordingtothemethodofImaietal.(1977,1988). All cytologicalpreparations were made fromsingle individuals cultured withbanana-agar-yeastmediaat18–20◦C. Cytological preparations were observed in an Olympus BX60® microscope equippedwithanOlympus U-MAD-3® camera.Suitablemitotic cellswereselectedandthendigitalizedusingtheImage-ProPlus® version 5.1 software. Furthermore, mitotic chromosomes from

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Fig.7.Drosophilabutantansp.nov.:maleparatype,rightwing,dorsalview.Scale bar=1mm.

individualsofbothsexesofDrosophilaguarajafromtheisofemale linecodedM28F1,establishedfromasinglegravidfemalecollected atdowntownUbatubaattheNorthcoastofthestateofSãoPaulo in July 2007, were obtained and analyzed as described above. Inthis case, themitoticcells wereobserved in aNikon Eclipse 80i microscope witha Digital Sight camera, and selectedcells were digitized using NIS-Elements AR software. All cytological imageswereprocessedinGIMP2.8.14(GNUImageManipulation Program).

Drosophilabutantansp.nov. (Figs.1–28)

Drosophilasp.W3,Vazetal.,2014:608+Supp.KeyS1. Materialexamined(6males,10females,depositedinMZUSP). Holotype male [ﬁrst-generation offspring of an isofemale line, codedI48F71,emerged10-22.X.1996,dissected]labeled:“Brasil – SP, São Paulo, Reserva Florestal [forest reserve] do IB-USP [InstitutodeBiociências–UniversidadedeSãoPaulo],Cidade Uni-versitária,28-30.VIII.1996,Ratcov&Vilelacoll./Drosophilabutantan [male symbol] Ratcov & Vilela/terminália ilustrada [illustrated terminalia]/HOLOTYPE/[glass microvial with terminalia in glyc-erin]”.Paratypes=onemaleandonefemale(photomicrographed) fromanundeterminedgenerationofthecitedisofemaleline,one wild-caughtfemale[motherofthecitedisofemaleline]plusits ﬁrst-generationoffspringbuttheholotype:4males[onedissected] and8females[twodissected]:samedataasholotype.

Type locality. Forest Reserve of IB-USP, Cidade Universitária “ArmandodeSallesOliveira”,districtofButantã,SãoPaulocity,state ofSãoPaulo,Brazil.

Diagnosis.Small coffee-brown ﬂy; eye wine-red; head,legs, scutumandscutellumcoffee-brown,dull;pleurablackishbrown, semi-dull;abdomenconspicuouslyshiny,tergites1–4anteriorly light brown, withbroad, welldelimited, blackish browndistal bands,tergites5–7usuallyentirelyblackishbrown;halter,cercus, epiproctandhypoproctlightbrown;anteriorscutellarsdivergent; winglightbrown,crossveindM-CuandapicesofR2+3,R4+5 and

Mveinsslightlyclouded,tipofMveinslightlyturnedposterad,C index2.84–3.29;valveofoviscaptapicallysomewhatpointedbut notsharply.

Description.Male (n=6)(Figs.1 and 2).Headbrown. Frontal length0.28(0.27–0.29)mm,frontalindex=0.89(0.85–0.92),top to bottom width ratio=1.72 (1.64–1.77). Frontal triangle sub-shining, about 83–100% of frontal length; ocellar triangle dark brown, about 33–36%of frontal length.Orbital plates subshin-ing, narrower adjacent to or2, anteriorly diverging from eye margin, about 92–100% of frontal length. Orbital setae black, or2 outsideor1 and or3; distanceof or3 toor1=80% ofor3 to vtm,or1/or3ratio=0.67,or2/or1ratio=0.33,postocellarsetae=63 (58–73)%,ocellarsetae=82(75–83)%offrontallength;vt index 1.00(0.90–1.11);vibrissalindex0.75(0.67–0.71).Faceandcheek shinylight brown. Carinalight brown, prominent, straight, not

apicallyblunt;distancebetweenapicalscutellarsetaeequidistant tothatbetweenapicalandbasalone;basalsetaestronglydivergent, apicalonescruciate;scutindex=1.04.Pleurashinybrown,sterno index=0.54,mediankatepisternalsetaabout50–63%ofanterior one.Halterlightbrownbasally,darkbrowndistally.Legsuniformly lightbrown;apicalsetaonprotibiaandmesotibia;preapicalseta onalltibiae.

Wing(Fig.7)lightbrownhyaline,crossveinsR-ManddM-Cu, andapicesofveinsR2+3,R4+5andMslightlyclouded,tipofvein

R4+5slightlycurvedposterad;length1.90(1.80–1.93)mm,length

to width ratio=2.02 (1.95–2.08). Indices: C=3.06 (2.84–3.29), ac=2.15(2.00–2.38),hb=0.49 (0.44–0.50),4C=0.79 (0.75–0.86), 4v=1.67(1.62–1.73), 5x=1.14(1.11–1.22), M=0.45 (0.42–0.46), prox.X=0.55(0.50–0.59).

Abdomen (Fig. 2) shiny, brownish black; tergites 1–4 light brownwithacharacteristicdistaldarkbrownband,welldelimited, mediallyinterrupted and laterallybroadened, reachinganterior marginoftergite;tergite5entirelyshinybrownishblack,exceptfor athinmedianlongitudinallightbrownstripeinsomespecimens, andtergite6entirelyshinydarkbrown.

Male terminalia (Figs. 8–18). Epandrium (Figs. 8, 9 and 16) almostbare,slightlymicrotrichoseonposteriordorsalarea;upper setaeabsent;ca.8lowersetae;ventrallobemostlymembranous (Figs.8and16),notcoveringsurstylus.Cercusslightly microtri-choseondorsocentralarea,linkedtoepandriumbymembranous tissue(Fig.8).Surstylusnotmicrotrichose,withabout7–8 cone-shapedprensisetae,about10long,strongoutersetaeandabout2 short,thin,mostlyinnersetae(Figs.8,9and16).Decasternumasin Figs.8and9.Hypandrium(Figs.10and17)aslongasepandrium, anteriormarginconvex;posteriorhypandrialprocessabsent; dor-salarchmediallypointedposterad,stronglysclerotized;gonopod notmicrotrichose,fusedtoparaphysis,bearingonelongsetaon medianinnermargin.Aedeagus(Figs.11–15and18)distallybiﬁd (indorsalandventralviews,Figs.11and15), bluntandslightly expandeddorsoventrally(inlateralview,Figs.13and18); subapi-callybearingadorsoventralmembranousareacoveredwithtiny spines;dorsalcleftca.1/3lengthofaedeagus(Fig.12); paraph-ysisnotmicrotrichose,fusedtogonopod,submediallybearingtwo setulaeadjacenttoproximalmargin(Figs.10and17).Aedeagal apodemeas longasaedeagus and fusedtoit, rod-shaped, dor-sodistallybiﬁd(Figs.11–15and18).Ventralrodtriangle-shaped, completelyfusedtoaedeagalapodeme(Figs.12–15,18).

Female(n=10)(Figs.3and4).Colordifferencefrommale:in somespecimens,colorpatternoftergite5issimilartothatofmale tergite4(compareFigs.2and4)withdarkbrowndistalband inter-ruptedbyalargelightbrowntriangle-shapedlongitudinalstripe runningfromtergite4to5,andcolorpatternoftergite6ofsome specimensnotbeingentirelydarkbrownbutbearingathinmedian lightbrownlongitudinalstripe.

Measurements: Frontal length 0.30mm; frontal index=0.87, topto bottomwidth ratio=0.61. Frontaltriangle about75–92% offrontallength.Ocellartriangleabout33–42%offrontallength. Orbitalplates about92–100% of frontallength.Distance of or3 toor1=67–125%of or3 tovtm,or1/or3ratio=0.65 (0.60–0.70), postocellar setae=67 (54–75)%, ocellar setae=91 (85–100)% of frontallength; vt index=1.04 (1.00–1.10);vibrissal index=0.61

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Surstylus Decasternum Dorsal arch Cercus Gonopod+paraphysis Hypandrium Epandrial lobe Aedeagus Aedeagal apodeme Ventral rod Epandrium 9 10 8 11 12 13 14 15

Figs.8–15.Drosophilabutantansp.nov.,maleholotype,terminalia:(8)epandrium,cerci,surstyli,anddecasternum,obliqueposteriorview;(9)surstyliand decaster-num,posteriorview;(10)hypandrium,gonopodsandparaphyses,posteriorview;(11–15)aedeagus+aedeagalapodeme,severalviewsfromdorsalthroughventral.Sale bar=0.1mm.

(0.44–0.75).Cheekindexabout7.43(6.67–10.50).Eyeindex=1.16 (1.10–1.24). Thorax length 1.12(1.10–1.17) mm. h index=1.00 (0.90–1.14).Transversedistanceofdorsocentralsetae229–267% of longitudinal distance; dc index=0.63 (0.53–0.72). Distance betweenapicalscutellarsetaeabout88–114%ofthatbetween api-calandbasalone;scutindex=1.01(0.94–1.12),sternoindex=0.54 (0.44–0.62), median katepisternal seta about 44–67% of

anteriorone.Winglength2.18(1.95–2.46)mm,lengthtowidth ratio=2.13 (1.95–2.28). Indices: C=3.21 (3.05–3.44), ac=2.26 (2.00–2.57),hb=0.47(0.42–0.50),4C=0.78(0.74–0.83),4v=1.70 (1.60–1.96), 5x=1.26 (1.10–1.44), M=0.49 (0.42–0.54), prox. X=0.56(0.48–0.71).

Terminalia (Figs. 19–22). Oviscapt valve double-walled, api-callyroundish, submediallyslightly expanded dorsad,ventrally

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Tergite 8

Epiproct

Hypoproct

Basal introvert Inner spermatecal capsule

Oviscapt valve 19

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Figs.16–20.Drosophilabutantansp.nov.,maleholotype(16–18)andfemaleparatype(19–20),terminalia:(16)epandrium,cerci,surstylusanddecasternum,obliqueposterior view;(17)hypandrium,gonopods+paraphyses,posteriorview;(18)aedeagus+aedeagalapodeme,leftlateralview;(19)tergite8,epiproctandhypoproct,oviscaptvalves, leftlateralview;(20)innerspermathecalcapsules,lateralview.Scalebar=0.1mm.

slightlyundulate,withca.16 discaland about6marginal, peg-like,mostlyroundish-tipped(neithersharplypointednorbearing a mediodorsal, pointed triangle-shaped process, as it occurs in D.nigrifemur),outerovisensilla;trichoid-likeinnerovisensilla:3 thindistallypositioned,and1long,slightlycurved,subterminal; innerwall(Fig.21,dashedline)proximallynarrow,distallyaswide asouter wall. Spermathecal inner capsule elongate, light bulb-shaped,sclerotized, distally notﬂatten (somewhat ﬂatten inD. nigrifemur),slightlywaistedsubbasally,furrowedatbasal1/7;basal introvertdeeplyinvaginated(butneitherreachingthetipnor dis-tallyexpanded,asit occursinD. nigrifemur)andsubproximally dilated.

Egg(n=1).Whitish,lengthca.0.51mm,bearing4thinﬁlaments ofequallength(0.43mm),slightlyshorterthanegg.

Puparium(n=1)(Figs.5and6).Reddishdarkbrown;hornindex about2.8,withca.18lightbrowntrachealbranchesperstalk;stalks ofanteriorandposteriorspiracleslightbrown.

Distribution.Thisspeciesisknownonlyfromthetypelocality (cityofSãoPaulo,stateofSãoPaulo,Brazil).

Etymology.Theepithetbutantan[alternativespellingofButantã] isanouninappositionandanallusiontothedistrictwherethetype localityislocated.

Ecology.Only9males(0.07%)of Drosophilabutantansp.nov. wereidentiﬁedamong13.057malesofspeciesbelongingtothe

Discal outer ovisensillum

Marginal outer ovisensillum

Subterminal inner ovisensillum

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Figs.21and22. Drosophilabutantansp.nov.,femaleparatype,terminalia:(21)leftoviscaptvalve,outerlateralview;(22)innerspermathecalcapsule,lateralview.Scale bar=0.1mm.

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X X X X X X X Y Y Y X X

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Figs.23–28.MitoticchromosomesofDrosophilabutantansp.nov.,(23–25)femaleand(26–28)malemetaphaseplatesfromlarvalneuroblasts.Thechromosomenumberis 2n=10(2R,2V,1D),withmetacentric(V-shaped)Xandtelocentric(rod-shaped)Ychromosomes.Sexchromosomesareindicatedinallcellsandarrowsindicatethesmall andfaintlystaineddotchromosomes.Scalebar=10␮m.

genusDrosophila sampled intwo years (1996–1997) by Ratcov (unpublished thesis) and Vilela from fermenting banana-yeast baitedtrapssetin thetype locality.It wascomparativelymore abundantin fournon-consecutive days of collectionconducted inDecember2006,inthesameforestreserve,byaspiratingflies frominflorescencesofGoeppertiamonophylla,where fourmales ofDrosophilabutantan,sp.nov.(1.29%),inadditiontosixfemales, werecollectedamong309malesofspeciesofDrosophilasampled. However,noneofthe137drosophilidsthatemergedfrom20 inflo-rescencesofGoeppertiamonophyllacollectedinthesamedateand sitebelongedtoDrosophilabutantansp.nov.Thus,thelarval breed-ingsitesofthisspeciesremainunknown.

Chromosomes(Figs.23–28).Drosophilabutantansp.nov.hasa karyotypeof 2n=10(2R, 2V, 1D),comprising twopairs of rod-shapedtelocentricchromosomesofdecreasingsize,twopairsof largeV-shapedmetacentrics,andonepairofbarelyrecognizable

dot(D)microchromosomes.TheV’sareoftwokinds,oneis approx-imatelyequal-armedwithbotheuchromaticarmsbeingobserved inpairsinmitoticcellsofbothsexes,andcorrespondingtoan auto-some,whiletheother,thelargestV’swithunequalarms,being theshortestarmtotallyheterochromaticandrepresentingtheX chromosome.Thesexchromosomesaredeﬁnitelyheteromorphic, whiletheXisalargeV,theYisatelocentric(rod-shaped) chro-mosome,shorterthantheXchromosome,totallyheterochromatic andoftencurvedratherthanbent,bearingasmallsatelliteatthe proximalregion(Figs.26–28).

Relationship.ItbelongstotheDrosophilaguaranispeciesgroup and is apparently closely related to Drosophila nigrifemur from Bolivia(DepartmentofLaPaz:MapiriandSanCarlos),redescribed and illustrated by Vilela and Bächli (1990: 116, 293C, 323E). Drosophilabutantansp.nov.alsosharessomesimilaritieswithD. alexandreiandD.guarajaregardingtheshapeandstructureofthe

29 X X X X Y Y Y X X X 32 33 34 30 31

Figs.29–34. MitoticchromosomesofDrosophilaguarajaKing,1947:(29–31)femaleand(32–34)malemetaphaseplatesfromlarvalneuroblasts.Thechromosome composi-tionis2n=10(3R,1V,1D),withtelocentric(rod-shaped)XandYchromosomes.Sexchromosomesareindicatedinallcellsandarrowsindicatethedotchromosomes.Scale bar=10␮m.

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Thetwopairsoflongerrodsoftenshowsatellitesandappearto havesubterminalcentromeres”.Theauthorpointedoutthat“The sexchromosomesareevidentlythesmallestrods”,however,our datasupportthattheXclearlycorrespondstothelargestrod chro-mosome,asitisshowninFigs.29–34.

Seven species of the guarani group showa diploid number of 12 chromosomes: D. araucana (see Brncic, 1957), D. griseo-lineata(see DobzhanskyandPavan, 1943; King,1947),D. guaru (see Dobzhansky and Pavan, 1943), D. limbinervis (see Clayton andWasserman,1957),D.maculifrons(reportedasD.guaramunu accordingtoVilelaand Bächli,1990, inDobzhanskyand Pavan, 1943,King,1947),D.ornatifrons(reportedasD.guaraniaccording toVilelaandBächli,1990,inDobzhanskyandPavan,1943,King, 1947),andD.subbadia(seeKing,1947).ThekaryotypesofD. butan-tansp.nov.andD.guarajadifferfromthoseofallotherspeciesof thegroupinshowingﬁveratherthansixpairsofchromosomes(or fourpairsofchromosomes,asinD.alexandrei).King(1947) pro-posedanexplanationforthereducedchromosomenumberinD. guaraja,andrelevanttothehithertoundescribedspeciesshown here.Hearguesthattwoautosomalrodshavebecomefusedby translocationwithresultinglossofoneofthecentromeres.Thesex chromosomesinD.butantansp.nov.are,likethosereportedforD. subbadiabyKing(1947),deﬁnitelyheteromorphic(X=V,Y=R).At present,thecytologicalobservationsshownheresupportthe mor-phologicaldistinctivenessamongD. butantansp.nov.andsome speciesofitsgroup.Forthetime being,Drosophila butantansp. nov.remainsunassigned toanyofthetwo subgroupscurrently recognizedwithintheguaranigroup.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

TheauthorsaregratefultoMr.FranciscoFlauzinoandDr.Lyria Mori,fortechnicalassistanceduringflycultureandchromosome preparation,respectively,toDr.DeniseS.Sheepmakerforproviding thephotostereomicroscopesystem.WealsothankMr.Fernando LucesforEnglishrevision,twoanonymousrefereesandthe edi-torforcorrectionsandsuggestionsthatimprovedthemanuscript. ThisstudywaspartlyfinancedbytheComisiónSectorialde Investi-gaciónCientífica(CSIC),UniversidaddelaRepública,andPrograma deDesarrollodelasCienciasBásicas(PEDECIBA),Universidaddela República–MinisteriodeEducaciónyCultura,UruguaytoBG.

Brncic,D.,1957.LasespeciesChilenasdeDrosophilidae.Colec.MonograﬁasBiol. Univ.Chile8,1–136.

Clayton,F.E.,Wasserman,M.,1957.Chromosomalstudiesofseveral speciesof Drosophila.Univ.TexasPubl.5721,125–131.

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