Can sibling species of the Drosophila willistoni subgroup be recognized through combined microscopy techniques?

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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

Can

sibling

species

of

the

Drosophila

willistoni

subgroup

be

recognized

through

combined

microscopy

techniques?

Rebeca

Zanini,

Maríndia

Deprá,

Vera

Lúcia

da

Silva

Valente

∗

ProgramadePós-Graduac¸ãoemBiologiaAnimal,LaboratóriodeDrosophila,UniversidadeFederaldoRioGrandedoSul,PortoAlegre,RS,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received6April2015 Accepted4August2015 Availableonline9October2015 AssociateEditor:MárciaS.Couri Keywords:

Crypticspecies

Drosophilawillistonisubgroup Drosophilapaulistorumcomplex Malegenitalia

Semispecies

a

b

s

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Inseveralarthropodgroups,malegenitaliaisthemostimportantfeatureforspeciesidentiﬁcation, espe-ciallyincrypticspecies.CrypticspeciesareverycommonintheDrosophilagenus,andtheNeotropical Drosophilawillistonispeciesgroupisagoodexample.Thisgroupcurrentlyincludes24speciesdivided intothreesubgroups:alagitans,bocainensisandwillistoni.Therearesixsiblingspeciesinthewillistoni sub-group–D.willistoni,D.insularis,D.tropicalis,D.equinoxialis,D.pavlovskianaandD.paulistorum,whichisa speciescomplexcomposedofsixsemispecies–Amazonian,Andean-Brazilian,Centroamerican,Interior, OrinocanandTransitional.Theobjectiveofthisstudywastocharacterizemalegenitaliaofthewillistoni subgroup,includingtheD.paulistorumspeciescomplex,usingscanningelectronmicroscopyandlight microscopy.Wealsotriedtocontributetotheidentiﬁcationofthesecrypticspeciesandtoaddsome commentsaboutevolutionaryhistory,basedonmalegenitaliacharacters.Despitebeingcrypticspecies, somedifferenceswerefoundamongthesiblings,includingtheDrosophilapaulistorumsemispecies.

Introduction

Male terminalia isone of themost importanttraits usedto identifycrypticspecies,whichareverycommonintheDrosophila Falléngenus.TheNeotropicalDrosophilawillistonispeciesgroupisa goodexampleofcrypticspeciation.Thisgroupincludes24species, whicharedividedintothreesubgroups:alagitans,bocainensisand willistoni(Bachli, 2015); the last of them showingvarious tax-onomiclevelswithsuccessivedegreesof reproductiveisolation (Robe et al., 2010). The willistonisubgroup includes six sibling species:D. willistoni Sturtevant, 1916,D. equinoxialis Dobzhan-sky,1946,D.insularisDobzhansky,1957,D.tropicalisBurlaandDa Cunha,1949,D.pavlovskianaKatritsisandDobzhansky,1967andD. paulistorumDobzhanskyandPavan,1949.Thesespeciesarealmost morphologicallyindistinguishablebasedonexternalmorphology, exhibitvaryingdegreesofprematingisolationandusuallydonot cross-hybridize(EhrmannandPowell,1982).Withinthesubgroup, Drosophila paulistorumisa species complex, oralsoreferred as a superspecies, composed of six semispecies (Dobzhansky and Spassky,1959; Perez-Salasetal., 1970).Thewillistonisubgroup also shows taxonomic differentiation at the subspecies level:

∗ Correspondingauthor.

E-mail:vera.valente@pq.cnpq.br(V.L.daSilvaValente).

D.willistonidifferentiatesintothewillistoniandquechuasubspecies (Ayala and Tracey, 1973); D. tropicalis contains the tropicalis and cubanasubspecies(Townsend, 1954);and D.equinoxialis is dividedintotheequinoxialisandcaribbensissubspecies(Ayalaetal., 1974).

AccordingthereviewofCordeiroandWinge(1995),thesibling groupisstillinanactiveprocessofspeciationandalllevelsofthis processcanbeobserved.Theauthorssuggesttwostepsof specia-tion:incipientisolation,representedbythesubspecies.Thesecond stepofspeciationisexempliﬁedbythesemispecies,whichshow severaldegrees ofreproductiveisolationrangingfromcomplete isolationtothepresenceoffertileoffspring(CordeiroandWinge, 1995)which wasobserved in the crossingsof the Transitional semispecieswiththeAndean-Brazilian and theCentroamerican semispecies(Ehrman,1961,1965).

D.willistonihasthebroaderdistributionofthegroup(Fig.1), spanningfromCentralMexicoandFloridatoSouthernBraziland NorthernArgentina, and fromthe Atlantictothe PaciﬁcOcean (DobzhanskyandPowell,1975;EhrmannandPowell,1982),even in areas of human disturbance (Valiati and Valente, 1996). D. willistoniisuninterruptedlydistributedover thisarea,exceptin deserts and high altitudes (Ehrmann and Powell, 1982). Other sibling specieshave narrowerdistributionswithinthe distribu-tionofD.willistoni,exceptD.insularis,whichisendemictoSaint Kittsand Saint Luciaof theAntillesIslands(Dobzhansky etal., 1957), andD. pavlovskiana,which hasbeenfoundonly oncein

http://dx.doi.org/10.1016/j.rbe.2015.09.006

0085-5626/© 2015 Sociedade Brasileira de Entomologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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D. willistoni D. equinoxialis D. paulistorum D. tropicalis D. insularis D. pavlowaskiana N Atlantic Ocean

Fig.1.GeographicdistributionoftheD.willistonispeciessubgroup,accordingto

Spasskyetal.(1971),Winge(1971),DobzhanskyandPowell(1975),Ehrmannand Powell(1982),SantosandValente(1990).

Guyana(Spasskyetal.,1971)andhasnotbeencollectedsincethen (Fig.1).

The D. paulistorum semispecies occur from Southern Brazil toCentral America (Guatemala) and Trinidad (Dobzhansky and Spassky, 1959) (Fig. 2).According toDobzhansky et al. (1964),

Amazonian Andean-brasilian Centroamerican Interior Orinocan Transitional N Atlantic Ocean

Fig.2. GeographicdistributionoftheD.paulistorumsuperespecies,accordingto

Spasskyetal.(1971),Winge(1971),DobzhanskyandPowell(1975),Ehrmannand Powell(1982),SantosandValente(1990).

whenthesemispecies’territoriesoverlap,theyapparentlydonot interbreed.Previousstudiessuggestedthatthedifferencesamong morphological,physiologicalandecologicaltraitswithinthe semis-peciesaretoosmalltodistinguisheachsemispecies(Pasteur,1970; Perez-Salas and Ehrmann, 1971) and could only berecognized byexaminationofthegenearrangementsontheirchromosomes (Kastritsis,1967;Rohdeetal.,2006)andbycrossingtests( Perez-SalasandEhrmann,1971).

Despite beinga traditionallystudied groupwithanexciting evolutionaryhistory,thereisalackofstudiesconcerningthe mor-phologyintheearlystagesofdevelopmentandalsoinadults.Some morphologicalstudiesofadultsweremadeafterthespecieswere described.Burla etal.(1949) presentedillustrationsof maxillar palpi, vaginal plates, spermatechae and hypandria of D. willis-toni,D. tropicalis,D.equinoxialis and D. paulistorum.Hsu(1949)

brieﬂydescribed D. willistoniand D. equinoxialis male genitalia, inadditionofsomespeciesofthealagitansandbocainensis sub-groups.Malogolowkin(1952)providedaverydetaileddescription ofthemale and femalegenitaliaofD. willistoni,D. equinoxialis, D. tropicalis, D. paulistorum and some species of the bocainen-sis subgroup. Spassky (1957) shown illustrations of hypandria, aedeagi,surstilyandprensisetaofﬁvesiblingspecies–D. willis-toni,D. tropicalis,D. equinoxialis, D.paulistorumand D. insularis.

Pasteur(1970)madeabiometricalcomparisonoffoursemispecies ofD.paulistorumregardingwingandtibialengths,wing-to-tibia ratios, wing sizeand number of prensiseta onsurstylus. Vilela andBächli(1990)redescribedD. willistoniandprovided several illustrationsofthemaleterminaliaofthisspecies.Eberhardand Ramirez(2004)presentedseveralScanningElectronMicroscopy (SEM) images of male and female terminalia of D. willistoni.

Rohde et al. (2010) provided photos of the hypandria of D. willistoni, D. equinoxialis, D. tropicalis and D. paulistorum with-outindicating thesemispecieswhilesuggestingtheimportance of this structure for their identiﬁcation. Recently, Souza et al. (2014) provided SEM images of D. willistoni, which was used asoutgroupofthephylogeneticreconstructionoftheD. saltans group. Civetta and Gaudreau(2015) shown photos of external malegenitaliaandaedeagusofD.willistoniandthesubspeciesD. willistoniquechua.Also,there areSEMimagesofmale termina-liaoffoursiblingspecies(D.willistoni,D.tropicalis,D.equinoxialis andD.paulistorum)availableinEmilioGoeldiMuseumdatabase (marte.museu-goeldi.br).

Burlaetal.(1949)onlyfoundslightmorphologicaldifferences thatwereinsufficientfortheidentificationofsingleindividuals, whileSpassky(1957)noteddifferencesinthemalegenitaliathat didpermitsuchidentification.Inotherway,Malogolowkin(1952)

described thegeneralmorphologyofthemale genitaliaoffour sibling species (D. willistoni, D. equinoxialis, D. tropicalisand D. paulistorum)asvery similarbutcompletelydifferentfrom non-sibling species.In addition, this author foundnodifferences in thepenisesofthesefoursiblingspecies.Spassky(1957),however, observedthattheshapesofthepenisesandtheirgonapophyses differedinthesiblingspecies.

With respect to D. insularis, only a few illustrations were presented by Spassky (1957). The previous descriptions and illustrations of D. paulistorum were based on Andean-Brazilian specimens once the specimens were collected in areas where onlythissemispeciesisfound(MogidasCruzes,SãoPaulo,Brazil in Burla et al. (1949) and Malogolowkin (1952); Tamandaré, Pernambuco,BrazilinRohdeetal.(2010)).Theremainingspecies, D.pavlovskiana,hasnotbeencollectedandisnolongeravailablein StockCenters.Thereisnodescriptionintheliteratureofthemale genitaliaofthisspecies.

In thisscenario, ourobjectivewastocharacterize and com-pare the male terminalia of the species of the D. willistoni subgroup,includingtheD.paulistorumsemispeciescomplex,using

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Table1

Speciesandstrainsusedinthisstudy.Thenumberedstrainsarerepresentedintheﬁgures.

Species Semispecies Populationsample Localities Source

D.equinoxialis[1] Mexico Apazapán/Mexico LeeEhrmanandYongKyuKim

D.equinoxialis FNT12 Belterra/Brazil MárioJosiasMüller

D.equinoxialis Tape03 Santarém/Brazil MárioJosiasMüller

D.equinoxialis 207018 BelémdoPará/Brazil MarluciaMartins

D.insularis[2] SL03 SaintLucia/LesserAntilles JeffreyR.Powell

D.insularis SL15 SaintLucia/LesserAntilles JeffreyR.Powell

D.tropicalis[3] 0801 SanSalvador/ElSalvador TucsonStockCenter

D.willistoni[4] WIP4 Salvador/Brazil AntônioR.CordeiroandHelgaWinge

D.willistoni Gdh4-1 Guadalupe/Caribe TucsonStockCenter

D.willistoni RibeirãoPreto RibeirãoPreto/Brazil CláudiaRohde

D.willistoni Mexico Apazapán/Mexico MargaretKidwell

D.willistoni DW-PoolIta Itapuã/Brazil AndréSchnorr

D.willistoni DW-PoolFaz SerradoCipó/Brazil CarlosVilela

D.willistoni CIP SerradoCipó/Brasil CarlosVilela

D.willistoni COR Coronilla/Uruguay BeatrizGo ˜ni

D.willistoni ISC SantaCatarina/Brazil DanieladeToni

D.willistoni DLA DoisLajeados/Brazil CláudiaRohde

D.willistoni MAS MorroSantana/Brazil AnaLauerGarcia

D.willistoni Joinville Joinville/Brazil JonasDöge

D.willistoni 17A2 EldoradodoSul/Brazil VeraL.S.Valente

D.paulistorum[5] Amazonian Paráq3 BelémdoPará/Brazil LeeEhrmannandYongKiuKim

D.paulistorum Tape60 Santarém/Brazil MárioJosiasMüller

D.paulistorum[6] Andean-Brazilian MLC SantaCatarina/Brazil MarcoGottschalk

D.paulistorum RIB RibeirãoPreto/Brazil VeraL.S.Valente

D.paulistorum RMT PortoAlegre/Brazil AnaLauerGarcia

D.paulistorum Pool-Bur Brasília/Brazil RosanaTidon

D.paulistorum Játon-Sacha JatonSacha/Equador MargaretKidwell

D.paulistorum[7] Centroamerican Lancetilla Lancetilla/Honduras LeeEhrmanandYongKyuKim

D.paulistorum[8] Interior INT Llanos/Colombia LeeEhrmanandYongKyuKim

D.paulistorum[9] Orinocan ORI Georgetown/Guyana LeeEhrmanandYongKyuKim

D.paulistorum[10] Transitional SantaMarta SantaMarta/Colombia LeeEhrmanandYongKyuKim

ScanningElectronMicroscopyandLightMicroscopy.Weattempt todescribethismorphologicaltraitwithinspeciesandﬁndfeatures thatdifferentiatethesiblingspeciesandsemispeciesofthe willis-tonisubgroupandverifyifthesespeciescanberecognizedbased oncharactersofthemaleterminalia.

Materialandmethods

Flystocks

Thestockswererearedinacornmealmedium(Marquesetal., 1966)ataconstanttemperatureandhumidity(17_±1◦C;60%rh). Someindividuals werepreservedin 70%ethanol, and mounted stubs and slides will be deposited in Fundac¸ão Oswaldo Cruz (Fiocruz) Collection. All strainsused in this studyare listed in

Table1.

Speciesrecognition

Species wereconﬁrmedwiththe Acph-1(Acid Phosphatase) electrophoresisprotocol(Garciaetal.,2006).Also,DNAsequences weregenerated andcomparedwithsequencesavailablein Gen-Bank–mitochondrialgenefragmentsCOI(CytochromeoxidaseI) (depositedbyGleasonetal.,1998),COII(CytochromeoxidaseII) (depositedbyRobeetal.,2010),andnucleargenesfragmentAdh (Alcoholdehydrogenase)(depositedbyGleasonetal.,1998and

Robeetal.,2010).Thesequencesobtainedwillbepresentedina furtherstudy.

Scanningelectronmicroscopy(SEM)preparationandobservation Male terminalia were treated with 10% KOH (Wheeler and Kambysellis,1966modiﬁedbyBächlietal.,2004)anddissected inglycerol.Terminaliaweredehydratedfor20–30swithacetone washesinthefollowingconcentrations:30%,50%,75%and100%. Theentireterminaliaandseparatedpartsweremountedinstubs withcarbon tapeand metalizedwithgoldin a BalzersSCD050 sputtercoater.Visualizationandimagecapturewereperformed in JSM6060 Scanning Electron Microscopein Centro de Micro-scopiadaUniversidadeFederaldoRioGrandedoSul.Weobserved approximately50 seven-day-oldspecimensof each speciesand semispecies.

Lightmicroscopypreparationandobservation

Maleterminaliawerepreparedaspreviouslydescribed.The ter-minalia,aedeagiandhypandriaweremountedinanon-permanent glycerinjellymedium(Klausetal.,2003).Theslideswereobserved andphotographedwithaCarl-ZeissStandardphasecontrast micro-scope.We analyzedthegenitalstructuresof7-day-oldmalesof eachspeciesandsemispecies(750individuals).

Terminologyandreferences

The morphological terminology used in this study follow

McAlpine(1981),Grimaldi(1990),VilelaandBächli(1990),Bächli et al. (2004) and Souza et al. (2014). Figures of external male

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Fig.3.GeneralexampleofexternalmalegenitaliaoftheD.willistonisubgroup (pic-tureofD.paulistorumAmazonian[5]).Scalebar50␮m.EP,epandrium;CE,cerci; SU,surstylus;PR,prensisetae;AE,aedeagus;VL,ventrallobe;HY,hypandrium.

genitaliaof D. willistoni subgroup, under light microscopy, are showninSupplementarymaterial1(S1).

Results

Epandrium,surstylusandprensisetae

Thecerciarenotfusedtotheepandrium(Figs.3and4;Fig.S1) inallthespeciesanalyzed.Thesurstylusiselongatedintoahook atthebottom,isnotmicropubescent,hasupto12prensiseta(also calledprimaryteeth)inD.paulistorumOrinocanandD.paulistorum Interior,andupto18prensisetainD.equinoxialis(Fig.5)in addi-tiontoonelargeprensisetaeandoneortwosetaontheventral hook.

Drosophilaequinoxialispresents18prensiseta,ninesmallerand ninelarger,andonesetaeontheventralhook(Figs.4Aand5A). D. insularis has approximately 17 prensiseta of equal size and onesetaeintheventralhook(Figs.4Band5B).D.tropicalishas 13–14crescentsizeprensisetaandonesetaeintheventralhook (Figs.4Cand5C).D.willistonialsohas13crescentsizedprensiseta andonesetaeintheventralhook(Figs.4Dand5D).

AmongtheD.paulistorumsemispecies,wefoundthatDrosophila paulistorumAmazonianhasa surstyluswith15 prensiseta,nine longer and six shorter, and one setae in the ventral hook (Figs.4Eand5E).D.paulistorumAndean-Brazilianhasasurstylus with15prensiseta,eightlongerandsevenshorter,andtwosetain theventralhook(Figs.4Fand5F).D.paulistorumCentroamerican hasasurstyluswith15prensiseta,eightlargerandsevensmaller, andtwosetaintheventralhook(Figs.4Gand5G).D.paulistorum Interiorhasasurstyluswith12crescentsizeprensisetaeandtwo setaintheventralhook(Figs.4Hand5H).D.paulistorumOrinocan hasasurstyluswith12prensisetaofapproximatelythesamesize andtwosetaintheventralhook(Figs.4Iand5I).D.paulistorum Transitionalhasasurstyluswith12prensisetaeofapproximately thesamesize,withonelargerprensisetaeinthemiddleandtwo setaintheventralhook(Figs.4Jand 5J).Thedistancebetween thesidesofsurstylusandhookscouldbeanartifactoftheSEM preparationandisnotconsideredadiagnosticcharacterforspecies identiﬁcation.

Fig.4.ScanningelectronmicroscopyofD.willistonispeciessubgroupexternalmale genitalia.Scalebar50␮m.ThestrainsareinbracketsandlistedinTable1.(A)D. equinoxialis[1];(B)D.insularis[2];(C)D.tropiacalis[3];(D)D.willistoni[4];(E)D. paulistorumAmazonian[5];(F)D.paulistorumAndean-Brasilian[6];(G)D. paulisto-rumCentroamerican[7];(H)D.paulistorumInterior[8];(I)D.paulistorumOrinocan [9]and(J)D.paulistorumTransitional[10].

Hypandrium

Thehypandriumissmallerthantheepandrium;itis approxi-mately1/4to1/3ofthesizeoftheepandrium.Thehypandriain allofthespeciesanalyzedhave,intheapicalregion,onepairof heavilysclerotizedmedianteeth,lobeswithparamediansetaon theapexandlateralextensions(Figs.6and7).Therelativesizeand thicknessofthemedianteeth,aswellasthesizeandshapeofthe lobes,varywithinthespecies(Figs.6and7).

In D. equinoxialis, the hypandrium is triangular, with well-developedtrapezoidallobesandlobesetaconvergenttoteeth.The teetharelargeandthick,twicetheheightofthelobes,alignedwith thelobes,anddonottoucheachother.Thelateralextensionsare veryprominenttowardthetop(Figs.6Aand7A).D.insularishas

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Fig.5.ScanningelectronmicroscopyofD.willistonispeciessubgroupsurstyliand prensiseta.Scalebar20␮m.ThestrainsareinbracketsandlistedinTable1.SU, Surstylus;PR,Prensisetaes;VH,Ventralhook.(A)D.equinoxialis[1];(B)D. insu-laris[2];(C)D.tropicalis[3];(D)D.willistoni[4];(E)D.paulistorumAmazonian[5]; (F)D.paulistorumAndean-Brasilian[6];(G)D.paulistorumCentroamerican[7];(H) D.paulistorumInterior[8];(I)D.paulistorumOrinocan[9]and(J)D.paulistorum Transitional[10].

atriangularhypandrium,withalmostabsentlobes.Thisspecies presentsoneortwo paramedianseta inthelobesandpresents thesmallerandmoreseparatedteeththatoccurinthewillistoni subgroup(Figs.6Band7B).D.tropicalispresentsatriangular hypan-drium,withverylargeroundlobesandsetaconvergenttoteethand almosttouchingthem.Italsohaslargethickteeth,twicetheheight ofthelobes,insertedslightlybelowthelobeline.Thelateral exten-sionsarelocatedunderthelobes(Figs.6Cand7C).D.willistonialso hasatriangularhypandrium,withsubtleroundlobes.Itsveryclose large,thickteetharethricetheheightofthelobesandareinserted farbelowthelobeline.Thelateralextensionsaresimilartothose ofD.tropicalis,butareadjacenttothelobes(Figs.6Dand7D).

In the D. paulistorum complex, D. paulistorum Amazonian presentsasquare-shaped hypandrium,dome-shapedlobes,and

slightlyconvergentseta.Thelobesalmosttoucheachotherandthe teetharelargeandslightlyseparated,insertedinthelobeline.The lateralextensionsareprominenttowardthetop,butlessthanin D.equinoxialis(Figs.6Eand7E).D.paulistorumAndean-Brazilian presents a square-shaped hypandrium, slightly square-shaped lobes,convergentsetaandveryclose,medium-sized,thinteeththat aretwicetheheightofthelobesandinsertedinthelobeline.There isavisiblegapbetweenthelobesandteeth.Thelateralextensions arealmostcontinuouswiththelobes(Figs.6Fand7F).D. paulis-torumCentroamericanpresentsarectangularhypandrium,which isthemostelongatedinD.willistonisubgroup,irregular-shaped lobes,convergentsetaand veryclose,medium-sized,thin teeth thataretwicetheheightofthelobesandinsertedbelowthelobe line.ThelateralextensionsaresimilartoD.willistonibutnearer tothelobes(Figs.6Gand7G).D.paulistorumInteriorisvery sim-ilartoD.paulistorumAndean-Brazilian,butthelateralextensions arenotcontinuouswiththelobesandthereis nogapbetween theteeth and lobes(Figs. 6H and7H).D. paulistorumOrinocan hypandriumisthesmallestofthesubgroup,issquare-shapedwith smallroundlobes,convergentsetaandmedium-sized,thinteeth thatarethricetheheightofthelobesandinsertedalittlebelow thelobeline.Lateral extensionsare expandedtoexternal sides ofthelobes(Figs.6Iand7I).D.paulistorumTransitionalpresents a square-shaped hypandrium, small round lobes very close to theteeth,convergent setaandlargethickteeththatarealmost thricetheheightofthelobesandinsertedinthelobeline.Lateral extensionsareprominenttowardthetop,higherthanthelobes (Figs.6Jand7J).

Aedeagus,aedeagalapodeme,paramereandlateralprojections In all of thespecies of the D. willistoni subgroup that have beenanalyzed,theaedeagusisdorsallymembranousandventrally directed downwards,asa birdbeak-like protusionatthedistal end(distiphallus),withtwolateralprojectionsattheanteriorhalf, coveredwithsometinyspinesthatarenotalwaysvisiblein prepa-rations.Theaedeagalapodemeisaslongastheaedeagus,isbar shapedandislinkedtotheaedeagusbyamembranoustissue.The parameresaresmoothandarealsolinkedtotheapodemebya membranoustissue. Theparamereanchors theaedeagus tothe hypandriumthroughthelateralexpansionsofhypandrium.

Themostnoticeabledifferencewithinthewillistonisubgroupis thedistalportionoftheaedeagus(distiphallus).Thisisvery promi-nentandcurvedinDrosophilatropicalis(Figs.8C,9Cand10C),long andstraightinD.willistoni(Figs.8D,9Dand10D),andstraightand shorterthanD.willistoniinD.equinoxialis(Figs.8A,9Aand10A). In D. insularis, this structure isthe shortestamongthe siblings (Figs.8B,9Band 10B).Thereare smallvariationsin sizewithin theD.paulistorumsemispecies,buttheshapeofthedistiphallusis uniqueineachincipientspecies(Figs.8E–J,9E–Jand10E–J).

Thelateralprojectionsexhibitsomedifferenceswithinthe sub-group, which are more notable in D. willistoni, D. tropicalis, D. equinoxialisandD.insularis.InD. equinoxialisandD.paulistorum Orinocan thedistal portionoflateralexpansions isrounded;in D. insularis,D. tropicalisand D.willistoniitispointy;andin the remainingD.paulistorumsemispecies,itisslightlypointy.

Theaedeagalapodemealsoshowsvariation;however,itisnot speciesspeciﬁc.InD.equinoxialis,D.insularis,D.tropicalisandD. willistoni,theaedeagalapodemeisrodshaped,without ornamenta-tioninthedistalportion(Figs.9A–Dand10A–D).IntheAmazonian, Andean-Brazilian,InteriorandTransitionalsemispecies,the aedea-galapodemeisalsorodshapedbutwithasmallroundedexpansion in the distal portion (Figs. 9E,F,H,J and 10E,F,H,J), and in the Centroamerican and Orinocan semispecies, there is a fan-like expansioninthedistalarea(Figs.9G,Iand10G,I).

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Fig.6. ScanningElectronMicroscopyoftheD.willistonispeciessubgrouphypandria.Scalebar50␮m.ThestrainsareinbracketsandlistedinTable1.LO,lobes;TE,teeth; LE,lateralextensions;SE,seta.(A)D.equinoxialis[1];(B)D.insularis[2];(C)D.tropicalis[3];(D)D.willistoni[4];(E)D.paulistorumAmazonian[5];(F)D.paulistorum Andean-Brasilian[6];(G)D.paulistorumCentroamerican[7];(H)D.paulistorumInterior[8];(I)D.paulistorumOrinocan[9]and(J)D.paulistorumTransitional[10].

Fig.7.HypandriaofD.willistonispeciesgroup.Scalebar0.1mm.ThestrainsareinbracketsandlistedinTable1.LO,lobes;TE,teeth;LE,lateralextensions;SE,seta.(A) D.equinoxialis[1];(B)D.insularis[2];(C)D.tropicalis[3];(D)D.willistoni[4];(E)D.paulistorumAmazonian[5];(F)D.paulistorumAndean-Brasilian[6];(G)D.paulistorum Centroamerican[7];(H)D.paulistorumInterior[8];(I)D.paulistorumOrinocan[9]and(J)D.paulistorumTransitional[10].

Fig.8.ScanningelectronmicroscopyofD.willistonispeciessubgroupaedeagi.Scalebar20␮m.ThestrainsareinbracketsandlistedinTable1.(A)D.equinoxialis[1];(B)D. insularis[2];(C)D.tropicalis[3];(D)D.willistoni[4];(E)D.paulistorumAmazonian[5];(F)D.paulistorumAndean-Brasilian[6];(G)D.paulistorumCentroamerican[7];(H)D. paulistorumInterior[8];(I)D.paulistorumOrinocan[9]and(J)D.paulistorumTransitional[10].

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Fig.9. Aedeagi,aedeagalapodemeandparameresoftheD.willistonispeciessubgroup;lateralview.Scalebar0.1mm.ThestrainsareinbracketsandlistedinTable1.AA, Aedeagalapodeme;DP,Distiphallus;LP,Lateralprojections;PA,Paramere.(A)D.equinoxialis[1];(B)D.insularis[2];(C)D.tropicalis[3];(D)D.willistoni[4];(E)D.paulistorum Amazonian[5];(F)D.paulistorumAndean-Brasilian[6];(G)D.paulistorumCentroamerican[7];(H)D.paulistorumInterior[8];(I)D.paulistorumOrinocan[9]and(J)D. paulistorumTransitional[10].

Fig.10.SEMofaedeagi,aedeagalapodemeandparameresoftheD.willistonispeciessubgroup;lateralview.Scalebar50␮m.ThestrainsareinbracketsandlistedinTable1. AA,Aedeagalapodeme;DP,Distiphallus;LP,Lateralprojections;PA,Paramere.(A)D.equinoxialis;(B)D.insularis;(C)D.tropicalis[3];(D)D.willistoni[4];(E)D.paulistorum Amazonian[5];(F)D.paulistorumAndean-Brasilian[6];(G)D.paulistorumCentroamerican[7];(H)D.paulistorumInterior[8];(I)D.paulistorumOrinocan[9]and(J)D. paulistorumTransitional[10].

Discussion

This study sheds new light on the identification of the D. willistoni sibling species subgroup, especially regarding the D. paulistorumcomplex.Wepresentedhereforthefirsttimeimages ofthemaleterminaliaofthesixsemispeciesoftheD.paulistorum. Now,themaleidentificationofthecrypticspeciesofthissubgroup

couldbeeasierandquickerthanenzymatic,molecularand chro-mosomalapproaches.

WefoundmajordifferencesespeciallyinD.willistoni,D. tropi-calis,D.equinoxialisandD.insularis.Whilethereisastrongsexual isolationwithinthesespecies,ithasbeenreportedthatthey occa-sionallyinterbreed(Dobzhanskyetal.,1957;WingeandCordeiro, 1963;Winge,1965;CordeiroandWinge,1995),andseveraldegrees

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ofreproductiveafﬁnityare presentbetweenthesibling species group(reviewedinCordeiroandWinge,1995).

OurfindingsareconsistentwiththeresultspresentedinSpassky (1957)regardingD.equinoxialis,D.insularis,D.tropicalis,D. willis-toni and D. paulistorum Andean-Brazilian. However, a notable aspectofthis comparisonisthatintraspecific variationwasnot observed in our results. We analyzed the male genitalia of D. equinoxialis,D.insularis,D.willistoniandD.paulistorum Andean-Brazilianfromseverallocalities(Table1),includingsomerecently collectedstrains,andnoremarkablecharactervariationwasfound (data not shown). This fact does not imply that there are no intraspecificvariation inthesespeciesand inotherspecies and semispeciesofthewillistonisubgroup.Also,laboratorystrainsmay have less charactervariation than found in nature.Burla et al. (1949),however,concludedthat“thevariabilityisgreatenough tomakeidentificationofsingleindividualshazardous”.

Burlaetal.(1949)founddifferencesinthehypandriaoffourof thesiblingspecies–D.willistoni,D.paulistorum,D.tropicalisandD. equinoxialis–anddescribedtheD.tropicalishypandriumassimilar tothatofD.paulistoruminshape,althoughlarger.Theseauthors alsostatedthattheD.willistonihypandriumisthemostdistinctive. IncontrastwiththeﬁndingsofBurlaetal.(1949)weobservedthat thehypandriaofD.insularisandD.tropicalisarethemostdistinctive withrespecttotheremainingspecies.D.willistoniseemstobemore similartotheD.paulistorumsemispeciesthantotheotherspecies. SomefeaturescouldonlybeobservedinSEM:D.willistoniandD. insularispresentedtwosetaintheapexofthelobes,onlyinone side(Fig.6B);Despitethelowfrequencyofthismodiﬁcation(1:50 inD.willistoniand2:50inD.insularis),itisaninterestingfeature. Thespecimenswiththischaracteristicdidnotpresentanyother peculiarity.

Pasteur(1970)consideredtheteethoftheclaspers(the pren-sisetaein the surstylus) tobethe singlecharacter ofthe male genitaliathat differentiatestheD. paulistorumincipientspecies. In our results(Fig.5), the number of prensisetae is consistent withtherangeofvaluespreviouslyobservedbyPasteur(1970). Weobservedtwogroupsregardingthenumberofprensisetae– thesemispeciesAmazonian,Andean-Brazilianand Centroameri-canwith15prensisetaeseachandCentroamerican,Interiorand Transitionalsemispecieswith12prensisetaeseach,althoughthe sizeandarrangementoftheprensisetaearenotthesameforeach one.Pasteur(1970)foundvariation in thenumberof prensise-taesineachsemispeciesfromdifferentlocalities.Inthisstudy,we observedD. paulistorumAndean-Brazilianfromseverallocalities andthenumberofprensisetaewasconstant,evenintherecently collectedstrains.

Inthepresentreport,weshowthatthereareseveral diagnos-ticcharactersofthemalegenitaliausefulfordifferentiatingthe speciesandeventhesemispeciesoftheD.willistonispecies sub-group.However,theaedeagusisnotthemostimportanttraitfor identifyingthesubgroup’scrypticspecies,asiscommoninother Drosophilaspeciesgroups.Inthestudiedspecies,thehypandrium seemstobethemaincharacterforspeciesidentiﬁcation.In addi-tiontothis,wesuggestthevisualizationoftheaedeagusunderlight microscopyforidentiﬁcationpurposes,sincethisisamembranous structureandmaybedeformedinSEM.

BasedonvisualobservationsofhypandriumD.insularisseems tobe the most distinctive species, related to the other sibling species.WithintheD.paulistorumsemispecies,themost dissim-ilaristhetransitional,especiallywithrespecttothehypandrium shape,surstylusandprensisetae.Suchvariationisinaccordwith theassumptionsofSpasskyetal.(1971)andDobzhansky(1970), statingthatthediversiﬁcationofthesemispeciesisapparentlystill inprogress.

Concerningtheevolutionaryrelationshipamongthewillistoni subgroup,anattempttophylogeneticreconstructionwasmade

usingthecharactersofthemaleterminaliaobservedanddescribed inthisstudy.Thereconstruction,however,wereinconclusive,since thegeneratedtreepresentedsomepolytomiesandlowsupport val-uesforthecharacters(datanotshown).Despiteofthedifferences observed,itispossiblethatthesecharactersdidnotaccumulated enoughdifferencestorepresenttheevolutionaryhistoryofthe sub-group.Nevertheless,thesecharacterscouldbeusefulinacombined phylogenyandwillbepresentedinafurtherstudy.

Althoughcompletereproductiveisolationisnotpresentwithin thespeciesandsemispeciesoftheD.willistonisubgroup(reviews inEhrmannandPowell(1982)andCordeiroandWinge(1995)), thenumberofdifferencesamongthemalegenitaliafoundinour observations is relevant, especially betweenthe D. paulistorum semispecies.Ininsects,therapiddivergenceinmalegenitaliaisso pronouncedthatevenrecentlydivergedsiblingspeciesshowahigh degreeofvariationinthemalegenitalia(Richards,1927;Liuetal., 1996;Song,2009),aswehaveobservedintheD.willistonispecies subgroup.Finally,inresponsetoourownquestion,siblingspecies oftheD.willistonisubgroupcanberecognizedthroughcombined microscopytechniques.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

WethanktoCNPq,CAPESandFAPERGS(process10/0028-7)for ﬁnancialsupport.WethanktoDr.HermesJoséSchmitz,Dr.Marco GottschalkandMsC.JeanLucasPoppeforprecioussuggestions.We thanktoDr.YongKyuKim,Dr.LeeEhrmann,Dr.JeffreyR.Powell andalltheothercollectorsfortheprovidedstrains.Wealsothank theanonymousreviewersforthesuggestions.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.rbe.2015.09.006.

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