Diversity of Drosophilidae (Insecta, Diptera) in the Restinga forest of southern Brazil

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REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution

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

Biology,

Ecology

and

Diversity

of

Drosophilidae

(Insecta,

Diptera)

in

the

Restinga

forest

of

southern

Brazil

Mayara

Ferreira

Mendes

a,b,∗

_,

_Felipe

_Berti

_Valer

c,d

_,

_Júlia

_Gabriela

_Aleixo

_Vieira

e,f

_,

Monica

Laner

Blauth

b

_,

_Marco

_Silva

_Gottschalk

a,b

a_Universidade_Federal_de_Pelotas,_Programa_de_{Pós-Graduac¸}_ão_em_Biologia_Animal,_Pelotas,_RS,_Brazil

b_Universidade_Federal_de_Pelotas,_Instituto_de_Biologia_(IB),_Departamento_de_Ecologia,_Zoologia_e_Genética_(DEZG),_Pelotas,_RS,_Brazil c_Universidade_de_São_Paulo,_Programa_de_{Pós-Graduac¸}_ão_em_Biologia_Celular_e_Molecular,_Ribeirão_Preto,_SP,_Brazil

d_Universidade_de_São_Paulo,_Faculdade_de_Medicina_de_Ribeirão_Preto,_Departamento_de_Biologia_Celular_e_Molecular_e_Bioagentes_{Patogênicos,}_São_Paulo,_SP,_Brazil e_Universidade_Federal_de_Pelotas,_Programa_de_{Pós-Graduac¸}_ão_em_{Fitossanidade,}_Pelotas,_RS,_Brazil

f_Universidade_Federal_de_Pelotas,_Faculdade_de_Agronomia_Eliseu_Maciel,_Departamento_de_{Fitossanidade,}_Campus_{Universitário}_Capão_do_Leão,_Pelotas,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14December2016 Accepted8May2017 Availableonline25May2017 AssociateEditor:GustavoGraciolli

Keywords: Biodiversityanalysis Communityecology Newdistributionrecord Pampabiome Taxonomicsurvey

a

b

s

t

r

a

c

t

AlthoughmembersofDrosophilidaearefrequentlythetopicofecologicalstudiesinBrazil,fewhave exploredRestingaor,untilonlyrecently,Pampabiomeenvironments.Thisstudyproposestodescribethe diversityandtemporalvariationoftheDrosophilidaeassemblagefromaRestingaforestofRioGrandedo Sul,Brazil.WeperformedmonthlycollectionsfromFebruary2013toJanuary2014usingyeasted banana-baitedtraps.Atotalof25,093individualsof46speciesweresampled.DrosophilasimulansandtheD. willistonisubgroupwerethedominanttaxa;D.polymorpha,D.immigrans,D.paraguayensisandZygothrica orbitaliswereofintermediateabundance,andtheother40specieswererare.Basedonsamplingeffort estimators,ourcollectionsweresufﬁcient.JaccardandMorisitaindicesevaluatedusingANOSIMreveal littlesimilarityinthecompositionofsamplesacrossmonths.Canonicalcorrespondenceanalysisshows thatthevariablesofmaximumandminimumtemperaturearethemainfactorsresponsiblefor differ-entiationofthespeciescompositionoftheassemblagethroughouttheyear,wherebycollectionsinthe coldestperiods(July,AugustandSeptember)arethosewithamoredifferentiatedcomposition.Inthese months,thedominanceofD.simulansandtheD.willistonisubgroupdecreaseswhileincreasedabundance oftheD.tripunctatagroup(asD.paraguayensis)andZ.orbitalisoccurs.Incomparisontootherstudies carriedoutinenvironmentsinsouthernmostBrazil,weobservedasimilarpatternofﬂuctuationin abun-danceovertheyear,withahigherabundanceofdominantspeciesinwarmermonthsandpopulation sizesdecreasingincoldermonths.

Introduction

Insectsplayanimportantroleinthediversityofnatural habi-tatsandareessentialforhabitatmaintenance(Didhametal.,1996;

Samways,2015).StudiesonDrosophilidae,awell-studiedDiptera

family,haveshedlightonspeciescoexistenceandtherelationship betweeninsectsandenvironments(ShorrocksandRosewell,1986;

SevensterandVanAlphen,1993;YamashitaandHijii,2003;Mata

etal.,2008),notingthatdrosophilidsaresensitivetochangesin habitatconditions,withexcellentpotentialasbioindicators(Mata

∗ Correspondingauthor.

E-mails:mayaramendes1993@hotmail.com(M.F.Mendes),fbvaler@gmail.com

(F.B.Valer),ju-aleixo@hotmail.com(J.G.Vieira),blauth.monica@gmail.com

(M.L.Blauth),marco.gottschalk@yahoo.com(M.S.Gottschalk).

etal.,2008,2010).MembersofDrosophilidaehavebeenstudied

throughoutBrazil,andtheirassemblagesarecharacteristicof dif-ferentecosystems(Martins,1987;ValandMarques,1996;Schmitz

etal.,2007;Dögeetal.,2008;Mataetal.,2008;Bizzoetal.,2010;

Rohdeetal.,2010;Poppeetal.,2014).

TheBrazilian coastischaracterizedbyRestingaforest,a par-ticularecosystemformedonsandyandnutrient-poorsoilsthatis characterizedbyheterogeneousvegetationwithastrongmarine influence.ThisecosystemismainlyassociatedwiththeAtlantic forestbiomeand isdistributed along theentireBrazilian coast, occupying80%ofthecoastalarea.TheRestingaforestmacroclimate fluctuatesaccordingtoitslatitudinallocation,beingmorestable thaninfieldareasbutvaryingmorewidelythanforestswithricher soils.ThestructureanddistributionofRestingaforestsarearesult ofdepositionalfeaturesdeterminedbysuccessivechangesinsea

http://dx.doi.org/10.1016/j.rbe.2017.05.002

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levelduringtheQuaternaryperiod(Lacerdaetal.,1982;Coutinho,

2006;Behlingetal.,2009;Magnagoetal.,2010).

InthestateofRioGrandedoSul,Brazil,theRestingaforesthasa particularphysiognomyandflorarelatedtothePampabiome.This ecosystemisconditionedbyatemperateclimate,whichcontrasts withthe tropical influence that predominates onthe northern coastofthestate(Waechter,1985;Bencke,2009).Recently,areas ofRestingahavebeenusedfor thedevelopmentof commercial forests,mainlyeucalyptusandpine,whichischangingthis envi-ronmentandgivingrisetoanewstructuralconfiguration(Fonseca

and Diehl, 2004).Overall, in additionto othernegative effects,

the lossof anynatural habitatcan cause a severe decrease in biodiversity,affectingtherateofpopulationgrowth,reducingthe lengthanddiversityofthefoodchainandchanginginteractions amongspecies(Forero-MedinaandVieira,2007).

DifferentenvironmentsofsouthernBrazilhavebeen character-izedintermsoftheirDrosophilidaefauna.However,mostresearch todatehasbeenconductedintheAtlanticforest,forestsand agri-culturalareasofthePampabiomeandurbanizedareas(Valente

andAraújo,1991;Silvaetal.,2005;Hochmülleretal.,2010;Garcia

et al., 2012; Poppe et al., 2012, 2013). Furthermore,studies of

RestingawereperformedinSantaCatarinaandSãoPauloStates

(BizzoandSene,1982;Bizzoetal.,2010)wheretheenvironmental

conditionsaredifferentduetotheAtlanticforestinﬂuence. There-fore,thepresentstudysoughttoreportthediversityandtemporal variationinDrosophilidaespeciesofRestingaforestin southern-mostBrazil,withagoalofjoiningeffortsincharacterizationofthe environmentsofthePampabiome.Moreover,investigationofthe speciesrichnessofthefaunaandﬂoraofdifferentenvironmentsis aprioritymeasureforstudiesfocusedonspeciesconservationand

Fig.1. Studiedarea.(A)MapofSouthAmerica,withBrazilingrayandthestateofRioGrandedoSulhighlighted.(B)MapofthestateofRioGrandedoSul,highlightingthe HortoBotânicoIrmãoTeodoroLuís(HBITL)withacircle.(C)SatelliteimageoftheRestingaforestofHBITL,withthepositionsofthetrapsshown(GoogleEarth).

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0.5 0.4 0.3 0.2 0.1 0.01 0 Relative abundance Species D. simulans D. willistoni subgroup D. polymorpha D. immigrans D. paraguayensis Zy . orbitalis D. mercatorum D. ornatifrons D. onca D. mediopunctata D. suzukii D. griseolineata D. neosaltans D. prosaltans D. schineri D. arassari D. melanogaster D. sturtevanti D. mediosignata D. hydei Za. indianus D. maculifrons

D. nappae D. repleta D. buscki

Zy . vittimaculosa Zy . ptilialis D. buzzatii D. cuaso D. mediostriata Hirtodrosophila sp. Zy . prodispar D. ananassae D. cardini D. pallidipennis Zy . dispar D. bipunctata D. flexa D. fuscolineata D. mediopicta D. nebulosa D. neocardini D. nigricuria D. papei D. montevidensis Leucophenga sp.5

Fig.2. RankabundanceplotoftherelativeabundancesofDrosophilidaespeciessampledintheHortoBotânicoIrmãoTeodoroLuis(HBITL),Brazil,fromFebruary2013to January2014.

understandingtheirtemporal dynamics.Accordingly,this study inaRestingaforestwithpermanentconservationareastatuswill assistinthemaintenanceofitsenvironmentalintegrityandsupport futurestudies.

Materialandmethods

Studiedarea

HortoBotânicoIrmão TeodoroLuis (HBITL)is locatedinthe municipality of Capão do Leão, Rio Grande do Sul, Brazil, at 31◦4748S,52◦1545W(Fig.1Aand B).HBITLhasbeena pro-tectedareasince1964,withapproximately25halocatedinthe Pampabiome (Guerra et al.,2015).It is composedof a mosaic of Restinga forest surrounded by wetlands and anthropogenic habitats, such as pasture and a few low buildings. The forest consistsofdifferentstrata:trees,shrubs,andherbaceousplants, withxeromorphic,succulent and thornyvegetation(Rodrigues, 2005).Largeﬁgtreesarecommon,andtheystandintheforest canopy.HBITLpresentsalonghistory ofanthropicinterference. Intendedforresearchandacademicpurposes,ithasbeenunder theresponsibilityoftheUniversidadeFederaldePelotassincethe 1960s.

AccordingtoKotteketal.(2006),theclimaticclassiﬁcationof this region is Cfa, presenting a mesothermal and super humid climate with no distinct dry season. The climatological nor-mals recorded in 2013 and 2014 included an average annual temperatureof17.2◦C(the lowestand highestaverage temper-atureswere 4.2◦C and 24.8◦C, respectively)and 81.6% relative humidity (the lowest and highest average relative humidities were 75.3% and 85.0%, respectively). The environmental data werecollectedfromEstac¸ãoAgroclimatológicadePelotas(2015)at 31◦5200S,52◦2124W. 60 50 40 30 20 10 0 0 20 40 60 80

Cummulative samples

Species richness

100 120 140

Fig.3.Randomizedaccumulatedcurveofobservedspecies(solidlines,barsare 95%conﬁdenceintervals)andbootstrap(dashedline)andMichaelis–Menten (dash-dottedlines)richnessestimatorsconstructedwithDrosophilidaespeciessampled intheHortoBotânicoIrmãoTeodoroLuis(HBITL),Brazil,fromFebruary2013to January2014.

Specimensamplingandidentiﬁcation

FromFebruary2013toJanuary2014,specimenswerecollected monthlyusing12trapsconstructedaccordingtoTidonandSene

(1988)andbaitedwithapproximately150gofbananaand1.5gof

dryyeast.Thetrapsweretiedtotreesataheightofapproximately 1.5manddistancedat60mfromoneanother(Fig.1C).Thetraps werekeptintheﬁeldfor3days.

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Thecollectedspecimens werepreservedin 70%ethanol and identifiedbased ontheirexternal morphologyaccordingtothe currentliterature.Maleterminaliaofsiblingspecieswere dissec-tedaccordingBächlietal.(2004)forspecies-levelidentification. Femalesofsiblingspecieswereidentifiedbyexternalmorphology and,whenpossible,thespecieslevelwasdeterminedaccordingto thequantitiesofmalesineachtrapforanalysispurposes. Vouch-ersweredepositedintheDrosophilidaecollectionoftheMuseude CiênciasNaturaisCarlosRitter(MCNCR).

Dataanalysis

Tocharacterizetheassemblage,weusedtheabsoluteand rela-tiveabundancesofeachspecies(niandpi,respectively)andthe

speciesrichness (numberofspecies inthesample, S).We veri-ﬁedthespatialautocorrelationofthespeciescompositionamong the traps in each samplingevent using Mantel tests with Jac-cardand Morisitaindices. Thesigniﬁcance level wascalculated with999permutations.Toperformthetests,geographic coordi-natesweredeterminedforthepositionofeachtrap.Thedistances betweentrapswerecalculatedbasedontheEuclideandistance. TheseanalyseswereconductedintheRprogramversion3.1.2(R

CoreTeam,2013)withtheade4package(EcologicalDataAnalysis,

2015).Aswedidnotobservestatisticallysigniﬁcantcorrelations betweenthedistancesandassemblagecompositionsinanyofthe monthsstudied(Supplementary material1),we usedthe com-positionofindividualtrapsasthestatisticalunitforsubsequent analyses(unlessotherwisestated).

Wegeneratedarank-abundancecurvetoevaluatedominance intheassemblagebasedonthetotalrelativeabundanceofspecies in all samples, and we constructed a randomized accumula-tioncurveoftheobservedspecieswith95%conﬁdenceintervals using EstimateS v.8.0 software (Colwell, 2006). Bootstrap and Michaelis–Mentenspeciesrichnessestimatorswereusedtoverify thesamplingeffort.

To evaluate the inﬂuence of temporal variation on the assemblage over thesampling period, we conducted canonical correspondenceanalysis(CCA) usingtheabsoluteabundanceof species collected in three or more samples or ni>10 and the

climaticvariables maximum temperature(TM), minimum tem-perature (Tm),relative humidity(RH) andprecipitation (PR)as independentvariables (Supplementarymaterial2).Thisanalysis wasconductedusingPAST3.0software(Hammeretal.,2001).For thestatisticalanalysis,weusedtheaveragevaluesofeach envi-ronmentalvariableoveraperiodof5days,3dayswiththetraps intheﬁeldand2dayspriortothecollections.Theenvironmental datawerestandardized(Zi=(Xi− ¯X)/S,whereXiisthevalueofthe

environmentalvariableforsamplei,Sisthestandarddeviationof thesamples,and ¯X istheaverageofthesamples).

Finally,weconductedSpearmancorrelationanalysisbetween thetotalabundanceandspeciesrichnessineachmonthandthe environmentalvariables.WeusedBonferronicorrectionto mini-mizetypeIerror.ThetestswereconductedusingPAST3.0software.

Results

Atotal of25,093individuals, belongingto46species infive genera of Drosophilidae, were collectedand identified, includ-ingthefirst record ofD. neosaltans forthe stateofRio Grande doSul.Twelvefemaleswereidentifiedasspeciesgroupsor sub-groupsandwerenotincludedinthestatisticalanalyses.Table1

presentstheabsoluteandrelativeabundancesofthespecimens identiﬁed.

Afterrankingspeciesbasedontheirrelativeabundances,we observed two dominant taxa in the assemblage, withpi>0.10:

Table1

AbundancesofDrosophilidaespeciessampledfromFebruary2013toJanuary2014 intheHortoBotânicoIrmãoTeodoroLuís(HBITL),Brazil.

Species ni pi Drosophilagenus Dorsilophasubgenus D.busckiigroup D.busckiiCoquillett,1901 5 a Drosophilasubgenus D.annulimanagroup

D.arassariCunhaandPavan,1947 27 0.001 D.schineriPereiraandVilela,1987 32 0.001 D.cardinigroup

D.cardiniSturtevant,1916 2 a

D.neocardiniStreisinger,1946 1 a

D.polymorphaDobzhanskyandPavan,1943 1587 0.063 D.coffeatagroup D.fuscolineataDuda,1925 1 a D.guaranigroup D.griseolineataDuda,1927 74 0.003 D.maculifronsDuda,1927 9 a D.ornatifronsDuda,1927 142 0.005 D.immigransgroup D.immigransSturtevant,1921 374 0.015 D.pallidipennisgroup

D.pallidipennisDobzhanskyandPavan,1943 2 a

D.repletagroup

D.buzzatiiPattersonandWheeler,1942 3 a

D.hydeiSturtevant,1921 10 a

D.mercatorumPattersonandWheeler,1942 170 0.007 D.nigricuriaPattersonandMainland,1943 1 a

D.oncaDobzhanskyandPavan,1943 129 0.005 D.papeiBächliandVilela,2002 1 a

D.repletaWollaston,1958 7 a

D.tripunctatagroup

D.bipunctataPattersonandMainland,1943 1 a

D.cuasoBächli,VilelaandRatcov,2000 3 a

D.mediopictaFrota-Pessoa,1954 1 a

D.mediopunctataDobzhanskyandPavan,1943 107 0.004 D.mediosignataDobzhanskyandPavan,1943 13 a

D.mediostriataDuda,1925 3 a

D.nappaeVilela,ValenteandBasso-da-Silva,2004 8 a

D.paraguayensisDuda,1927 370 0.015 D.montevidensisGo ˜niandVilela,2016 1 a

Siphlodorasubgenus D.ﬂexaLoew,1866 1 a Sophophorasubgenus D.melanogastergroup D.ananassaeDoleschall,1858 2 a D.melanogasterMeigen,1830 23 0.001 D.simulansSturtevant,1919 11,090 0.442 D.suzukiiMatsumura,1931 100 0.004 D.saltansgroup

D.neosaltansPavanandMagalhães,1950 57 0.002 D.prosaltansDuda,1927 36 0.002 D.sturtevantiDuda,1927 16 a D.willistonigroup D.nebulosaSturtevant,1916 1 a D.willistonisubgroup 10,336 0.413 Hirtodrosophilagenus Hirtodrosophilasp. 3 a Leucophengagenus Leucophengasp.5 1 a Zaprionusgenus Za.vittigergroup

Za.indianusGupta,1970 10 a

Zygothricagenus

Zy.ptilialisBurla,1956 4 a

Zy.dispargroup

Zy.dispar(Wiedemann,1830) 2 a

Zy.prodisparDuda,1925 3 a

Zy.orbitalisgroup

Zy.orbitalis(Sturtevant,1916) 307 0.012 Zy.vittimaculosagroup

Zy.vittimaculosaBurla,1956 5 a

Total 25,081

ni,absoluteabundance;pi,relativeabundances. a_p

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exoticD.simulansandtheNeotropicalD.willistonisubgroup(Fig.2). Theintermediaryspecies,at0.10≥pi>0.01,wereD.polymorpha,D.

immigrans,D.paraguayensisandZygothricaorbitalis.Theremaining 40species,atpi≤0.01,wererare.Amongthe46speciessampled,

12weresingletons(justonespecimencollected)andfour dou-bletons(onlytwospecimenscollected).Thus,40%ofthespecies wereoccasionallyorpoorlysampled.Fig.3 showsthe random-izedaccumulationcurveoftheobservedspecies(Sobs)with95%

conﬁdenceintervalsandtherichnesscurvesbasedonbootstrap andMichaelis–Menten estimators.Thebootstrap estimator pre-dicted51species,closetothespeciesrichness valuesobserved. Thisestimatorcurveisentirelywithintherangeofthe95% conﬁ-denceintervalsoftheSobscurve,exhibitinghighsimilaritybetween

them. Furthermore, the Michaelis–Menten estimator showed a lowerrichnessthanSobs,andthemeetingpointofthecurves

indi-catedtheinﬂectionpointoftheSobscurve(i.e.,thepointwherethe

speciesaccumulationratedecreases).Theseresultssuggestthatwe collectedthemajorityofthespeciesinthislocationwiththistype ofbaitedtrap.

Amongthefourclimaticvariablesconsideredinouranalysis, i.e.,themaximum(TM)andminimumtemperatures(Tm),relative humidity(RH)and precipitation(PR),TMandTmhad themost influenceonDrosophilidae speciesoverthestudiedyear.These variablesandPRwerehighlyrelatedtoaxis1ofCCA,whichexplains 52.7%ofthevariationinthedata(Fig.4).Axis2,correspondingto 43.5%ofthevariationinthedata,wasequallyrelatedtoTM,Tm andRH.IntheCCAplot,February,March,April,May,June,October, NovemberandDecember2013andJanuary2014occurredclose totheintersectionoftheaxes,whereD.simulans,theD.willistoni subgroupandD.polymorphawerethedominantspeciesandthe abundanceofD.neosaltans,D.prosaltansandD.suzukiiincreased (Fig.4).The firstthree species werepresent inall months,but theirabundancesalsodecreasedinJuly,AugustandSeptember, whenthetemperaturesdecreased(Fig.5).Inmonthswithlower temperatures,i.e.,JulyandAugust,Zy.orbitaliswasthedominant species,andtheabundanceofD.mediosignata,D.mediopunctata, D.melanogaster,D.paraguayensis,D.griseolineata,D.arassariandD. hydeiincreased.OtherspeciesofZygothricawerealsosampledin August,totaling226individuals.Incontrast,D.immigrans,D. merca-torum,D.repleta,D.nappae,D.sturtevanti,D.ornatifrons,D.schineri, D.onca,D.mediostriataandD.maculifronsdidnotshowmarked variationinabundanceduringthesampledyear.Precipitation(PR) hadlittleinfluenceonspeciescompositioninthisstudy.

When we analyzed the monthly variation in terms of the absolute abundance(Fig. 6A) and species richness (Fig. 6B) of Drosophilidae,weobservedahighabsoluteabundanceinwarmer months.The same pattern wasnot found for species richness. UsingtheSpearmantest,wecorrelatedabsoluteabundancewith climaticvariablesandobservedabundancetobepositively corre-latedwithTM(R=0.58,p=0.048)andTm(R=0.71,p=0.01)(Fig.7), whereasabsoluteabundancewasnot correlatedwiththeother twovariables(RH:R=−0.15,p=0.63;andPR:R=−0.46,p=0.14). Additionally,speciesrichnessshowednocorrelationwithany cli-maticvariable(TM:R=−0.03,p=0.92;Tm:R=−0.12,p=0.70;RH: R=−0.38,p=0.22;andPR:R=−0.57,p=0.053),thoughthepattern ofspeciesdominancechangedinthecoldestmonths,asobserved intheCCAplot.

Discussion

Southernexpansionofthedistributionofcollectedspecies

ThenewrecordofD.neosaltansexpandsitsgeographic distribu-tionlimittothelatitudeof31◦4748S(Silvaetal.,2005;Gottschalk

etal.,2008,2009;Garciaetal.,2012;Poppeetal.,2012,2014;Valer

etal.,2013;Depráetal.,2014;Hochmülleretal.,2010).Beforeour

survey,thesouthernmostrecordofD.neosaltanswasin Florianópo-lis,SC (27◦3549S, 48◦3258W), and thespecies wasrecorded moreofteninforestedareasoftheAtlanticforestbiome(DeToni

etal.,2007;Gottschalketal.,2007;Dögeetal.,2008).Inaddition,

thelimitsofthedistributionsofD.cuaso,D.fuscolineata,D.ﬂexa,D. maculifrons,D.nigricuriaandD.papeihavebeenexpandedfurther south.Thesespecies,whicharerareinsamplesfromdifferent stud-ies,werepreviouslyrecordedinthenorthernregionofthestateof RioGrandedoSul(Gottschalketal.,2009;Hochmülleretal.,2010;

Garciaetal.,2012;Poppeetal.,2012,2014;Valeretal.,2013,2016;

Depráetal.,2014).

TheDrosophilidaeassemblageofHBITL

Oftheﬁvegenera sampledin ourstudy,Drosophila wasthe richest.Becausethisgenusfrequentlyusesdecayingfruits,alarge numberofspeciesisexpectedwhensamplingisconductedusing yeastandbananabaits(reviewedinGottschalketal.,2008).We alsosampledonespecieseachofthegeneraHirtodrosophilaand Leucophenga,inadditiontoﬁvespeciesofZygothrica,whichare commonlyfoundinmushrooms,wheretheyfeed,layeggs,rear larvaeand/orcarryoutsexualcourtship(Grimaldi,1987;Valand

Kaneshiro,1988;Courtneyetal.,1990;Valeretal.,2016).The

cap-tureofspeciesofthesegenerawithbananabaitstypicallyoccurs sporadically.Finally,theDrosophilidaeassemblagealsoincluded thegenusZaprionus,representedbyoneexoticspecies,Za.indianus, whichisfrequentlycaughtbybananatraps.

We found seven exotic Drosophilidae species in HBITL, D. ananassae,D.busckii,D.immigrans,D.melanogaster,D.simulans,D. suzukiiandZa.indianus,amountingto11,604individuals,whichis slightlylowerthantheabundanceofnativespecies(13,488 indi-viduals).Thepresenceofexoticspecieshasbeenobservedinareas with different anthropization levels (Ferreira and Tidon, 2005;

DeTonietal.,2007;Gottschalketal.,2007;Garciaetal.,2008).

AlthoughHBITLiscurrentlyaprotectedareaandthereforelimited toresearch,thepresenceofexoticspeciescannonethelessbe asso-ciatedwithanthropicactioninHBITLanditssurroundingsinrecent decades.Inaddition,wesampledalargenumberofD.suzukii,an invasivespeciesrecentlyintroducedtoSouthAmerica;thisspecies wasﬁrstrecordedinBrazilinFebruary2013inthesouthernregion

(Depráetal.,2014).ThisspecieswassampledinOctober2013at

HBITL,locatedapproximately550kmfromthesiteofitsﬁrstrecord. The species sampled in our study represent approximately 40%of theDrosophilidaespecies recordedin thePampabiome

(Poppeetal.,2016).ThespeciesrichnessobservedinHBITL

cor-roboratesotherstudiesofDrosophilidaediversityusingthesame sampling methodin different areas of the Pampa and Atlantic forestbiomes ofthe stateof RS.These studies recorded26–53 species,withDrosophila beingthemostabundantand specious genus(Hochmülleretal.,2010;Garciaetal.,2012;Poppeetal.,

2012,2014).

Oursampledassemblagewascharacterizedbythedominance ofD.simulansandtheD.willistonisubgroup.Despitethefactthat itisanexoticspecies,D.simulansisfrequentlyfoundindifferent habitatsintheNeotropicalregion(Seneetal.,1980;Torresand

Madi-Ravazzi,2006;Bizzoetal.,2010;Schmitzetal.,2010;Garcia

etal.,2012),andahighabundanceofthisspecieshasalsobeen ver-iﬁedinareasofthePampabiome(Hochmülleretal.,2010;Poppe etal.,2012),aswellasinurbanizedareas(Gottschalketal.,2007;

Garciaetal.,2012).TheoccurrenceoftheD.willistonisubgroupas

thesecondmostabundanttaxondisagreeswiththeresultsofother studiesfromthePampabiome(Hochmülleretal.,2010; Poppe etal.,2012),whereitwasuncommonorrare,mostlikelybecause otherstudiesinthePampabiomewereconductedinareasof grass-landvegetationormoreanthropizedenvironments.Incontrast,the

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D. willistoni subgroup D. hydei D. immigrans D. repleta D. mercatorum D. griseolineata D. paraguayensis D. schineri D. ornatifrons D. nappae D. sturtevanti D. onca D. mediostriata D. simulans D. neosaltans D. prosaltans D. suzukii D. polymorpha D. maculifrons D. arassari D. melanogaster D. mediopunctata Zy. orbitalis D. mediosignata JAN RH MAY MAR JUN FEB TM TM Pr APR OCT

DEC _NOV SEP JUL

Axis 1

Axis 2

AUG

Fig.4.Canonicalcorrespondenceanalysisaxes1and2,showingtheorganizationofspeciesaccordingtoenvironmentalvariables(TM,maximumtemperature;Tm,minimum temperature;RH,relativehumidity;PR,precipitation)fromFebruary2013toJanuary2014.

1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00

Feb Mar Apr May Jun Jul Aug

Months

Relative abundance

Sep Oct Nov Dec Jan

D. simulans

D. willistoni subgroup

D. polymorpha

D. immigrans

D. paraguayensis

Zy. orbitalis

Other species

Fig.5.MonthlyrelativeabundanceofspeciesfromFebruary2013toJanuary2014intheHortoBotânicoIrmãoTeodoroLuís(HBITL),Brazil.

D.willistonisubgrouphasbeenwellrecordedinnatural environ-ments(Martins,1987;Saavedraetal.,1995),thoughitsabundance decreasesinurbanizedareas(Gottschalketal.,2007;Hochmüller

etal.,2010;Poppeetal.,2012).

Abioticvariables

Corroboratingtheliteraturetodate,thestudiedDrosophilidae assemblagerespondstoclimaticvariation (Martins, 1987;Mata

etal.,2008;Rohdeetal.,2010).Intemperate regionswithfour

well-markedseasons,abundanceof specimensis highinwarm months but with low diversity, whereas abundance is low in coldmonthsbutwithhighrichness(Petersen,1960;Araújoand

Valente,1981;Franckand Valente,1985;Saavedraetal.,1995;

DeTonietal.,2007;Gottschalketal.,2007;Poppe etal.,2013).

Oftheclimaticvariables,themaximumandminimum tempera-tureswerethosethatmostinﬂuencedtheassemblage,withthe minimumtemperature beingthemostdeterminantin termsof speciescomposition,asdescribedinotherstudiesconductedin dif-ferentenvironments(TorresandMadi-Ravazzi,2006;Bizzoetal.,

2010;Poppeetal.,2013).Inourstudy,14speciesexhibited

well-markedseasonality,withabundancevariationlargelyrelated to temperaturechanges(asevidencedbyCCA).However,inthearea studiedbyTorresandMadi-Ravazzi(2006),apositivecorrelation between species richness and rainfall was described. Associa-tionsbetweenDrosophilidaeassemblagesandrainfallwerealso

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A

7000 6000 5000 4000 3000 2000 1000 0 25 20 15 10 5 0

Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan

Feb Mar Apr May Jun Jul Months

Species richness

A

bsolute abundance

Aug Sep Oct Nov Dec Jan

B

Fig.6.Absoluteabundance(A)andabsoluterichnessofspecies(B)fromFebruary2013toJanuary2014intheHortoBotânicoIrmãoTeodoroLuís(HBITL),Brazil.

A

4.0 4.0 r=0.58, p=0.048 r=0.71, p=0.010 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 10 15 20

Maximum temperature - TM (

°C)

Minimum temperature - TM (

°C)

Absolute abundance (Log N)

25 30 0 5 10 15 20 25

B

Fig.7.Correlationanalysesbetweentheabsoluteabundanceofspeciesandthemaximumandminimumtemperatures.

strongerinstudiesperformedintheBrazilianCerrado,wherethe annualfluctuationin temperatureyearissmallerand theyears are characterizedby two seasons, one wet and one dry (Mata etal.,2008).AccordingtoDobzhanskyandPavan(1950), precipi-tationshouldbemoreimportantforDrosophilaspeciesabundance becauseprecipitationisessentialforthefloweringandfruitingof plantsusedasatrophicresourceforDrosophilidae.IntheHBITL area,precipitationhasa secondaryinfluenceontheassemblage compositionofDrosophilidaespecies(Fig.4),mostlikelybecause fluctuation in this abiotic variable is less marked in southern Brazil.

Onlythreetaxaweresampledduringall12monthsofcollection: D.simulans,D.polymorphaandtheD.willistonisubgroup.Thesetaxa showﬂuctuationintheirabundanceaccordingtoclimaticvariation. DrosophilasimulanswasdominantinApril,Novemberand Decem-ber2013,whereastheD.willistonisubgroupandD. polymorpha weredominantinJanuary2014,withtheD.willistonisubgroup reachinganabundancethatwasgreaterthan90%ofthetotalin

thatmonth.Thispatterndemonstratedthatthedominantspecies weremostabundantduringwarmermonths,withanaverage tem-peratureof25◦CinApril,November,andDecemberof2013and Januaryof2014.

ZygothricaorbitaliswasthedominantspeciesinJulyandAugust 2013,whenthemaximumandminimumtemperatureswerelow; forthis species specifically,itis assumed thata lackof trophic resourcesforimagoes(i.e.,mushrooms,Valeretal.,2016)causes themtovisitthebaitused.Thisfact,associatedwiththelow abun-danceofthedominantDrosophilaspecies,makesZy.orbitalisthe dominantspeciesinAugust.Inarecentstudybyourgroup,we sam-pledZy.orbitalisemergingprofuselyfromthefruitsofPsychotria sp.(Rubiaceae,Magnoliophyta),acommonbushpresentinHBITL (MayaraFerreiraMendes,unpublisheddata),whichmayindicate thatthisflyspeciesisattractedtothesefruitsforoviposition.When samplinginanareawithasimilarclimate,Garciaetal.(2012) ver-ifiedthesamepatterninwinter,withsubstitutionofthedominant speciesinwarmertimes.

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DrosophilidaeoftheRestingaforest

InBrazil,twostudiesfocusingonDrosophilidaefromRestinga environmentshavebeenperformed(BizzoandSene,1982;Bizzo et al.,2010).The areassampled byBizzoand Sene (1982) and

Bizzoetal.(2010)havecomparativelysparservegetationand a

greatermarineinﬂuence(Falkenberg,1999;Martinsetal.,2008). Moreover,duetolatitudinaldifferences,HBITLhasalower aver-agetemperatureinthecoldestmonth(approximately4◦C)than theareasstudiedbyBizzoandSene(1982)inSãoPaulo(46◦56W;

24◦14S)andbyBizzoetal.(2010)inSantaCatarina(48◦2749W;

27◦3821S),withaveragesinthecoldestmonthofapproximately 18◦C.Nonetheless,theaveragetemperatureinthewarmestmonth isthesameintheareascoveredbythesethreestudies (approxi-mately25◦C).Therefore,fewsimilaritiesinspeciescomposition wereobservedwhencomparedwithourdata,wherebythe domi-nantspecies,withtheexceptionofD.simulansandtheD.willistoni subgroup,weredifferentfromBizzoandSene(1982)andBizzo

et al.(2010).In theRestinga forestof São Paulo,theD. cardini

group(non-D.polymorpha)andD.sturtevantiwerecommon,and D. malerkotlianawasuncommon;in RestingaofSantaCatarina, Za.indianusandD.fumipenniswerecommonandD.nebulosawas uncommon.

Conclusion

Foremost,ourstudyincreasesknowledgeaboutDrosophilidae faunaintheRestingaforestofthePampabiome.Theassemblage speciesrichnessobservedandtherecordofD.neosaltansforthe stateofRioGrandedoSulsuggestthatHBITLisanimportantarea forthemaintenance ofthespeciesofthisfamilyinthis ecosys-tem.Temperaturewasfoundtobethepredominantinﬂuenceon theassemblage;D.simulansandtheD.willistonisubgroupdisplay positiverelationshipswiththisclimaticvariable,whereasthis rela-tionshipisnegativeforotherspecies.Duetodifferencesinlatitude andphysiognomy,theDrosophilidaetaxaobservedweredistinct fromthoseofRestingaforestsinnorthernareas.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

WewouldliketothankProf.Dr.VeraLúciadaSilvaValente, Prof.Dr.LucasPoppe,Prof.Dr.CristianoAgra Iserhard,Prof.Dr. RafaelAntunesDiasandtheanonymousreviewersfortheir help-fulcomments.ThisworkwassupportedbytheConselhoNacional deDesenvolvimentoCientíﬁcoeTecnológico(CNPq)undergrant n◦472973/2013-4.ThecollectionwasauthorizedbytheInstituto ChicoMendesdeConservac¸ãodaBiodiversidade(ICMBio),under permanentlicenseforcollectingbiologicalmaterialn◦25454-1.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.rbe.2017.05.002.

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