REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolutionw w w . r b e n t o m o l o g i a . c o m
Biology,
Ecology
and
Diversity
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,baUniversidadeFederaldePelotas,ProgramadePós-Graduac¸ãoemBiologiaAnimal,Pelotas,RS,Brazil
bUniversidadeFederaldePelotas,InstitutodeBiologia(IB),DepartamentodeEcologia,ZoologiaeGenética(DEZG),Pelotas,RS,Brazil cUniversidadedeSãoPaulo,ProgramadePós-Graduac¸ãoemBiologiaCelulareMolecular,RibeirãoPreto,SP,Brazil
dUniversidadedeSãoPaulo,FaculdadedeMedicinadeRibeirãoPreto,DepartamentodeBiologiaCelulareMoleculareBioagentesPatogênicos,SãoPaulo,SP,Brazil eUniversidadeFederaldePelotas,ProgramadePós-Graduac¸ãoemFitossanidade,Pelotas,RS,Brazil
fUniversidadeFederaldePelotas,FaculdadedeAgronomiaEliseuMaciel,DepartamentodeFitossanidade,CampusUniversitárioCapãodoLeão,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.
©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
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
0085-5626/©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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).
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 140Fig.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.
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. ap
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
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
A
7000 6000 5000 4000 3000 2000 1000 0 25 20 15 10 5 0Feb 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 20Maximum 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.
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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