REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolutionw w w . r b e n t o m o l o g i a . c o m
Systematics,
Morphology
and
Biogeography
High-level
phylogeographic
structuring
of
Neoleucinodes
elegantalis
Guenée
(Lepidoptera,
Crambridae)
in
Brazil:
an
important
tomato
pest
André
V.
P.
Maia
a,
Cícero
Almeida
b,∗,
Kleber
R.
Santoro
c,
João
L.
A.
Melo
c,
José
V.
Oliveira
a,
Raul
N.
C.
Guedes
d,
César
A.
Badji
caUniversidadeFederalRuraldePernambuco,DepartamentodeAgronomia,Recife,PE,Brazil
bUniversidadeFederaldeAlagoas,LaboratóriodeRecursosGenéticos,Arapiraca,AL,Brazil
cUniversidadeFederalRuraldePernambuco,UnidadeAcadêmicadeGaranhuns,Garanhuns,PE,Brazil
dUniversidadeFederaldeVic¸osa,DepartamentodeEntomologia,Vic¸osa,MG,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received15December2015
Accepted8March2016
Availableonline13May2016
AssociateEditor:HéctorVargas
Keywords:
Insectpest
Phylogeography
Smalltomatoborer
a
b
s
t
r
a
c
t
NeoleucinodeselegantalisisanimportanttomatopestinBrazil,occurringthroughoutthecountryand resultingineconomiclossesinagriculture.Inseveralspecies,biogeographicstudiesinBrazilindicatethe structuringofpopulations,followingtherefugemodel,withasplitbetweenthepopulationsofthe north-eastandthesoutheastregionsofBrazil.Theobjectiveofthisworkwastoanalyzethephylogeographyof N.elegantalisinBrazil,understandingitspopulationstructureandthedemographicpatterns.Larvaewere collectedfromeightlocationsthroughoutBrazil,andthemitochondrialcytochromecoxidasesubunit1 genewasanalyzed.Atotalof628bpin51individualswereobtained,showing12haplotypeswitha hap-lotypediversityof0.836.Spatialanalysisofmolecularvariance(SAMOVA)andclusteranalysisshowed twopopulations,indicatingpopulationstructuringbetweenindividualsfromthenortheast(population 1)andsoutheast(population2)regionsofBrazil.Phylogeneticanalysisindicatedthattheclades corre-spondingtothegroupsdefinedbySAMOVAhaveadivergencetimeof0.2–0.5millionyears,suggesting isolationduringclimaticeventsandaseparationofthetwopopulationscoincidingwiththepredicted refugestotheAtlanticforest.
PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeEntomologia.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Thesmalltomatoborer(NeoleucinodeselegantalisGuenée,1984, Lepidoptera:Crambridae)isapestthat hashadgreat economic impact in Brazil, Venezuela, and Colombia (Badji et al., 2003; Picanc¸oetal.,2007).Highinfestationlevelsofthispestmakethe fruitsunsuitableforconsumptionandindustrialprocessing(Badji etal.,2003; Benvengaet al.,2010;Picanc¸oet al.,2007).Losses resultingfromthedamagecanbeashighas90%ofthetotal pro-duction(Mirandaetal.,2005).
InBrazil,N.elegantalisisdistributedthroughoutthecountry, associatedwithhumanmigrationsintomato-producingregions. It extends from the cold regions of the southeast to the dry regionsofthenortheast,particularlyinareasofdryseasonalforests (Caatinga).These regions showdifferences in climate, topogra-phy,floristiccomposition,anddemographiceventsthatcanmodel thepopulationstructureofN.elegantalis.N.elegantalishasbeen
∗ Correspondingauthor.
E-mail:cicerocarlos@pesquisador.cnpq.br(C.Almeida).
controlledwiththeuseofchemical insecticidesand behavioral controlsusing sex pheromones (Badji et al., 2003).In controls usingsexpheromones,populationstructuringisveryimportantin determiningthespecificityofthepheromonetypes.Forexample, avariationincuticularhydrocarbonsassociatedwith geographi-caldistance(Bonellietal.,2015)wasdetectedinPolistesbiglumis (Hymenoptera:Vespidae).InN.elegantalis,unpublisheddata sug-gestthatpheromoneshavedifferenteffectsonthepopulationsof thesoutheastandthenortheastofBrazil,indicatingageographical associationwithpheromoneefficiency.Thesedifferencesmaybe associatedwithpopulationstructureduetotheinfluenceofclimate fluctuationsasthebiogeographicevents.
Biogeographicaleventssuchasclimatevariationsandchanges in habitat are significant factors in explaining the geographi-caldistributionofmany species,particularlyinclimacticevents in the Pleistocene. Using paleomodeling, Carnaval and Moritz (2008)showedthephylogeographiccenterofendemisminBrazil, suggesting distinct centers of endemism for different species, includingbutterflies.Thus,estimatesofthespatialdistributionof thepopulationsofN.elegantalis,includingdemographicpatterns andhistoricalpopulationparameters,enableanunderstandingof
http://dx.doi.org/10.1016/j.rbe.2016.03.004
0085-5626/PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeEntomologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
Phylogeographic studiesof insect species have beenconducted usingthecytochromecoxidasesubunit1(CO1)region,andithas becomeastandardizedregionfordispersionstudies.Theaimofthis studywastoanalyzethephylogeographicstructureofN.elegantalis inBrazilusingtheregionoftheCO1gene.Theresultsallowanalyze thehypothesisaboutpopulationstructuringresultingfromclimate changesthatmayhaveoccurredinthespecies,aswellas contribut-ingtoaclearerunderstandingofdistributionpatternsofspecies resultingfromsuccessiveclimaticcyclesduringthePleistocene.
Materialandmethods
SamplingandDNAextraction
Larvaewerecollectedineightlocations(51individuals) dis-tributedfromthesoutheasttothenortheastofBrazil(Fig.1).The sampleswerestoredin70%ethanolandconditionedat4◦C.Twoof thelocationswereinthesoutheast,inthestatesofSãoPauloand MinasGerais,andtheothersixlocationswereinthenortheastof Brazil,indryforestareasinthestateofPernambuco(Caatinga;map showninFig.1A).TheDNAwasextractedusingtheCTABprotocol (DoyleandDoyle,1987),quantifiedusingspectrophotometry,and analyzedforqualityusing1%agarosegel.
Amplificationandsequencing
TheregionofthemitochondrialCO1genewasamplifiedusing LepF1/LepR1 primers (Hebert et al., 2004).The reactionswere performed in a total of 50L, containing 5L reaction buffer, 1.5mMMgCl2,0.2mMdNTP,1.25UTaqDNApolymerase,0.5M
ofeachprimer,and100–150ngofDNA.Theamplificationwas per-formedwithaninitialdenaturationat94◦C for4min,followed by30cyclesat94◦C for40s,55◦Cfor35s,and72◦Cfor1min, andafinal extensionat74◦C for4min.ThePCRproductswere amplifiedusingaBigDye® Terminatorv3.1CycleSequencingKit (AppliedBiosystems®)inelectrophoresisina3500Genetic Ana-lyzer(AppliedBiosystemsInc.,FosterCity).
Analysisofthehaplotypes
Theforwardsequenceswereeditedusingthesoftwareprogram Mega5.2andalignedwithClustalWandMuscleimplementedin Mega5.2. Thealignmentwasperformedusingstandard adjust-mentsand manualoptimizationwhennecessary.Thehaplotype diversity(h)andnucleotidediversity(),alongwithFuandLi’s DandTajima’sDtests,werecalculatedusingDnaSP5.10.01 soft-ware(LibradoandRozas,2009).Estimateswithsignificantnegative valuesareexpectedfromtheFuandLi’sDandTajima’sDtestsin populationsthathaveundergonerecentdemographicexpansion.
Thebestadjustednucleotidesubstitutionmodelwasobtained usingthejModelTest2.1.4program(Darribaetal.,2012)tohelp selectthe molecularevolution model, and theHKY modelwas usedforposteriorphylogeneticinferenceusingBayesianAnalysis and MaximumLikelihood(ML). TheBayesiananalysiswas per-formedusingBeastv1.8.0(Drummondand Rambaut,2007)and theposteriordistributionwasapproximatedusingMarkovChain Monte Carlo (MCMC) for 50 million steps. The convergence of theparameterswascheckedusingTracer1.5software(Rambaut, 2009).Thetimetomostrecentcommonancestor(TMRCA)was calculatedassumingarelaxedmolecularclock(uncorrelated log-normal),followingtheparametersdescribedbyPapadopoulouetal. (2010):(ucld.stdev=0.2571;acoefficientofvariation=0.2609;and asubstitutionrateof0.0168permillionyearsago,Ma)(outgroup Lepidopterasp.–GenBank:JF843940).Genealogicalrelationships amongthehaplotypeswereestimatedusingthemedian-joining
1999).
Populationstructuring
Measurementsofpopulationdifferentiation(GSTandNST)were
calculatedusingDnaSP5.10.01(LibradoandRozas,2009).When theNSTestimatesweregreaterthanthoseoftheGST,
phylogeo-graphicstructuringwasassumed,withcloselyrelatedhaplotypes beingdetectedmorefrequentlyinthesameareathanremotely cor-relatedones.Thisapproachhasbeenusedinotherstudiestodetect phylogeographicalstructure(Guickingetal.,2011;Liuetal.,2012; Chiuetal.,2013).Aspatialanalysisofmolecularvariancewas con-ductedusingthespatialanalysisofmolecularvariation(SAMOVA) softwareprogram(Dupanloupetal.,2002).Thissoftwareuses sim-ulationtoidentifygroupsofpopulations(k)thataregeographically homogeneousandthosethatmaximizethedifferencesbetween groups,allowingvariationbetweenthegroups(FCT),betweenthe
locationswithineachgroup(FSC),andbetweenthelocationsin
rela-tiontothetotalsample(FST),tobeobtained.SAMOVAanalyses
wereconductedwith1000interactionsfork={2,...,8}groups. Aclusteranalysiswasconstructedusingthe“Bayesianapproach tophylogeographicclustering,”a Bayesianphylogeographicand ecologicalclustering(BPEC)package(Manolopoulouetal.,2011) implementedonRsoftware(Team,2012),usingtheparameters ds=0,maximumnumberofmigrations=5,and50millionstepsin MCMC.
Ecologicalnichemodeling
Ecological niche modeling was performed using MAXENT (Version 3.3.3k; Phillips et al., 2006). The climatic nichesused were the 19 BIOCLIM variable available in the WorldSIM data base(http://www.worldclim.org).Theenvironmentaldatacontain threedifferentperiods:bioclimlayersfortheperiodfrom1950to 2000ataresolutionof30arcs,thelastglacialmaximum(LGM; <21,000 years BP) in the climaticconditions at a resolution of 2.5arcmin,andthelastinterglacial(LIG:<120,000–140,000years BP)ataresolutionof30arcs.Toconstructtheecologicalniches, runswereconductedwiththeparametersconvergencethreshold (0.00001),maximumiterations(500),anddefaultprevalence(0.5). Thefigures wereproducedusingtheRsoftwareraster package (Team,2012).
Results
Haplotypedistributionandanalysis
TheregionoftheCO1genewassequencedin51individualsof N.elegantalis,andtheanalyzedfragmentswith628bpshowed17 polymorphicsiteswithatotalof12differenthaplotypes(Fig.1A), h=0.836±0.032 and =0.06608±0.00122. The haplotype dis-tributionshoweda cleardistinction betweenthesoutheastand northeast regionlocations in that haplotypesH6 and H10that occurred only in the populations from the southeast of Brazil, whereastheotherhaplotypesoccurredonlyinthenortheastregion (Fig.1A).Inthenortheastregionlocations,haplotypesH1andH5 occurredwithhigherfrequency,whereashaplotypesH2,H3,H9, andH12werelesscommon(Fig.1A).AccordingtotheNETWORK results,haplotypeH10hadthehighestnumberofsubstitutionsin relationtothemostfrequentlyoccurringhaplotypes(H1andH5) (Fig.1A).
Thephylogeneticanalysesshowedfourclades,twoofthem con-taininghaplotypesfromthesoutheastandtwoofthemwithonly haplotypesfromthenortheastofBrazil(Fig.1B).Theanalysisofthe
A
B
Haplotype networks H10 H2 H4 H1 H8 H6 H11 ES CM BE GA H4 H12 H2 H1 H7 H8 H5 H3 H11 H9 H6 H10Group B
Group A
Lepidoptera sp. (JF843940) 92 94 100 100 100 0.2 substitutions/site 2.00 1.75 1.50 1.25 1.00Million of years ago (Ma)
0.75 0.50 0.25 0.0 99 PT SJ CO SP
Haplotypes
H1 H2 H3 H4 H5 H6 H12 H11 H10 H9 H8 H7 H3 H5 H9 H7 H12Fig.1.DistributionpatternsofhaplotypesforN.elegantalis.(A)Frequencyofhaplotypesforthecytochromecoxidasesubunit1(CO1)regionbystudysitesandthehaplotype
networkobtainedbymedian-joining.Thestudysitesareindicatedbyletters,andthesizeofthegraphicsisproportionaltothesamplesize.Thecolorscorrespondtothe
haplotypesasdescribedbythelegend:Garanhuns(GA),Petrolina(PT),Coimbra(CO),Camocim(CM),EncruzilhadadeSãoJoão(ES),SãoJosédoR.Pardo(SP),Bezerros(BE)
andSãoJoão(SJ).(B)PhylogeneticanalysisforthehaplotypesusingtheBayesianapproach.Thisanalysisshowsthetimeofdivergencebetweenthehaplotypesinmillions
ofyears(Ma)(95%confidencesareindicatedbyboldhorizontalbars)andthegroupsdefinedbySpatialAnalysisofMolecularVariance(SAMOVA).Thesupportvaluesare
estimatedwithposteriorprobabilitiesBY(hereinpercentages).
TMRCAshowedthathaplotypeH10hadadifferentiationof approx-imately0.6Ma,andhaplotypeH6ofaround0.3Mainrelationtothe otherhaplotypes(Fig.1B).
Populationstructuring
The GST estimate (0.136) was lower than the NST estimate
(0.669),indicatingpopulationstructuring.Thespatialanalysisof molecularvariance resulted in structuring of two groups, with statisticalsignificance(FCT=0.77andFST=0.78,Table1),showing
that77.12% ofthe variation wasbetweenthegroups. Group A
consisted of locations in the northeast of Brazil (Garanhuns, Petrolina,Camocim,EncruzilhadadeSãoJoão,Bezerros,andSão João) and Group B was exclusively consisted of locations in thesoutheast ofBrazil (SãoPauloeMinasGerais)(Fig.1A and
Table2).HaplotypeanalysisshowedthatforGroupA,h=0.78and =0.0026,whereasforGroupB,h=0.5and=0.0079(Table2).
Clusteranalysisusing BPECshowedthreeclusters;one with southeastlocationsandothertwowithlocationsinthenortheastof Brazil(Fig.2).However,inthisanalysisapproach,colorsrepresent theclustersand the backgroundor shadedcolors show uncer-taintyabouttherespectiveclusters.In theresultsofthisstudy,
Table1
Spatialanalysesofmolecularvariance(SAMOVA)betweentwothegroups.
Sourceofvariation d.f. Percentageofvariation p-Value Amonggroupsa 1 77.12 FCT=0.77 <0.0001
Amongpopulationwithingroups 6 1.07 FSC=0.05 <0.0001
Withinpopulations 43 21.81 FST=0.78 <0.0001
Total 50 100.00
Foreachgroupsofpopulations,haplotypediversity(h),nucleotidediversity(),FuandLi’sD,Tajima’sDandnumbersofhaplotypes.
Groups Samplesize h Haplotypesno. FuandLi’sD Tajima’sD GroupA(GA,PT,CM,ES,BEeSJ) 42 0.78 0.0026 10 −0.95 −1.31 GroupB(COeSP) 9 0.50 0.0079 2 1.47* 1.67 Total 51 0.272 −0.58
thereisalackofclarityintermsofthetwoclustersinthe north-eastregionofBrazil;thus,theyareconsideredtogetherasasingle cluster.
Ecologicalnichemodeling
Thedistributionestimateshowsgoodspeciesdistribution rep-resentation(Fig.3).Comparingthepredictionsforthepresentand theLGM,weobserveadecreaseindistributioninthepresentwitha lossofhabitatsuggestingcontraction.Ontheotherhand, compar-ingtheLGMandLIGdistributions,theresultssuggestanexpansion ofthehabitatintheLGM.Ananalysisofthethreeecologicalniche predictionsshows anexpansionfromtheLIGtotheLGMand a retractionfromtheLGMtothepresent(Fig.3).
Fig.2. Theclusteranalysiswasbuiltusingthe“Bayesianapproachto
phylogeo-graphicclustering.”Thecolorsrepresenttheclusters,andthebackgroundorshaded
colorsindicateuncertaintyabouttherespectiveclusters.Thepointsmarkthe
samp-lingsites.
Discussion
N.elegantalishasbeenportrayedasanimportanttomatopest in Brazil,occurringthroughoutthecountrystretching fromthe southtothenorth.Itishighlyadaptabletodifferenttypesof envi-ronments,living incoldAtlanticforestregionstosemi-ariddry forestregions(Caatinga)ofthenortheastof Brazil.Accordingly, populationstudiesareextremelyimportantforunderstandingthe populationstructuresanddemographichistoryofthespecies.In thisstudy,theregionofthemitochondrialCO1genewassequenced in individuals from different locations distributed throughout Brazil for analyze the hypothesis about populationstructuring resultingfromclimatechanges,aswellascontributingtoaclearer understandingofdistributionpatternsofspeciesresulting from successiveclimaticcyclesduringthePleistocene.
Theresultsofthisstudydemonstrateaclearpopulation struc-ture, separating individuals originating in the southeast and northeastregionsof Braziland identifyingthetwopopulations. Thehaplotypesfoundinthesoutheastwerenotobservedinthe northeast nor were the haplotypes of the northeast observed in the southeast, showing that there has been no recent gene flow. This evidence is interesting because of the fact that N. elegantalisattacks thefruits,and commercializationcouldbe a dispersionfactor throughoutBrazil,which wasnot observedin thisstudy.Itispossiblethattheinsectisunabletocompleteits cycleunderfruit storageconditionsand/orthat eliminatingthe damaged fruit prior tomarketing impedes gene flow. Another explanation maybethat differences in sexpheromones hinder reproductionbetweenindividualsfromdifferentregions. Unpub-lisheddatasuggestthatbehavioralcontrolusingsexpheromonesis notmucheffectivewhenappliedtoindividualsofthesoutheastand northeastpopulations,suggesting differencesindistinct genetic groups.
The divergence time betweenthe two haplotypes from the southeast and the ten haplotypes from thenortheast of Brazil is 0.3–0.6Ma,suggesting tworefuges forthespecies.In theory, climaticfluctuationsinthePleistocenecouldhavecausedthe frag-mentationofhabitats,creatingisolatedfragmentsandresultingin refugesformanyspecies.TheresultsoftheTRMCAshowedthat cladescontaininghaplotypesofthesoutheastdivergedfrom hap-lotypesofthenortheastduringthePleistocene,indicatingthatthe populationstructureisaresultofclimaticvariations.The hypoth-esis of the emergence of refuges is validated by the resultsof ecologicalnichemodeling,inwhich thedistributionshowsthat therewasanexpansionfromtheLIGtotheLGMandacontraction fromtheLGMtothepresent,suggestingthatacontractionmay haveoccurredpriortotheLIG.
Theseparationbetweenpopulationsofthesoutheastandthe northeastofBrazilisobservedinmanystudies.Forexample,studies byMartins(2011)invertebratesshowaseparationbetween popu-lationsofthenorthandthesouthoftheAtlanticforestbetween 0.4and0.5Ma.Thecenterofendemismforbutterfliesshowsfour regions;twointhesoutheastandoneinthenortheast(stateof Pernambuco)(CarnavalandMoritz,2008).AstudyofN. elegan-talisinColombiashowsapopulationstructureassociatedwiththe AndesMountains,whichactasabarriertogeneflow(Diaz-Montilla etal.,2013),ascenariosimilartothisstudy.
20 0 –20 –40 –60 20 0 –20 –40 –60 20 0 –20 –40 –60 –100 –80 Now LGM LIG 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 –60 –40 –20 –100 –80 –60 –40 –20 –100 –80 –60 –40 –20
Fig.3.PotentialdistributionastheprobabilityofoccurrenceofN.elegantalisinthepresent(Now,0yearsBP),lastglacialmaximum(LGM;<21,000yearsBP),andlast
interglacial(LIG,120,000–140,000yearsBP).
Themovementordispersionofinsectsacrosslargegeographical distancesisveryimportanttoplantpestmanagementstrategies, includingthe identificationof breeds that couldhave different pheromonecompositionsand inwhichgeneticvariationscould differentiatethepest’sresponsetocontrolstrategies.For exam-ple,thepopulationstructureofinsectpestsenablesthedefinition ofpatternsof resistanceassociated withthegeographical envi-ronment(Labbeetal.,2005).Insectstudieshavedemonstratedan associationbetweengeneticvariationandcuticularhydrocarbon variation(Nestmaterecognitionmediator,acommonpheromone insocialinsects)(Dapportoetal.,2009;Dronnetetal.,2006).In this context,this study shows a populationstructure ofN. ele-gantalisassociatedwithgeographicaldistance.Futurestudiesare needed to study association betweeninterpopulation variation and pestcontrol strategies, suchasbehavioral and/or chemical insectcontrol,becausean understandingofdispersionpatterns andgeneticdiversityisnecessaryforeffectivecontrol,considering thattheadaptationabilityofanorganismdependsonitsgenetic variability.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
TotheUniversidadeFederaldoAlagoasforthelaboratoriesand scientificsupportandtheFundac¸ãodeApoioàPesquisadeAlagoas forfundingthisproject.
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