Genetic divergence of a newly documented population of the cecidogenous micromoth Eugnosta azapaensis Vargas & Moreira (Lepidoptera: Tortricidae) in the Atacama Desert of northern Chile

Short

Communication

Genetic

divergence

of

a

newly

documented

population

of

the

cecidogenous

micromoth

Eugnosta

azapaensis

Vargas

&

Moreira

(Lepidoptera:

Tortricidae)

in

the

Atacama

Desert

of

northern

Chile

Scott

Escobar-Suárez

_,

_Wilson

_{Huanca-Mamani}

_,

_Héctor

_A.

_Vargas

a_{UniversidaddeTarapacá,FacultaddeCienciasAgronómicas,DepartamentodeRecursosAmbientales,Arica,Chile} b_{UniversidaddeTarapacá,FacultaddeCienciasAgronómicas,DepartamentodeProducciónAgrícola,Arica,Chile}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received3May2017 Accepted31May2017 Availableonline15June2017 AssociateEditor:LucasKaminski Keywords: Asteraceae Baccharissalicifolia Chile DNAbarcodes

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EugnostaazapaensisVargasandMoreira,2015(Lepidoptera:Tortricidae)isamicromothnativetothe AtacamaDesertwhoselarvaeinducefusiformgallsinshootsofBaccharissalicifolia(Ruiz&Pav.)Pers. (Asteraceae).Thepresenceofthiscecidogenoustortricidwaspreviouslyrecordedonlyfromthetype locality,theAzapaValley,AricaProvince,northernChile.However,fusiformgallsonshootsofB.salicifolia wererecentlyfoundinChaca,anothercoastalvalleyoftheAtacamaDesert.Theadultsobtainedfrom thesegallswerepreliminarilyidentifiedasE.azapaensisbasedonmorphology.Subsequently,toassess anadditionalsourceofevidenceforthetaxonomicidentificationofE.azapaensisinthisnewlocality, sequencesoftheDNAbarcodefragmentofthecytochromeoxidasesubunitImitochondrialgenefromthe twolocalitieswereanalyzed.Fourhaplotypesweredetected,tworestrictedtoAzapaandtworestricted toChaca.Thegeneticdivergence(K2P)betweenhaplotypesofeachlocalitywas0.2–0.8%,whileitwas 1.1–1.4%betweenhaplotypesofdifferentlocalities,and8.7–13.5%betweentheChileanhaplotypesand otherspeciesofEugnostaHübner,1825.Inaddition,allthesequencesofAzapaandChacawereclusteredin awell-supportedgroupinaMaximumLikelihood(ML)analysis.Accordingly,divergenceandMLanalyses supportthemorphologicalidentificationofE.azapaensisintheChacaValley.Furthermore,although preliminary,theanalysessuggestthatthegeneticvariationofthepopulationsofthisinsectcouldbe geographicallystructured,apatternthatmustbeassessedinfurtherstudies.

©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Tortricidaeisoneofthemorediversefamiliesofmicromoths withmorethan10,000speciesdescribedworldwide,withthe high-estlevelofspeciesdiversityintheNeotropicalregion(Regieretal., 2012).Inarecentrevision,RazowskiandPelz(2010)recordedmore than80speciesfromChile,mostlybasedonthestudyofspecimens fromthecentralandsouth-centralareasofthiscountry.Incontrast, thespeciesfromthearidenvironmentsofnorthernChilehavebeen scarcelycollectedandstudied(Clarke,1987;BobadillaandVargas, 2015).Larvalfeedinghabitsarevariableinthisfamilyof micro-moths.Thelarvaeofmanyspeciesareleaf-tiersandleaf-rollers; othersboreintotwigs,feedonreproductiveorgans(seeds,flower partsorfruits),oraregallinducers(Haghanietal.,2014;Razowski andGiliomee,2014;BrownandNishida,2003).Thegall-inducing habithasbeendescribedforseveralgenera,mostlybelongingtothe

∗ Correspondingauthor.

E-mails:havargas@uta.cl,lepvargas@gmail.com(H.A.Vargas).

tribesCochylini,GrapholitiniandEucosmini(BrownandNishida, 2007).

EugnostaHübner,1825(Tortricinae:Cochylini)isaworldwide genuswithmorethan90speciesdescribed,about40ofwhichare fromtheNewWorld(Gilliganetal.,2014).Naturalhistoryhasbeen describedforafewspeciesofEugnosta,andintheNewWorldall aregallinducersinAsteraceae(Comstock,1939,1940;Goedenand Ricker,1981;Vargasetal.,2015).EugnostaazapaensisVargasand Moreira, 2015istheonly representativeofthegenuscurrently recorded in Chile.Its larvae inducefusiform galls onshoots of theshrubBaccharissalicifolia(Asteraceae)intheAtacamaDesert (Vargasetal.,2015).Thiscecidogenousmicromothwaspreviously knownonlyfromthetypelocality,theAzapaValley,AricaProvince. However,fusiformgalls wererecently detectedonshoots ofB. salicifoliaintheChacaValley,about30kmsouththetypelocality (Fig.1).TheadultsobtainedfromthesegallswereidentifiedasE. azapaensisbasedonmorphologyofthemaleandfemalegenitalia.

TheAtacamaDesertisthemostariddesertintheworld(Clarke, 2006),wherethecoastalvalleysofthenorthernmostpartofChile

http://dx.doi.org/10.1016/j.rbe.2017.05.004

0085-5626/©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Fig.1.ThestudyareainSouthAmerica(left)andthesamplingsites(right)ofEugnostaazapaensisintheAtacamaDesertofnorthernChile.ThetypelocalityAzapaValley (blackcircle)andthenewlydocumentedlocalityChacaValley(blacktriangle).

are recognized as valuable reservoirs of biodiversity for plants (LuebertandPliscoff,2006),birds(Estadesetal.,2007),mammals (Ossaetal.,2016)andinsects(VargasandParra,2009; Méndez-Abarca et al., 2012). Accordingly, it is important torecord the taxonomiccomposition accuratelyat alocal scale,especially in littlestudiedgroupssuchasmicromoths.

Theanalysisofdifferentsources ofevidence isimportantto reach sound taxonomic identifications (Dayrat, 2005; Schlick-Steineretal.,2010).Thisapproachisespeciallyimportantwhen morphologicallysimilar species are involved (Taft et al., 2016; WarrenandGrishin,2017),asisthecaseinmanygroupsof micro-moths(LandryandHebert,2013;Huemeretal.,2014;Kirichenko et al., 2015; Luz et al., 2015; Pereira et al., 2017).It hasbeen shownthattheanalysisoftheDNAbarcodefragment(sensuHebert etal.,2003)ofthecytochromeoxidasesubunitIisauseful com-plementtomorphologyforidentifyingsmallorganisms,including speciesofthefamilyTortricidae(Jaegeretal.,2013;Brownetal., 2014;Gilligan etal.,2016;Corleyand Ferreira,2017;Razowski etal.,2017).Accordingly,DNAbarcodesequenceswereanalyzed toassessthetaxonomicstatusofthenewlydiscoveredpopulation ofEugnostaintheChacaValley.

Sampling

Fusiform galls on shoots of B. salicifolia were collected in the Azapa (18◦3112S, 70◦1041W) and Chaca (18◦4856S, 70◦0907W)valleysbetweenMayandAugust2016.Thecollected gallswereplacedinplasticbagsandbroughttothelaboratoryand dissectedtoextractthececidogenouslarvae,whichwerekeptin 95%ethanolat−20◦_{CforDNAextraction.}

DNAextractionandsequencing

GenomicDNAwasextractedfromlarvaefollowingthe proce-duresdescribedinHuanca-Mamanietal.(2015)andsubsequently wassenttoMacrogenInc.(SouthKorea)foramplificationby poly-merase chain reaction (PCR) and sequencing with theprimers LCO-1490andHCO-2198(Folmeretal.,1994).Theamplification programwas:5minat94◦C,35cyclesof30sat94◦C,30sat47◦C, 1minat72◦C,andafinalelongationstepof10minat72◦C.

Sequenceanalysis

According to the procedures described by Hall (2013), the softwareMEGA6(Tamuraetal.,2013)wasusedtoperformthe sequence alignment by the ClustalW method, to estimate the sequencedivergencebytheKimura2-Parameters(K2P)method and to perform a Maximum Likelihood (ML) analysis. Besides the Chilean sequences reported here, the following DNA bar-codesequences (658bp) ofEugnosta withspecies identification weredownloadedfromBOLD(RatnasinghamandHebert,2007) fortheMLanalysis:E.beevorana(Comstock,1940) (LNAUU4001-15),E.brownana Metzler andForbes,2012 (LNAUU4004-15),E. busckana(Comstock, 1939) (LOCB682-06), E.erigeronana(Riley, 1881) (LILLA210-11),Eugnosta sp. (LNAUU3996-15),E. percnop-tila (Meyrick, 1933) (AFTOR133-12), E. sartana (Hübner, 1833) (LOFLA761-06).Inaddition,Aethesargentilimitana(Robinson,1869) (BBLPA769-10)was includedin theanalysis,as AethesBillberg, 1820isclosetoEugnosta(Regieretal.,2012).PrevioustotheML analysis,theGTR+Gwasselectedasthebestmodeltodescribethe substitutionpatternfollowingtheBayesianinformationcriterion.

H2 T – – T G A – – G – – 2 Azapa

H3 – A T – G A T A – A – 6 Chaca

H4 – A T – G A T A – A G 1 Chaca

a_{“–”indicatesnucleotideidentitytotheH1haplotype.} b _{Ts,transition;Tv,transversion.}

Thestatisticalsupportofthenodeswasassessedby1000bootstrap replicates.Thegenealogyof thehaplotypeswasinferredbythe Median-Joining(MJ)method(Bandeltetal.,1999)inthesoftware Network5.0.0.1(FluxusTechnologyLtd.).

Eleven DNA barcode sequences (658bp) were obtained; four from Azapa and seven from Chaca (GenBank accessions MF000408–MF000418),allofwhichwerecharacterizedby30.6% (A),39.1%(T),15.4%(C),14.8%(G).

Geneticdivergence

Elevenvariable siteswerefoundin thealignment of allthe sequencesofAzapaandChacavalleys,witheighttransitionsand threetransversions(Table1).Whenthesequencesofthedifferent localitieswerealignedseparatelyfivevariablesiteswerefoundin Azapa,whilejustonevariablesitewasfoundinChaca.The muta-tionsdeterminedthepresenceoffourhaplotypes,two(H1,H2) restrictedto Azapa,and two (H3,H4) foundonlyin Chaca.H1 andH2wererepresentedbytwoindividualseach,H3bysix indi-viduals,andH4byone(Table1).Thedivergence(K2P)was0.8% betweenhaplotypesofAzapaand0.2%betweenthoseofChaca.The divergencebetweenhaplotypesofdifferentlocalitieswas1.1–1.4%. Accordingly, the haplotypenetwork shows a lower number of mutationsbetweenhaplotypesofthesame(1–5)thanof differ-ent(7–9)localities(Fig.2).Thedivergencebetweenhaplotypesof

H1 H2 92 286 374 H4 H3 316 302 139 274 364 370 473 625

Fig.2. MedianjoiningnetworkofthehaplotypesoftheDNAbarcodefragment (658bp)ofthecytochromecoxidasesubunitI(COI)geneofEugnostaazapaensis fromAzapa(white)andChaca(black)valleys,AtacamaDesertofnorthernChile. H1,H2,H3,H4haplotypes;circlesproportionaltothefrequencyoftherespective haplotype;numbersbetweencirclesindicatesvariablesites;graytriangleamedian vector.

ChacaandAzapawithotherspeciesofEugnostaincludedintheML analysiswas8.7–13.5%.

DNAbarcodedivergenceabove2%(K2P)isgenerallysuggestive ofdifferentspeciesofLepidoptera(Hausmannetal.,2011). How-ever,casesofhigherintraspecificdivergencehavebeenreported, mostlyinvolvingwidelydistributedspecies(WiemersandFiedler, 2007;HausmannandHuemer,2011).Inaddition,casesof interspe-cificDNAbarcodedistanceslessthan2%alsohavebeendescribed, mostly dealing with morphologically close species (Kirichenko et al., 2015). Regardless of these exceptional cases, the diver-gencefoundbetweenhaplotypesoftheAzapaandChacavalleys (1.1–1.4%)appearstobeattheintraspecificlevel.Thissuggestion isreinforcedbythefactthatthelowestdivergencefoundbetween theotherspeciesofEugnostaanalyzedwas2.6%(E.busckanaandE. beevorana).

MLanalysis

ThealignmentfortheMLanalysisincludednineteenDNA bar-codesequencesof658bplengthwith166variablesites,108of whichwereparsimony informative.TheelevenAtacamaDesert sequences(Azapaand Chaca)wereclusteredwithhighsupport (Fig.3),suggestingthatallthesebelongtothesamespecies(E. aza-paensis).Inaddition,thesequencesofAzapawereclusteredwith highsupportasa subgroupintotheE.azapaensisclade(Fig.3), suggestingthatthegeneticvariationisassociatedwithgeographic distribution,although a similar scenariowasnot found forthe sequencesofChaca.

TherelationshipsofE.azapaensiswithcongenericswerenot resolvedintheanalysis.However,thiswasexpectedsinceonlya fewspeciesofEugnostaarerepresentedinBOLD,mostofwhichare Nearctic.TheNeotropicalE.argentinae(Razowski,1967)described fromArgentinaandE.ochrolemma(Razowski,1986)fromMexico aremorphologicallysimilartoE.azapensis(Vargasetal.,2015), but DNA barcode sequences of these species are unknown. In addition,thegallshapeof E.azapaensisis similartothatofthe NearcticE. busckana and E. beevorana. Interestingly, these two NearcticspecieswereclusteredwithhighsupportintheMLtree, althoughtheywerenotfoundtobeclosetoE.azapaensis.Evidently, additionalmorphological(adultandimmaturestages)and molec-ular(mitochondrialandnuclear)dataofotherNeotropicalspecies are necessary to understand the evolutionary relationships of E.azapaensis.

Furtherremarks

ThisisthefirstreportofE.azapaensisoutsidethetype local-ity. Galls of E. azapaensis were searched for on shoots of the onlyotherspeciesofBaccharisrepresentedinthestudyarea(B. scandens)withoutsuccess.Inaddition,surveysforgallsonB. sali-cifolia werecarried out on thewestern slopesof the Andes of

MF000412 Eugnosta azapaensis Chaca MF000413 Eugnosta azapaensis Chaca MF000411 Eugnosta azapaensis Chaca MF000410 Eugnosta azapaensis Chaca MF000409 Eugnosta azapaensis Chaca

MF000415 Eugnosta azapaensis Azapa MF000416 Eugnosta azapaensis Azapa MF000417 Eugnosta azapaensis Azapa MF000418 Eugnosta azapaensis Azapa MF000408 Eugnosta azapaensis Chaca MF000414 Eugnosta azapaensis Chaca LOCB682-06 Eugnosta busckana

LNAUU4001-15 Eugnosta beevorana LNAUU3996-15 Eugnosta sp.

AFTOR133-12 Eugnosta percnoptila LlLLA210-15 Eugnosta erigeronana

LNAUU4004-15 Eugnosta brownana LOFLA761-06 Eugnosta sartana

BBLPA769-10 Aethes argentilimitana 100 97 86 97 96 87 0.02

Fig.3. MaximumlikelihoodtreeofthesequencesoftheDNAbarcodefragment(658bp)ofthecytochromecoxidasesubunitI(COI)geneofEugnostaazapaensis(grayarea) fromAzapaandChacavalleys,AtacamaDesertofnorthernChile.Bootstrapsupportshigherthan70%areshownabovebranches.

the Parinacota Province, at about 3000m elevation, also with-out success. Thus E. azapaensis appears to be a host-specialist withageographicrangerestrictedtothelowelevation environ-mentsofthecoastalvalleysoftheAtacamaDesert.Thisgeographic pattern has been described for different insect groups (Porter, 1985;Howden,2008).Additionalcoastalvalleysinwhichthehost plantispresentmustbesurveyedinordertobetterunderstand thegeographicrangeofE.azapaensisalongthisextensivedesert area.

TheonlyDNAbarcodesequencepreviouslyavailableforE. aza-paensiswastheanalyzedintheoriginaldescriptionofthespecies (GenBankaccession KM023733.1; 622bp) which matches 100% withoneofthehaplotypesofAzapa(H2),whileH1,H3andH4are herereportedforthefirsttime.Thevariationfoundinthisstudy suggeststhattheDNAbarcodefragmentisavaluabletooltoassess thetaxonomicstatusofeventualnewpopulationsofEugnostaof theAtacamaDesert.AnalysisofsequencesofmitochondrialDNAis usefultoexploregeographicpatternsofgeneticvariation,atleastat apreliminarystage(Harperetal.,2008;Maita-Maitaetal.,2015; Velasco-Cuervoetal.,2016;Maiaetal.,2016).Inthisregard,an interestingfindingisthateachhaplotypeisrestrictedtoaspecific locality.Thisspatialsegregationofthegeneticvariationsuggests thatthearidconditionsoftheareaseparatingthevalleyscould beaneffectivebarrierforthedispersalofadultsofE.azapaensis, enhancingthegeneticdifferentiationofthelocalpopulationsof eachvalley.However,duetothelowdensityofgallsinthefield, onlyafewspecimenswerecollectedandanalyzedinthisstudy. Thusfurthersamplingandanalysesofadditionalmolecular mark-ers(Valadeetal.,2009;Seraphimetal.,2016)wouldbeneededto verifythispatternofgeographicstructureofthegeneticvariation inthedriestdesertoftheworld.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthankJohnW.Brown,LucasA.Kaminskiandtwo anonymousreviewersfor valuable commentsthat substantially improvedthefinal version of themanuscript, toPaula Escobar forprovidingFig.1andLafayetteEatonforcheckingtheEnglish. ThestudywassupportedbyProject9715-16fromUniversidadde Tarapacá.

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