REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolution w w w . r b e n t o m o l o g i a . c o mSystematics,
Morphology
and
Biogeography
Morphometric
and
molecular
differences
among
Calvertius
tuberosus
(Coleoptera:
Curculionidae)
populations
associated
with
Andean
and
coastal
populations
of
Araucaria
araucana
in
the
La
Araucanía
Region,
Chile
Luis
Huala
Jimenez
a,
Ramón
Rebolledo
Ranz
b,
Rubén
Carrillo
López
b,
Mario
Elgueta
c,
Marco
Paredes
Honorato
a,∗aUniversidaddeLaFrontera,DepartamentodeCienciasbásicas,Temuco,Chile
bUniversidaddeLaFrontera,DepartamentodeCienciasAgronomicasyRecursosNaturales,Temuco,Chile cMuseodeHistoriaNatural,ÁreadeEntomología,Santiago,Chile
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received25September2017 Accepted24December2017 Availableonline2February2018 AssociateEditor:AdrianaMarvaldi Keywords:
Araucariaforests ISSRmarkers Morphometry
a
b
s
t
r
a
c
t
Calvertiustuberosus(Curculionidae)livesexclusivelyonAraucariaaraucanatrees(commonlyknownas
pehuen)insouthernChile.Inthisstudy,morphometricandmoleculargeneticanalysesofAndeanand
coastalpopulationsofC.tuberosuswereperformedtoevaluateevolutionarydivergenceassociatedwith
thediscontinuityoftheAraucariaforestbetweenthecoastalandAndeanregions.SpecimensofC.
tubero-suswerecollectedinNahuelbutaNationalPark,VillaLasAraucarias,andMalalcahuelloNationalReserve
andwereclassifiedandstoredattheAnimalBiotechnologyResearchingLaboratory(LINBA),University
ofLaFrontera,Temuco,Chile.ThirteenmorphometricparametersandtheexpressionpatternsofISSR
(inter-simplesequencerepeat)markerswereanalyzed.Morphometricdatarevealedhighphenotypic
similaritybetweencoastalpopulations.Thegeneticanalysisrevealedahighsimilaritybetweencoastal
populations(geneticidentity,93%),whichweredifferentiatedfromtheAndeanpopulation(genetic
iden-tity,84%).ThisstudycontributesnewgenotypicandphenotypicdatafortheC.tuberosuspopulationsin
forestecosystemsofA.araucana,andclarifiestheassociationsbetweenthesecharacteristicsandthe
geographicdistributionsofpopulations.
©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen
accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
InChile,thereareover4200speciesofColeopterabelonging to97 families(Elgueta,2008),representing30%ofallinsectsin Chile,withlittlediversificationinmostgenera(Vergaraetal.,2006) andhighlevelsofendemismatthespecieslevel.Manyofthese generaaresharedwiththosefoundinAustraliaandNewZealand, insteadofSouthAmericantropicalzones(Arias,2000). Curculion-idaeisadiversefamily,withalmost4600generaand51,000species (Oberprieleretal.,2007).
Calvertiustuberosus(Fairmaire&Germain,1860)(Coleoptera: Curculionidae) is found between the Biobío (36◦4622S, 73◦0347W)andLaAraucanía(38◦5400S,72◦4200W)regions
∗ Correspondingauthor.
E-mail:[email protected](M.P.Honorato).
insouthernChile(Arias,2000),andisexclusivelyfoundon Arau-cariaaraucana((Mol)Koch,1869)(Pinales:Araucariaceae)trees, withafrequencyofalmost30%(Elguetaetal.,2008).
C.tuberosusisthelargestamongthe23speciesofcurculionids andotherrelatedfamilies,onthishost(Kuschel,2000).Adultswalk onthetrunkorfeedonleavesandsoftshoots,andlarvaearefound inthesubcorticalzoneinfallenorstandingtrees,wheretheyfeed onphloem,althoughtheyfrequentlybecomexylophagesattheend oftheirdevelopment(Barrigaetal.,1993;Morrone,1997;Kuschel, 2000;ElguetaandMarvaldi,2006).Morrone(1997)indicatedthat thisbeetleisasecondaryinvaderthatdoesnotattackhealthytrees, butentersbranchespreviouslyaffectedbybarkbeetles (Scolyti-nae).
Araucariaaraucanais anendemicspecies inSouthAmerican temperate forestsalong the Andes mountains from 37◦27S to 40◦03S(Morenoetal.,2011).Approximately97%ofits popula-tionsformextensivepureforests,oftenonsteepvolcanichills,and
https://doi.org/10.1016/j.rbe.2017.12.004
0085-5626/©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
areassociatedwithtemperaterainforestspecies(Hechenleitneret al.,2005).
Some relatively small and disjunct populations occur in the Nahuelbuta mountains at the Nahuelbuta National Park (33◦3700S,79◦0200W)andsurroundingareas,includingVilla LasAraucarias(38◦0017S,72◦5756W).Inthislatterarea, Arau-cariaarefoundinhighlyalteredenvironmentsdominatedbymixed forestofNothofagusspp.(Fagales:Nothofagaceae)andexotictrees, suchasEucalyptusglobulus(Labill)andPinusradiata(D.Don).
TheremainingAraucariaforestsbelongtoprivateownersand arepermanentlysubjecttohighlevelsofdisturbancebythe inade-quateextractionoftheirediblefruit,fires,logging,andsubstitution with commercial forest plantations (Donoso et al., 2006). The ecosystemsofboth areasarecharacterizedbya moist Mediter-raneanclimatewithdifferencesrelatedtoaltitudeandexposure; theirsoilsarecomposedofmetamorphicmaterialsand,insome locations,granite(Donosoetal.,2008).Ithasbeenproposedthat geographical isolationbetween coastaland AndeanA. araucana populationsresultsingeneticpopulationdifferentiation(Raffiand Dodd,1998).
Morphometry was among the first methods used in biodi-versityandphylogeneticstudiesandisstillapplied,despitethe widerangeofmoleculartechniquesusedcurrently(Wanek and Sturmbauer,2015).Morphometricanalysesareusedintaxonomy, butarealsousedincoevolutionandphylogeneticstudiesofdiverse groupsofinsects,suchasaphids,bees,grasshoppers,andbeetles (Sánchez-Ruiz and San Martín, 2000). Morphometric measure-mentsarewidelyusedinapproachesthatintegratesystematics withmoleculardataandcanleadtotaxonomicrevisions, compa-rabletophylogeniescreatedfromDNA.Whencorrectlyselected, morphometricparameterscanbeusedtoestablishphylogenetic relationships,especiallyforspeciesthatarenoteasytodistinguish owingtoalackofdiagnosticcharacters(PrzybycienandWaclawik, 2015).
Atthegeneticlevel,nucleotidesequencedifferencescanbeused tostudyevolutionaryrelationshipsamongspecies.Forinstance,
WoeseandFox(1977)classifiedprokaryotesbasedonribosomal genes.PhylogeniescreatedfromDNAsequencescanprovideuseful insightintotheevolutionaryhistoryofgenesandorganisms(Yang andRannala,2012).Duringevolution,geneticmaterialaccumulates mutationsthatpotentiallyresultinphenotypicchanges(Olsenand Woese,1993).
Inter-simplesequence repeats(ISSRs) area sensitivegenetic markerforstudiesofpolymorphism(BornetandBranchard,2004) withinpopulationsbasedontheabsenceorpresenceofagenomic elementandthelengthoftheamplifiedintermediary sequence (Zietkiewicz et al., 1994). ISSRs in the genomes of plants and animalsarehighlyvariableandthereforearecommonlyusedin populationgeneticstudies(Tikunovetal.,2003).ISSRanalysesdo notrequirehighconcentrationsofDNA,andprimerdevelopment doesnotrequirepreviousknowledgeofthegenomesequenceof theorganismunderstudy(Joshietal.,2000).Thehighdegreeof polymorphismandwidedistributionofmicrosatellitesenablethe detectionoflowlevelsofdifferentiation(Yua,2011).
OthermoleculartoolshavebeenusedtocharacterizeA. arau-canapopulations.Forexample,Marchellietal.(2010)studiedtheir possiblepre-Pleistoceneoriginusingchloroplastand mitochon-drialDNA sequences.Based onnuclearand mitochondrial gene sequenceanalyses, Sequeiraand Farrell(2001) investigatedthe phylogeneticrelationshipsandtheestimateddivergencetimesof barkbeetlesassociatedwithAraucariainAustraliaandSouth Amer-ica.Thestructure and geneticdiversityof populationsin South America have been studiedbased on the composition of foliar epicuticularwaxalkanes (RafiiandDodd,1998), RAPDmarkers (Bekessyetal.,2002),nuclearmicrosatellites(Martínetal.,2014), andAFLPs(Marconietal.,2011).
In this study, the morphological and genetic characteristics ofC.tuberosus specimensfromcoastalpopulations(Nahuelbuta NationalPark,hereafterNahuelbuta,andVillaLasAraucarias)and the Andean National Reserve (Malalcahuello National Reserve, hereafterMalalcahuello)of theLaAraucanía region,Chilewere evaluatedwithrespecttodifferencesamongA.araucana popula-tions.
Materialsandmethods Populations
C.tuberosuswerecollectedfromthebarkoffallenandstanding A.araucanathreesinthreeareasinthelaAraucaníaregion,i.e., Malalcahuello(38◦2421.56S,71◦3546.08W),VillaLasAraucarias (38◦2912.65S,73◦1541.13W),andtheNahuelbutaNationalPark (37◦4733.69S,72◦5953.36W).
Morphometricmeasurements
FortyspecimensfromVillaLasAraucarias(27male,13female), Nahuelbuta(30 male, 10 female), and Malalcahuello (32 male, 8female)wereincludedintheanalyses.For eachspecimen,13 parametersweremeasuredunderaLeicaEZ4stereoscopic mag-nifier(Wetzlar,Germany).Themorphometricparameterswereas follows:prothoraxlength,prothoraxbasewidth,prothorax max-imum width,elytra length,elytra base width,elytra maximum width,elytraminimumwidth,elytraapexwidth,pedicellength, flagellumlength,rostrum length,rostrum apexwidth, and ros-trumbasewidth (Fig.1).Additionally, coloration wasrecorded forspecimensineachpopulation.Thesemorphometric measure-mentswereusedtocreateadendrogramusingNei’s(1972)model implementedinPAST3.14(Hammeretal.,2001).
TotalDNAextractionfromC.tuberosus
Individualswerestoredat−80◦CattheAnimalBiotechnology
ResearchLaboratory(LINBA),UniversityofLaFrontera,Temuco, Chile,aftergrindingeachspecimeninaChinamortarfollowing treatmentfor10minwithUVlight,yielding1–2mgof homoge-nizedtissuefromeachinsect.Sampleswereprocessedusingthe AxyPrepMultisourceGenomicDNAMiniprepExtractionKit (Axy-genBiosciences,Tewksbury,MA,USA).
DesignofISSRmarkers
SeventeenISSRprimers(Table1)wereselectedbasedonthe methodsofKorpelainenetal.(2007).Sevenprimers(AC-T,CA-G, GA-C,AG-C,AC-C,CA-A,CAG)werepreparedattheAnimal Biotech-nologyResearchingLaboratory(LINBA)forPCRamplificationwith purifiedDNAofC.tuberosus,followingthemethodsofPérezde laTorre(2012),withmodifications.Theamplificationconditions wereasfollows:10minofdenaturationat95◦C,40sat90◦C(45 cycles),45sofreheatingat50◦C(45cycles),90sofinitialextension at72◦C(45cycles),10minoffinalextensionat72◦C.The reac-tionmixturecontainedthefollowing:10LofMaximaSYBRGreen qPCRMasterMix(2×),1LofDNA(200ng),1Lof17ISSRprimers, and8LofH2Oultra-pure(20-Lfinalvolume).Theamplification
reactionwasperformedusingaMultiGeneGradientThermocycler (LabnetInternationalInc.,Edison,NJ,USA).
PCRamplificationofISSRmarkers
Ofthe17primersevaluatedaccordingtotheirpatternsof poly-morphism,fivewereselected([AC]8-T,[GA]9-T,[GA]8-C,[GA]9-A,
10 9 12 11 13 3 1 5 6 4 7 8 2 Head Antenna
eye Front leg
Scutellum Pronotum Middle leg Hind leg Suture Striae Elytra
1, Prothorax length. 2, Prothorax base width. 3, Prothorax maximum width. 4, Elytra length. 5, Elytra base width. 6, Elytra maximum width. 7, Elytra maximum width. 8, Elytra ápex width. 9, Scape length. 10, pedicel and flagellum length.
11, Rostrum length. 12, Rostrum apex width. 13, Rostrum base width.
Fig.1.MorphologyofageneralizedCurculionidae,withthemorphometrics mea-surementsused(adaptedfromMarvaldiandLanteri,2005).
Table1
NamesandsequencesofISSRprimers(Korpelainenetal.,2007).
Names Sequences Names Sequences
AC-T [AC]8T CAA [CAA]5
CA-G [CA]9G CA-A [CA]9A
GA-T [GA]9T CAG [CAG]6
GA-C [GA]8C ATG [ATG]5
GA-A [GA]9A GA-C [GA]9C
AG-T [AG]8T AC-G [AC]9G
AG-C [AG]8C CA-T [CA]9T
AG-G [AG]9G GA-C [GAC]5A
AC-C [AC]8C
and[CA]9-G).ThePCRconditionswereasfollows:10Lof
Max-imaSYBRGreenqPCRMasterMix(2×),1LofDNA,1Lofeach ISSRprimer,and8Lofultra-pureH2O(finalvolume,20L).The
DNAamplificationprocedurewasperformedusinga MultiGene
GradientThermocycler(Fig.2).
IntheanalysisofISSRexpressionpatterns,onlypolymorphic andreproduciblebandswereconsidered,andvaluesof1or0were assignedtoindicatepresenceorabsence.Datawereclassifiedina binarymatrixandusedtoestimategeneticdistancesbasedonNei, 1972modelandthesedistanceswereusedtobuildadendrogram bytheUPGMAmethod(SneathandSokal,1975)withPOPGEN1.32 (Yehetal.,1999). 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 1000 800 700 600 500 400 300 200 100 900
Fig.2.ISSRpatternsofexpressionforC.tuberosusfromMalalcahuello(1-1,1-2, 1-3),VillaLasAraucarias(2-1,2-2,2-3)andNahuelbuta(3-1,3-2,3-3).
Results
A dendrogramwasgenerated basedonISSR-derived genetic distances,reflectingthemorphometricrelationshipsamong spec-imensobtainedfromthreepopulationsofC.tuberosus.Therewas greatermorphologicalsimilaritybetweenthespecimensobtained fromVillaLasAraucariasandNahuelbuta(bothcoastalmountain locations)thanbetweencoastaland Andeanpopulations. More-over,thespecimensobtainedfromMalalcahuelloexhibitedsimilar morphologicalfeaturestothoseofthespecimensobtainedfrom VillaLasAraucarias(Fig.3).
The specimens obtained from the Villa Las Araucarias and Nahuelbutapopulationspresentedamarkedblackordarkbrown color, while theMalalcahuello specimens exhibited a deepred color.
In the ISSR analysis, 45 bands were detected, of which 43 (96%)werepolymorphic,withasizeof250–1100bp(Fig.2).The coastalpopulations(NahuelbutaandVillaLasAraucarias) exhib-itedageneticidentityof93%.TheMalalcahuellopopulation(Andes Mountains)hadgeneticidentitiesof84%and87%incomparisons withtheVillaLasAraucariasandNahuelbutapopulations, respec-tively(Fig.4).
A genetic distance value of less than 0.07 was observed betweenthecoastalpopulations.Thegeneticdistancesbetween the Andeanpopulation(Malalcahuello) and theVilla Las Arau-cariasandNahuelbutapopulationswere0.17and0.14,respectively (Fig.4).
Discussion
Differencesinbodysizebetweenmalesandfemalesare com-monininsects;typically,femalesarelargerthanmales(Posadas etal.,2007).InCurculionidae,sexualdimorphismiscommoninthe rostrum,i.e.,thefemalerostrumisgenerallylargerandflatterthan thatofthemale(SotoandReyes,2014).Nevertheless,inC. tubero-sus,rostrumsizedidnotdiffersubstantiallybetweenfemalesand males;theobservedmorphometricvariationcouldbetheresultof geographicisolationamongC.tuberosuspopulations.
The morphometric analysis indicated the presence of geo-graphic variation; in particular, the coastal populations (Villa LasAraucariasand Nahuelbuta)exhibitedsimilarmorphological patterns, which differed from those of the Andean population (Malalcahuello)(Fig.3).Themorphologicalandgeneticsimilarity ofcoastalpopulationsofC.tuberosuscouldbeattributedtoamore recentbiogeographicseparationofthesepopulations.
Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Var Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Nah Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal Mal 1 2 1 3
Fig.3.Neighbor-joiningdendrogramofmorphometricsmeasurements;1.VillaLasAraucarias,2.Nahuelbuta,3.Malalcahuello.
Themorphological and geneticcharacteristics of thecoastal andAndeanpopulationsof C.tuberosus couldbeaconsequence ofthegeographicalseparationtheirhost,A.araucana,whichhas beenaffectedby large-scaleenvironmental changes.For exam-ple,sincePleistoceneglaciationevents,thetreepersistedinsmall
populationsduringtheLastGlacialMaximum(LGM)inthecoastal rangeofChileandsomeareasoftheAndesMountains(Villagrán, 2001;Martínetal.,2014),withadiscontinuousdistribution.The
C. tuberosus (Malalcahuello) C. tuberosus (Malalcahuello) C. tuberosus (Malalcahuello) C. tuberosus (Villa las araucarias)
C. tuberosus (Villa las araucarias)
C. tuberosus (Villa las araucarias)
C. tuberosus (Nahuelbuta) C. tuberosus (Nahuelbuta) C. tuberosus (Nahuelbuta) Araucarietus viridans UPGMA 0,84 (84%) 0,87 (87%) 0,07 0,93 (93%) Nei's, 1972 0,17 0,14
A
B
Fig.4.ISSRanalysis.(A):Dendrogramforthepopulationsstudied(controlgroupA.viridans).
substantialdistancebetweenpopulationssuggeststhepossibility ofpopulationgeneticchanges,suchasinbreeding,genedrift,and alteredgeneflow(Marchellietal.,2010).
Morerecentcatastrophicevents,suchastheVillarrica,Llaima, Lonquimay,andCopahuevolcaniceruptions(Heusseretal.,1988) andseriousdeforestationover thelastcentury,haveresultedin thedegradationorreplacementofabout120,000haofnative for-est(Herrmann,2006),leadingtofurtherdifferencesbetweenthe Andeanandcoastalpopulationsbyalackofavegetativecontinuum, limitinginsectdispersal.
DespiteacloserelationshipbetweenthepopulationsofVilla LasAraucariasandNahuelbuta,therewerecleargeneticand mor-phologicaldifferencesbetweenthespecimensof C.tuberosusin thepopulations.Currentdataindicatethatanthropicintervention atCordillera de Nahuelbuta(themountain range thatconnects both populations)hasresultedin over 70%lossof native vege-tation(Wolodarsky-FrankeandDíaz,2011)andproducedhabitat fragmentationandmoresubstantialpopulationisolation.
Differentiation in morphometric parameters between geo-graphically separated populations explains, to some extent, evolutionaryprocessesandisconsideredthefirststageinallopatric speciation(Oliveroetal.,2012).InC.tuberosus,theinterruptionof geneflowbetweenpopulationsindiversehistoricalperiodscould haveresultedinreproductiveisolation, whichwouldeventually leadtogeneticdifferentiationandevolutionarydivergence.
ThegeneticdifferencesandsimilaritiesamongpopulationsofC. tuberosus(Fig.4)canbecomparedwithgeneticvariationinits obli-gatehost,A.araucana.Thesecomparativeanalysesallowustoinfer thatchangesinthegeographicdistributionofAraucariainfluence
theobservedvariationinbothspecies,therebyexplaininggenetic variabilityincoastalandAndeanpopulations(Bekessyetal.,2002). TheISSRmarkersrevealedamoredistantrelationship(based ondistanceandgeneticidentity)betweenthecoastalpopulations (NahuelbutaandVillaLasAraucarias)andtheAndeanpopulation (Malalcahuello)thanbetweenthetwocoastalpopulations(Fig.4). TheseresultsareconsistentwithpreviousanalysesofA.araucana Andeanandcoastalpopulationsindicatingsignificantgenetic dif-ferences usingRAPDs (Bekessy et al.,2002)and microsatellites (Martínetal.,2014).SimilardifferencesbetweenAraucaria popu-lationshavebeendescribedbyRafiiandDodd(1997)basedonthe proportionalcompositionoffoliarepicuticularwaxalkanes.
Itisnotpossibletounderstandthenaturalworldwithout exten-siveknowledgeofthemorphologicalcharacteristicsoforganisms, whichplaycentralrolesinlifecycles,geographicaldistributions, identification,conservation,evolution,development,and delimi-tationofspecies (WortleyandScotland, 2006).Theinclusionof morphologicaldatasubstantiallyimprovestheresultsof molec-ularphylogeneticanalyses,provingtobeusefulnotonlyforthe resolutionoftaxonomicproblems,butalsoforcoevolution stud-iesandphylogeneticinferences(PrzybycienandWaclawik,2015). Thelinkageofmorphometricandmolecularanalysesispowerful forstudiesofpopulationstructure andtheadaptivesignificance of trait divergence (Lee and Lin, 2012), to identifybiotypes of thesamespeciesondifferenthosts(Fekratetal.,2014),andfor thegeographicdelimitationofspecies(SchwarzfeldandSperling, 2014). Therefore, integrative analyses including morphometric measurementsandDNAsequencesareneededforacomprehensive understandingoftheautoecologyofspecies.
Conclusions
Thisstudycontributesnewgenotypicandphenotypicdatafor theC.tuberosuspopulationsin forestecosystemsofA. araucana andclarifiestherelationshipsbetweenthesecharacteristicsand thegeographicdistributionsofthepopulations;accordingly,these findingsextendourknowledgeofC.tuberosuspopulationsinChile. Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest Acknowledgments
Thisresearchwasfunded byFONDEFD10I1038(Biodiversity informationnetworktoguidethepropertiesofscientificresearch insupportofpublicenvironmentalpolicies)andDIUFRODI14-0111 [Chemical and biological characterization of Maiten (Maytenus boaria)asa foodattractant of A. superciliosus(Coleoptera: Cur-culionidae)].DIUFRODI13-TD01supportedthedoctoralthesis. References
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