Genomic Mining of Phylogenetically Informative Nuclear Markers in Bark and Ambrosia Beetles

26 Pages·2016·2.66 MB·English

RESEARCHARTICLE
Genomic Mining of Phylogenetically
Informative Nuclear Markers in Bark and
Ambrosia Beetles
DarioPistone,SigridMugu,BjarteHenryJordal*
DepartmentofNaturalHistory,TheUniversityMuseum,UniversityofBergen,PB7800,NO-5020Bergen,
Norway
*[email protected]
a11111 Abstract
Deeplevelinsectrelationshipsaregenerallydifficulttoresolve,especiallywithintaxaofthe
mostdiverseandspeciesrichholometabolousorders.Inbeetles,themajordiversityoccurs
inthePhytophaga,includingcharismaticgroupssuchasleafbeetles,longhornbeetlesand
weevils.Barkandambrosiabeetlesarewoodboringweevilsthatcontribute12percentof
OPENACCESS thediversityencounteredinCurculionidae,oneofthelargestfamiliesofbeetleswithmore
than50000describedspecies.PhylogeneticresolutioningroupsofCretaceousagehas
Citation:PistoneD,MuguS,JordalBH(2016)
GenomicMiningofPhylogeneticallyInformative provenparticularlydifficultandrequireslargequantityofdata.Inthisstudy,weinvestigated
NuclearMarkersinBarkandAmbrosiaBeetles. 100nucleargenesinordertoselectanumberofmarkerswithlowevolutionaryratesand
PLoSONE11(9):e0163529.doi:10.1371/journal.
highphylogeneticsignal.APCRscreeningusingdegenerateprimerswasappliedto26dif-
pone.0163529
ferentweevilspecies.Weobtainedsequencesfrom57ofthe100targetedgenes.
Editor:BenJMans,OnderstepoortVeterinary
Sequencesfromeachnuclearmarkerwerealignedandexaminedfordetectingmultiple
Institute,SOUTHAFRICA
copies,pseudogenesandintrons.Phylogeneticinformativeness(PI)andthecapacityfor
Received:April13,2016
reconstructionofpreviouslyestablishedphylogeneticrelationshipswereusedasproxies
Accepted:September10,2016 forselectingasubsetofthe57amplifiedgenes.Finally,weselected16markerssuitable
Published:September26,2016 forlarge-scalephylogeneticsofScolytinaeandrelatedweeviltaxa.
Copyright:©2016Pistoneetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
Introduction
DataAvailabilityStatement:Allrelevantdataare
Inthepostgenomicera,obtainingwellresolvedandhighlysupportedmolecularphylogeniesof
withinthepaperanditsSupportingInformation
files. hyper-diverseeukaryoticlineagescontinuestorepresentamajorchallenge.Previousattempts
oninvestigatingphylogeneticrelationshipsinbeetleshavedemonstratedrecurrentproblemsin
Funding:Thisprojectwasfundedbygrant214232/
resolvingdeeperrelationshipssuchasthosebetweenthefourbeetlesuborders,butalsomuch
F20fromtheNorwegianResearchCouncil.The
funderhadnoroleinstudydesign,datacollection youngerdivergences[1–4].Oneofthemostproblematicgroupsincludestheweevils,where
andanalysis,decisiontopublish,orpreparationof themajorityoftribesandsubfamiliesremainunresolveddespiteconsiderableeffortsinassem-
themanuscript. blingmoleculardata[5–8].BarkandambrosiabeetlesinthesubfamilyScolytinaerepresenta
CompetingInterests:Theauthorshavedeclared weevillineagewheremuchefforthasbeeninvestedindevelopingmolecularmarkersforphylo-
thatnocompetinginterestsexist. geneticanalysis[9,10].Nevertheless,resolutionbetweenmanyCretaceousrelationships
PLOSONE|DOI:10.1371/journal.pone.0163529 September26,2016 1/26
GenomicMininginBarkandAmbrosiaBeetles
remainsratherlow[11],emphasizingthescarcenessofmolecularmarkerstoresolvethispar-
ticularphylogeny.
Sofar,thevastmajorityofphylogeneticstudiesonbeetleswerebasedonmarkerssuchas
ribosomalRNAsandmitochondrialcytochromeoxidaseIandIIgenes[8,12–15].Withthe
exceptionofnuclearribosomalgenes(18sand28srRNAs)aremostmarkersusefulforthereso-
lutionofCenozoicdivergences,showinglackofphylogeneticsignalforCretaceoustimeframes
[10].Inthelastyears,agrowingnumberofphylogeneticstudiesonbeetleshavestartedto
includenuclearproteincodinggenes,especiallyEF-1α,CAD,ArgK,andwingless[11,16,17],
whicharealsowidelyusedinotherinsecttaxa[18–21].However,arelativelylimitedamount
ofworkhasbeendonetodiscoverandselectadditionalnucleargenesforbeetlesystematics
[22,23–25],andallstudiestodatewerebasedonlessthan10molecularmarkers[26,27].
Therefore,obtainingahighdegreeofphylogeneticresolutioninbeetlesisdifficult;adirectcon-
sequenceofhighspeciesdiversityandalimitednumberofinformativemarkers.
Thefirststudiesontheutilityofproteincodinggenesininsectsystematicsdatebackto
morethan20yearsago[28–30].Theadvancementofinsectphylogenieshaslargelybeen
drivenbythedevelopmentofnewmarkersinLepidoptera[31].Atpresent,dozensofnuclear
markerscanbechosentoinvestigateLepidopteraphylogenyatvariousranks[18,32–36].
Hymenopteraisanothergroupwhereaconsistentnumberofnuclearmarkershavebeendevel-
oped[37–39].Althoughsimilarstudieshavebeencarriedoutinotherinsectgroupssuchas
Diptera[40–42],themajorityoftheremaininginsectorderspresentasituationmoresimilarto
Coleopterawithfewpublishedmarkersconservedacrossdifferentfamilies[43,44].Thus,
increasingthenumberofphylogeneticcharactersfromproteincodingnucleargenesisofman-
datoryimportanceforachievingrobustphylogenetichypothesesinbeetlesystematics.
Recently,theadventofnextgenerationsequencing(NGS)technologieshascontributedto
additionalground-breakingadvancementsinthesystematicsfield,profoundlyincreasingthe
levelofresolutioncomparedtopreviousphylogeniesbasedonsingleorfewgenes[45].Geno-
micandtranscriptomicdataobtainedfromNGSbasedresearchhasledtopredictiveinsect
phylogenies,whichnowmoreclearlyrevealkeyeventsininsectevolutionaryhistory[46–50].
Newdevelopmentsbasedonultra-conservedelements(UCEs)orRAD-sequencingwill
increaseresolutionalsoatlowertaxonomicranksininsects[51,52].However,thebenefitsof
NGSaregenerallycounterbalancedbythehighcostandcomputationallydemandinganalyses
ofsuchhighthroughputdata.Theutilityoffewwell-characterizedmarkersshouldnotbe
underestimatedastheyrepresentarapidandcosteffectiveapproachforresolvingsmallscale
phylogenies.
BarkandambrosiabeetlesinthesubfamilyScolytinaeconstituteagroupofhighlyderived,
smallwoodboringweevilscapableofexcavatinggalleriesintodifferentpartsofdeadtrees,
shrubsandbushes,aswellasinlianasandotherplanttissuesindifferentforesthabitats
throughouttheworld[53].Scolytinaeisgenerallyregardedasawell-supportedcladeofmore
than6000describedspeciesrepresentingapproximately12percentoftheentirediversityin
thefamilyCurculionidae[5,54,55].Atremendousvariabilityinlifecycles,reproductivestrate-
gies,matingsystems,hostplantsinteractions,feedingbehaviorandecologyhasbeendocu-
mented[56,57],whichmakesthisgroupofbeetlesparticularlyinterestingtostudyina
phylogeneticallycomparativecontext.PhylogeniesofScolytinaehavesofarreliedonacombi-
nationoffivemolecularmarkers(onemitochondrialandfournucleargenes)andeventually
morphologicalcharacters.GiventhehighdiversityofScolytinespecies,additionaldataare
neededtoobtainsufficientresolutionatdeepernodes.
Inordertoselectnewphylogeneticmarkers,100differentnucleargeneswerescreenedby
PCRusingdegenerateprimersandtestedinarestrictedbutrepresentativegroupofScolytinae
andotherweevils.Withtheaimofdevelopingslowlyevolvinggenes,thepropertiesofeachgene
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GenomicMininginBarkandAmbrosiaBeetles
fragmentwereevaluatedbasedonPCRamplificationandsequencingsuccessandtheirphyloge-
neticperformance.Thisstudyreportsonthedevelopmentandutilityof16novelmarkersfor
weevils,withaparticularfocusonbarkandambrosiabeetlesinthesubfamilyScolytinae.
MaterialsandMethods
We included18speciesofbarkandambrosiabeetlesand8additionalweevilsfromothersub-
familiesforprimerscreening(Table 1andS1Table).Thesebeetleswerecollectedbyoneofthe
authors(BHJ)duringfieldworkintropicalforests(1998–2012).Collectionpermitswere
requestedfromauthoritiesinUganda,Tanzania,Cameroun,SouthAfricaandMadagascar.
Ethicalguidelineswerefollowed.VoucherspecimensaredepositedintheColeopteracollection
oftheUniversityMuseumofBergen,UniversityofBergen,Norway.Allweevils,Platypodinae
andScolytinaespeciesusedinthisstudywerepreviouslydescribedinotherphylogeneticstud-
ies[7,11,58].
Theprocedureforprimerselectioncanbesummarizedasfollows:1)putativelysinglecopy
expressedsequencetags(ESTs) longerthan800basepairswereselectedinGenBankfortwo
differentbeetlespecies,Tribolium castaneumandDendroctonusponderosae;2)preliminary
Table1. Weevilspeciesincludedinthisstudy.
Species Code Subfamily Tribe Country
Brentidaesp. BrBre05 Brentidae(familiy) Brentinae Cameroon
Mesitesfusiformis CsMes01 Cossoninae Cossonini Spain
Pselactussp. CsPse01 Cossoninae Onycholipini Portugal(Madeira)
Larinussp. ClLar01 Lixinae Cleonini Russia
Portheteshispidus MoPor01 Molytinae Amorphocerini South-Africa
Platypusimpressus PlPla07 Platypodinae Platypodini Tanzania
Triozastusmarshalli PlTri02 Platypodinae Platypodini Cameroon
Chaetastustuberculatus TsCha02 Platypodinae Tesserocerini Cameroon
Pityophthorusmicrographus CoPit01 Scolytinae Corthylini Norway
Diamerusinermis/D.hispidus DiDia03/DiDia04 Scolytinae Diamerini Tanzania/Madagascar
Dryocoetesautographus DrDry01 Scolytinae Dryocoetini Russia
Ozopemonuniseriatus DrOzo02 Scolytinae Dryocoetini PapuaNewGuinea
Hylastesattenuatus HtHyt06 Scolytinae Hylastini Sweden
Hylesinusvarius HlHyl02 Scolytinae Hylesinini Sweden
Kissophagushederae HlKis01 Scolytinae Hylesinini Austria
Chaetopteliusvestitus ToCha01 Scolytinae Hylurgini Morocco
Dendroctonusterebrans/D.micans ToDen02/ToDen01 Scolytinae Hylurgini USA
Tomicuspiniperda ToTom01 Scolytinae Hylurgini Norway
Acanthotomicussp. IpAca01 Scolytinae Ipini Cameroon
Pityogenesquadridens IpPit03 Scolytinae Ipini Sweden
Premnobiuscavipennis PrPre01 Scolytinae Premnobiini SierraLeone
Camptocerusaenipennis ScCam02 Scolytinae Scolytini Guyana
Cnemonyxvismiacolens ScCne01 Scolytinae Scolytini Guyana
Scolytusintricatus ScScl02 Scolytinae Scolytini CzechRepublic
Xyleborusaffinis XyXyl00 Scolytinae Xyleborini Cameroon
Xyleborusmonographus XyXyl03 Scolytinae Xyleborini CzechRepublic
Degenerateprimersweredesignedonconservedregionsinthealignmentofinsectnucleotidesequencesthatwereavailablefromgenomicand
transcriptomicsources.Twoormoreconsecutivedegeneratesiteswerepreferentiallyavoidedaswellastheuseofcompletelydegeneratesites(N).Atotal
of274primersweredesigned(Table2-onlysuccessfulprimersreported).
doi:10.1371/journal.pone.0163529.t001
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BLASTsearcheswereperformedtodiscardunsuitablemarkers,basedontheevidenceformul-
tipleparalogouscopies(e.g.largegenefamilies)orambiguousgenomiccharacterization(e.g.
similarmatchingvaluesfordifferentproteins);3)availablesequencesforeachselectedgene
werealigned,includingannotatedgenomicandtranscriptomicsequencesfrommodelorgan-
isms(e.g.Drosophilamelanogaster,ApismelliferaandBombyxmori)todetermineintron-exon
structure;4)degenerateprimersweredesigned;5)aPCRscreeningwasrunandproductswith
theexpectedcorrectsize(albeithighlyvariableduetopresenceofintrons)weresequenced;6)
markersreachingaminimumPCRandsequencingsuccessof20%wereusedtoreconstruct
singlegenephylogenies(Bayesian)andtreeswerecomparedtopreviouslyestablishedand
well-supportedclades[5,7,10,11].
DNAwasextractedfromindividualspecimensusingDNeasyBlood&Tissuekit(Qiagen)
followingthemanufacturer’s instructions.ThePCRreactionmixturecontained2.5μl10xPCR
buffer(Qiagen),inwhichthefinalconcentrationofMgCl was2.0mM,200μMofeachdNTP
2
(SigmaAldrich),0.5μMofeachprimer,0.125unitsHotStartTaq1DNApolymerase(Qia-
gen),2μlDNA,withwateraddedtoafinalvolumeof25μl.Anegativecontrol(sterilewater)
wasincludedineachtest.ThePCRwasperformedusingaS1000TMThermalCycler(BIO-RAD
Laboratories,Inc.).Threestandardcycleprogramswereusedfortheinitialscreening:denatur-
ationstepat95°Cfor5minutes,35cyclesof30secondsat95°C,30secondsat48,52and58°C,
60secondsat72°C,andfinally5minutesextensionat72°C.Furtheroptimizationincludeda
gradientofannealingtemperaturesintherangeof44–62°C,modulatingtheextensiontime
dependingontheexpectedPCRproductlength,andMgCl concentration.We alsoconsidered
2
twodifferenttouch-downPCRprotocolsfortwoofthesegenes(seeTable 2fordetails).
PCRproductsweresequencedwiththesameprimersasthoseusedforamplification.DNA
sequencesofbothstrandswereobtainedusingtheBigDyeTerminator cyclesequencingready
reactionkit(AppliedBiosystemsInc.)usinganautomatedDNAsequencer(AppliedBiosys-
temsPrism3700)followingthemanufacturer’s instructions.
AllobtainedsequencesweresubmittedtoBLASTanalyses,acceptingacorrectgenetargetif
thecutoffvaluewasbelow1E-4.Allsequencesforeachgenewerealignedwithotherinsect
sequencesforapreliminaryNJanalysisinPAUP(cid:3)4.0[59]todetectdeviantsequences.The
sequenceswerecheckedbyeyeandusingBioedit7.2.5[60]andMAFFT[61]toaligngenefrag-
mentswithcomplexstructure,causedeitherbytothepresenceofindelsofcodingtriplets,or
lessfrequentlybylongintronsmarkedbyunusualexon-intronborderssuchasthemostcom-
monalternativesplicesiteGC—AG[62].
Intronsweretrimmedandthecodingfragmentsweretranslatedintoaminoacidsequences
usingBioedit7.2.5tocheckfortranslationalerrors(stopcodons).Allthesepreliminaryanaly-
seshadthepurposeofdetectingpseudogenesorearlysignsofpossibleparalogs(e.g.high
degreeofaminoacidsubstitutions).Inaddition,theaminoacidsequencesoftheselected
markerswereexaminedinOrthoDBv9toassessgeneorthology[63,64].Theorthologyfor
eachgenewasconfirmedbyclusteroforthologousgroups(COGs)comparisonamongarthro-
podsequencesinthedatabase.Ambiguousnucleotidepositionsinthecodingregionthatwere
difficulttoalignweretentativelyexcluded(inArr2andIap2)tocreateanalternativealignment
forcomparisons(seeresultsanddiscussion).
Phylogeneticanalyseswereperformedonunambiguouslyalignedsequencesobtainedfroma
minimumof5species.PhylogeneticinferencewasbasedonBayesianandmaximumparsimony
analyses,thelatterasimplementedinPAUP(cid:3)4.0.Nodesupportintheparsimonyanalyseswas
estimatedbybootstrapanalysesusing20randomadditionsofheuristicsearchesforeachof200
bootstrapreplicates.BayesianphylogeneticanalyseswereperformedinMrBayes3.2[65].The
mostappropriatemodelforbasefrequenciesandsubstitutionrateswasdeterminedbyjModelT-
est[66],usingtheAkaikeinformationcriterion(AIC).MrBayessearcheswererunforeachgene
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GenomicMininginBarkandAmbrosiaBeetles
Table2. Primersequencesandannealingtemperatureforthenuclearmarkersselectedinthisstudy. Furthermore,primersforadditionalgenesfor
lowerlevelphylogeneticsarereported.
Geneacronym Primerforward(5’-3’) Primerreverse(5’-3’) AnnealingT˚C
EF2 CGTTTCTAYGCBTTYGGHCGTG CCYTCYTTRGTGGCCCAYTGG TD58(10cy)44(25cy)
ATGATGGGYCGTTAYGTWGARGC TD58(10cy)44(25cy)
Hsp70 CAAGCYGACATGAAGCAYTGGCC CGGGTGATGGAGGTGTAGAARTC 58
GAYGGTATCTTYGARGTMAAGTC CGRCCYTTGTCRTTRGTGATGG 55
CCNC ATGGCTGGMAAYTTTTGGCARAG TCGAGCAGATARAAYTCRCAYTC 52
HDACRpd3 ATGAARCCSCACMGSATAMGSATGAC GTAGTCGTTRTARGGSAGYTCRTTGGC 53
GCCACSGAAGTYTCRTASGTCCA 53/50
Arr2 CGYGARGAGGAYGARGTYATGGG ACCATSGTRACYTCGCAATGYTGCAC 52
CTCAAARACKATRTTGTCGTCRTCGTC 52
Iap2 TGGAAYTAYGGRGACCAAGTRATGGC CCATCKGGCRTGYTCYGTCCAWGGATC 52
PABP1 CCRATTCGYATYATGTGGTC GAARGCRACAAAWCCRAAWCC 50
Prp1 ATGTCSGCKACTYTRGAYGCWGG GGRTASGTGTTRTCYTGCATYTC 44
CTR9 GAAGGYGATAARATGGAWCARGC TCGAAACAYTGKGCKGCATTTTC 52
RCC1 GGKTGYAATGACGARGGSGC CGGCCCAATTGTCCYTGYTC 52
SOD1 TCCACATYCAYGARTTYGGGG CCTTKKCCCAAATCATCMGG TD52(10cy)46(25cy)
TPI CGHAAATTCGTWGTYGGWGGHAACTGG CKGARCCYCCRTATTGRATTC 50
GGTGGHAACTGGAARATGAACGG 52
ADA2 GAYATGYTDGAYGTVCATGC ACAGGRCCRGCTTCRCCRCAATG 52
AARTTYAATGCCAAATAYAAYCC GGWCCRGCTTCACCRCARTGWGG 48/52
UBA5 TTGGKAGYGTAACWGCRGAAATG ATATGGCCWGARACSGCRTTTTC 52
Cda4 TACGARGARTGGGTKGGRGARATG AACCAATTMGTRTGRAASGGCATC 48
FEN1 GARGCCCCYTGYGARGCKGARGC TCACCATGCCYTCYTCRTCMGG 48
ACTB CTGAAGCCCCMTTGAACCCMAAGGC GAGATCCACATCTGYTGGAARGTGG
CXorf56 GAAGYATTGCRTGTTCSGAYAC GTCACMGAACTGAAYTTKCCC
eRF1 GTTGGCAGATGAATTTGGAACRGC CCRAABAGAGCTCCRTTACCATCC
U2AF ATYGCTGGATTWAAYGGRATGC TCTCKTCTRTGRTACTTRTCSGGWTC
MAD YAAYTTYCCWGCYATGRTWCC ACACCRTGRTTYTTWGCWCC
mp20 GACAAGGARGCCCARGARTGGATCG TCCCACAGRTCAACTGTYTGGAARAC
GGTCCGGGCCCAYTCRGRGTGCYTGTTAGG
5MP CATGACKTTTATGMGKGCKTTC CTTCYTCRGCGTTTTGWAGCC
Pi4k TGYTGYCCKTGYTGYTTYGG TGGTAYGGRTASGCYCGCC
Gel GAYGAGGGCSGGWTCSGCWGC AGGATRAAGCARTCRCCTTTGTTC
C1-THF CATYTRACYGGYGAYATYCATGC ACAGCYCCYGTKGCYCCCAAATC
alpha-Spec CAYGCHAATGCWTTCCATCARTGG GGYTGKCCYTCYTCWACCATYGG
AATS CATCAYACGTTTTTTGAGATG GCATGRTCNGCTAARACNCGRTARGCC
Hsp90 GATCATCAATATSTTCTACTC TCTCCGGTGATGWARTAGATG
dldE3 GGRGAYTGTATWCATGGRCC GCYTCRTTRATBARTTCRCC
CATCCWGAAGTKGGMTGGGTKGG
Mpgt AAACCSCTGTTYCCMGTTGCKGG GCMGTTTTYAACTGSGACCACC
NaK GGYGGTTTCGCSWTGYTGYGTGGATCGG GCGACGATGATACCGATCARGAAGATGACAGC
Fbox11 AATGCWTTRGCTGGWATYTGGG CCRCCRTGYTGACCRTGRTG
UDE AAGCCRGACACCGTWCCCGG CTGGCWTCRGGRCTGTACGCCC
GTPbp ATTARAAYGTAKCCATCGTTRCCCC GTGTTGATAATWGASGACTTGCC
CatL CACATTTACACTTTYAACCCRATG ACCARCTGTTYTTMACCARCCAGTA
TpC CTTCCCSCMGARCARATYGCCG CCTCSCCRGTCATCATCTCCATG
PGI GGCCCSCTKATGGTRACCGAAGC CCCAGCTCCACKCCCCATTGGTC
(Continued)
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Table2. (Continued)
Geneacronym Primerforward(5’-3’) Primerreverse(5’-3’) AnnealingT˚C
AcCoA GGTGTACTGCKGAYATTGGYTGGATCAC GGAAACSCAGCMGCKCCWGGYTTCAT
CATCAGRTGYCCKGASACGTTYARCAT
Ucdk GAGCACKGTWTGCAARCGYATWATGG CCYCTWGGAATRATRACATCAGC
PPO1 AAYCTSCACCAYTGGCAYTGGC CGGAASGTSCKCTCRAASGG
Prp6 AATCCSAATCATCCWCCGGCKTGG TTCTTCCAGYTTRGCSGCRGTWGTCC
Mxp TAMGSACRGCSTAYACSAACAC CGCTTGTGYTTCATSCKCCG
Npl4 CTCGYTGYGTSCAYTGCTC TCGCGCACYAGCGCCATRCAYTG
Cam1 GAYGGMGATGGCACRATYACTACC TCRTAATTGACCTGACCGTCRCC
STX1A ATGACYAARGAYAGATTRGCRGC GCCATRTCCATRAACATRTCRTG
TP120b TWGGRAATGTCAAYGTYTC AAGCTCAACCCKCKCCACATCC
CHS1 CATATMTTYTTCGAYGAYGC CAACGATCYTCKCCYTGATC
DDX49 AARGCTATACGARGAYCCWTATGG TGCCTGCYCTAGCWGTYCTYCC
GTF2H3 CTCGCATTTGATGCAGAAGGC CARATYGGRCTAAACTTGCA
IF3 ACTCGCTYTACAAAATGTTGGG CTTTSGTRTCGGCRATATGRATC
TIF6 GACACRATWCCSGTGGTSCATGC CTACCWCARTTWACYGTTCC
IDH TACAAYGTWGGAATWAARTGTGC CAMACAAARCCYCCYTCMGATTTC
Ecr GAAGTKATGATGTTCMGRATGGC GAWGCACATYTCDGARTTYTG
doi:10.1371/journal.pone.0163529.t002
separatelyandforconcatenateddatasets(8109bp–2702aa)usingthesuggestedmodelsfor
eachgenepartitionandamixedmodelforaminoacidsubstitution.Inbothcases,thesearch
consistedof2000000generationswithtwoindependentruns,eachwithfoursimultaneous
chains,andtreessampledevery1000generations.Theconvergencediagnostics(SDSF, PSRF)
andparametersampleplotswereevaluatedusingthesoftwareTracer1.6[67].
Anindirectmeasureofthephylogeneticsignalineachmarkerwasassessedthroughtopo-
logicalcongruencewithpreviouslywelldocumentedclades[5–7,10,11,68]whichwereused
toderiveaschemeofthecurrentclassificationofCurculionoidea(Fig1).Thesecladesbelong
tosixtribesofScolytinae(A=DryocoetiniincludingXyleborini,B=Ipini,C=Hylurgini
+Hylesinini,D=Scolytini)andthesubfamilyPlatypodinae(E).Rootingofthetreeswas
Fig1.SchematictreeshowingwellsupportedrelationshipsbetweentribeswithinthesubfamilyScolytinaeandother
weevilfamiliesandsubfamiliesconsideredinthisstudy.
doi:10.1371/journal.pone.0163529.g001
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dependentonthesequencesavailable,andusedinthefollowingorder:1)Brentidae,2)Platy-
podinae,3)Cossoninae,MolytinaeandLixinae,4)Scolytini[5,6].
Basicpropertiesofeachgene,includingtheoverallmeandivergenceofsequences(p-dis-
tance)andthevariationinfirst,secondandthirdpositions,werecalculatedforeachgenefrag-
mentusingMEGA6.0[69].Parsimonyinformativesiteswerecalculatedtogetherwiththe
homoplasyandretentionindices(respectivelyHIandRI–S2Table) usingPAUP(cid:3)4.0.Aphylo-
geneticinformativenessprofile(PI)wasobtainedforeachgeneusingPhyDesign[70],anon-
lineprogramdevelopedfromapreviousstudy[71].Substitutionratesforeachpositionwere
calculatedusingHyPhyimplementedinPhyDesign,selectingaK2Pmodel(basefrequen-
cies=0.25,transitions=2,transversions=1).TheinputtimetreewasobtainedusingBeast
v1.8.2[72],withtopologyconstraintsfollowingpreviouslypublishedphylogeniesofweevils
andScolytinae[5,6,11].Thetreewasreconstructedusingaconcatenateddatasetof16genes,
usingaGTR+I+Γmodelforeachgenepartition,andaYulespeciationprocess.We selectedan
uncorrelatedlognormalrelaxedmolecularclockanduseddefaultpriorsassuggestedbythe
authors(seeXMLS1fileinSupplementaryinformation).Twocalibrationpointswereused:
116MaforthenodesubtendingScolytinaeandotherweevilsubfamilies,and30Maforclade
A(Dryocoetini+Xyleborini).
Results
Sequenceswereobtainedfor57differentgenes,whereas43primersetsneveramplifiedthecor-
rectgene.Atotalof798sequenceswereobtained,butonly510ofthese(64%)wereunambigu-
ouslycharacterizedasbeetleorthologsinBLASTNsearch.Amongtheremaining288
sequences,53wereidentifiedasnon-beetlesequences(mainlyfrombacteria,fungiornema-
todesassociatedwithbeetles)withdifferentdegreeofconfidenceingeneidentity.Theremain-
ing235sequencesresultedinunreadableorpoorqualitysequenceswithoutaclearmatchin
GenBank(Evalue>1E-4,querycoverage<30%and/orlessthan30%identity).
Theevaluationofthe57markerswithreadablesequenceswasbasedonthenumberof
sequencesobtainedandtheirphylogeneticperformance.Whenonlyoneortwosequences
wereobtainedforagene(e.g.cathepsinL,troponinC,acetylcoenzimaAsynthetase,maxillope-
dia,calmodulin1),thephylogeneticutilitywasnotpossibletoassess.Otherexcludedmarkers
producedahighernumberofsequences,suchasodorantbindingprotein(8sequences)andgly-
cosidehydrolasefamily31(11),butthesewerelargelyunalignable.Anothergroupoffailed
markersproducedsequencesfromnon-targetorganisms,suchas6-phosphogluconatedehydro-
genaseoffungi,orphosphoglucoseisomeraseofbacteria.Atotalof23geneswerediscardeddue
tolowamplificationrates,highlevelsofnon-beetleamplification,orgenerallylowdegreeof
geneorthology.
Theremaining34genesshoweddifferingdegreeofPCRandsequencingsuccess(from5to
26sequencesobtained),andwerefurtherevaluatedbasedontheircapacitytorecoverknown
relationshipsatvarioustaxonomiclevels.Eighteenofthesemarkerswerefoundinsufficiently
informativeforhigherlevelphylogenetics,becausenomorethantwoofthepredefinedclades
werereconstructedcorrectly.However,mostdiscardedmarkersneverthelessrevealedsome
phylogeneticutilityatlowertaxonomiclevel;includingpopulations(seeS3Table forfurther
details).
We selected16genesthatrevealedarelativelyhighandstablePCRandsequencingsuccess
(from50to100%)asthebestcandidatesforScolytinaephylogenetics(Table 3).Alltheverified
sequencesobtainedinthisstudyweredepositedinGenBankdatabaseundertheaccession
numbersKX160539—KX160803(S1Table).ThespeciesXyleborusaffiniswasthemostsuc-
cessfulinPCRandsequencing(15outof16possiblesequencesobtained);theothersamples
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GenomicMininginBarkandAmbrosiaBeetles
Table3. PCRandsequencingsuccessfor16selectedgenes.
GENEACRONYM A B C D E F G H Total(%)
PABP1 4 3 6 3 3 4 2 1 26(100%)
TPI 4 2 6 - 2 2 2 - 18(69%)
UBA5 3 3 5 3 2 2 1 1 20(77%)
Iap2 3 3 1 2 1 4 2 - 16(62%)
SOD1 2 1 4 3 2 3 1 - 16(62%)
Prp1 3 3 5 1 3 1 2 - 18(69%)
ADA2 3 2 2 2 3 - 2 - 14(54%)
CTR9 2 2 4 2 - 1 2 - 13(50%)
CCNC 4 2 5 2 2 2 2 1 20(77%)
Cda4 2 1 4 - 3 1 1 1 13(50%)
HDACRpd3 3 1 4 - 2 2 1 - 13(50%)
Arr2 4 2 4 3 3 2 2 - 20(77%)
FEN1 3 2 4 2 1 - 2 1 15(58%)
EF2 2 2 3 2 3 - 2 - 14(54%)
Hsp70 1 1 5 2 1 1 2 1 14(54%)
RCC1 2 2 4 - 2 2 1 - 13(50%)
Thenumberofsequencesobtainedwasreportedforthefollowinggroups:A=Xyleborini+Dryocoetini,B=Ipini,C=Hylurgini+Hylesinini,D=Scolytini,
E=Platypodinae,F=otherCurculionidaesubfamilies,G=otherScolytinae,H=Brentidae.
doi:10.1371/journal.pone.0163529.t003
variedconsiderablyinthisrespectwithonly4sequencesobtainedforLarinussp.(S1Table).
Thetotalfragmentlength,thepresenceoflength-variableregions,andthenumberandposi-
tionofintrons,weremappedontheannotatedgenomesofT.castaneumandD.ponderosae
(eventuallytranscriptomicandgenomicdataofotherinsectspecies)tocreateamapofthe
genestructure(Fig2;seealsoTable 4).
OrthoDBanalysesshowedthat12outof16genesselectedinthisstudyarepresentinsingle
copyinmorethan70%ofthearthropodspeciescurrentlyinthedatabase(133).PABP1and
UBA5areinsinglecopyin96%ofthesespecies,followedbyHDACRpd3(95%),CCNC(94%),
Prp1(92%),TPI,CTR9andFEN1(90%),Cda4(89%),EF2(84%),RCC1(81%)andADA2
(74%).Onlyfivegenesarefrequentlyinmulti-copystatusinarthropodgenomes:Hsp70(single
copyonlyin2%ofthespeciesinthedatabase),Arr2(4.5%),Iap2(8.3%)andSOD1(22%).
ThebestevolutionarymodelforthemajorityofthegeneswasGTR+I+Γ,exceptforSOD1
andIap2inwhichSYM+I+ΓandGTR+Γwereselected.Bayesiananalysisoftheconcatenated
nucleotideandaminoaciddatafrom16genesshowedawellresolvedtreetopology(S1Fig)
withallexpectedcladesrecoveredwithmaximumsupport,exceptScolytini(pp=0.75).The
overalltreetopologywascorrectwiththeexceptionoffourweevilspeciesthatwerenested
insideScolytinaeasthesisterlineagetoHylurgini(weaklysupportedintheaminoacidanaly-
sis).Parsimonyanalysesoftheconcatenateddatasetrevealedsimilarresultsbothforthenucle-
otideandaminoaciddatasets,withallmajorcladesrecoveredwithmediumtohighbootstrap
support.However,thesub-familyScolytinaewasnotmonophyleticinrespecttotheother
advancedweevilspecies(S2Fig).
Singlegeneanalysesresultedinpartiallyresolvedphylogenies,mainlyrecoveringamono-
phyleticScolytinae,themajorityofthepredefinedsubgroupsofScolytinae(A-B-C-D),andthe
subfamilyPlatypodinae(Fig3).Allselectedgenesenabledthecorrectreconstructionofthe
mostrecentclade(A),with3genesobtainingthecorrectsistergroup(B).Noneoftheselected
genesshowedhighdegreeofincongruencethatreceivedhighnodesupport.Overallmean
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GenomicMininginBarkandAmbrosiaBeetles
Fig2.StructureofthePCRamplifiedgenefragments.Thegraphicsillustrateintron-exonpatternsin16markers
withcodingregionsshownasblackbarsandintronsasthinblacklines.Lengthvariablecodingregions(indels)were
coloredinlightgrey(Iap2andArr2).
doi:10.1371/journal.pone.0163529.g002
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GenomicMininginBarkandAmbrosiaBeetles
Table4. Geneinformation.
Acronym nucs aa Intron Intronrange(perintron)
PABP1 435 145 0 -
TPI 547 182 0–2 (457–51)(237–48)
UBA5 348 116 1 (94–48)
Iap2 672* 224* 1 (1131–50)
SOD1 213 71 0 -
Prp1 582 194 0–1 (258–55)
ADA2 624 208 2 (70–39)(105–53)
CTR9 627 209 0–1 (81–59)
CCNC 384 128 3 (200–69)(134–49)(71–58)
Cda4 410 136 0–3 (68–51)(63–56)(53)
HDACRpd3 858 286 3–5 (69–53)(70–54)(165–48)(564–54)(66–55)
Arr2 501* 167* 0–3 (110–51)(84–53)(158–55)
FEN1 417 139 1–3 (63–46)(55–42)(93–44)
EF2 621 207 1–2 (398–183)(702–84)
Hsp70 567 189 0–2 (61-?)(317–187)
RCC1 303 101 0–1 (250–51)
Foreachmarker,thelengthofthesequencedcodingregionisgivenasthenumberofnucleotidesand
aminoacids,togetherwiththenumberandlengthofintron(s).Thesymbol*indicatesgeneswithsequence
lengthvariabilityduetoexonicindels.
doi:10.1371/journal.pone.0163529.t004
divergenceinnucleotidesequenceswasreportedforeachcodonpositionforeachgene(S3
Fig).
SelectedgenesforScolytinaephylogeny
Polyadenylatebindingprotein1(PABP1). PABP1wasthemostsuccessfulmarker,with
sequencesobtainedfromall26species.Theamplifiedfragmentwas435bplong,containedno
introns,andtranslatedinto145aminoacids.Thephylogeneticanalysesrecoveredalmostall
pre-definedclades(Fig3a),butonlytwoofthemwerehighlysupported(B,pp=0.98;
E,pp=1).ThetribeScolytiniwasplacedoutsideapolytomyincludingtheremainingspecies
ofScolytinae,thesubfamilyPlatypodinaeandthevariousotherweevilsubfamilies.Noclear
evidenceofparalogsemergedfromtheanalyses.Preliminarystudiesindicatedincreasedphylo-
geneticperformancewithbroadertaxoncoverage.
Triose-phosphateisomerase(TPI). Acombinationoftwoprimerpairs(twoforward,one
reverse)resultedin67%PCRamplificationandsequencingsuccess.Thealignedfragments
consistedof547bpafterremovalofintrons,whichtranslatedinto182aminoacids.Two
intronswerelocatedinthisgenefragment(Fig2,Table 4).Thephylogenybasedonthismarker
confirmedthemonophylyofPlatypodinae(pp=1),whileScolytinaeformedalargepolytomy
includingtwoadvancedweevilspecies.FurthermorewasCossoninaemonophyletic(pp=1),
inadditiontoonescolytinesubgroup(A,pp=1),andsubgroupCalmostso(Fig3b).
Ubiquitin-likemodifieractivatingenzyme5(UBA5). TheUBA5genefragmentis348
bplongandtranslatedinto116aminoacids.Itwasamplifiedfrom20differentspecies(77%)
inallmaincladesandcontainedoneshortintroninallspecies.Thephylogenyrecoveredthe
monophylyofcladesAandEwithhighnodesupport(pp=0.99and1,respectively)while
cladeD(pp=1)hadScolytusintricatusexcluded.CladeBandCwereweaklysupported
(pp<0.95)andKissophagushederaewasnotincludedinHylurgini(Fig3c).
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