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25DNAbarcodingandspeciesdelimitationofChaitophorinae(Hemiptera,Aphididae)Xi-ChaoZhu1,2,JingChen1,RuiChen1,Li-YunJiang1,Ge-XiaQiao11KeyLaboratoryofZoologicalSystematicsandEvolution,InstituteofZoology,ChineseAcademyofSciences,No.
1-5BeichenWestRoad,ChaoyangDistrict,Beijing100101,P.
R.
China2CollegeofLifeScience,Univer-sityofChineseAcademyofSciences,ShijingshanDistrict,Beijing100049,P.
R.
ChinaCorrespondingauthors:Ge-XiaQiao(qiaogx@ioz.
ac.
cn);Li-YunJiang(jiangliyun@ioz.
ac.
cn)Academiceditor:R.
Blackman|Received8December2016|Accepted31January2017|Published14February2017http://zoobank.
org/D453CD7C-A688-4994-A31F-4469E66C31DCCitation:ZhuX-C,ChenJ,ChenR,JiangL-Y,QiaoG-X(2017)DNAbarcodingandspeciesdelimitationofChaitophorinae(Hemiptera,Aphididae).
ZooKeys656:25–50.
https://doi.
org/10.
3897/zookeys.
656.
11440AbstractChaitophorinaeaphidsarewidespreadacrossEurasiaandNorthAmerica,andincludesomeimportantagriculturalandhorticulturalpests.
So,accuraterapidspeciesidentificationisveryimportant.
Here,weusedthreemitochondrialgenesandoneendosymbiontgenetocalculateandanalyzethegeneticdistanceswithindifferentdatasets.
Forspeciesdelimitation,twodistance-basedmethodswereemployed,thresholdwithNJ(neighbor-joining)andABGD(AutomaticBarcodeGapDiscovery),andtwotree-basedap-proaches,GMYC(GeneralMixedYuleCoalescent)andPTP(PoissonTreeProcess).
Thegeneticinterspe-cificdivergencewasclearlylargerthantheintraspecificdivergenceforfourmolecularmarkers.
COIandCOIIgeneswerefoundtobemoresuitableforChaitophorinaeDNAbarcoding.
Forspeciesdelimitation,atleastonedistance-basedmethodcombinedwithonetree-basedmethodwouldbepreferable.
BasedonthedataforChaitophorussalinigerandLaingiapsammae,DNAbarcodingmayalsorevealgeographicalvariation.
KeywordsChaitophorinae,distance-basedanalysis,gnd,mitochondrialgenes,tree-basedanalysisZooKeys656:25–50(2017)doi:10.
3897/zookeys.
656.
11440http://zookeys.
pensoft.
netCopyrightXi-ChaoZhuetal.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
RESEARCHARTICLELaunchedtoacceleratebiodiversityresearchApeer-reviewedopen-accessjournal26IntroductionAphidsfrommorethan5,000species(Favret2016)feedonplantphloemdirectlyandspreadvariousplantdiseases(BlackmanandEastop2000),manyservingasimportanteconomicpests.
Theidentificationofaphidspeciesbasedonmorphologicalcharacteristicsfacestremendouschallengesduetotheircomplicatedlifecycle,polymorphism,phenotypicplasticity,andnumerousmorphs(ZhangandZhong1983,Foottitetal.
2009a).
ChaitophorinaelieswithinAphididae,andcomprisestwotribes,ChaitophoriniandSiphini,including196speciesandsubspeciesin12genera(RemaudiereandRemaudiere1997,Favret2016).
ThesubfamilyisdistributedmainlyinthePalaearctic(about80%ofspecies),andNearctic(Richards1972,Qiao1996,Liuetal.
2009,Wieczorek2010).
Mostspeciesinthissubfamilyaremonoeciousholocyclic,butsomespecies,suchasSipha(Sipha)flavaandSipha(Rungsia)maydis,maybeanholocyclicinregionswithmilderwinters(BlackmanandEastop2000,Wieczorek2010,WieczorekandBugaj-Nawrocka2014).
TheChaitophoriniismainlyassociatedwithplantsofthefamiliesSalicaceaeandAceraceae(BlackmanandEastop1994),whereastheSiphiniinfestplantsinthePoaceae,Cyperaceae,JuncaceaeandTyphaceae(BlackmanandEastop2006).
Additionally,individualspeciesoftenhavehighhostspecificity(BlackmanandEastop1994).
SpeciesidentificationofChaitophorinaeaphidscanbedifficultwhenbasedontheirmorphologicalcharacteristics.
TheChaitophorinihaveclearmorphologicaldifferencesbetweengenera,butChaitophorus(109knownspecies)andPeriphyllus(49knownspecies)havehighspeciesdiversity(Essig1952,HilleRisLambers1960,Pintera1987,Qiao1996);andthemorphologicaldifferencesbetweenspecieswithinthesegeneraarerelativelyslight,oftendependingonthechaetotaxyofthebodydorsumandappendages(Pintera1987).
IntheSiphini,bothamonggeneraandbetweenspecies,overlapandconvergenceofmorphologicalcharacteristicsarecommon,andgenusandspeciesidentificationarenoteasy.
Siphainparticular(11knownspecies)hasrelativelygreatdiversity,andspeciesidentificationcanbeaproblem.
DNAbarcodingbasedonashortfragmentofmitochondrialDNAcanprovideaneffectivetoolforspeciesdiagnosis.
Inanimals,the5'endofmitochondrialcytochromecoxidaseI(COI)witha658-bpfragmentwasselectedasastandardDNAbarcode(Hebertetal.
2003).
Thishasbeenwidelyusedforidentifyingunknownspecimensandtherapididentificationofspecies(Hebertetal.
2003,WangandQiao2009,Wangetal.
2013b,Wenetal.
2013).
Itspracticabilityandeffectivenesshavebeenrecognizedandacceptedinsomeinsectgroups,suchasDiptera(Schefferetal.
2006),Lepidoptera(Hajibabaeietal.
2006),Ephemeroptera(Balletal.
2005),Hemiptera(Leeetal.
2011),Coleoptera(LoblandLeschen2005),andHymenoptera(Smithetal.
2008).
Additionally,theapplicationrangewasexpandedtopestcontrolandquarantine(ArmstrongandBall2005,RatnasinghamandHebert2007,Naaumetal.
2012,Pelletieretal.
2012).
Foraphids,theDNAbarcodingapproachhasplayedanefficientroleintherapididentificationofspeciesonspecificplants(Naaumetal.
2012,Chenetal.
2013b,Wangetal.
2013a,Wangetal.
2013b,Wenetal.
2013,27Wangetal.
2015),theeffectivedistinctionofmorphologicallyindistinguishablespe-ciesandsubspecies(WangandQiao2009,Rebijithetal.
2013,CocuzzaandCavalieri2014,Bejietal.
2015,Kinyanjuietal.
2016),therecognitionofcrypticspecies(Re-bijithetal.
2013,Leeetal.
2015),andinspeciesclassification(CocuzzaandCavalieri2014,Mrozetal.
2015).
Likewise,DNAbarcodingmaybeusedinspeciesdiversityassessmentwithindifferentregions(Podmoreetal.
2015,Chenetal.
2016);andisapowerfultoolfortheidentificationofmulti-lifestages,differentmorphs,andbio-logicaldebris(ShufranandPuterka2011).
Crucially,ithasimprovedthemonitoringandcontrolofpestaphids.
Theidentificationofaphidspeciesisoftendifficultduetotheshortageofeasilydistinguishablemorphologicalcharacteristics,orfeatureconver-gence(WangandQiao2009).
Somechaitophorinespeciesareimportantagricultural,forestry,andhorticulturepests,forwhichaccurateidentificationisnecessary.
Attheauthors'lastcount(2016.
04.
06),researchershaveprovidedsomechaitophorineDNAbarcodingsequencesfor36speciestotheNCBIandfor49speciestotheBarcodeofLifeDataSystem(BOLD)(Foottitetal.
2008,Foottitetal.
2009b,Leeetal.
2011,Gwiazdowskietal.
2015).
However,theDNAbarcodingofthisgroupisinsufficient.
Inthiswork,wesequenced1,609sequencesfrom670samplesin8generafrombothtribesofChaitophorinae,basedonthreegenesfromtheaphidmitochondrialgenome,andonefromtheendosymbiontBuchnera.
Weemployedfourmethods(thresholdwithNJ,ABGD,GMYC,andPTP)toanalyzesequencediversitiesandgeneticdiver-gencesbetweendifferentspeciesandprobetheefficiencyofidentifyingspecies.
BasedonDNAbarcodingdata,wealsodiscusstheinfluenceofgeographicaldistributiononpopulationdifferentiation.
MaterialsandmethodsTaxasamplingandgeneselectionAllsampleswerecollectedintoandcryopreservedin95%or100%ethanol.
DNAfromoneindividualpersamplewasisolatedformolecularstudiesandthreetofiveindividualaphidspercollectionweremountedonmicroscopeslidesformorphologicalexamination.
Preservedaphidcolonieswereexaminedpriortopreparationtoensurethattheydidnotconsistofmultiplespecies.
VoucherspecimensforeachsamplewereidentifiedbyG.
X.
Qiaobasedonmorphologicaldiagnosticfeaturesusingstandardliterature-basedkeys(esp.
BlackmanandEastop1994,Pintera1987,Wieczorek2010)andbyacomparisonwithpreviouslyidentifiedspecimensintheNationalZoologicalMuseumofChina,Beijing.
Toavoidmutualinfluenceandtoensuretheindepend-enceofthedifferentresearchmethods,themorphologicalidentificationandmolecularresearchwereperformedindependently.
Allsamplesandvoucherspecimenswerede-positedintheNationalZoologicalMuseumofChina,InstituteofZoology,ChineseAcademyofSciences,Beijing,China.
Detailsofthesequencedtaxaandvoucherinfor-mationarelistedinSuppl.
material1.
28Threeaphidgenesweretargeted:mitochondrialcytochromeoxidasecsubunitI(COI),cytochromeoxidasecsubunitII(COII),andcytochromeb(Cytb),andoneaphidendosymbiontBuchneragenegluconate-6-phosphatedehydrogenase(gnd)(KimandLee2008,WangandQiao2009,Zhangetal.
2011,Chenetal.
2012).
DNAextraction,amplificationandsequencingTotalgenomicDNAwasextractedfromsingleaphid.
Individualaphidswereselectedfromtheethanol-preservedcandidateswithadestructiveDNAextractionprocedure.
Plumpadultsaretheidealexperimentalmaterial,buttheymustbeexaminedunderamicroscope(LeicaDM2500)toeliminateparasitizedindividuals.
TotalDNAwasextractedbyfollowingtheQuick-StartprotocolofDNeasyBlood&TissueKit(QIA-GEN,Dusseldorf,Germany)withasingleindividual.
TheDNAsolutionwasthenstoredat-20°Cforsubsequentmolecularexperiments.
Thepolymerasechainreaction(PCR)mixturefortheamplificationofCOI,COII,Cytb,andgndgenescomprised22μlofdoubledistilledwater(ddH2O),3μlof10*EasyTaqBuffer(+Mg2+)(TransGenBiotech,Beijing,China),2.
4μlof2.
5mM/800μldNTPs(TransGenBiotech),0.
6μlof10pmol/μlforwardandreverseprimers,0.
4μlof5U/μlEasyTaqDNAPolymerase(TransGenBiotech),and1μlofDNAsolutionforatotalvolumeof30μl.
ThePCRconditionsdifferedaccordingtothegeneandthespecificprimers,es-peciallytheannealingtemperature,whichwasthemostcriticalfactorinfluencingproductquality.
ThedetailedprimerinformationisshowninSuppl.
material2.
ThethermalsetupofprimerLepF/LepR(Foottitetal.
2008)orLCO1490/HCO2198(Folmeretal.
1994)forCOIgenefragmentwas:a5-minuteinitialdenaturationat95°Cfollowedby35cyclesof30-seconddenaturationat95°C,30secondsofan-nealingat50°C,a1-minuteextensionat72°C,anda10-minutefinalextensionat72°C.
Theprotocolforprimermt2993+(Stern1994)/A3772(Normark1996)fortRNA/COIImolecularmarkerwasasfollows:a5-minuteinitialdenaturationat95°Cfollowedby35cyclesof1-minutedenaturationat95°C,1minat42°C,1minat72°C,anda7-minutefinalextensionat72°C.
TheparametersofprimerCP1/CP2(Harryetal.
1998)forCytbamplificationwassimplifiedas:94°Cwith5min,and40cyclesof94°Cwith50s,48°Cwith1min,72°Cwith1.
5min,and72°Cwith10min.
ThesetupofprimerBamHI/ApaI(Clarketal.
1999)forBuchneragndgenewaspredigestedas:94°Cwith5min,and30cyclesof94°Cwith1min,55°Cwith30s,72°Cwith1min,and72°Cof10minfinalextension.
Theamplificationproductsweredetectedby1.
5%agarosegelelectrophoresis(AGE),andthenpurifiedusingEasyPureQuickGelExtractionKit(TransGenBiotech).
TheeligibleproductswerethensenttoTsingKeBiologicalTechnology,Beijing,ChinaorBGI,Shenzhen,Chinaforsequencing,whichwasrequiredtobebidirectional.
29SequenceeditionandalignmentThereturnedforwardandreversechromatogramswereloadedandthenassembledandeditedbySeqManinDNAStarsoftware(DNASTAR,Madison,Wisconsin,USA).
ThenucleotidesequenceswerefirstexaminedinNCBIbyBasicLocalAlignmentSearchTool(BLAST)(Altschuletal.
1990)totesttheiraffiliations.
Concurrently,fortheencodinggenefragments,wetranslatedtheassembledcontigsintoaminoacidsbyMEGA6(Tamuraetal.
2013)toexaminewhetherthesequenceswerecorrectandac-curate.
MultiplealignmentswereaccomplishedbyMAFFT(KatohandStandley2013),andthesequenceswerethenadjustedandtrimmedmanuallyinMEGA6.
Itisnotewor-thythatthesequencesamplifiedwithprimermt2993+/A3772coveredtheCOIIgenefragmentaswellasatRNA,whichwasthenremovedforsubsequentanalysis.
SpeciesdelimitationmethodsInadditiontosequencesfrom425samples,wedownloaded245COIand1COIIsequencefromNCBI.
Here,wedefinedthedatasetsasCOI-670(includingthewholeresearchgroupandNCBIsequences),COII-376(including375internalsequencesand1NCBIsequence),Cytb-413(newlygottenforthisstudy),gnd-396(newlyob-tainedsequences),andCOI-338,COII-338,Cytb-338,gnd-338,whichcontainedonlythespecimensthatacquiredall4genesequences.
Aneighbor-joining(NJ)(SaitouandNei1987)treewasconstructedbyMEGA6basedonthealignedsequences.
OnethousandbootstrapreplicationswerecalculatedtoassessthecredibilityoftheNJanalysis.
TheKimura2-parameter(K2P)modelofbasesubstitution(Kimura1980)wasselectedinpairwisedistancescalculation,andforthemoreaccuratecomparisonbetweensequences,thepairwisedeletionpatternwasselectedforgaps/missingdatatreatment.
Afterbootstrapconsensustreeswithboot-strapvaluesateachnodewereobtained,wecomputedthecondensedtreewitha50%cut-offvaluefortheconsensustree.
WhenanalyzingtheCOI-670tree,wechoseathresholdof2%(Foottitetal.
2009b)foraclusterstandard,whichhasbeenwellusedinaphids.
WithregardtotheCOII-376,COII-338,Cytb-413,Cytb-338,gnd-396andgnd-338NJtrees,wecalculatedonlytheclustertopologies.
TheAutomaticBarcodeGapDiscovery(ABGD)(Puillandreetal.
2012a)ap-proachisamodel-basedmethodfordelimitingspecies.
Basedontheexistenceofabarcodinggap(namelytheintraspecificdivergencesaresmallerthaninterspecificdivergences)andapriorintraspecificdivergence(p),theABGDprocedurefirstsortsthedatasetintoahypotheticalspecies,andthencomputesrecursivelywiththeprevi-ousgroupstoobtainaresultoptimizeduntiltherearenobetterpartitions.
WerantheABGDwithagraphicwebversion(http://wwwabi.
snv.
jussieu.
fr/public/abgd/abgd-web.
html).
First,wecalculatedthedistancevaluesamongsamplesbyusingMEGA6withp-distance,Jukes-Cantor(JC69)model,andK2Pmodelseparately,andthe30resultdataweresavedasCSVformatfile.
Wethenchose0.
055,whichhadbeensug-gestedforAphididae(Foottitetal.
2009b),asthepriorintraspecificdivergenceforCOI-670andCOI-338datasets,andweusedapvalueof0.
1,whichwasthedefaultandshowntobesufficientforanalysis,fortheotherdatasets.
Theotherparametersweremaintainedbydefaultforallanalyses.
TheGeneralMixedYuleCoalescent(GMYC)(FujisawaandBarraclough2013)isatree-basedapproachforthedelimitationofspecies.
WerantheGMYCmeth-odinRproject(availablefrom:https://www.
r-project.
org/)byusingthe"splits"package(availablefrom:http://r-forge.
r-project.
org/projects/splits).
Theinputtreewasrequiredtobestrictlyultrametricandbifurcating,whichmeanttherewasnozero-lengthbranch.
Here,weusedamaximumlikelihood(ML)treeastheinput.
Therefore,thehaplotypeswerecalculatedandgeneratedbyDnaSP(LibradoandRozas2009),andanMLtreewasconstructedbyRAxML(Stamatakis2006)withhaplotypedata.
Duetotheultrametricandbifurcatingrequirement,theMLtreewasconstructedwithr8s(Sanderson2003).
Theoutcometreemodifiedbyr8swasreadintothe"splits"Rpackage,andthedelimitingresultwasobtainedwithrelevantcommands.
ThePoissonTreeProcess(PTP)(Zhangetal.
2013)modelisanothertree-basedmethodforinferringputativespecies.
ThePTPapproachisacloserelativeoftheGMYCmethod,butitonlyneedsasimplephylogenetictreeasitsinputwithoutrequiringittobeultrametricandbifurcating.
AsanupdatedversionoftheoriginalPTP,thebPTPmethodwasemployedsimultaneouslytoseparatehypotheticalspe-cies,whichaddedaBayesiansupportvaluetothetree.
ThePTPandbPTPanalyseswererunonawebserver(http://species.
h-its.
org/ptp/)andthevalue500,000wasselectedforMCMCgenerations,withtheotherparameterssetbydefault.
Thein-puttreewasanMLtreeconstructedbyRAxMLwithGTRCATmodel.
However,weencounteredthesameproblemasSchwarzfeldandSperling(2015),namelythatthebPTPanalysisfailedtoshowconvergenceunder500,000generations(theupperlimitofthewebserver).
Therefore,onlythePTPresultisdisplayedanddiscussedbelow.
ResultsMorphologicalidentificationThe425samplescollectedbythegroupmembersinrecentyearswerecarefullyau-thenticatedwithmountedindividualsunderthemicroscope,andall425sampleswereidentifiedtospecies.
Thefewvoucherswithuncertainspeciesidentificationweresort-edintofeaturedclustersandweregiventheepithet"sp.
",whichmadethemconvenientforfurtheranalysis.
Atotalof75morphologicalspeciesweredeterminedfrom670wholesamples,and51wereidentifiedfromthe425mountedsamples.
31SequencealignmentTheCOIsequencesweretrimmedtoalengthof658bp,whichincluded365con-servedsites,293variablesitesand258parsimony-informativesites.
Thesequenceshadanaveragenucleotidecompositionof38.
0%T,17.
1%C,34.
4%A,and10.
5%G.
TheCOIIsequencesweretrimmedtoafinallengthof672bp,amongwhich399siteswereconserved,273siteswerevariable,and251siteswereparsimony-informative.
TheaverageT,C,A,Gcompositionsofthesesequenceswere38.
7%,14.
0%,39.
5%,and7.
8%,respectively.
TheCytbgenewas760bp,inwhichtherewere420conservedsites,340variablesitesand303parsimony-informativesites.
TheCytbsequencescon-sistedof41.
4%T,15.
3%C,34.
3%A,and9.
0%G.
Weobtainedatotallengthof807bpforthegndgenewithanaveragenucleotidecompositionof37.
8%T,9.
8%C,39.
5%A,and12.
8%G,amongwhichtherewere368conservedsites,439variablesitesand417parsimony-informativesites.
Acrossall4genes,astrongTandAnucleo-tidecompositionbiasexisted.
Fromatotalof425samples,425COIgenefragmentsequences,375COIIgenefragmentsequences,413Cytbgenefragmentsequences,and396gndgenefragmentsequenceswereacquired.
ThesuccessiveamplificationefficiencyofthosemarkersinorderwasCOI(100%)>Cytb(97%)>gnd(93%)>COII(88%).
GeneticdivergenceanalysisGeneticdivergenceswereassessedby5disparatemetricsamongandwithinspecies.
Fortheinterspecificdivergencesofcongenericspecies,wechosetheaverageinterspecificdistance,whichwascalculatedwithingenerathatcontainedmorethanonespecies,andthesmallestinterspecificdistance,whichmeanttheminimalvalueofinterspecificdistancewithingenerawithatleasttwospecies.
Whenevaluatingtheintraspecificdivergences,threevariables(averageintraspecificdistance,meantheta,andaveragecoalescentdepth)wereapplied.
Theaverageintraspecificdistancewastheaveragevalueofthegeneticdistancesbetweensampleswithinspeciesthathadatleasttwoindividu-als.
Themeanthetasignifiedamodifiedtheta,whichexpressedtheaveragepairwisedistancescoredforspecieswithmorethanoneobtainedrepresentative,bydislodgingimproperindividualsconcernedwiththeasymmetricalacquisitionofsamples.
Theaveragecoalescentdepth,namelytheaveragevalueofmaximumintraspecificdistance,wascalculatedforspeciesinwhichtherewerenofewerthantwosamples.
Allfiveinterspecificandintraspecificmetricsweredeterminedwithingeneraandspecies(Table1).
Theresultsofdifferentgenesanddatasetsshoweddistinctlyhighinterspecificdivergencesandlowintraspecificdistances.
ForDNAbarcodingthesmallestinterspecificdistanceandaveragecoalescentdepthwerethemostusefulandintuitiveparameters.
WithinChaitophorinae,thegeneticdivergencerangesofsmall-estinterspecificdistanceandaveragecoalescentdepthofCOI,COII,Cytb,andgnd32Table1.
Theinter-andintra-specificgeneticdistancesofcongenericspeciesofChaitophorinae.
InterspecificDistanceIntraspecificDistanceGenus/Dataset(no.
species/specimens)averageinterspecificdistancesmallestinterspecificdistanceaverageintraspecificdistancemeanthetaaveragecoalescentdepthChaitophorusCOI-670(38/534)0.
1158±0.
01910.
1015±0.
01780.
0070±0.
00600.
0083±0.
00570.
0126±0.
0126COII-376(25/283)0.
0956±0.
02460.
0853±0.
02070.
0017±0.
00190.
0025±0.
00190.
0060±0.
0054Cytb-413(25/323)0.
1233±0.
02810.
0971±0.
02600.
0049±0.
00660.
0058±0.
00680.
0219±0.
0357gnd-396(25/306)0.
0996±0.
03160.
0807±0.
02480.
0020±0.
00300.
0034±0.
00320.
0042±0.
0051COI-338(25/253)0.
1117±0.
02860.
0995±0.
02150.
0058±0.
00440.
0077±0.
00330.
0088±0.
0071COII-338(25/253)0.
0950±0.
02470.
0855±0.
02080.
0018±0.
00210.
0027±0.
00210.
0062±0.
0055Cytb-338(25/253)0.
1169±0.
03100.
0983±0.
02700.
0043±0.
00640.
0052±0.
00670.
0164±0.
0337gnd-338(25/253)0.
0843±0.
02640.
0811±0.
02550.
0014±0.
00160.
0025±0.
00140.
0032±0.
0033LambersaphisCOI-670(1/3)--0.
0040±0.
00280.
0060±0.
00000.
0060±0.
0000COII-376(1/3)--0.
0047±0.
00330.
0070±0.
00000.
0070±0.
0000Cytb-413(1/3)--0.
0053±0.
00120.
0053±0.
00120.
0070±0.
0000gnd-396(1/3)--0.
0007±0.
00050.
0010±0.
00000.
0010±0.
0000COI-338(1/3)--0.
0040±0.
00280.
0060±0.
00000.
0060±0.
0000COII-338(1/3)--0.
0047±0.
00330.
0070±0.
00000.
0070±0.
0000Cytb-338(1/3)--0.
0053±0.
00120.
0053±0.
00120.
0070±0.
0000gnd-338(1/3)--0.
0007±0.
00050.
0010±0.
00000.
0010±0.
0000PeriphyllusCOI-670(19/83)0.
1113±0.
02310.
1075±0.
02200.
0040±0.
01460.
0080±0.
01980.
0218±0.
0439COII-376(13/53)0.
0936±0.
02990.
0938±0.
02820.
0007±0.
00140.
0027±0.
00150.
0024±0.
0024Cytb-413(14/54)0.
0975±0.
02000.
0944±0.
01940.
0020±0.
00290.
0041±0.
00300.
0052±0.
0032gnd-396(14/54)0.
1256±0.
06690.
1292±0.
06020.
0004±0.
00100.
0016±0.
00140.
0007±0.
0012COI-338(13/53)0.
0971±0.
02580.
0985±0.
02480.
0019±0.
00320.
0056±0.
00320.
0044±0.
0035COII-338(13/53)0.
0936±0.
02990.
0938±0.
02810.
0007±0.
00140.
0027±0.
00150.
0024±0.
0024Cytb-338(13/53)0.
0974±0.
02030.
0935±0.
02060.
0020±0.
00290.
0041±0.
00300.
0052±0.
0032gnd-338(13/53)0.
1250±0.
06790.
1283±0.
06320.
0004±0.
00100.
0016±0.
00140.
0007±0.
0012TrichaitophorusCOI-670(3/3)0.
1233±0.
02000.
1233±0.
0200---COII-376(3/3)0.
1103±0.
01900.
1103±0.
0190---Cytb-413(2/2)0.
1040±0.
00000.
1040±0.
0000---gnd-396(3/3)0.
1427±0.
01620.
1427±0.
0162---COI-338(2/2)0.
0990±0.
00000.
0990±0.
0000---COII-338(2/2)0.
1190±0.
00000.
1190±0.
0000---Cytb-338(2/2)0.
1040±0.
00000.
1040±0.
0000---gnd-338(2/2)0.
1600±0.
00000.
1600±0.
0000---YamatochaitophorusCOI-670(3/3)0.
0043±0.
00090.
0043±0.
0009---COII-376(3/3)0.
0037±0.
00210.
0037±0.
0021---gnd-396(3/3)0.
0007±0.
00050.
0007±0.
0005---ChaetosiphellaCOI-670(3/24)0.
0515±0.
04180.
0693±0.
04900.
0149±0.
01280.
0197±0.
01110.
0185±0.
0165COII-376(3/24)0.
0372±0.
03680.
0563±0.
03990.
0083±0.
00510.
0091±0.
00470.
0090±0.
009033were(0.
0693–0.
1233,0.
0060–0.
0218),(0.
0563–0.
1110,0.
0024–0.
0070),(0.
0703–0.
1060,0.
0052–0.
0230),and(0.
0807–0.
1427,0.
0010–0.
0090),respectively.
Thefiguresabovewereobtainedacrossthewholedataset.
Toobtainamorereliableandcomparableanalysis,wecalculatedtheresultsofthe338-sampledatasets.
Thesmall-estinterspecificdistanceandaveragecoalescentdepthrangesofCOI-338,COII-338,Cytb-338,andgnd-338were(0.
0693–0.
0995,0.
0044–0.
0165),(0.
0563–0.
1190,0.
0024–0.
0090),(0.
0703–0.
1040,0.
0052–0.
0230),and(0.
0811–0.
1600,0.
0010–0.
0090),respectively.
Thecomputationsaboveshowedaproperlyhighsmallestinter-specificdistanceandacomparativelylowaveragecoalescentdepth.
Toobservetheoccurrencefrequencyofdifferentgeneticdivergences,wedrewthefrequencylinechartsofinter-andintra-specificgeneticdistancesbasedon338datasets(Figure1).
Eachgenewassignifiedinonechart:thetophalfwascalculatedwithallInterspecificDistanceIntraspecificDistanceGenus/Dataset(no.
species/specimens)averageinterspecificdistancesmallestinterspecificdistanceaverageintraspecificdistancemeanthetaaveragecoalescentdepthCytb-413(3/24)0.
0481±0.
04550.
0703±0.
04980.
0140±0.
01230.
0154±0.
01200.
0230±0.
0220gnd-396(3/23)0.
0521±0.
06080.
0887±0.
06270.
0084±0.
00680.
0107±0.
00580.
0090±0.
0090COI-338(3/23)0.
0512±0.
04220.
0693±0.
04900.
0147±0.
01280.
0202±0.
01070.
0165±0.
0145COII-338(3/23)0.
0371±0.
03690.
0563±0.
03990.
0083±0.
00520.
0091±0.
00480.
0090±0.
0090Cytb-338(3/23)0.
0480±0.
04570.
0703±0.
04980.
0142±0.
01240.
0158±0.
01210.
0230±0.
0220gnd-338(3/23)0.
0521±0.
06080.
0887±0.
06270.
0084±0.
00680.
0107±0.
00580.
0090±0.
0090LaingiaCOI-670(1/2)--0.
0640±0.
00000.
0640±0.
00000.
0640±0.
0000COII-376(1/2)--0.
0680±0.
00000.
0680±0.
00000.
0680±0.
0000Cytb-413(1/2)--0.
0620±0.
00000.
0620±0.
00000.
0620±0.
0000SiphaCOI-670(5/17)0.
0940±0.
02500.
0882±0.
03200.
0082±0.
01270.
0147±0.
01390.
0118±0.
0159COII-376(2/5)0.
1115±0.
00090.
1110±0.
00000.
0027±0.
00120.
0027±0.
00120.
0040±0.
0000Cytb-413(2/5)0.
1073±0.
00130.
1060±0.
00000.
0048±0.
00310.
0058±0.
00240.
0090±0.
0000gnd-396(1/4)--0.
0005±0.
00050.
0010±0.
00000.
0010±0.
0000COI-338(1/4)--0.
0033±0.
00210.
0040±0.
00170.
0060±0.
0000COII-338(1/4)--0.
0027±0.
00120.
0027±0.
00120.
0040±0.
0000Cytb-338(1/4)--0.
0048±0.
00310.
0058±0.
00240.
0090±0.
0000gnd-338(1/4)--0.
0005±0.
00050.
0010±0.
00000.
0010±0.
0000Notes:Interspecificdivergenceswerecalculatedusingtheaverageinterspecificdistanceandsmallestinterspecificdistance.
Intraspecificdivergenceswereevaluatedbytheaverageintraspecificdistance,meantheta,andaveragecoalescentdepth.
Theaverageinterspecificdistancewascalculatedwithingenerathatcontainedmorethanonespecies.
Thesmallestinterspecificdistancewasdefinedastheminimalvalueofinterspecificdistancewithingenerawithatleasttwospecies,theaverageintraspecificdistancewastheaveragevalueofthegeneticdistancesbetweensampleswithinthosespeciesthathadatleasttwoindivid-uals,themeanthetawasexpressedastheaveragepairwisedistancescoredfromspecieswithmorethanoneobtainedrepresentativesbydislodgingimproperindividualsconcernedwiththeasymmetricprocurementofsamples,andtheaveragecoalescentdepthwastheaveragevalueofmaximumintraspecificdistances.
34Figure1.
Frequencylinechartsofinter-andintra-specificgeneticdistancesbasedon338dataset.
Thex-axisrepresentsthegeneticdistance,andthey-axisrepresentstheoccurrencetimesinthewholegeneticdistancematrix.
Eachpeakwasadatapointwithcorrespondinggeneticdistanceandoccurrencetimes.
Thedatapointsonthegreenandredlinewerecalculatedwiththeinterspecificdistances,andthepointsonpurpleandbluelinewerecalculatedwiththeintraspecificdistances.
Theoverlapregion,whichwasthecrossingareaofinter-andintra-specificdivergence,isindicatedbythereddottedrectangle.
Eachgenewassignifiedinonechart:thetophalfwascalculatedwithallthe338samples;andthebottomhalfwasscoredbyeliminatingthequeriedsamplesofChaetosiphellalongirostris.
35Figure1.
Continue.
338samples;andthebottomhalfwasscoredbyeliminatingtwosamples(Nos.
25138and25161)ofChaetosiphellalongirostris.
Theoverlapregionwasindicatedbythereddottedrectangle.
NoobviousbarcodinggapwasfoundinthesesamplesacrossCOI,COII,Cytb,andgndgenes.
TheoverlapregionsofCOI,COII,Cytb,andgndinthetophalfwere0.
000–0.
031,0.
000–0.
023,0.
000–0.
045,0.
000–0.
018;andthose36inthebottomhalfwere0.
022–0.
031,0.
011–0.
023,0.
015–0.
045,0.
006–0.
018,re-spectively.
Itwasclearthattheoverlapregionwasmuchnarrowerbyeliminatingthequestionedvouchers,andthetotalfrequencywithinthatregionwasalsoreducedsig-nificantly(Figure1).
ThedataintheoverlapregionrepresentedsamplesthattheDNAbarcodingwouldfailtoidentify.
Therefore,alowertotalfrequencyinthatregionwasbetter.
TheorderofthetotalfrequenciesintheoverlapregionwasCOICytb(97%)>gnd(93%)>COII(88%).
Withinthe338-sampledataset,thedifferenceinvaluesbetweenthesmallestinterspecificdistanceandaveragecoalescentdepthwereunequalindifferentgroups.
ForChaitophorus,Periphyllus,andChaetosiphella(Table1),thedifferencevalueswereCOI>Cytb>COII>gnd,gnd>COI>COII>Cytb,andgnd>COI>COII=Cytb,respectively.
Fromtheoverlapregionandtotalfrequency(Figure1),COIandCOIIweresimilarwithanarroweroverlapregionandlessfrequencythanCytb.
Althoughtheoverlapspanofgndwassufficient,itstotalfrequencyinthatregionwasslightlylarger.
Therefore,theCOIandCOIIgenesmaybebettermarkersforDNAbarcoding.
Themostimportantfactorinchoosingthedelimitationmethodwastheidenti-ficationaccuracywithindifferentgenes.
Therefore,abetterapproachmeanshigheridentificationaccuracyandagreaterrangeofapplicationwithvariousgenes.
Theac-curacyofGMYC,PTP,andABGDwithinCOI-338,COII-338,Cytb-338,andgnd-338wereABGD(91.
30%)>GMYC(89.
36%)>PTP(85.
71%),GMYC(93.
33%)>PTP=ABGD(86.
67%),ABGD(83.
33%)>PTP(80.
43%)>GMYC(80.
00%),andABGD(93.
18%)>GMYC(92.
86%)>PTP(90.
24%),respectively(Table2).
InChaitophorinae,theABGDwasamuchbetteranalyticalmethod,andGMYCwasalsobetterthanPTPintree-basedapproaches.
ConsideringthattheanalysisofABGDrequiredpriorintraspecificdistance,atree-basedmethodneededtobeemployedconcurrently.
Atree-basedapproachshouldbecrosscheckedagainstanon-tree-basedapproachwithinspeciesdelimitationstudies(Fontanetoetal.
2015).
Asdifferentmethodsmayyieldinconformityconclusions(Carstensetal.
2013),theac-curateidentificationofspeciesrequiresfurtherintegrativeanalysis(Puillandreetal.
2012b,Panteetal.
2015).
Herein,abriefinvestigationofthemorphologicalcharac-teristicscombinedwithadistance-basedmethodofABGDandatree-basedmethodofGMYCmaybeaverysuitablepatternforspeciesdelimitationandtherapididen-tificationofChaitophorinae.
39DNAbarcodingmayrevealpopulationdifferentiationdrivenbygeographicaldis-tributionChaitophorussalinigerShinjiisanimportantpestonwillowsinEastAsia.
Basedonthetopologystructureandresultsofanalysiswithdifferentmethods(Figure2A,Suppl.
ma-terial4–11),allsamplesofthisspeciesweredividedintotwocladeswhichcouldberegardedastwodifferentspecies.
However,basedonthemorphologicalcharacteristics,allsamplesshouldbeChaitophorussaliniger.
Onexaminingthegeographicaldistributioninformationofallsamples,wefoundthatoneofthecladesconsistedoftwosamples(Nos.
17651and33320)ofC.
salinigercollectedfromNortheastChina,HeilongjiangProvince,andthevoucherofanothersequence(C.
saliniger-GU978785.
1,adownloadedsequencefromNCBI)fromtheKoreanPeninsula.
Thelocationsitesofthesethreesam-pleswereallatarelativelyhighlatitude.
Allsamplesintheothercladewerenotfromtheaforementionedregions.
So,withinC.
saliniger,apopulationdifferentiationemergedamongthesamplesfromdifferentlocations.
ThegeneticdivergencesbetweenthetwocladesforCOI,Cytb,andgndwere0.
043,0.
039,and0.
020,respectively,whichcouldberegardedasinterspecificdistances.
Althoughthemorphologicalcharacteristicsofallsamplesweresimilar,differentiationatthegenelevelseemstohaveoccurredbetweennorthernandsouthernpopulations.
Similargeneticdifferentiationbetweenthesepopu-lationshasalsobeendemonstratedforanucleargene,EF-1α(Fangetal.
2016).
Inasimilarmanner,twosamples(Nos.
17613and19950)ofLaingiapsammaeTheobaldweredividedintotwoindependentclades(Figure2B),aresultsupportedatallgenesandusingdifferentapproaches.
SampleNo.
17613wasfromJilin,northeast-ernChina,whereassampleNo.
19950wasfromXinjiang,northwesternChina.
Thegeneticdistancesofthreegenes(COI,COIIandCytb)betweenthetwosampleswere0.
064,0.
068,and0.
062,respectively,whichreachthelevelofspecies(WangandQiao2009).
Therefore,differentiationbetweennortheasternandnorthwesternpopulationsinL.
psammaeexists.
FromthetopologystructuresandtheconstructedconsequencesofthresholdwithNJ,ABGD,GMYC,andPTP,weobservedthatpopulationdifferentiationwasclearlypresentwithinbothC.
salinigerandL.
psammae.
Similarfindingshavebeenreportedinotheraphidspecies(Leeetal.
2011,Wangetal.
2011).
Theprominentdifferencesamongpopulationsmayevenbeanindicationofcrypticspecies(Bickfordetal.
2007).
Speciationisalongandcontinuousprocess,andcrypticspeciesarenoteasilyex-plained.
Withintheprocess,theincipientspeciesmayholdformillionsofyears(AviseandWalker1998).
Therefore,crypticspeciesneedfurtherstudywithmoresamples,combinedwithmorphologicalcharacteristicsandbiologicalinformation.
ConclusionsInthiswork,theDNAbarcodingofChaitophorinaeaphidswasinvestigated.
Threemitochondrialgenesandoneendosymbiontgenewereusedtocalculateandcompare40Figure2.
TheanalysisresultsofsomespeciesfromtheCOI-670dataset.
Theanalysisresultsbasedonothergeneswerealmostidentical.
TheNJtreewasconstructedbasedontheKimura2-parameter(K2P)modelwithabootstrapvalueover50%displayed.
Thegrayblocksbehindthetreerepresenttheputativespecies,whichmeansthatthetaxainthetreecorrespondingtoasingleblockareinoneputativespecies.
Thenumberofblocksexpressthenumberofputativespeciesusingthismethod.
AChaitophorussalinigerBLaingiapsammae.
41thegeneticdistanceswithindifferentdatasets.
Forthedelimitationofspecies,twodistance-basedmethods,thresholdwithNJandABGD,andtwotree-basedapproach-es,GMYCandPTPwereemployed.
Theinterspecificgeneticdivergencewasclearlygreaterthanintraspecificdivergenceinthefourmolecularmarkers.
Additionally,theCOIandCOIIgenesweremoresuitableasChaitophorinaeDNAbarcodingmarkers.
BasedonthedataforChaitophorussalinigerandLaingiapsammae,DNAbarcodingmayrevealpopulationdifferentiationdrivenbygeographicaldistribution.
AcknowledgementsWewereverygratefulforallthesamplescollectorsoftheirassistance,andappreciatedFen-DiYangformountedslidesmakingofallthevoucherspecimens.
ThisworkwassupportedbytheNationalNaturalSciencesFoundationofChina(Nos.
31620103916,31572307,31430078),andtheExternalCooperationProgramofBIC,ChineseAcad-emyofSciences(No.
152111KYSB20130012).
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80647Supplementarymaterial1TableS1Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:specimensdataExplanationnote:Sampleinformation.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
Supplementarymaterial2TableS2Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:Primerinformation.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
Supplementarymaterial3TableS3Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:TheanalysisresultswithABGDofalldatasets.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
48Supplementarymaterial4FigureS1Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:TheanalysisresultsofdatasetCOI-670.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
Supplementarymaterial5FigureS2Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:TheanalysisresultsofdatasetCOII-376.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
Supplementarymaterial6FigureS3Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:TheanalysisresultsofdatasetCytb-413.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
49Supplementarymaterial7FigureS4Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:Theanalysisresultsofdatasetgnd-396.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
Supplementarymaterial8FigureS5Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:TheanalysisresultsofdatasetCOI-338.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
Supplementarymaterial9FigureS6Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:TheanalysisresultsofdatasetCOII-338.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
50Supplementarymaterial10FigureS7Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:TheanalysisresultsofdatasetCytb-338.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.
Supplementarymaterial11FigureS8Authors:Xi-ChaoZhu,JingChen,RuiChen,Li-YunJiang,Ge-XiaQiaoDatatype:moleculardataExplanationnote:Theanalysisresultsofdatasetgnd-338.
Copyrightnotice:ThisdatasetismadeavailableundertheOpenDatabaseLicense(http://opendatacommons.
org/licenses/odbl/1.
0/).
TheOpenDatabaseLicense(ODbL)isalicenseagreementintendedtoallowuserstofreelyshare,modify,andusethisDatasetwhilemaintainingthissamefreedomforothers,providedthattheoriginalsourceandauthor(s)arecredited.