Phylogenetic
JournalofForestryResearch(2008)19(3):239-244
;D0Il0.1007/sl1676—008—004l一4
;Phylogeneticrelationshipsof18passerinesbasedonAdenylateKinase ;Intron5sequences
;GUOHui—yan.YUHui.xinBAISu—ying.MAYu—kun,
;MudanjiangNormalCollege,Mudanjiang157012,RChina
;Collegeof~lyeSciences,HeilongfiangUniversity,Harbin150080,RChina ;CollegeofWildlifeResources,NortheastForestryUniversity,Harbin150040,PRChina ;Abstract:The18speciesofbirdstudiedoriginallyareknowntobelongtomuscicapids,robinsandsylviidsofpasserines,butsomedis—
;putationsarealwayspresentintheirclassificationsystems.Inthisexperiment,phylogeneticrelationshipsof18speciesofpasserineswere
;studiedusingAdenylateKinaselntron5(AK5)sequencesandDNAtechniques.ThroughsequencesanalysisincomparisonwitheachotheL
;phylogenetictreefiguresof18speciesofpasserineswereconstructedusingNei曲bo卜
JoiningJ)andMaximum-Parsimony(MP)meth—
;odsTheresultsshowedthatsylviidsshouldbelistedasanindependentfamily,whilerobinsandflycatchersshouldbelistedintoMusci-
;capidae.Sincethephylogeneticrelationshipsbetweenlong—tailedtitsandoldworldwarb
lersarecloserthanthatbetweenlong-tailedtitsand
;parids,thelong—tailedtitsshouldbeindependentofparidaeandbecategorizedintoaegithalidae.MuscicapidaeandParidaeareknowntobe
;twomonophyliticfamilies,butSylviidaeisnotamonophyleticgroup.AK5sequenceshadbetterefficacyinresolvingcloserelationshipsof
;interspeciesamongintragenericgroups.
;Keywords:molecularphylogeny;AdenylateKinaselntron5:passeriformes;monophyly ;Introduetion
;Passeriformesarecomposedofmorethanhalfofalllivingbird
;species.Withintenseadaptiveradiations.theoriginandevolu.
;tionofPasseriformeshavebeenstudiedforalongtime(Chang
;etalJl992).Sincethedebateshavebeencontinuedonavian
;morphologicalclassificationsystems,itisverynecessarytouse
;moreexactandefficientmethodstoperfectaviansystematics.
;Studiesshowedthatmolecularclassificationsystemcanprovide
;muchinformationandsolvesomedivergentproblemsinthe
;aviansystematics(Neieta1.2002).
;TheexplosionuseOfDNAtechniquescanprovideagreatdeal
;ofnewdataforaviansystematists.Anewavianclassification
;systembasedonDNAhybridizationtechniqueswasfoundedby
;Sibley&Ahlquistfl990),inwhichPasseriformeswasdivided ;intotwosuborders:suboscinesandoscines.TheRestriction ;Foundationproject:TheprojectwassupportedbyHeilongjiangProvince ;PostdoctoralFund.
-08 ;Received:2008-02-22;Accepted:2008—03
;.
;N.ortheastni堕IIandSriner-V
;T.hejDnisayai.!abl呻:,/.E
;Biography:GUOHui-yan(1978-),female,
;NormalCollege,Mudanjiang157012,China.
;1:ws~hvl307@126.com
;2008
;n~erlink.com
;S!!魍pgAE_望
;Responsibleeditor:ZhuHong
;AssistantinMudanjiang
;FragmentLengthPolymorphism(RFLP)techniquehasbeen ;usedinavianphylogenetics.Inthistechnique,passerinemito- ;chondrialgenomeswerefoundtobegeneticallypolymorphic(Li ;eta1.2002).Threetitmicespecies(Parusbicolor,P.inornatus,P. ;wollweberi)intheNorthAmericawerestudiedbvGilland ;Slikasfl992)usingRFLP’andtheyfoundthatthesespecies
;originatedinthePlioceneorearlyPleistocene.Inaddition,DNA ;sequenceanalysishasalsobeenusedinaviansystematics,which ;providedmoreclassificationsystematicinformation.Adenylate ;KinaseIntron5(AK5)mayperfotinwellinrecoveringrelation—
;shipsfrominterspeciestointerfamily(Shapiroeta1.200l1.Inthe ;presentstudy.wefocusedtheattentiononthephylogenyOfl8 ;speciesofpasserinesincludingmuscicapids,robinsandsylviids. ;Thephylogeneticrelationshipswerestudiedamongl8speciesof ;passerinesusingAdenylateKinaseIntron5(AK5)sequences ;(Shapiroeta1.200l1.Comparedwithearlierclassificationsys—
;tems,newinsightswereprovidedabouttheirphylogeny. ;Materialandmethods
;Taxonsampling
;Samplesof18speciesofpasserineswerecollectedfromthe ;Mao’ershanMountain,HeilongjiangProvince,China(Table1).
;AK5sequencesofFalcofemoralis(AF307890)wasdownloaded ;fromGenBankasoutgroups.
;66@vahoo.comDNAextraction,PCRamplificationandsequencing ;TotalgenomicDNAwasextractedusingstandard
;垒Springer
;
;JournalofForestryResearch(2008)19(3):239-244
;phenol—chloroformextractions.Weobtainedtheprimersforam. ;plifngAK5sequencesfrompreviouspapers:AK5b+: ;5’ATTGACGGCTACCCTCGCGAGGTG3’.
;AK6c一:5’CACCCGCCCGCTGGTCTCTCC3’fShapiroeta1.
;2001).Theprimersarelocatedintheexon5andexofl6ofAde. ;nylateKinasegene.AK5PCRmixturescontained1.25.UBlend ;TaqDNApolymerase,10xbufferof5,2-mMMgCL2,
;200一oMdNTPs,0.4oMofeachprimer,and】0o.500n2ofDNA
;inafinalvolumeof50uL.Reactionmixturesweresubiectedto ;Table1.Speciesexaminedinthepresentstudy
;thefollowingPCRcyclingprotocol:withaninitial10minat ;94.C,followedby35cyclesof92.Cfor45s,56—60.Cfor60s.
;and72.Cfor60s,andafinalextensionafterthelastcycleat ;72.Cfor5min.PCRproductswerepurifiedwithQIAquickPCR ;purificationkit(Qiagen).MostofPCRproductsweresequenced ;directlyandfewofthemweresequencedaftercloning.Se. ;quencingwasperformedbytheTaqDyeDeoxyTerminatorCy—
;cleSequencingkit(ABI)andanalyzedonanautomatedABI ;sequencer.
;Dataanalysis
;SequenceswereproofedwithDNAStarsoftwarepackageand ;performedsimilaritysearchingwithBlastintheNCBI.Align. ;mentsweremadewithClustalxfHall2OO1)(Version1.8)and ;furtheranalyseswereconductedwithMega3.0.
;Phylogeneticanalyseswerefirstundertakenusing ;Neighbor—JoiningJ),andconductedwithPhylip.Thedata ;werealsoanalyzedusingtheMaximum.Parsimony(MP)method. ;KimuraTwo.ParameterModelwasusedforbasesubstitution ;analysis.Transitionsandtransversionswereallincludedandall ;threesubstitutionsiteswereused.Substitutionratesofthreesites ;wereassumedtobedifferent,whichfollowedtheFdistribution ;withitsshapecoe所cientof0.5.Phylogenetictreeswereana. ;1yzedby1000bootstrapreplicates.
;Resuits
;Sequenceanalysisandbasecomposition
;Withinsertionsanddeletions,AK5sequencesdifferinlength ;from451to530bp.AK5genesofallspeciesinthisstudyhave ;theGTandAGdoubletsitesthatmarkthebeginningandendof ;theintron(Breathnacheta1.1977;Mount1982;Kellereta1. ;垒Springer
;1985;Mounteta1.1992).Weobtained35AK5completese. ;quences.whichweredepositedintoGenbank.Theaccession ;numbersareasfollows:DQ116460.DQ119491-DQ119520and ;DQ365012一DQ365015.
;TheaveragecontentsofT,C,AandGinthe35AK5se-
;quenceswere16.8%,30.9%,22.4%,and29.9%,respectively. ;Thepercentagesofconservedsites.variableandparsi—
;mony.informativesiteswere63.4%,36.6%and22.7%.respec. ;tively.
;Indels(insenions/deletions)
;ConsiderablelengthvariationswerefoundamongtheAK5se. ;quencesexamined.Thesequencescontainnumerousindels, ;whichinvolvebothsingleandmultiplenucleotides.Mostindels ;arefromsingleevolutionaryeventsfi_e..insertionordeletionof ;asinglenucleotideorstringofnucleotides).Forexample,there ;werefourdeletionssharedbytheP.maior,P.ater,P.palustris ;andP.montanus(nucleotidesites:22—36.397—406,417—441.
;444—469).Consequently,thesefourspecieshavetheshortest ;AK5sequences.Therewereoneunique5bpinsertionfCCTAT. ;nucleotidesites32—36)andonelbpinsertions(G.nucleotide
;sites523)intheAK5sequencesofP.webbianusand1-I.rustica. ;respectively.Itisbelievedthattheseindelsrepresentunique ;evolutionaryeventsofunequivocalhomology,withnoreversa1. ;
;JournalofForestryResearch(2008)19(3):239-24424l ;Geneticdistances
;Geneticdistances(Table2)werecalculatedwithKimura ;Two—ParameterMode1.Theaveragegeneticdistancebasedon ;AK5sequencesamong18speciesofpasserinesis0.086.The ;geneticdistancebetweenTarsigercyanurusandLusciniaeyane ;wasthesmallest(0,045)andthelargest(0.162)appearedbe—
;tweenSittaeuropaeaandLusciniaeyane,Thegeneticdistances ;betweenSittaeuropaeaandotheroldworldwarblerswererela—
;tivelysmallerthanthatbetweenSittaeuropaeaandotherpas—
;serines,whichmeansthatSittaeuropaeahasarelativelyclose ;relationshipwitholdworldwarblers.Thegeneticdistancebe—
;tweenlong—tailedtitsandoldworldwarblerswassmallerthan ;thatbetweenlong—tailedtitsandparidsalso.whichmeansthat ;oldworldwarblershavecloserrelationshipwithlongtailedtits. ;Table2.PairwisedistancesbetweenAK5sequencesof18speciesofpasserinescomputedb
yKimuraTwo—parametermodel(Thelowertriangle
;numbersarepairwisedistancesandtheuppertrianglestandarderror) ;Phylogeneticanalyses
;Phylogenetictreesofthe18speciesofpasserineswerecon—
;structedbytheNJandMPmethods(Fig.11.Theresultsshowed ;thatthetwophylogenetictreefigureswerenotsimilarcom—
;pletely,buttheyallcomprisethreemajorclades.Thefirstmajor ;cladewasmadeupofoldworldwarblers,long—tailedtits,crow—
;tits,barnswallowandsi~ids.Thesecondwasparidsandthe ;thirdwasrobinsandflycatchers.
;Theclusteringsofthel8passerinespeciesareapproximately ;alikeinthetwotreefigureswithslightdifferences.Inthefirst ;majorcladePhvlloscopusinornatusandPhylloscopustro—
;ehiloideswereclusteredtogether,thenclusteredwithPv,f0一
;scopusproregulusandthenwithPhylloscopusfuscatus,Locus—
;tellalanceolatawasclusteredwithParadoxorniswebbianu.In ;theNJtreefigure,Aegithaloscaudatuswasclusteredwiththe ;fourwarblersabove,thenwithSittaeuropaea.ButintheMPtree ;figure,SittaeuropaeawasclusteredwithHirundorustica,then ;withAegithaloscaudatus.Inthesecondmajorclade,Paruspal- ;ustriswasclusteredwithParusmontanusformingonesmall ;clade.IntheNJtree,ParusmajorwasclusteredwithPar’tgsater
;tOformanothersmallclade,butintheMPtreefigure.Parus ;majorwasnotclusteredwithPaI’USater.Inthethirdmajorclade,
;FicedulamugimakiwasclusteredwithFicedulazanthopygia, ;andthenwithCyanoptilaeyanomelanatoformaclade.This ;cladethenclusterswithLusciniaeyaneandfinallywithTarsiger ;cyanurusintheMPtreefigure,butitclusterswithLuscinia ;eyanefirst,thenwithTarsigercyanurus(Fig.1). ;Diseussion
;Phylogeneticutility
;Frompreviousresearches,nuclearintronsweremainlyusedto ;revealmediumrelationshipsbetweenintergenericandinterfami—
;lies.RusselloandAmato(2004).studiedthephylogeneticrela—
;tionshipsofAmozana.usingthreenuclearintronsandthreemi—
;tochondrialgenesequences.Theresultsshowedthatnuclear ;intronscouldalsobeusedtoanalyzeintragenericrelationships. ;Alaterstudyshowedthatintronsequencescouldalsobeusedto ;analyzephylogeneticrelationshipsofcloselyrelatedspecies ;(Sladeeta1.1994).AstoAK5sequences.itmayperforillwellin ;recoveringrelationshipsamongjntermediatetodistantlyrelated ;congenericspeciesandbetweengenera(Shapiroeta1.2001). ;OIll”resultsshowedthatParispoultriesandParisMontanezwere
;difficultlydistinguishedonmorphologicaltraitsbuteasilydis—
;tinguishedwithanalysisOfAK5sequences.Therefore.AK5 ;垒Springer
;
;242JournalofForestryResearch(2008)19(3):239-244 ;sequencesalsohavefineeffectivenessinanalyzingrelationships ;amongcloserelatedcongenericspecies.
;Phylogeneficrelationships
;Withregardtorelationshipsamongmuscicapids,turdidsand
;sylviids,threemainopinionsarecoexisting.First,muscicapids, ;turdidsandsylviidsshouldbelistedintothreesubfamiliesrMus—
;cicapinae;TurdinaeandSylviinae)underthefamilyMuscicapi. ;dae(ZhengZuoxin2002);Second,muscicapidsandturdidsare ;listedintotwosubfamilies(MuscicapinaeandTurdinae)under ;thefamilyMuscicapidae.butsylviidsis1istedintothefamily ;Sylviidae(Sibidae,
;TurdidaeandSylviidae),(ZhengGuangmei2002;Howardeta1. ;2003).Itshouldbepointedoutthatrobinsareisolatedfrom ;TurdidaeandlistedintoSaxicolinaeunderMuscicapidaeinsome ;classificationsystems(Sibleyeta1.1990;Howardeta1.2003). ;butinothers,theyarestilllyinginTurdidae(ZhengZuoxin2002; ;ZhengGuangmei2002).Duetotheabsenceofotherspeciesof ;Turdidaeinourresearch.wearenotsurewhetherrobinsshould ;belistedintoSaxicolinae.whichisindependentofTurdidaeor ;not.AccordingtothephylogeneticanalyseiesconstructedwithAK5sequences ;rA:NJtree;B:MPtree)
;IntheclassificationsystemofSibley&Ahlquist(1990), ;paridsbelongtothesuperfamilySylvioidea.Theresultreveals ;thatparidshavecloserelationshipwithsylviids.Alstrometa1. ;(2006)studiedthesystematicstatusandthephylogeneticrela? ;tionshipsofsuperfamilySylvioidea,bycombiningmitochondrial ;CytbandmyoglobinintronIIsequences,andtheyreportedthat ;paridswereexcludedfromsuperfamilySylvioidea.Ttheircon? ;clusionsneedtobeexaminedfurtherduetohaving1esssample’s
;kindsandamountofparids.Inourstudy.theresultsfromgenetic ;distancebetweenparidsandsylviidsshowedthatparidsand ;sylviidshadlikelycloserrelationship.Fromphylogenetictrees ;垒Springer
;constructedbytwomethods,paridsalwaysgatherssylviidsfirst, ;soourresearchsupportSibley&Ahlquist’sclassifyingview?
;point.
;IntheclassificationsystemofSibley&Ahlquist(1990),Sini—
;daebelongstothesuperfamilySylvioidea.Butintheresultsof ;Sheldon&Gil1(1996).SiaidaewasclosetoSturnidaeand ;TurdidaeofthesuperfamilyMuscicapoidea(Sheldoneta1.1996). ;OurresultssupporttheopinionofSibley&Ahlquist.butfurther ;studyshouldbeconductedinthefuture.
;IntheNJtreefigure,HirundorusticaclusterswithLocustella ;lanceolataandParadoxorniswebbianuswith1owbootstrap ;
;JournalofForestryResearch(2008)19(3):239-244243 ;value(only32%1.IntheMPtreefigure,warblersfirstcluster ;withHirundorusticaandthenwithLocustellalanceolatawith
;highbootstrapvalue,whichmeansthattherelationshipbetween ;Hirundorusticaandwarblershasacloserrelationshipthanthat ;betweenLocustellalanceolataandwarblers.Alstrometa1.(2006) ;alsosupportedtheopinionhavingacloserrelationshipbetween ;Hirundorusticaandwarblers.
;IntheclassificationsystemofSibley&Ahlquistfl990).the ;taxonomicpositionofcrowtitsisinTimaliini.Sylviinae.Sylvii—
;dae,Sylvioidea,however,intheclassificationsystemofZheng ;Zuoxin(20021,crowtitsareinTimaliinaeandMuscicapidae.In ;anotherclassificationsystem,crowtitsturnouttobeParadoxor—
;nithidae(ZhengGuangmei2002).Inourresults,Paradoxornis ;webbianusclusterswithLocustellalanceolata,whichsupports ;theopinionofSibley&Ahlquist.
;Intheclassificationsystemof(ZhengZuoxin20021. ;1ong—tailedtitsjustbelongtoagenusunderparidae.Intheclassi—
;ficationsystemsofSibley&Ahlquistfl990)andSheldon&Gill ;(1996),long—tailedtitsarelistedinanindependentfamilyof ;aegithalidaeunderSylvioidea.Spicereta1.(2004)studiedthe ;phylogeneticrelationshipsofSylvioideausing16SrRNAse—
;quences.whoseresultssupportedtheopinionsofSibley& ;Ahlquistfl990)andSheldon&Gillf1996).Ourresultsalso ;supportedtheopinionthatlong—tailedtitsshouldbeindependent
;ofparidaeandbecategorizedintoaegithalidae.Alsoinourre—
;suits.therelationshipsbetweenlone—tailedtitsandoldworld
;warblershadcloserrelationshipthanthatbetweenlone—tailedtits
;andparids.Thismaybeaconsequenceoftherelativelysparse ;taxonsampinginthedataset.andmoreandmorescholarsem. ;phasizeddensenesssamplingandincreasingsamplequantity ;(Omlandeta1.1999;Rannalaeta1.1998).Thebootstraphum. ;bersoflong—tailedtitsandsylvioideaclusterinphylogenetic ;treesconst