Organizationand Variationofthe MitochondrialControlRegion inTwo VultureSpecies,Gypaetus barbatusand Neophronpercnopterus

S.ROQUES,J.A.GODOY,J.J.NEGRO,AND F.HIRALDO

FromtheEstacionbiologicadeDo~nana,CSIC,Pabello´ndel Peru,41013Sevilla,Spain.

AddresscorrespondencetoSe´verineRoquesattheaddressabove,ore-mail:.

We report thefirstentiremitochondrial DNA(mtDNA) controlregionsequences intwoendangeredvulturespecies, thebeardedvulture (Gypaetusbarbatus) andtheEgyptian vulture(Neophronpercnopterus). Resultsshowedthat the general organizationofvulturecontrolregionswasvery similar to other birds,withthree distinctdomains:aleft variabledomain(DI),acentralconservedone(DII)including theF,E,D,andCboxes,andarightdomain(DIII)containing theCSB1sequence.However,duetothepresenceof long tandemrepeats,vulturecontrolregionsdifferedfromother aviancontrol regionsbothinsizeandnucleotidecomposi- tion.TheEgyptianvulturecontrolregionwasfoundtobe the largest sequenced so far (2031bp), due to the simultaneous presenceofrepeatsin bothDI(80bp)and DIII (77bp). Lowvariationwasfoundin vulturecontrol regions, particularlyinG.barbatus,astheprobableresultof populations declinesinthelastfewcenturies.

Thevertebratemitochondrialgenome(mtDNA)isaclosed, maternallyinheritedcircularmolecule(15–20kb)composed ofabout37genescodingfor22transferRNAs(tRNAs),2 ribosomalRNAs(rRNAs), and13messengerRNAs (mRNAs).Although genetically conserved, gene order withinthemtDNAhasbeenshowntovary amongtaxa (Benschand Harlid2000; Desjardinsand Morais1990; Sacconeetal.2000).Withinthe mitochondrialgenome, differentregionsevolveatdifferentrates,withthecontrol region having the highest nucleotidesubstitution rate, probably duetothelackofcodingconstraints.Forthis reason, thisportionhasbeenuseful inagreatnumberof studiesatinter-andintraspecificlevels.

Thecontrolregionofbirds differsfromothervertebrates inthatitisflanked bythegenestRNAGlu andtRNAPhe (DesjardinsandMorais1990).Morerecently,Mindelletal. (1998)describedanewgeneorderinseveralbirdgroupsin which the control region is flankedby tRNAThr and

tARNPro.Studyoftheaviancontrolregionisofgrowing interest,mainlytoaddresspopulationstructureusingsmall partsofthecontrolregion(e.g.,Pitraetal.2000;Wenneberg

2001),butalsotodescribetheorganizationandvariationof theentireregionatseveraltaxonomiclevels(Desjardinsand Morais1990; Haringetal.2001; Sorensonetal.1999). RecentlyRuokonen andKvist (2002)extensivelydescribed thestructuralandevolutionarycharacteristicsof68avian species,butinformationisstill lackingonFalconiformes species.Lowerintra-and/or interspecific variationwas found in birds comparedto mammals,but more bird sequencesareneededto assessthe levelandextentof similarityatdifferenttaxonomiclevels.

Largesizevariationshavebeen describedinsomeavian

controlregionsasthe resultof tandemlyrepeatedsequences locatedindomainIordomainIII(e.g.,Bergetal.1995; RitchieandLambert2000).Suchtandemsequenceshave beendescribedinagreatvarietyoftaxa.Slippageduring DNAreplicationhas beenproposedas alikelymechanism forthegenerationoflengthpolymorphism(Weninketal.

1994),butitstillremainsunclearwhetherthoserepeatscould potentiallybeinformativeforphylogeneticpurposes.

The Egyptianvulture(Neophron percnopterus) and the

beardedvulture(Gypaetus barbatus) belongto the family Accipitridae(order Falconiformes),which includes the majorityoftheEurasianandAfricanbirdsofprey.These twospeciesclustertogetherinraptorphylogeneticrecon- structions(Seiboldand Helbig1995)and are therefore consideredtheclosestextantrelativesofeachother.Theyare twoof the most endangeredvulturespeciesinEurope, wheretheirpopulationshavedecreaseddrasticallysincethe beginningofthiscenturyduetohumancauses.Fewstudies ofgeneticvariability havebeeninvestigatedinEuropean vulturesexceptisolatedworksonphylogeneticrelationships betweenrelatedspecies(Mindelletal.1998;Wink1995)or populationstructureusingeithernuclearloci(Kretzmann

etal.2003;NegroandTorres1999)orasmallpartofthe controlregion(Godoyetal.2004).

In this articlewe characterizethe organizationand variabilityof the entire control regionsequencein N.

percnopterus andG.barbatus andcompareittootheravian controlregions.

Materialsand Methods

TissueSamples

Insertionsof mtDNA inthe nucleargenome(‘‘numts’’; Lopezet al.1994)havebeenobservedinseveralbirds species(Quinn1992,1997;SorensonandFleischer1996).In order toverifythattheamplificationsobtainedinthisstudy wereofmtDNAorigin,internalfragmentsofthecontrol regionwereamplifiedfromeitherbloodortissuerichin mitochondriaandcompared.Inaddition, maternal in- heritancewasevaluatedbysequencingmtDNAfragments infamilies (parentsandtheirprogeny).Allresultswere consistentwiththemtDNAoriginofvulturesequences.

SampleCollectionandDNAExtraction

Fivebeardedvultures(twofromthePyrenees,Spain;three fromtheformerSovietUnion)andsixEgyptianvultures (twofromtheIberianpeninsula,Navarra;twofromthe CanaryIslands;two from the Baleares, Menorca)were sequencedfor the entirecontrolregion.One additional samplecomesfromaseparatesubspecies, Neophron percnopterus ginginianus, fromsouth-central India.Genomic DNAwasextractedeitherfromblood stored inlysisbuffer (Seutinetal.1991)orfrozentissues(liver)usingalithium chloridemethod adapted from Gemmelland Akiyama (1996).

PolymeraseChainReaction(PCR)Primers andAmplification

Inordertodeterminegeneorderwithinvulturespecies,we initially attemptedtheamplificationoftheentirecontrol regionusingprimersdesignedfromthe consensussequence ofseveralbirdsspeciesintheknownflankingregions,the tARN-GluandtARN-Phegenes(BakerandMarshall1997). No amplificationproducts of the size expected were obtainedwiththeseprimersusingourvulturesamples.We thereforedesignednewprimersintheregionstThrFand tProRthat flankedthe newgeneorderrecentlydescribedby Mindelletal.(1998).Thistimeweamplifiedaproductofthe expectedsizeinG.barbatusandN.percnopterus.ThePCRwas performedin20ll reactionvolumescontaining 2.5mM MgCl2, 0.2mMdNTPs,1lMofeachprimer,1UTaq polymerase,andapproximately10–50ngoftotalDNA. The controlregionofN. percnopterus wasamplifiedusingPwo polymerase (RocheDiagnostics)followingthe manufac- turer’sprotocol. PCR conditionsweresimilarfor both speciesandincludedaninitialstepat928C for2min,34 cyclesat928Cfor30s,at648Cfor30s,andat728Cfor30s.

Followingthelastcycle,afinalextensionstepat728Cfor5 minwasperformed.

CloningandSequencing

Polymerasechainreactionproductsoftheentirecontrol regionwereextractedfromagarosegelsusingtheGFXGel ExtractionKit(AmershamLifeScience).G.barbatuspurified productswereclonedintoapMOSbluevector(CloningKit, Amersham Life Science)followingthe manufacturer’s instructions,whileN. percnopterus purifiedproductswere clonedinabluntendvectorpCAPsdigestedwithMluN1 andtransformedintoXL1BlueEscherichiacolicompetentcells (RocheDiagnostics). Plasmidsfrom both specieswere isolatedandpurifiedusingtheNucleospinplasmidextrac- tionkit(Macherey-Nagel)and1ll ofthefinaleluatewas loadedontoa1%geltoestimateDNAconcentration.We found morethan one lengthcontrolregionvariantper individual(sizeheteroplasmy)inbothvulturespecies,but asingleclonewassequenced.

Sequencingoftheclonesinboth directionswasfirst carriedoutusingflankinguniversalprimers(T7andU19,G. barbatus; S1andS2,N. percnopterus) using50–200ngof plasmidDNA.Sinceregionsdifficulttosequence(poly-C, secondarystructure)preventedthecorrectreadingofthe wholecontrolregion,wedecidedtoamplifyandsequence PCR fragments with internal primers. AmplifiedPCR productswerepurifiedusingtheGFXGelExtractionKit andabout50ngofPCRproductwassequencedusingthe ABIPrismBigDyeTerminatorCycleSequencingReady ReactionKit(AppliedBiosystem)inan ABIPrismTM310

GeneticAnalyzer.In G.barbatus, the majorpartof the mitochondrialcontrol region(;1450bp)wassequencedat leasttwice andwasthusdetermined withoutambiguities. However,duetoapoly-Cregion,roughlythefirst100bp werecorrectlyread foronlyoneoutoffive individuals.InN. percnopterus, allclonesweresuccessfullysequencedinboth strands.

SequenceAnalysis

Foreachofthespecies,sequenceswerealignedusingthe programSequencer4.1.2(GeneCodesCorp.)andcorrected manually.Nucleotidecompositionswerecomputedusing theprogramMega1.01(Kumaretal.1993).Theprogram DNAsp(Rozas andRozas1999)wasusedtocalculatevalues ofnucleotidediversity(p).Percentsimilaritiesbetweenboth vultureswereestimatedusingthealgorithmAlign inthe DnaStarsoftwarepackage(LasergeneInc.).

Results and Discussion

CharacterizationofBeardedandEgyptianVulture

ControlRegion

Gypaetus barbatus andNeophron percnopteruscontrolregions bothshowthegeneorderrecentlydescribedbyBenschand Ha¨rlid(2000)andMindelletal.(1998).Bothvulturespecies’

Figure 1. Generalschemeoftheorganizationofthelargestcontrolregionsequencesin(A)N.Percnopterusand(B)G.barbatus: ETAS1(stripedbox),conserved(blackboxes),complete(greyboxes),andincompleteRI(whiteboxes)repeatedsequences(R).

controlregionssharedmanyofthegeneralcharacteristics thathavebeenreportedinotherbirds (MarshallandBaker

1997;RuokonenandKvist2002).Generallythreedistinct domains are described in the control region, defined

arbitrarilyfrominformationongenetic variabilityorbase composition:the most variable, leftdomainI (DI); the conserved,centraldomain(DII);andtherightdomainIII (DIII) (Bakerand Marshall1997).Similarly thesethree distinctdomainswereobservedinvultures.The border betweenDIIandDIIIwasplacedbeforeCSB1,following Sbisaetal.(1997)(Figure1).Theoverallbasecompositions weresimilarbetweenbothspecies’controlregionsandin agreementwithpreviousobservationsinavianspecies,with AþT.GþC,aCT-richDII,andaDIIIverypoorinG andwithhighproportionsofATnucleotides(Bakerand

Marshall1997;RuokonenandKvist2002).However,the controlregionDIofN.percnopterusisnotACrich, asinmost birdspecies,butwasexceedinglyrichinthymidine(33.7%) duetothepresenceofatandemlyrepeatedregion.

Atthe 59endof theDI,wefoundaninterruptedpoly-C sequence(Figure2).Thisstructureseemstobeaconserved featureacrossmanyspecies andwasdescribedinbirds includingStruthioniformes,Galliformes,Falconiformes,and Sphenisciformes(Haringetal.2001;RitchieandLambert

2000).Althoughitcouldpotentiallyformastablehairpin structure (Quinn1992;QuinnandWilson1993),itsfunction

hasneverbeendetermined. Inaddition,conservedpalin- dromicmotifs59-TACAT-39(Sacconeetal.1991)and59- TATAT-3werefoundatthe59endofbothvulturespecies’ controlregions(Figure2).Thefour conservedF,E,D,and C boxeslocatedinthecentraldomainand theCSB1region wereidentifiedinbothspecies(Figures1and2).However, theconservedblocksCSB2andCSB3,previouslyreportedin othervertebratesandbelieved tobeinvolvedintheinitiation ofDNA synthesis,couldnotbemappedunambiguously (Sbisaetal.1997).Similarlytheseboxeswereabsentinother

Falconiformesand Ciconiiformesspeciessuch as Falco peregrinus(Mindelletal.1998), Buteobuteo(Haringetal.2001), andCiconia ciconia(Yamamotoetal.2000).Sbisaetal.(1997) identifiedtwolongconservedblocksinDI—ETAS1and ETAS2—potentially involvedinthecontrolofH strand synthesis.Interestingly,inboththe DIofG.barbatusandN. percnopterus, a31bplongsequenceofhighsimilarity(71%) withtheETAS1sequencecouldbeidentified(Figures1and

2).However,the ETAS2sequencecouldnot be found withoutambiguity.Intheirstudy,Sbisaetal.(1997)observed thattheETAS1sequencewascommoninmanyvertebrates species,but in contrast,the ETAS2,CSB2,and CSB3 conservedregionswereabsentinmanyofthem.

Oneofthemaincharacteristicofthevulturecontrol regionisitslargesize,duetothepresenceoflong tandem

repeats.RepetitivesequencesoccurinDIIIinbothspecies, butthemajordifferencebetweenspeciescomesfromthe presenceoftandemrepeatedsequences,alsointheDIofN. percnopterus (Figures1and2).Thereforethelengthofthe controlregionvariesamongindividualswithinspecies,with thesizedependingonthenumberoftandemrepeatsand also,withinindividuals (sizeheteroplasmy). According to this,thelength ofthecontrolregion inG.barbatusranges from1564bp(fiverepeats,DIII)to1750bp(sevenrepeats, DIII)andfrom1710bp(fourrepeats, DI;threerepeats, DIII) to2031bp(fourrepeats,DI;sevenrepeats,DIII) inN.

!

Figure 2. Nucleotidesequenceofthelargestmitochondrial controlregionin(A)N.percnopterusand (B)G.barbatus.Several conservedmotifswereidentifiedalongthisfragment:putative ETAS1elements(underlined),palindromicmotifs(TACAT, TATAT),boxes(F,E,D,C),blocks(CSB1),andrepeated sequences (*).GenBanknumbersareAY542899and AY542900.

percnopterus(Figure1).Comparedtootheravianspecies,the controlregionofN.percnopterusisthelargestsequencedso far (Delportetal.2002;Ruokonenand Kvist2002).

LevelsofSequenceVariationAmongandWithin

VultureSpecies

Becauseof the presenceof highinterspecific sequence variationin DI and DIII and the presenceof distinct tandemlyrepeatedsequences,thecontrolregionofthetwo speciescouldnotbealignedproperly.Wethereforecalculate sequencesimilaritybetweenportionsofthecontrolregion. Resultsshowedhighheterogeneityof percent similarity acrossthe controlregion.WithinDI, highinterspecific variabilitywasfound,butinterestingly,the first100bpwere well conserved(79%similarity).Inagreementwithprevious studies, thecentralDII,includingalltheconservedboxes(F, E,D,C,andCSB)washighlyconserved,with86%similarity. Intotal,980bpoutofthecommon1200bpofthecontrol regionpresented83%of thesimilaritybetweenbothvulture species. However,wefoundno sequencesimilarities betweentherepeatunitsinthetwovultures.

Withinspecies,controlregiondiversity waslow comparedtootheravianspecies(Delportetal.2002;Randi and Luchini 1998; Ruokonen and Kvist 2002). Low

intraspecific variabilitywasfoundinG.barbatuswith1.8% ofpolymorphicsitesalong1160bpofthecontrolregion(we excludetherepeatedsequencesfromtheanalysis).Roughly doublethisvariabilitywasfound inthe controlregion(1102 bp)ofN.percnopterus(4%).However,DIandDIIIwerethe mostvariable (3.3%and8.8%,respectively)andDIItheleast variable(0.8%in both species)domains,as previously reportedinmostavianspecies(MarshallandBaker1997; RuokonenandKvist2002).Apastbottleneckofvulture populationscouldexplainthisreducedvariability.Alterna- tively,the controlregioninvulturesmay not evolveas

rapidlyasinothertaxa(RuokonenandKvist2002).

TandemRepetitiveSequencesinDIandDIII

Allindividualsanalyzedshowedlongtandemlyrepeated sequences(Figures1and2). Bergetal.(1995)reportedthese longtandemrepeatswere frequentinCiconiiformesspecies, ranginginlengthfrom50to200bp andgenerallylocated2–

20bpfromthe59endofthe tRNAPhe.However,compared tootherbirdspecies(seeTable6inHaringetal.2001),the N.percnopteruscontrolregioncontainsthehighestpercentage (45.7%)ofrepeatedsequences,withlongrepeatspresent bothinDI (77bp)andDIII (80bp).Thesimultaneous existenceofbothoftheserepeatsinDIandDIIIisrarein birds, buthasbeenpreviouslyreportedinanotherraptor species,Falcoperegrinus(Mindelletal.1997,1998).

InG.barbatus,repeatedsequencesinDIIIconsistofan

incompleterepeat(RI)followedbyfiveorsevencopiesof a73bpsequencerepeatedintandem.Comparisonofrepeats withinandamongindividualsindicatedseveralinteresting features.First,inN.percnopterus, repeated sequencesinDI weremorevariablebothamongandwithinindividualsthan weretandemrepeatslocatedinDIII.Forexample,the80bp

repeats in DIII were identicalin most N. percnopterus individualsexcepttheonefromIndia,whilerepeatsinDI werehighlyvariable,both withinandamongindividuals. Similarly,inshrewspecies,nosequencevariationwasfound among DIIIrepeats fromallspecimens,whilemuchmore variationwasfoundamongDIrepeats(Fumagallietal. 1996; YamagataandNamikawa1999).

InG.barbatus,sequence variation intherepeatedregion waslow,bothamongandwithinindividuals.Theincomplete (RI)andfirstcompleterepeat(R1)weredistinctfromother repeats,butweresimilaramongalmostallindividuals, while adjacentremainingrepeatswithinan individualwereclosely related. Burokeretal.(1990)proposedamodelinwhich lengthmutationsmayoriginatethroughreplicationslippage duetoacompetitivedisplacementbetweenthetwostrands (H strand and D loop)in relationto stablesecondary structuresformedbytherepeated motifs.Thismodel generallyfitswellwithmostofthecasesdescribedtodate, but more complexmechanismswerealsoproposed to explainotherobservedpatternsofvariation(Brzuzan2000; Hoelzeletal.1993;RitchieandLambert2000).Inourstudy, wefoundthatdifferenceswerelargeramonghomologous repeatsbetweenindividuals than amongtandemrepeats withinanindividual,suggestingapatternofintraspecific concertedevolution(homogenizationofanarraythrough reiterated cyclesof insertion and deletion of repeats) (BroughtonandDowling1994;Rand1994).Thedifference inthelevelofvariationofrepeats betweenandwithinthe twovulturescouldbedueto eitheradifferentbalance betweenthepointmutationrate(generatingdiversityamong repeats) andtherateofinsertion/deletionofrepeats (implicatedinthehomogenizationofthearray)aspreviously proposed(Fumagallietal.1996;Hoelzeletal.1993),orto theintrinsiclowergenomevariabilityofG.barbatus.

Acknowledgments

Wearegratefulto N. CapoteandJ.Mun~ozforlaboratoryassistance, information,anddiscussionon thisproject,R.Antor (Foundationof BeardedVultureConservation,Zaragoza),theCaptiveBreedingProjectof theAlps,F.dePablo,O.Ceballos,J.A.Donazar,L.Gangoso,andC.J. Palaciosforprovidingsamples.ThisresearchwassupportedbyaMarie Curie FellowshipoftheEuropeanCommunityHostDevelopmentprogram undercontractno. HPMD-CT-2000-00009andbyprojectsPBREN-2000-

1556GLO andPB1997-1264oftheSpanishMinistryofScienceand

Technology.

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