Proc.Natl.Acad.Sci.USAVol.91,pp.2621-2624,March1994EvolutionMolecularevidencefortheoriginofbirds(evolution/tpod/amniote/phylogeny/systematics)S.BLAIRHEDGESDepartmentofBiologyandInstituteofMolecularEvolutionaryGenetics,208MuellerLaboratory,PennsylvaniaStateUniversity,UniversityPark,PA16802CommunicatedbyCharlesG.Sibley,December9,1993ABSTRACTThemajorgroupsofamniotevertebratesappearedduringarelativelyshorttimespanattheendofthePaleozoicEra,afactthathascauseddifficultyinestimatingtheirrelationships.Thefossilrecordsuggeststhatcrocodilansaretheclosestlivingrelativesofbirds.However,morphologicalcharactersandmolecularsequencedatafromlivingamnioteshaverepeatedlychaflengedthishypothesisbyindicatingabird-mammalrelationship.DNAsequencesfromfourslow-evolvinggenes(mitochondrial12Sand16SrRNA,tRNAVaI,andnucleara-enolase)nowprovidestrongstatisticalsupportforabird-crocodillanrelationship.Themajorgroupsoflivingamniotevertebratesarethemammals,birds,crocodilians,turtles,squamates(lizards,snakes,andamphisbaenians),andsphenodontids(tuataras).Thefirstamniotes(reptiles)appearinthefossilrecordabout300millionyearsago(mya),andby250myanearlyallofthemajorgroupshaddiverged(1).Mammalsarebelievedtorepresentthebasallineage,andtheirevolutionisseeninthefossilrecordasalongtransitionthroughgroupsofreptiles(synapsids)withprogressivelymoremammaliancharacter-istics,especiallyinjawanatomy.ThefossilbirdArchae-opteryxprovidesstrongsupportforalinkbetweenbirdsanddinosaurs,andcrocodiliansareseenastheclosestlivingrelativesofbirds.Therelationshipsofthesquamates,sphe-nodontids,andturtlesarelessclear,althoughallhavebeenplacedclosertobirdsthantomammals(1).Thisclassicalphylogenyofamnioteshasbeenchallengedbyrecentmorphologicalstudiesoflivingforms(2-4).Traitssuchasasingleaortictrunk,foldedcerebellum,scroll-liketurbinals,loopofHenle(kidney),adventitiouscartilage,andendothermyarefoundonlyinbirdsandmammalsandhavebeenproposedasevidenceforacloserelationship.Analysesoffossilandrecentmorphologicaldataindicatedthatsupportforabird-crocodilianrelationshiprestsprimarilyonthefossildata,andspecificallywithsomemammal-likereptilefossilsthatplacemammalsatthebaseoftheamniotetree(5).Molecularsequencedatafromthreegenes(myoglobin,(3he-moglobin,18SrRNA)havesupportedabird-mammalgroup-ing(6-8),butsequencedatafromseveralothergeneshavenot(8,9).Theseconflictingresultshavecreateduncertaintyaboutourabilitytoresolveamniotephylogeny.Toresolvethephylogeneticrelationshipsofthemajoramniotegroups,especiallytheoriginofbirds,IobtainedDNAsequencesoffourslow-evolvinggenesinrepresentativeamniotes.*MATERIALANDMETHODSCompletesequencesofthreeadjacentmitochondrialgenes(12SrRNA,16SrRNA,tRNAval)wereobtainedfromacrocodilian(Alligatormississippiensis),alizard(Sceloporusundulatus),asphenodontid(Sphenodonpunctatus),andaturtle(Trachemysscripta)andcomparedwithtetrapodse-quencesavailableinthedatabases:bird(Gallusgallus),mouse(Musmusculus),rat(Rattusnorvegicus),cow(Bostaurus),whale(Balaenopteraphysalus),seal(Phocavitu-lina),human(Homosapiens),andfrog(Xenopuslaevis).Thethreepublishedlungfishsequences(10)wereincludedtorootthetree.Forthemitochondrialgenes,thesequencedregioncorre-spondstosites648-3229inthehumansequence(11).The24primersusedforDNAamplificationandsequencingareasfollows[foreach,laboratoryname(L=lightandH=heavystrand),3'locationonthecompletehumansequence,and5'-*3'sequence(IUPACcode)aregiven]:12L1,1091,AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT;12L2,617,AAAGCAWRGCACTGAARATGCTWAGATG;12L3,1497,TGARGCRCGYACACACCGCCCGTCACCCTC;12L4,617,CAAAGCAYAGCACTGAAGATG;12L5,1093,GATTAGATACCCCACTATGC;12H1,1478,TGACTGCAGAGGGTGACGGGCGGTGTGT;12H2,1066,GCATAGTGGGGTATCTAATCCCAGTTTG;12H3,1194,CGRGGKKTATCGATTAYAGAACAGGCTCCTCTAG;12H4,1475,AGGGTGACGGGCGGTGTGTRCG;16L1,2606,CTGACCGTGCAAAGGTAGCGTAATCACT;16L2,2204,GGCCTAAAAGCAGCCACCTGTAAAGACAGCGT;16L2a,2021,CCAAACGAGCCTAGTGATAGCTGGTT;16L3,1854,AGCAAAGAYYAAMCCTYGTACCTTTTGCAT;16L4,2945,ACCAAGTTACCCTAGGGATAACAGCGCA;16L8,2604,TGACCGTGCRAAGGTAGCATAATMA;16L9,2511,CGCCTGTTTATCAAAAACAT;16L11,1802,GTACCGCAAGGGAAAGATGAA;16H1,3056,CTCCGGTCTGAACTCAGATCACGTAGG;16H2,2579,AGTGATTAC GCTACCTTTGCACGGTCAG;16H3,2813,GAGGTTTTT TTTTTCTCCAAGGTCGCCCCA;16H4,1825,ATGCAAAAGGTACRAGGKTTRRTCTTTGCT;16H5,2170,TTCTTTATWGGTGGCTGCTTTTAGGCCYAC;16H6,3259,GGATTTGAACCYCTGRKWWMAAGRKYTTARGYCTT;16H10,2582,TGATTACGCTACCTTTGCACGGT;16H11,1950,TCCCACTCTTTTGCCACAGAGA.Thecodingregionofthenuclearglycolyticenzymegenea-enolasefromacrocodilian(Alligatormississippiensis)wassequencedandcomparedwiththetetrapodsequencesavail-ableinthedatabases:duck(Anasplatyrhynchos),human(Homosapiens),rat(Rattusnorvegicus),andfrog(Xenopuslaevis).Thesequencedregioncorrespondstosites66-1251inthe1305-bpcodingportionofthehumana-enolasegene(12).ThesixprimersusedforDNAamplificationandsequencingareasfollows[foreach,name,3'locationonthecompletehumansequence,and5'-*3'sequence(IUPACcode)aregiven]:AE1,812,GACTTCAARTCYCCCGATGAYCCCAGCAGRTACAT;AE2,1252,CTGAAGTTYYTKCCRGCAAAGCKGGCC
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