October2000] ShortCommunications 1069

The Auk 117(4):1069-1078, 2000

Major Divisions in OscinesRevealed by Insertionsin the Nuclear Gene c-myc:A Novel Gene in Avian Phylogenetics

PER G. P. ERICSON,TM ULF S. JOHANSSON,1'2 AND THOMAS J. PARSONS3 •Departmentof VertebrateZoology, Swedish Museum of NaturalHistory, Box 50007, SE-104 05 Stockholm, Sweden; 2Departmentof Zoology,University of Stockholm,SE-106 91 Stockholm,Sweden; and 3U.S.Armed Forces DNA IdentificationLaboratory, Armed Forces Institute for Pathology,1413 ResearchBoulevard, Rockville,Maryland 20850, USA

The Passeriformesis a monophyleticgroup classificationof Sibley and Monroe (1990), the di- consistingof more than half of all living spe- chotomyis reflectedby the division of the oscines cies(Raikow 1982). The majorsplit of the into the parvordersCorvida and .The Pas- into the suboscinesand oscinesis well supportedby seridais further divided into the superfamiliesMus- morphologicalcharacters, although a few taxa (e.g. cicapoidea,, and Passeroidea. Acanthisittidae,New Zealandwrens) defy allocation The DNA-DNA hybridizationmethod as applied to either suborder(see Sibley and Ahlquist 1990). by Sibleyand Ahlquisthas been criticized on several Molecular analysescorroborate this dichotomyin grounds,and doubtsconcerning the validity of some passerines(Sibley and Ahlquist1990, Edwards et al. of their resultshave been raised (Cracraft1987, Hou- 1991). de 1987, Sarich et al. 1989, Sheldon and Bledsoe Some oscinefamilies are distinct,but convergent 1993).However, the currentlyfavored method in mo- evolutionapparently is commonand has obscured lecularsystematics, the comparisonof nucleotidese- phylogeneticrelationships, making the subdivision quences,so far has generatedfew phylogenetichy- of thisgroup based on morphologydifficult (Beecher pothesesat thishigh taxonomiclevel in oscines(but 1953, Tordoff 1954, Ames 1971, Raikow 1978, Bledsoe see Edwards et al. 1991, Groth 1998). 1988).In fact, eventhe delimitationsof mostfamilies Here,we presenta hypothesisof phylogeneticre- are uncertain,and only two families,the Alaudidae lationshipsamong oscines based on two previously () and the Hirundinidae ( and mar- undescribedinsertions in exon3 of c-myc.This hy- tins), are unambiguouslydefined (Mayr 1958).Con- pothesisdefines major groups of .C-myc is sequently,oscine relationships at thefamily level and a nuclear proto-oncogenethat encodesa protein aboveare insufficientlyknown, and all taxonomicar- transcriptionfactor that plays a crucialrole in the rangementsare controversial. regulation of cell proliferation and apoptosis(Bou- Besidesthe larksand swallows,three main groups chardet al. 1998).The sequenceof c-mycis highly of oscineshave been recognized based on morphol- conservedthroughout the vertebrates,especially ogy: (1) Old World insect-eatersand their relatives; comparedwith the morerapidly evolving mitochon- (2) New World insect-eatersand ; and (3) drial genes.Although no datesare known for splits crows, birds-of-paradise,and associatedfamilies between the evolutionarylineages studied herein, (Mayr and Greenway1956, Voous 1985). Before the someof themmight be very old, perhapseven of ear- advent of quantitativebiochemical methods, most ly Tertiary age (Feduccia1995). Mutational satura- systematistsrecognized these groups, and the major tion canreduce the resolving power of genesequenc- debate concernedhow they were related (Voous es and might be a problemwhen using mitochon- 1985).Although all combinationsof thethree groups drial genesto study ancientbranching events in havebeen advocated at onetime or another,a major issue is whether the crows and their allies constitute birds. In contrast,dissimilarities between c-myc se- the sister group to all other oscines,or are nested quencesincrease nearly linearly for evolutionarydi- within them.The fully developeddouble pneumatic vergenceswell beyond100 million yearsago (Gray- fossaein the proximalend of thehumerus present in beal 1994). To investigateearly avian divergences, many oscines,but not in crowsand alliesor in the we havesequenced about 500 base pairs of exon3 of suboscines(Bock 1962), suggests the existenceof a this genefor morethan 150 speciesrepresenting 65 cladeincluding all oscinesexcept crows and their al- nonpasserineand 36 passerinefamilies. Our results lies. This dichotomyhas been supportedby DNA- confirm the slow rate of evolutionof c-mycin birds. DNA hybridization studies (Sibley and Ahlquist The maximum sequencedivergence observed was 1990,Harshman 1994,Sheldon and Gill 1996).In the about11%, and only threeindels occurred. Only one indel, an insertion of four amino acids relative to the publishedchicken sequence, has been observed out- 4E-mail: [email protected] sidethe passerines. 1070 ShortCommunications [Auk, Vol. 117

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Methods.--Representativesof 46 passerinefamilies the hypothesisbased on DNA-DNA hybridizationof were selectedfor study (Table1). Specialemphasis a sister-grouprelationship between the Corvidaand wasplaced on samplingthe superfamily Passeroidea all other oscines. In most families, this extra amino sensuSibley and Ahlquist(1990). If not statedoth- acidis a threonine.However, it is a prolinein Hirundo erwise,the usage of familyand subfamily names fol- and Sylviaand a serinein Certhia,Carduelis, and Ic- lowsMorony et al. (1975).However, at higherlevels, terus. i.e. superfamiliesand parvorders,we use the termi- At position991, the ,Fringillidae, Em- nology of Sibley and Ahlquist (1990) to facilitate berizidae, Parulidae, and Icteridae share an addition- comparisonsbetween our resultsand their phylo- al insertion of three amino acids relative to the chick- genetichypotheses. en (Table2). The first two of theseare alwaysa serine We extracted genomicDNA from tissue or blood and a glycine.The third amino acid varies more usingstandard techniques of ProteinaseK/SDS di- amongthe families.Most taxa have a serine,but mo- gestionfollowed by phenol chloroformextraction tacillids (Motacilla and Anthus) have threonine; and ethanolprecipitation, or by QIAamp DNA ex- Geothlypis,Parula, Carpodacus, and Icterushave leu- traction kits following manufacturer'srecommen- cine; Conirostrumhas phenylalanine;and Carduelis dations.Amplification was performedwith primer hastryptophan. Some silent third-position variation pairs mycEX3A (CAAGAAGAAGATGAGGAAAT) in codoncoding also occursfor this third inserted and RmycEX3A (TTAGCTGCTCAAGTTTGTG), or amino acid. mycEX3D(GAAGAAGAACAAGAAGAAGATG) and Discussion.--Weconsider the passerinec-myc in- RmycEX3D(ACGAGAGTTCCTTAGCTGCT), devel- sertionsdescribed here to representtwo uniqueevo- oped by ThomasJ. Parsons.Sequencing was per- lutionary events,with no reversalsevident in the formedwith primersmycEX3A and RmycEX3A using taxa studied.This patternis stronglysuggested by Perkin Elmer Applied BioSystems373 or 377 auto- the extremerarity of indelsin c-mycexon 3 through- matedfluorescent sequencing instruments, and Per- out aviantaxa. For example, among 102 nonpasserine kin Elmer Applied BioSystemsPRISM terminatorcy- speciesstudied, representing65 families, only one cle sequencingkits with AmpliTaq FS polymerase indel has been observed. This insertion of four amino (eitherstandard rhodamine and BigDyechemistries acidsrelative to the chickensequence occurs at po- were employed).Sequence assembly was performed sition796, i.e. at a differentposition than the passer- using the Perkin Elmer Applied BioSystemsSe- ine insertionsreported here. The conservationin se- quenceNavigator or the DNASTARSeqMan II pro- quencelength of c-mycmay be dueto thefact the myc grams. Alignments of completed sequenceswere protein has a helix-loop-helixstructure that must performedby eye. Indicationsof sequencepositions form a heterdimericcomplex with the regulatory throughoutthis reportare relativeto the numbering Max protein. The centralregulatory role of mycin of the full-length protein-codingsequence of the cell division and developmentlikely would tolerate chicken(Watson et al. 1983). little functionalvariation (Bouchard et al. 1998,Eilers Results.--Nucleotidesequences of exon3 of c-myc 1999).Length changes may be rare owing to a re- havebeen studied in 80 speciesof suboscineand os- quirementfor radicalcompensatory changes in other cine passerines,representing 46 traditional families genes,with reversalsencountering an evolutionary (Table1). The sequencesvary from 498 to 510 bases hurdle of equivalentmagnitude. Table 2 indicates (correspondingto 166 to 170 aminoacids) in length that multiple amino-acid substitutionshave oc- as a consequenceof the presenceor absenceof two curredwithin the singleamino-acid insertion, with insertionsconsisting of one and threeamino acids, possiblythree substitutionsof prolinefor threonine respectively.These two insertionshave not beenob- and two substitutions of serine for threonine. This servedamong 65 nonpasseriformfamilies, but they furthersupports the low rateof indelmutations com- appearto exhibit consistenttaxonomic distributions pared with the alreadyslow rate of amino-acidse- within the Passeriformes(with no reversalsinferred quencesubstitution. Likewise, the third aminoacid on the portionsof the tree where relationshipsare of the three that are inserteddisplays substantial well established).Thus, they presumablyrepresent variationwithin relatedgroups, whereas the length unique and significantevolutionary events in pas- of insertion remains constant. serine evolution. The insertion involving a single amino acid ob- The ancestralstate in passerinesof no insertions servedin the c-mycsequence is a synapomorphyfor was observed in all nonpasseriformsinvestigated all oscinesthat we studied,except species in the par- and also was found in all suboscine and vorder Corvida (Fig. 1). This observationsupports families (Table 2). All oscine families representing the sister-grouprelationship of the corvidsand their theparvorder Passerida that we examinedpossessed alliesrelative to otheroscines, as suggested by DNA- an insertionof a singleamino acid at nucleotidepo- DNA hybridization (Sibley and Ahlquist 1990, sition 793 relative to the chickenc-myc sequence Harshman 1994, Sheldon and Gill 1996). Unfortu- (Watsonet al. 1983).The occurrenceof this insertion nately,only one representativeof the superfamily in all oscinepasserines except the Corvidasupports Menuroidea was available to us. 1074 ShortCommunications [Auk,Vol. 117

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Passerinestypically have 10 primaries, which is generallyagreed to be the ancestralcondition. In severaloscine families, the outermostprimary is sec- ondarilyreduced or lost,and species in thesegroups are effectivelynine-primaried. Which families are nine-primariedhas been a matter of considerable confusion,however. Some families that areregarded as "nine-primaried" include speciesin which the tenth primary is in fact present,although vestigial. A long-recognizedgroup of truly nine-primaried familiesis the so-called"New World nine-primaried oscines" that consist of the Parulidae, Emberizidae (Emberizinae,Thraupinae, Cardinalinae), and Icter- idae (Raikow1978, Feduccia 1996). Although not all of thesefamilies are confinedto the New World,they are concentrated there. All representativesof the New World nine-pri- maried oscinesthat we analyzed(Parulidae, Ember- izinae,Thraupinae, Cardinalinae, and Icteridae)pos- sessthe insertionof threeamino acids at position991 in the chickensequence. This is a strongindication of the sharedcommon ancestry of this group.More- over,the Fringillidaeand Motacillidae also share this insertion.The fringillids and motacillidsare includ- ed in the Passeroideaby Sibleyand Ahlquist(1990), along with the New World nine-primariedoscines. However, in other families in Passeroideaand stud- ied herein (Alaudidae, Nectariniidae,Dicaeidae, Es- trildidae, Passeridae,and Prunellidae),this insertion is absent.The c-mycdata thus supporta con- sistingof the New Worldnine-primaried oscines, the primarily Old World finches,and the wagtailsand . The Motacillidaehave a vestigial tenth pri- mary and traditionallyhave not beenthought to be closelyrelated to the New World nine-primariedos- cines,although cytochrome-b sequence data suggest them to be closer to the Emberizidae than are the Fringillidae(Groth 1998).Cytochrome-b sequence data also suggestthat the ten-primariedPasseridae are nestedwithin this cladeof emberizids,fringil- lids, and motacillids(Groth 1998).This arrangement is not supportedby c-mycdata, becausethe three speciesof Passeridae(= sensu Morony et al. 1975) we studied do not share the insertion of three aminoacids with the restof the group. It could be argued that the insertionsreported herein, as single characters,should not be afforded more weightthan othermolecular characters. How- ever, we believe that these insertionsrepresent uniqueevolutionary events of unequivocalhomolo- gy, with no reversal.As such,they presentpowerful evidenceregarding relationships within passerines that havebeen difficult to resolvebased on otherpo- tentially quite homoplasticcharacters. The greatly increasedsignificance of unique molecular rear- rangementshas been recognizedelsewhere (Batzer et al. 1996), and shared indels in protein-coding genespreviously have been interpreted as strong markersfor monophylyas long as the observations October2000] ShortCommunications 1077

Muscicapoidea, Sylvioidea, Motacillidae, Alaudidae, Fringillidae, Dicaeidae, Emberizidae, suboscines, Nectadniidae, Parulidae, Non-passerines Corvida Passeridae Icteridae ••. •. /• insertionofthree •. y aminoacids • .•' (= synapomorphyof a •. •' . subsetofPasseroidea) '• '• •' •\ insertionof one • y aminoacid • .•' (= synapomorphy • ofPasserida) FIG.1. Major divisionsof passerinesas indicated by insertionsof aminoacids in the nucleargene c-myc. Thefirst insertion is synapomorphicfor theparvorder Passerida (sensu Sibley and Ahlquist 1990), whereas all representativesof the New Worldnine-primaried oscines, the primarilyOld Worldfinches, and the Mo- tacillidae share a second insertion of amino acids.

are basedon wide taxonomicsampling (van Dijk et LITERATURE CITED al. 1999).We studiedsequences from morethan 110 families of passerinesand nonpasserines.The ex- AMES,P. L. 1971.The morphologyof the syrinxin tremelow frequencyof indelsin c-myc,and the tax- passerinebirds. Bulletin of the PeabodyMuse- onomicdistribution of insertionsthat we report,in- um of Natural History 95:151-262. dicatethat these should be consideredhighly signif- BATZER,g. g., S.S. ARCOT, J. W. PHINNEY, g. ALE- GRIA-HARTMAN, D. H. KASS, S. M. MILLIGAN, C. icantcharacters for elucidatingthe evolutionof pas- serines. KIMPTON, P. GILL, g. HOCHMEISTER, P.g. IOAN- NOU, R. J. HERRERA, D. g. BOUDREAU, W. D. Acknowledgments.--Tissueand blood sampleswere SCHEER,B. J. KEATS, P. L. DEININGER, AND M. kindly put at our disposalby the ZoologicalMuse- STONEKING. 1996. Genetic variation of recent um, Copenhagen(Peter Arctander, Jon Fjeldsfi, and Alu insertionsin human populations.Jornal of JaimeGarcia-Moreno); the Australian Museum, Syd- Molelular Evolution 42:22-29. ney (WalterBoles); and the Departmentof Zoology, BEECHER,W. J.1953. A phylogenyof theoscines. Auk Universityof Gothenburg(Staffan Andersson). The 70:270-333. othersamples used belong to the SwedishMuseum BLEDSOE,g. 1988.Nuclear DNA evolutionand phy- of NaturalHistory. Most of thesewere collected dur- logeny of the New World nine-primariedos- ing extensivefieldwork in Paraguayas part of col- cines. Auk 105:504-515. laboration with the Museo Nacional de Historia Nat- BOCK,W. J. 1962.The pneumaticfossa of the humer- ural del Paraguay,San Lorenzo.The Direccionde us in the Passeres. Auk 79:425-443. ParquesNacional y Vida Silvestreissued the neces- BOUCHARD, C., P. STALLER, AND M. EILERS. 1998. sary collectingand exportspermits in Paraguay.The Control of cell proliferationby myc.Trends in MagnusBergvails Stiftelse, Olle och Signhild Engkv- Cell Biology8:202-206. ists Stiftelser, and the Swedish Natural Science Re- CRACRAFT,J. 1987. DNA hybridizationand avian searchCouncil (grant B-AA/ BU 01913-304)funded phylogenetics.Evolutionary Biology 21:47-96. this project.Michael J. Braunand the Smithsonian EDWARDS, S. V., P. ARCTANDER, AND A. C. WILSON. Laboratoryof MolecularSystematics provided initial 1991.Mitochondrial resolution of a deepbranch materialsupport to TJPand stronglyencouraged in- in the genealogicaltree for perchingbirds. Pro- vestigationof thephylogenetic potential of thec-myc ceedingsof theRoyal Society of LondonSeries B gene for resolving avian relationships.Gullevi 243:99-107. Bergkvistand Mari K•illersj6provided advice and EILERS,g. 1999. Control of cell proliferatin by myc assistancein thelaboratory. The paper has benefitted family genes.Molecules and Cells 9:1-6. significantlyfrom the commentsof ScottV. Edwards, FEDUCCIA,g. 1995. Explosiveevolution in Tertiary FrederickH. Sheldon,and an anonymousreviewer. birds and mammals. Science 267:637-638. 1078 ShortCommunications [Auk, Vol. 117

FEDUCCIA,A. 1996.The origin and evolution of birds. lecular systematics,1970s to 1990s.Annual Re- Yale University Press,New Haven, Connecticut. view of Ecologyand Systematics24:243-278. GRAYBEAL,A. 1994.Evaluating the phylogeneticutil- SHELDON,E H., AND E B. GILL. 1996. A reconsider- ity of genes:A searchfor genesinformative ationof songbirdphylogeny, with emphasison about deep divergenciesamong vertebrates. titmice and their sylvioid relatives.Systematic SystematicBiology 43:174-193. Biology 45:473-495. Gr•Oq'H,J.G. 1998.Molecular phylogenetics of finches SIBLEY,C. G., AND J. E. AHLQUIST.1990. Phylogeny and sparrows:Consequences of characterstate and classificationof birds. YaleUniversity Press, removal in cytochromeb sequences.Molecular New Haven, Connecticut. Phylogeneticsand Evolution10:377-390. SIBLEY,C. G., AND B. L. MONROE. 1990. Distribution HARSHMAN,J. 1994.Reweaving the tapestry:What and taxonomyof birds of the world. Yale Uni- can we learn from Sibley and Ahlquist (1990)? versity Press,New Haven, Connecticut. Auk 111:377-388. TORDOFF,H. B. 1954.A systematicstudy of the avian HOUDE,P. 1987. Critical evaluationof DNA hybrid- family Fringillidaebased on the structureof the skull. Miscellaneous Publications Museum of ization studiesin avian systematics.Auk 104:17- 32. ZoologyUniversity of Michigan81:7-41. VAN DIJK, M. A.M., E. PARADIS, E CATZEFLIS, AND MAYR,E. 1958.The sequenceof songbirdfamilies. Condor 60:194-195. W. W. DE JONG.1999. The virtues of gaps:Xe- narthran (Edentate) supported by a MAYR, E., AND J. C. GREENWAY.1956. Sequenceof unique deletionin •xA-crystallin.Systematic Bi- passerinefamilies (Aves).Breviora 58:1-11. ology 48:94-106. MORONY, J. J., Jr., W. J. BOCK, AND J. FARRAND,JR. Voous, K. H. 1985. Passeriformes.Pages 440-441 in 1975. Reference list to the birds of the world. A dictionaryof birds (B. Campbelland E. Lack, American Museum of Natural History, New Eds.).T. and A.D. Poyser,Carlton, United King- York. dom. RAIKOW,R. J. 1978. Appendicular myology and re- WATSON, D. K., E. P. REDDY, P. H. DUESBERG,AND t. lationshipsof the New Worldnine-primaried os- S. PAPAS.1983. Nucleotidesequence analysis of cines(Aves: Passeriformes). Bulletin of the Car- the chickenc-myc gene reveals homologous and negieMuseum of Natural History 7:1-43. uniquecoding regions by comparisonwith the RAIKOW,R. J. 1982.Monophyly of the Passeriformes: transforming gene of avian myelocytomatosis Test of a phylogenetichypothesis. Auk 99:431- virus MC29, delta gag-myc.Proceedings of the 445. National Academy of SciencesUSA 80:2146- SARICH, V. M., C. W. SCHMID, AND J. MARKS. 1989. 2150. DNA hybridizationas a guideto phylogenies:A critical analysis.Cladistics 5:3-32. Received1 June1999, accepted28 April 2000. SHELDON, E H., AND g. H. BLEDSOE.1993. Avian mo- Associate Editor: F. H. Sheldon

TheAuk 117(4):1078-1080,2000

SpecializedExtrapair Mating Display in WesternBluebirds

JANISL. DICKINSON,1 KEN KRAAIJEVELD,AND FEMMIESMIT-KRAAIJEVELD HastingsNatural History Reservation and Museum of VertebrateZoology, University of California, 38601 EastCarmel Valley Road, Carmel Valley, California 93924, USA

Western Bluebirds (Sialia mexicana)are socially (Dickinsonand Akre 1998).DNA fingerprintinghas monogamous,maintain long-termpair bonds,and revealed that more than 45% of females have at least share equally in biparental care (Dickinson et al. one offspring sired by a male outsidethe family 1996). Femalesoften have extrapair young in their group and that 19% of offspringare siredby extra- nestseven though males exhibit kin-based winter so- pair males(Dickinson and Akre 1998).Paired males ciality and sometimeshelp at the nestsof relatives follow their matesclosely during the receptivepe- riod, a behavior that dramaticallyreduces the fre- quencyof extrapaircopulation (EPC) attempts (Dick- E-mail: [email protected] inson and Leonard 1996, Dickinson 1997). As a con-