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JFieldOrnithol


871961032016DOI101111/jofo12132OvercomingchallengestomorphologicalandmolecularidenticationofEmpidonaxycatchersacasestudywithaDuskyFlycatcherErinLHeller1KevinCRKerr2NorFDahlan3CarlaJDove3andEricLWalter

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1 J.FieldOrnithol. 87(1):96–103,2016DOI:10
J.FieldOrnithol. 87(1):96–103,2016DOI:10.1111/jofo.12132 Overcomingchallengestomorphologicalandmolecular identiÞcationof Empidonax ßycatchers:acasestudy withaDuskyFlycatcher ErinL.Heller, 1 KevinC.R.Kerr, 2 NorF.Dahlan, 3 CarlaJ.Dove, 3 andEricL.Walters 1,4 1 DepartmentofBiologicalSciences,OldDominionUniversity,Norfolk,Virginia23529,USA 2 TorontoZoo,361AOldFinchAvenue,Toronto,OntarioM1B5K7,Canada 3 DivisionofBirds,SmithsonianInstitution,P.O.Box37012,MRC116,Washington,D.C.20013,USA ABSTRACT.Flycatchersinthegenus Empidonax areamongthemostdifcultaviantaxonomicgroupsto identifytospecies.Observersoftenrelyoncallsorsongsintheeldordetailedmorphometricsinthehandto identifyspecies.InJanuaryandFebruary2013,wetwicecapturedan Empidonax ycatcherattheVirginiaZooin Norfolk,Virginia.Afterbeingunabletoidentifytheycatchertospecieslevelusingmorphometricsandphotographs, weextractedDNAfromtwotailfeatherscollectedduringthesecondencountertoidentifytheindividualgenetically. Comparisonofcytochrome c oxidaseI(COI)withreferencesequencesintheBarcodeofLifeDatabase(BOLD) suggestedthatthespecimenhada � 99.8%probabilityofplacementasaDuskyFlycatcher( ). AdditionalcomparisonsofNADHdehydrogenasesubunit2(ND2)toreferencesequencesinGenBank,however, suggestedthatthespecimenwasaPineFlycatcher( EmpidonaxafÞnis ),aspeciesnotrepresentedinBOLDand connedgeographicallytoasmallareainMexicoandGuatemala.AfteranalyzingbothCOIandND2from additionalvoucheredspecimens,thebirdcaughtinVirginiawasdeterminedtobeaDuskyFlycatcher.Wealso suspectthatsomeofthesequencesinGenBankmightderivefromincorrectlyidentiedspecimensorotherwise couldrepresentoverlookedpseudogenes.Becausetheputativeidentication,basedonGenBanksequences,would haverepresentedtherstrecordofPineFlycatcherfromtheUnitedStates,ourresultsreinforcetheneedforcarefully vettedandtaxonomicallycomprehensivemoleculardatabasestoallowdenitiveconclusionsaboutsampleidentity. RESUMEN. RetossobrelaidentiÞcaci « onmorfol « ogicaymoleculardepapamoscasdelgenero Empidonax :uncasodeestudiocon Empidonaxoberholseri LospapamoscasEmpidonaxseencuentranentreelgrupodeavesm ´ asdif ´ cilesdeidenticartaxon ´ omicamente aniveldeespecies.Paraidenticarestosp ´ ajaros,losobservadoresdependen,particularmente,delcantoodelas llamadasodedetallesquesepuedenobservarcuandotienenelaveenlamanoEneneroyfebrerodel2013, endosocasionescapturamosun Empidonax ´ ogicodeVirginia,Virginia.Nopudimosidenticarla especieutilizandofotograf ´ asorasgosmorfom ´ etricosyatalesefectosextrajimosADNdedosdelasplumasdel rabo,paratratardeidenticarelsegundoindividuogen ´ eticamente.Lacomparaci ´ ondelaoxidasaccitocromica I(COI),conlareferenciasecuencialenlabasededatosdel“C ´ odigodeBarrasdelaVida”(BOLD)sugiri ´ oque elesp ´ ecimenten ´ a � 99.8%deprobabil

2 idaddeserun Empidonaxoberholseri .Noobst
idaddeserun Empidonaxoberholseri .Noobstante,unacomparaci onde NADHdehidrogenasa,subunidad2(ND2)delBancoGen ´ etico(GenBank)suger ´ aqueelaveera Empidonax afÞnis ,unaespeciequenoestabarepresentadaenBOLDygeogr ´ acamenteconnadaaun ´ areapeque ˜ nade M ´ exicoyGuatemala.LuegodeanalizarelCOIyelND2deespec ´ menesadicionales,sedetermin ´ oqueelave capturadaenVirginiaera E.oberholseri .SospechamosquelamismasecuenciaenelGenBanksehab ´ aobtenidode aquelaidenticaci ´ onputativa,basadoenlasecuenciadelGenBank,hubierarepresentadoelprimerregistrode un E.afÞnis enlosEstadosUnidos,nuestrosresultadosapoyanlanecesidaddetenermuchocuidadoconlasbases molecularesparapermitirlaidenticaci ´ onconclusivademuestras.Senecesitaunarevisi ´ onmolecularlogeogr ´ aca delos Empidonax pararesolverambig ¨ uedadeshaplot ´ picas. Keywords: Barcoding,BOLD, EmpidonaxafÞnis , Empidonaxoberholseri ,GenBank Empidonax morphologicallysimilarinsectivorouspasser- inesinthefamilyTyrannidae(Sedgwick1993, JohnsonandCicero2002).Becauseoftheirnon- 4 Correspondingauthor.Email:ewalters@odu.edu descriptplumage,theseycatchersaredifcult toidentifyvisually(Phillipsetal.1966,Johnson andCicero2002,Novitchetal.2015).Ofthe 15 Empidonax speciesfoundinNorthAmerica, manyhaverangesthatoverlap.Rangeoverlaps becomemorepronouncedduringfallandspring migrationwhenindividualsaretypicallyless C  2016AssociationofFieldOrnithologists 96 Vol.87,No.1 IdentiÞcationof Empidonax Flycatchers 97 Fig.1.DuskyFlycatcher( Empidonaxoberholseri )caughton27January2013(left)and23February2013 (right)inamist-netattheVirginiaZooinNorfolk,Virginia(photocredit:ErinL.Heller). vocal(Sedgwick2001).Thisleadstofurther challengesincorrectlyidentifyingindividualsto species.Here,wesummarizetheidentication processforanunknown Empidonax ycatcher capturedduringthewinterinVirginia,USA.We describetheprocessbywhichweconrmedits identityandhighlightthedifcultyassociated withmorphologicalambiguitiesanderrorsin geneticrepositoriesforthegenus Empidonax . METHODS On27January2013,wecapturedan Em- pidonax ycatcher(Fig.1)inamist-netat theVirginiaZoo(Norfolk,VA;36.8786 ° N, 76.2774 ° W).Thebirdwasnotbandedbecause identicationwasnotcertain.An Empidonax ycatcherwasagaincaughtatthesamelocation on23February2013,presumablythesame individualwecapturedinJanuary.Whilebeing removedfromthenet,thebirdshedfourtail rectricesthatwecollectedforlaterexamination. Again,wephotographedthebird(Fig.1)before releasingit. Twooffourrectricescollectedwereusedfor geneticanalysisattheFeatherIdenticationLab (SmithsonianInstitution,Washington,D.C.). Calamiwereexcisedfromtherectricesusing sterilescissorsandDNAextractionmethodsfol- lowedDoveetal.(2013).Noother Empidonax specimenswerehandledinthelabatthistime. Usingtheextract

3 edDNA,afragmentofthe cytochrome c oxidas
edDNA,afragmentofthe cytochrome c oxidaseI(COI)geneknownasthe “DNAbarcode”wasampliedusingpolymerase chainreaction(PCR).Thegenefragmentwas sequencedandcomparedagainstalibraryof knownDNAbarcodesintheBarcodeofLife Database(BOLD)usingtheBOLDIdentica- tionSystem(RatnasinghamandHebert2007; http://www.boldsystems.org/),whichincluded 11ofthe15 Empidonax species.Wethen sequencedNADHdehydrogenasesubunit2 (ND2),forwhichall15 Empidonax taxawere representedinGenBank. BecausetheDuskyFlycatcherwasonlyrep- resentedinGenBankbyasinglespecimen,we sequencedbothCOIandND2genesfromve additionalmuseumspecimensofDuskyFly- catchersandalsofromvemuseumspecimensof thecloselyrelatedPineFlycatcher( Empidonax afÞnis ;Table1).PCRandsequencingmeth- odsfollowedDoveetal.(2008)forCOIand Doveetal.(2013)forND2.Weconstructed 98 E.L.Helleretal. J.FieldOrnithol. Table1.SamplesusedforcomparisonwiththeBarcodeofLifeDatabaseandGenBank. Identied MuseumnumberspeciesDateLocalityPutativespecies UWBM82521 E.afÞnis 11August2006LosMimbres,Durango,MX E.afÞnis UWBM82897 E.afÞnis 13May2006Xocomanatlan,Guerrero,MX E.oberholseri UWBM99693 E.afÞnis 23January2009Ixtlan,Oaxaca,MX E.afÞnis UWBM99851 E.afÞnis 31January2008Teopisca,Chiapas,MX E.afÞnis UWBM107797 E.afÞnis 25April2003Bola ˜ nos,Jalisco,MX E.hammondii UWBM80199 E.oberholseri 9June2003CleElumWA E.oberholseri UWBM80291 E.oberholseri 10August2003BlueLake,WA E.oberholseri UWBM85193 E.oberholseri 27May2004MendocinoNatlForest,CA E.oberholseri UWBM90450 E.oberholseri 16June2010LookingGlass,OR E.oberholseri UWBM104552 E.oberholseri 16July2002CassHousePeak,NV E.oberholseri N/AUnknown23February2013Norfolk,VA E.oberholseri UWBM,UniversityofWashingtonBurkeMuseum. maximumlikelihoodtreeswithadditional Empidonax sequencesfromGenBankusing PhyML3.0(Guindonetal.2010)andadded FuscousFlycatcher( Cnemotriccusfuscus )se- quencesfromGenBankasanoutgroup. RESULTS TheCOIsequence(516bp)fromtheun- knownbirdmatchedareferencesequencein BOLDforDuskyFlycatcherwiththeexception ofonenucleotide,andhada99.8%probability ofplacementbasedontheBOLDIdentica- tionSystem(RatnasinghamandHebert2007). DuskyFlycatchersarecloselyrelatedtoPine Flycatchers(JohnsonandCicero2002),aspecies notincludedintheBOLDlibrary.Sequences fromJohnsonandCicero(2002)demonstrated thatthesetwotaxawerevirtuallyidenticalat the3  endofCOI,butweremoreeasilydis- tinguishedbyND2.Wesubsequentlyamplied theND2sequencefortheunknownbirdand compareditwithsamplesinGenBank,which resultedinaclosermatchtothesinglePineFly- catchersequenceinthatdatabase.Thus,based onBOLDmatches,theunknownspecimenwas aDuskyFlycatcher,whereas,basedonGenBank andND2,itwasaPineFlycatcher.BoththeCOI andND2sequencesfromtheunknownbirdare providedinSupplementaryTableS1. WeproducedCOIsequencesforallbut oneDuskyFlycatcher(UWBM104552)and onePineFlycatcher(

4 UWBM99851),and ND2wasproducedforallbuton
UWBM99851),and ND2wasproducedforallbutonePine Flycatcher(UWBM99693)specimen.All newsequencesfromvoucheredmuseumspec- imensweresubmittedtoGenBank(acces- sionnumbers:KT806379-KT806395).Speci- menUWBM107797,originallyidentiedas aPineFlycatcherwhencollected,wasiden- tiedasaHammond’sFlycatcher( E.ham- mondii )basedonCOI(Fig.2).However, theND2sequenceampliedforthisindivid- ualwasuniquelydivergentandcouldrepre- sentanuclear-mitochondrialpseudogene(see Discussion). NoneoftheveputativeDuskyFlycatcher specimensmatchedthetwoND2sequences fromGenBankforDuskyFlycatcher(Fig.3). TheND2sequencefromGenBankforthe PineFlycatcherbelongedtoacladecontain- ingtheunknownspecimen,fourofthenew DuskyFlycatchersequences,andoneofthe newsequencesforthePineFlycatcher(UWBM 82897).TheremainingtwonewPineFlycatcher sequencesformedtheirownuniqueclade,sister totheaforementionedclade.TheCOIresults largelymirroredthoseofND2(Fig.2),with theadditionthatUWBM99693alsojoined thenewPineFlycatcherclade,althoughwith COIitappearstobethesistercladetoHam- mond’sFlycatcher.Ultimately,theCOIand ND2sequencesfromtheunknownVirginiabird matchedcladesthatprimarilyincludeDusky Flycatcher. DISCUSSION TheCOIsequencefromourunknown specimenunambiguouslymatchedareference Vol.87,No.1 IdentiÞcationof Empidonax Flycatchers 99 0.03 E. flaviventris E. traillii E. wrightii UWBM 85193 EO E. alnorum C. fuscus Pseudogene 1 E. alnorum Pseudogene 2 E. oberholseri E. hammondii E. virescens E. flaviventris Pseudogene 1 UWBM 107797 EA UWBM 80291 EO E. virescens Pseudogene 1 UWBM 80199 EO E. alnorum Pseudogene 1 E. fulvifrons UWBM 82897 EA E. occidentalis Cnemotriccus fuscus UWBM 90450 EO UNKNOWN UWBM 99693 EA UWBM 82521 EA E. minimus E. difficilis ? O A H Fig.2.Maximumlikelihoodtreebasedoncytochrome c oxidaseI(COI)for11ofthe15NorthAmerican Empidonax ycatchers,includingHammond’sFlycatcher( E.hammondii ),DuskyFlycatcher( E.oberholseri ), LeastFlycatcher( E.minimus ),GrayFlycatcher( E.wrightii ),Buff-breastedFlycatcher( E.fulvifrons ),Willow Flycatcher( E.traillii ),AlderFlycatcher( E.alnorum ),Yellow-belliedFlycatcher( E.ßaviventris ),Pacic-slope Flycatcher( E.difÞcilis ),CordilleranFlycatcher( E.occidentalis ),AcadianFlycatcher( E.virescens ),anda sequencefromtheFuscousFlycatcher( Cnemotriccusfuscus )asanoutgroup.Fivesequencesfromtwoclades ofpseudogenesarealsoincludedforreference.ThetreewasruninPhyML3.0usingageneraltime- reversibleevolutionarymodel(Guindonetal.2010).Thesufx“EA”indicatesspecimensidentiedupon collectionasthePineFlycatcher( E.afÞnis );thesufx“EO”indicatesthoseidentiedas E.oberholseri . Thegraybarsidentifycladesbelievedtobelongto E.afÞnis (A), E.oberholseri (O),or E.hammondii (H). Thescalerepresentssubstitutionspersite.GenBankaccessionnumbersforsequencesnotgeneratedinthis studyincludeAY666171

5 ,DQ432910,DQ432913,DQ432914,DQ433602,DQ4
,DQ432910,DQ432913,DQ432914,DQ433602,DQ433606,DQ433613, DQ433617,DQ433622,DQ433624,GU116555,GU116559,HM033459,andHM396218.BOLD ProcessIDnumbersforadditionalpseudogenesequencesincludePTYRN010-11,PTYRN020-12,and PTYRN023-12. 100 E.L.Helleretal. J.FieldOrnithol. 0.03 UWBM 104552 EO E. affinis UWBM 85193 EO UWBM 99851 EA UWBM 82897 EA UWBM 80291 EO UWBM 107797 EA UWBM 90450 EO UWBM 80199 EO UWBM 82521 EA UNKNOWN E. hammondii E. minimus E. wrightii E. atriceps E. fulvifrons E. difficilis difficilis E. d. insulicola E. occidentalis E. flavescens E. flaviventris E. virescens E. albigularis E. alnorum E. traillii Cnemotriccus fuscus ? O A H E. oberholseri ? Fig.3.MaximumlikelihoodtreebasedonNADHdehydrogenasesubunit2(ND2),includingall15North American Empidonax ycatchersplusasequencefromtheFuscousFlycatcher( Cnemotriccusfuscus )asan outgroup.ThetreewasruninPhyML3.0usingageneraltime-reversibleevolutionarymodel(Guindon etal.2010).Thesufx“EA”indicatesspecimensidentieduponcollectionas E.afÞnis ;thesufx“EO” indicatesthoseidentiedas E.oberholseri .Thegraybarsidentifycladesbelievedtobelongto E.afÞnis (A), E. oberholseri (O),or E.hammondii (H).Thescalerepresentssubstitutionspersite.Speciesnamesareprovidedin Fig.2.GenBankaccessionnumbersforsequencesnotgeneratedinthisstudyincludeAF447624-AF447629, AF447649,AY030124,AY030125,AY143209-AY143234,andDQ294544. Vol.87,No.1 IdentiÞcationof Empidonax Flycatchers 101 sequenceforDuskyFlycatcherinBOLD,but only11of15 Empidonax specieswererepre- sentedinthedatabase.ComparinganND2 sequencefromtheunknownspecimenrevealed amatchtoapurportedPineFlycatchersequence inGenBank,but,basedonanalysisofadditional specimens,thesinglevoucheroftheputative PineFlycatcher(FMNH393990)wasprobably originallymisidentied. Forthetwospecieswefocusedon,we wouldhaveexpectedtorecovertwodistinc- tiveclades,onerepresentingeachspecies,but, instead,wefoundve.Possibleexplanations forthisincludemisidentiedspecimensand nuclear-mitochondrialpseudogenes.Thefor- merishighlyprobablegiventhatmanyofthe voucherspecimenswerecollectedinpartsof MexicowherethewinterrangesofNorthAmer- icanmigrantspeciesoverlaptherangesoflocal endemics(Johnson1963,MortonandPereyra 1985).Foronespecimen,bothfactorsmight havebeenatplay.TheputativePineFlycatcher (UWBM107797)producedaCOIsequence thatmatchedthoseofHammond’sFlycatcher, butitsND2sequence,thoughcloselyalliedto Hammond’sFlycatcher,wasunique.Giventhe expectedlinkagebetweenCOIandND2,this suggeststhatoneofthetwoND2haplotypes (“H”inFig.3)couldrepresentanuclear- mitochondrialpseudogene.Thisisfurthersup- portedbythevariationinbranchlengthbecause nuclear-mitochondrialpseudogenestendtoex- hibitslowersubstitutionratesthanmitochon- drialgenes(Bensassonetal.2001). Theotherextrahaplotypescouldalsobeat- tributedtonuclear-mitochondrialpseudogenes;

6 intheabsenceofgeographicisolation,mu- t
intheabsenceofgeographicisolation,mu- tationaloneisanunlikelyexplanationgiven thedegreeofdivergence.Ascanofthese- quences,however,didnotrevealthetypi- calhallmarksofpseudogenes(e.g.,indelsand frameshiftmutations),butsimilar“cryptic” nuclear-mitochondrialpseudogeneshavebeen identiedpreviouslyfromthisavianfamily(Kerr 2010,StoeckleandKerr2012).Basedonits basalpositiononthetree(Fig.3),wesuggest thattheND2haplotypereportedfor E.ham- mondii byJohnsonandCicero(2002)ismore likelyanuclear-mitochondrialpseudogene.Of thethreeremaininghaplotypes,one(UWBM 90450)isuniqueanddiscussedfurther.Ofthe othertwo,oneconsistsofthreePineFlycatcher specimens,andtheotherincludesallofthe DuskyFlycatcherspecimensaswellastwo PineFlycatcherspecimensandourunknown specimen.ThePineFlycatcherND2sequencein GenBank(accessionnumberAY143209)could befromamisidentiedspecimen.PutativePine FlycatcherUWBM82897wascollectedin southernMexico,andre-extractionconrmed thebird’splacementinaclusterwithDusky Flycatcher,suggestingthatthisspecimentoo mighthavebeenmisidentied.Therearerecords ofDuskyFlycatchersfromthatpartofMexico atthattimeoftheyear,supportingtheplau- sibilityofamisidentication(Sedgwick1993). Hybridizationandintrogressionareunlikelyex- planationsbecausethebreedingrangesofthese twospeciesdonotoverlap(Nelson1901,AOU 1983,Godfrey1986). PutativeDuskyFlycatcherUWBM90450 haduniquesequencesforbothCOIandND2, butbothwerestillmostsimilartotheexpected speciesidentity(DuskyFlycatcher).Although thiscouldrepresentamutation,multiplecopies oflongandconvincingpseudogeneshavebeen recoveredinothercongeners,suchasAlderFly- catchers( E.alnorum ;StoeckleandKerr2012), andsothepresenceofpseudogenesremainsa possibleexplanation.Nonetheless,becausewe conrmedthatsequencesfromourunknown specimenunequivocallymatchedthesequences ofspecimenscollectedwithinthebreedingrange ofDuskyFlycatchersinNorthAmerica,we believethatourunknownspecimenisaDusky Flycatcher.Misidenticationisthemostpar- simoniousexplanationforthenon-complying PineFlycatcherspecimensfromMexico. ThisistherstrecordofaDuskyFlycatcher fromVirginia(RottenbornandBrinkley2007, eBird2015).However,vagrantsofthisspecies havebeenrecordedatseveralotherlocationsin easternNorthAmerica,includingPennsylvania (1969,Uhrich2000),Ontario(1993,Ridout 1994),Delaware(2002,Burgieletal.2002), threerecordsinAlabama(2009,AlabamaOr- nithologicalSociety2011),andGeorgia(2012, GeorgiaOrnithologicalCommittee2013). Ourresultsreinforcetheneedforcarefully vettedandtaxonomicallycomprehensivemolec- ulardatabasestoallowdenitiveconclusions aboutsampleidentity.Deeperandmorecom- prehensivegeneticreviewofthisandother Tyrannidgeneraisstillwarranted.Giventheir propensityforharboringnuclear-mitochondrial pseudogenes,extremecareshouldbeexercised 102 E.L.Helleretal. J.FieldOrnithol. whenem

7 ployingmitochondrialmarkerswith Tyrant&#
ployingmitochondrialmarkerswith Tyrantycatchers.Measurescanbetakento avoidinadvertentlysequencingpseudogenes (SorensonandQuinn1998),butduetotheir crypticnatureinthisgroup,wholegenomic sequencingmightbenecessarytorevealthe trueextentofmitochondrialtranslocationthat hasoccurredinthisfamily.Ultimately,further studyisneededtodocumentgeographicaland genomicvariationinthe Empidonax genusand renemorphologicaldistinctionsbetweenthe species. ACKNOWLEDGMENTS WethankR.SweeneyandJ.LotzoftheVirginia Zooforgrantingusaccesstomist-netandbandbirds, thevolunteereldassistants(A.Johnson,L.August, E.Cali,andS.Haskell)whoassistedonthedayswhen theDuskyFlycatcherwascaught,andE.Brinkleywho assistedinattemptingtovisuallyidentifythebirdand providedvaluableinputthataidedinwritingthispaper. WealsothankS.Birks,GeneticResourcesManager,at theBurkeMuseuminSeattle,WA,forsendingspecimens, D.Barshisandthreeanonymousreviewersforcomments onapreviousversionofthismanuscript,andG.Ritchison forhelpfulsuggestionstoimprovethemanuscript.The eldcomponentofthisstudywassupportedbyawardsto ELHbytheVirginiaAcademyofScienceSmallProject ResearchFundsandtheVirginiaSocietyofOrnithology JJMurrayResearchAward,andthroughstart-upfunds toELWprovidedbyOldDominionUniversity.The SmithsonianFeatherLabissupportedbyInteragency AgreementswiththeU.S.AirForce,Navy,andFederal AviationAdministration.Theresearchreportedherewas conductedunderpermitsfromOldDominionUniversity, U.S.DepartmentoftheInterior,U.S.FishandWildlife Service,andtheVirginiaDepartmentofGame&Inland Fisheries. LITERATURECITED A LABAMA O RNITHOLOGICAL S OCIETY [online].2011.Al- abamaBirdRecordsCommitteereviewlist. http:// www.aosbirds.org/ABRCreports/abrc_review% 20list_march%202011_update.pdf � (Accessed10 July2015). A MERICAN O RNITHOLOGISTS ’U NION (AOU).1983. Check-listofNorthAmericanbirds,6thed.Ameri- canOrnithologists’Union,Washington,D.C. B ENSASSON ,D.,D.X.Z HANG ,D.L.H ARTL , AND G.M.H EWITT .2001.Mitochondrialpseudogenes: evolution’smisplacedwitnesses.TrendsinEcology andEvolution16:314–321. B URGIEL ,J.C.,R.O.P AXTON , AND D.A.C UTLER .2002. Hudson-Delaware.NorthAmericanBirds56:158– 161. D OVE ,C.J.,N.F.D AHLAN , AND S.V.D ROVETSKI .2013. MtDNAND2sequenceidentiesStreakedHorned Lark( Eremophilaalpestrisstigata )frombirdstriketo USAirForceF-15atPortlandInternationalAirport, Oregon.ConservationGeneticResources5:997– 999. ———,N.C.R OTZEL ,M.H EACKER , AND L.A.W EIGT . 2008.UsingDNAbarcodestoidentifybirdspecies involvedinbirdstrikes.JournalofWildlifeManage- ment72:1231–1236. EBIRD[online].2015.eBird:anonlinedatabaseof birddistributionandabundance.eBird,Ithaca,NY. http://www.ebird.org � (Accessed10July2015). G EORGIA O RNITHOLOGICAL S OCIETY [online].2013. GeorgiaOrnithologicalChecklists&RecordsCom- mitteeActivity. http://www.gos.org/gcrc-a

8 ctivity- 2013 � (Accessed10July20
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