JOURNALOFAVIANBIOLOGYJOURNALOFAVIANBIOLOGY351351432004JOURNALOFAVIANBIOLOGY3522004 thegenusAerodramuswhichincludesecholocatingspeciesfromotherswiftlettaxainthegeneraHydrochousBrooke1972Medway ID: 187849
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TheevolutionofecholocationinswiftletsJ.JordanPrice,KevinP.JohnsonandDaleH.ClaytonPrice,J.J.,Johnson,K.P.andClayton,D.H.2004.Theevolutionofecholocationinswiftlets.J.AvianBiol.35:135Theabilitiesofsomecave-nestingswiftletstoecholocatehastraditionallybeenusedtoseparatethegenusAerodramus JOURNALOFAVIANBIOLOGYJOURNALOFAVIANBIOLOGY35:135143,2004JOURNALOFAVIANBIOLOGY35:2(2004) thegenusAerodramus,whichincludesecholocatingspecies,fromotherswiftlettaxainthegeneraHydrochous(Brooke1972,MedwayandPye1977).ThisschemelargelydependsonecholocationfirstappearingintheimmediateancestorsoftheAerodramusclade,however,onlytwoofthethreespeciescurrentlyrecognizedashavebeenshowntolackthisability(CranbrookandMedway1965,Medway1967,Fenton,1975).Thepresenceorabsenceofecholocationinthethirdspecies,Collocaliatroglodytes,hasbeenconsideredanopenquestion(ChantlerandDriessens1995,Chantleretal.2000),yetitiscrucialforunder-standinghowecholocationevolvedinthisaviangroup(MedwayandPye1977).HerewereportforthefirsttimethepresenceofecholocationinC.troglodytes,thepygmyswiftlet.Recordingsofecholocationclicks,aswellastissueformolecularanalyses,wereobtainedfromaC.troglodytesrepresentativecollecteddirectlyoffitsnestintotaldarkness30metersinsideacave(Fig.1).ApreviousmolecularanalysisoftheswiftletsbyLeeetal.(1996)usingpartialcytochrome)sequencessuggestedthatthegeneraAerodramusarenotsistercladesand,accordingly,thatC.troglodytesrelativelydistantlyrelatedtoecholocatingAerodramustaxa.Giventhisproposedphylogeny,ourfindingwouldsuggestthatecholocationevolvedindependentlyinthetwoclades,asthisabilityisunlikelytohavearisenonceandthenbeenlostrepeatedly(MedwayandPye1977).However,theevidencepresentedbyLeeetal.(1996)againstswiftletmonophylywasnotstrong.Inthisstudy,weconstructamolecularphylogenyfortheswiftletsusingsequencedatafromtwomitochon-drialgenes,cytandNADHdehydrogenasesubunit2(ND2),andusethistreetoreconstructtheevolutionofecholocationusingournewdataconcerningthisabilityinthegenus.Ourprincipalobject-ivesareto:(1)testthemonophylyofswiftlets,andinsodoingconfirmthephylogeneticpositionofC.troglodytesrelativetothegenusAerodramus,and(2)investigatetheoriginofecholocationandchangesintheacousticstructureoforientationsounds(e.g.,singleclicksversusdoubleclicks)duringswiftletevolution.SequencingDNAwasextractedfrommuscleorbloodtissuefrom60individualsrepresenting38speciesandsubspeciesofswiftsandswiftlets(Table1).TaxonomyfollowsChan-tleretal.(2000).Atreeswift,Hemiprocnecomata,wasusedasanoutgrouptorootthetree.WeusedPCRtoamplifyaportionofthecyt-geneandalloftheND2geneusingtheprimersL14841(Kocheretal.1989)andH4a(Harshman1996)forcytandL5215(Hackett1996)andH6313(JohnsonandSorenson1998)forND2.ReactionconditionsfollowedJohnsonandClay-ton(2000).TissuefromHydrochousgigas,thewaterfallswiftlet,wasobtainedfromthestudyofLeeetal.(1996),butwewereunabletoobtainanyPCRamplificationfromthissample.DirectsequencingofPCRproductswasperformedforeachgeneusingthePCRprimersandtheinternalprimersH15299(Kocheretal.1989)andL15517(JohnsonandSorenson1998)forcytandL5758s-GGYTGAATRGGACTWAACCARAC-3)andH5766s(5-GATGAGAAGGCYAGGATTTTTCG-)forND2.SequencingwasperformedasdescribedbyJohnsonandClayton(2000).Wereconciledcomplemen-tarystrandsandalignedsequencesacrossspeciesusingSequencher(GeneCodes,Madison,Wisconsin).PortionsoftheflankingtRNAgeneswereincludedinthealignment,butindelswereinfrequentmakingalignmentstraightforward.Wefoundnoevidencefornuclearcopies,basedonthelackofindels,stopcodons,ormultiplepeaksinoursequencinganalyses(Sorensonand Fig.1.VouchernestofapygmyswiftletCollocaliatroglodyteslocatedintotaldarknessapproximately30metersinsidetheentranceofBaletecaveonSamalIsland,Philippines.Thenest,whichwasanchoredtoaverticalwallwithcopiousamountsofsalivacement,wascomposedofÞnevegetableÞbersheldtogetherwithsaliva.ItcontainedtwoeggsbeingincubatedbyabirdremoveddirectlyfromthenestforpositiveidentiÞcation.Toourknowledge,thisistheÞrstillustrationofthenestofthespecies.IllustrationbySarahBush.JOURNALOFAVIANBIOLOGY35:2(2004) Quinn1998).Wetreatedgapsasmissingdatainallanalyses.Thealignedregionforcyttotaled1058bpandthatforND2totaled1078bp.ThesesequenceshavebeendepositedinGenBankundertheaccessionnum-bersAY294424toAY294483andAY204486toAY294545. Table1.Tissuesamplesincludedinthephylogeneticanalysis.Taxon*VoucherInformationGeographicLocationAerodramussalangananatunaeDMT002Gomantongcaves,Sabah,MalaysiaAerodramussalangananatunaeDMT047Gomantongcaves,Sabah,MalaysiaAerodramussalangananatunaeDMT048Gomantongcaves,Sabah,MalaysiaAerodramusfuciphagusDMT027Gomantongcaves,Sabah,MalaysiaAerodramusfuciphagusDHC40Gomantongcaves,Sabah,MalaysiaAerodramusfuciphagusgermaniDHC04BalambanganIs.,Sabah,MalaysiaAerodramuselaphrusDHC59SeychellesAerodramuselaphrusDHC60SeychellesAerodramuselaphrusDHC61SeychellesAerodramusfrancicusDHC52MauritiusAerodramusfrancicusDHC53MauritiusAerodramusspodiopygiusassimilisDHC36Suva,FijiAerodramusspodiopygiusassimilisDHC37Suva,FijiAerodramusspodiopygiusspodiopygiusDHC31WesternSamoaAerodramusspodiopygiusassimilisDHC39Suva,FijiAerodramusanikorensispalawanensisDHC01BalambanganIs.,Sabah,MalaysiaAerodramusanikorensispalawanensisDHC06BalambanganIs.,Sabah,MalaysiaAerodramusmearnsiSEA116Mindanao,PhilippinesAerodramusbartschiDHC77Oahu,HawaiiAerodramussawtelliW3Atiu,CookIslandsAerodramusanikorensislugubrisUWBM58708RennellIs.,SolomonsAerodramusmaximuslowiDHC117MadaiCaves,Sabah,MalaysiaAerodramusmaximuslowiDHC120MadaiCaves,Sabah,MalaysiaAerodramusmaximuslowiDMT040Gomantongcaves,Sabah,MalaysiaAerodramusmaximuslowiDMT042Gomantongcaves,Sabah,MalaysiaAerodramusmaximuslowiDHC03BalambanganIs.,Sabah,MalaysiaAerodramusbreirostrisDHC66TangkubanPrahu,Java,IndonesiaAerodramusterraereginaeterraereginaeDHC20TullyGorge,Queensland,AustraliaAerodramusterraereginaeterraereginaeDHC30TullyGorge,Queensland,AustraliaAerodramusterraereginaeterraereginaeDHC28TullyGorge,Queensland,AustraliaAerodramuswhiteheadiCMNH37000Mindanao,PhilippinesCollocaliaesculentacyanoptilaDHC88LahadDatu,Sabah,MalaysiaCollocaliaesculentacyanoptilaDHC97LahadDatu,Sabah,MalaysiaCollocaliaesculentacyanoptilaDMT051Sandakan,Sabah,MalaysiaCollocaliaesculentacyanoptilaDMT050Sandakan,Sabah,MalaysiaCollocaliaesculentacyanoptilaDMT057Selangor,MalaysiaCollocaliaesculentacyanoptilaDMT059Selangor,MalaysiaCollocaliaesculentabagoboATP92.131Mindanao,PhilippinesCollocaliaesculentabagoboATP92.280Mindanao,PhilippinesCollocaliaesculentamarginataFMNH358301Sibuyan,PhilippinesCollocaliaesculentamarginataFMNH358303Sibuyan,PhilippinesCollocaliaesculentabeckiUWBM60227IsabelIs.,SolomonsCollocaliaesculentanitensMSP068NewGuineaCollocalialinchiDHC72Bogor,Java,IndonesiaCollocaliatroglodytesFMNH358312Sibuyan,PhilippinesApusaffinisSMG3775PakistanApusapusPL05UnitedKingdomApusmelbaCTCSouthAfricaCypsiurusbalasiensisDHC45KualaLumpur,MalaysiaCypsiurusparLSUB34256SouthAfricaHirundapuscaudacutusUWBM46916RussiaChaeturapelagicaMCP96.1343USAChaeturaELB05Oregon,USAChaeturachapmaniJWF86.065BrazilChaeturacinereiSML1187PeruNeafrapuscassiniPRS2081CentralAfricanRepublicCypseloidesnigerCTC02USAStreptoprocnezonarisPRS791VenezuelaCypseloidesphelpsiGFB3015VenezuelaHemiprocnecomataCMNH38185Philippines*TaxonomyfollowsChantleretal.(2000).ATPA.TownsendPeterson;CMNHCincinnatiMuseumofNaturalHistory;CharlieCollins;DHCDaleClayton;DMTDanTompkins;ELBEvelynBill;FMNHFieldMuseumofNaturalHistory;GFBGeorgeBarrowclough;JWFJohnFitzpatrick;LSULouisianaStateUniversity;MSPMichaelPutnam;PatLee;PRSPaulSweet;SEASarahAl-Tamimi;SMGSteveGoodman;SMLScottLanyon;UWBMUniversityofWashingtonBurkeMuseum;WGrahamWragg.JOURNALOFAVIANBIOLOGY35:2(2004) PhylogeneticanalysesPAUP*(Swofford2002)wasusedforallanalysesofthemoleculardata.Weconductedapartitionhomogeneitytest(Farrisetal.1994,1995,Swofford2002)toevaluatepossibleevidenceforconflictingsignalbetweenthecyt-andND2datasets.Becausetherewasnoevidenceforsignificantconflict(P0.48),weconductedallfurtheranalysesonthecombineddata.Wefirstanalyzedthecombineddatasetusingunorderedparsimonywith100randomadditionTBRreplicates.Toassessthesensitiv-ityofthetreetocharacterresampling,webootstrappedthedataset(Felsenstein1985)using1000bootstrapreplicates.Toassessthesensitivityofthetreetopologytomethodofanalysis,wefurtheranalyzedthedatausingmaximumlikelihood.Weestimatedthebestfitmodelthatcouldnotberejectedinfavorofasimplermodel(HuelsenbeckandCrandall1997)usingModeltest(PosadaandCrandall1998).Thisanalysisrevealedthatamodelincorporatingunequalbasefrequencies,ageneraltimereversiblesubstitutionmatrix,invariantsites,andrateheterogeneityaccordingtoagammadistributionG)couldnotberejectedinfavorofsimplermodels.Weusedtheparametersestimatedbythisanalysisinmaximumlikelihoodsearchesofthecom-bineddata(10randomadditionreplicateswithTBRbranchswapping).Wealsoused100bootstrapreplicatestoassessthesensitivityofthetreetopologytocharacterAnalysisofecholocationsoundsTape-recordingsofswiftletecholocationclickswereobtainedinthefieldbyD.H.Claytonandfromavarietyofothersources(Table2).OrientationsoundsofCollocaliatroglodytesAerodramusbreirostriswererecordedafterreleasingsingleindivi-dualsintodarkrooms.IndividualsofCollocalialinchiC.esculentawerealsoreleasedintodarkroomstoensurethattheycouldnotecholocate.Asinpreviousstudiesoftheselatterspecies(Medway1967,Fenton1975),thebirdsproducednoaudibleclicksduringthesetrialsandrepeatedlyflewintoobstacleswhendeprivedofvisualcues.Mostrecordingsofotherecholocatingspecieswereoffree-flyingbirdsenteringorleavingtheirroostcaves.Soundsofseveraltomanyindividualsweretypicallypresentonarecording.Foreachtaxon,weselectedafewoftheleastdistortedexamplesofecholocationclicksforanalysis.WedidnotobtainrecordingsoftheAustralianswiftlet,Aerodramuster-raereginae(formerlyCollocaliaspodiopygia).However,echolocationinthisspecieshasbeendescribedindetailinpreviousinvestigations(e.g.,GriffinandThompson1982,SuthersandHector1982,SmythandRoberts1983,Marchantetal.1999),soweincludedthisinformationinourstudy.Inall,weexaminedtheecholocationclicksof12ofthe16Aerodramusincludedinthemolecularanalysis,aswellastheclicksofC.troglodytesSpectrogramsofswiftletecholocationsoundsweregeneratedusingCanarysoundanalysissoftware(Ver-sion1.2.4,CornellLaboratoryofOrnithology;samplingfrequency22.05kHz,frequencyresolution349.7Hz,temporalresolution11.6ms,93.75%overlapofframesinsuccessivetransforms).Weclassedswiftletsintothosewithsingleclicksandthosewithdoubleclicksbyvisualinspectionofon-screenspectrograms.Fortaxawithdoubleclicks,wemeasuredthepausebetweenthetwopulses,orintraclickinterval,forseveralexamplesfromeachindividual.Previousanalyseshavesuggestedthatintraclickintervalscanvarywithinanindividual(SuthersandHector1982)anddonotdifferconsistently Table2.Recordingsofecholocationsoundsincludedinthestudy.TaxonGeographicLocationRecordist/Source*CollocaliatroglodytesPhilippinesD.H.ClaytonAerodramuselaphrusSeychellesD.H.ClaytonAerodramuselaphrusSeychellesA.Gretton(NSA-28945)AerodramusfrancicusMauritiusD.H.ClaytonAerodramusspodiopygiusspodiopygiusSamoaR.I.Orenstein(LNS-20510)AerodramusterraereginaeQueensland,AustraliaFromliteratureAerodramusbreirostrisJava,IndonesiaD.H.ClaytonAerodramussalangananatunaeSabah,MalaysiaBBC(NSA-CDR16/17)AerodramusanikorensispalawanensisPalawan,PhilippinesC.CollinsAerodramusbartschiHawaii,USAD.H.ClaytonAerodramussawtelliAtiu,CookIslandsJ.H.Fullard(9recordings)AerodramusmaximuslowiSabah,MalaysiaBBC(NSA-MB6/17)AerodramusmaximuslowiSabah,MalaysiaBBC(NSA-CDR16/24)AerodramusmaximuslowiSarawak,MalaysiaLordMedway(NSA-22458)AerodramusfuciphagusgermaniPahang,MalaysiaA.B.VandenBerg(LNS-36421)AerodramusfuciphagusSabah,MalaysiaBBC(NSA-CDR16/19)BritishMuseumNationalSoundArchive;LNSMacaulayLibraryofNaturalSounds,CornellLaboratoryofOrnithology;BBCBritishBroadcastingCorporation.ClickcharacteristicsobtainedprimarilyfromSuthersandHector(1982).JOURNALOFAVIANBIOLOGY35:2(2004) amongsomespecies(MedwayandPye1977),however,nopreviousstudyhascomparedthistraitacrossalargenumberofswiftlettaxa.Wedidnotincludemeasuresoffrequencycharacteristicsinouranalysisbecausethissoundcomponentismorelikelytobeinfluencedbysuchfactorsasacousticcharacteristicsoftherecord-ingenvironment,thedistancebetweenthebirdandmicrophone,andcharacteristicsofthevariousrecord-ingequipmentused.Free-flyingbirdswererecordedunderconditionsrangingfromsmallcaveswithen-tranceslessthan1mindiametertoenormouscavernsmorethan100mtall.Variationintheseconditionsbetweenrecordingswouldhaveobscuredanyactualdifferencesinfrequencymeasuresbetweentaxa,likethosedocumentedinthevocalizationsofothertaxa(PriceandLanyon2002a).Wemappedthepresenceorabsenceofecholocationandaspectsofclickstruct-ureontothemolecularphylogenyusingsimpleparsi-monyinMacClade(Version4,MaddisonandMaddisonResultsOfthe1058sitesforcyt,401(37.9%)werevariableand315(29.8%)werepotentiallyphylogeneticallyinforma-tive.Geneticdivergencesrangedfrom0.0%(betweenmembersofthesamesubspecies)to15.1%(betweenHemiprocneandsomemembersoftheingroup).Ofthe1078sitesforND2,528(49.0%)werevariableand417(38.7%)werepotentiallyphylogeneticallyinformative.GeneticdivergencesforND2rangedbetween0.0%andUnweightedparsimonyanalysisofthecombinedgeneregionsproduced12trees,theconsensusofwhichisshowninFig.2.Althoughthereareseveralmostparsimonioustrees,theconsensusisgenerallywellresolvedandbootstrapsupportformostnodesisgenerallyhigh.Importantly,boththegeneraAerodra-musaremonophyletic(bootstrap100%and99%,respectively).Inaddition,unliketheresultsofLeeetal.(1996),swiftlets(Aerodramus)arerecoveredasamonophyleticgroupwithstrongsupportWithinAerodramus,therearethreestronglysup-portedclades:whiteheaditerraereginaemaximusbreirostris,andtheremainderofAerodramus.Somespeciesdonotappeartobemonophyletic.AerodramusanikorensispalawanensisissistertoA.mearnsi,while.lugubrisissistertoA.sawtelliA.bartschiissistertoA.salangananatunaetheexclusionofA.f.germani,althoughthisisonlyweaklysupported(53%).Betweensubspecies,geneticdivergenceisoftenconsiderable,rangingfrom1.8%A.fuciphagusA.f.germani3.7%betweenanikorensislugubris.palawa-.Withinasubspecies,incontrast,geneticvariationisgenerallylessthan0.5%,withtheexceptionoftwo2%divergenthaplotypesofA.spodiopygiusC.troglodytesissistertotheremainderofthegenus.MonophylyofCollocaliaesculentastronglysupported(78%).However,geneticdivergencebetweensubspeciesofC.esculentaisconsiderable,rangingfrom3.0%betweenC.e.bagoboC.e.marginatato5.8%betweenC.e.beckiC.e.cyanoptila.LikeinAerodramus,divergencewithinsub-speciesgenerallyrunslessthan0.5%.Forthemostpart,monophylyofotherswiftgeneraissupported.However,thereisstrongsupportforpara-phylyof,withStreptoprocnezonarissistertaxonofCypseloidesnigerwith100%bootstrapStreptoprocneformthesistertaxontoallotherswiftswithstrongsupport(100%),whichagreeswithprevioussuggestionsthatthesetaxaconstituteaseparatesubfamily(Brooke1972).Maximumlikelihoodanalysisproducedasingletreethatwaswellresolvedandwellsupportedbybootstrapanalyses(Fig.3).Inmostrespectsthistreeissimilartotheparsimonytree,differingmostlyinthearrangementofsomeweaklysupportednodes.Importantly,swiftletsaremonophyletic(96%)asareAerodramus(100%)and(100%).Otherrelationshipsdiscussedabovealsoappearinthemaximumlikelihoodtree.MappingecholocationontothemoleculartreeshowsthatthisabilityeitherevolvedatthebaseoftheswiftletsandwasthenlostonthebranchleadingtoC.linchi,oraroseindependentlyintroglodytesandthegenusAerodramus(Fig.4).Asfoundinpreviousstudies(Medway1959,MedwayandPye1977,Fullardetal.1993),AerodramusmaximusA.sawtellidifferfromotherswiftletsinproducingecholocationpulseswithasingleclickdesign(Fig.5).Allotherecholocatingtaxaproducedoubleclicks,C.troglodytes.Basedonourreconstructionsofthisfeatureonthemoleculartree(Fig.4),singleclicksappeartobeaspecializationthatevolvedindependentlyA.maximusA.sawtelli,whereasthedoubleclicksofotherAerodramustaxaandofC.troglodytesrepre-sentthemoreancestralcharacterstate.Intraclickintervalsmeasuredinthisstudyvariedamongspeciesnomorethantheydidwithinindividuals.Aerodramustaxawithdoubleclickshadintervalsofapproximately15to20ms(mean16.8ms,SEms),whichagreeswiththeresultsofasmallersurveybyMedwayandPye(1977)butisslightlylowerthanvaluesmeasuredforAerodramusterraereginaebySuthersandHector(1982;18to25ms).Interestingly,thedoubleclicksofC.troglodyteshaveameanintraclickintervalof16.9ms,wellwithintherangemeasuredinAerodramusspecies(compareFig.5bandc).JOURNALOFAVIANBIOLOGY35:2(2004) Phylogeneticanalysisofcombinedcyt-andND2sequencesforswiftsandswiftletsproducedtreesthatweregenerallywellsupportedandresolved(Figs.2and3).Monophylyofswiftletswasstronglysupported,aswasthereciprocalmonophylyofAerodramus.Withourdiscoveryofecholocationintroglodytes,ourphylogenyrevealsthattheevolutionofthisabilityintheswiftletshastracedanhistoricalpatternmuchdifferentthanpreviouslythought.Asaresult,echolocationcannolongerbeconsideredausefulcharacterforseparatingthesetwogenera.Inapreviousstudy,Leeetal.(1996)concludedthatanabilitytoecholocateevolvedonceintheimmediateancestoroftheAerodramusgenus.Echolocationinwasatthattimeunknownandtheirmolecularanalysisindicatedparaphylyofswiftlets.Incontrasttothoseresults,ourdataindicatethatecholocationisunlikelytohaveevolvedinthisway.Rather,echolocationaroseeither(1)onceatthebaseoftheswiftletsandwassecondarilylostonthebranchleadingtoC.linchi,or(2)twiceindependentlyamongswiftlets.Ourphylogenydoesnotresolvewhichofthesepossibilitiesismorelikely(Fig.4).However,thestrikingsimilaritiesbetweentheorientationclicksofCollocaliatroglodytesandthoseofmostAerodramustaxa(Fig.5)suggestthatthesesoundsarehomologousratherthanindependentlyderived.Moredetailedcom-parisonsofclickstructureinwhichrecordingsaremadeFig.2.Consensusof12mostparsimonioustrees(length0.428)producedfromcombinedanalysisofcyt-ND2sequencesforswiftsandswiftlets.Numbersassociatedwithbranchesindicatepercentsupportfrom1000bootstrapreplicates.Branchlengthsareproportionaltothenumberofreconstructedchanges. JOURNALOFAVIANBIOLOGY35:2(2004) undermuchmorestandardizedconditions,aswellascomparativeanalysesofsyringealmorphology,areneededtofurtherinvestigatethispossibility.Singleclickshaveevolvedfromdoubleclicksonatleasttwoseparateoccasionsduringswiftletevolution.Intheory,asingleclickdesignmightbemoreeffectiveforacousticorientationthanadoubleclickbyallowingbirdstoavoidpotentialpulse-echooverlap(Fullardetal.1993).However,producingsingleclicksmightalsorequirespecialphysiologicalmechanisms.Detailedstu-diesbySuthersandHector(1982)ofthephysiologyofclickproductionsuggestthatthedoublenatureofthesesoundsisabyproductofthemannerinwhichtheyareproduced.Eachdoubleclickistheresultofabirdmomentarilyclosingitssoundproducingorgan,thesyrinx,inthemidstofproducingalongersqueak-likesound,thusseparatingthisvocalizationintotwobriefclickswithhighbandwidthandabruptonsetandoffsettimes.Producingasingleclickprobablyinvolvestheadditionalstepofsuppressingoneofthesesounds(SuthersandHector1982).Evolutionarychangesinclickdesign,aswellasthepossiblelossofthisabilityinsometaxa,thereforemightreflecttrade-offsindifferentswiftletlineagesbetweenthephysiologicalcostsofclickproductionandtheneedforaneffectivenavigationThestronglysupportedmonophylyofswiftlets95%)inthisanalysisisperhapssurprisinggivenFig.3.Mostlikelytree(L16,830.3715)resultingfromcombinedlikelihoodanalysisofcyt-andND2generegionsunderamodelwithbasefrequencies(A0.3294,C0.0657,T0.2023),generaltimereversiblesubstitutions(A-C0.3922,A-G13.3884,A-T0.2016,C-T6.2253,G-T1.0),gammashapeparameter1.1988,andfractionofinvariant0.5008.Numbersassociatedwithbranchesindicatesupportfrom100bootstrapreplicates.Branchlengthsareproportionaltothelengthoptimizedunderthelikelihoodmodel(scaleindicated). JOURNALOFAVIANBIOLOGY35:2(2004) thatapreviousphylogeneticanalysisofpartialcyt-sequences(Leeetal.1996)resolvedswiftletsaspara-phyletic,withAerodramusbelongingtoseparateclades.However,Leeetal.(1996)analyzedonly406bpofthecyt-gene,lessthan20%ofthenumberofbasepairsincludedinthepresentstudy.SinceparaphylyoftheswiftletswasnotstronglysupportedintheLeeetal.(1996)analysis,itseemslikelythatthedifferenceinresultsbetweentheirstudyandoursisexplainedlargelybythedifferenceinthenumberofbasepairsanalyzed.Cautionshouldthusbeexercisedwhendrawingconclu-sionsfromweaklysupportedtreesbasedonalimitednumberofbasepairs.AmorerecentanalysisofswiftletphylogenybyThomassenetal.(2003)usinglongercyt-(1143bp)fromalimitednumberoftaxahasprovidedfurthersupportforswiftletmonophyly,inagreementwithourfindings.Unlikeourstudy,however,theysuggestparaphylyofAerodramusbyplacingthenon-echolocatingwaterfallswiftletHydrochousgigasthatgenus,aresultthatcouldindicateeitherthelossofecholocationinH.gigasortheappearanceofecholoca-tionmultipletimesindifferentAerodramustaxa(Tho-massenetal.2003).Supportforthisplacementofgigaswasweak,however,andtheanalysisbyThomassenetal.(2003)sampledonlyfourAerodramusspeciesanddidnotincludedatafromCollocaliatroglodytes.Inclu-sionofthe406bpsequenceofH.gigasfromthestudyofLeeetal.(1996)inthecurrentanalysisresultedinaweaklysupportedsisterrelationshipbetweenAerodra-musHydrochous,stillrecoveringswiftletmonophylyandthemonophylyofAerodramus.However,sincewewereunabletoreamplifytheHydrochoustissueforaproperinvestigation,wetreattheseresultswithcautionandrefrainfromdrawingconclusionsaboutecholoca-tionbasedonthisspecies.Perhapssequencingadditionalgenesinthisenigmaticswiftletwillhelpresolveitsrelationshipsandprovideaclearerpictureofhowecholocationhasevolvedinthegroup.Swiftletshavebeenperhapsthemosttaxonomicallydifficultgroupofbirds,andthisisevidencedbyourresults.Althoughmanyspeciesrelationshipsarewellresolved,severalspeciesareshowntobeparaphyletic.AdditionalsamplingofspeciesandsubspeciesinAero-dramusisneededtoresolvemanyoftheseoutstandingtaxonomicissues.Ontheotherhand,deepphylogeneticrelationshipsamongmanyoftheswiftsarestronglysupportedbyourdata,unlikemanypreviousstudiesofmitochondrialgenesinbirds(e.g.,JohnsonandClayton2000,Klickaetal.2000,Johnson2001,PriceandLanyon2002b).Thisresultunderscoresthestrongpotentialformitochondrialgenesequencestoanswerotherunresolvedquestionsinswiftsystematics.AcknowledgementsWethankthefollowingindividualsandinstitutionsforprovidingtissuematerialforthisstudy:CharlieCollins,MikePutnam,AmericanMuseumofNaturalHistory, Fig.4.Thepresenceofecholocationandtwotypesofecholocationsounds,singleclicksanddoubleclicks,recon-structedontothemaximumlikelihoodphylogenyofswiftlet Fig.5.Spectrogramsofecholocationsoundsproducedby(a)Aerodramussawtelli,(b)A.bartschiand(c)Collocaliatroglo-.ThedoubleclicksofA.bartschi(b)andC.troglodytesaresimilarinacousticstructure,yetbotharecomparativelydifferentfromthesingleclicksofA.sawtelli(a),whichissistertaxontoA.bartschi(recordingsbyJ.H.Fullard(a)andD.H.Clayton(bandc)).JOURNALOFAVIANBIOLOGY35:2(2004)