doi 10.1098/rspb.2001.1717

Phylogeneticevidencefor colourpattern convergenceintoxicpitohuis: Mu ¨ llerianmimicry in birds? JohnP.Dumbacher * and RobertC. Fleischer Molecular Genetics Laboratory,National Zoological Park, Smithsonian Institution,Washington, DC 20008, USA Birdspecies inthe genus Pitohui arechemically defended by a potentneurotoxic alkaloid in their skinand feathers. Thetwo most toxicpitohui species, the hoodedpitohui ( Pitohuidichrous )andthe variablepitohui (Pitohuikirhoce phalus ),aresometimes strikinglypatterned and,in certain portionsof their geographical ranges,both species sharea nearlyidentical colour pattern, whereasin otherareas they do not. MÏ llerian mimicry (the mutualresemblance oftwo chemically defended prey species) is commonin some other animalgroups and Pitohui birds havebeen suggested as one of the most likelycases inbirds. Here, we examinepitohui plumage evolution in the contextof a well-supportedmolecular phylogeny and use a maximumlikelihood approach to test forconvergent evolution in coloration. W eshowthat the `mimetic’ phenotypeis ancestralto both species andthat the resemblance inmost races is better explainedby a sharedancestry .One largeclade of P.kirhocephalus lost this mimetic phenotypeearly in their evolution andone race nested deepwithin this cladeappears to have re-evolved this phenotype.These latter ¢nd- ingsare consistent withthe hypothesisthat MÏllerian mimicry is drivingthe evolutionfor a similar colourpattern between P. dichrous,but onlyin this oneclade of P.kirhocephalus . Keywords: Pitohui;toxicbirds; molecularsystematics; mitochondrialDNA ;MÏllerian mimicry; New Guinea

(Mayr1 941;Rand& Gilliard1 967).Fourdi¡ erent 1.INTRODUCTION P.kirhocephalus subspecies innorthwest NewGuinea, Birdspecies inthe endemic NewGuinea genus Pitohui southeast New Guineaand on the WandammenPeninsula containpotent defensive toxins in their skinand feathers almostperfectly resemble the colourpattern of P. dichrous (Dumbacher et al.1992,20 00).Evidencesuggests that (¢gure 1 ).Thiscolour pattern is abrick-red bellyand these toxinsmay defend pitohuis from human hunters backthat contrasts witha jet-blackhead, wings and tail (Majnep& Bulmer 1977;Kocher-Schmid 1991,1993),as andis hereafter calledthe `type1’ plumagepattern. Inter- wellas ectoparasites (Mouritsen &Madsen 1994; vening P.kirhocephalus races canhave strikingly di¡ erent Dumbacher 1999).Pitohuitoxins belong to the well- colourpatterns from P. dichrous,forexample some have knownbatrachotoxin family of compounds, which attack greyor brown hoods and a lighterback, some havea voltage-dependentsodium channels (Daly & Spande more tawnybreast andbelly plumage, some areshades of 1986).Becausemost higheranimals have these sodium light grey^brownthroughout and still others aredi¡ erent channelsand because the channelsare highly conserved, shadesof bright ferruginous throughout (¢ gure 1 ).From it is expectedthat these toxinscould defend against a their distributionit appearsthat multiple P.kirhocephalus widevariety of natural predators, including other visual races couldhave independently evolved the type1 predatorssuch ashawksand some snakes. plumagepattern viaselection forMÏ llerian mimicry of The genus Pitohui containssix species, ofwhich Pitohui P. dichrous (Diamond1 992;Dumbacher et al. 1992). kirhocephalus and Pitohuidichrous aresigni¢ cantly more MÏllerian mimicry isthe mutualresemblance ofmultiple, toxicthan the others (Dumbacher 1997;Dumbacher et al. chemicallydefended prey species (Sheppard et al. 1985), in 2000). Pitohuidichr ous sometimes carries enoughtoxin to whichthe mimics bene¢t bysharing the cost ofeducating irritate buccalmembranes andcause sneezing and watery predators(MÏ ller 1879).MÏllerian mimicry iscommonin eyes ifmerely handled(Salvadori 1 881;Kocher-Schmid insects, such asbutter£ ies, andpitohuis may represent a 1991). Pitohuikirhoce phalus and P. dichrous alsohave bolder veryrare case ofMÏ llerian mimicry inbirds (Dumbacher plumagepatterns; P. dichrous hasa brick-red bellyand &Pruett-Jones1996) .However,an alternative hypothesis backthat contrasts witha jet-blackhead, wings and tail. isthat the type1 plumagepattern resemblance isahistor- Contrastingcolour patterns arecommon among toxic icalartefact dueto a sharedprimitive colourpattern animalsand are often thought to serve aswarning or (Dumbacher et al. 1992). `aposematic’signals for visually orientated predators Performingdirect tests forcolour pattern selection and (Cott 1940;Owen 1 980). MÏllerian mimicry between P.kirhocephalus and P. dichrous Theplumage coloration of P.kirhocephalus varies iscurrently impossible becausepitohuis’ natural habitat is tremendouslyacross its geographicrange and as manyas remote andthe crucialportions of their rangesare 20morphologicallydiagnosable subspecies arerecognized currently inaccessible.However, we can use aphyloge- netic approachfor examining colour pattern evolutionin *Author forcorrespondence ([email protected]) . P.kirhocephalus and P. dichrous.If the type1 colourpattern

Proc. R.Soc.Lond . B (2001) 268, 1971^1976 1971 © 2001The RoyalSociety Received 15February2001 Accepted 24April 2001 1972J .P.Dumbacher andR. C.Fleischer MÏllerian mimicry in birds?

Figure1. Map of Pitohuikirhocep halus subspeci¢c rangesshown with P.kirhocephalus phenotypes.This unusual`checkerboard ’ distributionof plumagetypes has puzzled ornithologists (Beehler et al. 1986), yet P.kirhocephalus isusuallytreated as asingle specieson thebasis of similarityin theirsong and habits, clinal variation between some races and because no tworaces have been foundin sympatry. Pitohuidichrous showslittle geographical variation throughout its range and its plumage pattern is identical to that of P.k.dohertyi (f)foundon theWandammen Peninsula. The subspeciessampled for this study are marked with letters. `Type1’ phenotypes(ranges shown in orangeand green in b, c,f, h, i,p, q)areconsidered potential mimics of P. dichrous. The pitohuiimages were taken from Beehler et al. (1996). (© 1986Princeton University Press.) ispleisiomorphicor ancestral for the potentiallymimetic subspecies, then this providesno phylogenetic evidence forMÏ llerian mimicry .Alternatively,ifthe type1 colour pattern is derivedmultiple times fromnon-black-hooded ancestors, then this pattern is consistent withMÏ llerian mimicry andis takenas phylogenetic evidence for mimicry. Here, weuse DNA sequence datafrom two mitochon- drialgenes and two nuclear gene introns inorder to reconstruct aphylogenetichypothesis for P. dichrous and P.kirhocephalus . We sampled P.kirhocephalus tissue from10 recently collectedPapua New Guineaindividuals and an additional27 museum skins.Analyses of the entire Pitohui Figure2. Head colour value versus back ^headcontrast of genusshow that the P. dichrous and P.kirhocephalus races pitohuiplumage. The Munsell`value’ measures brightness, forma clade( J.P.Dumbacher andR. C.Fleischer, with zerobeing pure black (`jet black’, 1.46) and 10 being unpublisheddata) .Wetherefore included10 P. dichrous in purewhite. Back ^headcontrast is measuredas the Munsell orderto help polarize characters within P.kirhocephalus , backcolour value minus the head value. Letters reference andwe used two Pitohuiincertus (white-belliedpitohui) Pitohuikirhocep halus subspeciesfrom ¢ gures1 and3 and individualsin order to root the tree. numbersin parenthesesindicate the number of individuals ofeachsubspecies represented by eachpoint. Note that type-1-patternedsubspecies clearly cluster with Pitohui 2.MATERIAL AND METHODS dichrous. Wemeasuredpitohui plumage colour by matching their head,back, wings, belly ,tailand rump colours to those of stan- birdspecies ( Diamond1 966,1972) ,yetin manyplaces we dardcolour charts and translated their colours into Munsell visited,local New Guineans did not distinguish between type 1 notation(Smithe 1 975).Weillustratethe colour and pattern P.kirhocephalus and P. dichrous species(Herowana Village, Eastern similarityby plotting the value of head colour against the HighlandsProvince, Bonua Village, Central Province and contrastof head colour and back colour in ¢gure2. Those KakoroVillage, Gulf Province, Papua New Guinea) .Inareas P.kirhocephalus racesthat cluster with P. dichrous have type 1 wherethe two species overlapped but were not similar, the local plumagepatterns and are considered potential mimics in our peopleinvariably recognized them as distinct species. analyses.It is important to note that the type 1 racesare also similarin theiroverall plumage colour and plumage pattern (a) DNAisolation andsequencing throughoutthe body and not just on the hood and back. This is DNAwas isolated from museum skins using standard ancient perhapsbest illustrated by noting that local New Guinean DNAtechniques in aDNAisolation laboratory in which no huntersare renowned for their ability to distinguish between modernDNA or post-polymerase chain reaction (PCR) samples

Proc. R.Soc.Lond . B (2001) MÏllerian mimicry in birds? J.P.Dumbacher andR. C.Fleischer 1973 arehandled ( Cooper et al.1996;Sorenson et al.1999).Asmall dataset, estimates of relative support are similar using either toepad fragment (2 mm 5 mm 0.5mm) was digested over- globalor local methods, but we reportestimates based upon £ £ night at 56 8Cin lysisbu¡ er (1 mM Tris,EDT A,DTT and locallikelihoods because they are more conservative. proteinasek, pH 8.0) ,phenoland chloroform extracted and dialysedin centrifuge-assistedCentricon 1 tubes(Amicon, 3. RESULTS Beverly,MA, USA).Existingprimers were modi¢ ed slightly in orderto amplify a 307bp region in cytochrome b (Kocher et al. Our phylogeneticanalysis yielded a singlemost likely 1989)and a 196bpmitochondrial DNA region that includes tree (¢gure 3) .When rootingwith P. incertus,ourbest tree portionsof tRNA-lysineand A TPase8genes (L9051 and H924 1) containeda monophyletic P.kirhocephalus ,but bootstrap (Greenberg et al.1998;Sorenson et al.1999).Wealsoampli¢ ed a supportfor this nodewas weak (47 .1%).Thephylogeny 109bp fragment of aldolase intron G (primersald2f, 5 0- identi¢ed three highlydivergent P.kirhocephalus clades: a ATCCAGGAGAACGCCAACAC-3 0 and ald3r, 50-AACCCT north-coastclade (bootstrap support 97 .6%),asouth- CACAGGGATTCTCC-3 0)anda 211bpfragment of adenylate coastclade (93.8%) and a West-PapuanIsland clade kinaseintron 5 (primersAK51 78f,5 0-CTCTACACGGCCCAA (99.9%).Theaverage HKY85-corrected (Hasegawa et al. GAGAC-30 andAK5291 r,5 0-GGGGCGATCTATGGACAG-3 0). 1985)pairwise distances forcytochrome b are 0.077 PCR reactionswere prepared in aDNA-freehood and a single betweenthe north-coastand south-coast clades,and PCR reactionwas run using Perkin-Elmer ( FosterCity ,CA, 0.086between the north-coastand W est-PapuanIsland USA)T aqGoldDNA polymerase ( 10min at 94 8C before the clades,indicating a deepphylogenetic divergence between cyclingbegan and then 45 cyclesat 92 8C for 60 s, 50 8C for 45 s these groups.By assuming a passerine molecularclock and 72 8Cfor45 sdenaturing,annealing and extension tempera- rate of0.016sequence divergenceper millionyears (Myr) tures,respectively) .The PCR productswere puri¢ ed and forthis portionof cytochrome b (Tarr& Fleischer 1993; sequencedusing dideoxy chain termination using recommended Fleischer et al.1998)and by subtracting the average Applied BiosystemsInc. (F osterCity ,CA,USA)protocols and within-cladepairwise distances (Wilson et al. 1885; Nei sequencereactions were cleaned using sephadex resin and run 1987),wecan estimate approximatelythe time that these onan ABI 373-stretchautomated sequencer. cladesdiverged. The estimated divergenceof the north- coastand south-coast cladesis 0.077between ( 70.017% (b) Phylogeny andancestral character state withinclades) .Dividingthis by0.01 6per Myr results in reconstruction anageof divergence of ca.3.7Myr.Forthe north-coast / Sequenceswere aligned and edited with S EQUENCHER 3.1.1 West-PapuanIsland split weestimate ca.4.6Myr. Thus, software( GeneCode,Ann Arbor,MI, USA).Sequenceswere these three majorclades probably formed ca.3.5^4.6Myr exportedin Nexusformat to M ACCLADE 4.0( Maddison& ago. Maddison2000) in which sequencesfrom di¡ erent genes were Wereconstructed the evolutionof the type1 phenotype concatenatedfor each individual. The completeDNA data usingthis phylogeny.Our analysissuggested that the * matrixwas analysed using P AUP 4.0(Swo¡ ord 1 999).Weused probabilitiesof gains and losses oftype 1 plumagewere maximum-likelihoodheuristic searches with atwo-parameter approximatelyequal ( ¬/­ 0.825)and that the di¡er- ˆ modelof sequenceevolution ( Hasegawa et al.1985).Weusedthe ences werenot statistically signi¢cant (maximum- successiveapproximations method ( Huelsenbeck1 998)in order likelihoodratio test, d.f. 1 and p 0.783).Becausemost ˆ ˆ toobtain a singlebest-¢ t treeand parameter estimate (see type 1 P.kirhocephalus subspecies belongedto the south- ¢gure3) (transition: transversion ratio 5.097).Weaccepted coastclade, our analysis suggests that the ancestorof the ˆ thistree as the best working hypothesis for the relationships of south-coast cladealso had the type1 plumagepattern P.kirhocephalus subspecies. (relativesupport 0.999 for typ e1plumage).Therefore,the Wethenreconstructed the evolution of the type 1 phenotype resemblance between P. dichrous andthe south-coast ontothis phylogeny using a maximum-likelihoodapproach with P.kirhocephalus isbest explainedby shared ancestry rather thecomputer program D ISCRETE 4.0(Pagel 1 999 a,b). Because thanby convergent evolution. nothingis known about the genetic basis for colour pattern in However,in the north-coastand W est-PapuanIsland pitohuis,we madeno starting assumptions and used the clades,1 0ofthe 11 P.kirhocephalus subspecies havenon-type 1 phylogenyfor estimating ancestral states and evolutionary para- plumagewith the exceptionof the type1 subspecies Pitohui meters. DISCRETE requiresa fullyresolved dichotomous tree, so kirhocephalus dohertyi ,whichis foundon the Wandammen unresolvednodes were arbitrarily resolved for analyses. Pensinsula(¢ gure 1 f).Therelative support for non-type 1 DISCRETE estimatedthe probabilities of gains ( ¬)andlosses ( ­ ) plumagein the ancestorof both groups is 0.541.Our withoutrestrictions and also by restricting to ¬ ­ . The esti- analysesindicate that the ancestorof the north-coastclade ˆ matesof gainsand losses were very similar to each other and to hadnon-type 1 plumage(relative support ca. 1.0). This estimateswhen ¬ wasrestricted to equal ­ .The one-parameter P.k. dohertyi subspecies is phylogeneticallynested deep model(e.g. setting ¬ ­ )didnot lead to a signi¢cant reduction withinthe north-coastclade. These maximum-likelihood ˆ in likelihoodwhen comparedto the unrestricted two-parameter analysesindicate that the immediate ancestors of model(maximum-likelihood ratio test, LR 0.075561 and P.k. dohertyi hadnon-type 1 plumagepatterns andthat ˆ p 0.783),soall subsequent analyses used the simpler one- P.k. dohertyi recently re-evolvedthe type1 phenotypeafter ˆ parametermodel ( Pagel1 999 b).Weusedthe method outlined by it hadbeen lost some 3.5^6Myr ago. Mooers& Schluter(1999) for estimating ancestral character Inorder to test whetherthe tree topologysigni¢ cantly statesusing D ISCRETE 4.0.Relative support for ancestral supportedthe secondarygain of the type1 phenotypein characterstate estimations is based upon `local’ likelihood the north-coastclade, we searched for the nextshortest estimates (sensu Pagel1999) .Wecomparedthe relative support tree that didnot require additionallosses orgains of the * basedupon ` global’likelihoods as given by P AUP 4.0.F orthis type1phenotype.The best alternativetree placed

Proc. R.Soc.Lond . B (2001) 1974J .P.Dumbacher andR. C.Fleischer MÏllerian mimicry in birds?

Figure3. The totalevidence maximum-likel ihoodphylogram showing the relationships of Pitohuikirhocep halus and Pitohuidichrous . Branchesshorter than 10 78 werecollapsed to polytomies. Numbers above branches represent bootstrap support in 1000 maximum-likelihood,fast-addition bootstrap replications performed by P AUP*.The letterspreceding taxon names correspond tothetaxon ranges in ¢gure1. The mostparsimonious plumage character state reconstruction is shown, with type1 branches shownin greyand non-type 1 branchesshown in black.Maximum-likelih oodrelative support for character state reconstructionsisshownwith piediagrams drawn adjacent to the ancestors of importantclades. Numbers in parentheses indicatethe number of individualssequenced with identicalhaplotypes.

P.k. dohertyi asthe sister taxonto the north-coastclade merely aresult ofshared ancestral character states in andwas signi¢ cantly longer (¢ ve additional steps) P.k. dohertyi . (Templetonnon-parametric test, p 5 0.0253).Wetherefore Bycontrast, ourdata indicate no convergent evolution concludethat boththe tree topologyand the character between P. dichrous and P.kirhocephalus inthe south-coast reconstructions indicateconvergent evolution between clade. Pitohuikirhoce phalus meridionalis and Pitohuikirhoce phalus P.k. dohertyi and P. dichrous.Thus,phylogenetic evidence uropygialis lookremarkably similar toeach other and refutes the nullhypothesis that plumagesimilarity is to P. dichrous,yetthey are found at opposite ends ofthe

Proc. R.Soc.Lond . B (2001) MÏllerian mimicry in birds? J.P.Dumbacher andR. C.Fleischer 1975 island,at least 2000kmapart. Our datasuggest that direct test ofmimicry .Wearecurrently unableto perform P.k. meridionalis and P.k. uropygialis areclosely related and the di¤cult tests that arenecessary forimplying direct possiblysister taxa,and have an average HK Y85- selection, particularlyin many of the remote andpoliti- corrected pairwisedistance (Hasegawa et al.1985)of only callysensitive localitieswhere pitohuis live. However ,we 0.012incytochrome b.Ahistoricallong- distancedisp ersal cano¡ er thatthe most promisingarea to conduct such eventbetter explainstheir unusualdistribution. studies wouldbe on the WandammenPeninsula where When consideringevidence of long-distancedispersal in P.k. dohertyi and P. dichrous livesympatrically .Second,as the south-coastclade, we were concerned that dispersal nothingis knownof the geneticbasis oftheir colour andgene introgression might account for the type1 pattern, it is possiblethat some otherfactor ,geneticor plumage of P.k.dohertyi .However,three independently ecological,may be responsiblefor the reversalto the type1 segregatinggenetic loci provided no evidence of gene£ ow phenotypein P.k. dohertyi .Third,multiple P.kirhocephalus fromthe south-coastclade. Mitochondrial genes, as well subspecies showsexual plumage dichromatism, primarily asphylogeneticallyinformative transitions ineach nuclear inthe south-coast cladeand females havea slightlylighter geneintron (adenylate kinase ( C T)andaldolase head,wings and tail. I tis possiblethat bothtype 1 and $ (G A)),clearlyidentify P.k. dohertyi asa member ofthe non-type1 phenotypeswere retained in the ancestors of north-$ coastclade and suggest that the type1 phenotype P.k. dohertyi species throughdichromatism, whichwould evolvedsecondarily from non-type 1 ancestors. a¡ect ourcharacter reconstruction ina waythat is di¤cult topredict. Fourth,although we found no phylogenetic evidencefor mimicry inthe south-coast clade,selection for 4.DISCUSSION mimicry maystill bestrong and may be important for Our phylogeneticevidence argues that onemajor clade maintainingthe colourresemblance overseveral million of P.kirhocephalus type1 subspecies evolvedtheir plumage years.Again, direct studies ofselection willbe necessary in pattern fromtype 1-patterned ancestors. Inthese cases, orderto test this hypothesis. ourphylogeny therefore providesno evidence of conver- Convergentevolution explained less ofthe overall gentevolution or supportfor MÏ llerian mimicry . geographicalvariation in P.kirhocephalus than we Our datafor the oneremaining type 1 subspecies predicted. Our dataimply that multiple factorsmay be P.k. dohertyi aremore consistent withthe hypothesisof workingconcurrently in order to produce the extreme MÏllerian mimicry .ForMÏllerian mimicry tobe atenable morphologicaland genetic variation in this group.These explanationfor the convergentevolution of the type1 factorsinclude vicariance events (such asthe formationof phenotypein P.k. dohertyi ,twoadditional conditions must the central mountainranges which isolated the north- bemet. First, bothspecies must carrydefensive chemicals. coastand south-coast clades) and long- distancedispersal Inevery locality from which they have been sampled (whichappears to have distributed the sister subspecies (Dumbacher 1997;Dumbacher et al.2000),both P. dichrous P.k.meridionalis and P.k.uro pygialis toopposite ends ofthe and P.kirhocephalus carrypotent alkaloid toxins, although island). their toxinconcentrations vary greatly .Second,the ranges Why is Pitohui mimicry notmore commonin N ew of P. dichrous and P.kirhocephalus must overlap.Although the Guineaand why has the north-coastclade lost the type1 twosp ecies havesomewhat di¡ erent altitudinaldistri- plumage?Wehypothesizethat variationin their toxin butions,they are expected to overlap broadly from ca. 500 concentrations,the degreeof sympatry between Pitohui to1 100m altitude(Rand & Gilliard1 967;Beehler et al. species andvariation in predator guilds may all a¡ ect the 1986;Coates 1 990).Where collectingtrips havebeen strength ofselection forbright coloration and mimicry .For made, P. dichrous and P.kirhocephalus areoften sympatric. example,in surveys along the northcoast near Madang Forexample, museum specimens atthe American Town(Dumbacher 1997), P. dichrous wasless toxicthan Museum ofN aturalHistory andAcademy Of Natural Pitohuikirhoce phalus brunneicaudus sothere maybe noselec- Sciences ofPhiladelphia document sympatry in the tiveadvantage in P.kirhocephalus resembling P. dichrous. In WeylandMountains, along the IdenburgRiver ,Cyclops the nearbyFinisterre Mountains,where P. dichrous was Mountains,Hollandia, Ifaar Lake Sentani, Adelbert highlytoxic, P.kirhocephalus didnot extend into the moun- Mountains,Madang, Huon Peninsula, Herowana Village, tains,so the twospecies werenot sympatric. In localities Karimui andSo’ obo.W ealsoestimate that over50% of wherevisually hunting predators are rare, selection might the WandammenPeninsula, where P.k.dohertyi is found, betooweak for driving plumage mimicry despite the high lies inthis middlealtitude range from 50 0to1 100mand toxicityof models. P. dichrous hasalso been collected on the Wandammen Peninsula.W etherefore expectthat these twosp ecies have The authorsthank the American Museum of Natural History abroadoverlap in this region. (G.Barrowclough and J. Cracraft) ,theAcademy of N atural Six of the 20 P.kirhocephalus subspecies resemble Sciencesof Philadelphia ( L.Joseph),theSmithsonian National P. dichrous toa reasonabledegree. Of those six,we only Museumof N aturalHistory ( G.Graves and S. Olson)and the foundstrong phylogenetic evidence that refutes the null BritishMuseum (R. Prys-Jones)for allowing us tosample tissue hypothesisof resemblance dueto shared ancestry in frommuseum skins. W ethankF .Bonaccorso,B. A. Iova, P.k. dohertyi .Althoughthis is notconclusive evidence for H.Wasel,I. S. Majnep,M. Silakum,the Papua New Guinea MÏllerian mimicry ,these dataare consistent withthe NationalMuseum and Art Gallery ,CraterMountain WMA, theResearch and Conservation F oundationof Papua New hypothesisthat MÏ llerian mimicry helpeddrive plumage Guinea,Christensen Research Institute and the Papua New evolutionon the WandammenPeninsula. Although our GuineaNational Parks for support with ¢eldtissue collections dataare consistent withthe mimicry hypothesis,we andE. M. Draperfor laboratory assistance. W ethank suggestcaution for several reasons. First, this is nota D.Swo¡ ord for statistical advice, M. Pagelfor providing the

Proc. R.Soc.Lond . B (2001) 1976J. P.Dumbacher andR. C.Fleischer MÏllerian mimicry in birds?

program DISCRETE,twoanonymous reviewers for their useful Kocher,T .D.,Thomas, W .K.,Meyer,A., Edwards,S. V., commentsand critiques, and Princeton U niversityPress for PÌÌbo, S., Villablanca,F .X.&Wilson,A. C. 1989Dynamics allowingus to reprint pitohui images in ¢gure1 .The ¢eldwork ofmitochondrial DNA evolution in animals:ampli¢ cation wasfunded by the Hinds F undof theUniversity of Chicago, the andsequencing with conservedprimers. Proc.N atlAcad. Sci. ChristensenResearch Institute and the N ationalGeographic USA 86,6196^6200. Society.The laboratorywork was funded by the Smithsonian Kocher-Schmid,C. 1991 Ofpeopleand p lants:a botanicalethno- Institutionand the F reedF oundation.J.P .D.wassupported by graphyof N okopoVillage,Madang and Morobe Provinces, P apuaNew theU niversityof Chicago, National Institutes of Health and Guinea.BaslerBeitrÌ ge zur Ethnologie: Band 33. Basel, U.S.Department of Education,Graduate Assistance in Areasof Switzerland:Ethnologisches Seminar der UniversitÌ t und NationalNeed training grants, the Smithsonian Institution and MuseumfÏ r VÎlkerkunde,in Kommissionbei W epfund Co. Friendsof the National Zoo. Kocher-Schmid,C. 1993Birds of Nokopo. Muruk 6, 1^15. Maddison,W .P.&Maddison,D. R. 2000 MacClade:analysis of phylogenyand character evolution .Sunderland,MA: Sinauer REFERENCES Associates,Inc. Beehler,B. M., Pratt,T .K.&Zimmerman,D. A. 1986 Birds of Majnep,I. S. &Bulmer,R. 1977 Birdsof my K alamcountry . New Guinea.PrincetonUniversity Press. Aukland,New Zealand: Aukland University Press. Coates,B. J.1990 TheBirds of N ewGuinea .PortMoresby ,Papua Mayr,E. 1941 Listof N ewGuinea Birds: a systematicand faunal list NewGuinea: Robert Brown Press. ofthe birds of N ewGuinea and adjacent islands . New York: Cooper,A. J.,Rhymer, J .,James,H., Olsen, S., McIntosh,C., AmericanMuseum of NaturalHistory . Sorenson,M. &Fleischer,R.1996Ancient DNA and island Mooers,A. O.& Schluter,D .1999Reconstructing ancestor endemics. Nature 381, 484. stateswith maximumlikelihood: support for one- and two- Cott,H. B. 1940 Adaptivecoloration in animals .London:Mathuen. ratemodels. Syst. Biol. 48, 623^633. Daly,J.W.&Spande,T .F.1986Amphibian alkaloids: chemistry , Mouritsen,K. N.& Madsen,J. 1994 T oxicbirds: defence pharmacology,andbiology .In Alkaloids:chemical and biological againstparasites ? Oikos 69, 357^358. perspectives ,vol.4 (ed.S. W.Pelletier),pp.1^274. New Y ork: MÏller, F .1879 Ituna and Thyridia:aremarkablecase of mimicry Wiley. in butter£ies. Trans.Entomol. Soc. Lond. 1879, xx^xxix. Diamond,J. M. 1966Zoological classi¢ cation system of a primi- Nei, M. 1987 Molecularevolutionary genetics .NewY ork:Columbia tivepeople. Science 151,1102^1104. UniversityPress. Diamond,J. M. 1972 Avifaunaof the eastern highlands of N ew Owen,D .1980 Camou£age and mimicry .Universityof Chicago Guinea.LosAngeles, CA: The NuttallOrnithological Club. Press. Diamond,J. M. 1992R ubbishbirds are poisonous. Nature 360, Pagel,M. 1999 a Discrete.Reading,UK. 19^20. Pagel,M. 1999 b The maximumlikelihood approach to recon- Dumbacher,J .P.1997The ecologyand evolution of chemical structingancestral character states of discrete characters on defensein theavian genus Pitohui, PD thesis.University of phylogenies. Syst. Biol. 48, 612^622. Chicago,IL, USA. Rand,A. L.&Gilliard,E. T.1967 Handbookof N ewGuineabirds . Dumbacher,J. P .1999The evolutionof toxicity in pitohuis.I. London:W eidenfeld& Nicolson. E¡ectsof homobatrachotoxin on chewing lice (order Salvadori,T .1881 Ornitologiadella P apuasiae delleMolucche . Socio Phthiraptera). Auk 116, 957^963. Residentedella R. Accademiadelle Scienze di Torino,Parte Dumbacher,J .P.&Pruett-Jones,S. 1996A vianchemical Seconda. defenses. Curr.Ornithol. 13, 137^174. Sheppard,P .M., Turner,J .R.G.,Brown, K. S., Benson,W .W. Dumbacher,J. P .,Beehler,B. M., Spande,T .F.,Garra¡o, H. M. &Singer,M. C. 1985Genetics and the evolution of &Daly,J.W.1992Homobatrachotoxin in thegenus Pitohui: Muellerianmimicry in Heliconius butter£ies. Phil.T rans.R. Soc. chemicaldefense in birds? Science 258, 799^801. Lond. B 308, 433^610. Dumbacher,J .P.,Spande,T .&Daly,J.W.2000Batrachotoxin Smithe,F .B. 1975 Naturalist’scolorguide .NewY ork:The American alkaloidsfrom passerine birds :asecondtoxic bird genus Museumof Natural History . (Ifritakowaldi ). Proc.N atlAcad. Sci. USA 97,12970^12975. Sorenson,M. D.,Cooper, A., Paxinos,E. A., Quinn,T .W., Fleischer,R.C., McIntosh,C. E.&Tarr,C. L.1998Evolution James,H. F.,Olsen,S. L.&Fleischer,R. C. 1999 avolcanicconveyor belt: using phylogeographic reconstruc- Relationshipsof the extinct moa-nalos, £ ightlessHawaiian tionsand K -Ar-basedages of the Hawaiian Islands to waterfowl,based on ancient DNA. Proc.R. Soc.Lond . B 266, estimatemolecular evolutionary rates. Mol. Ecol. 7, 533^545. 2187^2193. Greenberg,R., Cordero,P .J.,Droege, S. &Fleischer,R. C. Swo¡ord, D. L. 1999 PAUP* 4.0b2:p hylogeneticanalysis using 1998Morphological adaptation with nomitochondrial DNA parsimony.Sunderland,MA: SinauerAssociates, Inc. di¡erentiation in thecoastal plain swamp sparrow . Auk 115, Tarr,C. L.&Fleischer,R.C. 1993Mitochondrial DNA varia- 706^712. tionand evolutionary relationships in theAmakihi complex. Hasegawa,M., Kishino,H. &Yano,T .1985Dating of the Auk 110, 825^831. human^apesplitting by a molecularclock of mitochondrial Wilson,A. C., Cann,R. L.,Carr, S. M., George,M. & DNA. J.Mol. Evol. 21, 160^174. Gyllensten,U .B. 1885Mitochondrial DNA and two Huelsenbeck,J. P .1998Systematic bias in phylogeneticanalysis: perspectiveson evolutionary genetics. Biol.J .Linn.Soc. 26, is the Strepsiptera problemsolved ? Syst. Biol. 47, 519^537. 375^400.

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