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 humanhunters 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, form aclade( 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 notdistinguish 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