The Auk 116(3):629-639, 1999

A MOLECULAR PHYLOGENY OF THE BLACKBIRDS (ICTERIDAE): FIVE LINEAGES REVEALED BY CYTOCHROME-B SEQUENCE DATA

SCOTT M. LANYON • AND KEVIN E. OMLAND 2 JamesFord Bell Museum of NaturalHistory, University of Minnesota,1987 UpperBuford Circle, St. Paul, Minnesota 55108, USA

ABSTRACT.--NewWorld blackbirds (Icteridae) have long served as model systems for stud- iesof avianecology, evolution, and behavior.However, this workhas been conducted in the absenceof a strongphylogenetic hypothesis for the group.We sequenced 890 base pairs (bp) of the mitochondrialcytochrome-b gene for 28 of the 29 icteridgenera and subgenerarec- ognizedby Blake (1968).We found strongevidence of five lineagesof blackbirds:grackles and allies;caciques and oropendolas;orioles; and allies;and a monotypiccup- nestingcacique lineage. However, we foundlittle supportfor any furtherstructure among thesefive lineages and no strongevidence supporting monophyly. Our resultsset the stagefor forthcomingwork on relationshipswithin lineagesand forhigher-level studies that addressblackbird monophyly and relationshipsamong lineages. Received 9 February 1998, accepted6 November1998.

FEW GROUPS OF have been as well stud- sequencedata from the cytochrome-bgene to ied by evolutionary ecologistsas the New derive a phylogenyfor the describedgenera World blackbirds(Icteridae). exhibit a and subgeneraof blackbirds. wide range of behaviors(brood parasitism, co- Fewstudies have included enough taxa, char- loniality,polygyny, monogamy, delayed breed- acters,and methodologicalrigor to provide ing, vocal mimicry, long-distancemigration), strong hypothesesof relationshipsamong morphologies (sexual dichromatism, sexual blackbird genera, and little agreementexists size dimorphism,delayed plumage matura- amongthese previous studies. The most com- tion) and ecologies(range of diets and habi- prehensiveattempt to addressgeneric relation- tats).Furthermore, icterids often have served as shipswithin the Icteridaewas the morpholog- modelsfor the study of evolutionarygenetics ical work of Beecher(1951). Based on extensive and molecular evolution. These characteristics, dissectionsof skull muscles,Beecher concluded and the ease with which blackbirds are ob- that the cowbirds (Molothrus)were the most served,have made them popular study organ- primitiveblackbird and that three other isms. lineageswere derivedfrom a cowbird-likean- Despitethe impressivenumber of studiesof cestorLack of explicittree-building methods, blackbirds,knowledge of the phylogeneticre- and the absence of a data matrix, make it dif- lationshipswithin the group is inadequate. ficult to interpret his results in a modern Thisgap in knowledgelimits the abilityof evo- framework. Freeman and Zink (1995) pub- lutionary biologists to apply comparative lisheda studythat includeda datamatrix and methodsin this assemblage(e.g. Brooksand provided explicit statements of analytical McLennan1991, Harvey and Pagel1991). Here, methods. They analyzed restriction-enzyme we developa phylogeneticframework to serve cleavagesites in the mitochondrialgenome of as the foundationfor the study of behavioral, 47 blackbirdspecies to derivea phylogenyfor morphological,and ecologicalevolution in the group.However, their study did not pro- blackbirds.First, we brieflyreview past studies vide strongresolution on the identity of the that have addressedgeneric affinities within blackbirds. Next, we use mitochondrial DNA majorlineages within blackbirds.They identi- fied one major cladethat consistedof Icterus, Cacicus,Psarocolius, and . Most other • E-mail: [email protected] studieshave included only a few taxa and pro- 2Present address: National ZoologicalPark, Ge- vided little resolutionof blackbirdlineages or netics Laboratory, 3000 Connecticut Avenue NW, theirrelationships (e.g. Smith and Zimmerman Washington,D.C. 20008,USA. 1976,Raikow 1978,Sibley and Ahlquist1990).

629 630 LANYONAND OMLAND [Auk, Vol. 116

Finally,many classificationsof birds haveim- primer that had been limiting in the secondstage plied genericaffinities within blackbirds.For PCR. DNA sequenceobtained from sequencingone example,Blake (1968) placed the Bobolink(Dol- strand was confirmed by sequencingthe comple- ichonyxoryzivorus) in its own subfamily,sepa- mentary strand. An additionalsegment of the cytochrome-bgene rate from the rest of the blackbirds.Although was amplifiedwith primersL15042 (5'-ATCTGCA- suchclassifications lack accompanyingjustifi- TCTACCTACACATCGG-3'; B3) and H15767 (5'- cation,we discussthem in light of our cyto- GATGAATGGGTGTTCTACTGGTTG-3';B4), which chrome-bphylogeny. target a 726-bp fragmentoverlapping that of the fragmentproduced with B1 and B2. Then, single- METHODS strandedamplification products were producedand sequencedwith primersB3, B4, and B5 (L15243;5'- Taxa.--Wechose taxa from 28 of the 29 generaand ACCCTAGTAGAATGAGCCTGAGG-3')as described subgenerarecognized by Blake(1968) to adequately above.Alternatively, the double-strandedamplifica- samplehigher-level relationships within the black- tion productwas freed of remainingnucleotides and birds (Hypopyrrhuswas not represented;Appendix primers using a glass powder suspension(Gene- 1). One individual from each taxon was sequenced. Clean, Bio101,Inc.) and sequenceddirectly by the Our primary intent was to resolvegeneric affinities, protocolof Thein(1989), modified by the additionof so we concentratedour sequencingefforts on more 10% dimethyl sulfoxideto annealing,labeling, and speciesrather than more individuals.The sistertax- terminationreactions. See Lanyon (1994) for further on of the Icteridaeis not known with certainty (Rai- details on laboratorymethods. kow 1978, Bledsoe1988, Sibley and Ahlquist 1990). In total, an 890-bp region from the cytochrome-b Therefore,we chosefive outgrouptaxa representing gene was successfullysequenced and visually threeother speciose families closely related to black- alignedfor 60 blackbirdtaxa and 5 outgroups.Ow- birds (Thraupidae:Thraupis episcopus; Emberizidae: ing to occasionalambiguous banding, compressions Melospizamelodia; Cardinalidae: Passerina cyanea, Sal- on sequencinggels, and difficultyreading sequences tatorcoerulescens, Spiza americana). Within the black- at the ends,19 bases, on average,were not scoredfor birds, we usethe nomenclatureof Sibleyand Monroe eachtaxon. With few exceptions,these unscored ba- (1990),except in the caseof Icterusbullockii, for which ses were at sites that were otherwise uninformative we follow AOU (1998). We used Sibley and Monroe within the ingroup.The sequenceshave been depos- (1990) becauseof its worldwide coverageand be- ited in GenBank under accession numbers AF089004 causeit is a recentand widely availablecheck-list. to AF089068. Appendix1 listsvoucher information for all thespec- Analysisof sequencedata.--Data were analyzed us- imens used in this study. With 60 ingroup taxa and ing PAUP*(versions 4d56 to 4d64 providedby D. L. 5 outgrouptaxa, our main objectiveswere to attempt Swofford). Becausetransversions and transitionsac- to definemajor lineages within theblackbirds, define cumulate at different rates and, therefore, are not ex- the relationshipsamong these lineages, and begin to pectedto be equally phylogeneticallyinformative, testblackbird monophyly. we choseto differentiallyweight these two classesof Laboratorymethods.--DNA was extracted using charactertransformations. The weighting scheme standardphenolchloroform protocols (Maniatis et al. was obtained by reconstructing transformations 1982).In the initial phaseof the study,a 307-bpseg- from the cytochrome-bdata set on three different ment of the mitochondrialcytochrome-b gene was random trees using MacClade3.04 (Maddisonand amplifiedwith the so-called"universal" primers (B1 Maddison 1992).All three reconstructionsproduced and B2;Kocher et al. 1989).Double-stranded ampli- very similar transitionbiases (2.40, 2.42, and 2.30), fications(dsPCR) were performedusing 30 to 35 cy- whichwe roundedoff to the nearestinteger. As a re- cles of denaturation at 93øC (1 min), annealing at sult, our primary analysesare basedon a 2:1 trans- 52øC(1 min), and extensionat 72øC(2 min). Five mi- version:transition weighting (hereafter, 2x weight- crolitersof the dsPCR product were run on a 3% ing). Weconducted five heuristicsearches with ran- NuSieve agarosegel. The amplified PCR product dom additionof sequencesusing the TBRalgorithm, wasexcised from the gel and meltedin 300uL of wa- and treeswere rootedusing the five outgrouptaxa. ter. A dilution of this solution was then used as tem- All equallyparsimonious trees resulting from the 2 x platein a 100-uLasymmetric PCR to generatesingle- weightedanalysis were combinedto producea ma- strandedDNA templatefor direct sequencing(Gyl- jority-rule consensustree. To evaluatethe degreeto lensten and Erlich 1988). Excessprimers, salts, and which the cytochrome-bdata were saturated,we free nucleotideswere removed by threecycles of cen- plottedtransitions versus transversions for third po- trifugal dialysis (Centricon-30,Amicon). The PCR sitions,and first and secondpositions combined us- productwas then sequencedby the dideoxymethod ing the SAVEDISTcommand in PAUP*. (Sangeret al. 1977) using a commerciallyavailable To estimatethe degreeto which the topologyre- kit (Sequenase,United StatesBiochemical) with the sultingfrom theseparsimony analyses depended on July1999] BlackbirdLineages 631 characterand taxoncomposition, we usedtwo data Amblycercusis a monotypiclineage, this sup- manipulations.Bootstrapping (Felsenstein 1985) ex- port is de factobecause it was excludedfrom aminesthe degreeto which topologyis dependent the otherlineages at thosejackknife and boot- on the charactercomposition of the data set. Jack- straplevels.) In general,genetic distances with- knifingtaxa (Lanyon1985) determines the degreeto whichtopology is dependenton the taxonomiccom- in lineageswere lower than thosebetween lin- positionof the data set.We conducted100 bootstrap eages (Table 1). replicationswith heuristicsearches and randomad- Despitethe strongsupport for the existence dition of taxa. We jackknifedtaxa by deletingeach of the five lineages,our analysesdid not re- taxa one at a time using commandlines in PAUP*. solvefurther structure among the five lineages. The outputfrom these65 jackknifepseudoreplicates The parsimonyanalysis with 2x weightingof wasthen synthesized using two programswritten by transversionsidentified 72 equallymost parsi- S. M. Lanyon for DOS (availableupon request). monioustrees (two islandsof 12 and 60 trees, To explorethe degreeto which our resultsare de- respectively).The majority-ruleconsensus of pendenton our a prioriassumptions concerning the thesetrees includes structure among the five bestway to analyzethese cytochrome-b data, we also conductedequally weighted searches and analyses lineages (grackles, (caciques/oropendolas, with six-parameterweighting (Williams and Fitch meadowlarks),(Amblycercus, orioles)). This ad- 1989).We determinedthe six-parameterweights by ditional structuredid not receiveany statistical mappingchanges onto the shortestequally weighted supportin the 2x weightedanalysis. However, tree and thenused the negativenatural log of these Amblycercusgrouped with the oriolesin 78%of changefrequencies following Cunningham (1997). thejackknife replications in theequally weight- We used the same analytical approachoutlined ed analyses.The six-parameterweighting re- above for both of these additional weighting suitedin 78%jackknife support for a cacique, schemes. oriole, meadowlarkgroup, and 82% jackknife Todetermine whether alternative phylogenetic hy- pothesesdiffered significantly from eachother, we supportfor a cacique,oriole, , Am- usedthe Kishino-Hasegawatest (1989) in PAUP*.We blycercusgroup. None of thesegroupings, nor evaluatedthe likelihoodscores of the treesresulting any otherstructure among the otherlineages, fromour threeweighting schemes. We also (1) tested was supportedin morethan 50% of the boot- Blake's(1968) hypothesis that Dolichonyxis sisterto strap replicationsin any of the weighting all otherblackbirds assuming the other relationships schemes. asdetermined by our2x weightedtree and (2) eval- The2x weightedand six-parameterweight- uated Freeman and Zink's (1995: figure la) tree us- ed jackknifeanalyses revealed 94 and 95%sup- ing the 36 taxacommon to bothstudies. For all tests, port, respectively,for blackbirdmonophyly. we used a nested set of models of sequenceevolu- However,monophyly of the Icteridaewas not tion, beginningwith a simplemodel assuming the strongly supported,with bootstrapanalyses same2:1 transition:transversionratio used by the parsimonyanalysis with empiricalnucleotide fre- revealingless than 50% supportfor blackbird quencies.We then testedthe topologiesusing more monophylyin all weightingschemes. This is complex models (transition:transversionratio esti- perhapsbest reflected by thefact that in the2x mated, general-timereversible, and general-timere- weightedanalysis, the small island of most- versible with rate heterogeneity;Swofford et al. parsimonioustrees showed the blackbirdsas 1996). non-monophyletic,whereas the larger island showsthem as monophyleticoIn the equally RESULTS weightedanalysis, the majority-ruleconsensus showedthe blackbirdsas non-monophyleticin We found strongevidence of five lineagesof a differentway. However,the statisticalsup- blackbirds:(1) gracklesand allies, (2) caciques portwas not strong for eitherof thesearrange- and oropendolas,(3) orioles,(4) meadowlarks ments.Genetic distances mirror the branching and allies,and (5) cup-nestingcaciques (Fig. 1). patternsin theserespects (Table 1). Although Monophylyof eachof theselineages was robust distancesto theoutgroups were not on average in all manipulationsand weightingschemes greaterthan distancesamong ingroup lineag- employed.With 2x weighting,all fivelineages es, no one ingrouplineage had lower genetic received100% jackknife support, and all of the distancesto the outgroups. lineagesexcept the meadowlarksand alliesre- We found no significantdifferences among ceivedat least94% bootstrapsupport. (Because the shortesttrees created by any of the three 632 LANYONAND OMLAND [Auk, Vol. 116

GRACKLES AND ALLIES

94 100

94 CACIQUES AND OROPENDOLAS 100

ORIOLES 100 100

7O MEADOWLARKS AND ALLIES 100

CUP-NESTING CACIQUES Amblycercus holosericeus

Outgroups FIG.1. Five lineagesof Icteridsas revealed by parsimonyanalysis of cytochrome-bsequences. Statistical supportfor lineagesindicated by bootstrapvalues (above branches) and jackknifedtaxon values (below branches).Values shown are basedon the analysiswith 2x weightingof transversions. weightingmethods (Kishino-Hasegawa test, P any of the alternativeshortest trees that we > 0.05). Furthermore,using this approachre- foundregardless of the modelof sequenceevo- vealed no significantsupport or rejectionof lution used for the test. In contrast, the alter- blackbirdmonophyly. The small island of 2 x native trees based on the previous work of weighted trees that showedthe blackbirdsas Blake (1968) and Freeman and Zink (1995) non-monophyleticwas not significantlydiffer- were significantlyworse fits to the sequence ent than the largeisland supporting monophy- data (P -• 0.001),again regardless of the model ly. Therewere no significantdifferences among used.

TABLE1. Meanpercent divergence within (on diagonal)and between(above diagonal) the five lineagesof Icteridaeand outgroups.Standard deviations in distancesare in parentheses.

Grackles Caciquesand Meadowlarks Cup-nesting and allies oropendolas Orioles and allies caciques Outgroups Grackles and allies 7.8 (1.3) 10.8 (0.7) 10.3 (0.6) 10.6 (0.8) 9.5 (0.5) 10.5 (0.9) Caciquesand oropendolas 7.8 (1.2) 9.9 (0.7) 10.8 (1.1) 9.4 (0.6) 10.6 (0.8) Orioles 7.1 (1.3) 11.0 (1.0) 9.3 (0.7) 10.4 (0.8) Meadowlarks and allies 9.1 (1.9) 9.7 (1.2) 10.9 (1.1) Cup-nestingcaciques -- 11.5 (1.3) Outgroups 9.5 (0.9) July 1999] BlackbirdLineages 633

Grackles and Allies Agelaius phoeniceus assimilis ß Agelaius phoeniceus phoeniceus Agelaius tricolor Agelaius humerails O0 ß Agelaius xanthomus '•o 99 I Euphagus carolinus ß Euphagus cyanocephalus 55 Quiscalus lugubris Quiscalus major Quiscalus mexicanus Quiscalus niger ? Quiscalus quiscula Dives warszewiczi Molothrus aeneus Molothrus ater ? Molothrus bonariensis 72 lOO Scaphidura oryzivora Molothrus rufoaxillaris Agelaius cyanopus 58 ,;, 1100[•--{•ß Agelaius xanthophthalmus Agelaius thilius Agelaius icterocephalus Agelaius ruficapillus Pseudoleistes guirahuro Pseudolelstes vlrescens •o• •oo I Agelaius flavus 94 Molothrus badius 100 ß Oreopsar bolivianus Amblyramphus holosericeus ß Curaeus curaeus 77 Gnorimopsar chopi Gymnomystax mexicanus Macroagelaius imthurni 55 Lampropsartanagrinus Nesopsar nlgerrimus F[c.2. Detailof relationshipswithin the grackle and allies lineage based on a majorityrule consensus of the2x weighedtrees. Statistical support for lineagesbased in 2x weightingindicated by bootstrapvalues (abovebranches) and jackknifed taxon values (below branches). Nodes supported by fewerthan 50% of the pseudoreplicatesare indicatedwith a questionmark.

The relationshipswithin the four polytypic Basecomposition (26.7% adenine,35.0% cy- blackbirdlineages are shownin Figures2 to 5. tosine,13.3% guanine and 24.9%thymine) was These trees are our best estimate of the rela- similarto that reportedfor otherbird groups tionshipsamong these taxa. However, the com- (e.g. Burns 1998). Third-positiontransitions pletenessof taxonsampling varies among lin- showedstrong evidence of saturation(Fig. 6). eages.We countedthe numberof speciesthat However, plots of first- and second-position shouldprobably be includedin theselineages transitions showed no evidence of saturation. basedon the placement of theircongeners. Our study included34 of 41 gracklesand allies,8 of DISCUSSION 20 caciquesand oropendolas,10 of 25 orioles,6 of 9 meadowlarksand allies, and the single We found strong evidenceof five lineages speciesof cup-nestingcacique. within the Icteridae. However, as with several 634 LANYONAND OMLAND [Auk, Vol. 116

Caciques and Oropendolas Cacicus cela Cacicus uropygialis Cacicus chrysonotus 1• Psarocolius angustifrons Psarocolius atrovirens Psarocolius decumanus 100 Gymnostinops bifasciatus Cacicus solitarius

Fig. 3. Detail of relationshipswithin the caciqueand oropendolalineage (see Fig. 2 legend). previousmolecular and nonmolecularstudies, to all othericterids. The cytochrome-b data pro- we wereunable to resolvethe branchingorder videno supportfor eitherof thesepossibilities. among lineages.Moreover, we did not find Of Beecher'sthree blackbirdlineages, the cy- strongsupport for monophylyof the Icteridae. tochrome-bdata contradictall but the lineage Futurehigher-level studies will be aimedat ad- consistingof the oropendolasand caciques. dressingthese latter two issues(J. Klicka et al. Our findingsalso disagreewith severalas- unpubl. data). Furthermore, more detailed pectsof publishedclassifications. In particular, studieswithin lineagesare being completed for Blake (1968) recognizedtwo major lineages the orioles(Omland et al. 1999)and the grack- within the Icteridae:Dolichonychinae (contain- les and allies (Johnsonand Lanyon1999; also ing only Dolichonyx)and Icterinae(containing seeLanyon 1994), and detailedanalysis of the all other blackbirds; also see AOU 1983:722, in othertwo lineagesare planned. which Dolichonyxis placedin its own tribe). Our findingsmostly disagree with the few The currentstudy doesnot supportthis ar- previousstudies of blackbird systematics.Bee- rangement,and Blake'shypothesis was a sig- cher's(1951) major finding was that cowbirds nificantly worse fit to the sequencedata. Al- were primitive blackbirdsfrom which three thoughour data showthat Dolichonyxis very major radiations were derived: orioles, mead- distant from all other blackbirds in cyto- owlarks,and agelineblackbirds; grackles; and chrome-bsequence (mean sequence divergence oropendolasand caciques.Beecher's conclusion = 11%), Dolichonyx,Xanthocephalus, Sturnella, could be interpreted in two ways in modern and nevertheless seem to be more close- terms. Either he believed that cowbirds were ly relatedto eachother than they are to the re- the actual ancestorsof all other blackbirds, in mainingblackbirds. These taxa form the mead- which casecowbirds would be paraphyletic owlark and allies lineage with at least 70% with respectto all othericterids, or that cow- bootstrap support. Within this lineage, the birds were remnantsof an early lineage,in meadowlarksthemselves (Leistes and Sturnella) which case cowbirds would be the sister taxon form a strongly supported monophyletic

Orioles Icterus cayanensis cayanensis 98 Icterus cayanensisperiporphyrus lOO Icterus spurius Icterus bullockii Icterus nigrogularis 59 . Icterus leucopteryx lOO Icterus parisorum lOO ? Icterus graceannae lOO lOO Icterus mesomelas Icterus icterus

Fig. 4. Detail of relationshipswithin the oriole lineage (seeFig. 2 legend). July1999] BlackbirdLineages 635

Meadowlarks and Allies 60 Dolichonyxoryzivorus

100 98 Leistesmilitaris •91 78 100I 100I XanthocephalusSturnellabellicosaxanthocephalus 1O0 100 I Sturnellamagnaneglecta Fig. 5. Detail of relationshipswithin the meadowlarkand allieslineage (see Fig. 2 legend). group.Conversely, bootstrap support for plac- Finally, our resultsdiffer from Freemanand ing the Bobolinkand Yellow-headedBlackbird Zink's mitochondrialrestriction-site study in (Xanthocephalusxanthocephalus) with the mead- three important respects(Freeman and Zink owlarks,or with eachother, is not that strong 1995: figure lb; their well-supported tree). in any weightingscheme (-•70%). Thesetwo First, their data indicatethat the Troupial (/c- taxa are distant from eachother (8.1% uncor- terusicterus) does not groupwith the otherori- rectedcytochrome-b sequence divergence) and oles and may be basal to all other blackbirds from meadowlarks(->9.0%) and other icterids (althoughthey mentionthat this finding was (->8.2). "not commonto all the equallylikely treesan- The DNA-DNA hybridizationstudy of Sibley alyzed").In contrast,we foundstrong support and Ahlquist(1991: figure 384) includedonly for themonophyly of Icterus,with thegenus be- sevengenera of blackbirds(all of whichwe ex- ing monophyleticin at least97% of bootstrap amined) and resolved six nodes. The cyto- replicationsunder all threeweighting schemes. chrome-bdata support only oneof thesenodes: The monophylyof the oriolesis further sup- a clustercontaining Cacicus and Psarocolius.We portedby additionaltaxon sampling and a sec- note,however, that only three of the seventaxa ond mitochondrialgene (Omland et al. 1999). were radioactivelylabeled, making it unclear Second,the stronglysupported tree of Free- how the six nodes were derived (Lanyon man and Zink (1995) included an oriole, ca- 1992b). cique,and oropendolaplus meadowlarkclade.

0.25

o•0.20

.> ßo 0.15

c: 0.10 o

0.05

0.00 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 Overall transversion divergence

F•G.6. Saturationplot showingthird positiontransitions plotted as a functionof overalltransversion divergence. 636 LANYONAND OMLAND [Auk, Vol. 116

This arrangement,especially the inclusionof Agelaiusdemonstrated by Lanyon (1994). Sim- meadowlarks, is not supported by our data. ilarly,our studyconfirms the paraphylyof Mol- Third, Freemanand Zink (1995)found that the othrus(Lanyon 1992a). In both cases,the earlier Dolichonyx/Xanthocephalusclade grouped with work based on a more limited number of taxa the Red-winged Blackbird (Agelaiusphoeni- is supportedby the morecomplete taxon sam- ceus).Our datacontradict this grouping in that pling in our study.The non-monophylyof Age- the latterwas in the grackleand allieslineage laius and Molothrus underscores the need to es- and the former in the meadowlark and allies tablishphylogenetic relationships before con- lineage,both with bootstrapsupport. However, ductingcomparative studies. these are not strong disagreementsbecause Surprisingly,we did not find strongevidence bootstrap analysis of Freeman and Zink's for blackbirdmonophyly. Monophyly was not (1995)matrix resultedin only a few nodeswith supportedby a majority of bootstrapor jack- bootstrapsupport that exceeded50%, noneof knife replications, outgroup taxa branched whi& conflictedwith our lineages.Further- with the ingroupin shortesttrees, genetic dis- more, when we combined our data set with the tanceswere no greaterto outgrouptaxa than restriction-site data, the total-evidence tree among ingroup lineages,and monophyletic supportedthe samefive lineages defined by cy- treesdid not havesignificantly better fits to the to&rome-b alone. However, the Kishino-Hase- sequencedata. However,although no strong gawa test showedthat Freeman and Zink's support existedfor blackbirdmonophyly, nei- (1995)best estimateof blackbirdrelationships therdid strongsupport exist for anyof the out- was a significantlyworse fit to the sequence groupsbran&ing consistentlywithin the Icter- data. idae.Furthermore, Raikow's (1978) morpholog- Felsenstein(1978) and others have shown ical work providedat leastone morphological thatlong bran&es can be attractedto ea& oth- synapomorphyfor the blackbirds.Specifically, er in parsimonyanalysis. We found that both he stated that "In the forelimb the Icteridae Dolichonyxand Xanthocephalusare distantfrom show a modificationof the flexor digitorum ea& other (8.1%uncorrected cyto&rome-b se- profundus in whi& the caudalborder of the quence divergence) and from meadowlarks muscleis narrowed."Although this is not a (->9.0%) and other icterids (->8.2). Thus, the profound modification, it occurs in all forms placementof thesetwo taxa could be affected examined,and this consistency suggests that it by long-branchattraction in eitherthe Freeman is a useful &aracter for reliablydefining the and Zink (1995)study, our study,or both. Re- family.The lack of resolutionamong blackbird gardless,our data show that Xanthocephaluslineages, and the lackof supportfor blackbird xanthocephalusdid not share a recentancestor monophyly,suggest two possibleexplanations with Agelaiusphoeniceus because these that are not mutually exclusive.The first is that show 10.3% sequencedivergence in cyto- cyto&rome-bis too saturatedin third positions &rome-b. to reliablyrecover these relationships (Fig. 6). The distant relationshipbetween Agelaius Second,it is possiblethat the blackbirdswere phoeniceusand Xanthocephalusxanthocephalus is formed and diversifiedduring a rapid radia- oneof our mostsurprising findings. These taxa tion among all nine-primaried oscines.Future havelong been considered to be closelyrelated work using sequencingand other lines of in- for the purposesof behavioraland ecological quiry are neededto addressthese hypotheses studies(e.g. Willson 1966,Miller 1968,Orians and further resolvehigher-level relationships within the Icteridae. However, our current find- and Christman1968). Most classifications place ingsprovide a strongfoundation for su& work the two generaadjacent to ea& other (Blake at higher levels,as well as detailed studies 1968, Morony et al. 1975, Sibley and Monroe within the five lineagesof blackbirds. 1990), and Lack (1968) assumeda close rela- tionshipwhen discussingthe ecologicalsimi- ACKNOWLEDGMENTS laritiesof thesetaxa with the ploceidEuplectes. Our resultsindicate that the ecologicalsimilar- This researchwas madepossible by contributions ities between Agelaiusand Xanthocephalusare from many individuals, foundations,and govern- convergent. mentagencies. For assistance in the fieldwe thankS. Our data set corroboratesthe polyphylyof Cardiff, A. Castillo,H. Colon,J. Fitzpatrick,A. Kirk- July1999] BlackbirdLineages 637 cormell, N. Klein, M. Lentino, E McGill, G. Morales, data partitionsa reliable indicatorof phyloge- A. T. Peterson,J. V. Remsen,T. Schulenberg,D. Stotz, netic accuracy.Systematic Biology 46:464-478. L. Whittingham, and D. Willard. For generously FELSENSTEIN,J. 1978. Casesin which parsimonyand loaning tissue samplescollected by staff and stu- compatibilitymethods will be positively mis- dentswe thank the LouisianaState University Mu- leading. SystematicZoology 27:401-410. seumof Zoology.For laboratory assistance we thank FELSENSTEIN,J. 1985. Confidence limits on phyloge- M. A. Rogers,P. Austin, J. Hall, and J. Briggs. For fi- hies:An approachusing the bootstrap.Evolu- nancial assistance SML is indebted to the National tion 39:783-791. ScienceFoundation (grants BRS-8614240and BRS- FREEMAN,S., AND R. M. ZINK. 1995.A phylogenetic 8508361to J. W. Fitzpatrick),the Eppley Foundation, study of the blackbirdsbased on variationin mi- and the Ellen Thorne Smith Fund. Laboratory work tochondrialDNA restrictionsites. Systematic Bi- was conducted in the Field Museum's Biochemical ology 44:409-420. SystematicsLaboratory supported by the Pritzker GYLLENSTEN,U. B., AND H. A. ERLICH. 1988. Gener- Foundation Endowment. Our work would not have ationof single-strandedDNA by thepolymerase been possiblewithout the assistanceand encourage- chain reaction and its application to direct se- ment providedby the followingindividuals and in- quencingof the HLA-DQA locus.Proceedings of stitutions: J. V. Reinsen, Jr., Louisiana State Univer- the National Academyof SciencesUSA 85:7652- sity; P. Vanzolini, Museu de Zoologia,Universidade 7656. SaoPaulo; S. Simha,Department of Natural Resourc- HARVEY,P. H., ANDM.D. PAGEL.1991. The compar- es, Conservation Division, Jamaica; E. Cardona, De- ative method in evolutionary biology. Oxford partamento de RecursosNaturales, Puerto Rico; M. University Press,Oxford. Lentino, Sociedad Conservacionista Audubon de JOHNSON,K. P., AND S. M. LANYON. 1999. Molecular Venezuela; L. Gonzalo Morales, Universidad Central systematicsof the gracklesand allies,and the ef- de Venezuela;A. Castillo, Museo Noel Kempff Mer- fectof additionalsequence (cyt b and ND2). Auk cado, Universidad de Santa Cruz, Bolivia; I. Franke, 116:759-768. Museo JavierPrado, Peru; and the United StatesFish KISHINO, H., AND M. HASEGAWA.1989. Evaluation of and Wildlife Service. We also thank J. Klicka, K. John- the maximum likelihood estimate of the evolu- son,R. Zink, and two anonymousreviewers for com- tionary tree topologiesfrom DNA sequence mentson the manuscript.David Swoffordallowed data, and the branchingorder in Hominoidea. us to use test versionsof PAUP*. KEO was supported Journal of Molecular Evolution 29:170-179. by a National ScienceFoundation PostdoctoralFel- KOCHER, t. D., W. K. THOMAS, A. MEYER, S. V. ED- lowshipin BiosciencesRelated to the Environment. WARDS,S. PAABO, E X. VILLABLANCA,AND A. C. WILSON.1989. Dynamics of mitochondrialDNA LITERATURE CITED evolution in mammals: Amplification and se- AMERICAN ORNITHOLOGISTS' UNION. 1983. Check- quencingwith conservedprimers. Proceedings list of North American birds, 6th ed. American of the National Academyof SciencesUSA 86: 6196-6200. Ornithologists'Union, Washington,D.C. AMERICAN ORNITHOLOGISTS' UNION. 1998. Check- LACK,D. 1968.Ecological adaptations for breedingin list of North American birds, 7th ed. American birds. Chapmanand Hall, New York. Ornithologists'Union, Washington, D.C. LANYON,S. M. 1985. Detectinginternal inconsisten- BEECHER,W. J. 1951. Adaptationsfor food-gettingin cies in distancedata. SystematicZoology 34: the American blackbirds. Auk 68:411-440. 397-403. BLAKE,E. R. 1968. Family Icteridae.Pages 138-202 in LANYON,S. M. 1992a.Interspecific brood parasitism Check-list of birds of the world, vol. 14 (R. A. in blackbirds (lcterinae):A phylogeneticper- Paynter, Jr., Ed). Museum of Comparative Zo- spective.Science 255:77-79. ology,Cambridge, Massachusetts. LANYON,S. M. 1992b.Book review.Phylogeny and BLEDSOE,A. H. 1988. Nuclear DNA evolution and classificationof birds:A study in molecularevo- phylogenyof the New World nine-primariedos- lution. Condor 94:304-307. cines. Auk 105:504-515. LANYON,S. M. 1994.Polyphyly of the blackbirdge- BROOKS,D. R., ANDD. A. MCLENNAN.1991. Phylog- nusAgelaius and the importanceof assumptions eny,ecology, and behavior:A researchprogram of monophylyin comparativestudies. Evolution in comparativebiology. University of Chicago 48:679-693. Press,Chicago. MADDISON, W. P., AND D. R. MADDISON. 1992. BURNS,K. J. 1998.Molecular phylogenetics of the ge- MacClade:Analysis of phylogenyand character nus Piranga:Implications for biogeographyand evolution, version 3.04. Sinauer Associates, Sun- the evolutionof morphologyand behavior.Auk derland, Massachusetts. 115:621-634. MANIATIS, T., E. E FRITSCH,AND J. SAMBROOK.1982. CUNNINGHAM,C. W. 1997. Is congruencebetween Molecular cloning.A laboratorymanual. Cold 638 LANYONAND OMLAND [Auk, Vol. 116

Spring Harbor Laboratory,Cold Spring Harbor, and classificationof birds. YaleUniversity Press, New York. New Haven, Connecticut. MILLER,R. S. 1968. Conditionsof competitionbe- S1BLEY,C. G., AND B. L. MONROE, JR. 1990. Distri- tween Red-wingedand Yellow-headedblack- butionand taxonomyof birdsof the world.Yale birds. Journalof Ecology37:43-62. University Press,New Haven, Connecticut. MORONY, J. J., w. J. BocK, AND J. J. FARRAND.1975. SMITH,J. K., AND E.G. ZIMMERMAN.1976. Biochem- Reference list of the birds of the world. Ameri- ical geneticsand evolutionof North American can Museum of Natural History, New York. blackbirds, family Icteridae. Comparative Bio- OMLAND, K. E., S. M. LANYON, AND S. J. FRITZ. 1999. chemistryand Physiology53B:319-324. A molecularphylogeny of the New World ori- SWOFFORD,D. L., G. J. OLSON,P. J. WADDELL,AND D. oles (Icterus):The importanceof dense taxon M. HILLIS.1996. Phylogeneticinference. Pages sampling.Molecular Phylogeneticsand Evolu- 407-514 in Molecular systematics(D. M. Hillis, tion 12:224-239. C. Moritz, and B. K. Mable, Eds). Sinauer Asso- OR1ANS,G. H., AND G. M. CHRISTMAN. 1968. A com- ciates, Sunderland, Massachusetts. parative study of the behavior of Redwinged, THEIN,S. L. 1989.A simplifiedmethod of direct se- Tricolored, and Yellow-headed blackbirds. Uni- quencingof PCRamplified DNA with sequenase versity of California Publicationsin Zoology 84: T7 DNA polymerase.United StatesBiochemical 1-81. Comments 16:8-9. RAIKOW,R. J. 1978. Apendicular myology and rela- WILLIAMS,P. L., ANDW. M. FITCH.1989. Finding the tionshipsof the new world nine-primariedos- minimal changein a given tree. Pages453-470 cines(Aves: Passeriformes). Bulletin of the Car- in The hierarchyof life (B. Fernholm,K. Bremer, negie Museum of Natural History 7:1-43. and H. Jornvall,Eds.). Elsevier, New York. SANGER, E, S. NICKLEN, AND A. R. COULSON. 1977. WILLSON,M. E 1966.Breeding ecology of theYellow- DNA sequencingwith chain-terminatinginhib- headed Blackbird.Ecological Monographs 36: itors. Proceedingsof the National Academyof 51-77. Sciences USA 74:5463-5467. S1BLEY,C. G., AND J. E. AHLQUIST.1990. Phylogeny AssociateEditor: A. J. Baker

APPENDIX.Voucher information for the specimenssequenced in thisstudy. Museum abbreviations: AMNH - AmericanMuseum of Natural History; ANK = Museode Historia Natural "Noel Kempff Mercado," SantaCruz, Bolivia;ANSP = Academyof Natural Sciences,Philadelphia; Cuba = Museode HistorieNat- ural, Havana, Cuba;FMNH = Field Museum of Natural History; MPEG - Muesu ParaenseEm'lio Goeldi, Belem, Brazil.

Species Museum number Agelaiuscyanopus FMNH 334636 Agelaiusfiavus FMNH 330747 Agelaiushumeralis No voucherspecimen Agelaiusicterocephalus FMNH 339772 Agelaiusphoeniceus assimilis Cuba Agelaiusphoeniceus phoeniceus FMNH 341893 Agelaiusruficapillus FMNH 330775 Agelaiusthilius FMNH 334615 Agelaiustricolor LSUMZ 130833 Agelaiusxanthomus No voucherspecimen Agelaiusxanthophthalmus FMNH 324095 Amblycercusholosericeus LSUMZ 98900 Amblyramphusholosericeus FMNH 334662 Cacicus cela FMNH 324080 Cacicuschrysonotus LSUMZ 103278 Cacicus solitarius FMNH 324091 Cacicusuropygialis ANSP 182884 Curaeus curaeus AMNH 826156 Dives warszewiczi LSUMZ 113959 Dolichonyxoryzivorus FMNH 334721 Euphaguscarolinus FMNH 333317 Euphaguscyanocephalus FMNH 341985 Gnorimopsarchopi FMNH 334679 July 1999] BlackbirdLineages 639

APPENDIX. Continued.

Species Museum number Gymnomystaxmexicanus FMNH 339743 Gymnostinopsbifasciatus FMNH 324076 Icteruscayanensis cayanensis MPEG 40.357 Icteruscayanensis periporphyrus FMNH 334609 Icterus bullockii FMNH 341938 Icterusgraceannae ANSP 181810 Icterus icterus FMNH 324092 Icterusleucopteryx FMNH 331149 Icterus mesomelas ANSP 181806 Icterusnigrogularis FMNH 339736 I cterusparisorum FMNH 341943 Icterusspurius FMNH MEX 8495 Lampropsartanagrinus LSUMZ 125586 Leistes militaris FMNH 334657 Macroagelaiusimthurni FMNH 339783 Molothrus aeneus LSUMZ 130743 Molothrus ater FMNH 350707 Molothrus badius FMNH 330801 Molothrus bonariensis FMNH 334768 Molothrusrufoaxillaris FMNH 330805 Nesopsarnigerrimus FMNH 331150 Oreopsarbolivianus FMNH 334687 Passerinacyanea FMNH 341743 Psarocoliusangustifrons FMNH 324068 Psarocolius atrovirens FMNH 324106 Psarocolius decumanus FMNH 324065 Pseudoleistesguirahuro FMNH 330795 Pseudoleistes virescens FMNH 330796 Quiscaluslugubris FMNH 339797 Quiscalusmajor FMNH 341918 Quiscalus mexicanus FMNH 341975 Quiscalusniger FMNH 331153 Quiscalusquiscula FMNH 341733 Saltator coerulescens ANK 170 Scaphiduraoryzivora FMNH 324097 Spizaamericana FMNH MEX 2733 Sturnella bellicosa ANSP 178.118 Sturnellamagna FMNH 339780 Sturnellaneglecta FMNH 330039 Thraupisepiscopus FMNH 339708 Xanthocephalusxanthocephalus LSUMZ 126564 Zonotrichia melodia FMNH 341624