Syst. Biol. 47(4):589–603, 1998

CanW eightingImprove Bushy Trees?Models of Cytochrome b Evolutionand the Molecular Systematics of Pipitsand W agtails(A ves: )

GARY VOELKER1 AND SCOTT V. EDWARDS Burke Museumand Department of Zoology,Box 353010, University of Washington,Seattle, Washington 98195, USA

Abstract.—Among-site ratevariation ( a )andtransition bias( k )havebeen shown, most often asin- dependent parameters,to beimportantdynamicsin DNAevolution. Accounting forthese dynamics should result in better estimates of phylogenetic relationships. Totest this idea,we simultaneously estimated overall (averagedover all codonpositions) andcodon-speciŽ c values of a and k , using maximumlikelihood analysesof cytochrome b datafrom all generaof andwagtails (Aves: Motacillidae), andsix outgroup species, usinginitial trees generatedwith default values. Estimates of a and k were robust to initial tree topology andsuggested substantial among-site rate varia- tion even within codonclasses; a waslowest (largeamong-site rate variation) atsecond-codon and highest (low among-siterate variation) atthird-codon positions. Whenoverall values were applied, there were shifts in tree topology anddramatic and statistically signiŽcant improvements in log- likelihood scores of trees comparedwith the scores fromapplication ofdefault values. Applying codon-speciŽc values resulted in yet anotherhighly signiŽ cant increase in likelihood. However, al- thoughincorporating substitution dynamicsinto maximumlikelihood, maximumparsimony ,and neighbor-joininganalyses resulted in increases in congruence amongtrees, there were onlyminor improvements in phylogenetic signal,and none of the successive approximationstree topologies were statistically distinguishable fromone another by the data.W esuggestthat the bushlike na- ture of manyhigher-level phylogenies in birdsmakes estimating the dynamicsof DNAevolution less sensitive to tree topology but alsoless susceptible to improvement via weighting.[Character weighting; cytochrome b;maximumlikelihood; Motacillidae; ratevariation; systematics; transition bias.]

Aprincipal goalof systematics is to gen- In lieu ofcongruence tests,many system- eratethe bestpossible estimateof the in- atistsuse parsimonyand likelihood scores, terrelationshipsof a group oforganisms, orbootstrapping (Felsenstein, 1985),as “in- andmolecular techniques areincreasingly ternal”tests of the robustness.T oimprove providing systematistswith the meansfor aphylogenetic signal,as measured by ei- doing so.T otestthe strengthof molecu- thercongruence or“ internal”tests, a recent larphylogenies, systematistshave often re- trendhas been toapply aprioriweight- lied oncongruence withother estimates of ing schemeseither toindividual characters phylogeny (Miyamotoand Fitch, 1995). Fre- (e.g., downweighting fast-changingthird quently thishas involvedcomparing molec- positions)or individual transformations ularphylogenies withphylogenies based (e.g., downweighting transitions).Although onmorphological data (e.g., Austin,1996; the conditionsunder whichweighting im- Nunn andCracraft, 1996), but increasingly, provestrees is not fully explored (Huelsen- testsof congruence arebeing used tocom- beck et al.,1994), weighting canconsider- pare severalmolecular estimates of phy- ably improvethe congruence andinternal logeny withone another(e.g., Hackett,1996; robustnessof phylogenetic estimates(Chip- Yoderet al., 1996). Unfortunately ,mosttax- pindale andWiens, 1994; Miyamoto et al., onomicgroups do not have previous esti- 1994;Allard and Carpenter, 1996; Y oderet matesof phylogenetic relationships,either al.,1996; Edwards and Arctander, 1997). morphologicalor molecular, to permit tests However,twogeneral concernsabout the ofcongruence. applicationof weighting schemeshave been raised:(1) Many schemesare ad hoc andhave not been basedon empirical pat- ternsderived fromthe dataunder studyor 1 Present address(and address for correspon- dence): BarrickMuseum, Box 454012, University of fromthose of relatedgroups, and (2) some NevadaLas V egas,Las V egas,Nevada 89154, USA; weighting schemesdo nottake much infor- E-mail:[email protected]. mationfrom the datainto account (Allard 589 590 SYSTEMATICBIOLOGY VOL. 47 andCarpenter, 1996; W akeley,1996;Y ang, odspermit rejection ofthis model in favor 1996a;Edwards, 1997; Purvisand Bromham, ofmore detailed models by suchmethods 1997;Sullivan andSwofford, 1997). These asthe likelihood ratiotest (LRT; Goldman, concernshave been particularlyacute in 1993)(Kishino andHasegawa, 1989; Sulli- avianmolecular systematic studies, many van, 1996). ofwhich apply arbitrarilychosen weights Even if phylogenetic estimatesare not im- toparticular characters or transformations provedby accountingfor differences in char- (e.g., Lanyon,1994; Ellsworth et al.,1996; acterevolution, measuring the dynamicsof Nunn andCracraft, 1996; Richman, 1996; DNAsequences isinteresting in itsown but see alsoEdwards and Wilson, 1990; Ed- rightand can provide useful descriptionsof wardset al., 1991; Cooper et al.,1992;Austin, the molecularevolutionary process. For in- 1996;Edwards, 1997; Houde et al.,1997). For stance,although the gene forcytochrome b example, manyrecent avianstudies alter- isby farthe mostwidely used gene in avian natelyinclude orexclude thirdpositions, or systematics,the dynamicsof this gene have third-positiontransitions, without consider- only begun tobe described in (e.g., ing intermediateweights or treatments for GrifŽths, 1997). Thus, most systema- these sites. tistsemploy verygeneralandadhoc weight- Neglecting toincorporate details of se- ing schemes,typically based on estimates quence evolution,or taking an all-or- fromother data sets. This tendency has nothingapproach to weighting, canbe prob- contributedto questions about whether cy- lematic;for example, completely ignoring tochrome b isa useful phylogenetic marker someclasses of data (e.g., third-codonpo- in avarietyof taxa (Graybeal, 1993; A viseet sitions)or transformations (e.g., transitions) al.,1994; Meyer ,1994). canresult in lossof phylogenetic informa- Weuse the pipits,, and tionat some hierarchicallevels (e.g., Cracraft (Aves:Motacillidae) to explore the above andHelm-Bychowski, 1991; Simon et al., ideas.Despite anessentially global distribu- 1994;Y oderet al.,1996; Moore and DeFil- tion,no explicit morphologicalor molecu- ippis, 1997).In the past,ignorance of the larphylogeny existsfor this group. Tradi- valuesto apply tothese parametersslowed tionally,Žve genera havebeen recognized implementationof character and transfor- withinthe family. Anthus (pipits) isthe most mationweighting schemes.New maximum diverse,including about40 species (Clancey, likelihood (ML)methodsfor estimating dy- 1990;Sibley andMonroe, 1990), and is also namicsof gene regions(e.g., Sullivan et al., themostwidespread, with species occurring 1995;Y ano,1996b) may provide asolution onevery majorlandmass except Antarctica. toat least some of these concerns,as they Motacilla (wagtails)includes 10species and cansuggest weighting schemesthat employ isdistributed from Africa through Eurasia to dynamicsestimated directly from the data. Alaska. Macronyx (longclaws)consists of an An advantageof employing MLmethods Africanradiation of 10 species.Two mono- isthat they provide statisticallyrejectable typic genera, Tmetothylacus (golden ) hypotheses of sequence evolution(Gold- and Dendronanthus (forestwagtail) are re- man,1993) and tree topologies(Kishino and strictedto eastern Africa and eastern Asia, Hasegawa,1989; Huelsenbeck et al.,1996; respectively.In additionto these genera, Sullivan andSwofford, 1997). For example, hasrecently been resurrected the Hasegawa–Kishino– Y anomodel with onthe basisof limited morphological data gammadistribution (HKY85 G ;Hasegawaet (Cooper,1985; Clancey ,1990)as a link be- al.,1985), in whichthere isa single transi- tween Macronyx and Anthus;twospecies, tion:transversion(ts:tv) ratio ( k ) and a sin- Macronyxsharpei and Anthus chloris have gle parameterincorporating rate variation been united in thisgenus. The only state- amongsites ( a ),hasproven asurprisingly mentof phylogenetic relationshipsamong versatiledescriptor of DNA sequence evolu- motacillidgenera isthe limiteddescrip- tion(Hasegawa et al.,1985; Goldman, 1993; tionof Cooper (1985). Although Cooper Yang,1996a). But in principle, MLmeth- suggested that Dendronanthus and Motacilla 1998 VOELKERAND EDWARDS—ESTIMATES OF MOTACILLIDPHYLOGENY 591 formeda sisterclade to a cladein which different tree-building methods,we then in- Tmetothylacus and Anthus were mostclosely corporatethese ratedynamics a posteriori relatedand linked to Macronyx via Hemi- togenerate new treesof motacillid genera. macronyx,he providedno dataor character Ourapproach can be termedsuccessive ap- matrixto support this. proximationsin sofar as we incorporate Inthispaper ,weuse the approachof suc- informationgleaned froman initialtree into cessiveapproximations, based on ML esti- subsequent phylogenetic analyses,although matesof cytochrome b dynamicsof our data, itdiffers fromtraditional successive approx- todetermine the phylogenetic relationships imationapproaches because ouremphasis is of motacillidgenera. Successive approxi- notdirectly on character weights but rather mationapproaches (Farris, 1969), in which ontransformation weights and distributions characterweights are determined by using ofrates among sites. somefunction ofcharacter consistency on aninitial tree, are most often discussedin MATERIALS AND METHODS the contextof parsimony but canin principle be applied in aMLcontextas well (Yang, TaxaInvestigated and DNA Extraction 1994).W eŽrstapply ana prioriweight- Wesequenced 1035base pairs (bp) of ing scheme tocytochrome b sequence data the mitochondrialcytochrome b gene from toestimate initial trees and then use these 12motacillid taxa, as well asfrom 6 out- treesto estimate the dynamicsof our data group taxa(T able 1).Frozen tissues were set,speciŽ cally the transitionalbias ( k ) and availablefor most of these taxa,and mito- among-siterate variation ( a ).Using three chondrialDNA (mtDNA) wasisolatedfrom

TABLE 1.Species, collection locality,andmuseum andvoucher numberfor avian specimens used in this study.

Family,species Origin Collectiona andvoucher number Troglodytidae Salpinctesobsoletus Washington:Grant County UWBM 56992 Cinclidae Cinclusmexicanus Washington:Whatcom County UWBM 56993 Prunellidae Prunellaatrogularis Russia:Gorno-Altay Republic UWBM 46573 Turdidae Catharus fuscescens Washington:Kittitas County UWBM 56991 Motacillidae Dendronanthusindicus Russia:Amurskaya Oblast’ UWBM 45241 Motacilla ava Russia:Sakhalinskaya Oblast’ UWBM 47504 Motacillacinerea Kazakhstan:Alma-Ata Oblys UWBM 46556 Motacillaalba Russia:Gorno-Altay Republic UWBM 46304 Motacillacapensis SouthAfrica: KwaZulu/NatalProvince UWBM 53145 Motacilla aviventris Madagascar FMNH 352834 Tmetothylacustenellus Kenya:Coast Province LACM 87914 Macronyxcapensis SouthAfrica: KwaZulu/NatalProvince UWBM 52793 Macronyxcroceus SouthAfrica: KwaZulu/NatalProvince UWBM 52806 Hemimacronyx chloris SouthAfrica: EasternCape Province UWBM 52814 Anthusbrachyurus SouthAfrica: KwaZulu/NatalProvince UWBM 52901 Anthuscaffer SouthAfrica: KwaZulu/NatalProvince UWBM 52810 Bombycillidae Bombycillagarrulus Alaska:Houston UWBM 53989 Passeridae Passer griseus SouthAfrica: OrangeFree State UWBM 52748 aUWBM= University of WashingtonBurke Museum; FMNH =Field Museum of NaturalHistory; LACM= LosAngeles CountyMuseum. 592 SYSTEMATICBIOLOGY VOL. 47 these specimens viaa cesiumchloride gra- at94°C, 30 s at 50°C,and30 s at72 °C repeated dient (Dowling et al.,1990) to minimize the for35 cycles.AmpliŽ ed productswere pre- chanceof amplifying nuclearmitochondrial paredas templatesfor automated sequenc- sequences. Tissuesof Motacilla aviventris ing by puriŽcation and concentration in were storedin buffer, andtotal genomic 22 m lofwater after three passesthrough DNAwasobtained by Chelex extraction Ultrafree-MC Žltersby centrifugation(Mil- (Walshet al.,1991). Although oursample lipore). Twomicrolitersof the puriŽed and of Tmetothylacus wasfrom a specimen pre- concentratedPCR product was used asa servedin alcohol/formalin,the DNAwas templatein a10- m lDyeDeoxy Cycle Se- successfully extractedvia Chelex. Wewere quencing reaction(ABI), alongwith one of able toamplify andcombine smallersec- the aboveprimers, according to the man- tionsof cytochrome b fromthis specimen, ufacturer’sinstructions(Perkin-Elmer). Af- each ~ 400 bp. Dendronanthus DNA was ob- tercycle sequencing, productswere placed tainedby snipping apiece offoot pad from a oncoarse-grained Sephadex columnsand museum skin andextracting the DNAwith cleaned ofexcess nucleotides via centrifu- acommercialkit (Tissue Protocol,QiAmp, galpassage through the columns(1500 rpm Qiagen;see Mundy et al.,1997). for7 min). Bothlight andheavy strands ofthe entire 1035-bpfragment considered Cytochrome b Isolation,AmpliŽ cation, and were readwith an ABImodel373 automated Sequencing sequencer. Sequences were aligned unam- ForpuriŽ ed mtDNAsamples, the seg- biguously by eye by using Genetic Data mentof cytochrome b consideredhere Environment(developed andmaintained wasampliŽ ed asa single unit viathe by S.Smith,with compilation of programs polymerasechain reaction (PCR) withuse by variousauthors; available free from ofprimers L14841 (Kocher et al.,1989) ftp.bio.indiana.edu,in molbio/unix/GDE). andH16065(Helm-Bychowski andCracraft, 1993).These twoprimers were alsoused in PhylogeneticAnalysis variouscombinations with primers L15114, Choiceof outgroups was difŽcult because L15299,L15609, H15547 (Edwards et al., the pipits andwagtailshave been allied with 1991),and H15299 (Hackett, 1996) to am- nofewer than12 passerinefamilies (see Sib- plify DNAfromthe remaining taxain over- ley andAhlquist, 1981). W etherefore in- lapping segments.For sequencing, weused cluded in thisstudy six of these potential the aboveprimers, the primer H15915(Ed- outgroups,two of which, accentors (Prunel- wardsand Wilson, 1990), and the following lidae) andOld W orldsparrows (Passeridae), primersdesigned speciŽcally for this study were suggested asbeing closeto pipits and (identiŽed by numbers correspondingto the wagtailson the basisof DNA hybridization 3’position in domesticfowl sequence; De- evidence (Sibley andAhlquist, 1981, 1990). jardinsand Morais, 1990): Wearbitrarilyrooted all trees to an Amer- L15086(5’ -CTCTGT AGCTCACATATGCC-3’), icandipper ( Cinclusmexicanus ;Cinclidae), L15376(5’ -CTAGCAGAA TGAGCCTGAGG-3’), which represents afamily thatfalls close to L15616(5’ -GTTGCCCTAACCCTA TTCTC-3’), but hasnot been proposedas asistergroup L15811(5’ -CCCCT ACTCCACACATCAAA-3’), topipits, and allowed all other potential out- H15345(5’ -GTAAT AACGGTAGCTCCTCA-3’), group familiesto “ oat”in the phylogenetic H15671(5’ -GGTGTGAAGTTTTCTGGGTC-3’ ), analyses. H15853(5’ -GGCGGAAGGTTA TTGATC-3’). Nucleotide compositionfor each codon positionand for the entire region sequenced, Wealsoused: aswell ascompositional bias, was deter- mined by using MOLPHY(Adachi and L15350(5’ -TT ACAAACCTATTCTCAGC-3’) Hasegawa,1996). The a (among-siterate designed byJ.Klicka. variation)and k (transitionbias) parame- Fragmentswere ampliŽed in 100- m l PCR ters(see Yang,1996a) were determined for reactions;ampliŽ cation conditions were 30s eachcodon position and for the entire se- 1998 VOELKERAND EDWARDS—ESTIMATES OF MOTACILLIDPHYLOGENY 593 quence by using PAML(Yang,1996b), and overall k and a estimatesto generate anew treeswere determined by variousmethods tree andlog-likelihood score.In asecond (see below). In PAML, weused the HKY85 G analysis,we used codon-speciŽc estimates modelof DNA evolution,which allows of k and a togenerate log-likelihood scores fortwo substitution rates (transitions vs. foreach position by utilizing the tree de- transversions),as well asunequal basefre- rived fromthe Žrstweighted analysisand quencies (Hasegawaet al., 1985). then summedthese scoresfor an overalllog- Maximumparsimony (MP) analyseswere likelihood score.There arecurrently noac- performed withP AUP* 4.0.054d(written by cessible methodsfor estimating ML treesby DavidL. Swofford;results published with assigningspeciŽ c valuesof k and a to spe- permission).In the initialanalysis, all sites ciŽc sites,so weinsteadconŽ ned ouranaly- were treatedas unordered characterstates. sesto estimating the log-likelihood onother Although k wasincorporated into subse- trees. quent analysesvia step-matrices applied to Totestwhether the log-likelihood scores the entire sequence (i.e., one ts:tvvalue for providedby eachlevel ofanalysis were sig- allsites) and to each codon position (i.e., a niŽcantly different, weemployed the LRT different ts:tvvalue foreach codon position), (Goldman,1993). This method tests whether a couldnot readily beincorporatedinto par- morecomplex (or parameter-rich) models simonyanalyses. The heuristicsearch op- provide signiŽcantly better explanationsof tionwith random addition and 10 replicates the observeddata than do simpler mod- wasused foreach weighting scheme.Cladis- els.The teststatistic follows a chi-square ticsignal was determined foreach clade by distribution,with degrees offreedom de- bootstrapping(Felsenstein, 1985). pendent onthe difference in the number Using PAUP*, aneighbor-joining (NJ; ofparameters used between models.Thus, Saitouand Nei, 1987)method was used to accordingto our methodology ,twotests are Žtatree toa matrixof T amura–Nei dis- possible:the log-likelihood scorefrom the tances,which permit unequal basefrequen- initialestimate of phylogeny versusthat cies.Global or codon-speciŽ c a and k val- fromthe estimatederived by using overall ues were incorporatedinto subsequent NJ rates of k and a ,andthe scorewith a sin- analysesby using the MLdistanceoption. gle k and a versusthe scorewith codon- Forthe codon-speciŽc analysis,we gener- speciŽc variables.These comparisonsare ateda distancematrix for each codon posi- nested models(J. Huelsenbeck, pers.comm.; tionbased on codon-speciŽ c a and k values J.Felsenstein, pers.comm.) and so areappro- andthen summedthese distancesto gen- priately testedby using LRT.W eused the eratedistances based on codon-speciŽ c pa- Kishino–Hasegawa (1989) test to determine rameters;direct calculation of such distances whether sometree topologieswere signiŽ- iscurrently unavailablein PAUP*. cantlyworse than others. W eused Rohlf’s An initialML analysiswas done with (1982;as implemented in PAUP*) consensus PHYLIP (Felsenstein, 1993),using default indices tomeasure whether treesgenerated options(F84 model; Felsenstein, 1984)with by using estimatedparameters were more three jumbles oftaxon order. This tree was congruent thanthose trees generated by us- identicalin topologyto one generated in ing default parameters. PAUP* withthe closelyrelated HKY85 G model.W etherefore used the HKY85 G RESULTS modelin allsubsequent MLanalysesbe- causeit describes the dynamicsof many MitochondrialCytochrome b Sequences and genes well (Goldman,1993; Y ang,1996a) Parameters andbecause the parameterestimates from Wealigned 1035-bpsamples without in- PAML were determined under the same sertionsor deletions (sequences tobe sub- model.W eused PAUP* toconduct further mittedfor separate publication). Because MLanalyses.In theŽrstanalysis,we lumped mtDNAwas isolated for most specimens, togetherall codon positions and used the nuclearcopies are less likely forthese taxa 594 SYSTEMATICBIOLOGY VOL. 47 but havea greaterchance of ampliŽ ca- TABLE 3. Values of a and k estimated fromthe initial tion from Tmetothylacus and Dendronanthus . maximumparsimony (MP), neighbor-joining (NJ), and However,because there were noalignment maximumlikelihood (ML)tree topologies. problems,and no nonsense codonswere Method a k present, weareconŽ dent thatthe genes used in thisstudy were mitochondrial. MP 0.305 4.580 NJ 0.307 4.556 Forthe 1035nucleotide sites,447 were ML 0.308 4.543 variable,and 289 were parsimony-informa- tiveacross all taxa. Across ingroup taxa, 299sites were variable,and 159 were distribution,in whichmost sites have inter- parsimony-informative.Of the parsimony- mediaterates and few siteshave very low informativesites, 62 (21.4%) were atŽ rst-, orvery high rates.Third positions also had 25(8.6%) were atsecond-, and 202 (70%) much higher k valuesrelative to Ž rst-and were atthird-codon positions. The un- second-codonpositions (T able 2).These dy- correctedpercent sequence divergence be- namicsare illustrated in Figures 1and2.The tween motacillidtaxa varied from 3.77% leveling off ofthe curve oftransitions ver- to15.07%. Sequence divergence between sustransversions for third positions (Figure motacillidtaxa and outgroups ranged from 1)implies saturationat these sitesin some 14.20%to 22.32%. First-codon positions comparisons.Removal of all third-position showedthe leastamount of base-compos- sitesfrom the dataset resulted in 26equally itionalbias, followed by the second-and parsimonioustrees and caused Motacillaand third-codonpositions (T able 2).Third-codon Anthus tobecome paraphyletic,suggesting positionsshowed a moderateamount of thatthe thirdpositions contain some sig- base-compositionalbias (0.192, G-T poor), nal.Indeed, third-position-onlytransver- whichis similar in directionfor all sequences sionparsimony (where third-position (Table 2). transversionsare upweighted relativeto Overall k and a valuesvaried very little othersubstitutions at all codon positions; acrossthe initialtree topologiesgenerated see Yoderet al.,1996) retained Anthus and by eachmethod of analysis (T able 3;see Motacilla asmonophyleticgenera. below). Asone wouldexpect fora protein- codinggene, the analysesshowed substan- tialrate heterogeneity amongall sites. The PhylogeneticAnalyses low a valuesfor Ž rstand second positions An equally weighted (i.e., unweighted) (Table 2)suggestthat most sites have either parsimonyanalysis resulted in three most- very lowsubstitution rates or are invariable, parsimonioustrees of 1174 steps (Fig. 3a), whereasa few sitesexist with very high rates which variedonly in their placement of (Sullivan et al.,1995; Y ang,1996a). Third po- Motacillacinerea , ava, and alba relative to sitions,however, showed a higher value eachother .Passeridaeis placed asthe closest of a (Table 2),suggesting amoreuniform outgroupto Motacillidae in avery pectinate

TABLE 2.Overall andcodon-speciŽ c dynamicsof the cytochrome b geneacross all taxaestimated byusing PAMLand the unweighted MLtree (Fig.3c).

Codon position Dynamic First Second Third All Meanbase composition a 24,24,22, 30 13,20, 42, 25 3, 45, 6, 46 13,28,25, 34 Meannucleotide bias 0.004 0.061 0.192 0.030 a 0.299 0.152 1.352 0.308 k 4.493 3.175 21.512 4.543 aBasecomposition is presented as%G,%A, %T,%C averagedover all sequences. 1998 VOELKERAND EDWARDS—ESTIMATES OF MOTACILLIDPHYLOGENY 595

FIGURE 2.Codon-speciŽ c analysisof among-site FIGURE 1.Codon-speciŽ c plots of observed num- ratevariation in the motacillid cytochrome b sequence bersof transitions versus transversions forthe motacil- data. lid mitochondrial cytochrome b gene.

(ladder-like) tree.This tree wasnotwell sup- bustto uncorrected, Kimura two-parameter ported,as indicated by bootstrapanalysis, (Kimura,1980) and ML distancemethods. in which allintergeneric relationshipswere Becauseof its placement with Macronyx unresolved (i.e., < 50%bootstrap support and Tmetothylacus rather than Anthus, Hemi- [BS]), aswere mostfamilial relationships macronyx issupported asa validgenus in (Fig. 3a). thisanalysis. The tree suggeststhat Dendron- AMPstep-matrixanalysis that weighted anthus and Motacilla aresisters. Prunelli- tranversionsby the overall k value (Table 2) daehere ismost closely related to Motacil- resulted in asingle most-parsimonioustree, lidae.As in the MPanalysis,there isstrong whichdiffered fromthe equally weighted supportthat Prunellidae andPasseridae are analysis(Fig. 3a)only in the switchingof the closerto Motacillidae than are the otherout- Macronyx and Tmetothylacus nodes.There group taxaconsidered here. wasslightly better bootstrapresolution of An NJanalysisperformed withoverall relationships,in thatall pipit andlongclaw rates of k and a did notresult in changes genera were grouped togetherwith moder- in tree topology,regardlessof which dis- ate(60%) BS. Relationshipsbetween genera tancemeasure was used. However,NJ anal- onthis node, as well asbetween thisnode ysisusing distancesgenerated fromcodon- and Dendronanthus and Motacilla, nonethe- speciŽc k and a values(Fig. 3e) changed lesshad low BS. the relationshipsof all Motacilla from those AnMP analysisemploying codon-speciŽc in the initialNJ analysis(Fig. 3b);unfortu- step-matrices(i.e., weighting eachposition nately,the availableprograms did notper- by itsrespective k value) produced asingle mita bootstraptest in thiscase. However, most-parsimonioustree whosetopology because species samplingcan have a large wasnonpectinate (Fig. 3d).The number of impacton tree topology(Lecointre et al., nodessupported at > 50%both within 1993),and because we included only half Motacillidaeand among the closestout- of all Motacilla species in thisanalysis, nei- groupswas one greaterthan those of the thertree topologycan truly beconsidereda equally weighted bootstrapanalysis (Fig. better estimateof relationships within this 3a). . Resultsof the NJanalysisutilizing default InitialML analysisin PAUP* (HKY85 G values(Fig. 3b) stronglysupport the mono- model) resulted in atree withlog likelihood phyly ofMotacillidae. This result was ro- of-7154.06. This tree (Fig. 3c)differed from 596 SYSTEMATICBIOLOGY VOL. 47

FIGURE 3.Phylogenetic relationships amongmotacillid generabased on default parameters(a– c) andon overall parametersestimated forcytochrome b (d–f). Numbers at nodesrepresent BS > 50%for 100 replicates. the initialNJ tree only in that Dendronanthus , An MLanalysisemploying the overall a rather than Motacilla,wasbasal to the pipits and k valuesof 0.308 and 4.493 (T able 2),re- andlongclaws.Passeridae was placed asthe spectively,resulted in achange in tree topol- closestoutgroup to Motacillidae. ogy (Fig. 3f) fromthe initialML estimate 1998 VOELKERAND EDWARDS—ESTIMATES OF MOTACILLIDPHYLOGENY 597

(Fig. 3c). Hemimacronyx wasplaced assis- dex.The averagecongruence between the ter to Tmetothylacus ,andPrunellidae was unweighted MP,NJ,andML topologieswas placed asthe closestoutgroup to Motacil- 0.502(T able 5),whereas the averagecongru- lidae;this tree isotherwise consistent with ence between the weighted MP,NJ,andML the initialML estimate,indicating robust- topologieswas 0.679 (T able 5).MP proved nessof ML tomodel ofevolution.In addition sensitiveto which parameters were used;NJ tothese tree changes,there wasa signiŽcant andML were not(Table 5). improvementin likelihood scoreunder the new modelas compared with that for the DISCUSSION default modelon this new tree (Table 4). Although thissecond ML tree isa sig- Cytochrome b Dynamics niŽcant improvement of relationships in Table 3showsthat the estimatesof k (tran- comparisonwith the initialtree, we won- sitionbias) and a (among-siterate varia- dered whether ananalysisutilizing aneven tion)were robustto the initialtree chosen; morecomplex model of evolution, based on thisis not always the case.Y ang(1996a) ourpicture of evolutionarydynamics of andW akeley (1996)showed that these rates cytochrome b (Table 2),provided an im- couldbe seriouslyunderestimated if the tree provedŽ tofthe datato this tree topology? topologyused toestimate the ratesis com- Totestthis idea, we summedthe likeli- pletely wrong.Incorrect estimates may de- hoodsof each codon position on the second pend onhow many wrong long branches MLtree by using codon-speciŽc dynamics; arein the tree being used;in ourmotacil- we obtainedyet anotherhighly signiŽcant lidsand many other family-level trees(see improvementin log-likelihood score(T able below) there arefew long internalbranches. 4).Unfortunately ,we areunable todeter- Itremains to be seen howgeneral the rela- mine whether thiscodon-speciŽ c likelihood tionshipbetween sensitivityto tree topology scorecould be coincidentwith a further andbranch lengths is. change in tree topologywith available pro- Asexpected fora protein-coding gene like grams;P AUP* doesnot yet acceptmulti- cytochrome b,there wasample evidence for ple valuesof a (D.Swofford,pers. comm.), variationin substitutionrate among codon andPHYLIP isunable toassign speciŽ c val- positions(Figs. 1and2; T able 2).Interest- ues of a tospeciŽ c sites(M. Kuhner,pers. ingly,wefound substantialamong-site rate comm.).Given the improvementin score variationwithin each codon position, the when codonsare analyzed independently ratevariation being mostextreme withinthe fromone another,a change in topologyis Žrstand second positions (T able 2).Within- entirely possible. codon-positionrate variation is probably Wewere unable toreject statistically(via due tofunctional constraints on protein the Kishino–Hasegawa test) any of the tree products;attempts to assess these functional topologiespresented in Figure 3asbeing constraintshave only recently been under- signiŽcantly worse estimates of phyloge- takenfor birds (e.g., GrifŽths, 1997). k also netic relationships.There is,however, an in- variedsubstantially between codonposi- creasein congruencebetween default (orun- tions,with third positions showing very weighted) andweighted tree topologies,as high biascompared with Ž rstand second determined byRohlf’s(1982)consensus in- positions(T able 2).

TABLE 4.Likelihood ratio tests ofdifferences in log-likelihood estimates between increasingly more complex models of cytochrome b evolution utilizing Motacillidae andoutgroup data.

Estimatedvalues of k and a Log likelihood SigniŽcantly better estimate? None(default options) -7154.06 Overall -6699.99 yes; P < < 0.001 Codon-speciŽc -6195.41 yes; P < < 0.001 598 SYSTEMATICBIOLOGY VOL. 47

TABLE 5.Measures of congruence amongtree topologies in Figure3, using Rohlf’ s (1982)consistency index.

Default Estimated MP NJ ML MP NJ ML Default MP — NJ 0.508 — ML 0.338 0.660 — Estimated MP 0.286 0.702 0.638 — NJ 0.460 0.915 0.596 0.638 — ML 0.286 0.702 0.870 0.761 0.638 —

Ourexploration of among-site rate varia- The dramaticimprovements of likelihood tion(Fig. 2)suggests that removing entire seen while using increasingly complexmod- classesof characters may not be the best els (i.e., default valuesvs. overall dynamics courseof action, because somechanges in vs.codon-speciŽ c dynamics)suggests that thosesites appear unsaturated and thus may cytochrome b evolutioncannot be described bephylogenetically informative(e.g., third aseither a“fast”or a “slow”gene (Meyer, positions,Fig. 2).Some sitesand changes 1994)nor as one withan overallhigh transi- withinsuch classes can be evolving more tionbias. Codon-speciŽ c dynamicsthus rep- slowlythan some sites in nonsaturated resenta better descriptionof cytochrome b classesof characters (Fig. 1).For example, 59 evolutionthan do the overalldynamics em- third-positionsites with one change show ployed in moststudies with this gene. lesslability than do 82 Ž rst-and second- Wefound differences in basecomposition positionsites with two or more changes per between codonpositions but littledifference site(Fig. 2). in basecomposition within any given codon Although we showed(T able 4)that a positionacross motacillid species; interspe- modelincorporating speciŽ c parameters ciŽc variationin basecomposition of third wasan improvementover the use ofa sin- positionswas the mostextreme (notshown). gle k and a ,the datain Figure 2suggest Ourbase composition Ž ndings were similar otherfruitful lines ofanalysis. A rate-speciŽc tothose reported from other avian studies, modelthat could account for site-speciŽ c andbase composition at each codon posi- heterogeneity regardlessof codon position tionwas found tovary little across many mightprove abetter estimatorof phylogeny distantlyrelated bird lineages, suchas Ram- anddynamics.W ebriey explored thisidea phocelus tanagers(Hackett, 1996), birds-of- by placing allsites into one ofthree cate- paradise(Nunn andCracraft, 1996), and gories:slow sites (those changing 0to2 steps severalsuboscine and oscine taxa in Fig. 2),medium sites(3 to 4 steps),and fast (Edwardset al., 1991), as well nonpasser- sites(5 to 8 steps).W eagainused PAML with ine taxa(e.g., somecranes [Krajewski and the MLtree toestimate k and a foreach cate- King, 1996]and woodpeckers [Cicero and goryand then used these valuesto estimate Johnson,1995]). This similarity in basecom- likelihood scoreson the tree in Figure 3fby positionamong diverse avianlineages in- using PAUP*. Bysummingthese scores,we directlysuggests similarity in cytochrome b obtaineda likelihood scoreof -6108.593, a re- dynamics(Sueoka, 1992;Jermiin et al.,1995). ductionof 85 units from the codon-speciŽc Equilibrium basecompositions are proba- score(T able 2).Although thisimprovement bly due topatterns of directional mutation maybe nonsigniŽcant (these scoresare not pressure (Sueoka, 1992),and the composi- comparableby use ofthe LRT),the trend tionof sites with very biasedcompositions doessuggest that Ž ner partitioningof sites mayre ect the underlying mutationspec- wouldalmost surely improveŽ tofmodels trummore faithfully thansites with an even todata and possibly to tree topology. basecomposition (Jermiin et al.,1995). Thus 1998 VOELKERAND EDWARDS—ESTIMATES OF MOTACILLIDPHYLOGENY 599 basecomposition can be auseful surrogate daethan did default values(Fig. 3a:3 nodes forsubstitution dynamics, unless patternsof > 60%,Fig. 3d:7 nodes > 60%),and total selectionvary widely acrossspecies, which BSwithinMotacillidae improved from 583 isunlikely forcytochrome b.Thissimilarity when using default parametersto 673 and in basecomposition raises the exciting pos- 656with use ofoverall and codon-speciŽ c sibility thatpatterns of base substitution in parameters,respectively .Incorporatingthe cytochome b aresimilar across birds, a pre- estimated k value alsoincreased congru- dictionthat can be testedwith further anal- ence ofMP toother tree topologies(Table 5) yses. andresulted in greateraverage congruence The k valuesestimated for other birds amongmethods as well (althoughthis does havevaried by studyand gene region notnecessarily imply correctrelationships; (Cooper et al.,1992; Austin, 1996; Krajew- Felsenstein, 1993).Our results suggest that skiand King, 1996;Edwards, 1997), and in- aposterioriweighting hasimprovedthe de- evitably these empirically derived values tectionof signal in ourmotacillid data. (1:3.8–1:9 for all codon positions in the cy- Despite improvementin signal,we were tochrome b segment examined) differ from unable toreject statisticallyany of the trees thosemost commonly used in otherstudies in Figure 3viathe Kishino–Hasegawa (1989) (e.g., 1:2,1:5, 1:10). Edwards (1997) found test.This may be afunction ofrapid evo- thatestimates of k and a varied,depend- lution,rather than some inherent problem ing onthe taxonomiclevel investigatedand ofusing cytochrome b in phylogenetic anal- the depth ofthe tree.Because no bird stud- ysis.Branch lengths linking Dendronanthus ies haveyet applied standarderrors to es- and the non-Anthus pipits toother genera timates of k ,wedonotknow the extentto arerelatively short compared with other which these resultsare compatible with one branches,perhaps suggesting thatrapid anothernor how much ourestimates would evolution(similar to a starphylogeny) has change were wetoexamine, say ,cytochrome occurredin thisgroup andthus confound- b variationwithin Anthus alone.In addition, ing anyestimation of phylogenetic relation- althoughprevious ML estimatesof k in birds shipsfrom cytochrome b. areto be commended,none ofthe previ- Ourinability todetermine whether ousestimates assumed among-site rate vari- Prunellidae orPasseridae is closer to ation.Because W akeley (1993)showed that Motacillidae(see below) mayalso be a k and a arenot independent andneed tobe function ofrapid evolution. In their treat- estimatedsimultaneously ,these earlieresti- mentof Passeridae, Sibley andAhlquist matesare compromised. (1990)placed Motacillinaebetween Prunel- Doesincorporating these dynamicsim- linae andPasserinae, stating that the diver- prove phylogenetic signaland consistency? gences withinPasseridae were soclose to- Yesand no. NJ provedresistant to whether gether thatthey couldnot be certainof the default orestimated parameters were em- exactsequence ofbranchings. Sibley and ployed (Fig. 3;Table 5).However, using es- Ahlquist(1990) also suggested thatthese timateddynamics in MLanalysesis clearly branchingsof passeridlineages occurred superiorto using default values(T able 4), withina period of5 millionyears and that andin ourMP analyses,the number ofnodes the branchingsmay be represented asa supportedat > 50%increased from 9, de- multifurcation.Other studies (Bleiweiss et termined by using default values,to 10, de- al.,1994; Sheldon andGill, 1996) have ex- termined by using overallrates or codon- perienced similardifŽ culties in resolving speciŽc rates(Fig. 3).Using overallrates higher-levelavianrelationships and have ar- providedless support within Motacillidae gued thatshort internodal distances sug- thandid codon-speciŽc rates(6 resolved gestsshort time intervals between the di- nodesvs. 7). When weincreasedthe criteria vergences ofmajor groups. Thus, the lack forresolution to 60% BS, overallor codon- ofresolution for the groupsconsidered here speciŽc ratesalso provided for higher levels shouldnot be broadlyinterpreted asanother ofsupport for nodes within the Motacilli- strikeagainst using cytochrome b in molecu- 600 SYSTEMATICBIOLOGY VOL. 47 larsystematics, despite thefactthat poor res- themselves,and, if ourpipit resultsare gen- olutionof many above-species-level treesin eral,the shortbranches that seem toplague birdsby cytochrome b isbecoming common- manyavian phylogenies mayprove resis- place.Indeed, weknowthat the poorreso- tantto improvement via weighting. lutionin ourtrees is not strictly a function ofcytochrome b,because thisgene hasbeen PhylogeneticRelationships shownto resolve branches that are older All methodsof phylogenetic reconstruc- thanor approximatelyequal to(e.g., Moore tionconsidered here supportthree groups andDeFilippis, 1997)those in thisstudy .A withinthe Motacillidae: Dendronanthus , litmustest for poorly resolved groups will wagtails (Motacilla),andthe pipits plus betouse adifferent, e.g.,nuclear, gene on longclaws (Anthus, Tmetothylacus,Hemi- thesamegroup todetermine whether phylo- macronyx,Macronyx ).BothNJ andML genies derived fromthem are well-resolved stronglysupport (and MPweakly sup- andthus able todiscriminate between bad ports)the assertionfrom a morphologi- gene orbad trees/ groupsin previousphy- calstudy (Cooper, 1985) that the yellow- logenetic estimates. breastedpipit isnot an Anthus, and thus The mostfrequently employed alternative wesuggestthat it is reasonable to place this tochoosing an a prioriweighting scheme is species in Hemimacronyx .Interestingly,re- touse the default aprioriweighting scheme movingthis species from Anthus places all ofequal weight (i.e., unweighted) atallsites; members ofthe pipit andlongclaw group thus,it bears repeating thatthe apriorias- thatare almost entirely yellow ventrally sumptionof equal weightsis itself astrong togetheras close relatives ( Tmetothylacus, assumption(Swofford etal., 1996) that can Hemimacronyx,Macronyx ). itself be morearbitrary than weights based MP (Fig. 3a,d) failsto support, and NJ onempirical and theoretical considerations andML (Figs. 3e,f) conict regarding, the (Simon et al.,1994). Many studiescriticiz- placement of Dendronanthus .The BSofthe ing the use ofcytochrome b arein effect say- initialNJ tree ismoderate (Fig. 3b).De- ing thatthe methodsof analysis and our un- spite aresemblance to Motacilla in over- derstandingof cytochrome b dynamicsare allappearance of plumage, mostbehavioral inadequate.Cytochrome b maywell have andmorphological similarities support the complexdynamics that result in fewer sites placement of Dendronanthus near Anthus, thatare parsimony-informative by standard assuggested by the MLtree.For example, methods,but ML estimatesof ts:tv and other when onthe ground, Dendronanthus resem- rates,obtained from the relevantsequence bles apipit (Ali andRipley ,1973);also, like dataand applied aposteriori,appear to be some Anthus,itspends much timein trees, one wayof improving estimates of phy- isassociated with woodlands, and is not logenetic relationships(e.g., Sullivan and closelyassociated with water, as Motacilla is Swofford,1997; this study). Although not (Neufeldt, 1961).Also, Dendronanthus eggs problem-free (e.g., Yang,1996), additional resemble thoseof Anthus hodgsoni (Neufeldt, beneŽts of using MLtoestimate phyloge- 1961).Based on these resemblancesto An- netic relationshipsfrom sequence dataare thus,wesupportthe placement of Dendron- itsinsensitivity to problems such as base anthus suggested by the MLtree (Fig. 3f). compositioninequalities, multiple substi- All successiveapproximation analyses tutions,models of DNAsubstitution,and supportPrunellidae asthe nearestout- number oftaxa— problems that are inher- group toMotacillidae, although Passeridae ent in othermethods (Huelsenbeck, 1995; iscloser in somedefault analyses(Fig. 3). Perna andKocher, 1995; Sch oniger¨ andvon Bothof these Žndings areconsistent with Haeseler,1995; Strimmer and von Haeseler , the Žndings fromDNA– DNAhybridiza- 1996;Swofford etal., 1996; see alsoNay- tion,which suggested acloserelationship lorand Brown, 1997). Even so,the ability between these three lineages (Sibley and toimprove trees via weighting seemscrit- Ahlquist,1981, 1990; see above).All meth- icallydependent onthe natureof the trees odssupport Prunellidae andPasseridae as 1998 VOELKERAND EDWARDS—ESTIMATES OF MOTACILLIDPHYLOGENY 601 being closerto Motacillidae than are other for“ higher”nonpasserines:Reevaluating akeypor- outgroupsconsidered here, but amore- tion ofthe avianfamily tree. Mol.Phylogenet. Evol. detailedanalysis of familial relationships is 3:248–255. CHIPPINDALE, P. T., AND J. J. WIENS.1994.W eighting, necessarybefore adeŽnitive statementcan partitioning, andcombining characters in phyloge- be made. netic analysis.Syst. Biol. 43:278–287. CICERO, C., AND N. K. JOHNSON.1995.Speciation in sapsuckers( Sphyrapicus ):III.Mitochondrial-DNA se- ACKNOWLEDGMENTS quence divergence atthe cytochrome- b locus. Auk G.V.is very grateful to S.Rohwer forstarting him 112:547–563. onthe motacillid path.This work beneŽted greatly CLANCEY,P.A.1990.A review ofthe indigenous fromdiscussions/ communications with J.Felsenstein, pipits (genus Anthus Bechstein: Motacillidae) ofthe D.Swofford,J. Sullivan, P.Beerli, andJ.Huelsenbeck.S. Afrotropics. DurbanMus. Novit. 15:42–72. Rohwer, R.Huey ,andthree anonymousreviewers con- COOPER, A., C. MOURER-CHAUVIRE, G. K. CHAMBERS, tributed valuable comments andcriticisms. G.V.thanks A. VON HAESELER, A. C. WILSON, AND S. PA¨ ABO¨ . A.Berruti, T.Cassidy ,R.Nuttall, andthe Leitch, Pien- 1992.Independent origins ofNew Zealandmoas and aar,and Whipp families forŽ eld assistance andhospi- kiwis. Proc. Natl.Acad. Sci. USA89:8741–8744. tality while in SouthAfrica; V.Molkov,V.Khrokov,and COOPER,M.R.1985.A reveiw of the genus Macronyx N.Borisova forthe samein Kazakhstanand Russia; andits relationship to the yellow-bellied pipit. Hon- andthe permitting agencies of all three countries for eyguide 31:81–92. their consideration andassistance. W ethankD. Swof- CRACRAFT, J., AND K. HELM-BYCHOWSKI.1991.Parsi- fordfor permission to use PAUP*, andK. Garrett(Los monyand phylogenetic inference usingDNA se- Angeles CountyMuseum) andD. Willard andT. Schu- quences:Some methodological strategies. Pages184– lenberg(Field Museum of NaturalHistory) for samples 220 in Phylogenetic analysisof DNA sequences (M. of species criticalto this research.Throughout this work M.Miyamotoand J. Cracraft,eds.). Oxford Univ . G.V.hasbeen supported byanEddyGraduate Fellow- Press, New York. ship in Ornithologyand the LeadersFiveFellowshipfor DEJARDINS, P., AND R. MORAIS.1990.Sequence and Molecular Systematics,both from the BurkeMuseum. geneorganisation of the chicken mitochondrial While collecting specimens forthis researchin Russia, genome:A novel geneorder in highervertebrates. 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