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: Motacillidae) 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 DNA evolution. Accounting for these dynamics should result in better estimates of phylogenetic relationships. Totest this idea,we simultaneously estimated overall (averagedover all codonpositions) andcodon-speci c values of a and k , using maximumlikelihood analysesof cytochrome b datafrom all generaof pipits 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 was lowest (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 signicant improvements in log- likelihood scores of trees comparedwith the scores fromapplication of default values. Applying codon-specic values resulted in yet anotherhighly signi 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 anotherby the data.W esuggestthat the bushlike na- ture of manyhigher-level phylogenies in birdsmakes estimating the dynamicsof DNA evolution less sensitive to tree topology but alsoless susceptible to improvement via weighting.[Character weighting; cytochrome b;maximumlikelihood; Motacillidae; ratevariation; systematics; transition bias.] Aprincipal goalof systematicsis 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 of thismodel 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). of which 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; DNA sequences 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 birds(e.g., ing intermediateweights or treatments for Grifths, 1997). Thus, most bird 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 nothing approachto 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,wagtails, and longclaws 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,ve 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 10species.Two mono- isthat they provide statisticallyrejectable typic genera, Tmetothylacus (golden pipit) 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, Hemimacronyx hasrecently been resurrected the Hasegawa–Kishino– Y anomodel with onthe basisof limitedmorphological data gammadistribution (HKY85 G ;Hasegawaet (Cooper,1985; Clancey ,1990)as a link be- al.,1985), in which there 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 motacillidgenera. 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 ourdata, 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 of ratesamong sites. somefunction of characterconsistency on aninitial tree, are most often discussedin MATERIALS