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Home , California quail, Callipepla, Montezuma quail, Scaled quail

The Auk 115(2):394-403, 1998

MOLECULAR SYSTEMATICS OF THE SCALED COMPLEX (GENUS )

ROBERT M. ZINK 1 AND RACHELLE C. BLACKWELL JamesFord Bell Museum of NaturalHistory, 100 EcologyBuilding, University of Minnesota, St. Paul, Minnesota 55108, USA

ABSTRACT.--Weobtained 1,040 bp of sequencefrom the mitochondrialDNA (mtDNA) genescytochrome b (cyt b; 736 bp) and NADH-subunit2 (ND2; 304 bp) to addressphylo- geneticrelationships among the four speciesin the ScaledQuail complex. Quail (Callipeplacalifornica) and Gambel'sQuail (C. gambelii)were sistertaxa, whereasthe rela- tionshipsof theElegant Quail (C. douglasii)and (C.squamata) were unclear; they might be sisterspecies, or ElegantQuail might be the sisterto California plus Gambel's .A third, less-likelyalternative predicts a contemporaneousorigin of ElegantQuail, ScaledQuail, and the ancestorof Californiaand Gambel'squail. The latterphylogenetic hy- pothesis,however, matches Hubbard's (1973) biogeographic model. Irrespective of which biogeographichypothesis is correct,calibration of mtDNA geneticdistances suggests that the speciationevents are mucholder than the late Pleistocenedates given by Hubbard.Cal- ibrationof the rate of mtDNA (cyt b, ND2) evolutionbased on datingof fossilremains of the extinctspecies cooki suggested a rate of 2% per millionyears. (Colinusvirginianus), (Oreortyxpictus), and Montezuma Quail (Cyrtonyx montezumae)were successivelymore distantly related to the ScaledQuail complex.Phylo- genetictrees derived from allozymes(Gutierrez et al. 1983) and mtDNA sequenceswere topologicallyidentical, suggesting that bothtypes of genetrees recover the speciestree. Re- ceived28 March 1997, accepted9 October1997.

THE SCALEDQUAIL COMPLEX(Hubbard 1973) Slowinski 1995, Klicka and Zink 1997). Some- includes four : (Calli- what unusual,at least by modernstandards, peplacalifornica), Gambel's Quail (C. gambelii), was the initial suppositionof a basaltrichoto- ScaledQuail (C. squamata),and ElegantQuail my. (C. douglasii).These species occur throughout We used sequencedata from the mitochon- arid regionsof the southwesternUnited States drial DNA (mtDNA) cytochromeb (cyt b) and and north of the Isthmus of Tehuante- NADH-subunit 2 (ND2) genes to test Hub- pec (Fig. 1). Hubbard (1973) proposedan evo- bard's(1973) hypothesis. Our data confirma lutionaryscenario involving at leasttwo glacial close relationshipbetween California Quail cyclesto accountfor the evolutionand biogeo- and Gambel'sQuail, and a possiblesister-taxon graphichistory of thesequail. During the Illi- relationshipbetween Scaled Quail andElegant noian Glacial a widespread ancestor was Quail. We also assessedcongruence between thoughtto havebeen isolated into three taxa phylogenetichypotheses derived from mtDNA termed " pre-californica/ gambelii," " pre-doug- sequencesand allozymevariation (Gutierrez et lasii," and "pre-squamata."Their rangesex- al. 1983). pandedduring the Sangamoninterglacial and during the WisconsinGlacial californicaand METHODS gambeliisplit into distinct species,whereas douglasiiand squamatadid not becomefurther We sequencedthree CaliforniaQuail (from Baja subdivided.Thus, Hubbardpresented an ex- California),three Gambel's Quail (),four plicit and testablehypothesis for the evolution ScaledQuail (New Mexico),and three ElegantQuail and distributionof thesefour species.Typical (,Mexico), and one eachof the followingout- of suchexplanations was the assumptionthat group species:Northern Bobwhite(Colinus virgini- the lastbout of glaciationplayed a rolein spe- anus),Montezuma Quail (Cyrtonyxmontezumae), and ciation (Berminghamet al. 1992, Zink and MountainQuail (Oreortyxpictus). Specimen voucher numbersand detailson collectinglocalities are given with sequenceinformation in the Genbankaccession E-mail: [email protected] (nos.AFO28750 to 28782;see Table 2). Sequencedata

394 April 1998] MolecularSystematics ofQuail 395 'rnia Quail (C. californica) J Gambel'sQuail (C.gambelii)

Scaled Quail (C. squamata)

Elegant Quai ( C. douglasii) F•c. 1. Distributionof speciesin the ScaledQuail complex(from Hubbard 1973). for the aboveingroup and outgroupspecies consti- 1996) were performed and run on 6% acrylamide tute Data SetA. For comparisonwith allozymedata gels.Cyt b wassequenced in bothdirections with the of Gutierrezet al. (1983),we usedonly sequencedata amplification primers and one internal primer, for a 736 bp segmentof cyt b from Kornegayet al. H15299 (Hackett 1996). ND2 was sequencedonly (1993;Genbank accession code) for Chukar (Alectoris with H5578, because L5215 did not work as a se- chukar;L08378), JapaneseQuail (Coturnixcoturnix; quencingprimer. Sequences were alignedwith the L08377), and Silver (Lophura nycthemera; published chicken sequence (Desjardins and Morais L08380,as a substitutefor theRing-necked Pheasant 1990);no gaps were detected. [Phasianuscolchicus]), and we obtainedsequence data We computed basic sequencestatistics, Kimura's of LesserPrairie-Chicken (Tympanuchus pallidicinc- (1980) two-parameterdistances (K2P), and neigh- tus)from J. G. Grothand G. E Barrowclough(unpubl. bor-joining(NJ) trees with MEGA (Kumar et al. data). Sequencedata for thesespecies plus thosein 1993). We used PAUP (Swofford 1993) to conduct Data Set A constitute Data Set B. We also determined maximumparsimony searches (branch and bound) the sequencefor a smallsegment of cyt b andthe ad- of thedata, with weightsof 1:1and 1:2 for transitions joining t-RNA•eu from samplesof study skinsfor two and tranversions,to bootstrap(Felsenstein 1985) the additional species,Banded Quail (Philortyxfasciatus; unweightedcharacters 1,000 times, and to compute Bell Museumornithology catalogue number 10947) g-valuesas measuresof phylogeneticsignal (Hillis and Singing Quail (Dactylortyxthoracicus; Bell Mu- 1991, Hillis and Huelsenbeck1992, Kallersjoet al. seum 14958), to assesstheir relationshipsto the 1992). PAUP* (Swofford pers. comm.) was used to ScaledQuail complex. computelog-determinant distances (log-der), a con- We used standard methods to extract DNA from servative estimate of mtDNA genetic distances tissueand study skins (Ellegren1992, Hillis et al. (Swofford et al. 1996). Maximum-likelihoodtrees 1996,Zink et al. 1997)and the polymerasechain re- were estimated with PHYLIP (Felsenstein1993). action (PCR; Saiki et al. 1988, Palumbi 1996) to am- Competingtree topologieswere tested with the plify mtDNA. PrimersL14841 (Kocheret al. 1989) Kishino-Hasegawa(1989) maximum likelihood test. and H4a (Harshman1996) amplified an approxi- MacClade (Maddison and Maddison 1992) was used mately 1,100bp segmentof cyt b. A 400 bp portion to evaluatethe lengthsof alternativetopologies. We of ND2 wasamplified with L5215and H5578 (Hack- used Mantel's (1967) test as implemented in NTSYS ett 1996).Manual sequencingreactions (Hillis et al. (Rohlf 1992) to comparematrices of Rogers'(1972) 396 ZINK AND BLACKWELL [Auk, Vol. 115

TABLE 1. Distribution of variation at nucleotide spectively)were: Adenine (27.1%, 34.0%), Thy- sitesfor cytochromeb (Cyt b) and NADH-subunit 2 (ND2); TS transition, TV - transversion. mine (25.2%, 22.8%), Cytosine (33.7%, 32.2%), and Guanine (14.0%, 11.0%). Of the 1,040 First Second Third alignedbases, 241 were variable,and 135 were Gene region TS TV TS TV TS TV potentiallyphylogenetically informative; vari- able sites were mostly third positiontransi- Cyt b 30 7 8 0 92 33 ND2 6 3 7 2 42 11 tions (Table 1). A total of 33 amino acids was variable (11 were parsimonyinformative). Sequencedivergence (K2P distances;Table 2) ranged from 0.0019 (within California Quail genetic distances derived from allozyme compari- and Gambel's Quail) to 0.1635 (Montezuma sons (Gutierrez et al. 1983) and 1og-detdistances; Quail vs. Mountain Quail). Among the four significancevalues were basedon 9,999 randomma- trix permutations.The absolutenumbers of transi- speciesof the ScaledQuail complex,sequence tions and transversionsat first and third positionsof divergenceaveraged 0.054 -+ SD of 0.016, and codonswere plotted against1og-det distance to eval- ranged from 0.022 (California vs. Gambel's uate saturation.To assessmolecular-rate heteroge- quail)to 0.069(Scaled vs. Gambel'squail). The neity,we comparedthe log likelihoodsof maximum- Northern Bobwhite was closer to the Scaled likelihoodtrees computed with and withoutthe as- Quail complex(average K2P distance = 0.085 sumption of a molecular clock (PHYLIP routines + 0.002) than was the Mountain Quail (0.115 _+ DNAMLK and DNAML, respectively)by doubling the difference between the two likelihood estimates 0.004) or the MontezumaQuail (0.151 + 0.004). Saturation was not evident at first and third and determiningits chi-squareprobability (df = n - 2, where n = number of species;Felsenstein 1993). positions(Fig. 2), nor at the nearly invariant secondposition (not shown).Log likelihoods

RESULTS derived from analyseswith and without the as- sumption of a molecular clock did not differ DataSet A.--We obtained1,040 bp of mtDNA significantly(P > 0.10), indicating an absence sequencefor theScaled Quail complexand out- of molecular-rateheterogeneity among taxa. groups,including 304 bp from ND2 and 736bp A significantg•-value of -0.63 suggestedsig- from cyt b. Fromthe 17 individualssequenced, nal in the data(Kallersjo et al. 1992).The short- we identified 11 haplotypes.Percentage base est tree among 1,000 random trees, 460 steps, compositionwas biasedin a manner typical of was significantlylonger than those inferred and othervertebrates where guanineres- from maximum-parsimonyanalysis, which idues are uncommon,especially at third posi- producedtwo equally parsimonioustrees (Fig. tions.Overall basepercentages (cyt b, ND2, re- 3) of length348 with sitesunweighted, and one

TABLE2. Kimura (1980)two-parameter distance values among taxa. Distances are in the upperright matrix, and standard errors are in the lower left matrix.

on a I 2 3 4 5 6 7 8 9 10 11

I -- 0.0029 0.0555 0.0644 0.0661 0.0607 0.0608 0.0608 0.0808 0.1499 0.1189 2 0.0017 -- 0.0587 0.0677 0.0694 0.0640 0.0640 0.0641 0.0819 0.1537 0.1213 3 0.0077 0.0079 -- 0.0643 0.0630 0.0575 0.0576 0.0576 0.0864 0.1598 0.1155 4 0.0083 0.0086 0.0083 -- 0.0019 0.0217 0.0217 0.0217 0.0848 0.1533 0.1150 5 0.0084 0.0087 0.0082 0.0014 -- 0.0216 0.0216 0.0216 0.0865 0.1536 0.1157 6 0.0080 0.0083 0.0078 0.0047 0.0046 -- 0.0019 0.0019 0.0865 0.1477 0.1112 7 0.0081 0.0083 0.0078 0.0047 0.0047 0.0014 -- 0.0019 0.0866 0.1463 0.1123 8 0.0081 0.0083 0.0078 0.0047 0.0046 0.0014 0.0014 -- 0.0845 0.1489 0.1101 9 0.0094 0.0095 0.0098 0.0097 0.0098 0.0098 0.0098 0.0097 -- 0.1463 0.1147 10 0.0134 0.0137 0.0140 0.0137 0.0136 0.0133 0.0132 0.0134 0.0132 -- 0.1635 11 0.0116 0.0118 0.0115 0.0114 0.0115 0.0112 0.0113 0.0112 0.0115 0.0145 --

•Species,with Genbanknumbers in parentheses:1, ScaledQuail i (AFO28753-55);2, ScaledQuail 2 (AFO28756-58);3, ElegantQuail (AFO28750-52);4, Gambel'sQuail 4 (AFO28759-61);5, Gambel'sQuail 6 (AFO28762-64);6, CaliforniaQuail 3 (AFO28765-67);7, California Quail 5 (AFO28768-70);8, California Quail (AFO28771 73); 9, Northern Bobwhite(AFO28774-76); 10, MontezumaQuail (AFO28777 79); 11, Mountain Quail (AFO28780-82). April 1998] MolecularSystematics of Quail 397

80- Despite use of many primer pairs for PCR, we onlysucceeded in obtaining135 bp of cyt b and t-RNA•u sequence(Appendix) from P.fas- ciatus and D. thoracicus,because DNA from the 60- 45-year-oldstudy skins had degraded.Com- parisonof these135 bp from all ingroupspe- ciesrevealed that D. thoracicuswas very distant 40- fromthe Scaled Quail complex (>6%), whereas O•oø ee P.fasciatus was approximatelythe same dis- tance (2.7%) as the Northern Bobwhite(2.0%) o • [] fromthe ingroup.Thus, we foundno evidence 20- • thateither P. fasciatus or D. thoracicuswas closer to theingroup (or a part of it) andwould there- fore be more appropriateoutgroups, than the Northern Bobwhite. 0-• o Wenoted a discrepancyin the cyt b sequence for Gambel's Quail between our data and that depositedin Genbank (accessionL08382) by Log-Det Kornegayet al. (1993).In particular,their nu- FIG. 2. Plot of the numbers of transitions and cleotidesequence beginning at aminoacid 135, transversionsat first and third positionsversus log- CATGAGGGC, almost certainly should be det. Cyt b and ND2 datacombined. Open squares are CCATGAGGG, which is consistent with most first-positiontransitions, closed squares are first-po- of theother galliforms that they sequenced and sition transversions,open circlesare third-position the threeGambel's Quail that we sequenced. transitions,and closed circles are third-position DataSet B.--Species showed considerable di- transversions. vergence(151 of 736base positions were phy- logeneticallyinformative), with K2P distances of up to 20% (datanot shownbut can be com- tree(1 = 692)identical to thatin Figure3A with the2:1 weighting scheme. Maximum likelihood puted from sequencesin Genbank).Therefore, for this phylogeneticanalysis, we weighted and NJ (distance= log-det or K2P) analyses transversions 2:1 over transitions. Maximum- supportedthe topologyin Figure3A; however, the topologyin Figure3B (weighted1 = 700) parsimonyanalysis of cyt b sequencesrevealed did not have a significantlyworse log-likeli- a singletree with thetopology in Figure3A for hoodratio (P = 0.62;Kishino and Hasegawa the speciesalready discussed, with the follow- [1989]test). Within the ScaledQuail complex, ing taxaadded as successively more distant sis- the sister-speciesrelationship between Califor- ter taxa:A. chukar,C. coturnix,and L. nycthemera nia Quail and Gambel'sQuail is highly sup- (with T. pallidicinctusas the root). ported (bootstrappercentage = 100%)regard- less of analyticalapproach. Scaled Quail and DISCUSSION ElegantQuail weresupported as sister species in 51% of the bootstrapmaximum-parsimony Phylogenyand classification.--The sister-taxon treesand 62% of thebootstrap NJ trees. relationshipof CaliforniaQuail and Gambel's Becausedistant outgroups can biasrooting Quailis unambiguous,with thebootstrap sup- (Smith 1994),we removedMountain Quail and port at 100%;sequences of thesespecies differ Montezuma Quail and used only Northern by lessthan 2.5% (Table 2). The morphologyof Bobwhite,the sister species of theScaled Quail these two speciesis similar (Holman 1961, complex,as an outgroup.Maximum-parsimo- 1964, Hudson et al. 1966), and some authors ny analysisof unweightedsequence data re- havesuggested that they are conspecificor to- coveredthe topologyin Figure3A (treelength getherform a superspecies(Mayr and Short 168),and that in 3Bwas one step longer Thus, 1970,AOU 1983).Sequence data do not resolve useof the two moredistant outgroups yielded the relationshipsof ElegantQuail and Scaled a secondtree, and thereforemore ambiguity, Quail. The two alternativetopologies (Fig. 3) but thetrees were very similar in length. resultedfrom differentplacements of the root 398 ZINK AND BLACKWELL [Auk, Vol. 115

3,9• 9,• ScaledQuail - 1 ScaledQuail -2 ElegantQuail Gambel'sQuail-4 27,•• 12,1•,/ -'"'•Gambel's Quail-6

A BobwhiteQuail ••' MountainQuail • Montezuma Quail

ScaledQuail- 1 ScaledQuail- 2 ElegantQuail Gambel'sQuail- 4

Bobwhite Quail MountainQuail Montezuma Quail F•c. 3. Two equallyparsimonious trees of length348; ci = 77, ri = 68, and rc = 53. Usinga 2:1 TV:TS ration,the log-likelihoodwas -3003.338of topologyA and -3005.607of topologyB. TopologyA wasfound byNJ and the 2:1 weighted maximum parsimony analysis. Numbers represent synapomorphies and numbers in bold (topologyA only) are bootstrapproportions derived from 5,000replications. on the ingroup(Scaled Quail complex)tree. Of likely with extant quail and phylogenetic the threepossible unrooted trees that existfor placementsof ElegantQuail and ScaledQuail four taxa,topology A is bestsupported (Fig. 4). remain uncertain. Distantoutgroups can result in the rootbeing Weconsider the two topologiesin Figure3 as drawn artifactuallyto long branches(Smith viablehypotheses. A thirdhypothesis, contem- 1994).The two rootingsin Figure4 (denotedby poraneousspeciation, is discussedbelow. The arrows) occur on relatively long branches. topologiesin Figure 3 couldresult from spe- Hence,our outgroupsmight be inappropriate ciation events being closely spaced in time, for rootingthe treebecause the nearestspecies whichinhibits their recovery(Lanyon 1988), or to the ScaledQuail complex,the NorthernBob- from inadequatedata. One mightfavor closely white, differson averageby 8.5%from the in- spacedspeciation events, a so-called"star phy- group.Our analysisof shortsegments of se- logeny,"because phylogenetic resolution oc- quencefrom P.fasciatus and D. thoracicuspro- curred at both basal and terminal nodes. Such videsno better choices for outgroups,although a situationsuggested to Lara et al. (1996)that the formeris muchcloser to the ingroup.Thus, the genesused were capable of resolvingrela- unambiguous outgroup rooting appears un- tionships.However, no theory predictsthat April 1998] MolecularSystematics ofQuail 399

theMountain Quail). Principles of phylogenetic Gambel'sQuail• i= lo8 • ScaledQuailsystematics (Hennig 1966,Wiley 1981) applied CaliforniaOuailJ f • ElegantQuail to Figure3 invalidatethis nomenclatureunless the Northern Bobwhite is also included in Cal- lipepla,a suggestionmade by Mayr and Short (1970).Our data do not rule out a genusthat combinesCallipepla, Colinus, and Oreortyx.Al- Gambel'sScaledQuailJ• I=131 • CaliforniaElegantQuailQuailthough it would be inappropriatein our opin- ion (Johnsonand Zink 1983)to uselevels of ge- netic distanceto resolvetaxonomic categories, such a combinedgenus would be genetically Garnbel'sQuail• i=131 j ScaledQuail heterogeneous (pairwise distances up to ElegantQuailJ • CaliforniaQuail12.0%) relative to otheravian genera studied to date. The other speciesof FIC. 4. Three possibletrees for ingroup taxa (all shouldbe examinedin depth prior to generic conspecifichaplotypes from Figure 2 were used to revision. determine tree length [numbers]but for clarity are not shown).Topology A (top panel) is the best ex- Ratesof molecular evolution in quaiL--Theonly planation of the data. calibrationof allozymedistances with a rela- tively old New World fossilwas by Gutierrez et al. (1983)for the samegalliform birds that we studied.They concluded that an extinctspecies, speciationevents must be equally spacedand recoverablethroughout the durationof a lin- Cyrtonyxcooki (congeneric with Cyrtonyxmon- eage.Lack of phylogeneticresolution for Ele- tezumaeand whichwas examined by Gutierrez gant and Scaled quail likely resulted from et al. [1983]and us), indicatedthat this genus closely spacedspeciation events at an inter- had beenevolving independently from other mediate period during the evolutionof New New World galliformsfor at least 16 million World quail. Although more sequencedata years (MY). However, Marten and Johnson might allow phylogeneticresolution, we note (1986)obtained unpublished information from that thesequail speciesare relativelydifferen- paleontologistsfamiliar with the depositional tiated,with manysynapomorphies and typical environmentsof the samespecimen of C. cooki levelsof homoplasy(Fig. 3). Therefore, it would that suggestedit couldbe as young as 7 to 9 take a differentquality (e.g.genes encoded in MY. Thus,estimates of the ageof the fossildif- the nucleus)of sequencedata, rather than sim- fer by a factor of two. Marten and Johnson ply more mtDNA data, to shift the balanceof (1986) used the estimateof 7 to 9 MY to cali- synapomorphiesto favor significantlyone to- brate an allozyme clock. pologyover the other(but seeOtto et al. 1995). Calibrations of molecular clocks assume a Previous classifications(e.g. AOU 1957) roughly uniform rate of nucleotidesubstitu- placed California Quail Gambel'sQuail and tion,which was supported for our data.The av- Elegant Quail in Lophortyx,with only the erage log-det distancefrom C. montezumaeto ScaledQuail in Callipepla.The topologyin Fig- the ScaledQuail complexis 0.154(0.148 for cyt ure 3B is consistentwith this arrangement.A b data alone).Assuming that C. cookiis cor- tree (not shown)in which ScaledQuail is sister rectlyplaced in a phylogeneticcontext (see Gu- to California Quail plus Gambel'sQuail is four tierrez et al. 1983),the rate of mtDNA sequence stepslonger than the mostparsimonious tree, divergence(cyt b aloneor combinedwith ND althoughit is not significantly"worse" accord- 2) is 1 to 2%,depending on the ageof thefossil. ing to the Kishino-Hasegawa(1989) maximum- This is consistent with estimates derived from likelihoodtest. Yet, this topologyoccurred in independentcalibrations of a numberof other no methodof treebuilding, including neighbor avian taxa (citationsin Klicka and Zink 1997). joining,maximum parsimony (unweighted or Given the apparentconvergence of suchesti- weighted),or maximumlikelihood. Therefore, mateson 2% per MY, this figure is perhaps we do not considerit to be a viablehypothesis. morelikely than the estimateof 1%per MY de- Mayr and Short(1970) used the genusname rived from assumingan age of 16 MY for C. Callipeplafor the singlespecies of Oreortyx(i.e. cooki.We stress,as do others(Avise 1994), that 400 ZtNK AND BLACKWELL [Auk, Vol. 115 owing to a variety of factors,rate calibrations Phylogeneticcongruence ofallozyme and mtDNA are best viewed as heuristictools only. data.--Mantel's test indicated that the matrices Biogeography.--Theconsensus of the two of allozyme and mtDNA distanceswere signif- equallyparsimonious trees (Fig. 3) yieldsa tri- icantly congruent(P < 0.0001;matrix correla- chotomy (not shown), exactly as outlined in tion = 0.84). In addition, Gutierrez et al. (1983: Hubbard's(1973) scenario. This consensustree figure2) presenteda treethat was topologically is six stepslonger than the shortesttrees, but it identicalto that basedon cyt b data alone.How- is not significantlyworse than eithertopology ever, large distancesamong taxa for both in Figure 3A (Kishino-Hasegawatest, P = 0.2). mtDNA and allozymesmight makephylogeny The increasedlength of the consensustree over reconstructionrelatively unambiguous;unfor- the two equally parsimonioustrees illustrates tunately, Gutierrez et al. (1983) lacked speci- why many taxonomistsdo not interpret con- mensof ElegantQuail. Nonetheless,the con- sensustrees phylogenetically (Swofford et al. gruenceof allozymesand mtDNA for this set 1996). That is, the consensustree indicatesun- of taxa, as well as for many others(e.g. Zink et certaintyabout the placementsof ElegantQuail al. 1991, Zink and Dittmann 1991, Zink and and Scaled Quail, not necessarilythat a tri- Blackwell1996, Zink et al. 1998), suggestthat chotomyis the best explanationof the data. allozymesand mtDNA effectivelyrecover phy- Thus, Hubbard's(1983) hypothesisof a basal, three-waysplit of a commonancestral species logeneticsignal; i.e. both recoverthe species tree. is not supported by parsimony but it is not ruled out by maximumlikelihood. Zink et al. Recentstudies have noted that amplification (1998) have favored contemporaneousspecia- and sequencingof nuclearcopies of mitochon- tion for other aridland avian lineages,and we drial genes(Zhang and Hewitt 1996)can bias consideras a third viable (albeit lesslikely) hy- phylogeneticinference. The congruencebe- pothesisthe contemporaneousorigin of Ele- tween nuclear (allozymes) and mtDNA data gant Quail, ScaledQuail, and the commonan- setsrender it lesslikely that we accidentallyse- cestor of California Quail and Gambel's Quail. quencednuclear copies of cyt b or ND2 in quail. Resolutionof general area relationshipsre- Alternatively,if we did sequencenuclear cop- quirescomparison of phylogeneticpatterns in ies, they must have been transposedrecently multiple lineages, including quail, Polioptila into the nucleusso as not to confoundphylo- (Zink and Blackwell 1998), Pipilo (Zink et al. geneticinference. The observationsthat our se- 1998), and Toxostoma(Zink et al. unpubl. data). quencescontained no stop codons,and that Given the precedingdiscussion of rates of they exhibited a typical distributionof varia- molecular evolution,the timing of speciation tion at first, second,and third codonpositions, eventsenvisioned by Hubbard (1973) is too re- provide further evidenceagainst nuclear con- cent.It is extremelyunlikely that the 2.2% di- tamination(Zhang and Hewitt 1996). vergence between California and Gambel's quail evolved in only 40,000 to 100,000years, concomitant with the onset of the Wisconsin ACKNOWLEDGMENTS glacialperiod. A rate of 2% per MY alsowould alter the timing of speciationevents shown in We thank A. Fry, J. Klicka and O. Rojas-Sotofor Gutierrezet al. (1983:figure 5). The dateof sep- field assistance.H. Tordoff,E Gill, and D. Delehanty arationof Mountain Quail from the remaining provided specimens.Permits were provided by SE- specieswould be about 6 MY rather than the DESOL, USDA, and USFW. Lab assistancewas pro- vided by T. Line, J. Van Pilsum, A. Pavlova,and M. 12.6MY asshown. Separation of NorthernBob- Ginos. S. Russell and Sr. Porchas assisted with col- white would date to 4.1 MY rather than 7.0 MY, lecting in Sonora.A. Navarro and B. Hernandez as- and California Quail and Gambel'sQuail sep- sistedwith permits.Funding was providedby NSF arated from their commonancestor approxi- grantDEB~9317945. We thankA. Baker,E. Randi,J. mately1 MYA. Thesedates are hypothesesbut Klicka,K. Johnson,S. Lanyon,and an anonymousre- are consistentwith other data (Zink and Slo- viewerfor commentson the manuscript.D. Swofford winski 1995,Klicka and Zink 1997) that sug- gave permission to use a prereleaseversion of gestthat most extantavian speciesoriginated PAUP*,and J. Grothand G. Barrowcloughprovided in the late Plioceneor early Pleistocene. unpublisheddata. April1998] MolecularSystematics ofQuail 401

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