Comparison of Phenotypic and Genetic Differentiation in South American Antwrens (Formicariidae)

COMPARISON OF PHENOTYPIC AND GENETIC DIFFERENTIATION IN SOUTH AMERICAN ANTWRENS (FORMICARIIDAE)

SHANNON J. HACKETT AND KENNETH V. ROSENBERG Museumof NaturalScience and Department of Zoologyand Physiology, LouisianaState University, Baton Rouge, Louisiana 70803 USA

ABSTRACT.--Weexamined patterns and levels of genetic,morphometric, and plumagevari- ation in Myrmotherulaantwrens and related antbirds (Formicariidae). We derived matricesof distancesindependently for proteins, qualitative plumage traits, and measurementsof study skins.Our goalswere to assessconcordance between the morphological(morphometric and plumage)and geneticpatterns, to estimatephylogenetic relationships, and to test monophyly of Myrmotherula.Myrmotherula is not monophyletic.Analysis of allozymesshows that although the "gray" and "streaked"plumage types are closelyrelated, the "checker-throated" Myrmotherulaspecies are not closelyrelated to the gray/streakedclade. Plumage divergence exceedsdivergence in protein genes, whereas change in external morphology has been relatively conservative.Antwren speciationhas been accompaniedby differentiationof plumage types rather than by entry of speciesinto new regionsof morphometricspace. Relatively large geneticdistances among and within Myrmotherulaspecies imply that thesetropical taxa are older and more geographicallysubdivided than are mostNorth Americanspecies. Neither plumage nor morphometricsalone correctlypredicted the genetic relatednessamong the taxa. We urge that taxonomic revisions be accompaniedby molecular data, especially in species-richtropical families. Received5 June1989, accepted26 December1989.

RECONSTRUCTIONof the history of lineagesis are shared with other small antbirds in a num- a central aim of evolutionarybiology. Compar- ber of related genera; indeed, no unique mor- ative biochemicaltechniques provide powerful phological traits support Myrmotherula as a methods to assessgenetic relationships among monophyleticgroup. The natural history and species. Questions remain about the relative vocal behavior suggest the division of Myr- utility of molecular versus morphological ap- motherulainto several ecological groups (e.g. proaches(Felsenstein 1985; see papers in Pat- Gradwohl and Greenberg 1984, Remsen and terson 1987). In particular, it is important to Parker 1984, Rosenberg in press). Questions establish if molecules and morphology give concerning the pattern of appearanceof phe- concordantpictures of pastevolutionary events, notypic innovations (e.g. the various plumage and to define the problems and limitations of typesor foragingmodes) can be addressedonly thesemethods. Few studieshave integratedboth in the context of an estimateof genealogical morphological and molecular approachesin relationshipsamong species (Felsenstein 1985). analysesof the evolutionary history of organ- The diversity of plumage types and ecological isms, and the empirical relationship between habits makes this avian group a test casefor the morphological variation and genealogy re- comparisonof patternsof morphologicand ge- mains poorly understood. netic evolution. As a casestudy using multiple data sets,we analyzedlevels of genetic,morphometric, and METHODS plumagesimilarity amongmembers of a single large genus of antwrens (Myrmotherula)and We analyzed tissuesamples of 18 Myrmotherula among other antbirds of uncertain relatedness species,and 11other formicariid genera (mostly small antwrens). Grailariaquitensis was selectedas the out- to Myrmotherula.Several featuresof this genus groupbecause Grailaria is part of a distantlyrelated facilitate comparison of genealogy and mor- subgroupof formicariidsthat may deservefamilial phology. The speciesin Myrmotherulaare sim- status(Sibley and Ahlquist 1985). ilar in size and shape,but occuras three distinct Specimens,scientific names, and collectinglocali- plumage groups:checker-throated, gray, and tiesof the 29 speciesexamined are listed in Appendix streaked(Table 1). Someof theseplumage traits 1. We used four individuals of M. haematonota(one

473 The Auk 107:473-489. July 1990 474 HACKETTAND ROSENBERG [Auk, Vol. 107

T^BLE1. Plumage types, weight, and tail measure- this study). However, Archie et al. (1989) challenged ments of 5 males for each Myrmotherulaspecies. this assertion. We suggest that the conservatismof Specieswith * are consideredin this study;PS, avian allozyme divergence,fixed or nearly fixed al- PD, NB, and SB indicate sample locality (see Ap- lozymes unique to certain groups in this study, and pendix 1). low heterozygositymay minimize the sample-sizebias predictedby Archie et al. (1989).Protein electropho- Weight Tail resis followed standard procedures (Johnson et al. Species Plumagetype (g) (ram) 1984).Samples of heart, liver, and musclewere taken M. brachyura* streaked 7.3 17.6 within 3 h of death and stored in liquid nitrogen M. obscura* streaked 7.1 18.0 (-196øC). Tissueextracts were prepared by grinding M. sclateri* streaked 8.6 23.6 samplesof heart, pectoral muscle, and liver in 1 ml M. klagesi streaked M. surinamensis* streaked 8.7 24.8 of grinding buffer (6% sucrose,0.01% NAD, 0.01% NADP, 0.01% DTT, deionized water) with a tissue M. ambigua streaked M. cherriei streaked homogenizer.The mixture was spun in a Sorvall RC- M. longicauda* streaked 8.3 36.0 5B centrifuge (Sorvall rotor SM 24) at 16,000rpm for M. gularis checker-throated 30 rain, and the resulting supernatantfrozen (-70øC) M. gutturalis checker-throated for subsequentelectrophoretic experiments. Buffer M. fulviventris* checker-throated 10.2 34.3 conditions,running conditions,and buffer recipesare M. leucophthalma* checker-throated 9.3 39.5 available (from Hackett) on request. All gels were M. haematonota 11.5% starch. Each locus was scored on at least two (NB)* checker-throated 8.0 36.8 M. haematonota different buffer types to minimize hidden variation (SB)* checker-throated 8.5 34.9 (Hackett 1989). M. haematonota Protein electrophoresisfollowed standard proce- (PD)* checker-throated 8.7 36.7 dures (Johnsonet al. 1984). Twenty-six enzyme sys- M. haematonota tems representing32 genetic loci were resolved.En- (PS)* checker-throated 9.4 41.5 zymes were assayedusing proceduresoutlined by M. ornata * gray a 9.6 33.5 Harris and Hopkinson (1976), with slight modifica- M. erythrura* checker-throated 10.8 42.5 tions. Ten enzymeswere monomorphicand fixed for M. guttata gray the same allele across all taxa examined: Malate de- M. hauxwelli* 10.3 25.7 gray hydrogenase-1and 2 (EC 1.1.1.37),isocitrate dehy- M. erythronotus gray M. axillaris* gray 6.8 35.4 drogenase-2 (1.1.1.42), fumarate dehydrogenase M. schisticolor * gray 8.6 36.3 (4.2.1.2),glutamate dehydrogenase(1.1.1.47), glycer- M. sunensis gray aldehyde-3-phosphatedehydrogenase (1.2.1.12), di- M. longipennis* gray 8.9 33.2 aphorase( 1.6.*.*), hemoglobin,glutathione reductase M. minor gray (1.6.4.2), and superoxide dismutase-2 (1.15.1.1). Pu- M. iheringi gray fine nucleosidephosphorylase (NP; 2.4.2.1) was elim- M. grisea* gray 8.4 34.9 inated from the analysisdue to inconsistentbanding M. unicolor gray patterns.Alleles at a locuswere codedby their mo- M. behni* gray bility from the origin. The most anodal allele was M. urosticta gray M. menetriesii* gray 8.0 29.3 designated"a," the next mostanodal "b," and so on. M. assimilis* gray 9.3 29.1 The computer program BIOSYS-1 (Swofford and Selander 1981) was usedto computegenetic distances • Placementin "gray" group reflectsmale plumageof Myrmotherula ornata meridianalis;males of other races have rufous backs, and female (Nei 1978, Rogers 1972), estimate Distance-Wagner plumagesuggests inclusion in "checker-throated"group (seetext). trees (Farris 1972, 1981), and derive a UPGMA phenogram (Sneath and Sokal 1973). The "multiple ad- from eachof four localities)to assessintraspecific geo- dition criterion" of Swofford (1981) was used in the graphicvariation. There were a total of 32 operational Distance-Wagnerprocedure because it generallyfinds taxonomic units (OTUs). Nomenclature follows Mo- treesof better fit to the original distancematrix (Farris tony et al. (1975).All tissuespecimens were obtained 1981). The maximum number of trees held at each from the LouisianaState University Museumof Nat- step in the DistanceWagner procedurewas set at 20. ural Science (LSUMNS) Frozen Tissue Collection. Trees were rooted at the outgroup Grailariaquitensis. Voucher specimens(skin and skeletons) for tissue We usedPHYLIP (Felsenstein1986) to analyze cla- samplesare housedin the LSUMNS. distically the allele distribution.Alleles were coded Electrophoresis.--Oneindividual per OTU was used as present (1) or absent (0) (Mickevich and Mitter for the analysisof geneticvariation. Gorman and Ren- 1981).Only phylogeneticallyinformative alleleswere zi (1979) suggestedthat one or few individuals per used (alleles present in two or more taxa). The data taxonprovide robustestimates of geneticdistances as set, which consisted of 56 characters (alleles), was long as the number of loci examinedis reasonably analyzedwith the MIX programof PHYLIP. PENNY, high and heterozygosityis low (conditionsmet by a program to find all mostparsimonious trees implied July1990] EvolutionofAntwrens 475

by the data, couldnot be usedbecause of the large ficients. This matrix was then used to construct a number of taxa in this study. UPGMA phenogramthat summarizesoverall plum- There is no generally acceptedmethod for coding age similarity (NTSYS, Rohlf et al. 1974). Maximum allelesfor cladisticanalysis. The use of presence/ab- parsimonyanalysis of plum.age characters (using the sencecoding of alleles has been criticized for a num- computerprogram PAUP; Swofford1985) produced ber of reasons(but see Rogersand Cashnet 1987 for the samegroupings as the phenetic analysis. a defenseof this type of coding),and a more appro- Comparisonofgenetic, morphometric, and plumage data priate method of coding may be to treat the locus as sets.--Wedescribed overall similarity among datasets a character with its alleles as unordered character states in three ways.First, Mantel's (1967) test was used to (Buth 1984, Swofford and Berlocher 1987). We chose describethe similarity in overall structurebetween not to order alleles, becauseordering of alleles im- eachpair of distancematrices. Second, we performed pliesknowledge of evolutionaryrelationships among Spearmanrank correlationson paireddistances among specificalleles that is usuallylacking in mostelectro- taxa.Finally, to estimatethe magnitudeof divergence phoreticstudies (Mickevich and Mitter 1983).One of of morphometricand plumage(male only) characters the difficultiesin coding loci as multistate-unordered relative to genetic characters,we comparedscaled charactersis the treatmentof polymorphismsin taxa, OTU by OTU distancematrices for eachof the three a commonresult of electrophoreticdata (seeAppen- data sets.To scaleeach matrix, the largestdistance in dix 2). We realize the limitations of presence/absence the matrix was assigneda value of 1, and eachother coding of alleles and of using the locus as the char- distancewas then dividedby the largestdistance. The acter,and we chooseto presentresults treating alleles outgroup was omitted from the scaling processto as characters. avoid compressionof distancesbecause of the pres- Morphometrics.--Onfive adult males of each OTU, ence of a single large distance. we measuredlength of closedwing, wing-tip extension, tarsuslength, length of middle toe, exposed RESULTS culmenlength, bill depth,bill width, and tail length (Baldwin et al. 1931);body weight was also used as a Plumage.--Distance matrices based on male morphometriccharacter. From charactermeans, a ma- and female plumage characteristicsare avail- trix of taxonomic distances (Sneath and Sokal 1973; able from the authors.Phenetic analyses of male available from the authorsupon request)was derived plumage characteristicsdistributed the Myr- and used to constructa UPGMA phenogram to sum- motheruIaspecies among three majorgroups (Fig. marizeoverall morphometric similarity (NTSYS, Rohlf 1). The first group included five MyrmotheruIa et al. 1974).In addition, we subjectedthe correlation matrix from character means of all taxa except the speciesthat are mainly white- or yellow-streaked outgroup(Grailaria quitensis) to principal components throughout with black (hereafter, "streaked" analysis(PCA) using SAS (SAS Institute 1982) to iden- Myrmotherula group). Drymophila devillei, Hy- tify major axes of morphometricvariation. In this pocnemiscantator, and Herpsilochmusrufimargina- analysis,cube root of body weight wasused as a mor- tus,which are alsoboldly patternedor streaked, phometric measureand, to assessvariation in shape were most similar to this group. The second independent of body size, all other measurements majorgroup consistedof primarily gray species, were divided by the cuberoot of body weight. Factors mostwith blackthroats and white-tipped wing were subjectedto varimax rotation, which maximizes coverts,a plumage pattern common in the For- the spreadof the variableloadings among the factors micariidae. Eight Myrmotherula species fell and thus facilitatesinterpretation (Johnsonand Wich- ern 1982), but does not affect relative positions of within this group (hereafter, "gray" Myrmoth- OTUs in PC space. erulagroup), along with Hylophylaxpoecilonota, Plumage.--Variationin male and female plumage Dysithamnusmentalis, and Pygiptilastellaris. Myr- characteristicswas assessedqualitatively from study motherulaaxillaris, along with Microrhopiasquix- skins.Body regions (throat, breast,back, crown, wing, ensisand Formicivorarufa, is aligned with this and tail) were treated as characters, with unordered group but is more extensivelyblack with prom- characterstates being the coloror patternin that body inent white markings on the flanks or tail. region(e.g. the character"throat" had characterstates A third group consistedof four Myrmotherula of white, yellow, black, checkered,and so on [see species(including the four populations of M. Appendix 3]). Although these characterstates were haematonota)that had brown or rufous upper- somewhatarbitrary (e.g. severalsubtle shades of gray or brown were considered equivalent), this repre- parts,a checkeredthroat-patch, and buffy wing sentsa conservativeassessment of plumage variation spots(hereafter, "checker-throated"Myrmoth- amongthese species. From thesedata, we constructed erulagroup). Finally, Thamnophilusdoliatus and OTU by OTU pairwise distance matricesseparately Terenurahumeralis, with unique plumage char- for malesand females,based on simple matchingcoef- acteristics,formed a fourth group. Analysis of 476 •4^CKETrAND ROSœNBœRG [Auk, Vol. 107

I I I 1 ,• 0.50 0.00

PLUMAGE DISTANCE Fig.2. Pheneticanalysis (UPGMA) of variationin female plumage.Cophenetic correlation = 0.841.PS, Fig. 1. Phenetic(UPGMA) analysisof variationin PD, NB, and SB after Myrmotherulahaematonota in- male plumage.Cophenetic correlation = 0.898. PS, dicatesample locality (seeAppendix I). PD, NB, and SB after Myrmotherulahaematonota in- dicatesample locality (seeAppendix 1). maining species,with the exceptionof Micro- femaleplumage resulted in similar,but lesswell rhopiasquixensis, formed a third, poorlydefined defined,groups of species(Fig. 2). Thefirst group group. Femalesof the gray Myrmotherulaspp. of streakedMyrmotherula and similarspecies re- were most similar to each other, and Pygiptila mained nearly the sameas in the analysisof stellariswas identical in all plumage characters males;these speciesare weakly sexuallydi- to M. menetriesii. morphic.All remainingspecies were various Morphometrics.--Myrmotherulaspecies varied shadesof brown, buff, or rufous, a typical pat- little in overall mass (7-11 g) and body pro- tern in female antbirds. The secondgroup, con- portions,especially compared with other ant- sisting of checker-throatedMyrmotherula spp. birds (Figs. 3-5). Most variation among Myr- alongwith M. hauxwelli,M. axillaris,and M. or- motherulaspecies was in tail length (Table 1). nata,was defined primarily by the presenceof Althoughthe checker-throatedspecies were all buffy wing spots.Female M. ornatahave a relatively long-tailed, this group overlapped checkered throat like that of the male checker- with both streaked and gray Myrmotherula throatedspecies. In addition,M. ornataexhibits species(Fig. 3A). Membersof the threeplumage markedgeographic variation in male plumage, groups did not differ consistentlyin tarsus with somepopulations having a rufous back; length or wing shape. M. longipenniswas ex- thus, its inclusion in the gray group basedon ceptionallylong-winged, and M. hauxwelliwas male plumage (Table 1) is tenuous. The re- long-legged. The only univariate morphomet- July1990] EvolutionofAntwrens 477

70'

60'

50'

40'

20'

10'

I I I I I I• I 5 10 15 20 25 30 70

Weight (g)

0.70 '

0.65-

o2e 0.60'

• 0.45'

I I I I I 0.10 0.15 0.20 0.25 0.30

RelativeBill Depth (Bill depth / Billlength) Fig. 3. Morphometric variation in Myrmotherulaspp. and related antbirds with respectto size and tail length (A), and relative toe length and bill shape(B). Trianglescorrespond to checker-throatedMyrmotherula spp.,squares correspond to streakedMyrmotherula spp., closedcircles correspond to gray Myrmotherulaspp., and open circlescorrespond to non-Myrmotherulaspecies. Numbers for taxa correspondto numbersin Ap- pendix 1. tic measuresthat separated the Myrmotherula Principal componentsanalysis for all taxa re- plumage groups were relative bill depth and suited in five rotated factors with eigenvalues relative toe length (Fig. 3B).The checker-throat- greaterthan 1.0, which explained83.0% of the ed species (including M. ornata) had consis- total variance (Table 2). Axes defined by factors tently deeper bills and larger feet than other 1 and 2 showed relatively clear separationof Myrmotherula.However, the gray and streaked the three Myrmotherulaplumage groups (Fig. speciesoverlapped even on thesemeasures. 4). Factor1, which representedoverall size and 478 HACKETTAND ROSENBERG [Auk,Vol. 107

-1.25 •0•0 O25 1,00 1.75 2.6O 3.25 4.00

Fig. d•. Distribution of 30 Formicariid taxa in Prin- cipal Componentsspace, based on 9 morphometric measures(includinõ weiõht). See text for interpre- tation of factors.Numbers and codesas in Fig. 3.

relative bill depth, separated the checker- throated speciesfrom all other Myrmotherula except M. hauxwelli; the gray and streaked groups overlapped completely on this factor. Factor2 representedrelative wing length and shape (long, pointed vs. short, rounded). The gray and streakedMyrmotherula species overlapped little along this factor, whereas the I I checker-throatedspecies all showed interme- diatescores. A nearlyidentical dispersion of the threeplumage groups existed along factor 3 (not Fig. 5. UPGMA phenogram of 9 morphometric illustrated), which representedrelative tarsus characters(including body weight) based on taxo- and tail length. Of the 10 outgroup species,4 nomic distancesfor 31 Formicariid taxa. Cophenetic correlation = 0.965. PS, PD, NB, and SB after Myr- overlappedwith the Myrmotherulaspecies along motherulahaematonota indicate sample locality (see factor 1, and all overlappedwith Myrmotherula Appendix 1). along factor 2. The overall morphometricsimilarity within Myrmotherulais reflected in the UPGMA phe- from allopatricpopulations averaged 0.060. Av- nogram(Fig. 5) basedon the taxonomicdistance erage distancewithin the genusMyrmotherula matrix (available from authors) derived from as a whole (0.329) was similar to the average the raw measurements.Among the other ant- within the family Formicariidae (0.365) as a bird species, HerpsiIochmusrufimarginatus, whole (see Table 3 for a summary of genetic Microrhopiasquixensis, Hypocnemis cantator, and distancesin Neotropical birds; matrix of pair- Terenurahumeralis, clustered among the Myr- wise Rogers'[1972] and Nei's [1978]genetic dis- motherulaspecies. The remaining specieswere tancesare available from the authors upon re- either larger in size or longer tailed, and clus- quest). tered outside the Myrmotherulagroup. There- The cladisticanalysis of alleles basedon the fore, morphometricmeasures failed to define maximum parsimonycriterion (Fig. 6) divides clearlyMyrmotherula apart from other small ant- the Myrmotherulaspecies into two majorgroups. wrens. The first majorgroup includedall the gray and Genetic.--Of the 32 loci examined, 22 (69%) streakedspecies, except for M. assimilis,which varied amongthe speciesanalyzed (Appendix clustered with several non-Myrmotherulataxa. 2). Nei's (1978)average genetic distances within Therewas no clearseparation between gray and variousplumage groups were roughly similar streakedMyrmotherula species. The secondma- (Table3): checker-throatedMyrmotherula, l• = jor group included all checker-throatedMyr- 0.186;gray Myrmotherula, /• = 0.208;streaked motherula,Microrhopias quixensis, and Pygiptila Myrmotherula,/•= 0.136.Distances among sin- stellaris.MyrmotheruIa ornata clearly belongs in gle individuals of Myrmotherula haematonota the checker-throatedgroup. July1990] EvolutionofAntwrens 479

TABLE2. Factor loadingsof size-transformedmorphometric characters of antbirds.Principal components analysisof variable means using Varimax rotation. All variableswere divided by the cube root of body weight.

Variable Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Weight 0.883 0.026 0.160 -0.094 0.080 Wing length 0.130 0.912 0.123 0.089 0.081 Wing-tip extension -0.165 0.867 -0.254 -0.012 0.005 Tarsuslength 0.004 -0.240 0.793 -0.384 0.087 Toe length 0.271 -0.193 -0.042 -0.555 -0.515 Bill length 0.080 0.024 -0.085 -0.054 0.916 Bill depth 0.874 -0.053 0.016 0.366 -0.072 Bill width 0.226 -0.006 -0.037 0.829 -0.059 Tail length 0.226 0.111 0.814 0.331 -0.235 Eigenvalue 2.166 1.968 1.226 1.107 1.003 Proportion of variance 0.241 0.219 0.136 0.123 0.111

The UPGMA phenogramand Distance-Wag- tion,whereas morphometric evolution may have ner tree (not shown, available from the authors) been lessextensive than protein evolution. differed only slightly from the cladogramin topology. The separationof Myrmotherulainto DISCUSSION two majorgroups remained, although relationships within these clustersand with non-Myr- The currently recognizedgenus Myrmothe- motherulataxa changedslightly. Myrmotherula rula was defined by the morphologicalsimilar- fulviventris was genetically most divergent ity of its members(Sclater 1858). We haveshown within the checker-throatedgroup in the dis- that this grouping does not reflect a genetic tance analyses.Regardless of the type of data phylogenyand that a systematicrevision of the analysisperformed, the genusMyrmotherula was small antwrens is necessary(Rosenberg and not monophyletic.It would add 21 stepsto the Hackett in prep.). phylogeny in Fig. 6 to make Myrmotherula Comparisonof evolutionin genesand morpholo- monophyletic. gy.--Protein evolution in thesebirds has been Comparisonof genetic,morphometric, and plum- paralleled more closelyby variation in plumage age distancematrices.--Mantel's (1967) test (all characteristicsthan by variation in size and t-valueshighly significant,P < 0.001)indicated shape.For example,the checker-throatedplum- that these matrices have some structure in com- agepattern is uniqueamong antbirds; however, mon, and implied that the pattern of genetic the checker-throatedspecies are only weakly differentiationamong these taxa paralleled dif- differentiated morphometrically from other ferencesin morphometricsand plumage.Sim- small antwrens. Our phylogeny suggeststhat ilarly, all pairwisecorrelations of genetic,mor- the checker-throatedgroup is a clade separate phometric,and plumagedistances among these from all other Myrmotherula species. The taxawere significant,but correlationswere low streakedgroup also representsa geneticclade, (from 0.34 between morphometricand genetic but it probably arose relatively recently from distancesto 0.58between genetic and plumage within the graylineage. As with the grayplum- distances). age type, the streakedpattern occursin other Scaling of the three distancematrices indi- clades(e.g. by our criteria Hypocnemiscantator cateddifferences in relative magnitude of char- was identical in plumageto Myrmotherulascla- acter evolution in morphology and protein teri). Without prior knowledge of genetic re- genes (Fig. 7). In each Myrmotherulaplumage lationships,these plumage traits could not be type and acrossall taxaexamined, plumage dis- usedto predict genetic relatednessamong the tanceswere relatively higher than geneticdis- plumagetypes. For example,we could not have tances,whereas morphometric distanceswere determinedthat the streakedgroup was derived lowest.We suggestthat plumageevolution may from gray ancestorswithout independent ge- have been more extensivethan protein evolu- netic data. Also, the placement of M. ornata 480 HACKETTAND ROSENBERG [Auk,Vol. 107

TABLE3. Meangenetic distances (/•; Nei 1978)by taxonomicrank for Neotropicalbirds. Ranges are given only for taxa examined in this study.

Num- ber of com- pari- Taxonomiclevel sons /• + SD Range Reference Within species Myrmotherulahaematonota 6 0.060 + 0.014 0.032-0.066 This study Mionectesoleagineus I 0.01 Capparella and Lanyon 1985 Cyclarhisgujanensis 6 0.014 + 0.009 Johnson et al. 1988 Vireolanius leucotis 3 0.024 + 0.021 Johnson et al. 1988 Hylophilusochraceiceps I 0.00 Johnson et al. 1988 Chiroxiphiapareola I 0.066 Capparella 1988 Pipra coronata 10 0.019 + 0.015 Capparella 1988 Glyphorynchusspirurus 10 0.025 +_0.025 Capparella 1988 Myrmoborusmyotherinus 6 0.031 + 0.034 Capparella 1988 Pithysalbifrons 6 0.003 + 0.002 Capparella 1988 Within genus Myrmotherulaa 210 0.329 + 0.145 0.032-0.617 This study checker-throated 28 0.186 + 0.129 0.032-0.438 This study gray 28 0.208 + 0.080 0.049-0.336 This study streaked 10 0.136 + 0.068 0.033-0.277 This study Heliodoxa 21 0.240 + 0.088 Gerwin 1987 Synallaxis 6 0.19 + 0.05 Braun and Parker 1985 Cranioleuca 10 0.08 + 0.03 Braun and Parker 1985 Schizoeaca I 0.13 Braun and Parker 1985 Mionectes I 0.08 Capparella and Lanyon 1985 Hylophilus• 20 0.302 Johnson et al. 1988 Vireo a 142 0.291 Johnson et al. 1988 Pipra 10 0.101 Capparella 1988 Within family Formicariidae 496 0.365 + 0.129 0.032-0.742 This study Bucconidae b 28 0.32 + 0.09 Lanyon and Zink 1987 Galbulidae b 6 0.52 + 0.11 Lanyon and Zink 1987 Capitonidaeb I 0.22 Lanyon and Zink 1987 Ramphastidae• 10 0.32 + 0.07 Lanyon and Zink 1987 Trochilidae 91 0.625 + 0.215 Gerwin 1987 Vireonidae 322 0.354 Johnson et al. 1988 Cotingidae• 3 0.531 + 0.104 Lanyon 1985 Rupicolidaeb 28 0.338 + 0.097 Lanyon 1985 Tyrannidaeb 105 0.473 + 0.161 Lanyon 1985 Thesegenera are not monophyletic. Thesegenetic distances are underestimatesbecause only conservativeloci were used in the calculations.

within the checker-throated clade was uncer- Morphometric conservatismis a characteris- tain. In a similar comparisonof genetic and tic of the family Formicariidae;antbirds seem morphologicaldifferentiation in Australo-Pa- to sharea similar body and bill shape,and vary puan scrubwrens (Sericornis),Christidis et al. primarily in overall size. Body weight varied (1988) concludedthat externalmorphologic fea- more than other morphometric characters,but turesare not concordantwith their geneticphy- morphometricsimilarity was not simply an ef- 1ogeny, and that similarities in plumage rep- fect of similar body sizes. Furthermore, our resent "unresolved plesiomorphies and assessmentof plumage variation was conser- homoplasies."Once we have a genetic phylog- vative. If we recognized more subtle differences eny, we can trace the pattern of evolution of in color, the disparity between morphometric plumage and morphometric traits within lin- and plumage similarities would have been eages. greater. The reasonplumage evolution hasbeen July1990] EvolutionofAntwrens 481

Fig.6. Parsimonyanalysis of allelicvariation in 32 Formicariidtaxa. PS, PD, NB, and SBafter Myrmotherula haematonotaindicate sample locality (seeAppendix 1).

more extensive than morphometric evolution Pamilo and Nei 1988).For example,if speciation is complex.Phenotypic canalization may deter- events occurredin burstswith subsequentlong mine ways in which closely related taxa can periodsof independentevolution, then molec- diverge, in this case limiting morphometric ular evidence of monophyly may not be re- change in antbirds to size variation on the same tained in extant descendants(Lanyon 1988). In basicshape. In the Parulidae,plumage also seems the absenceof molecularmarkers of monophy- highly variable relative to morphometricvari- ly, key innovations (behavioral, morphologic, ation, whereas plumage variation seemscon- or ecological) may provide evidence of mono- servativein Furnariidae. Comparisonof genetic phyly. In our study, the checker-throatedphe- divergence with plumage and morphometric notypeand specializeddead-leaf foraging were variation is a way of quantifying "mosaic"evo- key innovations consistentwith our molecular lution (Mayr 1970). The estimate of genealogical relationships producedfrom protein electrophoresisis likely a better estimateof phylogeny than one based 0.9- on comparisonsof morphology, although this 0.8- viewpoint is controversial (Donoghue et al. 0.7- 1989). Morphological traits are influenced by 0.6- an unknown number of genes, each with an 0.5 0.4- unknown contribution to the phenotype, and 0.3- by the effects of natural selection, which can obscurephylogeny through convergence. Also, morphological traits are not likely to be independent,because they are controlled by the same M P G M P G M P G M P G STREAKED GRAY CHECKER OVER ALL genes (Schaffer 1986, Schluter 1984). The ge- THROATED TAXA netic basesof allozyme differencesare clearer; -- Myrmotherula -- the loci are generally independent and rela- Fig. 7. Comparison of scaled distances within tively free from the effectsof natural selection. plumagegroups and over all taxa for genetic (G), This doesnot imply, however, that biochemical morphometric (M), and male plumage (P) distance estimatesare without problems (Lanyon 1988, matrices. 482 HACKETTAND ROSENBERG [Auk, Vol. 107 phylogeny. For data sets that lack molecular Amazon River is comparableto thosefound by supportfor monophyly, a combinationof bio- Capparella (1988) for other taxa. chemicaland other(morphological, behavioral, An implication of the high geneticdistances ecological)analyses may be necessaryto pro- is the probableincreased age of tropical forest ducerobust estimates of phylogeneticrelation- taxa relative to North American taxa. Even ships. though dating speciation events using molec- Disparity between phenetic and cladistices- ular data is controversialand requiresa number timates of relatednessexist. In our genetic data of assumptions(e.g. a molecularclock), such an set,M. fulviventriswas the mostdistinct member exercisecan be informative.Using a calibration of the checker-throatedgroup based on phe- of one unit of Nei's (1978) genetic distanceas netic analysis, whereas the cladistic analysis roughly equivalent to 26 million years of in- placed it within this clade (Fig. 6). Severalaut- dependent evolution (Gutierrez et al. 1983), we apomorphiesin M. fulviventrisinflated its ge- can make a conservativeestimate of timing of netic distance from other checker-throated divergence events within these lineages. With species,which placed it outside these species few exceptions,most species-level splits among in our pheneticanalysis. Such discrepancies may these antbird taxa occurred at least 2.5 million result from variations in the rate of protein evo- yearsbefore present. Within eachplumage type, lution or from phenogram instability (Archie a few sistertaxa probably differentiated during et al. 1989). Our cladisticanalysis used shared- the Pleistocene(e.g. Myrmotherula longipennis vs. derived alleles, and autapomorphies have no M. menetriesiiand M. obscuravs. M. sclateri). effect on the species'placement. Thus, in spite Within the checker-throatedclade, all currently of minor discrepancies,both cladisticand phe- recognized speciesare old, with only popula- netic analysesproduced similar major groups tion-level differentiation being of Pleistocene of speciesand demonstratedthat Myrmotherula origin (i.e. within M. haematonota).The checker- is not monophyletic. throatedclade itself hasevolved independently Levelsof geneticdifferentiation.--Average ge- from other lineagesfor approximately9 million neticdistance (0.329) in the genusMyrmotherula years. Our results, and those of Capparella is artificially high becausethe taxa averagedto (1988),contradict Haffer's (1974, 1985, 1987) hy- get this estimatedo not form a monophyletic pothesisthat diversification of Amazonian taxa group. Such overestimatesare not unique to is largely Pleistocenein origin. this study (Johnsonet al. 1988), and caution The low levels of genetic differentiation re- should be exercisedwhen reporting and com- ported for temperate-zone birds have raised paring genetic distances. questionsabout the utility of starch-gelelectro- The averagegenetic distanceof 0.060 within phoresisof proteins for documentingpatterns M. haematonota exceeded the mean value re- of population differentiation (see Zink 1986). ported between some temperate zone conge- The comparablygreater genetic distancesfor nericoscine species ([5 = 0.0440;Barrowclough Neotropical species (at all taxonomic levels) 1980).Genetic distances within other speciesof outlined in Table 3 demonstrate that protein tropical birds are of approximately the same electrophoresispermits analyses of Neotropical magnitudeas that within M. haematonota(Table bird populations. 3). We submit that speciesof Neotropical birds Our study has implications for several areas are structured differently from those of tem- of avian systematics.First, current taxonomy, perate birds. Factorsthat could increasegenetic especiallyof many species-richtropical groups, differentiation in Neotropical birds include may be inadequate, and studies of Neotropical weak dispersalability, smalllong-term effective birds may be biased by a taxonomy that does population sizes,and increasedage of the taxa. not reflect evolutionarypatterns. Morphologi- Capparella (1988) demonstratedthe effects of cal analyses should be compared with molec- riverine barriers, suchas the Amazon and Napo ular-basedphylogenies in revising classifica- rivers, on the genetic population structureof tions, especially at the generic level. Finally, Neotropical birds. In contrast, there are no other characteristicsof species(ecological, vo- known North American avian taxa phenotyp- cal, and behavioral) must be studied to deter- ically differentiatedacross even the Mississippi mine how reliable they are as predictorsof evo- River. The geneticdistance (0.066) between two lutionary relationships.For example, Christidis M. haematonotapopulations sampled acrossthe et al. (1988)found that patternsof geographical July1990] Evolutionof Antwrens 483

distributionand foragingniche were consistent phylogenetic reconstruction. Annu. Rev. Ecol. with a protein-basedphylogeny in casesin Syst. 20: 430-460. which morphologicalevidence was contradic- Fracas,J. S. 1972. Estimatingphylogenetic trees from tory. In Myrmotherulaantwrens, groups based distance matrices. Am. Nat. 106: 645-668. on foraging specializationand vocalizationsalso 1981. Distancedata in phylogeneticanalysis. correspondto our estimatesof genetic relat- Pp. 3-23 in Advancesin cladistics,vol. 1 (V. A. Funk and D. R. Brooks, Eds.). New York, New edness(Rosenberg and Hackettin prep.). York Botanical Gardens.

ACKNOWLEDGMENTS FELSENSTEIN,J. 1985. Phylogeniesand the compar- ative method. Am. Nat. 125: 1-15. We thank the LouisianaState University Museum 1986. PHYLIP (Phylogenyinference pack- of Natural Sciencefor financial supportand the mu- age), version 3.0. Seattle,Univ. Wash.,Dep. Ge- seumpersonnel for collectingthe tissuesamples used netics. in this study. V. Lancasterprovided excellent com- GERWIN,J. g. 1987. Evolutionaryhistory of hum- puter assistance;K. J. Burns and B. Copeland helped mingbirds, a molecular perspective.M.S. thesis. with computerdata entry. We benefited from advice Baton Rouge, Louisiana State Univ. and comments on antbirds from M. Isler, P. Isler, T. GORMANßG. C., & J. RENZIJR. 1979. Genetic distance A. Parker III, J. V. Remsen, and D. Stotz. E. Theriot, and heterozygosityestimates in electrophoretic R. M. Zink, J. V. Remsen Jr., D. Watt, and A. J. Baker studies:effects of samplesize. Copeia 1979: 242- provided helpful commentson this manuscript. 249. GRADWOHL,J. & R. GREENBERG.1984. Search behav- LITERATURE CITED ior of the Checker-throatedAntwren foragingin aerial leaf litter. Behav. Ecol. Sociobiol. 15: 281- ARCHIE,J. W., C. SIMONß& A. MARTIN. 1989. Small 285. sample size does decreasethe stability of den- GUTI•RREZ,R. J., R. M. ZINK, •r $. Y. YANG. 1983. drograms calculated from allozyme-frequency Genic variation, systematic,and biogeographic data. Evolution 43: 678-683. relationshipsof some galliform birds. Auk 100: BALDWIN, S. P., H. C. OBERHOLSER,•r L. G. WORLEY. 33-47. 1931. Measurements of birds. Cleveland, Ohio, Sci. Publ. Cleveland Mus. Nat. Hist. HACKETT,S.J. 1989. Effectsof varied electrophoretic conditionson detectionof evolutionarypatterns BARROWCLOUGH,G.F. 1980. Genetic and phenotypic in the Laridae. Condor 91: 73-90. differentiation in a wood warbler (genus Den- droica)hybrid zone. Auk 95: 691-702. HAFFER,J. 1974. Avian speciation in tropical South America. Publ. Nuttall Ornithol. Club 14. BRAUN,M. J., & T. A. PARKERIII. 1985ß Molecularß ß 1985. Avian zoogeographyof the Neotrop- morphological, and behavioral evidence con- ical lowlands. Pp. 113-146 /n Neotropical orni- cerning the taxonomicrelationships of "Synal- thology (P. A. Buckley,M. S. Foster,E. S. Morton, laxis"gularis and other synallaxines.Pp. 333-346 R. S. Ridgely, and F. G. Buckley,Eds.). Ornithol. in Neotropical ornithology (P. A. Buckley, M. S. Monogr. 36. Foster,E. S. Morton, R. S. Ridgely,and F. G. Buck- ley, Eds.).Ornithol. Monogr. 36. 1987. Biogeography of Neotropical birds. BUTH,D.G. 1984. The applicationof electrophoretic Pp. 105-150 in Biogeographyand quaternaryhis- data in systematicstudies. Annu. Rev. Ecol.Syst. tory in tropical America (T. C. Whitmore and G. 15: 501-522. T. Prance, Eds.). Oxford, Clarendon Press. CAPPARELLA,A. P. 1988. Genetic variation in Neo- HARRIS, H., & D. A. HOPKINSON. 1976. Handbook of tropicalbirds: implications for the speciationpro- enzyme electrophoresis in human genetics. Am- cess.Proc. Int. Ornithol. Congr. 19: 1658-1664. sterdam, North Holland Publ. Co. ß& S. M. LANYON. 1985. Biochemical and mor- JOHNSON,N. K., R. M. ZINK, & J. A. MARTEN. 1988. phometric analyses of sympatric, Neotropical, Genetic evidence for relationshipsin the avian sibling species,Mionectes macconnelli and M. ole- family Vireonidae. Condor 90: 428-445. agineus.Pp. 347-355 in Neotropicalornithology --, G. F. BARROWCLOUGHß& J. A. MARTEN. (P. A. Buckley, M. S. Foster,E. S. Morton, R. S. 1984. Suggestedtechniques for modern avian Ridgely, and F. G. Buckley, Eds.). Ornithol. systematics.Wilson Bull. 96: 543-560. Monogr. 36. JOHNSON,R. A., & D. W. WICHERN. 1982. Applied CHRISTIDIS,L., R. $CHODDE,& P. R. BAVERSTOCK.1988. multivariatestatistical analyses. Englewood Cliffs, Genetic and morphological differentiation and New Jersey,Prentice-Hall Inc. phylogeny in the Australo-Papuanscrubwrens LANYON, S. M. 1985. Molecular perspective on (Sericornis,Acanthizidae). Auk 105: 616-629. higher-level relationships in the Tyrannoidea DONOGHUE,M. J.,J. A. DOYLE,J. GAUTHIER,A. G. KLUGE, (Aves). Syst.Zool. 34: 404-418. & T. ROWE. 1989. The importanceof fossilsin ß 1988. The stochastic mode of molecular evo- 484 HACKETTAND ROSENBERG [Auk, Vol. 107

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APPENDIX1. Scientificnames, species codes, and collectinglocalities of specimens.LSUMNS tissuenumbers are in parenthesesafter scientific name.

Species Code Locality

I. Myrmotherulahauxwelli (B4128) MHAUX Peru:Dpto. Loreto;lower Rio Napo region 2. M. fulviventris(B2299) MFULV Panama:Prov. Darien; E slope Cerro Pirre 3. M. leucophthalma(B9223) MLEUC Bolivia:Dpto. Pando;S Cobija 4. M. haematonota(B1986) MHPS Peru: Dpto. Pasco;Villa Rica-PuertoBermudez Hwy. 5. M. haematonota(B9043) MHPD Bolivia:Dpto. Pando;S Cobija 6. M. haematonota(B4579) MHSB Peru:Dpto. Loreto;S bank Rio Amazonas,SSW mouth Rio Napo 7. M. haematonota(B6964) MHNB Peru:Dpto. Loreto;N Rio Amazonas,85 km NE Iquitos 8. M. erythrura(B5474) MERYT Peru: Dpto. San Martin; NE Tarapoto 9. M. ornata (B9502) MORN Bolivia:Dpto. Pando;S Cobija 10. M. schisticolor(B2124) MSCHI Panama: Prov. Darien; NW Cana I I. M. longipennis(B8877) MLONP Bolivia:Dpto. Pando;S Cobija 12. M. menetriesii(B9759) MMENE Bolivia:Dpto. Pando;S Cobija 13. M. grisea(BI132) MGRIS Bolivia: Dpto. La Paz; Rio Beni 14. M. behni(B7453) MBEHN Venezuela: TF Amazonas; Cerro de la Neblina 15. M. assimilis(B7370) MASSI Peru: Dpto. Loreto; Isla Pasto,Rio Amazonas 16. M. axillaris(B5468) MAXIL Peru: Dpto. San Martin; NE Tarapoto 17. M. obscura(B4908) MOBSC Peru: Dpto. Loreto; S Rio Amazonas 18. M. surinamensis(B2229) MSURI Panama:Prov. Darien; E. slopeCeror Pirre 19. M. longicauda(BI091) MLONC Bolivia:Dpto. La Paz; Rio Beni 20. M. sclateri(B9715) MSCLA Bolivia:Dpto. Pando;S Cobija 21. M. brachyura(B4722) MBRAC Peru:Dpto. Loreto;S Rio Amazonas 22. Drymophiladevillei (B9350) DDEV Bolivia:Dpto. Pando;S Cobija 23. Microrhopiasquixensis (B9294) MQUIX Bolivia:Dpto. Pando;S Cobija 24. Terenurahumerails (B4964) TI-IUME Peru: Dpto. Loreto; S Rio Amazonas 25. Herpsilochmusrufimarginatus (B2162) I-IRUFI Panama:Dpto. Darien; NW Cana 26. Pygiptilastellaris (B9703) PSTEL Bolivia:Dpto. Pando;S Cobija 27. Thamnophilusdoliatus (B1488) TDOL Bolivia:Dpto. SantaCruz; S SantaCruz 28. Dysithamnusmentalis (B2003) DMENT Peru: Dpto. Pasco;Villa Rica-PuertoBermudez Hwy 29. Formicivorarufa (B6821) FRUFA Bolivia:Dpto. Beni; N Yacumo 30. Hypocnemiscantator (B9140) I-ICANT Bolivia:Dpto. Pando;S Cobija 31. Hylophylaxpoecilonota (B8905) HPOEC Bolivia:Dpto. Pando;S Cobija 32. Grailariaquitensis (B357) GQUIT Peru:dpts. Piura-Cajamarca; Cerro Chinguela 486 H^cr.•rTAND ROSENBERG [Auk,Vol. 107 July1990] EvolutionofAntwrens 487 488 HACKETTAND ROSENBERG [Auk, Vol. 107 July1990] EvolutionofAntwrens 489

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