GENETIC VARIATION IN PICIFORM : MONOPHYLY AND GENERIC AND FAMILIAL RELATIONSHIPS

SCOTT M. LANYON • AND ROBERT M. ZINK Museumof Zoology,Louisiana State University, Baton Rouge, Louisiana 70803 USA

ABSTRACT.--Inter-and intrafamilial relationshipswithin the New World Piciformeswere examined through an electrophoreticanalysis of 20 protein-coding loci (19 of which varied between taxa). One individual from each of 26 speciesrepresenting 25 genera and 5 families was analyzed;Momotus momota (, Momotidae) was usedas an outgroup.Al- though levels of genetic differentiation were high (the mean Nei's unbiaseddistance was 1.07), the data proved useful for phylogeneticinference. The jackknife techniquewas used to estimatethe robustnessof phylogenetichypotheses. At the interfamilial level, the results suggestthe following groupings:[I[(Bucconidae) (Galbulidae)]{(Picidae)[(Capitonidae) (Ram- phastidae)]•]].These results were consistentwith hypothesesof familial relationships pro- posedby two recent cladisticanalyses of morphologicalcharacter complexes (Simpson and Cracraft 1981, Swierczewski and Raikow 1981). Our data challenge the currently accepted monophylyof the ,however, in much the sameway asdo DNA-DNA hybridization data. Agreement among independently derived hypothesesof interfamilial relationships suggestsconfidence in our knowledge of evolutionarypatterns among piciform taxa. Hy- pothesesof intrafamilialrelationships, some of which agreedwith morphologicalpatterns obtainedin other studies,were presented.This study showsthat starch-gelelectrophoresis may be usefulat higher taxonomiclevels. Received 10 November1986, accepted 4 June 1987.

RECENTyears have witnesseda plethora of fast rate of change) of a synapomorphicchar- biochemical systematic studies (Avise and acter state,such as a particular allele. Aquadro 1982).These studies have demonstrat- Nonetheless,the potential of biochemical ed that molecular characters have both advan- methods for the inference of evolutionary his- tagesand limitationsfor phylogenyreconstruc- tory is widely appreciated(Buth 1984,Wilson tion (Lanyon 1985b).There are severaltypes of et al. 1985, Sibley and Ahlquist 1986) because limitations.For example,molecular methods are they allow directaccess to geneticinformation. scaledependent. That is, one would generally A framework of quantitative models provides not usestarch-gel electrophoresis of proteinsto objectivemethods for estimatingphylogenies investigaterelationships of higher taxa (Buth (Felsenstein1982). Probably few phylogenetic 1984)because often taxashare no alleles,which estimates,however, irrespectiveof the data or contributes no phylogenetic information. In methods used to construct them, are immune other instancesmolecular methodsmay be un- to bias. Recently,statistical or quantitative tests ableto resolveevolutionary patterns because of have been developedto assessthe accuracyof the relationshipbetween the nature and rate of a particularphylogenetic pattern (e.g. Temple- characterevolution (e.g. allelic substitutionsoc- ton 1983, Felsenstein1985, Lanyon 1985a).Fur- curring more or lessuniformly over time) and thermore, systematicstudies that comparepat- the nature of the evolutionary history itself (Fi- terns of morphological, biochemical, and ala and Sokal1985). For example,if cladogenetic behavioralvariation may lead to more robust events are close in time and lineages are short- estimatesof evolutionary history(Wagner 1961, lived before fragmentation (speciation), then Miyamoto1981, Lanyon and Lanyon 1986). Such there is a low probability of the origin (and multidisciplinary approachescan exploit posi- retention through evolutionary time, given a tive aspectsof eachsuite of characters,and per- mit evaluation of whether consistent patterns emerge.A disadvantageof this approachis that • Present address: Division of Birds, Field Museum it requiresinvestigators to becomefamiliar with of Natural History, RooseveltRoad at Lake Shore many different techniques,which can increase Drive, Chicago,Illinois 60605 USA. greatly the time required to completestudies 724 The Auk 104: 724-732. October 1987 October1987] PiciformBiochemical Systematics 725 of even small assemblages.It is possibleto sim- BUCCONIDAE ulate such a multidisciplinary study by inte- GALBULIDAE grating independentlyor collaborativelygath- ered data setsof several investigators. CAPITONIDAE We examined phylogenetic patterns in elec- trophoretic characters(allozymes) in the - RAMPHASTIDAE formes.Recent hypotheses (Fig. 1) of piciform PICIDAE relationships have been based on hindlimb musculature (Swierczewski and Raikow 1981), INDICATORIDAE osteologicalcharacters (Simpson and Cracraft Fig. 1. Cladogram of higher-level relationships 1981),and the anatomyof the feeding apparatus supportedby Simpsonand C racraft(1981) and Swier- (Burton 1984). We comparephylogenetic pat- czewski and Raikow (1981). A = Ramphastoidea,B = ternsderived from analysisof our allozyme data Picoidea, C = Pici, D = Galbulae, and E = Piciformes. set with thosederived from analysesof muscle Derived characterssupport each node. and skeletal variation. We illustrate the impor- tance of examining the robustnessof phylo- genetic estimatesthrough the use of the jack- (1971),adjusted to pH 6.5].Twenty proteins(see Table knife technique (Lanyon 1985a).Furthermore, 1), presumedhomologous across taxa, were identified this is one of the few systematicapplications of using protein-specificassays outlined by Harris and Hopkinson (1976). During the initial survey of loci, protein electrophoresisat higher taxonomic the mobility of each character(=electromorph) across levelsin birds (Lanyon 1985b);we suggestthat the 27 taxa was recorded relative to a standard. All the limits of the techniquefor avian systematics alleles of similar mobility were then compared in are unknown. side-by-sidetests on additional gels to ensure the ac- curacyof character-statedesignations. METHODS Data analysis.--We used the computer program BIOSYS-1(Swofford and Selander 1981) to compute Taxonselection.--The 27 tissuesamples (represent- Rogers'(1972) and Nei's (1978) geneticdistances and ing 25 generaand 5 familiesand 1 outgroup;see Table to estimate phylogenetic patterns using the UPGMA 1) analyzed in this study were collectedover a period and distanceWagner procedures.The computer pro- of 3 yr by personnelof the LouisianaState University gram PHYLIP (Felsenstein1985) was usedto produce Museum of Zoology. From 2 to 7 individuals, eachin Fitch-Margoliash(F-M) trees.These distance analyses a different ,were selectedto representeach of yield phylogenetic information becauseof the pre- the five traditionallyrecognized New World piciform dominantly stochasticmanner in which the charac- families [Bucconidae(), Galbulidae (jaca- ters evolve (Kimura 1983). We note, however, that mars),Capitonidae (barbers), Ramphastidae (), ratesof nucleotide substitutionsmay differ between and Picidae()]. We maximizedthe num- lineages(Avise and Aquadro1982, Britten 1986), which ber of species-leveltaxa used, instead of individuals, resultsin reducedaccuracy of phylogenetic hypoth- to estimatelevels and patternsof genetic variation esesderived usingdistance analyses (Felsenstein 1978, within and among families. The use of a single in- 1982). dividual/taxon to estimategenetic distancesis suffi- To estimatethe degreeof error in phylogeniescon- cient when investigatingrelationships at higher taxo- structedby our analysesof geneticdistances, we em- nomic levels (Gormanand Renzi 1979, Lanyon 1985b). ployed a jackknife analysis(Lanyon 1985a),in which The outgroup(Momotus momota, Momotidae) was se- pseudoreplicatedistance matrices were created(each lected from the presumed sister group, the Coraci- consistingof all possiblecombinations of n - 1 taxa, iformes (Simpsonand Cracraft 1981, Swierczewski where n = the total number of taxa under consider- and Raikow 1981; but see Olson 1983). ation), and F-M trees generated.A single strict con- Electrophoresis.--Homogenateswere preparedfrom sensustree was then produced to identify the nodes pooledsamples of liver and pectoralmuscle: 0.5-1.0 that were consistentwith all analyses. g of mincedtissue and an equalvolume of Cleland's Values reported are means ñ SD. Reagentwere centrifugedat 12,000rpm for 60 min, the supernatantremoved and preservedat - 70øC,and the pellet discarded.Homogenates were applied to RESULTS 12% horizontal starch gels using filter-paper wicks. Two discontinuousand two continuousbuffer sys- Allelicfrequencies.--Inspection of the pattern tems were used to isolate proteins using horizontal of electromorphicvariation at the 20 loci (Table starch-gelelectrophoresis [buffers 8-10 of Aquadro 1) revealsconsiderable variation among the taxa. and Avise (1982); and Tris-maleate of Selander et al. An average of 5.3 + 2.1 alleles/locus was ob- 726 LANYONAND ZINK [Auk, Vol. 104

T^BLE1. Distribution of electromorphs(denoted by lowercaseletters) in 26 piciform taxa and 1 outgroup (Mornotusrnornota, Momotidae). Taxa 2-9 representthe family Bucconidae,10-13 the Galbulidae, 14-20 the Picidae, 21-22 the Capitonidae, and 23-27 the Ramphastidae.

LOCUSa,b

Genus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1. Momotus momota g b b e c f b e b g e f h cd c c b ab b a 2. Buccocapensis e a a d c f c cf f e e d gh a e c ab c b a 3. Nystalusradiatus e a a d c f c e g e e d i a e c d bc b a 4. Malacoptilafusca e a d d a f c e d e e d i c e a d c b a 5. Micromonacha lanceolata e a c d c f c e f e f d i c e a d c b a 6. Nonnula rubecula e a d d c a c e f e e d h c e a d c b a 7. Hapaloptilacastanea e a d d c f a e f e e d i c e a d bc c a 8. Monasanigrifrons e a a d c f c e f e c d i c d b d c b a 9. Chelidopteratenebrosa e a a d c f d e f e c d i b d c d c b a 10. aIbirostris f a d d c g a e e e f f d c e a d b b a 11. Jacameropsaurea f b e d c b a e a d g g h d e a d b b a 12. Brachygalbasalmoni f a d e c e a e e g g d j c e a d b b a 13. Galbalcyrhynchusleucotis f a a d c b a g g e e f f ac e a d b b a 14. Carnpephilushaematogaster b c f f b g c b e a b d g c f c c d b a 15. Picoides scalaris b c f c b g c e h f a k c c d c c c a a 16. Sphyrapicusvarius b c f c c g c b h fad f a c d c c c b a 17. Picoides villosus a c f c b g c bd h f a k c c d c c c b a 18. Melanerpesaurifrons ab c f c d g d b h b ac d bc c d c c c b a 19. Colaptesauratus b c f c d g c g h f a g cd c e c c c b a 20. Picumnus borbae b c d c b g c g h h b f d d f c bc c b a 21. Capitoniger h c f d b g c g f f c k i c b a b c b a 22. Eubucco bourcierii h c f d b g c g i a c k h c b b b c b a 23. Selenideraspectabilis h c f b a g c g c a e d d c b a b c b a 24. Aulacorhynchusprasinus h c f b a g c g f c c f d d b a b c b a 25. Andigenahypoglauca dh c f b a g e g c c ce f f c b b b c b a 26. Pteroglossuscastanotis h c f b a g c g c c c g d c b a b c a a 27. Rarnphastossulfuratus h c d a a g c g c e c f d a b b b c b a • Loci in are: Pgm-1 (E.C. 2.7.5.1), Mdh-l,2 (1.1.1.37),Got-I (2.6.1.1), Ck-2 (2.7.3.2), Eap (3.1.3.2), Pgi (5.3.1.9), Est "D" (3.1.1.1), Sod-I (1.15.1.1),Gda (3.5.1.2),6-Pgd (1.1.1.44),Peptidase C, B (3.4.11),Mpi (5.3.1.8),Ldh-l,2 (L1.1.27), ldh-l,2 (1.1.1.42),Fum (4.2.1.2),and Lap (3.4.11.1). bTwo letterssignify a heterozygousgenotype. served;only one locus(Lap) wasmonomorphic analyses(Fig. 2) and Fitch-Margoliashanalysis acrossall taxa. Ten other loci were examined (not shown) producedthe samebranching se- but were too variable to warrant continued quences for the five families. Furthermore, the analysisfrom a logisticalstandpoint. branchingsequence was completelystable to Geneticdistances.--A summary of Nei's (1978) the jackknife analysis.Three hypothesesof in- geneticdistances (Table 2) showsaverage ge- terfamilial relationships proposed indepen- neticdistances within andbetween major groups dently by Swierczewskiand Raikow (1981)and (the completematrix is available from the au- by Simpsonand Cracraft(1981)were supported. thors). Becausethe distance values in Table 2 The Bucconidaeand Galbulidae were identified were basedon conservativeloci,theyare under- as sister taxa, supporting a hypothesis previ- estimatesof the actual level of geneticdiffer- ously defined by four osteologicalcharacters entiation in piciform birds;this shouldbe con- (Simpson and Cracraft 1981), six myological sideredwhen comparingour resultswith values characters(Swierczewski and Raikow1981), and reported in the literature. The distancesare, similarities in conalbumins and ovalbumin nevertheless,greater than those reported for (Sibleyand Ahlquist 1972). Although these taxa comparisonsat comparabletaxonomic levels in have consistentlybeen identified as close tel- other birds (Barrowclough1980). The average atives, the evidence presented here reveals Nei (1978)genetic distance among all piciform markedgenetic dissimilarity (D = 0.80 + 0.22), taxa was 1.07. and a considerableamount of time probablyhas Distanceanalysis and interfamilialphylogenetic elapsed since they shareda commonancestor. patterns.--TheUPGMA and distanceWagner This is in contrastto the Capitonidaeand Ram- October1987] PiciformBiochemical Systematics 727

TABLE2. Nei's(1978) genetic distance (+ SD)between major groupings of piciformtaxa. Entries in thediagonal arecomparisons between taxa within eachgrouping. Sample sizes are the numberof pairwisecomparisons.

Motmot Puffbirds Jacamars Barbets Toucans Woodpeckers

Motmot -- Puffbirds 1.15 + 0.10 0.32 + 0.09 (Bucconidae) n = 7 n = 28 Jacamars 1.08 + 0.08 0.80 + 0.22 0.52 + 0.11 (Galbulidae) n = 4 n = 32 n = 6 Barbets 1.57 1.12 + 0.24 1.50 + 0.20 0.22 (Capitonidae) n = 2 n = 16 n=8 n=l Toucans 1.55 + 0.25 1.38 + 0.29 1.59 + 0.37 0.48 + 0.07 0.32 + 0.07 (Ramphastidae) n = 5 n = 40 n = 20 n = 10 n = 10 Woodpeckers 1.55 + 0.19 1.38 + 0.22 1.75 + 0.36 1.26 + 0.49 0.98 ñ 0.19 0.44 + 0.18 (Picidae) n = 7 n = 56 n = 28 n = 14 n = 40 n = 21

phastidae, also recognized as sister taxa, be- branching sequence of families shown in tween which the average intergeneric distance Fig. 2. was considerablysmaller (D = 0.48 + 0.07). IntrafamiIiaIphylogenetic relationships.--To in- The monophyly of the Capitonidaeand Ram- vestigate relationships within piciform fami- phastidae,supported by two myological char- lies, we assumedthat each family represented acters (Swierczewski and Raikow 1981) and one a monophyletic assemblage.A jackknifed F-M osteologicalsynapomorphy (Simpson and Cra- strictconsensus tree was producedfor eachfam- craft 1981), was also supportedby the electro- ily and was rooted using the remaining taxa. At phoretic analysis. We suggest that capitonids this lower level of taxonomic investigation, we and ramphastidsare asclosely related asare the detectedtree instability. Jackknifingallowed us jacamargenera (D = 0.52 + 0.11). to expose inconsistencieswithin the distance The electrophoreticdata supported a mono- matrix, identified as unstable nodes, and to iso- phyletic assemblageconsisting of the Picidae, late reliable nodeswithin families (Figs. 3-5). Capitonidae, and Ramphastidae.This hypoth- Four nodes were retained for the eight buc- esis has been supported previously by four conid genera(Fig. 3a). The hypothesisthat Buc- osteological characters, six myological syn- co and NystaIusrepresent sister taxa was sup- apomorphies, and the mobility of an s-Mdh ported [Peters (1964) and Cottrell (1968) zymogram (Avise and Aquadro 1987). A close consideredthem to be congeners].Swierczew- relationshipbetween capitonidsand picidswas ski and Raikow (1981) identified NystaIusand suggestedby analysis of egg-white proteins CheIidopteraas being more closelyrelated to each (Sibley and Ahlquist 1972), but ramphastids other than either is to MaIacoptiIa or NonnuIa were not analyzed. In contrast,Peters (1964) (Fig. 3b). This was neither supported nor re- placedthe capitonidsand ramphastidswith the futed by the electrophoretic investigation, bucconidsand galbulids rather than with the which failed to resolvethe branching sequence picids.Sibley and Ahlquist (1985)suggested that for these taxa. The electrophoretic characters the New World capitonidswere more closely also identified MaIacoptiIa,NonnuIa, HapaIoptiIa, related to ramphastidsthan to Old World cap- and Micromonachaas a monophyletic assem- itonids. We had tissuesamples only from New blage within which Micromonachais the sister World capitonids,and we cannot comment on taxon to the remaining three genera. Finally, the monophyly of the Capitonidae. we note that the distance between Bucco and To evaluate the familial relationships sup- NystaIus,which are currently considered con- ported by this study, one specimenfrom each generic (A.O.U. 1983),is comparableto the dis- of the five families (plus Momotus)was selected tance between CheIidopteraand Monasa (D = at random and a Fitch-Margoliash analysiscon- 0.418 and 0.387, respectively). The latter two ducted. Ten iterationswere performed, and in taxa have not previouslybeen suggestedas sis- all casesthe results(not shown) supportedthe ter taxa (or congeners)but have been placed 728 LANYONAND ZINK [Auk,Vol. 104

8 .66 Momotus .20 .10 • .20 Bu½co

ß2 .20 Ch©lldoptera

• .20 MlcromonachaNonnula

.30 Galbula

.10 .40 Galbalcyrbynchus .45 Jacamerops Campephllus .12 P. scalarIs

.05 -- Sphyraplcus Colapt©s Melanerpes .48 Plcumnus .20 .19 • .20 EubuccoCaplto 03. .20Selenldera Aulacorbynchus

.3• Ramphastos

Momotus .05 Bucco

.05

Chelldoptera Mlcromonacha .O9

.07 Nonnula .04 .07 Brachygalbe

Galbalcyrhynchus .25 .O8

.O8

.07 Sphyraplcu$ Melan©rp©s

.23 Plcemnus .06

Euhucco .08

Aulacorhynchus

Ramphastos

Fig. 2. UPGMA (a) and optimized distanceWagner (b) treesderived using Rogers'genetic distancemea- surescalculated from the matrix of electromorphs(Table 1). The copheneticcorrelation coefficientfor the UPGMA phenogramis 0.93, indicating a reasonablygood fit of the branchingdiagram to the distancematrix. Farris' "f" for the distanceWagner tree is 14.2. The values on the diagramsrepresent branch lengths. The distanceWagner tree was rooted using Momotusas an outgroup.See legend to Table 1 for family membership of taxa. together at the end of the family in recent clas- tential lack of confidencein the branching pat- sifications(Peters 1964, A.O.U. 1983).Although tern depicted(it might, after all, be correct),the genetic distance cannot be an absolute measure allelic frequency data do provide insight into of taxonomicstatus, our data suggestscrutiny galbulid evolution. The relatively great genetic of the postulated close relationship between distancebetween galbulid genera, 0.52 +__0.11, Buccoand Nystalus. suggeststhat the four genera analyzed herein None of the galbulid phylogenetic hypoth- are from distinct, relatively old lineages.These esesgenerated by the distanceanalyses (Fig. 2) lineagesmight have originated closein time. was retained after jackknifing. Despite this po- Two nodes were consistentlysupported for October1987] PiciformBiochemical Systematics 729

/ / Aulecorhynchus --•- / /-..... I .• MalacoptllaMlcromonacha I \ •_.... Selenldere I •- ..... Hapaloptlla I •" Monasa

% • • Bucco b ' Auiacorhytlchus

I Nonnula Ramphastos

• Chelldoptera % • Nystalus ½ Fig. 3. (a) Jackknifedstrict consensusFitch-Mar- goliash tree for the Bucconidae.All other taxa were usedas a compositeoutgroup to root the tree. Dotted • Aulacorhynchus lines indicate portionsof the topology that were un- • SelenlderaPteroglossus stableto the jackknifemanipulation. (b) Phylogenetic relationships within the Bucconidae supported by Fig. 4. (a) Jackknifedstrict consensusFitch-Mar- Swierczewskiand Raikow's (1981) analysisof hind- goliashtree for the Ramphastidae.All other taxawere limb musculature. used as a compositeoutgroup to root the tree. Dotted lines indicateportions of the topologythat were un- stableto the jackknife manipulation.(b) Phylogenetic relationshipswithin the Ramphastidaesupported by the five ramphastid taxa examined (Fig. 4a). Swierczewskiand Raikow's (1981) analysisof hind- Ramphastoswas identified asthe outgroupto the limb musculature. (c) Phylogenetic relationships remaining genera,and Selenideraand Pteroglos- within the Ramphastidaesupported by Haffer's(1974) suswere identified as sister taxa (note, however, analysis of , vocalizations, and biogeogra- that theseresults differ from the distanceWag- phy. ner tree shown in Fig. 2b). Our findings conflict with those of Swierczewski and Raikow (1981), who concludedthat Pteroglossusand Ramphastos were unresolved. Lastly, Picoidesvillosus and P. were sistertaxa (Fig. 4b), but are consistentwith scalaris were identified as sister taxa, as one Haffer's (1974) phylogeny for part of the family would expectfrom their congenericstatus. Sev- (Fig. 4c). Haffer (1974) suggestedthat the low- eral hypothesessuggested by Short's classifi- land generaSelenidera and Pteroglossuswere sis- cation and the cladogramdeveloped by Swier- ter taxa on the basis of vocal similarities. czewski and Raikow (1981) cannot be addressed Three apparently robust phylogenetic hy- here. Specifically, we lack a consistently sup- potheseswere generated for the seven wood- ported branching sequence for Melanerpes, pecker taxa analyzed (Fig. 5a). The identifica- Sphyrapicus,Colaptes, and Picoides.Additional tion of Picumnusas the sister group to the study of patternsof protein variation promises remainingforms is consistentwith Short's(1982) insightsinto picid relationships. classificationand the findings of Swierczewski and Raikow (1981) (Fig. 5b). Campephiluswas DISCUSSION shown to be the next lineage to arise (relative to thosesurveyed), as suggested by Swierczew- Polyphylyof the Piciformes.--Sibleyand Ahl- ski and Raikow (1981). In the classification of quist (1972, 1985), Olson (1983), and Burton Short (1982) the relationships among the Me- (1984)have suggestedthat the Piciformesmight lanerpini, Campetherini, and Campephilini be polyphyletic. Theseauthors suggest that the 730 LANYONAND ZINK [Auk, Vol. 104

eral sourcesof bias causephylogenetic trees to be unstable or unreliable. A particular phylo- genetic estimate is dependent on the samples of individuals, characters, and taxa used in its construction;different samplesof each might result in different phylogenetic estimates.In addition, the particular algorithm used has in- herent assumptions,such as an averageuniform rate of change (UPGMA) or parsimony (dis- tanceWagner). It usuallyis not possibleto know the nature of character evolution and therefore to select the appropriate algorithm. For some algorithms (e.g. distance Wagner), several equivalent or nearly equivalent treescan result. Thus, methodsof testing robustnessof treesare being developed (Templeton 1983, Felsenstein 1985, Lanyon 1985a). It is sometimesnot appreciatedthat methods f $phyraplcusof producing branching diagrams do sowithout any implied confidencein the branching pat- tern of either the entire tree or parts thereof. Colapres Often, a tree's structuredepends on few char- Campephllus acters, and sampling additional charactersor individuals shifts branching patterns. System- ••i , PlcumnusMelanerpes atists should evaluate routinely the robustness Fig. 5. (a) Jackknifed strict consensusFitch-Mar- of particular nodes in phylogenetic estimates. goliashtree for the Picidae.All other taxa were used Several factors merit comment, however. The as a compositeoutgroup to root the tree. (b) Phylo- patternsobtained from the first passof an al- geneticrelationships within the Picidaesupported by gorithm (e.g. UPGMA) might be correctin their Swierczewskiand Raikow's (1981) analysisof hind- entirety; such a conclusionwould be enhanced limb musculature. if a similarbranching pattern was obtainedfrom an independent data set. Becausesome nodes Galbulae(Bucconidae and Galbulidae)might be of the phylogeny might be basedon a single or more closelyrelated to someCoraciiformes than very few characters,such nodes might disap- either is to the Picae (Picidae, Ramphastidae, pear wrongly with some resamplingmethod, and Capitonidae). We did not specificallyad- suchas jackknifing. In other words, an ancestral dressthis question.We can considerthe ques- branch might be very short lived, with time for tion of monophyly, however, under the as- few character-statetransitions. If one happened sumption of a strong positive correlation to analyze the (derived) charactersthat define between genetic distanceand time since diver- sucha branch,the fact that only a few characters gence. The UPGMA phenogram, which as- (could) support the branch might causeit to be sumesa strongtime correlation,places Momotus collapsed in tests of robustness.In addition, as the sister taxon to the Galbulae. The reason other methods of testing the stability or confi- for this topologycan be seenfrom the summary dence of branching diagrams, such as boot- of genetic distances(Table 2). The averagedis- strapping(Felsenstein 1985), might indicatein- tancesbetween Momotusand the galbulid and stability in a consensustree obtained by jack- bucconid genera (1.08 + 0.08 and 1.15 + 0.10, knifing. Therefore, we note that our branching respectively)are considerablysmaller than the patternsfor all taxa might be correct,but we averagedistance between the Picaegenera and limit interpretationto thosepatterns for which Momotus (D = 1.55 + 0.19). Therefore, our find- someconfidence is indicatedby jackknifing.We ings are consistentwith the possibilitythat the suggestthat at least one test of the robustness order Piciformes(sensu Peters 1964) is polyphy- of branchingpatterns be employed,along with letic. conservative interpretation of patterns. Few Testingthe stabilityof phylogenetictrees.--Sev- avian systematicstudies have used explicit, ob- October1987] PiciformBiochemical Systematics 731 jective methods to examine the robustnessof in this study. We also thank J. C. Avise and C. F. phylogenetichypotheses (Lanyon 1985b). Aquadrofor providing a prepublicationcopy of their Lastly,we note the ongoing debateconcern- manuscript. R. L. Cann, A. P. Capparella, J. Cracraft, ing the inferenceof phylogeniesfrom distance J. A. Gerwin, M. S. Hafner, S. J. Hackett, and J. V. matrices (e.g. Farris 1986, Felsenstein 1986). Remsen,Jr., commented constructively on the manu- script. Resolution of this issue, we suspect,will not alter our primary conclusions. LITERATURE CITED Suggestionsfor furtherwork on piciform relation- AMERICAN ORNITHOLOGISTS' UNION. 1983. Check-list ships.--Asa result of this study and indepen- of North American birds, 6th ed. Washington, dent analysesof two very different suites of D.C., Amer. Ornithol. Union. characters(Simpson and Cracraft 1981, Swier- AQUADRO,C. F., & J. C. AVISE. 1982. Evolutionary czewskiand Raikow 1981),a set of reliable hy- geneticsof birds. VI. A reexaminationof protein pothesesof systematicrelationships has been divergenceusing varied electrophoreticcondi- identifiedfor the Piciformesat the family level tions. Evolution 36: 1003-1019. and above. With the exception of the Picidae, AVISE,J. C., &: C. F. AQUADRO.1982. A comparative however, for which Short (1982) constructed a summary of genetic distancesin the vertebrates. relatively detailed classification,relationships Pp. 151-185 in Evolutionarybiology, vol. 15 (M. within the piciform families remain obscure. Hecht, B. Wallace, and R. Prans, Eds.). New York, Plenum Press. Using the reliable hypothesesof familial rela- , &--. 1987. Malate dehydrogenaseiso- tionshipsto identify proper outgroupsand the zymesprovide a phylogenetic marker for the Pic- preliminaryintrafamilial hypothesespresented iformes(woodpeckers and allies). Auk 104:324- here and by Swierczewski and Raikow (1981) 328. as a foundation, studiesof phylogeneticrela- BARROWCLOUGH,G.F. 1980. Geneticand phenotypic tionshipswithin each piciform family may be differentiation in a wood warbler (genus Den- designed. For example, it will be useful to de- droica)hybrid zone. Auk 97: 789-798. termine whether New World capitonids are BRITTEN,R. J. 1986. Ratesof DNA sequenceevolu- more closely related to ramphastidsor to Old tion differ between taxonomic groups. Science 231: 1393-1398. World capitonids(see Sibley and Ahlquist 1985). BURTON,P. J. K. 1984. Anatomy and evolution of Severaltaxa representing the rangeof variation the feeding apparatusin the avian orders Cora- within the Picidae should be used as a com- ciiformes and Piciformes. Bull. Brit. Mus. Nat. posite outgroup. Hist. (Zool.) 47: 331-443. A criticalhigher-level systematic question that BUTH,D.G. 1984. The applicationof electrophoretic remainsto be answereddefinitively is whether data in systematicstudies. Ann. Rev. Ecol. Syst. the Piciformesare monophyletic. Allozymic data 15: 501-522. suggestthat the traditional Piciformesmay be COTTRELL,G.W. 1968. The generaof puffbirds. Bre- polyphyletic (Fig. 2a), although further assess- viora, No. 285. ment of this questionmust await a study in FARRIS,J. S. 1986. Distances and statistics. Cladistics 2: 144-157. which a compositeoutgroup is constructedfrom FELSENSTEIN,J. 1978. Casesin which parsimony or closelyrelated orders.We note also the useful- compatibility methods will be positively mis- nessof electrophoresisof proteins (allozymes) leading. Syst.Zool. 27: 401-410. for estimating phylogenetic relationships at 1982. Numerical methodsfor inferring evo- higher taxonomic levels, levels at which the lutionary trees. Q. Rev. 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