324 ShortCommunications [Auk,Vol. 104

ination. CaptiveBreeding of Diurnal Birdsof Prey LAKE,P. 1975. Gamete production and the fertile 1: 3-5. period with particular reference to domesticated FuJII,S., & T. TAMURA.1963. Locationof spermsin . Symp. Zool. Soc. London 35: 225-244. the oviducts of the domesticfowl with special MERO, K. N., • F. X. OGASAWARA. 1970. Dimensions reference to storageof spermsin the vaginal of uterovaginal sperm-storagetubules of the gland. J. Fac.Fish. Anim. Husbandry,Hiroshima chicken and their possiblesignificance in sperm Univ. 5: 145-163. release. Poultry Sci. 49: 1304-1308. GILBERT,A.B. 1979. Femalegenital organs.Pp. 237- PAL, D. 1977. Histochemistry of the utero-vaginal 360 in Form and function in birds, vol. I (A. S. junction with specialreference to the sperm-host King and J. McLelland, Eds.). New York, Aca- glandsin the oviduct of the domesticduck. Folia demic Press. Histochem.Cytochem. 15: 235-242. HATCH,S. A. 1983. Mechanismand ecologicalsig- VAN DRIMMELEN,G. C. 1946. "Spermnests" in the nificanceof sperm storagein the Northern Ful- oviduct of the domestic hen. J. S. African Vet. mar with reference to its occurrence in other Med. Assoc. 17: 43-52. birds. Auk 100: 593-600. HOWARTH,B. 1971. Transportof spermatozoain the Received14 March 1986, accepted23 September1986. reproductive tract of turkey hens. Poultry Sci. 50: 84-89.

Malate DehydrogenaseIsozymes Provide a PhylogeneticMarker for the Piciformes( and Allies)

JOHN C. AVISEAND CHARLESF. AQUADRO• Departmentof Genetics,University of Georgia,Athens, Georgia 30602 USA

Nuclear genesencode two major forms of malate of approximatelyequal intensity" (Kitto and Wilson dehydrogenase(MDH; E.C. 1.1.1.37)in birds:S-MDH 1966). This distinctive S-MDH pattern was subse- (or MDH-1), which is found in the soluble fraction quently observedin a Japanesewoodpecker (Kuroda of the cell cytoplasm;and M-MDH (or MDH-2), which et al. 1982:fig. 3) and in three speciesof North Amer- is housedin mitochondria(Karig and Wilson 1971). ican woodpeckers(Aquadro and Avise 1982). Under standard starch-gel electrophoretic condi- Phylogeneticrelationships of the woodpeckersand tions, S-MDH typically migrates toward the anode allieshave been the subjectof intensedebate (Simp- while M-MDH migratescathodally. In termsof gen- son and Cracraft 1981, Swierczewski and Raikow 1981, eral zymogram appearance and electromorph fre- Olson 1983, Raikow and Cracraft 1983, Sibley and quenciesacross avian taxa, both S-MDH and M-MDH Ahlquist 1986). In most traditional taxonomic lists, are very conservativeevolutionarily (Kitto and Wil- six families are included in the Piciformes: Galbuli- son 1966,Aquadro and Avise 1982,Kuroda et al. 1982). dae (),Bucconidae (), Capitonidae In particular, S-MDH assayedin pH 7.0 gels exhib- (barbers),Indicatoridae (),Ramphasti- ited the same common electromorphin representa- dae (),and Picidae (woodpeckers).Recently, tives of 22 avian orders (Kitto and Wilson 1966). Al- this classificationhas been challengedon the grounds though additional S-MDH variation was detected that the assemblagemay be polyphyletic(Olson 1983, under the multiple electrophoreticconditions em- Sibleyand Ahlquist 1986).Raikow and Cracraft(1983), ployed by Aquadro and Avise (1982), S-MDH re- however, defendedthe validity of the synapomorphs mains extremely conservative in comparison with (hypothesized shared-derived traits) used to infer most other enzyme systems. monophyly of the Piciformes.Additional questions Kitto and Wilson (1966) first noticed an unusual concernthe relationshipsof true piciform families to S-MDH zymogram pattern in three speciesof - one another.In an effort to help resolvesome of these formes. Unlike the typical avian S-MDH pattern, phylogeneticissues, we surveyedmembers of all the which consistsof an intense major band with one or taxa listed above, plus other possiblepiciform allies, two much lighter anodic subbands,the Piciformes for occurrenceof the distinctiveand obviously de- were "exceptionalin having two S-MDH subbands rived S-MDH pattern. Frozen tissuesamples (usually heart, liver, or mus- • Present address: Section of Genetics and Devel- cle) were homogenized separatelyin an equivalent opment,Cornell University, Ithaca,New York 14853 volume of grinding solution (0.01 M Tris, 0.001 M USA. EDTA; pH 6.8) and centrifuged at 49,000 g. Electro- •,pril 1987] ShortCommunications 325

r+

Fig. I. Representative S-MDH zymogram pat- ternsin variousspecies of "piciforrn" birds. From left to right: (I) Melanerpescarolinus (Picidae); (2) ruficauda(Galbulidae); (3) Veniliornisnigriceps (Pici- dae); (4) Melanerpescarolinus (Picidae); (5) cruentatus(Picidae); (6) Piculusrivolii (Picidae); (7) and (8) Chelidopteratenebrosa (Bucconidae); (9) Monasani- grifrons(Bucconidae); (10) Melanerpescarolinus (Pici- dae); (11) Andigena cucullata (Ramphastidae); (12) Pteroglossuscastanotis (Ramphastidae); (13) culminatus(Ramphastidae); (14) Malacoptilasemicincta (Bucconidae).The 3-band S-MDH pattern is evident in lanes I, 3-6, and 10-13. The typical S-MDH pat- tern in nonpiciforrn birds is pictured in fig. I of Aquadro and Avise (1982) and is like that in lanes 2, 7-9, and 14 above. Fig. 2. Two (amongseveral) possible phylogenies for "piciform"birds. (A) Inferred from a traditional classificationproposed by Peters(1948). (B) Proposed phoresisof supernatantextracts involved horizontal by Simpsonand Cracraft(1981) and independently starchgels (12.5%),run at 75 mA for 5-7 h. The fol- by Swierczewskiand Raikow (1981). The postulated lowing amine-citrate (A-C) buffer system was em- origin of the derived 3-band S-MDH zymogram is ployed routinely: electrodebuffer, 0.04 M citric acid, indicated by the black rectangle. pH adjustedto 6.1 with N-(3-aminopropyl) morpho- line (purchasedfrom Aldrich Chemicals);gel buffer, I to 19 dilution of electrode buffer. This systemwas pectoralmuscle, leg muscle,heart, liver, gizzardwall, chosen because,among 12 buffers utilized previ- intestine wall, eye, and brain. There were no ob- ously, it provided the best resolution for S-MDH vious, differential tissuespecificities for any S-MDH (Aquadro and Avise 1982). Nonetheless,other buffer isozymes(although all bands were darkest in the conditions yielded comparable results. MDH was muscleand heart samples). stained using the recipe of Selander et al. (1971). In the woodpecker-type S-MDH zymogram, all The avian material consistedof 29 speciesthat rep- three bandswere invariably present,and the middle resented 22 of the 74 piciforrn genera (30%) recog- band stained most intensely. Thus, the gel pattern nized in most current checklists. In addition, mem- gives the impression of "fixed heterozygosity" for bers of Coliiformes and several families of S-MDH, which is known to be a dimeric molecule in Coraciiforrnes(possible relatives of the Piciforrnes) birds. One plausibleexplanation is that woodpeckers were surveyed(Table I). The samplesoriginated from possessa gene duplication for S-MDH, such that the three continents: Africa, North America, and South middle band in the zymogram representsan inter- America (Table I). locusheterodimer, flanked by the two respectivein- S-MDH zymograms.--Thedistinctive "woodpeck- iralocus homodimeric products. Similar zymogram er-type" S-MDH electrophoresedwith the arnine-ci- patternsand assemblyof interlocushybrid molecules trate buffer exhibits three major bands (Fig. I), the are known in somefishes that carry an S-MDH gene two most anodal of which apparently correspondto duplication(Bailey et al. 1970,Wheat et al. 1971).If the "two subband"zymogram pattern originally no- the S-MDH gene is also duplicated in woodpeckers, ticed by Kitto and Wilson (1966). This pattern is individuals detectably heterozygousat either locus readily distinguishablefrom the usual S-MDH pat- shouldexhibit a 6-bandzymogram (three homo- and tern in other birds, which on A-C gels consistsof a three heterodimers).Unfortunately, despiteassay of single intense band, with occasional,much lighter more than I00 piciform specimens(Table I), we did surrounding bands observableupon heavy staining not detect any heterozygotes.This may not be sur- (Fig. 1). prising, however, because S-MDH is essentially In both the ( pubescens) monomorphic in most avian (Aquadro and and (Colapiesauratus), we assayed Avise 1982). 326 ShortCommunications [Auk, Vol. 104

T^BI•E1. Woodpeckersand possiblenonpasseriform allies assayedfor presencevs. absenceof the unique S-MDH isozymepattern.

Unique "woodpecker" S-MDH n Continent of collection zymogram? Piciformes Picidae (woodpeckers) Colaptesauratus • 3 North America Yes Dryocopuspileatus 1 North America Yes Melanerpescarolinus 34 North America Yes Melanerpeserythrocephalus 7 North America Yes Melanerpesformicivorus 11 North America Yes Picoidespubescens 21 North America Yes Picoides scalaris 5 North America Yes Picoides villosus 5 North America Yes Sphyrapicusvarius 2 North America Yes Melanerpescruentatus 1 South America Yes rivolii 1 South America Yes Veniliornisnigriceps 1 South America Yes Veniliornispasserinus 1 South America Yes Campetheraabingoni 1 Africa Yes Dendropicosfuscescens 1 Africa Yes Indicatoridae(honeyguides) minor 2 Africa Yes Capitonidae (barbers) Lybiustorquatus b 1 Africa Yes Trachyphonusvaillantii 3 Africa Yes leucomelas 1 Africa Yes Rhamphastidae(toucans) Andigenacucullata 1 South America Yes Pteroglossuscastanotis 1 South America Yes Ramphastosculminatus 2 South America Yes Ramphastostoco • 1 South America Yes Bucconidae(puffbirds) Chelidopteratenebrosa 2 South America NO Malacoptilasemicincta 1 South America No Monasanigrifrons 1 South America No Nonnularuficapilla 1 South America No Nystalusstriolatus 1 South America No Galbulidae (jacamars) Galbularuficauda 1 South America No Alcedinidae () Cerylealcyon 1 North America No Bucerotidae () Tockuserythrorhynchus 2 Africa No Tockusfiavirostris 1 Africa (rollers) Coracias caudata 1 Africa No Momotidae (motmots) Momotus momota • ! South America No Phoeniculidae (wood-hoopoes) Phoeniculuspurpureus 2 Africa No April 1987] Short Communications 327

T^nLE 1. Continued.

Unique "woodpecker" S-MDH Taxon n Continent of collection zymogram?

Upupidae (hoopoe) Upupaepops • 1 Africa No Coliiformes Coliidae (colies) striatus 3 Africa No indicus 1 Africa No

One specimenalso assayedby Kitto and Wilson (1966). Assayedby Kitto and Wilson (1966) but not in the present study.

On the other hand, from available data we cannot If true, the probability of secondaryloss of the pat- exclude the possibility that some "epigenetic" phe- tern presumablywould be low. nomenon, such as posttranslational protein modifi- Cladistic analysesof skeletal morphology (Simp- cation, might account for the multiplicity of S-MDH son and Cracraft 1981) and limb myology (Swier- bandsin woodpeckers.For example,subbands of al- czewski and Raikow 1981) of the Piciformes pro- cohol dehydrogenasein Drosophilaresult from the duced the phylogeny shown in Fig. 2B. The observed binding of a NAD-carbonyl complex to the protein distribution of the 3-band S-MDH pattern is sup- (Everseet al. 1971). Regardlessof its particular mo- portive of the Pici composedof Ramphastidae, lecular basis,the unique S-MDH zymogram pattern Capitonidae, Indicatoridae, and Picidae. Together should provide an informative phylogenetic marker. with resultsfrom DNA-DNA hybridizations(Sibley S-MDH and piciformphylogeny.--The distinctive, 3-band S-MDH zymogramwas present in all species of Picidae, Indicatoridae, Capitonidae, and Ramphas- T^nLE2. Avian taxa (orders and passeriformfami- tidae assayed,but not in the Bucconidaeor Galbuli- lies) previously assayedfor S-MDH isozyme pat- dae (Table 1; see examples in Fig. 1). The "wood- terns. No assayedrepresentatives of these groups pecker-like" S-MDH pattern was absent from all six exhibited the S-MDH pattern characteristic of families of Coraciiformes,and from the two repre- woodpeckers. sentativesof the Coliiformes (Table 1). Together with Tinamiformes a Passeriformes d taxa previously assayed(Table 2), S-MDH has now Rheiformesa Eurylaimidae• been surveyed in 26 of the approximately 27 living Apterygiformesa Pipridae• avian orders,and in 16 familiesof Passeriformes.Only Struthioniformes• Tyrannidaea in a subsetof the Piciformes--woodpeckers,honey- Gaviiformes a,b Pittidae a guides, barbets, and toucans--has the distinctive Podicipediformes• Hirundinidae a,b 3-band S-MDH pattern been observed. Sphenisciformes• Corvidaea,b One traditional and widely recognized classifica- Procellariiformes a Paridae b tion of Piciformes(by Peters 1948; see also Wetmore Pelicaniformes• Troglodytidaeb 1960) implies the phylogeny shown in Fig. 2A. It is Ciconiiformes• Muscicapidae-,b a,• Mimidae •,b incompatible with the observed taxonomic distribu- Falconiformes•,b Bombycillidaeb tion of the 3-band S-MDH pattern, provided this a,b Sturnidae -,• MDH pattern has not been lost secondarilyby the Gruiformes •,• Picathartidae • commonancestor of jacamarsand puffbirds.The like- Charadriiformes • Nectariniidae • lihood of such secondary loss is difficult to assess Columbiformes •,• Emberizidae •,b critically. Note that the 3-band S-MDH pattern must Psittaciformes • Passeridae •,• be very old, however, becauseit is present in four Cuculiformes •,• families and in both Old and New World Piciformes. Strigiformes•,• Sibleyand Ahlquist (1985,1986) estimated that some • of thesegroups diverged nearly 80 million yearsago. Apodiformes• The earliest known referable to the true Pici Trogoniformes• (as defined below) are about 50 million years old (S. • From Kitto and Wilson (1966), L. Olson pers. comm.).In any event, the retention of • From Aquadro and Arise (1982). • From Kuroda et al. (1982: fig. 3). the unusual S-MDH genotype over such time spans a Olson(1983) suggested that the closestliving relativesof the Pici suggeststhat it may have someadaptive significance. (as in Fig. 2B) may be found amongthe Passeriformes. 328 ShortCommunications [Auk,Vol. 104 and Ahlquist 1986), and further analyses of mor- cleotideswith substratesof pyridine nucleotide phology (Olson 1983),the monophyleticstatus of the linked dehydrogenases.Bioorgan. Chem. 1: 207- Pici now seems well established. Most of the further 233. debateabout piciform phylogeny concernswhether KARIG, L. M., & A. C. WILSON. 1971. Genetic varia- the Galbulaeand Pici are sistergroups (as arguedby tion in supernatantrealate dehydrogenaseof Raikow and Cracraft 1983), or whether each has clos- birds and reptiles. Biochem. Genet. 5: 211-221. estavian relatives elsewhere (as argued by Olson1983, KITTO, G. B., & A. C. WILSON. 1966. of and Sibley and Ahlquist 1986). realate dehydrogenasein birds. Science 153: Wiley (1981) noted that "one true synapomorphy 1408-1410. is enough to define a unique genealogicalrelation- KURODA, N., R. KAKIZAWA, H. HORI, Y. OSAKA, N. ship. The problem is that the synapomorphieswe USUDA, •r S. UTIDA. 1982. Evolution of mito- hypothesizemay or may not be true synapomor- chondrial realate dehydrogenasein birds. J. Ya- phies." Much of the debateabout piciform phylog- mashina Inst. Ornithol. 14: 1-15. eny has centered on whether a few particular mor- OLSON,S. 1983. Evidencefor a polyphyleticorigin phological features reflect shared ancestry or of the Piciformes. Auk 100: 126-133. convergence.Sibley and Ahlquist (1983) arguedthat PETERS,J. J. 1948. Check- of the world, quantitative DNA-DNA hybridization values indi- vol. 6. Cambridge,Massachusetts, Harvard Univ. catetrue cladisticrelationships because they are pro- Press. portionalto the relative timesof divergencebetween RAIKOW,R. J., & J. CRACRAFT.1983. Monophyly of taxa.They alsopredicted that qualitativemorpholog- the Piciformes:a reply to Olson. Auk 100: 134- ical or genetictraits, properly interpreted, "will prove 138. to be congruentwith the DNA evidencein all cases" SELANDER,R. K., M. H. SMITH, S. Y. YANG, W. E. (Sibley and Ahlquist 1986). In this instanceat least, JOHNSON, & J. B. GENTRY. 1971. Biochemical the derived S-MDH genotype(as well as certainpre- polymorphism and systematicsin the viously studiedmorphological traits) are indeed con- Peromyscus.I. Variation in the old-field mouse gruent with the DNA-hybridization data in predict- (Peromyscuspolionotus). Stud. Genet. VI, Univ. ing monophyletic status for a Pici assemblage Texas Publ. 7103: 49-90. composedof the toucans,barbets, honeyguides, and SIBLEY,C. G., & J. E. AHLQUIST.1983. Phylogeny and woodpeckers. classification of birds based on the data of DNA- We thank Michael Braun and Herb Dessauer of the DNA hybridization. Pp. 245-292 in Current or- Louisiana State University (LSU) Museum of Frozen nithology, vol. 1 (R. F. Johnston,Ed.). New York, Tissues,and the following collectorsof the South Plenum. Americanbirds: J. V. Reinsen,T. S. Schulenberg,D. ß & --. 1985. The relationshipsof some A. Weidenfield, S. M. Lanyon, and A. P. Capparella. groups of African birds, based on comparisons The LSU collectionswere made possibleby cooper- of the geneticmaterial, DNA. Pp. 115-161in Proc. ation of the Bolivian governmentand the Direcci•n Intern. Syrup.African Verts. (K.-L. Schuchmann, de Ciencia y Tecnologla,through Gast•n Bejerano, Ed.).Bonn, West Germany, Publ. A. KoenigMus. and the Academia Nacional de Ciencias, La Paz. We , & --. 1986. Reconstructingbird phy- thank Alan Kemp and staffof the TransvaalMuseum, logeny by comparingDNA's. Sci. Amer. 254: 82- Pretoria, South Africa. We also appreciate the efforts 93. of StorrsOlson, Phil Angie, Nancy Joste,Peter Stan- SIMPSON,S. F., & J. CRACRAFT.1981. The phyloge- gel, and Bobby Crawford, who provided specimens. netic relationshipsof the Piciformes( Aves). StorrsOlson made many helpful suggestionsfor the Auk 98: 481-494. manuscript.Work was supportedby NSF grantsBSR- SwIERCZEWSKI,E. V., & R. J. RA•KOW. 1981. Hind 8217291 and BSR-8603775. limb morphology,phylogeny, and classification of the Piciformes. Auk 98: 466-480.

LITERATURE CITED WETMORE, g. 1960. A classification for of the world. Smithsonian Misc. Coil. No. 139. AQUADRO,C. F., & J. C. AVISE. 1982. Evolutionary WHEAT, T. E., W. F. CHILDERS, E. T. MILLERß •r G. S. geneticsof birds. VI. A reexaminationof protein WHITT. 1971. Genetic and in vitro molecular divergenceusing varied electrophoreticcondi- hybridization of realate dehydrogenase iso- tions. Evolution 36: 1003-1019. zymesin interspecificbass (Micropterus) hybrids. BAILEY, G. S., A. C. WILSON, J. E. HALvER, & C. L. Anita. Blood Grps. Biochem. Genet. 2: 3-14. JOHNSON. 1970. Multiple forms of supernatant WILEY,E. D. 1981. Phylogenetics.New York, John realatedehydrogenase in salmonidfishes. J. Biol. Wiley and Sons. Chem. 245: 5927-5940. EVERSE,J., L. ZOLL, L. KAHAN, & N. KAPLAN. 1971. Received18 July 1986, accepted26 September1986. Addition products of diphosphopyridine nu-