PHYLOGENETIC RELATIONSHIPS AMONG CRANES (GRUIFORMES: GRUIDAE) BASED ON DNA HYBRIDIZATION CAREY KRAJEWSKI• ZoologicalMuseum, Department of Zoology,University of Wisconsin,Madison, Wisconsin 53706 USA ABSTRACr.--Iused a DNA-DNA hybridizationmethod to generatemore than 1,200pairwise comparisonsamong speciesof the family Gruidae (cranes)and an outgroupLimpkin (Ara- midae). A matrix of genetic distancesincluded averagedelta Tmvalues for all cells, and reciprocalsamong cranes,based on 3-10 replicateexperiments per cell. I chosedelta T• as the appropriate dissimilarity measure becausevirtually all homologoussingle-copy DNA sequencesin cranegenomes were sufficientlysimilar to form hybrid duplexesunder standard experimental conditions. The normalized percent hybridization (NPH) values approached 100 for all crane speciespairs. The delta Tmdata departed slightly from metricity as a result of experimentalvariation associatedwith the measurementof small geneticdistances. The DNA data, analyzed with a best-fit tree approachand checkedfor internal consistencyby jackknifing,support the traditional view that crowned cranes(Balearica) are the mostancient lineageof extantgruids. The enigmaticSiberian Crane (Grus leucogeranus) appears as the sister group of the remaining species,which fall into four closelyrelated groups.Bugeranus and Anthropoidesare sistergroups. The three speciesof AustralasianGrus (antigone, rubicunda, and vipio)form a clade, as do five predominantly PalearcticGrus (grus, monachus, nigricollis, japo- nensis,and americana).The SandhillCrane (G. canadensis),while clearlya memberof the gruine clade, is an old lineage without close relatives. Received21 March 1989, accepted18 May 1989. THE CLASSIFICATIONSof Peters (1934) and Wet- terials that cranes diverged from a common more (1934) establishedthe traditional family- ancestorwith limpkins (Aramidae) in the late level distinction of cranes (Gruidae) within the Paleocene. Order Gruiformes. This arrangement, though Archibald (1975, 1976) performed the first not universally accepted (Cracraft 1973), has comprehensiveanalysis of cranes based on a persistedin mostrecent revisions(e.g. Wetmore coherent set of characters.His study of crane 1960, Storer 1971, Sibley et al. 1988). Opinions unison calls (stereotypedbehavior patterns in- regarding speciesaffinities within the Gruidae volved in pair-bonding) led him to identify have varied greatly. Following Peters (1934), clusters of similar species(Fig. 1). Archibald's most workers have acceptedthe existenceof 14 work verified the distinctness of crowned cranes extant speciesin four genera, though Walkin- (Balearica)and suggestedan unexpected rela- shaw's(1964) designationof a 15th specieshas tionship between Bugeranuscarunculatus (Wat- gained many adherents. The crowned cranes tled Crane) and Grus leucogeranus(Siberian (Baleatica)are commonly placed in the Balear- Crane). Archibald included leucogeranusas a icinae, apart from the remaining species(Gru- member of Bugeranus, and recommended inae; Brodkorb 1967) based on their lack of ster- "SpeciesGroup" statusfor the remaining Grus hal excavation and tracheal convolution. and Anthropoidesclusters. Wood (1979), like Ar- Balearicines,apparently the more ancient lin- chibald, found a high level of similarity be- eage, are abundantly represented by Tertiary tween Wattled and Siberian cranes, which, aside fossil materials from western Eurasia (Brodkorb from complicated multivariate resemblances, 1967). Gruine fossilsappear in the Miocene of lack the exaggerated tracheal convolutions Europe, but are best represented in North present in other gruines. American Pliocenedeposits and by a scattering Ingold et al. (1987)attempted to resolvecrane of Pleistoceneremains worldwide (Johnsgard relationships with allele-frequency analysis. 1983). Cracraft (1973) inferred from these ma- Their speciesarrangement was a major depar- ture from traditional views, although Balearica Current address:Museum SupportCenter, Smith- appearsdistinct from gruines.Their work in- sonian Institution, Washington, DC 20560 USA. volved small sample sizes, and only approxi- 603 The Auk 106: 603-618. October 1989 604 C^R•¾KRAJEWSI<I [Auk,VoL 106 G. grus tions are given in Krajewski(1988) and Springer and G. nigricollis Kirsch (1989). DNA was recovered from erythro- cyteslysed with sodiumlauryl sulfatein TNE buffer, G. monachus purified by repeatedphenol/chloroform extractions, G. japonensis and treated with proteaseand RNase (Maniatis et al. G. americana 1982).Native DNA was fragmentedinto short strands G. canadensis (100-2,000 bp) using high-frequency sound. Frag- ment size distributions were monitored for each sam- G, vipio pie by agarosegel electrophoresisand comparison G. ontigone with commercial size markers. G. rubicunda Single-copy sequences(scDNA) were recovered from eachspecies using the reassociation-kineticand G. [eucogeronus hydroxyapatitecolumn-chromatography methods of Bugeranus Kohne and Britten (1971). Sheared DNA was boiled A. virgo and incubatedat 60øCin 0.48 M phosphatebuffer (PB) A. paradiseo to a Cotof 200, diluted to 0.12 M PB, applied to hy- droxyapatite(HAP) columnsat 60øC,and single-copy Baleor/co sequenceseluted in 20 ml of 0.12M PB.Samples were Fig. 1. Crane relationshipsas indicatedby overall dialyzed for 24-48 h, then frozen and lyophilized. similarity in their unison calls (Archibald 1975;fig- The methodemployed to produceradioactive tracer ure redrawn from Wood 1979). The horizontal axis is DNA was derived from the general iodination pro- arbitrary, showing only Archibald's view of nested tocols of Commorford (1971), Davis (1973), Tereba similarity levels. and McCarthy (1973), Orosz and Wettour (1974), A1- tenberg et al. (1975), Scherbergand Refetoff (1975), mately two birds per specieswere assayedfor Chan et al. (1976),Anderson and Folk (1976),Prensky allele-frequencies. (1976),and Sibleyand Ahlquist (1981).Modifications of these proceduresare noted below. Basedon a very limited DNA hybridization Lyophilized,single-copy DNAs were rehydratedin study of the cranes, Ingold (1984) concluded a small volume (25-100 •1) of 0.2 M NaAc (pH 5.7), that substantial genetic divergence exists be- transferred to a 1.5 ml microfuge tube, vortexed for tween putative Balearicaspecies, and that G. leu- 30 s, and centrifuged at 3,000 g for 30 s. Reaction cogeranuswas not particularly "close" to Buge- mixtures were prepared by combining 100 •g of ranus,contra Archibald (1975, 1976) and Wood scDNA with enough 0.2 M NaAc (pH 5.7) to make a (1979). The latter conclusion seems difficult to solution at 0.77 •g/•l, as well as 5 •1 of 0.002 M KI defend in light of Cracraft's (1987) criticisms and 10.4•1 bromcresolgreen dye (BCG),in 1 ml stop- regarding the inadequaciesof partial genetic peredsepturn vials. Samples were adjustedto pH 4.7- distancedata for resolving relationships. 4.8 with 0.2 M NaAc at pH 4.0. Iodinationswere usually carried out for a set of I derive a phylogeny for the cranesbased on eight DNA samples.The isotope(•25I, Amersham IMS- a complete matrix of pairwise DNA hybridiza- 300) was obtained in 5 mCi amounts (ca. 10 •1), diluted tion comparisons.Previous avian work with this with 340 •1 of 0.2 M NaAc and 10 •1 of 0.001 M KI, technique (see referencesin Sibley et al. 1987) and allowed to equilibrate for 1 h. Eachtracer sample has focused on high-level taxonomy, though received 40 •1 of isotope solution (0.625 mCi), fol- Sheldon (1987a) used it to resolve intrafamilial lowed by 60 •1 of 0.0018 M thallium (III) chloride relationshipsamong herons (Ardeidae). Crane (T1C13).Samples were incubated at 60øCfor 15 rain, relationshipsare predominantly those of con- then cooled in an ice bath for 5 min. The pH of the genericspecies, and previouswork hasleft some reaction mixtures was raised above neutrality by ad- doubt as to the resolving power of DNA hy- dition of 30 •1 1.0 M Tris, sampleswere heated again at 60øC for 10 rain, and chilled on ice for 5 min. After bridization at this level. ! will show that the cooling, sampleswere dialyzed against4 1 of "iodin- method provides limited resolution among ation buffer" (Sibley and Ahlquist 1981). cranes, yet, with numerous replicate compari- DNA hybridswere preparedwith 0.5 •g tracer DNA sons,it is capable of defining clustersof phy- and 250 •g sheared,native DNA ("driver") in 0.48 M logenetically related species. PB. Hybrids were boiled and incubated at 60øC to a Cotof at least5,600, diluted to 0.12 M PB,and applied METHODS to HAP columns in an automated Thermal EIution Device. Column temperatures were raised to 60øCand DNA hybridization.--Thebiochemical protocol for three 8-ml fractions of 0.12 M PB were collected. Ad- DNA hybridization was essentiallythat describedby ditional fractions were collected at 2øC intervals from Sibley and Ahlquist (1981, 1983). Detailed descrip- 64øC to 94øC, and a final fraction was collected at 98øC. October1989] CranePhylogeny 605 Elution fractionswere assayedfor radioactivityin a PackardAuto-Gamma 5000 Seriesgamma counter. Two tracer DNA preparations were employed for every crane species,with replicate interspecieshy- brids arranged in experimental sets of 25. Each ex- •6f Gc perimental set was defined by one or two reference ("homologous")hybrids (i.e. tracer and driver DNAs from the same extraction of the same individual bird), with which all interspecies("heterologous") hybrids were compared.Three to 10 replicate heterologous hybrids were examinedfor each pairwise combina- tion of species(reciprocals treated separately). The