Reassociation kinetic analysis of nuclear DNAs from Balearica pavonina (Black-crowned Crane) and Grus leucogeranus (Siberian Crane) shows that the nuclear genome of each contains at least 75% single-copy sequences. The kinetic data show the haploid genome of each species to be 1.0-1.5 pg. Neither DNA-DNA hybridization of single-copy nuclear sequences under conditions of reduced stringency nor albumin micro-complement fixation (MC'F) separated the three crane genera Grus, Anthropoides, or Bugeranus in our experiments. These three genera were separable from Balearica by both techniques. The DNA and MC'F results compare favorably with a parallel electrophoretic study. We conclude that Balearica and Grus separated between 10.0 and 6.2 MYBP (million years before present), as determined from MC'F and electrophoretic data, which is consistent with fossil evidence for cranes. Received 7 November 1988, accepted 3 May 1989.

Department of Zoology, Miami University, Oxford, Ohio 45056 USA, and 2Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802 USA THE CRANES (Aves: Gruidae) are a widely dis- tributed group of 15 species in four genera (Johnsgard 1983). Interest in the cranes has in- creased as many natural populations have be- come endangered. Recent systematic investi- gations examined the unison call as a taxonomic character (Archibald 1976a, b), a variety of ex- ternal morphological and skeletal characters (Wood 1979), and proteins using starch gel elec- trophoresis (Ingold et al. 1987a, b). Comparison of Wood's and Archibald's taxo- nomic schemes indicates that the Siberian Crane (Grus leucogeranus) clusters with the genus Bu- geranus and not with Grus. Wood (1979) found the Siberian Crane to be very similar to the Whooping Crane (G. americana) in external fea- tures but more similar to the Wattled Crane (Bugeranus carunculatus) in skeletal characters. In contrast, the Siberian Crane is more similar electrophoretically to the Whooping Crane than it is to the Wattled Crane (Ingold et al. 1987a). Wood (1979) attributed the similarity between the Siberian and Whooping cranes to conver- gence that resulted from selection in similar $ Present address: Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17604 USA. 595 ecological niches. The proper taxonomic posi- tion of the Siberian Crane is of intense interest (Archibald pers. comm.) because it and the Whooping Crane are endangered in the wild. Cross-fostering and possible hybridization be- tween species make information on relation- ships important. There has also been controversy regarding composition of the genus Balearica (crowned cranes). Some investigators (White 1965, Snow 1978) have suggested that Balearica represents one species with four well-defined subspecies, whereas Walkinshaw (1964) maintained that there are two species, each of which consists of two well-defined subspecies. The studies of Ar- chibald (1976a), Wood (1979), and Ingold et al. (1987b) provide strong support for Walkin- shaw's (1964) contention that two species of Bal- earica are distinct and not subspecies. The study of this family offers an exceptional opportunity for the comparison and evaluation of various molecular techniques commonly uti- lized in studying phylogenetic relationships. We present data from DNA-DNA hybridization and albumin micro-complement fixation (MC'F) to measure genetic divergence among 14 species of cranes and compare our data with results derived by electrophoretic techniques (Ingold et al. 1987a). We find that all three of these diverse experimental approaches produce con- gruent patterns of evolutionary relationships. METHODS Materials.--We studied 9 species of Grus, 1 species of Bugeranus, and 2 species each of Balearica and An- thropoides (Appendix). Most were captive individuals housed at the International Crane Foundation, Bar- aboo, Wisconsin; the specimens of G. americana, how- ever, were from the captive flock at the U.S. Fish and Wildlife Patuxent Research Center, Laurel, Maryland. House Sparrows (Passer domesticus) and Common Yel- lowthroats (Geothlypis trichas), used as outgroups for the DNA-DNA hybridization and MC'F, respectively, were collected near Oxford, Butler County, Ohio. DNA isolation.--Blood was collected from two in- dividuals in each of 15 species (Table 1). High mo- lecular weight DNA was obtained from the combined erythrocyte nuclei of both individuals using a phenol extraction procedure (Vaughn et al. 1982), which in- cluded RNAase and pronase digestions. The purified DNA was sonicated to obtain short fragments, which were sized by agarose gel electrophoresis using pBR322 DNA cut with HaeIII as a standard (Southern 1979). This procedure yielded fragments averaging 380 nu- cleotide pairs in length (range: 250-500 nucleotide pairs). Sample purity was determined by the 260/230 absorption ratio. A sample with a ratio that is greater than or equal to 2/1 is considered to be pure. The purity of our samples ranged from 2.16/1 to 2.45/1. DNA reassociation kinetics.--Kinetics of reassociation were determined for genomic DNA of the two species to be used as probes, B. pavonina and G. leucogeranus, utilizing standard techniques (Britten et al. 1974, Laird and McCarthy 1969). We have previously described in detail our modifications of these methods, and our second-order reaction kinetic calculation procedures (Vaughn 1975). Balearica and Grus were selected be- cause they represent two different subfamilies of cranes (Brodkorb 1967); G. leucogeranus was used in anticipation of clarifying its questionable phyloge- netic position within the family. DNA labeling and preparation of single-copy compo- nent.--We labeled sonicated total nuclear DNA of B. pavonina and G. leucogeranus with 3H-dCTP (23 Ci/ mmole, 1 mCi/ml; New England Nuclear) by the nick translation technique (Rigby et al. 1977) to a specific activity of 1,200 cpm/ng and 630 cpm/ng, respec- tively, as determined by liquid scintillation counting in Scinti-verse II cocktail (Fisher). Single-copy nucle- ar DNA probes for the two species were isolated by denaturing and renaturing the labeled DNAs of each species to a Cot of 200. Prior kinetic analysis showed this to be sufficient to permit renaturation of virtually all repeated sequences but very little of the single- copy component (Fig. 1). Because the labeled DNAs were in small quantity, DNA fractionation was car- ried out on 0.3 g hydroxylapatite (HAP, Bio-Rad) col- umns which had previously been treated with 100 of sonicated, denatured salmon sperm DNA to block all irreversible DNA binding sites. Single-stranded single-copy DNAs were eluted with 0.12 M sodium phosphate buffer (PB) at 60øC, after a 0.03 M PB wash step. The single-copy probes were examined for pos- sible contamination with repetitive or snap-back se- quences (Galau et al. 1976) and for their ability to form stable duplexes, by hybridization of probes to unlabeled homoduplex nuclear DNAs in excess (driv- er DNA) at Cot values of ca. 50-5,000 at a driver/probe ratio of about 1000:1. Hybridization and DNA fractionation by thermal hydroxylapatite chromatography.--Homoduplex and heteroduplex hybrids were formed by mixing labeled and unlabeled DNA at a driver/probe ratio of 1,000: 1. Two-hybridization reactions were performed for each species pair comparison (30 comparisons). We were limited to small sample sizes because of the small amounts of DNA available from these endan- gered birds. Reaction mixtures were sealed in glass capillary tubes, denatured for 3 min in a boiling water bath, incubated in 0.50 M PB at 55øC to a driver Cot of 5,000 and frozen quickly at -20øC until samples could be fractionated on HAP columns. The 55øC in- cubation temperature represents a condition of low- ered stringency and was used to detect the more di- vergent cross-reactive DNA sequences expected to be present in heteroduplex reactions (Rice 1972), and permitted comparison of a greater percentage of each genome. Samples were adjusted to 0.03 M PB prior to loading DNAs onto water-jacketed columns equil- ibrated to 0.03 M PB and 55øC. The 0.3 g HAP bed was determined previously to be more than sufficient to bind all the DNA contained in each reaction tube. Very short DNA fragments incapable of binding to HAP were eluted with 0.03 M PB; the column was equilibrated to 0.12 M PB prior to thermal elution. Column temperature was raised in 5øC increments to a final temperature of 95øC. Once each new tem- perature was reached, the column was allowed to equilibrate for 2 min before the fractions were eluted. Column temperature was monitored with a therm- istor probe (Bailey) rated to be linearly sensitive to the nearest 0.1øC. At each temperature, two 2.5-ml fractions were collected manually by elution with 0.12 M PB. After the 95øC elution step, the column was washed with three 1.2-ml fractions of 0.50 M PB to remove any remaining double-stranded DNA. Each fraction was mixed with 15 ml of scintillation cocktail (Scinti-verse II, Fisher) and counted in a liquid scin- tillation counter to an error of < 10%. We determined that, under these conditions, quenching was propor- tional for all samples. DNA data analysis.--Hybridization data were ana- lyzed by the T5oH statistic (Kohne 1970, Bonner et al. 1981). T50H and Tm values were obtained by plotting the cumulative counts per minute eluted vs. temper- ature on normal probability paper (Knittel et al. 1968) and fitting a linear regression line to the data points that made up the major DNA component. This tech- O nique accommodates those DNA sequences that have diverged to such an extent that they no longer hy- bridize between distantly related species (Sibley and 25 Ahlquist 1983), and the results are expected to be more nearly linear with true genealogical distance constructions than analysis by ATto values. A ToH values were corrected by averaging the re- ciprocal heterologous hybridization values obtained x 75 between Balearica pavonina and Grus leucogeranus. When B. pavonina was the labeled taxon, the uncorrected A TsoH was 5.0, and when G. leucogeranus was the labeled o 1OO taxon, the uncorrected A ToH was 4.5; the corrected A ToH was 4.8. The remaining values were corrected as follows: When B. pavonina is the labeled taxon, 4.8/ 5.0 = 0.96. The uncorrected A T0H for B. pavonina vs. G. americana is 4.7. We calculated the corrected A T0H value as 4.7 x 0.96 = 4.5. All other A T50H values derived from the hybridization of labeled B. pavonina DNA were similarly adjusted. When G. leucogeranus is the labeled taxon, 4.8/4.5 = 1.07, so all A Ts0H values derived from labeled G. leucogeranus DNA were cor- rected by multiplying those values by 1.07. Micro-complement fixation.--Antisera were prepared to serum albumin purified from 1 ml each of plasma 1OO from Bugeranus carunculatus from Africa and Grus leu- cogeranus from Asia. Each antiserum was made in two 10-1 female New Zealand white rabbits by established procedures (Maxson and Szymura 1979) and individ- ual rabbit antisera were pooled in inverse proportion Fig. 1. to their MC'F titers. A total of 4-8 mg of albumin was administered to each rabbit over the 13-week im- munization period. All rabbit antisera were directed primarily against albumin as evidenced by immu- noelectrophoresis with whole plasma (Prager et al. 1974). Additional MC'F tests with pure albumin and whole plasma gave identical results. The titers and slopes of the pooled antisera were 5,000 and 400 in both cases. Micro-complement fixation analyses were per- formed at standard conditions (Champion et al. 1974, Maxson et al. 1979) and reported as immunological distance units (IDU). For albumin, it has been esti- mated that 1 unit of ID is roughly equivalent to 1 amino acid difference between the albumins com- pared (Maxson and Wilson 1974, Maxson and Maxson 1986). Sequence evolution in albumin can be trans- lated into a divergence time for when the lineages compared diverged. In birds, 10 IDU stochastically accumulate every 16-17 million years (Prager et al. 1974, Prager and Wilson 1975) of lineage indepen- dence. RESULTS DNA reassociation kinetics of nuclear DNAs.-- DNA reassociation curves for various avian 10 -1 10 ¸ 101 10 2 10 3 10 4 0  25  5o 75 I I I I 10 ¸ 101 10 2 10 3 10 4 Equivalent Cot, mole-$ec / liter Genomic renaturation kinetic curves (--O--) for the cranes B. pavonina (Top) and G. leu- cogeranus (Bottom). The upper curve in each panel (--O--) shows the results of hybridization of the la- beled single-copy probe to the driver genomic DNA. The Cot 1/2 for the DNA component is indicated by a vertical bar for each curve. Each curve is the cal- culated theoretical second-order reaction kinetic plot that best fits the data points. species (Shields and Straus 1975, Epplen et al. 1978, Burr and Schmike 1980, Wagenmann et al. 1981) reveal three more-or-less distinct ki- netic components that correspond to the highly repetitive, moderately repetitive, and single- copy DNA fractions. The moderately repetitive component, which was not critical in our study, is usually difficult to resolve clearly from the single-copy component. These three compo- nents were not all clearly distinguishable in reassociation curves for crane DNAs (Fig. 1), and we suspect that an unresolved, moderately repetitive component is present. This fraction would not, however, be expected to have con- taminated the single-copy fraction used as a probe since genomic DNAs were reassociated TABLE 1. Thermal stability (øC) of hybrids formed between 3H-single-copy DNA and total genomic DNAs and average single-copy sequence divergence in cranes. AT T5oH AT&oH AT T5oH ATsoH 3H-single-copy Balearica pavonina vs. Balearica pavonina 0.0 79.7 0.0 B. regulorum 2.3 77.4 2.3 Grus americana 4.7 75.0 4.7 G. leucogeranus 4.2 74.7 4.8 G. grus 4.4 74.2 5.3 G. canadensis 5.7 74.0 5.5 G. japonensis 6.0 73.7 5.8 G. monacha 4.1 73.3 6.1 G. antigone 6.7 73.0 6.4 Anthropoides paradisea 6.0 72.9 6.5 A. virgo 5.3 72.8 6.6 Bugeranus carunculatus 5.9 72.7 6.7 Grus rubicunda 5.5 72.5 6.9 G. vipio 5.5 72.3 7.1 Passer domesticus 20.1 36.5 41.5 H-single-copy Grus leucogeranus vs. Grus leucogeranus 0.0 78.4 0.0 G. americana 0.8 77.6 0.9 G. grus 2.3 76.1 2.5 G. canadensis 2.5 75.9 2.7 G. japonensis 2.9 75.5 3.1 Anthropoides virgo 2.6 75.3 3.3 Grus rubicunda 2.7 75.2 3.4 G. vipio 2.7 75.1 3.5 G. monacha 3.3 74.9 3.7 Bugeranus carunculatus 3.4 74.9 3.7 Anthropoides paradisea 2.5 74.5 4.2 Grus antigone 4.1 74.3 4.4 Balearica pavonina 3.7 73.9 4.8 B. regulorum 4.8 72.1 6.7 Passer domesticus 16.8 41.1 39.8 to Cot 200, which is sufficient to renature any presumptive moderately repetitive component. Britten and Kohne (1968) state that the central two-thirds of a Cot curve should be a straight line. The slope of this line can be evaluated by determining the ratio of the values of Cot at the ends of the straight line, with the ratio being ca. 100. The two Cot curves (Fig. 1) show this approximation. The test for purity of the iso- lated single-copy probes (Fig. 1) affords confi- dence that our probe contained primarily sin- gle-copy DNA. The Cot 1/2 is virtually identical to that of the presumptive single-copy com- ponent within the genomic DNA of each species. Both crane species contain up to 75% single- copy DNA, which is sharply resolved from the other kinetic components and comparable to other avian species (Epplen et al. 1978). The haploid genome size is determined to be ca. 1.0-1.5 pg for both crane species examined, based on the observed Cot 1/2 values of ca. 1 x 103 for their single-copy component (Laird 1971, Crain et al. 1976). This estimate is only approx- imate because an internal standard was not used and it is not possible to make meaningful com- parisons of this type between different labora- tories. The small difference in Cot 1/2 between the two species is not significant. Our estimates of genomic size values are comparable to the 1.4-1.7 pg values determined for other cranes lOC 75 c 5c 25 (B)/ 60 70 80 90 100 60 Tempenatune (øC) Y=-227.0. X + 3.6& R 2= 0.97 o 50 70 80 go :99 ;)5 BO 5O 2O 00 Fig. 2. A representative thermal elution curve for the heteroduplex G. leucogeranus and A. virgo reaction: (A) traditiona! curve and (B) regression line for major components plotted on normal probabi!ity paper. (Rasch and Kurtin 1975, Biederman et al. 1982) and other birds (Shields and Straus 1975, Ep- plen et al. 1978, Wagenmann et al. 1981). Hybridization of single-copy DNAs with nuclear DNAs.--The hybridization and thermal stabil- ity determinations are expressed as Ts0H and Z Ts0H (Table 1). A representative thermal elution curve, for the hybrid pair G. leucogeranus/A. vir- go, is presented in Fig. 2. Within the family, the /X Ts0H values range from 0.9øC to 7.0øC. With Grus as the reference, the two Balearica species are most divergent (œ = 5.8), and G. americana is the most similar (0.9). When Balearica is used as a reference, the other three genera are at a mean distance of 6.0 and the two Balearica species have a/X T0H of 2.3øC. Micro-complement fixation.--Albumin immu- nological distance values for cranes are pre- sented in Table 2. MC'F curves for the recipro- cal homologous comparisons are presented in Fig. 3. In both experiments the homologous antiserum concentration was used for each an- tigen. The difference in fixation observed with the Bugeranus antiserum reflects an immuno- TABLE 2. Albumin immunological distances be- tween Grus leucogeranus, Bugeranus carunculatus and the other 12 species of cranes and Geothlypis trichas. ID to G. ID to B. leuco- carun- Species compared gernaus culatus Grus grus 0 2 G. monacha 0 2 G. canadensis 1 0 G. japonensis 1 2 G. americana 0 2 G. vipio 0 3 G. antigone 1 6 G. rubicunda 0 2 G. leucogeranus 0 4 Bugeranus carunculatus 0 0 Anthropoides paradisea 0 2 A. virgo 0 5 Balearica pavonina 9 10 B. regulorum 11 10 Geothlyphis trichas 76 76 logical distance of 4 units. The reciprocal dis- tance of 0 is evidenced by identical peak heights obtained with the Grus antiserum. The values for comparisons between the species of Grus, 100- .Bugeranus ,6rus // \ ' 2'5 ' 160 'tOO B__ugeranus o6rus ' ' 100 'z,60 Albumin (rig) Fig. 3. Micro-complement fixation curves obtained with antiserum to B. carunculatus albumin homologous reaction; --O--, heterologous reaction) and antiserum to albumin of G. leucogeranus homologous reaction; --O--, heterologous reaction). In all four curves, the antiserum dilution was 1:5000 of the antiserum pool. The concentration of albumin was estimated from average concentrations of albumin in avian plasma. Using Bugeranus albumin antiserum and Grus albumin as antigen, there is 15% less complement fixation than in the homologous reaction. This corresponds to an immunological distance (ID) of 4 units. Using antiserum to to Grus, the same amount of complement fixation is obtained with Bugeranus albumin as with the homologous albumin, indicating a distance of 0 ID. Thus the average reciprocal distance is 2 IDU. Anthropoides, and Bugeranus range between 0 and 6 immunological distance units (IDU), whereas the two species of Balearica are an average of 10 IDU from the other species. DISCUSSION Published DNA-DNA hybridization studies for nonpasserine birds are becoming more com- mon (Sibley and Ahlquist 1983, 1985; Sibley et al. 1988; Sheldon 1987a, b; Madsen et al. 1988). DNA-DNA hybridization data for cranes are limited [but see Krajewski, this issue--ed.]. Sib- ley and Ahlquist (1985) obtained a A T50H be- tween Grus and Anthropoides of only 0.7. Because such low values are subject to a relatively high percentage of error, we chose to use conditions of lower stringency to detect the more diver- gent cross-reactive sequences. This is a useful alternative because birds have been shown to have a slower rate of protein evolution (Prager et al. 1974) and, as a result, have a high potential for interspecific hybridization (Prager and Wil- son 1975). The lower stringency gives greater A T50H values. Micro-complement fixation data for other nonpasserine serum albumins are similar to our crane data (Prager et al. 1974; Prager and Wilson 1975, 1976). In their comparisons of ducks in the genus Anas, Prager and Wilson (1975) ob- tained serum albumin IDUs of 0.0 and 2.0 while intergeneric values within the order ranged from 8 to 13. Intra-ordinal serum albumin IDUs >13 have been reported for the Galliformes (Prager and Wilson 1975). When nonpasserine birds are compared with passetines, serum al- bumin IDUs typically range from 42 to 93 (? = 68; Prager and Wilson 1976); an average value of 76 IDU was obtained in a comparison of the cranes to Geothylpis trichas (Parulinae). When the DNA-DNA hybridization (Table I) and MC'F data sets (Table 2) are compared with the electrophoretic data (Ingold et al. 1987a), Grus, Anthropoides, and Bugeranus show very lit- tle genetic differentiation whereas the genus Balearica is relatively distinct. The close rela- tionship among Grus, Anthropoides, and Buge- ranus is more obvious when we compare their genetic distances (Nei's [1978] D ranged 0.00- 0.12) to those of other nonpasserines. Barrow- clough et al. (1981) obtained genetic distance values of 0.11-0.61 in 7 species (each in a dif- ferent genus) in the family Procellariidae. In- gold et al. (1984) obtained values of 0.39 and 0.53 when comparing Zenaida macroura (Mourn- ing Dove) with Columba livia (Rock Dove) and C. f. fasciata (Band-tailed Pigeon), respectively. Behavioral (Archibald 1976a, b) and morpho- logical (Wood 1979) data on evolutionary re- lationships within the cranes differ somewhat from the biochemical data presented here. The most consistent relationship from all data sets is the separation of Balearica from the other three genera. Balearica is usually placed in the subfamily Balearicinae while the remaining genera are included in the subfamily Gruinae (Brodkorb 1967). The DNA-DNA hybridization data (Table I) are consistent with the idea that there are two distinct species of crowned cranes. It is not possible, however, to separate Bugeranus and Anthropoides from Grus using the present biochemical data. Both Archibald (1976a, b) and Wood (1979) suggest that the Siberian Crane should be con- sidered congeneric with Bugeranus and placed in that genus. DNA-DNA hybridization and electrophoretic data show the Siberian Crane is most closely related to the Whooping Crane. These two species are very similar in their ex- ternal features, breed in similar habitats, and have breeding ranges separated only by the Be- ring Strait (Johnsgard 1983). Archibald (1976a, b) showed that these two species have very dis- similar unison calls and therefore are not closely related. We believe, however, that the unison call, which is part of the courtship display, is not a good character for determining relation- ships. Based on the genetic distance, the Sibe- rian and Whooping cranes most likely split very recently, and one would expect their courtship behaviors to diverge rapidly if we are to accept the function of pre-mating reproductive isolat- ing mechanisms. We believe that Bugeranus and Anthropoides might be considered congeneric with Grus. Sib- ley and Ahlquist (1982) proposed that birds are oversplit at the supraspecific level, which may be the case for the cranes. Systematic consid- erations based on DNA-DNA hybridization will require larger sample sizes and a more complete study. Even though we lack a complete matrix of DNA-DNA hybridization values, we feel that we can comment on relationships among the above species as all three data sets give similar results. Reciprocal pairwise comparisons were made only between two species and no out- group was used as a probe. Therefore, we have no information on the extent to which A T5oH values within subfamilies truly represent ge- netic distance or are instead artifacts of differ- ences in rates of evolution, genome size, sample purity, fragment length, or conditions between experimental trials. Crane systematics may be further enhanced by analysis of mitochondrial DNA which has been shown to evolve 5-10 times faster than nuclear DNA (Wilson et al. 1985, Avise 1986). ACKNOWLEDGMENTS We thank G. Archibald and the staff of the Inter- national Crane Foundation and J. Carpenter and S. Derrickson of the Patuxent Wildlife Research Center for providing us with crane blood. Neal Mundahl drew the figures. National Science Foundation grant BSR-8303966 and a Steenbock Award from the Wis- consin Society for Ornithology to Ingold supported the DNA laboratory work. Micro-complement fixa- tion analyses were partially supported by National Science Foundation grant BSR-8319969 and the Uni- versity of Illinois Department of Genetics and De- velopment to Maxson. We thank P. Houde and an anonymous referee for helpful comments on the manuscript. LITERATURE CITED ARCHIBALD, G. W. 1976a. The unison call of cranes as a useful taxonomic toolß Ph.D. dissertation. Ith- aca, New York, Cornell Univ. ß 1976b. Crane taxonomy as revealed by the unison callß Proc. Int. Crane Workshop 1: 225- 251. AVlSE, J. C. 1986. 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Scientific name Common name Grus grus Common Crane G. monacha Hooded Crane G. canadensls Sandhill Crane G. japonensis Red-crowned Crane G. americana Whooping Crane G. vipio White-naped Crane G. antigone Sarus Crane G. rubicunda Brolga G. leucogeranus Siberian Crane Bugeranus carunculatus Wattled Crane Anthropoides virgo Demoiselle Crane A. paradlsea Stanley Crane Baleatica pavonina Black-crowned Crane B. regulorum Gray Crowned Crane Geothlypis trichas Common Yellowthroat Passer domeshcus House Sparrow a Common names for cranes are those used by the International Crane Foundation (Archibald pers. comm.).