Cooke, F.

Quinn, T.W.

White, B.N.

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January 1990] Short Communications 199 Molecular Sexing of Geese Using a Cloned Z Chromosomal Sequence with Homology to the W Chromosome THOMAS W. QUINN? 2 FRED COOKE, • AND BRADLEY N, WHITE I :Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada At present, sexing of monomorphic birds and of the young of many dimorphic species is limited to surgical techniques (such as endoscopy), to chromo- somal analyses, or to cloacal eversion. Surgery carries risks associated with high stress levels, infection, and anesthesia; and, in most cases, it can be performed only on mature birds and by trained personnel (Prus and Schmutz 1987). Chomosomal analysis is tedious and requires identification of the Z and W chromo- somes (females are Z/W and males Z/Z) against a background of (often numerous) microchromosomes, as well as the establishment of cell cultures. This can be difficult with samples taken under field conditions. While karyotyping has been used to sex Wt•ooping Cranes (Grus americana; Biederman et al. 1982), it is not a very practical technique for a larger number of birds, and the success rate has been shown to be rel- atively poor in Psittacines (Prus and Schmutz 1987). Cloacal sexing is reliable, but it is generally limited in its applicability to waterfowl. We report the isolation of a DNA segment of the Snow Goose (Chen caerulescens) Z chromosome, a seg- ment that shows hornology to the W chromosome and that allows a new approach to sex determination us- ing DNA extracted from small blood samples. Blood is a very convenient tissue to sample and transport, and it consistantly ields high molecular weight DNA, even after storage at room temperature for more than one week (Quinn and White unpubl.). To our knowl- edge, this is the first report of the cloning of a unique segment of the avian sex chromosome. Related mo- lecular studies have concentrated on repetitive se- quences on the avian W chromosome (Tone et al. 1984, Kodama et al. 1987) or 6n sex-specific fragments de- tected with M13 DNA (Longmire et al. 1988). We have described the construction of a genomic library and isolation of DNA probes from the Lesser Snow Goose (Chen caerulescens caerulescens) (Quinn and White 1987) and applied such probes to pedigree anal- yses (Quinn et al. 1987, Quinn et al. 1989). One of these probes (DQSG10, 14 kb) is the focus of this study. Blood samples (1-5 ml) were taken from 119 live birds captured at La Perouse Bay near Churchill, Man- itoba (58ø24'N, 94ø24'W). We extracted DNA follow- ing the procedure of Quinn and White (1987). The sex of each bird was determined by cloacal eversion 2 Present address: Department of Biochemistry, University of California, Berkeley, California 94720 USA. (Taber 1971) at the time of blood sample collection. Samples of DNA were digested with the restriction endonuclease Taql, electrophoresed, and blotted as described (Quinn and White 1987). The autoradio- graph resulting from the hybridization of DQSG10 to a blot which included 5 male and 7 female DNA samples is given as Fig. 1. The surprising feature of this and other autoradiographs produced using this probe was a weak 2.1 kb band present in the female samples but absent in the male samples. In total, 55/57 adult females had this band, while all 26 adult males lacked it. Thus, a 98% concordance was found between the field sexing and the presence/absence of the band in female/male adult birds. We tested an additional 36 juveniles and found a concordance of 92%. Although not •erified by dissection, the most likely explanation for the discrepency between clo- acal and "molecular" sexing arose from recording or cloacal sexing errors in the field. Banded adults re- captured (and resexed) from one year to the next at La Perouse Bay provided a rough estimate of that error rate. Overall, the recorded sex differed between the first capture and the second capture in 268 of 5,352 (5%) of those cases recorded between 1968 and 1988. There was no evidence of any allelic counterparts to the 2.1 kb band as would be expected if this were a restrictiot• fragment length polymorphism (RFLP). We concluded that this band represented a fragment of (female specific) W chromosomal DNA with hornol- ogy to DQSG10. There was evidence that DQSG10 itself was derived from the Z chromosome. Two RFLPs are apparent in Figure 1. One, locus DI, is represented by the 4.4 kb and the 4.0 kb alleles as described previously (Quinn and White 1987). The males appeared homozygous for the 4.4 kb allele (lanes 1, 2, 4, and 8), heterozygous (lane 7), or homozygous for the 4.0 kb allele (not shown). No heterozygous females were found, but rather females appeared hemizygous and carried either the 4.4 kb allele (lanes 3, 5, 6, 10, and 12) or the 4.0 kb allele (lanes 9 and 11). The second polymorphic locus (DIII) was undescribed previously and shows a 2.6 (DIII-1) and a 2.3 (DIII-2) kb allele. The 2.3 kb allele was identical in size to a constant band present in all samples examined. The males were either homo- zygous for the 2.6 kb band, in which case the 2.6 and 2.3 (constant) bands were of equal intensity (lanes 1, 2, 4, and 7); heterozygous, in which case the 2.3 kb bands were of much higher intensity (lane 8); or homozygous for the 2.3 kb band, in which case the 2.6 kb band was absent (not shown). No heterozygous females were found, and again they appeared hemi- 200 Short Communications [Auk, Vol. 107 I 2 3 4 5 6 7 8 9 101112 5.4 4.4 4.0 ,2.8 '2.6 2.3 2.1 1.2 0.8 Fig. 1. Autoradiograph of DNA from male and female Snow Geese probed with DQSG10. Genomic DNA (5 •g) extracted from the blood of adult Lesser Snow Geese (Chen caerulescens caerulescens) was di- gested with 15 units of TaqI for 3 hours. After elec- trophoresis in 1% agarose, these were transferred to Genescreen Plus membrane (New England Nuclear) by the method of Southern (1975). This blot was probed with DQSG10 as described (Quinn and White 1987) and washed twice in 2 x SSC for 5 rain at 20øC, and three times in 0.2x SSC, 0.1% SDS for 30 rain at 65•C. zygous for either the 2.6 kb band (lanes 3, 9, 11, and 12) or the 2.3 kb band (lanes 5, 6, and 10). By using the p•sence of the 2.1 kb band to identify females and its absence to identify males, we were able to screen a total of 71 males and 104 females with this probe. The sample included an additional 56 in- dividuals obtained from hunter-killed birds, which were collected originally in British Columbia and the state of Washington for a different study and which had not been sexed in the field. In all cases, the rel- ative band intensities and patterns were consistent with the interpretation of the data of Fig. 1. For both polymorphic regions (DI and DIII), the genotypes of the two sexes were consistent with the male possess- ing two alleles and the female being hemizygous (Ta- ble 1). No cases of females with two different alleles at one locus were found. Out of 41 samples hetero- zygous for either DI or DIII or both, there were no cases where the 2.1 kb band was present. If the null hypothesis is made that females are homozygous for these RFLPs, then the chance of observing such a deviation from expected Hardy-Weinberg equilibri- TABLE 1. Distribution of alleles at two loci in males and females. DQSG10-DI and -Dill allele frequen- cies and genotype distribution within the two sexes were calculated under the assumption (null hy- pothesis) that both have two copies at this locus and are in Hardy-Weinberg equilibrium. This was evaluated using the G-test (Sokal and Rohlf 1969); *=P•0.001. Geno- Ob- justed a pect. ed b type served (f) (f) Male • DI-I,1 52 52.5 52.5 1,2 18 18.5 17.1 3.2 (NS') 2,2 1 1.5 1.4 DIII-I,1 36 36.5 37.8 1,2 32 32.5 28.0 4.4 (NS) 2,2 3 3.5 5.2 Female a DI-I,1 84 84.5 68.2 1,2 0 0.5 32.0 101.3' 2,2 20 20.5 3.8 DIII-I,1 78 78.5 58.5 1,2 0 0.5 39.0 116.2' 2,2 26 26.5 6.5 ß Yate's correction. b Assumes Hardy-Weinberg equilibrium. • G = 2(•f•ln f, - 2.30259 •f, log •). a Sexing based on the presence (9) or absence (8) of the 2.1 kb band. ' NS: not significant at P = 0.05, df = 2. um is much less than 0.001 for both regions (Table 1). Hence, we conclude that females are hemizygous. The second form of evidence of the Z chromosomal origin of DQSG10 was the intensity of the constant 5.4 kb band that, after correcting for the amount of DNA loaded per track, showed a male to female ratio of 2:1 (Table 2). We conclude that the DQSG10 probe is derived from the Z chromosome and, as described earlier, has hornology to the W chromosome. The occurrence of regions of crosshomology between the sex chromo- somes has also been noted in humans (Page et al. 1982,1984; Geldwerth et al. 1985) and may result from either transposition or recombination events between the X and Y chromosomes, or from sequence conser- vation from an ancestral pair of homologues (Ohno 1967). These results demonstrate the utility of the general approach of using chromosomal probes for sex de- termination in birds. Sampling requires minute amounts of blood, as we routinely extract 40 times more DNA than used for this analysis from 50 #1 of blood (Quinn and White 1987). Such small volumes of blood carry little risk to the individual sampled, even in the case of small birds (Strangel 1986). In the Snow Goose, a female (W chromosome) specific band January 1990] Short Communications 201 TABLE 2. Densitometric analysis of putative Z chromosomal autoradiographic bands in males and females. Densities were estimated for the 5.4 kb band of Figure 1 by taking the average tracing of three scans (LKB Ultroscan XL Laser Densitometer) and interpolating the area under the curve. As a control for variability in total DNA loaded, another scan was taken of the constant 2.9 kb band produced on an autoradiograph by hybridizing the same blot with the autosomal probe DQSG12. DQSG10 Control Adjusted area a area •' area Track Sex (a) (b) (a/b) Summary 1 M 0.30 2.33 0.13 Male: •m = 0.134 2 M 0.28 1.94 0.14 sm= 0.0055 3 F 0.11 1.81 0.06 4 M 0.26 2.03 0.13 Female: :• = 0.066 5 F 0.17 2.70 0.06 s t = 0.0079 6 F 0.09 1.48 0.06 7 M 0.33 2.57 0.13 8 M 0.31 2.28 0.14 9 F 0.14 2.02 0.07 œ•/• = 2.03 10 F 0.12 1.86 0.06 t c = 16.6 (P << 0.001) 11 F 0.17 2.07 0.08 12 F 0.17 2.43 0.07 Area under densitometric tracing of the 5.4 kb (constant) band. Area under densitometric tracing of the 2.9 kb (constant autosomal) band of the same blot probed with DQSG12 (Quinn and White 1987). Using the pooled standard eviation of s = 0.0070, to test the null hypothesis that œ• = ;?f with 10 degrees of freedom (Sokal and Rohlf 1969). can be scored directly to identify the sex. In this and other species, relative intensities of hybridization sig- nal to the Z chromosome on Southern or dot blots could provide the same information. Preliminary data show that this probe also hybridizes to DNA from members of the Passeriformes, Galliformes, and Cha- radriiformes. The DQSG10 probe will allow us to isolate more clones from both the Z and the W chromosomes. Clones from the W chromosome represent DNA mol- ecules that are inherited maternally like mitochon~ drial DNA but that "travel" in the nuclear rather than mitochondrial cellular compartment. A W chromo- some probe would make sex identification possible on the basis of presence or absence of signal on a dot blot. It would then be practical to determine the sex of hundreds of birds over a short time (2 days). Such probes will also allow comparisons of levels and pat- terns of polymorphisms between the sex chromo- somes and autosomal chromosomes over the range of the Lesser Snow Goose, and they will be useful in questions concerning sex determination and rates of molecular evolution on the different chromosomes ACKNOWLEDGMENTS We are grateful to Don Rainey, Tom Barry, Dave Smith, Gary Grigg, Mike Davison, Ken Abraham, Jon Lewis, the Canadian Wildlife Service, and the U.S. Department of Fish and Wildlife for assistance in col- lecting Snow Goose samples; to Sue Hannon, Peter Boag, Pat Weatherhead, Dov Lank, and Ann Gibbins for collecting other avian samples; to Joyce Marsolais for assistance in the laboratory; to Dov Lank for timely assistance with data analysis; and to three anonymous reviewers for helpful comments. These studies were supported in part by the Natural Sciences and Engi- neering Research Council of Canada (NSERC) grant A8527, by the Canadian National Sportsmen's Fund grants 5-R22 and 6-R61, and by the Canadian Wildlife Service contracts KN 107-4-4137 and KN 107-5-4161. Quinn was supported by a NSERC Postgraduate Fel- lowship, an Ontario Graduate Scholarship; and a Queen's Graduate Fellowship. LITERATURE CITED BIEDERMAN, B. M., C. C. LIN, E. KuYT, & R. C. DREW- IEN. 1982. Genome of the Whooping Crane. J. Fiered. 73: 145-146. GELDWERTH, D., C. BISHOP, G. GUELLAEN, g. KOENIG, G. VERGNAUD, J.-L. MANDEL, & J. WEISSENBACH. 1985. Extensive DNA sequence homologies be- tween the human Y and the long arm of the X chromosome. EMBO J. 4: 1739-1743. KODAMA, H., H. SAlTOH, M. TONE, $. KUHARA, Y. $A- KAKI, & S. MIZUNO. 1987. Nucleotide sequences and unusual electrophoretic behavior of the W chromosome-specific repeating DNA units of the domestic fowl, Gallus gallus domesticus. Chromo- soma 96: 18-25. LONGMIRE, J. L., A. K. LEWIS, N. C. BROWN, J. M. BUC•CING•AM, L. M. CLAP, X, M.D. JON•S, L. J. MEINCKE, J. MEYNE, R. L. RATLIFF, F. A. RAY, R. P. WAGNER, & g. K. MOYZIS. 1988. Isolation and molecular characterization of a highly polymor- phic centromeric tandem repeat in the family Falconidae. Genomics 2: 14-24. 202 Short Communications [Auk, Vol. 107 OHNO, S. 1967. Sex chromosomes and sex-linked genes. New York, Springer. PAGE, D. C., M. E. HARPER, J. LOVE, & D. BOTSTEIN. 1984. Occurrence of a transposition from the X-chromosome long arm to the Y-chromosome short arm during human evolution. Nature 311: 119-123. ß B. DE MARTINvILLEß D. BARKER, A. WYMAN, R. WHITEß U. FRANCKE, &D. BOTSTEIN. 1982. Single- copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromo- somes. Proc. Natl. Acad. Sci. USA 79: 5352-5356. PRus, S. E., & S.C. SCHMUTZ. 1987. Comparative efficiency and accuracy of surgical and cytoge- netic sexing in Psittacines. Avian Diseases 31: 420-424. QUINN, T. W., & B. N. WHITE. 1987. Identification of restriction-fragment-length polymorphisms in genomic DNA of the Lesser Snow Goose (Anser caerulescens caerulescens). Mol. Biol. Evol. 4: 126- 143. ß J. C. DAVIESß F. COOKEß & B. N. WHITE. 1989. Genetic analysis of offspring of a female-female pair in the Lesser Snow Goose Chen caerulescens caerulescens). Auk 106: 177-184. ß J. S. QUINNß F. COOKE, & B. N. WHITE. 1987. DNA marker analysis detects multiple maternity and paternity in single broods of the Lesser Snow Goose. Nature 326: 392-394. SOKAL, R. R., & F. J. ROHLF. 1969. Biometry. San Franciscoß W. H. Freeman and Co. SOUTHERN, E.g. 1975. Detection of specific se- quences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. STRANGEL, P.W. 1986. Lack of effects from sampling blood from small birds. Condor 88: 244-245. TABER, R. D. 1971. Criteria of sex and age. Pp. 325- 401 in Wildlife management techniquesß 3rd ed. (R. H. Giles, Ed.). Washingtonß D.C., The Wildlife Society. TONEß M., Y. SAKAKI, T. HASHIGUCHI, & S. MIZUNO. 1984. Genus specificity and extensive methyl- ation of the W chromosome-specific repetitive DNA sequences from the domestic fowl, Gallus gallus domesticus. Chromosoma 89: 228-237. Received 14 April 1989ß accepted 9 September 1989. Evidence for Vocal Learning by a Scrub Jay TOM WEBBER • AND ROSEMARY A. STEFANI 2 •Florida Museum of Natural Historyß University of Floridaß Gainesvilleß Florida 32611 USA, and 2Division of Environmental Studiesß University of California, Davisß California 95616 USA The vocalizations of corvids, other than their faint- ly uttered and seldom heard whisper song, are typ- ically referred to as calls rather than songs (Hardy 1983, Goodwin 1986). This convention reflects a wide- spread impression that these calls are more simple than the primary advertising songs of many other oscines and are delivered in a different way. Corvids mimic the sounds of other bird species and even the sounds of inanimate objects (Goodwin 1986), but the evidence that they learn their distinctive species-spe- cific calls is sketchy. We present evidence that a cap- tive Scrub Jay (Aphelocoma coerulescens superciliosa) from California learned two calls from Florida Scrub Jays (A. c. coerulescens). A California jay was captured near Carmichael, Sac- ramento Countyß as a late-stage nestling (23 June 1979) and hand-raised. On 5 August 1979, it was given to the Florida Museumß where it lived in a large aviary with other wild-caught Scrub Jays from Florida and California (Webber and Cox 1987). In the following account, we refer to this California jay as "Red" be- cause of its red leg bands. In mid-January 1981ß Webber noticed that a male Florida Scrub Jay (pink leg bands: "Pink") in the avi- ary was pairing with a female ("Green") from Cali- fornia. Pink passed food to Greenß called in response to her flights, and flew back and forth in front of her while calling loudly. Green gave the rattle call (Web- ber 1984) in response to Pink's flights. This behavior is typical of Scrub Jays when they form pairs in the wild (Webber 1984). Other jays in the aviaryß includ- ing Red, often called and flew in response to Pink and Green's pair-forming behavior, as wild Scrub Jays do when a new pair forms in a neighboring territory (Webber 1984). Red gave two kinds of calls (referred to as A [Fig. la] and B [Fig. ld]) that, to Webberß sounded identical to those of Pink (Fig. lb, e) and another male Florida Scrub Jay ("Yellow") in the same aviary. Webber recorded the calls of Red, Pinkß and Yellow on six days from 11 January to 29 March 1981. These recordings include at least 138 calls of type A by Pink and 40 by Red, as well as at least 100 calls of type B by Pinkß 36 by Red, and 7 by Yellow (Florida Museum of Natural History Bioacoustics Archives master tapes 905-907). Webber (1984) found no calls of types A or B in a 12-month study of Scrub Jay calls in Los Angeles County, California (FSM masters 633-637B, 639A- 641B). Stefani (pers. obs.) also found no calls of types A or B in a 3-month study of Scrub Jay vocalizations in Davisß California, 28 miles from Carmichael. We think it unlikely that wild Scrub Jays in Carmichael give calls A and B. Wild Scrub Jays in Florida give calls (FM masters 

Molecular Sexing of Geese Using a Cloned Z Chromosomal Sequence with Homology to the W Chromosome

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Molecular Sexing of Geese Using a Cloned Z Chromosomal Sequence with Homology to the W Chromosome

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