Birds are difficult to sex. Nestlings rarely show sex-linked morphology and we estimate that adult females appear identical to males in over 50% of the world's bird species. This problem can hinder both evolutionary studies and human-assisted breeding of birds. DNA-based sex identification provides a solution. We describe a test based on two conserved CHD (chromo-helicase-DNA-binding) genes that are located on the avian sex chromosomes of all birds, with the possible exception of the ratites (ostriches, etc.; Struthioniformes). The CHD-W gene is located on the W chromosome; therefore it is unique to females. The other gene, CHD-Z, is found on the Z chromosome and therefore occurs in both sexes (female, ZW; male, ZZ). The test employs PCR with a single set of primers. It amplifies homologous sections of both genes and incorporates introns whose lengths usually differ. When examined on a gel there is a single CHD-Z band in males but females have a second, distinctive CHD-W band.

Sex differences in the foraging behaviour of adults have been observed in a number of sexually size‐dimorphic birds, and the usual inference has been that these sex‐specific differences are driven primarily by differences in body size. An alternative explanation is that foraging differences result from sex differences unrelated to size, such as sex‐specific nutritional requirements. To examine these alternative hypotheses, the foraging behaviour of parents was compared between two sympatric and congeneric species of seabird, the Brown Booby Sula leucogaster, which is highly sexually size‐dimorphic (females 38% larger) and the Red‐footed Booby S. sula, in which sex differences in body size are less marked (females 15% larger). Using temperature and depth loggers, we found that there were highly significant differences in the foraging trip durations and diving behaviour of male and female Brown Boobies. These sex differences were less marked in Red‐footed Boobies. Thus, our interspecies comparison revealed that the magnitude of the difference between the sexes matched the sexual size dimorphism of the species, providing support for the size hypothesis.

Sex identification is a problem in research and conservation. It can often be solved using a DNA test but this is only an option if a sex-specific marker is available. Such markers can be identified using the amplified fragment length polymorphism (AFLP) technique. This is usually a taxonomic method, as it produces a DNA fingerprint of 50-100 PCR bands. However, if male and female AFLP products are compared, sex-specific markers are confined to the heterogametic sex and can rapidly be identified. Once a marker is found, AFLP can be used to sex organisms directly or the marker can be sequenced and a standard PCR test designed.

Sex allocation studies seek to ascertain whether mothers manipulate offspring sex ratio prior to ovulation. To do so, DNA for molecular sexing should be collected as soon after conception as possible, but instead neonates are usually sampled. Here, we aim to identify and quantify some of the problems associated with using molecular techniques to identify the sex of newly laid avian eggs. From both fertilized and unfertilized chicken (Gallus gallus) eggs, we sampled (1) the blastoderm/disc, (2) vitelline membrane and (3) a mixture of (1) and (2). Thus, we replicated scenarios under which contaminated samples are taken and/or unfertilized eggs are not identified as such and are sampled. We found that two commonly used molecular sexing tests, based on the CHD-1 genes, differed in sensitivity, but this did not always predict their ability to sex egg samples. The vitelline membrane was a considerable source of maternal and probably paternal contamination. Fertile eggs were regularly assigned the wrong sex when vitelline membrane contaminated the blastoderm sample. The membrane of unfertilized eggs was always female, i.e. maternal DNA had been amplified. DNA was amplified from 47 to 63% of unfertilized blastodiscs, even though it was highly unlikely that DNA from a single haploid cell could be amplified reliably using these polymerase chain reaction (PCR) techniques. Surprisingly, the blastodiscs were identified as both males and females. We suggest that in these cases only maternal DNA was amplified, and that 'false' males, Z not ZZ, were detected. This was due to the reduced ability of both sets of primers to anneal to the W chromosome compared to the Z chromosome at low DNA concentrations. Overall, our data suggested that estimates of primary sex ratios based on newly laid eggs will be appreciably inaccurate.

Methods for the identification of the sex of bird species without external sexual dimorphism are specially important in field studies and for captive breeding of endangered taxa. We confirmed the accuracy of a polymerase chain reaction (PCR)‐based method to identify the sex in three disparate avian orders that included 31 species of parrot, two species of toucan, and eight species of curassow, for which many individuals were previously sexed. In each case, two DNA fragments were amplified in females and one in males with the use of a single set of primers. This method was also tested on unsexed birds of 13 other species of parrot and five species of toucan. The same kind of polymorphism was detected in each. The PCR products of parrots and toucans could be separated in simple agarose gels, while the curassows' products could only be distinguished in acrylamide gels. An advantage of this DNA test is that samples of blood or feathers can be easily collected and stored at room temperature, which is of particular importance for studies of wild birds. Zoo Biol 17:415–423, 1998. © 1998 Wiley‐Liss, Inc.