Richard Griffiths

A DNA test to sex most birds.

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 worlds 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 identification in birds using two CHD genes

In theory, birds should control the sex ratio of the offspring they produce. In practice, we have very limited evidence to support this idea because of our difficulty in sexing nestling birds. In addition, extinction is facing an increasing number of birds. Our ability to help includes captive breeding which, again, is difficult if male and female adults cannot be recognized. Here we describe the discovery of a W-linked gene in the Great tit (Parus major). It is named CHD-W (chromodomain-helicase-DNA-binding protein W-linked), it is highly conserved and it is W-chromosome linked in a range of bird species. These birds also possess a second, non-W-linked CHD gene (CHD-NW). A single, simple polymerase chain reaction technique based on both genes can be used to identify the sex in a wide variety of birds.

Sex-specific foraging behaviour in a monomorphic seabird

Sexual differences in the foraging behaviour of parents have been observed in a number of sexually sizedimorphic birds, particularly seabirds, and the usual inference has been that these sex–specific differences are mediated primarily by differences in body size. To test this explanation, we compared the foraging behaviour of parents in a monomorphic seabird species, the northern gannet Morus bassanus. Using specially designed instruments and radio telemetry we found that individuals of both sexes were consistent in the directions and durations of their foraging trips. However, there were significant differences in the foraging behaviour of males and females. Female gannets were not only more selective than males in the areas where they foraged, but they also made longer, deeper dives and spent more time on the sea surface than males. As the sexes are morphologically similar in this species, then these differences are unlikely to have been mediated by body size. Our work highlights the need to investigate sexual differences in the foraging behaviour of seabirds and other species more closely, in order to test alternative theories that do not rely on differences in body size.

A CHD1 gene is Z chromosome linked in the chicken Gallus domesticus

Chromo-helicase-DNA binding 1 (CHD1) is a conserved protein with a putative role in chromatin architecture. Single homologues have been found in mouse, Drosophila and yeast. In birds the situation is different as they possess two homologues. One is known to be W-linked, we show the second, closely related gene is linked to the Z sex chromosome. The basic structure of the Z-linked gene is similar to the homologous genes, however, it does possess an additional, internal 88 amino acid hydrophilic domain, rich in glutamic acid and lysine. Studies on pairs of genes sex-linked in mammals suggests rapid divergence of DNA sequence and function. We suggest the DNA sequences of CHD-W and CHD-Z do not follow this pattern.

Increased reproductive effort results in male-biased offspring sex ratio: an experimental study in a species with reversed sexual size dimorphism

Adaptive sex–ratio theory predicts that parents should overproduce the more beneficial offspring sex. Based on a recent experimental study of lesser black–backed gulls, we tested this hypothesis with the great skua, Catharacta skua, a bird species closely related to gulls but where females are the larger sex. When in poor body condition, the gulls overproduced daughters, the smaller and more viable sex under those circumstances. To discriminate between a mandatory physiological overproduction of female (i.e. non–male) eggs versus the overproduction of the smaller and presumably more viable sex, we conducted an egg–removal experiment with the great skua. Since the males are smaller, larger size and being male are separated. Through egg removal we induced females to increase egg production effort. Eggs were sexed using a DNA–based technique. Manipulated pairs produced a significant male bias at the end of the extended laying sequence, while the sex ratio in the control group did not differ from unity. Our results present an example of facultative sex–ratio manipulation and support the hypothesis that in sexually dimorphic birds parents overproduce the smaller sex under adverse conditions.

The use of amplified fragment length polymorphism (AFLP) in the isolation of sex‐specific markers

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.

Primary sex ratios in birds: problems with molecular sex identification of undeveloped eggs

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.

Subtle manipulation of egg sex ratio in birds.

Mothers are predicted to overproduce male or female eggs when the relative fitness gains from one sex are higher and outweigh the costs of manipulation. However, in birds such biases are often difficult to distinguish from differential embryo or chick mortality. Using a molecular technique to identify the sex of early embryos, we aim to determine the effect of maternal nutrition on zebra finch (Taeniopygia guttata) egg sex ratios after 2 days of incubation, which is as close to conception as is currently possible. We found no overall bias in the sex ratio of eggs laid and sex did not differ with relative laying order under any diet regime. However, mothers on a low-quality diet did produce a female bias in small clutches and a slight male bias in large clutches. On a high-quality diet, mothers produced a male bias in small clutches and a female bias in large clutches. Those on a standard diet produced a roughly even sex ratio, irrespective of clutch size. These observed biases in egg sex are partly in line with predictions that, in this species, daughters suffer disproportionately from poor rearing conditions. Thus, when relatively malnourished, mothers should only rear daughters in small broods and vice versa. Sex-ratio patterns in this species therefore appear to be subtle.

Evidence of male-bias in capture samples of marbled murrelets from genetic studies in British Columbia

We report a significant male-bias (1.8:1) in Marbled Murrelets (Brachyramphus marmoratus) captured with floating mist nets during the breeding season ovcr four years (1994-1997) at Theodosia Inlet, Desolation Sound, British Columbia. There was little evidence for marked annual or diurnal variation in male-bias, or for variation due to flight direction of birds when they contacted thc net (inland vs. out to sea). In contrast, samplcs of adult and juvcnile Marbled Murrelets captured using a night-lighting technique at Desolation Sound in 1997 were not male-biased. We believe that the most likely explanation for our results is that thcre are sex-specific differences in behavior of Marbled Murrelets during the breeding period, such that more males than females are flying between marine foraging and inland ncsting areas.

EXTRA-PAIR PATERNITY IN THE COMMON MURRE

Abstract Multi-locus DNA fingerprinting was used to estimate the frequency of extra-pair paternity in the Common Murre (Uria aalge), a colonial, sexually monomorphic seabird that breeds at very high densities and in which extra-pair copulation is frequent. Common Murres produce a single chick. We detected 6 cases of extra-pair paternity in 77 families (7.8%). This value was higher than the proportion of successful extra-pair copulations (1.6%) estimated from behavioral data from an earlier study of the same population.