Contrasting levels of nucleotide diversity on the avian Z and W sex chromosomes.

Sex chromosomes may provide a context for studying the local effects of mutation rate on molecular evolution, since the two types of sex chromosomes are generally exposed to different mutational environments in male and female germ lines. Importantly, recent studies of some vertebrates have provided evidence for a higher mutation rate among males than among females. Thus, in birds, the Z chromosome, which spends two thirds of its time in the male germ line, is exposed to more mutations than the female-specific W chromosome. We show here that levels of nucleotide diversity are drastically higher on the avian Z chromosome than in paralogous sequences on the W chromosome. In fact, no intraspecific polymorphism whatsoever was seen in about 3.4 kb of CHD1W intron sequence from a total of >150 W chromosome copies of seven different bird species. In contrast, the amount of genetic variability in paralogous sequences on the Z chromosome was significant, with an average pairwise nucleotide diversity (d) of 0.0020 between CHD1Z introns and with 37 segregating sites in a total of 3.8 kb of Z sequence. The contrasting levels of genetic variability on the avian sex chromosomes are thus in a direction predicted from a male-biased mutation rate. However, although a low gene number, as well as some other factors, argues against background selection and/or selective sweeps shaping the genetic variability of the avian W chromosome, we cannot completely exclude selection as a contributor to the low levels of variation on the W chromosome.

Molecular evolution of the avian CHD1 genes on the Z and W sex chromosomes.

Genes shared between the nonrecombining parts of the two types of sex chromosomes offer a potential means to study the molecular evolution of the same gene exposed to different genomic environments. We have analyzed the molecular evolution of the coding sequence of the first pair of genes found to be shared by the avian Z (present in both sexes) and W (female-specific) sex chromosomes, CHD1Z and CHD1W. We show here that these two genes evolve independently but are highly conserved at nucleotide as well as amino acid levels, thus not indicating a female-specific role of the CHD1W gene. From comparisons of sequence data from three avian lineages, the frequency of nonsynonymous substitutions (K(a)) was found to be higher for CHD1W (1.55 per 100 sites) than for CHD1Z (0.81), while the opposite was found for synonymous substitutions (K(s), 13.5 vs. 22.7). We argue that the lower effective population size and the absence of recombination on the W chromosome will generally imply that nonsynonymous substitutions accumulate faster on this chromosome than on the Z chromosome. The same should be true for the Y chromosome relative to the X chromosome in XY systems. Our data are compatible with a male-biased mutation rate, manifested by the faster rate of neutral evolution (synonymous substitutions) on the Z chromosome than on the female-specific W chromosome.

Male-biased mutation rates revealed from Z and W chromosome-linked ATP synthase alpha-subunit (ATP5A1) sequences in birds.

Whether the mutation rate differs between sexes has been a matter of discussion for years. Molecular analyses of mammals have indicated that males mutate more often than females, as manifested by the faster rate of neutral sequence evolution on the Y chromosome than on the X chromosome. However, these observations can as well be interpreted as specific reduction of the X chromosome mutation rate, which would be adaptive because of reducing the number of slightly deleterious recessive mutations exposed in hemizygote males. Recently, data from birds have suggested that vertebrate mutation rates may indeed be male-biased. In birds, females are the heterogametic sex (ZW), and analyses of the Z-linked CHD1Z gene have shown that it evolves faster than its W-linked and thus female-specific homologue, CHD1W. We have now studied the second avian gene known to exist in a copy on the nonrecombining regions of both the Z and the W chromosome, viz., the ATP synthase alpha-subunit (ATP5A1). In independent comparisons of three pairs of bird species from divergent lineages, intron sequences of the Z-linked copy (ATP5A1Z) were consistently found to evolve faster than the W-linked copy (ATP5A1W). From these data we calculated male-to-female mutation rate ratios (alpha) of 1.8, 2.3, and 5.0 in Galliform, Anseriform, and Ciconiiform lineages, respectively. Therefore, this study provides independent support for a male-biased mutation rate in birds.

Sex ratio and fledging success of supplementary-fed Tengmalm's owl broods.

A nest box population of Tengmalm's owls (Aegolius funereus) in northern Sweden was studied to investigate the effects of extra food on the sex ratio between hatching and fledging in this sexually size-dimorphic species. The brood size and brood sex ratio of supplementary-fed and control broods were compared. Newly hatched nestlings were blood sampled and sexed by polymerase chain reaction (PCR) amplification of the sex-linked CHD1Z and CHD1W genes. The brood sex ratio at hatching was strongly male biased (65%); this was also the case in broods where all eggs hatched (72%). There was no relationship between hatch order and sex ratio, and hatching sex ratio did not vary significantly with laying date. Brood size decreased between hatching and fledging, but did not differ between fed and control broods at either stage. Brood sex ratio did not differ between hatching and fledging, and fledging sex ratio did not differ between fed and control broods. It was concluded that, at least during the year in which the study was carried out, feeding had no effect on brood reduction, and that male and female nestlings did not show any differential mortality. The mechanisms behind the male-biased sex ratio at hatching, and any possible adaptive reasons for it, are not known.

The avian egg as a test system for endocrine disrupters: effects of diethylstilbestrol and ethynylestradiol on sex organ development.

Many environmental contaminants are known or suspected to interfere with hormonal function in animals. In vivo test methods to detect and characterize chemicals that disrupt the endocrine system are therefore urgently needed. In this study, we assessed the usefulness of abnormalities of the reproductive organs as test endpoints for estrogenic activity of xenobiotics in Japanese quail embryos. Two synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), were injected into the yolks of embryonated eggs. At a dose as low as 2 ng EE2/g egg, all male embryos became feminized, containing ovary-like tissue in the left testis. The extent of feminization of the testes was determined by measuring the relative area of the ovary-like component. Persistent Müllerian ducts (oviducts) in male embryos, and malformations of the Müllerian ducts in females occurred at 2 ng EE2/g egg and higher doses. DES was approximately one-third to one-tenth as potent as EE2. The morphological changes studied were dose-dependent, indicating that they are useful as test endpoints for estrogenic activity. Feminization of the left testis in males proved to be the most sensitive endpoint. We propose the quail egg as a simple in vivo test system for estrogenic compounds.

Evolution of the avian sex chromosomes from an ancestral pair of autosomes.

Among the mechanisms whereby sex is determined in animals, chromosomal sex determination is found in a wide variety of distant taxa. The widespread but not ubiquitous occurrence, not even within lineages, of chromosomal sex determination suggests that sex chromosomes have evolved independently several times during animal radiation, but firm evidence for this is lacking. The most favored model for this process is gradual differentiation of ancestral pairs of autosomes. As known for mammals, sex chromosomes may have a very ancient origin, and it has even been speculated that the sex chromosomes of mammals and birds would share a common chromosomal ancestry. In this study we showed that the two genes, ATP5A1 and CHD1, so far assigned to the female-specific W chromosome of birds both exist in a very closely related copy on the Z chromosome but are not pseudoautosomal. This indicates a common ancestry of the two sex chromosomes, consistent with the evolution from a pair of autosomes. Comparative mapping demonstrates, however, that ATP5A1 and CHD1 are not sex-linked among eutherian mammals; this is also not the case for the majority of other genes so far assigned to the avian Z chromosome. Our results suggest that the evolution of sex chromosomes has occurred independently in mammals and birds.

Identification of a mutation in the low density lipoprotein receptor gene associated with recessive familial hypercholesterolemia in swine.

Elevated blood plasma cholesterol (hypercholesterolemia) is a major risk factor for coronary artery disease (CAD) in humans. Genetic dissection of polygenic lipid and lipoprotein disorders in swine, a key animal model for the study of familial hypercholesterolemia (FH) and CAD, led to the isolation of a monogenic subphenotype (FH-r), that is inherited in the recessive (r) manner. A genome scan mapped the FH-r locus close to the centromere of chromosome 2. Comparative mapping showed that this region shares homology with a part of human chromosome 19 that harbors the low density lipoprotein receptor (LDLR) locus, and therefore suggested LDLR as the prime candidate gene for FH-r. Cloning and sequencing of hepatic LDLR cDNA from two FH-r/r and one normal (N/N) animals disclosed a single missense mutation (R84C) in a region that corresponds to human exon 4. The C84 mutation cosegregates invariantly with hypercholesterolemia, which strongly suggests that this mutation is responsible for the observed hyperlipidemia.

Expansion of the pig comparative map by expressed sequence tags (EST) mapping.

We have used a PCR-based approach for the genetical and physical mapping of 34 transcripts isolated from a porcine small intestine cDNA library. All but one gene were regionally localized by using a somatic pig-rodent cell hybrid panel, and 12 genes were mapped by linkage analysis of single-stranded conformational polymorphisms developed in 3' untranslated regions of transcripts. For 20 of the transcripts, the human homolog has already been mapped. This study thus represents a significant contribution to the pig comparative map. Some important findings were that we could clarify the extent of a previously identified inversion event in a region of conserved synteny between SSC6q and HSA1p, that SSC14q does contain a region homologous to HSA1, a situation not clear from earlier ZOO-FISH studies, and that the homology between SSC17 and HSA20 includes the p-arm of HSA20.

Male-driven evolution of DNA sequences in birds.

Assuming that new mutations arise mainly during DNA replication, sequence evolution in mammals has been seen as 'male driven' (ref. 1) because of the many more cell divisions in spermatogenesis than in oogenesis. Molecular support for this idea has been obtained from the observation of higher substitution rates in genes on the Y than on the X chromosome of primates and rodents, which are species with male heterogamety, but has not been confirmed by the reciprocal analysis of organisms with female heterogamety. The recent suggestion that an intrinsic reduction in the X-chromosome mutation rate may be confounded with male effects in previous comparisons, and the paradoxical finding of low levels of polymorphism on the primate Y chromosome indicate that the idea of male-biased mutation rate needs to be re-examined. We have analysed the molecular evolution of the gene CHD, which is present on the Z and W sex chromosomes of birds. The substitution rate at synonymous positions, as well as in intron DNA, was considerably higher on the Z chromosome than on the female-specific W chromosome, with an estimated male-to-female bias in mutation rate (alpha m) of 3.9-6.5. Thus, evolution appears to be male driven in birds--a situation that supports a neutral model of molecular evolution.