Segregation of sperm mitochondria in two- and four-cell embryos of the blue mussel Mytilus edulis: Implications for the mechanism of doubly uniparental inheritance of mitochondrial DNA.

Species of the family Mytilidae have 2 mitochondrial genomes, one that is transmitted through the egg and one that is transmitted through the sperm. In the Mytilus edulis species complex (M. edulis, M. galloprovincialis, and M. trossulus) there is also a strong mother-dependent sex-ratio bias in favor of one or the other sex among progeny from pair matings. In a previous study, we have shown that sperm mitochondria enter the egg and that their behavior during cell division is different depending on whether the egg originated from a female- or male-biased mother. Specifically, in eggs from females that produce mostly or exclusively daughters, sperm mitochondria disperse randomly among cells after egg division. In eggs from females that produce predominantly sons, sperm mitochondria tend to stay together in the same cell. Here, we extend these observations and show that in 2- and 4-cell embryos from male-biased mothers most sperm mitochondria are located near or at the cleavage furrow of the major cell, in contrast to embryos from female-biased mothers where there is no preferential association of sperm mitochondria with the cleavage furrow. This observation provides evidence for an early developmental mechanism through which sperm mitochondria are preferentially channeled into the primordial cells of male embryos, thus making the paternal mitochondrial genome the dominant mtDNA component of the male germ line.     

The fate of paternal mitochondrial DNA in developing female mussels,Mytilus edulis: implications for the mechanism of doubly uniparental inheritance of mitochondrial DNA.

Species of the marine mussel family Mytilidae have two types of mitochondrial DNA: one that is transmitted from the mother to both female and male offspring (the F type) and one that is transmitted from the father to sons only (the M type). By using pair matings that produce only female offspring or a mixture of female and male offspring and a pair of oligonucleotide primers that amplify part of the COIII gene of the M but not the F mitochondrial genome, we demonstrate that both male and female embryos receive M mtDNA through the sperm and that within 24 hr after fertilization the M mtDNA is eliminated or is drastically reduced in female embryos but maintained in male embryos. These observations are important for understanding the relationship between mtDNA transmission and sex determination in species with doubly uniparental inheritance of mitochondrial DNA.     

Sex-specific effects of hybridization on reproductive fitness in Mytilus

Blue mussels of the genus Mytilus form extensive hybrid zones in the North Atlantic and elsewhere where the distributions of different species overlap. Mytilus species transmit both maternal and paternal mtDNA through egg and sperm, respectively, a process known as doubly uniparental inheritance (DUI), and some females produce offspring with extremely biased sex ratios. These two traits have been shown to be linked and maternally controlled, with sex determination involving nuclear–cytoplasmic interactions. Hybridization has been shown to disrupt DUI mitochondrial inheritance and sex ratio bias; however, the effect of hybridization on reproductive fitness has not previously been examined. We investigated this effect in M. edulis × M. trossulus crosses through histological examination of mature F1 progeny, and spawning of F1 hybrids to monitor survival of their progeny through to the D stage of larval development. For progeny produced from mothers with a strong bias toward female offspring (often 100%) in pure-bred crosses, there was a clear breakdown in female dominance of progeny and significantly more hermaphrodites in the hybrid crosses produced from sperm with the M-tr1 mitotype. We also found significant sex-specific differences among hybrid progeny, with females producing normal eggs while males and hermaphrodites evidenced impaired gonadal development with significantly greater numbers of Sertoli cells, phagocytic hemocytes, and degenerating germ cells, all associated with gonad resorption. Males from crosses where DUI was disrupted and where male progeny were homoplasmic for the female mtDNA were the most severely compromised. Allelic incongruity between maternal and paternal mitotypes in hybrid crosses was associated with significant disruption of male gonadal development.     

Male-Dependent Doubly Uniparental Inheritance of Mitochondrial DNA and Female-Dependent Sex-Ratio in the Mussel Mytilus Galloprovincialis

We have investigated sex ratio and mitochondrial DNA inheritance in pair-matings involving five female and five male individuals of the Mediterranean mussel Mytilus galloprovincialis. The percentage of male progeny varied widely among families and was found to be a characteristic of the female parent and independent of the male to which it was mated. Thus sex-ratio in Mytilus appears to be independent of the nuclear genotype of the sperm. With a few exceptions, doubly uniparental inheritance (DUI) of mtDNA was observed in all families fathered by four of the five males: female and male progeny contained the mother's mtDNA (the F genome), but males contained also the father's paternal mtDNA (the M genome). Two hermaphrodite individuals found among the progeny of these crosses contained the F mitochondrial genome in the female gonad and both the F and M genomes in the male gonad. All four families fathered by the fifth male showed the standard maternal inheritance (SMI) of animal mtDNA: both female and male progeny contained only the maternal mtDNA. These observations illustrate the intimate linkage between sex and mtDNA inheritance in species with DUI and suggest different major roles for each gender. We propose a model according to which development of a male gonad requires the presence in the early germ cells of an agent associated with sperm-derived mitochondria, these mitochondria are endowed with a paternally encoded replicative advantage through which they overcome their original minority in the fertilized egg and this advantage (and, therefore, the chance of an early entrance into the germ line) is countered by a maternally encoded egg factor.     

Sex determination in the androdioecious plant Datisca glomerata and its dioecious sister species D. cannabina.

Datisca glomerata is an androdioecious plant species containing male and hermaphroditic individuals. Molecular markers and crossing data suggest that, in both D. glomerata and its dioecious sister species D. cannabina, sex is determined by a single nuclear locus, at which maleness is dominant. Supporting this conclusion, an amplified fragment length polymorphism (AFLP) is heterozygous in males and homozygous recessive in hermaphrodites in three populations of the androdioecious species. Additionally, hermaphrodite x male crosses produced 1:1 sex ratios, while hermaphrodite x hermaphrodite crosses produced almost entirely hermaphroditic offspring. No perfectly sex-linked marker was found in the dioecious species, but all markers associated with sex mapped to a single linkage group and were heterozygous in the male parent. There was no sex-ratio heterogeneity among crosses within D. cannabina collections, but males from one collection produced highly biased sex ratios (94% females), suggesting that there may be sex-linked meiotic drive or a cytoplasmic sex-ratio factor. Interspecific crosses produced only male and female offspring, but no hermaphrodites, suggesting that hermaphroditism is recessive to femaleness. This comparative approach suggests that the hermaphrodite form arose in a dioecious population from a recessive mutation that allowed females to produce pollen.     

POLLEN PERFORMANCE AND SEX-RATIO EVOLUTION IN A DIOECIOUS PLANT

There has been a proliferation of studies, in a variety of taxa, that have detected sex-linked or cytoplasmic genes that enhance their own transmission via sex-ratio distortion. One of the most important parameters influencing the dynamics of these elements is the magnitude of their transmission advantage. In many systems, the mechanism of sex-ratio distortion is to abort X- or Y-bearing gametes. With this mechanism, the transmission advantage associated with sex-ratio distortion is diminished when the production of male gametes limits offspring production or when competition among the gametes of different males is intense. In this study, we analyzed the outcome of pollen competition between males that produced different sex ratios in the dioecious plant, Silene alba, and estimated how the sex-ratio bias influenced the transmission properties of the sex chromosomes. We varied the intensity of pollen competition by controlling the quantity of pollen used in crosses and used a combination of single-male pollinations and pollen mixtures to evaluate the effects of multiple paternity. Paternity in pollen mixtures was estimated using allozymes. Sex-ratio bias was directly influenced by the quantity of pollen, but the magnitude of this effect was small. The relative performance of pollen from different males varied substantially, especially when there was multiple paternity. Specifically, males with biased sex ratios sired far fewer offspring of either sex in pollen mixtures. In crosses involving single males, however, these “sex-ratio” males produced the same number of offspring as other males, so the female bias caused a significant transmission advantage for X-linked genes. X-linked genes could enhance their transmission via sex-ratio distortion in Silene populations, but the magnitude of this transmission advantage will depend on the ecological circumstances that influence the opportunity for multiple paternity.     

Sex-Ratio Bias in Alates of Polygyne Colonies of the Red Imported Fire Ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis> Buren (Hymenoptera: Formicidae) in China

A sex-ratio bias was studied in alates of natural polygyne colonies of Solenopsis invicta Buren in southern China. The results showed that at the population level, the numbers of male and female alates were nearly equal, even though there was a strong bias toward producing one particular sex at the nest level. For example, 88.89% of the nests sampled were strongly biased toward males or females. In particular, three bias types were observed: extreme female bias, extreme male bias, and a moderate bias. Future studies should address the factors that lead queens to produce strongly biased sex ratios.     

Determinisme des anomalies de sex ratio á hérédité paternelle chez le crustacé amphipode Orchestia gammarellus Pallas

Summary

In the amphipod crustacea Orchestia gammarellus (heterogametic species: 2AXY male, 2AXX female), two kinds of sex ratio bias are recorded, hi the first category (thelygeny linked with intersexuality) a parasitic protozoa modifies the sexual phenotype of genetic males and can transform them into intersex males or functional females. This leads to the occurrence of viable 2AYY males and females.

In a second kind of sex ratio bias, males cause hereditary shifts of sex ratio. These ‘paternal sex ratio’ (psr) traits are transmitted by the male at each generation. Psr-f males cause an excess of females, psr-m males an excess of males.

The psr-m trait has a strictly patroclinous mode of transmission, but females from psr-f strains intervene in the expression of psr-f trait. Intra-sib matings are characterized by an excess of males. This characteristic seems to be linked with the age of the female. It disappears during successive brood. A relation between the psr-m and psr-f trait is observed: some psr- m males give psr-f males in the their progeny.

The analysis of crosses between psr-f or psr-m males and YY females allows to discard meiotic drive or sex lethal mortality as causes for the psr traits. Our results are best explained if we suppose that psr-f and psr-m males are XX and that extrachromosomal hereditary factors or transposable genetic elements intervene in the determinism of the psr traits: a psr-m factor able to masculinize all the embryos and a psr-f factor able to masculinize embryos if present in a sufficient amount.     

Quantitation of the male and female types of mitochondrial DNA in a blue mussel, Mytilus galloprovincialis, using real-time polymerase chain reaction assay

The system termed doubly uniparental inheritance (DUI) of mitochondrial transmission to progeny has been reported in Mytilus. Under DUI, it has been thought that males have both paternally (M type) and maternally (F type) transmitted mitochondrial DNA (mtDNA), and females have only F type. However, the presence of M type in females has been reported. To clarify the ratio of M type to F type mtDNA in female and male tissues to further our understanding of mitochondrial transmission, we developed a procedure to measure the copy numbers of the two types of mtDNA in Mytilus galloprovincialis using a real-time polymerase chain reaction assay. The following results were obtained by this method. In females, the copy numbers of M type mtDNA detected in adductor muscle, gonad and eggs were approximately 10 000-fold lower than those of F type. In males, F type dominated in adductor muscle, as in the female tissue. However, copy numbers of M type mtDNA were approximately 1000-fold higher than those of F type in gonad and 100 000-fold higher than those of F type in sperm. We examined the quantity relationship between the two types of mtDNA and the transmission mechanism of mtDNA in M. galloprovincialis.     

Meiotic drive alters sperm competitive ability in stalk-eyed flies.

Meiotic drive results when sperm carrying a driving chromosome preferentially survive development. Meiotic drive should therefore influence sperm competition because drive males produce fewer sperm than non-drive males. Whether meiotic drive also influences the competitive ability of sperm after ejaculation is unknown. Here we report the results from reciprocal crosses that are designed for estimating the sperm precedence of male stalk-eyed flies (Cyrtodiopsis whitei) with or without X-linked meiotic drive. We find that nearly half of all sex-ratio males, as compared with 14% of non-sex-ratio males, fail to produce young in a reciprocal cross. Furthermore, the proportion of progeny sired by a sex-ratio male in a female jointly inseminated by a non-sex-ratio male was less than expected from the number of sperm transferred. These effects are not due to differential sperm storage by females because, after a single mating with a sex-ratio male, all females stored sperm and because two sex-ratio males share paternity after jointly mating with a female. In addition to demonstrating a new mechanism of sperm competition, these results provide insight into the maintenance of sex-ratio polymorphisms. Sex-ratio males have less than one-half the fertility of non-sex-ratio males, as is required in order for frequency-dependent selection on males to produce a stable sex-ratio polymorphism.     

The Effects of Natural Hybridization on the Regulation of Doubly Uniparental Mtdna Inheritance in Blue Mussels (Mytilus Spp.)

Blue mussels in the Mytilus edulis species complex have a doubly uniparental mode of mtDNA inheritance with separate maternal and paternal mtDNA lineages. Female mussels inherit their mtDNA solely from their mother, while males inherit mtDNA from both parents. In the male gonad the paternal mtDNA is preferentially replicated so that only paternal mtDNA is transmitted from fathers to sons. Hybridization is common among differentiated blue mussel taxa; whenever it involves M. trossulus, doubly uniparental mtDNA inheritance is disrupted. We have found high frequencies of males without and females with paternal mtDNA among hybrid mussels produced by interspecific matings between M. galloprovincialis and M. trossulus. In contrast, hybridization between M. galloprovincialis and M. edulis does not affect doubly uniparental inheritance, indicating a difference in the divergence of the mechanisms regulating mtDNA inheritance among the three blue mussel taxa. Our data indicate a high frequency of disrupted mtDNA transmission in F(1) hybrids and suggest that two separate mechanisms, one regulating the transmission of paternal mtDNA to males and another inhibiting the establishment of paternal mtDNA in females, act to regulate doubly uniparental inheritance. We propose a model for the regulation of doubly uniparental inheritance that is consistent with these observations.     

Male Transmission of Linear Plasmids and Mitochondrial DNA in the Fungus Neurospora

One of the general rules of heredity is that in anisogamous matings genetic elements in organelles are inherited maternally. Nevertheless, there are cases of paternal transmission, both as rare exceptions, and as regular modes of inheritance. We report two new cases of paternal transmission in crosses of the model fungus Neurospora. First, we show leakage of a linear plasmid from males, the first case in fungi and the second in eukaryotes. Transmission frequencies ranged from 1% to 15% in different crosses, but some crosses showed no detectable male transmission. Second, we show leakage of male mitochondrial DNA (mtDNA), the second case in fungi. Some of the resulting progeny have only the male mtDNA type, but some are heteroplasmons. Heteroplasmons show novel restriction fragments attributable to recombination or rearrangement. Heteroplasmy of mtDNA through male transmission has not been reported previously in any eukaryote. In addition we have shown paternal leakage of circular mitochondrial plasmids, supporting another reported case. In a male bearing a linear and a circular plasmid, these plasmids and the mtDNA are transmitted in different combinations. These results show a potential for mitochondrial segregation and assortment during the sexual cycle in anisogamous fungi, pointing to more potential avenues for novel associations between genomic compartments, and between genomic and extragenomic elements.     

Sexual selection favors female-biased sex ratios: the balance between the opposing forces of sex-ratio selection and sexual selection

In a verbal model, Trivers and Willard proposed that, whenever there is sexual selection among males, natural selection should favor mothers that produce sons when in good condition but daughters when in poor condition. The predictions of this model have been the subject of recent debate. We present an explicit population genetic model for the evolution of a maternal-effect gene that biases offspring sex ratio. We show that, like local mate competition, sexual selection favors female-biased sex ratios whenever maternal condition affects the reproductive competitive ability of sons. However, Fisherian sex-ratio selection, which favors a balanced sex ratio, is an opposing force. We show that the evolution of maternal sex-ratio biasing by these opposing selection forces requires a positive covariance across environments between the sex-ratio bias toward sons (b) and the mating success of sons (r). This covariance alone is not a sufficient condition for the evolution of maternal sex-ratio biasing; it must be sufficiently positive to outweigh the opposing sex-ratio selection. To identify the necessary and sufficient conditions, we partition total evolutionary change into three components: (1) maternal sex-ratio bias, (2) sexual selection on sons, and (3) sex-ratio selection. Because the magnitude of the first component asymmetrically affects the strength of the second, biasing broods toward females in a poor environment evolves faster than the same degree of bias toward males in a good environment. Consequently, female-biased sex ratios, rather than male-biased sex ratios, are more likely to evolve. We discuss our findings in the context of the primary sex-ratio biases observed in strongly sexually selected species and indicate how this perspective can assist the experimental study of sex ratio evolution.     

Sex determination in crayfish: are intersex Cherax quadricarinatus (Decapoda, Parastacidae) genetically females?

In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1:1 sex ratio in the F1 progeny. Crosses between intersex individuals and females yielded a 1:3 (male:female) sex ratio in most crosses. According to our hypothesis, one-third of the females produced in a cross of a female with an intersex animal should be WW females. The hypothesis was tested by crossing normal males with F1 females, which were progeny of intersex fathers. These crosses yielded almost 100% females, a finding that conforms to the above-suggested sex determination model for C. quadricarinatus and the female WZ genotype of intersex individuals.     

Single-locus complementary sex determination in the inbreeding wasp Euodynerus foraminatus Saussure (Hymenoptera: Vespidae)

The Hymenoptera have arrhenotokous haplodiploidy in which males normally develop from unfertilized eggs and are haploid, while females develop from fertilized eggs and are diploid. Multiple sex determination systems are known to underlie haplodiploidy, and the best understood is single-locus complementary sex determination (sl-CSD) in which sex is determined at a single polymorphic locus. Individuals heterozygous at the sex locus develop as females; individuals that are hemizygous (haploid) or homozygous (diploid) at the sex locus develop as males. sl-CSD can be detected with inbreeding experiments that produce diploid males in predictable proportions as well as sex ratio shifts due to diploid male production. This sex determination system is considered incompatible with inbreeding because the ensuing increase in homozygosity increases the production of diploid males that are inviable or infertile, imposing a high cost on matings between close relatives. However, in the solitary hunting wasp Euodynerus foraminatus, a species suspected of having sl-CSD, inbreeding may be common due to a high incidence of sibling matings at natal nests. In laboratory crosses with E. foraminatus, we find that sex ratios and diploid male production (detected as microsatellite heterozygosity) are consistent with sl-CSD, but not with other sex determination systems. This is the first documented example of sl-CSD in a hymenopteran with an apparent natural history of inbreeding, and thus presents a paradox for our understanding of hymenopteran genetics.     

Heritable determinants of left-right bias in the rat

The offspring of matings of rats having opposite or same-sided turning biases were tested for turning biases as adults and the degree of similarity to the parents' biases assessed. There were significant and equivalent tendencies for the male offspring to have the same bias as the male parent and the opposite bias as the female parent. Although, overall, female offspring were distributed randomly with respect to the parents' biases, a significant tendency for female offspring to have biases opposite those of the female parent was apparent in litters having more males than females. Based on reports indicating a relationship between the sex ratio of a litter and levels of testosterone in female fetuses, it was suggested that $\text{in utero}$ exposure to testosterone reverses the coding of a heritable female influence and induces a tendency for the offspring to have biases opposite those of the female parent. The origins of sidedness in the rat appear to involve a complex interaction between heredity and hormones.     

Uniparental Inheritance of Mitochondrial Genes in Yeast: Dependence on Input Bias of Mitochondrial DNA and Preliminary Investigations of the Mechanism

In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes, which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsp5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These classes of models are distinguished genetically by the fact that the first predicts that the output frequency of a given allele among the progeny of a large number of zygotes will approximately equal the average input frequency of that allele, while the second class predicts that any input bias will be amplified in the output. The data suggest that bias amplification does occur. We hypothesize that maternal inheritance of mitochondrial or chloroplast genes in many organisms may depend upon a biased input of organelle DNA molecules, which usually favors the maternal parent, followed by failure of the minority (paternal) molecules to replicate in many or all zygotes.     

Female‐dependent transmission of paternal mtDNA is a shared feature of bivalve species with doubly uniparental inheritance (DUI) of mitochondrial DNA

Several species from a number of bivalve molluscan families are known to have a paternally transmitted mitochondrial genome, along with the standard maternally transmitted one. The main characteristic of the phenomenon, known as doubly uniparental inheritance (DUI), is the coupling of sex and mtDNA inheritance: males receive both genomes but transmit only the paternal to their progeny; females either do not have the paternal genome or, if they do, they do not transmit it to their progeny. In the families Mytilidae and Veneridae, both of which have DUI, a female individual is either female‐biased (it produces only, or nearly so, female progeny), male‐biased (it produces mainly male progeny) or non‐biased (it produces both genders in intermediate frequencies). Here we present evidence for a same pattern in the freshwater mussel, Unio delphinus (Unionidae). These results suggest that the maternal control of whether a fertilized egg will develop into a male or a female individual (and the associated feature of whether it will inherited or not inherit the paternal mtDNA) is a general characteristic of species with DUI.     

Mitochondria are inherited from the MATa parent in crosses of the basidiomycete fungus Cryptococcus neoformans

Previous studies demonstrated that mitochondrial DNA (mtDNA) was uniparentally transmitted in laboratory crosses of the pathogenic yeast Cryptococcus neoformans. To begin understanding the mechanisms, this study examined the potential role of the mating-type locus on mtDNA inheritance in C. neoformans. Using existing isogenic strains (JEC20 and JEC21) that differed only at the mating-type locus and a clinical strain (CDC46) that possessed a mitochondrial genotype different from JEC20 and JEC21, we constructed strains that differed only in mating type and mitochondrial genotype. These strains were then crossed to produce hyphae and sexual spores. Among the 206 single spores analyzed from six crosses, all but one inherited mtDNA from the MATa parents. Analyses of mating-type alleles and mtDNA genotypes of natural hybrids from clinical and natural samples were consistent with the hypothesis that mtDNA is inherited from the MATa parent in C. neoformans. To distinguish two potential mechanisms, we obtained a pair of isogenic strains with different mating-type alleles, mtDNA types, and auxotrophic markers. Diploid cells from mating between these two strains were selected and 29 independent colonies were genotyped. These cells did not go through the hyphal stage or the meiotic process. All 29 colonies contained mtDNA from the MATa parent. Because no filamentation, meiosis, or spore formation was involved in generating these diploid cells, our results suggest a selective elimination of mtDNA from the MATalpha parent soon after mating. To our knowledge, this is the first demonstration that mating type controls mtDNA inheritance in fungi.     

Nestling sex ratios in a population of Bluethroats Luscinia svecica inferred from AFLP™ analysis

We studied the sex ratio of Bluethroat Luscinia svecica broods using AFLPs. Our aim was to test whether there is a bias towards males that could be explained by sexual selection theories, or conversely, a bias towards females that could help explain the female-biased sex ratio among juveniles observed at a wintering site. The AFLP technique was reliable in sexing the nestlings from even small initial DNA quantities. Given the large number of polymorphic markers that can be obtained for each primer combination, the probability of detecting a W-chromosome-linked fragment is reasonably high. As a consequence, this method could be used in other species for sex-ratio studies and for other genetic purposes. Among 246 nestlings, we found an overall proportion of males of 50.8% at hatching and the sex-ratio variation using broods as independent units was not significantly different from expectation under a binomial distribution. None of the parental and environmental variables tested changed significantly the deviance to the model. Thus, sex determination in the Bluethroat seems to match the classical Mendelian model of a 1:1 sex ratio and cannot explain the biased sex ratio towards juvenile females found at the wintering site.