S08.1: Avian sex ratio distortions: The myth of maternal control

Abteilung Sinnesbiologie, Institut für Biologie, Humboldt-Universität, Invalidenstr. 43, D-10115 Berlin, Germany, fax 30 2093 8491, e-mail sven.krackow@rz.hu-berlin.de

With female heterogamety, the primary sex ratio is determined at the first meiotic division shortly before ovulation, in birds. Up to now, there is no evidence of any physiological mechanism leading to hatching sex ratio deviations. The primary follicles in prophase I are already supplied with considerable amounts of yolk and chromosomal segregation distortion appears to be highly constrained by the mechanism of meiotic division. Also, genetic interests of gametes strongly oppose any maternal attempt at control of the sex ratio. Hence, sex ratio manipulation by preferential ovulation of Z- or W-ova is argued to be implausible. More probably, to avoid any further loss of investment, sex ratio adjustment should take place early after ovulation and fertilisation via sex-specific termination of embryonic development before initiation of egg shell formation. I suggest that the widely reported influence of the position within the egg laying sequence on offspring sex could potentially serve as an explanation for a wide range of sex ratio effects found in birds. The sequence effect, in turn, might be caused by changes in concentrations of yolk testosterone over the laying sequence. This effect could be sex-specific if non-dosage compensated gene products of Z-chromosomes counteracted any adverse effects on embryonic survival of deviating testosterone levels.

'Significant variation in the sex ratio at hatching is unusual in birds.' (Clutton-Brock 1986)

Since T.H. Clutton-Brock's above conclusion, there has been a considerable flow of reports on avian sex ratio distortions that has now become further stimulated by the advent of molecular sexing techniques (e.g. Appleby et al. 1997; Bradbury et al. 1997; Ellegren et al. 1996; Graves et al. 1993; Griffiths et al. 1996; Lessells & Mateman 1998; May et al. 1993; Millar et al. 1992; Nakamura et al. 1990; Quinn 1999; Westerdahl et al. 1997; Zimmer et al. 1997). Factors invoked in clutch sex ratio adjustment are diverse, including prey abundance (Appleby et al. 1997; Arnold 1989), rainfall (Burley et al. 1989), season (Dijkstra et al. 1990; Zijlstra et al. 1992), habitat quality (Komdeur 1996; Wiebe & Bortolotti 1992), year of study (Tella et al. 1996), clutch sequence (Heinsohn et al. 1997), parental condition (Decoux 1997), clutch size (Dijkstra et al. 1998), diet (Bradbury & Blakey 1998), feeding regimen (Kilner 1998), maternal age (Blank & Nolan 1983) and dominance status (Leonard & Weatherhead 1996), mate quality (Svensson & Nilsson 1996) and sexual attractiveness (Ellegren et al. 1996). Though such effects are sometimes regarded as being of adaptive significance (see accompanying symposium papers), they are either unique (i.e. there is no other study of a respective factor in a particular species) or inconsistent between studies. Hence, doubts have been raised as to their functional explanation (e.g. Cooke & Harmsen 1983; Fiala 1981; Koenig & Dickinson 1996; Krackow 1993; Leroux & Bretagnolle 1996; Ryder & Termaat 1987; Tella et al. 1996).

The unavoidable reporting bias in favour of significant results, in addition, renders the significance of scattered, weakly significant findings of slightly deviating sex ratios questionable (sensu Festa-Bianchet 1988). Furthermore, evidence relates to sex ratios at hatching at the earliest, thereby leaving the mechanisms leading to such deviations obscure. Hence, there is no evidence of how birds manipulate the sex ratio; notwithstanding the widespread prejudice for maternal control because of female heterogamety in birds (Oddie 1998). However, without knowledge of the mechanism of sex ratio adjustment, the costs of sex ratio deviations - and hence, their adaptive value - cannot be assessed. Knowledge of the mechanism would also help to distinguish false positive from false negative statistical results, as well as separate causal from correlational influences (intercorrelation of potentially influential variables is a common finding in sex ratio studies, e.g., Lessells et al. 1996).

My aim is to evaluate physiological events before egg laying that might be involved in sex ratio variations in response to maternal or environmental factors, and briefly comment on implications of a possible mechanism for functional explanations. I am not specifically concerned with overall sex ratio skews that might exist (Bednarz & Hayden 1991; Bradbury et al. 1997; Gowaty & Lennartz 1985; Ligon & Ligon 1990; McIlhenny 1937) and be adaptive (Emlen et al. 1986, but see Lessells & Avery 1987; Gowaty 1993, but see Blums & Mednis 1996; Olsen & Cockburn 1991, but see Newton & Marquiss 1979). The following argument, of course, does not preclude any other pathway for sex ratio distortions.

To reduce the costs of manipulation (sensu Maynard Smith 1980), sex ratio adjustments should be carried out as early as possible. With female heterogamety in birds, this would imply the selective production of Z- and W-containing ova rather than sperm selection, as in mammals. To achieve sex-selective ovulation, the maternal organism could either distort sex-chromosome segregation to the first polar body, or dispose of oocytes retaining the 'unwanted' sex chromosome (Oddie 1998).

However, sex is determined shortly before ovulation (about half an hour in the chicken, Gallus domesticus; Sturkie 1986) when the first meiotic division proceeds from late prophase I, as commonly found in vertebrates (Gilbert 1997). At that time, the oocyte of the primary follicle is already provisioned with a significant amount of yolk, weighing about 15-18 g in chickens (Sturkie 1986). Disposing of 'wrong-sex' follicles would, therefore, imply some loss of energy already invested, and, furthermore, a delay until the next ovulation (Emlen 1997), as the primary follicle suppresses further development of other follicles during the last days prior to ovulation (Sturkie 1986). The same would apply to selective ovicide as a measure of sex ratio manipulation after ovulation, with energetic costs increasing with developmental stage. Hence, distortion of chromosomal segregation seems superior to disposal of 'wrong-sex' follicles within the ovary or to selective ovicide after ovulation.

There appear to be considerable obstacles to distortion of segregation of the sex chromosomes at the first meiotic division. Metaphase I chromosomes aggregate near the cell surface and, during anaphase I, the spindle apparatus is already directionally determined, i.e. one centrosome is situated in a membrane protrusion that forms the first polar body (Swanson et al. 1981). Hence, the sex of the oocyte is determined when chromosomes have attached to the spindle apparatus during metaphase I. The attachment of kinetochores to the spindle apparatus, on the other hand, appears to be a purely stochastic process (Nicklas 1997). If the directionality of the spindle apparatus pre-determines sex, but the distribution of W- and Z-chromosomes to either side of the spindle is random, sex-chromosome specific polar body formation could not occur.

One might argue that anaphase I need not be accomplished with pre-determined polar body formation, i.e. the two centrosomes could, in theory, be free to move in either direction, after separation of chromosomes. However, this would clearly bear the risk of non-disjunct haploid nuclei. Hence, to avoid meiotic failure, a mechanism assigning the centrosomes to ovum and polar body, respectively, prior to separation of chromosomes seems highly adaptive. Likewise, pre-ovulatory yolk-acquisition and late meiosis are probably related to optimal timing of these activities rather than to sex ratio adjustment (Chandra 1991).

But even if there was a possible mechanism allowing sex chromosomes to segregate non-randomly, there is a strong functional argument against maternal sex ratio adjustment through segregation distortion. Reiss (1987) has pointed out that the sex chromosomes of gametes from the heterogametic sex have no genetic interest in sex ratio deviation, regardless of maternal optima. Gametic autosomes would agree on sex ratio distortions only if the expected fitness of a member of one sex would exceed that of the other sex by at least a factor of three (Reiss 1987). Such a strong fitness effect seems implausible in most avian populations. Hence, any maternal attempt at control of the gamete sex ratio would have to overcome opposing genetic responses by the gamete genome.

The parent-gamete conflict would also predict strong selection for ova to avoid sex-identification by the mother (Reiss 1987), which renders suppression of ovulation of the 'wrong sex' follicles an improbable measure of sex ratio manipulation. Potentially negative effects of atretic primary follicles on subsequent ovulations might further subtract from the value of this measure, though such effects have not yet been studied. The rarity of naturally occurring atretic primary follicles (D'Herde et al. 1996; Saidapur 1978; Waddington et al. 1985) would be in agreement with these conjectures.

After ovulation, sex-specific prevention of fertilisation (i.e. of ova bearing a particular sex-chromosome) appears to be impossible for the maternal organism because sperm is released continuously from the sperm storage tubules from before the time of the first ovulation (Birkhead 1995). Hence, sperm are available in the infundibulum, where fertilisation takes place within minutes of ovulation, at all times.

After fertilisation, there is much less conflict over the optimal sex ratio between parent and offspring than between parent and gamete (Reiss 1987; Trivers 1974). The (autosomal) offspring optima deviate only slightly from maternal ones (Trivers 1974), and sex ratio adjustment to parental optima is expected under a range of conditions (Eshel & Sansone 1994). Furthermore, sex-specific dispensation of further investment and tubal re-absorption appear to be, hypothetically, easily achievable physiological mechanisms exclusively under maternal control. After initiation of uterine egg shell formation (about 4-5 hours following ovulation in hens; Sturkie 1986), manipulation would imply the loss of high amounts of yolk, albumin, and calcium as effective re-absorption of calcified eggs seems unfeasible. Hence, sex ratio adjustment would be expected to commence shortly after ovulation (and fertilisation) but before the egg enters the uterus. Loss of ova by re-absorption would be in agreement with the finding that about 5-40% of ova shed are not oviposited in chickens ('internal laying'; Sturkie 1986; Wood-Gush & Gilbert 1970).

Among the many factors reported to affect avian sex ratios, only the effect of laying sequence has repeatedly been identified in a considerable number of species and populations (see Krackow (1995b) for a review; Clotfelter 1996; Dzus et al. 1996; Kilner 1998). This effect appears to imply that the probability of becoming male differs for eggs in different positions in the egg laying sequence. Such a trend is not seen in all species or populations, and trends may differ between them or even during different stages of the breeding season (Krackow 1995b).

Sequence effects might cause many of the above-mentioned sex ratio distortions: first, biases in early laid eggs need not be balanced by later ones, hence, any differences in clutch size as well as slopes of trends would produce differences in mean sex ratios. Secondly, if either ova very early or late in the sequence would be disadvantaged, skewed clutch sex ratios would result (cf. Krackow 1995a). Temporal limitation of the period of responsiveness of tubal and/or uterine support of egg development in response to a multitude of environmental and maternal factors does not seem implausible. E.g., nutritional effects as well as deviating reproductive hormone levels due to environmental disturbances might shorten this period.

Egg yolk testosterone levels have been shown to increase over the egg laying sequence (Schwabl 1993) and to vary with photoperiod in canaries (Serinus canaria, Schwabl 1996a), and to depend on breeding population density in house sparrows Passer domesticus (Schwabl 1997). Such trends may also differ between species, being absent in Zebra Finches Poephila guttata (Schwabl 1993), and, while levels increase over the laying sequence in canaries (Schwabl 1993), they appear to decrease in the Cattle Egret Bubulcus ibis (Schwabl et al. 1997). In canaries, yolk testosterone levels correlate positively with maternal testosterone concentrations during the main yolk deposition interval, while maternal testosterone levels appear to decrease over the egg laying sequence (Schwabl 1996a).

Obviously, if testosterone levels affected the sex ratio, a sequence effect on sex ratios would result. The strength and shape of such a trend would then depend on a any factor that affected maternal hormone levels that are relevant for yolk testosterone deposition. Even mate attractiveness may alter the endocrine status of the mother, because the yolk testosterone trend in canaries depends on the presence of a male (Schwabl 1993). How could yolk testosterone levels interfere with clutch sex ratios?

In principle, any Z-chromosome specific embryonic gene activity that interacted with yolk testosterone to affect embryo-survival could cause testosterone-dependent sex ratio shifts. This is because, in birds (and in most other female-heterogametic ZW-systems, in contrast to most male-heterogametic XY-systems), the Z-chromosome is not dosage-compensated (Chandra 1991; Jablonka & Lamb 1988). This means that enzymes and other Z-chromosomal gene products are generally found in higher concentrations in male than in female embryos (Baverstock et al. 1982, but see Dominguez-Steglich & Schmid (1993) for an opposite finding of female chicken consistently producing higher levels of Z-chromosome specific ornithine transcarbamylase than male ones). A sex difference would, of course, hold for W-specific gene products, if there were any (cf. Chandra 1994). Inasmuch as embryonic non-dosage compensated Z-chromosomal gene expression could counteract adverse effects of deviating testosterone levels on embryo development, mortality could be sex-specific.

The drawback of this view is that there is direct evidence neither for yolk testosterone nor for embryonic gene activity playing any significant role in pre-laying egg development. Maternal supply of mRNA and gene products is enormous and sufficient for egg formation (Chandra 1991). However, the activities of the egg-shell gland appear to be triggered by hormonal events at about the time of ovulation (Sturkie 1986). This may indicate that some hormonal pathway could potentially mediate effects of embryonic genotype or yolk testosterone on the probability of successful egg-shell formation. Also, low Ca2+-diet induces a decrease or even cessation of laying in hens, but laying in Ca2+-deprived hens is considerably prolonged when treated with a pituitary extract (Sturkie 1986). That means that suppression of egg laying in response to low Ca2+ is hormonally regulated and not a mere physical process.

While significant effects on embryonic development have been determined at hatching (Schwabl 1993; Schwabl 1996b), it is not known whether the natural variation in yolk hormone levels could actually lead to developmental failure before egg-shell formation. Admittedly, counter-acting non-dosage compensated Z-chromosmal gene products have not yet been described.

Most sex ratio deviations in birds are slight and inconsistent between studies (see Introduction). This would, of course, be anticipated if such reports reflect statistical artefacts (sensu Festa-Bianchet 1996). However, egg sequence effects are widespread and could easily result in slight sex ratio biases in response to a range of conditions (see above). Hence, at least some of the phenomena are most probably real rather than artefactual. But that does not automatically imply adaptation.

Some non-dosage compensated Z-chromosomal gene effect that caused slight sex ratio deviations need not be viewed as an adaptive event: on the one hand, it seems probable that non-dosage compensation has evolved for other reasons than to allow maternal sex ratio manipulation (cf. Chandra 1991; Jablonka & Lamb 1988). On the other hand, slight sex ratio deviations imposed by such a constraint might not be counter-selected, as such responses to environmental variables might merely increase genotypic sex ratio variance rather than shifting the overall-sex ratio, which is known to be selectively neutral in reasonably large populations (Kolman 1960). Some loss in energetic investment as a consequence of early egg loss would, therefore, trade-off with the benefits of conserving non-dosage compensation.

Adaptive sex ratio adjustment would require the costs of manipulation to be offset by the benefits. As outlined above, cost-free mechanisms seem not to be available in birds, but selective termination of investment before egg shell production seems not to involve insurmountable costs. Hence, such a mechanism would be expected to be exploited for adaptive sex ratio adjustment if the selection pressure for sex ratio manipulation was appropriately high. Accordingly, it has been argued that the extraordinarily strong sex ratio response to habitat quality found in Seychelles warblers Acrocephalus sechellensis (Komdeur 1996) has evolved in response to exceptionally high benefits due to the peculiar breeding biology of that species (Emlen 1997).

In Seychelles warblers, parental fitness is increased by the presence of helping, predominantly female offspring in good quality habitats, but decreased in poor quality habitats due to the effects of local resource competition; and males disperse earlier than females (Komdeur 1996). Hence, managing to manipulate the sex ratio in order to produce helping female offspring in good habitats and early dispersing males in poor habitats would be adaptive. Sex ratio manipulation might be of much higher adaptive value in this monotocous (i.e. one-egg clutch) species than in polytocous species where a delay of laying within the egg sequence might be more costly than a short delay of a single-egg clutch, and the production of offspring of the helping sex would occur with higher probability by chance (Emlen 1997).

Another extreme case of sex ratio adjustment has been reported in the Eclectus parrot Eclectus roratus (Heinsohn et al. 1997), where mothers produce long sequences of two-egg clutches each containing young of the same sex at fledging. There is no hint as to the adaptive value of this (Heinsohn et al. 1997), nor of the mechanism involved. A strong egg sequence effect, as described above, is, of course, equivalent to a strong sex ratio shifts in birds with very small clutches.

Knowledge of the physiological mechanism behind sex ratio variation is crucial in understanding slight sex ratio deviations, in order to separate real phenomena from statistical coincidence and to assess the adaptive value of adjustments. Unfortunately, there is no evidence of a mechanism of clutch sex ratio adjustment in birds. Physiological reasoning favours mechanisms working after ovulation and before egg shell formation, where slight deviations might result from reproductive constraints. Such sex ratio deviations per se do not automatically imply counter-selection, i.e. may be viewed as non-adaptive by-products of physiological constraints. However, the case of the Seychelles warbler strongly suggests that the absence of more examples of clear adaptive sex ratio manipulation in birds does not reflect an insurmountable physiological constraint, but rather the absence of sufficiently high selection pressure for sex ratio manipulation to evolve in most species.

Critical comments of C.M. Lessells and J.S. Quinn on an earlier version of this manuscript are kindly acknowledged. The author was supported by a grant of the Deutsche Forschungsgemeinschaft (KR 1290/3-1) during this work.

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