Yolk Androgens Research Articles

Migratory constraints on yolk precursors limit yolk androgen deposition and underlie a brood reduction strategy in rockhopper penguins

Hormonally mediated maternal effects link maternal phenotype and environmental conditions to offspring phenotype. The production of lipid-rich maternal yolk precursors may provide a mechanism by which lipophilic steroid hormones can be transported to developing yolks, thus predicting a positive correlation between yolk precursors in mothers and androgen levels in eggs. Using rockhopper penguins (Eudyptes chrysocome), which produce a two-egg clutch characterized by extreme egg-size dimorphism, reversed hatching asynchrony and brood-reduction, we examined correlations between circulating concentrations of the primary yolk-precursor vitellogenin (VTG) and levels of yolk androgens. Previous work in Eudyptes penguins has shown that egg-size dimorphism is the product of migratory constraints on yolk precursor production. We predicted that if yolk precursors are constrained, androgen transport to developing yolks would be similarly constrained. We reveal positive linear relationships between maternal VTG and androgens in small A-eggs but not larger B-eggs, which is consistent with a migratory constraint operating on the A-egg. Results suggest that intra-clutch variation in total yolk androgen levels depends on the production and uptake of yolk precursors. The brood reduction strategy common to Eudyptes might thus be best described as the result of a migratory constraint.

Long‐term fitness consequences of high yolk androgen levels: sons pay the costs

Summary1. Early growth conditions, for example hormonal environment during embryonic development, may have long‐lasting effects on behaviour and phenotype, with subsequent fitness consequences. Yolk androgens have been shown to affect various offspring traits in the short‐term, but fitness consequences for either offspring or parents, a prerequisite for studying the adaptiveness of this maternal effect, are poorly known in the wild.2. We experimentally elevated yolk androgen levels of whole clutches in a wild population of collared flycatchers (Ficedula albicollis) and investigated the long‐term effects of yolk androgens on offspring local recruitment, parental return rate, and timing and success of breeding in both parents and offspring in the breeding season(s) following the manipulation.3. Yolk androgen elevation lowered the local recruitment rate of male, but not female offspring, but had no effect on any breeding parameters of offspring of either sex. Furthermore, yolk androgen elevation of the clutch had no effect on the return rate or any breeding parameters of the parents.4. Our results indicate that high yolk androgen levels may impose a potential direct fitness cost for male offspring, but no long‐lasting additional indirect fitness costs for parents (direct costs of transferring androgens to eggs not considered). Such a sex‐specific cost on offspring could constrain yolk androgen deposition and select for sex‐specific deposition mechanisms.5. As yolk androgen levels in this population are heritable, our results on long‐term fitness effect of yolk androgens support the idea that hormone‐mediated maternal effects may evolve under selection and thereby affect evolutionary processes.

Relationships between yolk androgens and nest density, laying date, and laying order in Western Burrowing Owls (Athene cunicularia hypugaea)

Increases in yolk androgens within and among avian clutches have been correlated with decreased incubation time, increased aggression within a nest, increased begging behaviour, decreased immune response, and decreased life span. Although the mechanisms that lead to variability in yolk androgens within and between clutches are not completely known, yolk androgens can be a function of both social and environmental conditions. We were interested in if and how nesting density, laying date, and laying order influenced yolk androgens in Western Burrowing Owls ( Athene cunicularia hypugaea (Bonaparte, 1825)) in which nest density varies considerably. In 2006 and 2007, we used radioimmunoassay to quantify the concentrations of testosterone, 5α-dihydrotestosterone, and androstenedione in the egg yolks from one early and one late-laid egg in 47 nests of Burrowing Owls located in the Morley Nelson Snake River Birds of Prey National Conservation Area in southern Idaho. Nesting density had no detectable effect on yolk androgens. Yolk androgens varied temporally and peaked in the middle of the laying season while being low before and after this time period. Within nests, late-laid eggs had higher testosterone and dihydrotestosterone than early-laid eggs; adrostendione exhibited a similar pattern in one but not both years of our study. It is possible that the seasonal pattern in yolk androgens that we observed is related to aspects of mate quality for females or declining chances of fledging success for later nesting females, whereas rises in egg androgens between early and late eggs within clutches could reflect a mechanism to assist nestlings from late-laid eggs that hatch one to several days after their siblings to better compete for resources within the nest or promote survival in the presence of larger siblings.

Growth and innate immunity are not limited by selection for high egg testosterone content in Japanese quail

The effects of maternal androgens on fitness-related traits of offspring are generally assumed to be epigenetic adaptations to the environment that may be encountered by the next generation. Possible constraints of high yolk androgen transfer are still not understood, although a suppressed immune response in offspring is frequently considered. The aim of our study was to examine the innate immune defence in high (HET) and low egg testosterone (LET) lines of Japanese quail, which differ in the hormonal milieu of their eggs, thus providing a good physiological model for the study of androgen-mediated maternal effects. Acute phase response was induced by a lipopolysaccharide injection in 12-day-old quail and plasma corticosterone and the heterophil:lymphocyte ratio were measured at 1 and 3 h post-treatment. Basal levels of non-specific antibodies (IgY) were determined in the circulation. We found that HET quail were heavier than LET quail from the second week of age, indicating enhanced post-hatching growth. At 1 h post-lipopolysaccharide challenge, plasma corticosterone concentrations increased in the HET but not in the LET line. The heterophil:lymphocyte ratio rose in both lines at 3 h post-immune challenge, with a more pronounced response in HET quail. Moreover, HET chicks displayed higher IgY levels than LET chicks, suggesting either enhanced passive immunoprotection or stimulated endogenous antibody production. In conclusion, our data demonstrate that the genetic selection for high egg testosterone content positively influences growth and, simultaneously, does not limit the acute phase response in young quail.

Food conditions affect yolk testosterone deposition but not incubation attendance

In many bird species with hatching asynchrony, yolk androgens increase across the laying sequence. This has been hypothesized to represent a compensatory mechanism for disadvantages of later-hatching chicks – via positive effects of yolk androgens on early competitiveness and growth. However, the costs and benefits of this compensatory strategy probably depend on environmental factors determining the survival chances of the chicks such as the food conditions, which should, therefore, influence maternal yolk androgen deposition.We studied the consequences of manipulated food conditions on the expected level of hatching asynchrony in canaries (Serinus canaria) assigning females to either a low (=LQ) or high quality (=HQ) diet. We measured the incubation behaviour (as incubation attendance) and the yolk androgen deposition in order to investigate whether and how females modulate hatching asynchrony in relation to the food conditions.Females on a HQ diet laid larger and heavier clutches, showed a stronger increase in yolk testosterone content towards the last-laid eggs, but did not alter their incubation attendance. Thus, females on a HQ diet seem to favour the survival of later hatching chicks, as indicated by their yolk testosterone deposition pattern. However, females on a HQ diet laid larger clutches and might need to compensate more in order to achieve a similar degree of hatching asynchrony than females on a LQ diet, given the lack of plasticity in incubation attendance. This suggests that canary females respond to food manipulations mainly via changes in clutch size rather than by altering the degree of hatching asynchrony.

Mind the gap: the ratio of yolk androgens and antioxidants varies between sons and daughters dependent on paternal attractiveness

Females are expected to partition resources between offspring in a context-dependent way to maximise total fitness returns from a reproductive attempt. Female zebra finches (Taeniopygia guttata) vary the allocation of yolk androgens and antioxidants among offspring. Importantly, the balance between androgens and antioxidants in yolks may be more important than their independent absolute amounts in terms of fitness consequences for developing young. Therefore, we tested whether the relative allocation of these two resources in yolks varies according to either the Trivers–Willard, positive or compensatory maternal investment hypothesis. We manipulated male attractiveness using coloured leg bands (red-banded males appear attractive; green-banded males, unattractive) and measured yolk androgens and antioxidants in each egg, egg sex, clutch sex ratio and female condition. While female zebra finches manipulated the balance of androgens and antioxidants within and between clutches in response to mate attractiveness, offspring sex and their own condition, they did not do so in a way that consistently followed any of the hypotheses. Mothers paired with unattractive males allocated a larger antioxidant/androgen ratio to daughters than sons. This pattern was reversed when paired to an attractive male; sons received a larger antioxidant/androgen ratio than daughters. We also found offspring sex ratio decreased with increasing female condition for unattractive males, but not for attractive males. However, without knowing the fitness consequences of the balance of different egg constituents, it is difficult to interpret the patterns consistently in terms of the Trivers–Willard, compensatory and positive investment hypotheses.

Geographical Variation in Egg Mass and Egg Content in a Passerine Bird

Reproductive, phenotypic and life-history traits in many animal and plant taxa show geographic variation, indicating spatial variation in selection regimes. Maternal deposition to avian eggs, such as hormones, antibodies and antioxidants, critically affect development of the offspring, with long-lasting effects on the phenotype and fitness. Little is however known about large-scale geographical patterns of variation in maternal deposition to eggs. We studied geographical variation in egg components of a passerine bird, the pied flycatcher (Ficedula hypoleuca), by collecting samples from 16 populations and measuring egg and yolk mass, albumen lysozyme activity, yolk immunoglobulins, yolk androgens and yolk total carotenoids. We found significant variation among populations in most egg components, but ca. 90% of the variation was among individuals within populations. Population however explained 40% of the variation in carotenoid levels. In contrast to our hypothesis, we found geographical trends only in carotenoids, but not in any of the other egg components. Our results thus suggest high within-population variation and leave little scope for local adaptation and genetic differentiation in deposition of different egg components. The role of these maternally-derived resources in evolutionary change should be further investigated.

Female great tits do not alter their yolk androgen deposition when infested with a low-transmittable ectoparasite

In birds as in many other taxa, parasites can have deleterious effects on offspring development. Therefore, avian mothers have evolved responses to counteract parasite virulence in offspring via transgenerational defense mechanisms that is the transfer of immune-enhancing substances such as antibodies to their eggs. Another maternal pathway is suggested by the finding that infested great tit mothers produced eggs with lower androgens, since these yolk androgens are immunosuppressive and potentially affect parasite susceptibility of the nestlings. However, whether this pathway is a specific adaptation to infestation with parasites that affect the offspring or an epiphenomenon of lower androgen production in the female due to the parasite effects on the mother itself is as yet unclear. In this study we infested female great tits (Parus major) with sheep ticks (Ixodes ricinus), which are nonnidicolous ectoparasites with low vertical transmission capability, and evaluated the effects on yolk androgen deposition. Tick-infested females did not significantly reduce their deposition of androgens (androstenedione (A4) and testosterone) compared to tick-reduced females, which is in contrast to a previous study showing a lowered deposition of A4 and testosterone when females were exposed to the nidicolous hen flea. Thus, females alter their hormone deposition, and thus likely offspring phenotype, when exposed to parasites that also form the parasitic environment of their offspring, but not when temporarily infested with the field-dwelling sheep ticks with low transmission capability. This suggests that selection favored the evolution of an adaptive transgenerational effect by acting mainly on the parasite-induced maternal effect.

A Place to Hide in the Home-Cage Decreases Yolk Androgen Levels and Offspring Emotional Reactivity in Japanese Quail

An animal's emotional responses are the result of its cognitive appraisal of a situation. This appraisal is notably influenced by the possibility of an individual to exert control over an aversive event. Although the fact that environment controllability decreases emotional responses in animals is well established, far less is known about its potential trans-generational effects. As the levels of avian yolk hormones can vary according to the mother's environment, we hypothesized that housing environment of mothers would modulate the quality of her eggs and in turn her offspring's behaviour. Two groups of female Japanese quail were constituted: a group that had access to a place to hide in their home-cage (Hd, n = 20) and a group that had nowhere to hide (NoHd, n = 20) when stressed. Both groups were submitted to daily human disturbances for a twenty-day-period. Hd females produced eggs with both less testosterone and androstenedione than did NoHd females. The emotional and social reactivity of Hd females' offspring were lower and their growth was slower than those of NoHd females' offspring. Our results show that a minor difference in housing environment had substantial effects on eggs and offspring. The presence of a shelter probably helped quail to cope with daily human disturbances, producing less reactive offspring. This transgenerational effect caused by an opportunity to hide could lead to applications in care of laboratory animals, conservation biology and animal welfare.

Negative effects of yolk testosterone and ticks on growth in canaries

Maternal yolk hormones in bird eggs are thought to adjust the offspring to the post-hatching environment. This implies that the effects of maternal yolk hormones should vary with the post-hatching environment, but to date such context-dependency has largely been ignored. We experimentally increased yolk testosterone concentrations in canary eggs and simultaneously manipulated the post-hatching context via an experimental tick-infestation of the chicks. This allows us to evaluate the context-dependency of hormone-mediated maternal effects, as it has previously been shown that ectoparasites alter the maternal yolk androgen deposition. The experimental tick infestation reduced growth in chicks from sham-treated eggs, indicating harmful effects of this ectoparasite in canaries. Chicks from testosterone-treated eggs were not affected in their development by ticks, suggesting lower ectoparasite vulnerability. But this may also be due to the fact that experimentally elevated yolk testosterone levels impaired growth even under parasite-free conditions. This contrasts previous studies, but these studies often manipulated first laid eggs, while we used eggs of subsequent laying positions. Later laid eggs are presumably of lower quality and contain higher yolk testosterone concentrations. Thus, the effects of elevated yolk testosterone on growth may be dose-dependent or vary with the egg quality, suggesting prenatal context-dependency.

Differential Maternal Testosterone Allocation among Siblings Benefits Both Mother and Offspring in the Zebra Finch Taeniopygia guttata

Parents are selected to preferentially invest in the offspring with highest reproductive value. One mechanism for achieving this is the modification of competitive asymmetries between siblings by maternal hormones. In many organisms, offspring value varies according to birth position in the brood, which determines survival chances and competitive advantage over access to resources. In birds, variation in yolk androgen allocation over the laying sequence is thought to modulate dominance of senior chicks over junior brood mates. We tested this hypothesis in zebra finches, which show a naturally decreasing pattern of within-clutch testosterone allocation. We abolished these within-clutch differences by experimentally elevating yolk testosterone levels in eggs 2-6 to the level of egg 1, and we assessed fitness measures for junior offspring (eggs 2-6), senior offspring (egg 1), and their mothers. Testosterone-injected eggs hatched later than control eggs. Junior, but not senior, chicks in testosterone-treated broods attained poorer phenotypic quality compared to control broods, which was not compensated for by positive effects on seniors. Mothers were generally unaffected by clutch treatment. Thus, naturally decreasing within-clutch yolk testosterone allocation appears to benefit all family members and does not generally enhance brood reduction by favoring senior chicks, in contrast to the widely held assumption.

Effects of divergent selection for yolk testosterone content on growth characteristics of Japanese quail

Effects of yolk androgens on postnatal growth of offspring have been widely studied but their physiological role in the growth control is not fully understood due to an inconsistency in obtained results. We investigated androgen-mediated maternal effects on postnatal growth in relation to endocrine control mechanisms using two lines of Japanese quail divergently selected for high (HET) and low (LET) egg testosterone (T) content. Embryonic growth did not differ between the lines. During the growth period HET quail were heavier and displayed longer tarsi as compared with LET quail, with more pronounced line differences in males than females. HET males were heavier than LET males from the age of 2 weeks, reached the age of maximum growth rate earlier, and displayed higher asymptotic body weight than LET males. Accelerated growth in HET males was not accompanied by increased postembryonic plasma T concentrations. Plasma triiodothyronine levels did not differ between lines while plasma thyroxine levels were decreased in HET as compared with LET female chicks. Line differences in body weight disappeared in adult quail suggesting that yolk androgens, increased in a physiological way, resulted in stimulation of juvenile growth rate in precocial Japanese quail under stable social and environmental conditions.

Patterns of maternal yolk hormones in eastern screech owl eggs (Megascops asio)

Owl clutches typically hatch asynchronously, and brood size hierarchies develop. In this study, we describe intra-clutch variation of testosterone, androstenedione, estradiol, and corticosterone in Eastern screech owl egg yolks. In order to assess whether these hormones may have originated in the follicle, we also characterize variation of testosterone, androstenedione, and corticosterone within the exterior, intermediate, and interior regions of the yolk. Concentrations of testosterone and androstenedione were distributed relatively evenly across egg lay order with the exception of first-laid eggs that had significantly lower concentrations of both androgens than eggs later in the laying sequence. Corticosterone and estradiol did not vary with laying order. Our results suggest that when food is abundant, yolk hormones are deposited in patterns that minimize sibling differences except to reduce dominance by the first-hatching chick. Testosterone and androstenedione concentrations varied throughout the yolk, while corticosterone was evenly distributed throughout the yolk. This supports a follicular origin for both yolk androgens, and an adrenal origin for yolk corticosterone.

Yolk androgen deposition without an energetic cost for female rockhopper penguins: a compensatory strategy to accelerate brood reduction?

Whether androgen deposition in eggs is physiologically costly for female birds has remained a crucial but unsolved question, despite a broad use of this assumption in functional studies. We tested whether females depositing high androgen concentrations experienced higher mass losses than females depositing low androgen concentrations. Analysing female body mass change during egg formation in rockhopper penguins (Eudyptes chrysocome chrysocome), we observed no energetic cost to androgen deposition. Nevertheless, lighter females laid eggs with higher yolk androgen concentrations. This relationship existed only for the second-laid egg (B-egg), but not for the first-laid egg (A-egg). Since the B-egg is usually the first to hatch and the only one to produce a fledging chick, we hypothesize that differential yolk androgen deposition may be an adaptive strategy for females to affect brood reduction.

Yolk androgens in great tit eggs are related to male attractiveness, breeding density and territory quality

Females can adaptively adjust phenotype of their offspring via deposition of various compounds into eggs, including androgens and other hormones. Here, I investigated how egg yolk androgens (testosterone and androstenedione) related to environmental conditions and parental traits in the great tit (Parus major) across three breeding seasons. Male and female traits studied included age, condition and multiple feather ornaments, both carotenoid- and melanin-based (carotenoid and UV chroma of yellow breast feathers, area of black breast band and white cheek immaculateness). Yolk mass increased with laying temperature, laying date and area of male black breast band. Concentration of androgens increased with breeding density, territory quality and carotenoid chroma of male yellow breast feathers and was higher in mates of 1 year old as compared to older males. Yolk androgens were not related to any of the female traits analysed. These patterns were thus consistent with (1) social and environmental effects on yolk mass and composition and (2) both positive and negative differential allocation strategies of resource allocation in females. Overall, male traits were the most important predictors of egg yolk characteristics in this socially monogamous songbird.

Yolk androgen deposition in rockhopper penguins, a species with reversed hatching asynchrony

To maximize fitness, females should invest optimally in the siblings within a litter or brood and adapt this investment to environmental conditions. Chick mass and yolk androgens have been shown to influence the outcome of sibling competition. In birds, asynchronous hatching plays a major role in this process and often leads to brood reduction. We studied maternal deposition of yolk androgens in eggs of southern rockhopper penguins (Eudyptes chrysocome chrysocome). Contrary to other avian models, laying and hatching sequences do not coincide in this species, which exhibits reversed hatching asynchrony. This provides a unique model to test whether the first egg to hatch (B-egg), which is the most likely to survive, differs in composition from the second egg to hatch (A-egg). We found that B-eggs had higher egg masses, yolk masses, yolk androgen concentrations and total yolk androgen amounts than A-eggs. This was observed consistently for the three androgens analyzed (testosterone, androstenedione and 5α-dihydrotestosterone). Laying date affected androgen deposition into A- and B-eggs differently. Interestingly, late clutches had proportionally higher androgen levels in the B-egg compared to the A-egg than early clutches. We discuss these results in relation to the chronology of egg formation and the potential effect of the observed differences on embryo development and brood reduction.

Digit ratios have poor indicator value in a wild bird population.

Early androgen exposure is known to have long-lasting effects on phenotype, behaviour and even fitness, but difficulties in measuring the exposure hinders the study of its importance in evolutionary context. Digit ratios have been highlighted as a potential easy-to-measure indicator of early steroid exposure, as they have been suggested to reflect steroid, mainly testosterone levels during prenatal development. However, evidence for digit ratios reflecting early steroid levels is weak, as experimental studies, especially in wild populations, are scarce. We studied the association between maternally derived yolk androgens and digit ratios (2D:4D, 2D:3D and 3D:4D) using both correlative data and a rather high level of experimental elevation of yolk androgens in a passerine bird, the pied flycatcher (Ficedula hypoleuca). We also examined whether digit ratios have indicator value in an evolutionary context by studying correlations between digit ratios and reproductive traits, secondary sexual traits and exploratory behaviour. We did not find any association between digit ratios and yolk androgen level either in correlative or experimental data. Digit ratios were neither related to any of the reproductive and secondary sexual traits or exploratory behaviour measured. There was, however, a sex difference in 2D:3D and 3D:4D of adult birds (due to second and fourth digits being shorter in females), which was not apparent in fledglings or captivity-raised juveniles. This suggests that either the sex difference may develop as late as during the sexual maturation for breeding. These results indicate that, in this species, digit ratios are not reliable markers of maternally derived yolk androgen exposure and that they bear little relevance as correlates of the adaptive traits we measured.

Yolk androstenedione, but not testosterone, predicts offspring fate and reflects parental quality

Yolk androgen deposition is a widely investigated maternal effect in birds, but its adaptive value is at present unclear. The offspring fitness correlates of natural yolk androgen levels are virtually unknown, whereas manipulations largely focused on testosterone and neglected other androgens. We determined yolk concentrations of the 2 dominant androgens, androstenedione and testosterone, from all eggs in collared flycatcher clutches and followed the fate of individual offspring from these eggs in a crossfostering experiment. Yolk concentration of androstenedione was much higher than that of testosterone. Offspring from eggs with relatively higher androstenedione concentrations within a clutch were relatively large after hatching, grew slower thereafter, and had a higher recruitment rate in subsequent years. The increase of androstenedione with laying order and its within-clutch variance were negatively correlated with a condition-dependent female ornament, perhaps indicating compensatory hormone deposition into later hatching eggs by females in low condition. Yolk testosterone variation within or among clutches was not related to any measured aspect of offspring or parental quality. Our results suggest that in some species, especially those with much more androstenedione than testosterone in the yolk, androstenedione and not testosterone may be the yolk androgen with a long-term function and adaptive deposition pattern. Key words: androgen, female ornament, growth, maternal effect, recruitment. [Behav Ecol]

Yolk hormones and sexual conflict over parental investment in the pied flycatcher

Female birds might be able to manipulate the parental effort of their male partner through elevated transfer of hormones to the eggs, since these hormones affect many chick traits that males might use as cues for adjusting the level of their investment. We experimentally studied whether female pied flycatchers Ficedula hypoleuca could manipulate male investment via yolk androgens. There is much more variation in yolk androgen levels between females than within clutches, and in order to change the androgen levels of the eggs, we swapped whole clutches between nests. To estimate the androgen levels of the clutch, we measured the androgen content of a single egg per clutch. Females did not succeed in manipulating male effort using yolk androgens, since there was no relationship between the division of parental care within a pair and either original or foster egg androgen levels. One of these relationships should have occurred if females were manipulating males. The proportion of feeding visits by the male was higher when the male was old (55%) than when he was young (45%) and females laid eggs with higher androgen levels when mated with a young male. Young males did not exhibit any responses to yolk androgen levels either, which indicates that females cannot exploit their effort more than that of old males. We suggest that females may allocate yolk androgens to adjust the growth trajectories of the chicks to poor growing conditions when mated with young males that are poor providers or occupying a poor territory.

Egg‐yolk androgen and carotenoid deposition as a function of maternal social environment in barn swallowsHirundo rustica

Evidence is mounting that female animals use egg‐yolk compounds (e.g. steroids, antioxidants) to adaptively engineer the quality of their offspring as a function of several maternal and environmental factors. Though adjustments to yolk allocation have been well‐characterized as a function of parental phenotypes, we know very little about how an individual's social environment influences yolk composition. Here, we consider how two types of yolk compounds, androgens and carotenoids, relate to the maternal social environment during the egg‐laying period, controlling statistically for known correlations between various aspects of parental quality and egg yolk compounds. Barn swallowsHirundo rustica erythrogasterbreed in groups of highly variable size and spacing, allowing us to test whether or not the social environment is correlated with these maternal effects. We found no relationship between carotenoid levels in eggs as a function of colony size, colony density, or nearest‐neighbor distance. However, eggs from females in larger groups had lower concentrations and total amounts of yolk androgens than those from females in smaller, less dense social settings. Our results counter previous predictions and literature, showing that females breeding in large groups deposit more androgen in eggs, mechanistically, because they compete more with conspecifics and have higher circulating androgen levels themselves and, functionally, because it could be advantageous for their offspring to show high androgen‐mediated competitive abilities early in life. Instead, because group size in this species is governed largely by site fidelity and the availability of old nests for re‐use, and because reproductive output does not differ as a function of group size, it may be that competition is greater for limited nests in small groups, thus elevating androgen levels. Further, yolk androgens were previously shown to be affected by male quality, and the greater concentrations and amounts of yolk androgens in smaller sites may reflect differential allocation to darker males found at these sites.