Polymorphic male color morphs visualized with steroids in monomorphic females: a tool for designing analysis of sex-limited trait inheritance.

In diploid animals, males and females share most of the genome (except sex-specific elements, such as sex chromosome genes), yet despite sharing the underlying genes that hard-wire traits, males and females may differ in their phenotypes when traits are controlled by proximate mechanisms, such as hormones. In color polymorphic species where coloration is only expressed in one sex, the design of studies of the inheritance of color and coevolved morph-specific traits (e.g. territorial vs sneaker strategies, skewed energetic investment in territorial defense vs sperm production, etc.) is compromised as the expression of morph-coding genes is only visualized in one sex. Here, we circumvented this problem by first characterizing oxidative stress traits in both sexes and then using testosterone implants in females to expose their otherwise 'silent' coloration. Males of our model species are highly territorial and exhibit morph-specific levels of aggression, whereas females are non-territorial and display very low levels of aggression. Interestingly, reactive oxygen species levels were found to be morph specific regardless of sex, despite considerable differences in lifestyle. Males and females did differ remarkably, however, in superoxide levels depending on whether they sported a colored throat bib or not, a trait also used in male sexual signaling. Females with throat bibs had significantly lower levels of superoxide than females without a bib, which was not the case for males.

Net superoxide levels: steeper increase with activity in cooler female and hotter male lizards.

Ectotherms increase their body temperature in response to ambient heat, thereby elevating their metabolic rate. An often inferred consequence of this is an overall upregulation of gene expression and energetic expenditure, and a concomitant increased production of reactive oxygen species (e.g. superoxide) and, perhaps, a shortened lifespan. However, recent work shows that this may be a superficial interpretation. For example, sometimes a reduced temperature may in fact trigger up-regulation of gene expression. We studied temperature and associated activity effects in male and female Australian painted dragon lizards (Ctenophorus pictus) by allowing the lizards to bask for 4 h versus 12 h, and scoring their associated activity (inactive versus active basking and foraging). As predicted, long-basking lizards (hereafter 'hot') showed heightened activity in both sexes, with a more pronounced effect in females. We then tested for sex-specific effects of basking treatment and activity levels on the increase in net levels of superoxide. In males, short-baskers (hereafter 'cold') had significantly more rapidly decreasing levels of superoxide per unit increasing activity than hot males. In females, however, superoxide levels increased faster with increasing activity in the cold than in the hot basking treatment, and females earlier in the ovarian cycle had lower superoxide levels than females closer to ovulation. In short, males and females differ in how their levels of reactive oxygen species change with temperature-triggered activity.

Predictors of telomere content in dragon lizards.

Telomeres shorten as a consequence of DNA replication, in particular in cells with low production of telomerase and perhaps in response to physiological stress from exposure to reactive oxygen species, such as superoxide. This process of telomere attrition is countered by innate antioxidation, such as via the production of superoxide dismutase. We studied the inheritance of telomere length in the Australian painted dragon lizard (Ctenophorus pictus) and the extent to which telomere length covaries with mass-corrected maternal reproductive investment, which reflects the level of circulating yolk precursor and antioxidant, vitellogenin. Our predictors of offspring telomere length explained 72 % of telomere variation (including interstitial telomeres if such are present). Maternal telomere length and reproductive investment were positively influencing offspring telomere length in our analyses, whereas flow cytometry-estimated superoxide level was negatively impacting offspring telomere length. We suggest that the effects of superoxide on hatchling telomere shortening may be partly balanced by transgenerational effects of vitellogenin antioxidation.

Sex-specific SOD levels and DNA damage in painted dragon lizards (Ctenophorus pictus).

When groups of individuals differ in activities that may influence the production of reactive molecules, such as superoxide, we expect selection to result in congruent upregulation of antioxidant production in the group(s) most at risk of suffering concomitant erosion of essential tissue and biomolecules, such as DNA. We investigate this in a (near) annual lizard species, the Australian painted dragon (Ctenophorus pictus), in which males and females have fundamentally different lifestyles, with males being overtly conspicuous and aggressive, whereas females are placid and camouflaged. When kept in identical conditions to females in captivity, males had higher levels of superoxide dismutase (SOD) through the activity season, which is consistent with selection for a higher capacity of superoxide antioxidation and a lower level of DNA damage than females. Males, however, lacked the clear negative, linear relationship between SOD and DNA erosion observed in females, suggesting that female upregulation of SOD results in a more predictable antioxidation and a more immediate target for selection. Lastly, we analysed aspects of female reproduction from a DNA erosion perspective. Females closer to ovulation, hence with less remaining, circulating vitellogenin, had higher superoxide levels. Furthermore, a multiple regression analysis showed that females that produced more clutches over time suffered more DNA erosion, whereas females with higher SOD levels suffered less DNA erosion.

Basal superoxide as a sex-specific immune constraint.

There is increasing evidence that reactive oxygen species (ROS), a group of unstable and highly reactive chemical molecules, play a key role in regulating and maintaining life-history trade-offs. Upregulation of ROS in association with immune activation is costly because it may result in an imbalance between pro- and antioxidants and, hence, oxidative damage. Previous research aimed at quantifying this cost has mostly focused on changes in the pro-/antioxidant balance subsequent to an immune response. Here, we test the hypothesis that systemic ROS may constrain immune activation. We show that systemic, pre-challenge superoxide (SO) levels are negatively related to the strength of the subsequent immune response towards the mitogen phytohaemagglutinin in male, but not female painted dragon lizards (Ctenophorus pictus). We therefore suggest that systemic SO constrains immune activation in painted dragon males. We speculate that this may be due to sex-specific selection pressures on immune investment.

Afternoon T: testosterone level is higher in red than yellow male polychromatic lizards.

Recent work on within-species polymorphism across a broad range of taxa has renewed and considerably increased the attention to this classic evolutionary area, notably in lizard species where colors covary with reproductive strategies. We demonstrate elsewhere that red-headed males beat yellow-headed males in staged contests for females in the Australian painted dragon lizard Ctenophorus pictus. This morph difference in behaviour is linked to what appears to be a convention of red dominance in male-male interactions set very early in ontogeny, long before coloration has developed. In the current note, we investigate the relationship between time of day, which is directly linked to vigilance time in territorial males, and plasma levels of testosterone and corticosterone. We show that red males have higher testosterone levels in late afternoon following a day of territory patrolling and a non-significant trend in plasma corticosterone levels that decline with time of day. In conclusion, there are significant differences in testosterone profile between the two color morphs, providing a potential proximate link to the behavioural differences between them.

Oxidant trade-offs in immunity: an experimental test in a lizard.

Immune system functioning and maintenance entails costs which may limit investment into other processes such as reproduction. Yet, the proximate mechanisms and 'currencies' mediating the costs of immune responses remain elusive. In vertebrates, up-regulation of the innate immune system is associated with rapid phagocytic production of pro-oxidant molecules (so-called 'oxidative burst' responses). Oxidative burst responses are intended to eliminate pathogens but may also constitute an immunopathological risk as they may induce oxidative damage to self cells. To minimize the risk of infection and, at the same time, damage to self, oxidative burst activity must be carefully balanced. The current levels of pro- and antioxidants (i.e. the individual oxidative state) is likely to be a critical factor affecting this balance, but this has not yet been evaluated. Here, we perform an experiment on wild-caught painted dragon lizards (Ctenophorus pictus) to examine how the strength of immune-stimulated oxidative burst responses of phagocytes in whole blood relates to individual oxidative status under control conditions and during an in vivo immune challenge with Escherichia coli lipopolysaccharide (LPS). Under control conditions, oxidative burst responses were not predicted by the oxidative status of the lizards. LPS-injected individuals showed a strong increase in pro-oxidant levels and a strong decrease in antioxidant levels compared to control individuals demonstrating a shift in the pro-/antioxidant balance. Oxidative burst responses in LPS-injected lizards were positively related to post-challenge extracellular pro-oxidants (reflecting the level of cell activation) and negatively related to pre-challenge levels of mitochondrial superoxide (suggesting an immunoregulatory effect of this pro-oxidant). LPS-challenged males had higher oxidative burst responses than females, and in females oxidative burst responses seemed to depend more strongly on antioxidant status than in males. Our results confirm the idea that oxidative state may constrain the activity of the innate immune system. These constraints may have important consequences for the way selection acts on pro-oxidant generating processes.

A significant component of ageing (DNA damage) is reflected in fading breeding colors: an experimental test using innate antioxidant mimetics in painted dragon lizards.

A decade ahead of their time, von Schantz et al. united sexual selection and free radical biology by identifying causal links between deep-rooted physiological processes that dictate resistance to toxic waste from oxidative metabolism (reactive oxygen species, ROS), and phenotypic traits, such as ornaments. Ten years later, these ideas have still only been tested with indirect estimates of free radical levels (oxidative stress) subsequent to the action of innate and dietary antioxidants. Here, we measure net superoxide (a selection pressure for antioxidant production) and experimentally manipulate superoxide antioxidation using a synthetic mimetic of superoxide dismutase (SOD), Eukarion 134 (EUK). We then measure the toxic effect of superoxide in terms of DNA erosion and concomitant loss of male breeding coloration in the lizard, Ctenophorus pictus. Control males suffered more DNA damage than EUK males. Spectroradiometry showed that male coloration is lost in relation to superoxide and covaries with DNA erosion; in control males, these variables explained loss of color, whereas in EUK males, the fading of coloration was unaffected by superoxide and unrelated to DNA damage. Thus, EUK's powerful antioxidation removes the erosion effect of superoxide on coloration and experimentally verifies the prediction that colors reflect innate capacity for antioxidation.

Digit ratio, color polymorphism and egg testosterone in the Australian painted dragon.

Variation in exposure to sex hormones during early development contributes to phenotypic plasticity in vertebrate offspring. As a proposed marker for prenatal sex hormone exposure and because of their association with various physiological and behavioral characteristics, digit ratio and/or digit length have received notable interest within the field of evolutionary ecology. However, the validity of digit measures as a proxy of prenatal sex hormone exposure is controversial and only few studies have provided direct evidence for the link between digit development and prenatal sex hormones. Here, we report morph- and sex-specific variation in digit ratio in wild painted dragon lizards (Ctenophorus pictus). Lizards expressing a yellow bib have significantly larger third-to-fourth toe ratios (3D:4D) than lizards without a bib. Males have significantly smaller 3D:4D than females. Furthermore, we show that experimental elevation of yolk testosterone significantly increases 3D:4D in hatchling painted dragon lizards, but has no influence on hatchling size. Our results provide direct and indirect evidence for the involvement of prenatal sex steroids in digit development and it is suggested that digit ratio may be used as a biomarker for prenatal steroid exposure in this reptilian species. As such, digit ratio may provide a useful tool to study temporal or spatial differences in the proximate hormonal mechanisms modulating physiological and behavioural phenotypes.

Testing the quality of a carrier: a field experiment on lizard signalers.

In the Australian painted dragon lizard (Ctenophorus pictus), males occur in two different morphs with respect to gular color, with or without a yellow bib. Males without a bib lost within-clutch paternity significantly more often to rivals than bibbed males. Thus, it appears that bibs identify some phenotypic advantage linked to competitive ability. To test whether this could be related to whole-organism capacity to withstand an increased workload (due to better health and vigor, or evolved differences in self-maintenance), we implanted males with a lead pellet (loaded), Styrofoam pellet (controls), or sham-operated males without implants (shams), and compared male categories with respect to how they maintained body mass during the mating season. Somewhat unexpectedly, bibbed males consistently lost more body weight across all treatments and controls, although we could not verify that this translated into higher mortality in this short-lived animal (about 80% survive for one year only). However, bibbed males may invest more into "mating success" than nonbibbed males, which agrees with our experimental results and paternity data.

Free radicals run in lizard families.

In the ageing individual, the production of reactive oxygen species (ROS) accelerates with cell senescence. Depending on the heritability of the underlying processes that determine net ROS levels, this may influence ageing per se and its evolutionary direction and rate of change. In order to understand the inheritance and evolution of net ROS levels in free-ranging lizards, we used flow cytometry together with ROS-sensitive fluorogenic probes to measure ROS in lizard blood cells. We measured basal levels of (i) non-specific ROS (superoxide, singlet oxygen, H2O2 and peroxynitrite), (ii) superoxide specifically and (iii) superoxide after CCCP treatment, which elevated ROS production in the mitochondria. The cumulative level of non-specific ROS was higher in adults than juveniles and superoxide level showed high heritability and variability among families. We suggest that the evolution of ROS dynamics may be ROS species specific and perhaps depend on the relative degree of uni- or biparental inheritance of ROS main regulatory pathways.

Sons are made from old stores: sperm storage effects on sex ratio in a lizard.

Sperm storage is a widespread phenomenon across taxa and mating systems but its consequences for central fitness parameters, such as sex ratios, has rarely been investigated. In Australian painted dragon lizards (Ctenophorus pictus), we describe elsewhere that male reproductive success via sperm competition is largely an effect of sperm storage. That is, sperm being stored in the female reproductive tract out-compete more recently inseminated sperm in subsequent ovarian cycles. Here we look at the consequences of such sperm storage for sex allocation in the same species, which has genetic sex determination. We show that stored sperm have a 23% higher probability of producing sons than daughters. Thus, shifts in sex ratio, for example over the reproductive season, can partly be explained by different survival of son-producing sperm or some unidentified female mechanism taking effect during prolonged storage.

Mating system variation and morph fluctuations in a polymorphic lizard.

In polymorphic male painted dragon lizards (Ctenophorus pictus), red males win staged contests for females over yellow males, and yellow males have greater success in staged sperm competition trials than red males. This predicts different reproductive strategies in the wild with red males being more coercive or better mate guarders than yellow males. Yellow males would be expected to sire more offspring per copulation and have a greater proportion of offspring from clutches with mixed paternity. However, here we show using microsatellites that the frequency of mixed paternity in the wild is low (< 20% on average across years), that all morphs on average have the same number of offspring sired per year, and that mating system variation (polyandry vs. monandry) is strongly correlated with perch density on male territories. Furthermore, a logistic regression on male successful vs. unsuccessful mate acquisition showed that red males were under negative selection when they dominated the population, which suggests ongoing frequency dependent selection on male colouration.