Sex determination was once thought to be unstable in ectothermic vertebrates, but several highly diversified groups of non-avian reptiles, such as iguanas sensu lato (Pleurodonta), appear to have conserved sex chromosomes. However, this statement has been criticized as being based on a parsimonious conclusion from limited sampling. Here, we tested sex chromosome homology in a further 35 species of pleurodont lizards, mainly of the family Anolidae. We demonstrated sex chromosome homology based on the comparison of the gene dosage of X-specific genes by quantitative PCR. Including these new data, the homology of sex chromosomes has so far been reported in 92 out of over 1,200 recently recognized species of Pleurodonta. The very conserved sex chromosomes in all but one family are at least as old as the basal split of Pleurodonta (estimated over 90 million years). In the case of the family Corytophanidae, all genera share different XX/XY sex chromosomes, which are thus over 25 million years old. We discuss the efficiency and limitations of the approach used for tests of the homology of sex chromosomes, as well as the reasons for the evolutionary stability of sex chromosomes in some lineages. We argue that to obtain a complete picture, all extant species should be tested for homology; however, until we reach this ambitious goal, parsimonious estimates in lineages where fragmentary data suggest stability of sex chromosomes, as in Pleurodonta, are substantiated.

Reproduction entirely without males is exceptionally rare among vertebrates. Recently, an enigmatic all-female lineage of casquehead lizards, initially thought to be a captive anomaly, appeared in zoos. During a targeted expedition to Honduras, we discovered two wild, asexually reproducing populations near the type locality of Laemanctus julioi, a species originally described from a single female. Using a de novo phased long-read genome and cytogenetics, we reveal these lizards are clonal hybrids, originating from two geographically separated sexual species that diverged 12 million years ago. We confirmed two independent clonal lineages, one matching the zoo population. To form clonal eggs, these lizards duplicate their genome before meiosis. Experimental breeding with a male from a paternal species produced non-viable offspring with increased genome size, suggesting these clones established themselves by remaining isolated from parental species. As the largest-bodied known vertebrates reproducing exclusively without males, these lizards demonstrate that radically different reproductive strategies can remain undetected even in highly conspicuous animals.

INTRODUCTION:Sex-specific genotype and early organization can influence the expression of sexually dimorphic traits in vertebrates. We tested these hypotheses in male-typical behaviour and rapid change to bright colouration in the veiled chameleon (Chamaeleo calyptratus) with XX/XY sex chromosomes. METHODS:Hormonal manipulations included castration with and without testosterone replacement and testosterone administration in females. RESULTS:Long-term testosterone treatment induced male-typical sexual behaviour and an ability to switch to bright colouration in females, while castration suppressed these traits in males. These observations document that elevated testosterone alone is sufficient for the expression of these traits in both males and females. Surprisingly, high testosterone levels led to indiscriminate courtship behaviour, with frequent mating attempts directed at conspecifics regardless of their sex and testosterone level in both home cages and neutral arenas. This unexpected behaviour suggests that visual cues, such as body and head-casque size, may not reliably guide sex recognition during short distance encounters. CONCLUSION:The dependence of the male-typical sexual behaviour and colour change on the elevated androgen levels contrast sharply with earlier results on skeletal traits (body size and head-casque size), which are fully developed in castrated males, demonstrating that the ontogeny of the sex-typical phenotype involves different mechanisms in the emerging model species of chameleons.

Endothermic and ectothermic amniotes differ in the timing of reproductive onset, with reptiles initiating reproduction before reaching final body size. Long-term consequences of maternal effect for early reptile offspring are poorly explored. We conducted growth experiments to compare the growth of offspring produced by young and older females of gecko Paroedura picta . Young, not fully grown females lay smaller eggs leading to production of smaller offspring. These offspring undergo accelerated growth and ultimately reach a comparable sex-specific final body length as do offspring of older females. Final body length is thus canalized with respect to the maternal effect on egg size. Notably, the offspring of young mothers have a tendency towards larger body mass. Ontogeny of the offspring of young females shares similarities with that of mammalian offspring with low birth weight or early malnutrition, exhibiting catch-up growth and a predisposition to obesity. We highlight the important consequences of early reproduction for offspring in animals that initiate reproduction prior to reaching final body size. Both life-history models and conservation practices should take into account that female lizards might produce the most fit offspring only between reaching their final body length and the onset of reproductive senescence.

Ectothermic vertebrates such as reptiles were assumed to be indeterminate growers, which means that there is no terminal point in time or size for growth in their lifetime. In recent years, evidence for the determinate nature of growth in lizards has accumulated, necessitating a re-examination of models of their ontogeny and evolution of sexual size dimorphism (SSD). In the female-larger gecko Paroedura vazimba , we monitored post-embryonic growth over a period of 15 months. After hatching, females grew faster than males but also reached their final body size, that is, closed growth of their vertebrae, earlier than males. The closure of bone growth in females correlates with the onset of reproductive maturation. We compared this pattern with the previously minutely studied, male-larger species Paroedura picta , where we documented determinate growth as well. We propose a model to explain the evolutionary switches in the direction of SSD in lizards based on bipotential effects of ovarian hormones on growth. In this model, male growth is assumed to require no male-specific growth modifier, such as sex-limited hormonal regulators, while growth is feminized by ovarian hormones in females. Low levels of ovarian hormones can promote bone growth, but high levels associated with maturation of the reproductive organs promote senescence of bone growth plates and thus cessation of bone growth. We suggest that models on growth, life-history and evolution of body size in many lizards should acknowledge their determinate nature of growth.

The prevalence of environmental sex determination (ESD) in squamate reptiles is often overestimated in the literature. This is surprising because we have reliable data demonstrating ESD in only a few species. The documentation of ESD in three species of geckos presented here has significantly increased our knowledge, given that satisfactory evidence for ESD existed in only eight other gecko species. For the first time, we document the occurrence of ESD in the family Sphaerodactylidae. Our finding of unexpected variability in the shapes of reaction norms among geckos highlights that traditional descriptions using parameters such as pivotal temperature, that is, temperature producing a 50:50 sex ratio, are unsatisfactory. For example, the gecko Pachydactylus tigrinus lacks any pivotal temperature and its sex ratios are strongly female-biased across the entire range of viable temperatures. We argue for the effective capture of the relationship between temperature and sex ratio using specific nonlinear models rather than using classical simplistic descriptions and classifications of reaction norms.

Squamate reptiles have been considered to be indeterminate growers for a long time. However, recent studies demonstrate that bone prolongation is stopped in many lizards by the closure of bone growth plates. This shift in the paradigm of lizard growth has important consequences for questions concerning the proximate causes of sexual size dimorphism. The traditional model of highly plastic and indeterminate growth would correspond more to a long-term action of a sex-specific growth regulator. On the other hand, determinate growth would be more consistent with a regulator acting in a sex-specific manner on the activity of bone growth plates operating during the phase when a dimorphism in size develops. We followed the growth of males and females of the male-larger Madagascar ground gecko (Paroedura picta) and monitored the activity of bone growth plates, gonad size, levels of steroids, expression of their receptors (AR, ESR1), and expression of genes from the insulin-like growth factor network (IGF1, IGF2, IGF1R, and IGF2R) in livers. Specifically, we measured gene expression before the onset of dimorphic growth, at the time when males have more active bone growth plates and sexual size dimorphism was clearly visible, and after a period of pronounced growth in both sexes. We found a significant spike in the expression of IGF1 in males around the time when dimorphism develops. This overexpression in males comes long after an increase in circulating testosterone levels and sexual maturation in males, and it might be suppressed by ovarian hormones in females. The results suggest that sexual size dimorphism in male-larger lizards can be caused by a positive effect of high levels of IGF1 on bone growth. The peak in IGF1 resembles the situation during the pubertal growth spurt in humans, but in lizards, it seems to be sex-specific and disconnected from sexual maturation.

(1) In contrast to mammals and birds, reptiles have been considered as indeterminate growers, whose growth reflects differential allocation of resources to growth versus other energetically demanding processes such as reproduction. (2) We monitored the growth and activity of bone growth plates, hormonal profiles, and reproductive activity in males and females of the male-larger gecko Paroedura picta. We show that growth plates fuse in this species in a sex-specific manner. The more abrupt epiphyseal closure and more pronounced growth deceleration in females coincide with the increased activity of their reproductive organs. (3) We conclude that at least some lizards are determinate growers whose sexual size dimorphism is potentially driven by ovarian hormones. The major difference in growth between endothermic and ectothermic amniotes appears to be in the magnitude of growth before and after the first reproduction, not in the mechanistic processes such as senescence of growth plate cells.

The study of energy allocation is essential in understanding the regulation of major life history traits. It is often assumed automatically that the limitation of an energy budget or higher allocation to a single trait affect all life history traits. This assumption was inherently included in influential models of ontogenetic growth. We aim to challenge this perspective by focusing on growth in lizards. Summarizing the results of a series of long-term manipulative experiments in the Madagascar ground gecko (Paroedura picta), we show that although growth is generally assumed to be highly plastic in reptiles and other ectothermic vertebrates, it is at least in this species largely canalized and does not seem to be affected by energy limitations under several experimental conditions. Diet restriction, resulting in lower allocation to fat storage and reproduction, and the allocation to energetically demanding traits such as reproduction in both sexes and tail regeneration had little if any effect on structural growth. We document that sexual size dimorphism does not emerge in the ontogeny of the studied species directly due to differential allocation to structural growth in males and females. Instead, sex-specific growth trajectories are driven by a signaling of ovarian hormones as the key proximate mechanism shaping sex-specific allocation decisions during ontogeny. We suggest that the large degree of canalization of the structural growth can reflect hierarchy in energy allocation with the structural growth being prioritized to investment in other traits. The prioritized allocation to structural growth can reflect selective advantage of reaching a final, optimal size for a given sex as fast as possible.

Proximate control of the development of sexual dimorphism is still hotly debated in reptiles. In some squamates, many male-typical exaggerated traits including body size were assumed to be controlled by masculinization by male gonadal androgens. We performed a manipulative experiment to test the importance of this mechanism in the development of pronounced sexual differences in body size and size of head casque in the chameleon Chamaeleo calyptratus . Castrated males attained male-typical body size highly deviating from the body size of control females. Ontogenetic allometries of casque size on head length revealed that sexes depart considerably in casque growth later in the ontogeny; however, castrated males still follow male-typical casque growth. Paradoxically, exogenous testosterone led in females to slight increase of casque size, which might reflect interference with the feminizing effects of female gonadal hormones. The results in males strongly suggest that masculinization by male gonadal androgens during growth is not required for the development of sexual dimorphism in body size and casque size in the chameleon. The ontogeny of sexually dimorphic body size and exaggerated traits in at least some squamates is likely controlled by other proximate mechanism, possibly by feminization by ovarian hormones.

Within-species variation in the number of neurons, other brain cells and their allocation to different brain parts is poorly studied. Here, we assess these numbers in a squamate reptile, the Madagascar ground gecko (Paroedura picta). We examined adults from two captive populations and three age groups within one population. Even though reptiles exhibit extensive adult neurogenesis, intrapopulation variation in the number of neurons is similar to that in mice. However, the two populations differed significantly in most measures, highlighting the fact that using only one population can underestimate within-species variation. There is a substantial increase in the number of neurons and decrease in neuronal density in adult geckos relative to hatchlings and an increase in the number of neurons in the telencephalon in fully grown adults relative to sexually mature young adults. This finding implies that adult neurogenesis does not only replace worn out but also adds new telencephalic neurons in reptiles during adulthood. This markedly contrasts with the situation in mammals, where the number of cortical neurons declines with age.

Frequent independent origins of environmental sex determination (ESD) are assumed within amniotes. However, the phylogenetic distribution of sex‐determining modes suggests that ESD is likely very ancient and may be homologous across ESD groups. Sex chromosomes are demonstrated to be old and stable in endothermic (mammals and birds) and many ectothermic (non‐avian reptiles) lineages, but they are mostly non‐homologous between individual amniote lineages. The phylogenetic pattern may be explained by ancestral ESD with multiple transitions to later evolutionary stable genotypic sex determination. It is pointed out here that amniote ESD shares several key aspects with sequential hermaphroditism of fishes such as a lack of sex differences in genomes, biased population sex ratios, and potentially also molecular mechanism related to general stress responses. Here, it is speculated that ESD evolves via a heterochronic shift of the sensitive period of sex change from the adult to the embryonic stage in a hermaphroditic amniote ancestor. Also see the video abstract here https://youtu.be/q2mjtlCefu4 .

Facultative parthenogenesis in vertebrates is believed to be exceptional, and wherever documented, it always led to single‐sex progeny with genome‐wide homozygosity. We report the first challenge to this paradigm: frequent facultative parthenogenesis in the previously assumed sexually reproducing tropical night lizard Lepidophyma smithii results in offspring of both sexes and preserves heterozygosity in many loci polymorphic in their mothers. Moreover, we documented a mixture of sexually and parthenogenetically produced progeny in a single clutch, which documents how cryptic a facultative parthenogenesis can be. Next, we show that in the studied species, 1) parthenogenetically produced females can further reproduce parthenogenetically, 2) a sexually produced female can reproduce parthenogenetically, 3) a parthenogenetically produced female can reproduce sexually, and 4) a parthenogenetically produced male is fully fertile. We suggest that facultative parthenogenesis should be considered even in vertebrates with frequent males and genetically variable, heterozygous offspring.

The nature and hormonal control of cues used for recognition of sex and reproductive status of conspecifics remain largely unstudied in reptiles. It has been proposed that production of a female attractiveness pheromone controlled by female ovarian hormones (and which is suppressed by male gonadal androgens) is necessary to elicit courtship in males. In the case of leopard gecko (Eublepharis macularius), it has been suggested that an individual is recognized as a male and attacked unless it produces female-specific stimuli in its skin and that females are attacked, not courted, while shedding. We tested the reactions of males to control males and control shedding and non-shedding females, castrated males, females treated with exogenous androgens (testosterone and dihydrotestosterone), and prepubertal individuals. The individuals with high androgen levels (i.e., control males and hormone-treated females) were attacked while animals in all the other groups were courted. Our results indicate that in leopard gecko hormonally controlled pheromones advertising female attractiveness are not required and that sex discrimination is based on the presence or absence of cues dependent on masculinization by male gonadal steroids.

Body size is a fundamental trait correlated with nearly every aspect of animal life. It is influenced by numerous genetic and non-genetic factors. Despite its central importance, proximate mechanisms of intra- and interspecific variability in body size are still not well understood even in such a largely studied group as reptiles. For our study, we concentrated on the gecko species Paroedura picta. We investigated whether differences in sexual size dimorphism and in final and asymptotic snout-vent length (induced by a range of incubation and rearing temperatures) are correlated with differences in the number of presacral vertebrae. Moreover, we tested whether changes in this number were associated with evolutionary changes in sexual size dimorphism and body size in the genus Paroedura. We found that the variation in the number of presacral vertebrae is very limited both intra- and interspecifically, ranging between 26 and 28 vertebrae with most individuals possessing the modal number of 27. We conclude that changes in the number of vertebrae do not contribute to developmental plasticity or evolutionary changes in body size nor, in contrast to some other squamate lineages, to sexual size dimorphism.

Cell size plays a role in evolutionary and phenotypically plastic changes in body size. To examine this role, we measured the sizes of seven cell types of geckos (Paroedura picta) reared at three constant temperatures (24, 27, and 30 °C). Our results show that the cell size varies according to the body size, sex and developmental temperature, but the pattern of this variance depends on the cell type. We identified three groups of cell types, and the cell sizes changed in a coordinated manner within each group. Larger geckos had larger erythrocytes, striated muscle cells and hepatocytes (our first cell group), but their renal proximal tubule cells and duodenal enterocytes (our second cell group), as well as tracheal chondrocytes and epithelial skin cells (our third cell group), were largely unrelated to the body size. For six cell types, we also measured the nuclei and found that larger cells had larger nuclei. The relative sizes of the nuclei were not invariant but varied in a complex manner with temperature and sex. In conclusion, we provide evidence suggesting that changes in cell size might be commonly involved in the origin of thermal and sexual differences in adult size. A recent theory predicts that smaller cells speed up metabolism but demand more energy for their maintenance; consequently, the cell size matches the metabolic demand and supply, which in ectotherms, largely depends on the thermal conditions. The complex thermal dependency of cell size in geckos suggests that further advancements in understanding the adaptive value of cell size requires the consideration of tissue-specific demand/supply conditions.

In vertebrates, male-typical sexual behavior (MSB) is largely controlled by gonadal androgens, however, the mechanism of this control is believed to vary among species. During immediate activation MSB is tightly correlated with circulating levels of androgens, while the organization of MSB by a hormonal event at a specific developmental period, early in ontogeny or during puberty, has been postulated in other lineages. Here, we put forward an alternative concept of "temporal organization". Under temporal organization longer exposure to circulating androgens is needed for the onset of MSB, which can continue for a long time after the levels of these hormones drop. We tested this concept through long-term monitoring of MSB in females and castrated males of the leopard gecko (Eublepharis macularius) in response to experimental changes in testosterone levels. Several weeks of elevated testosterone levels were needed for the full expression of MSB in both treatment groups and MSB diminished only slowly and gradually after the supplementation of exogenous testosterone ended. Moreover, despite receiving the same application of the hormone both the progressive onset and the cessation of MSB were significantly slower in experimental females than in castrated males. We suggest that the concept of temporal organization of MSB can parsimoniously explain several earlier discrepancies and debatable conclusions on the apparent variability in the hormonal control of MSB in vertebrates, which were based on behavioral testing at a few subjectively selected time points. We conclude that long-term and continuous behavioral testing after hormonal manipulations is needed to understand the regulation of MSB in vertebrates.

Sexual size dimorphism (SSD) reflects sex-specific solutions to the allocation of energy among growth, reproduction and survival; however, the proximate mechanisms behind these solutions are still poorly known even in vertebrates. In squamates, sexual differences in body size used to be attributed to direct energy allocation to energetically demanding processes, largely to reproduction. In addition, SSD is assumed to be controlled by specific endogenous mechanisms regulating growth in a sex-specific manner, namely masculinization by male gonadal androgens or feminization by ovarian hormones. We performed a manipulative growth experiment in females of the male-larger gecko Paroedura picta in order to test the reproductive cost hypothesis, the male androgen hypothesis and the ovarian hormone hypothesis. Specifically, we investigated the effect of total ovariectomy, prepubertal ovariectomy, unilateral ovariectomy, and total ovariectomy followed by exogenous estradiol, dihydrotestosterone or testosterone treatment, on female growth in comparison to males and reproductively active females. The present results and the results of our previous experiments do not support the hypotheses that SSD reflects direct energy allocation to reproduction and that male gonadal androgens are involved. However, all lines of evidence, particularly the comparable growth of reproducing intact and unilaterally ovariectomized females, were concordant with the control of SSD by ovarian hormones. We suggest that feminization of growth by female gonadal hormones should be taken into consideration as an endogenous pathway responsible for the ontogeny of SSD in squamates.

Previous work on lizards has shown that many sexually dimorphic traits depend on testosterone (T), but the details of this control can vary among species. Here, we tested the role of T on the expression of morphological, physiological, and behavioral traits in Lichtenfelder's gecko (Goniurosaurus lichtenfelderi), from the lizard family Eublepharidae notable for interspecific variation in sexually dimorphic traits and the mode of sex determination. Experiments included three groups of males (intact control, surgically castrated, castrated with T replacement) and two groups of females (intact control, T supplemented). In males, castration caused reductions in 1) the size of hemipenes, 2) offensive aggression, 3) male sexual behavior in a neutral arena, 4) activity of precloacal glands, and 5) loss of male chemical cues for sex recognition. These reductions were not observed in castrated males with T replacement. Interestingly, castrated males performed sexual behavior in their home cages, which shows that the effect of T depends on the environmental context. Notably, tail vibration, previously reported as a courtship behavior in other eublepharids, is displayed by males of G. lichtenfelderi during interactions with conspecifics of both sexes, suggesting an evolutionary shift in the meaning of this signal. In females, T induced growth of hemipenes and male-typical courtship but did not induce precloacal pore activity, aggression, or mounting. In comparison to previous reports on Eublepharis macularius, our results indicate that effects of T do not depend on the mode of sex determination. Further, our results extend our understanding of the complexity of control of male traits and illustrate how lability in the effects of T can be a general mechanism causing evolutionary changes in the components of suites of functionally correlated traits.