Complex ontogeny of sexual size dimorphism in a female-larger gecko: Implications of determinate growth for lizard body size and life-history evolution.

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.

Catch-up growth and overweight adults in the offspring of young gecko mothers resembling low birth weight infants.

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.

Scaling of erythrocyte shape and nucleus size among squamate reptiles: reanalysis points to constrained, proportional rather than adaptive changes.

Small erythrocytes might be beneficial for blood rheology, as they contribute less to blood viscosity than large erythrocytes. We predicted that rheological disadvantages of larger erythrocytes could be alleviated by relatively smaller nucleus size in larger cells allowing higher flexibility and by more elongated shape. Across squamate reptiles, we found that species with larger erythrocytes tend to have smaller ratio of nucleus size to cell size (N : C ratio), but that larger erythrocytes tend to be rounder, not more elongated. Nevertheless, we document that in fact nucleus area changes with erythrocyte area more or less linearly, which is also true for the relationship between cell length and cell width. These linear relationships suggest that nucleus size and cell size, and cell width and cell length, might be constrained to largely proportional mutual changes. The shifts in widely used N : C ratio and elongation ratio (cell length/cell width) with cell size might be misleading, as they do not reflect adaptive or maladaptive changes of erythrocytes, but rather mathematically trivial scaling of the ratios of two variables with a linear relationship with non-zero intercepts. We warn that ratio scaling without analyses of underlying patterns of evolutionary changes can lead to misinterpretation of evolutionary processes.

Development of male-larger sexual size dimorphism in a lizard: IGF1 peak long after sexual maturity overlaps with pronounced growth in males.

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.

Sex chromosomes in meiotic, hemiclonal, clonal and polyploid hybrid vertebrates: along the 'extended speciation continuum'.

We review knowledge about the roles of sex chromosomes in vertebrate hybridization and speciation, exploring a gradient of divergences with increasing reproductive isolation (speciation continuum). Under early divergence, well-differentiated sex chromosomes in meiotic hybrids may cause Haldane-effects and introgress less easily than autosomes. Undifferentiated sex chromosomes are more susceptible to introgression and form multiple (or new) sex chromosome systems with hardly predictable dominance hierarchies. Under increased divergence, most vertebrates reach complete intrinsic reproductive isolation. Slightly earlier, some hybrids (linked in 'the extended speciation continuum') exhibit aberrant gametogenesis, leading towards female clonality. This facilitates the evolution of various allodiploid and allopolyploid clonal ('asexual') hybrid vertebrates, where 'asexuality' might be a form of intrinsic reproductive isolation. A comprehensive list of 'asexual' hybrid vertebrates shows that they all evolved from parents with divergences that were greater than at the intraspecific level (K2P-distances of greater than 5-22% based on mtDNA). These 'asexual' taxa inherited genetic sex determination by mostly undifferentiated sex chromosomes. Among the few known sex-determining systems in hybrid 'asexuals', female heterogamety (ZW) occurred about twice as often as male heterogamety (XY). We hypothesize that pre-/meiotic aberrations in all-female ZW-hybrids present Haldane-effects promoting their evolution. Understanding the preconditions to produce various clonal or meiotic allopolyploids appears crucial for insights into the evolution of sex, 'asexuality' and polyploidy. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

Ontogeny, phylogeny and mechanisms of adaptive changes in evaporative water loss in geckos.

Body size dependence of metabolic rate, body surface and scale morphology complicate disentangling the contribution of these characteristics to adaptive changes in total evaporative water loss (TEWL) of reptiles. To separate adaptive changes from size-related dependence, we compared intra- and interspecific scaling of several candidate traits in eyelid geckos (Eublepharidae), a group exhibiting large variation in body size and TEWL. The intraspecific allometry of TEWL of a eublepharid species fits the geometric surface-mass relationship. However, evolutionary shifts to both higher and lower evaporation were strongly correlated with habitat aridity and cannot be explained by shifts in body size alone. The intraspecific allometry of standard metabolic rate is nearly the same as the interspecific allometry. Unlike for mammals and birds, this pattern rules out respiratory water loss as a driver of the adaptive changes in TEWL among eublepharids. Scale morphology was independent of TEWL variation as well, but the correlation between cutaneous water loss and TEWL suggests a crucial role of skin permeability in adaptation to habitat aridity. Our analyses demonstrate how powerful a comparison between intra- and interspecific allometries can be for detecting body size-dependent mechanisms of adaptive changes in ecophysiological traits correlated with body size.

Variability in vertebral numbers does not contribute to sexual size dimorphism, interspecific variability, or phenotypic plasticity in body size in geckos (Squamata: Gekkota: Paroedura).

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.

Not all cells are equal: effects of temperature and sex on the size of different cell types in the Madagascar ground gecko Paroedura picta.

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.

Bayesian Phylogenetic Estimation of Clade Ages Supports Trans-Atlantic Dispersal of Cichlid Fishes.

Divergence-time estimation based on molecular phylogenies and the fossil record has provided insights into fundamental questions of evolutionary biology. In Bayesian node dating, phylogenies are commonly time calibrated through the specification of calibration densities on nodes representing clades with known fossil occurrences. Unfortunately, the optimal shape of these calibration densities is usually unknown and they are therefore often chosen arbitrarily, which directly impacts the reliability of the resulting age estimates. As possible solutions to this problem, two nonexclusive alternative approaches have recently been developed, the "fossilized birth­death" (FBD) model and "total-evidence dating." While these approaches have been shown to perform well under certain conditions, they require including all (or a random subset) of the fossils of each clade in the analysis, rather than just relying on the oldest fossils of clades. In addition, both approaches assume that fossil records of different clades in the phylogeny are all the product of the same underlying fossil sampling rate, even though this rate has been shown to differ strongly between higher level taxa. We here develop a flexible new approach to Bayesian age estimation that combines advantages of node dating and the FBD model. In our new approach, calibration densities are defined on the basis of first fossil occurrences and sampling rate estimates that can be specified separately for all clades. We verify our approach with a large number of simulated data sets, and compare its performance to that of the FBD model. We find that our approach produces reliable age estimates that are robust to model violation, on par with the FBD model. By applying our approach to a large data set including sequence data from over 1000 species of teleost fishes as well as 147 carefully selected fossil constraints, we recover a timeline of teleost diversification that is incompatible with previously assumed vicariant divergences of freshwater fishes. Our results instead provide strong evidence for transoceanic dispersal of cichlids and other groups of teleost fishes.

An energetic perspective on tissue regeneration: The costs of tail autotomy in growing geckos.

Tail autotomy is a crucial antipredatory lizard response, which greatly increases individual survival, but at the same time also compromises locomotor performance, sacrifices energy stores and induces a higher burden due to the ensuing response of regenerating the lost body part. The potential costs of tail autotomy include shifts in energy allocation and metabolic rates, especially in juveniles, which invest their energy primarily in somatic growth. We compared the metabolic rates and followed the growth of juvenile males with and without regenerating tails in the Madagascar ground gecko (Paroedura picta), a nocturnal ground-dwelling lizard. Geckos with intact tails and those that were regrowing them grew in snout-vent-length at similar rates for 22weeks after autotomy. Tail regeneration had a negligible influence on body mass-corrected metabolic rate measured at regular intervals throughout the regenerative process. We conclude that fast-growing juveniles under the conditions of unrestricted food can largely compensate for costs of tail loss and regeneration in their somatic growth without a significant impact on the total individual body mass-corrected metabolic rate.

The complete mitochondrial genome of the Madagascar ground gecko Paroedura picta (Squamata: Gekkonidae).

We sequenced the complete mitochondrial genome of the Madagascar ground gecko Paroedura picta (Squamata: Gekkonidae). The mitogenome is 17 220 base pairs long and conforms to the typical vertebrate gene composition and arrangement, i.e. 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes and 1919 bp long control region. We reconstructed phylogenetic relationships of P. picta and representatives of nine other genera from the family Gekkonidae and calculated mean p-distances for all 13 protein-coding mitochondrial genes. The lowest mean p-distances were found in cytochrome oxidase subunit I and III genes (COI and COIII) indicating their usefulness for elucidating deeper phylogenetic relationships.

Endogenous control of sexual size dimorphism: Gonadal androgens have neither direct nor indirect effect on male growth in a Madagascar ground gecko (Paroedura picta).

Changes in the effect of gonadal androgens on male growth are considered as a possible mechanism allowing shifts in magnitude and even direction of sexual size dimorphism in vertebrates, particularly squamate reptiles. Positive effects of gonadal androgens on male growth were found in several male-larger species of lizards. Contrastingly, we document that in the male-larger Madagascar ground gecko (Paroedura picta) gonadal androgens do not affect male growth under constant thermal conditions. However, the absence of a thermal gradient might prevent the potential indirect effect of gonadal androgens on growth via the influence of circulating hormones on an individual's thermoregulation and hence metabolic rate. In order to study this, we monitored the growth and body temperature of socially isolated sham-operated and castrated males of the same species in a thermal gradient. We also compared the oxygen consumption and activity between the treatment groups in the open field to test the effect of gonadal hormones on these traits potentially affecting growth. Even under a thermal gradient we found no effect of gonadal androgens on growth rate or final body dimensions. Castration also did not significantly affect oxygen consumption or activity in the open field test. Together with our previous findings, we can exclude both the direct effect of male gonadal androgens on the ontogeny of sexual size dimorphism via the influence on the growth axis, and the indirect influence of gonadal androgens acting on the ontogeny of SSD through the effect on thermoregulation, metabolic rate and activity.

Ontogeny of metabolic rate and red blood cell size in eyelid geckos: species follow different paths.

While metabolism is a fundamental feature of all organisms, the causes of its scaling with body mass are not yet fully explained. Nevertheless, observations of negative correlations between red blood cell (RBC) size and the rate of metabolism suggest that size variation of these cells responsible for oxygen supply may play a crucial role in determining metabolic rate scaling in vertebrates. Based on a prediction derived from the Cell Metabolism Hypothesis, metabolic rate should increase linearly with body mass in species with RBC size invariance, and slower than linearly when RBC size increases with body mass. We found support for that prediction in five species of eyelid geckos (family Eublepharidae) with different patterns of RBC size variation during ontogenetic growth. During ontogeny, metabolic rate increases nearly linearly with body mass in those species of eyelid geckos where there is no correlation between RBC size and body mass, whereas non-linearity of metabolic rate scaling is evident in those species with ontogenetic increase of RBC size. Our findings provide evidence that ontogenetic variability in RBC size, possibly correlating with sizes of other cell types, could have important physiological consequences and can contribute to qualitatively different shape of the intraspecific relationship between metabolic rate and body mass.

Neither male gonadal androgens nor female reproductive costs drive development of sexual size dimorphism in lizards.

Sexual size dimorphism (SSD) is an extensively studied phenomenon in animals, including reptiles, but the proximate mechanism of its development is poorly understood. The most pervasive candidates are: (1) androgen-mediated control of growth, i.e. a positive effect of gonadal androgens (testosterone) on male growth in male-larger species, and a negative effect in female-larger species; and (2) sex-specific differences in energy allocation to growth, e.g. sex with larger reproductive costs should result in smaller body size. We tested these hypotheses in adults of the male-larger lizard Paroedura picta by conducting castrations with and without testosterone implants in males and manipulating reproductive status in females. Castration or testosterone replacement had no significant effect on final body length in males. High investment to reproduction had no significant effect on final body length in intact females. Interestingly, ovariectomized females and females with testosterone implants grew to larger body size than intact females. We did not find support for either of the above hypotheses and suggest that previously reported effects of gonadal androgens on growth in male lizards could be a consequence of altered behaviour or social status in manipulated individuals. Exogenous testosterone in females led to decreased size of ovaries; its effect on body size may be caused by interference with normal ovarian function. We suggest that ovarian factors, perhaps estrogens, not reproductive costs, can modify growth in female lizards and may thus contribute to the development of SSD. This hypothesis is largely supported by published results on the effect of testosterone treatment or ovariectomy on body size in female squamates.

Overlap of female reproductive cycles explains shortened interclutch interval in a lizard with invariant clutch size (Squamata: Gekkonidae: Paroedura picta).

Variable clutch size is unambiguously an ancestral state in reptiles. Only several lizard lineages have evolved so-called invariant clutch size, where all females lay just one or two eggs per clutch. This mode of reproduction is characteristic for geckos. In some gecko lineages, decreased fecundity in a single clutch is compensated by conspicuous shortening of interclutch intervals. The proximate mechanism of high clutch frequency in these geckos is not known. Here, we document that three subsequently laid clutches develop simultaneously in females of the Madagascar ground gecko (Paroedura picta). The extremely short interclutch intervals in this species-even as short as a week-thus could be attributed to the overlap of female reproductive cycles. Such overlap should be associated with altered female hormonal cycles. Based on measurements of hormonal levels, we suggest that cycles of estradiol and progesterone during reproductive cycles of females in P. picta are largely independent. Thus, in contrast to the presumable ancestral reptile state, higher levels of progesterone do not seem to interfere with vitellogenesis in this species. We discuss potential consequences of this derived mode of reproduction, such as possible simultaneous maternal transfer of nutrients and other yolk components to several subsequent clutches.

Mitochondrial DNA variation reveals recent evolutionary history of main Boa constrictor clades.

We sequenced a 1114-bp fragment of cytochrome b gene in six subspecies (115 samples) of Boa constrictor and detected 67 haplotypes. Our analyses revealed the presence of two distinct clades, one from Central America (CA) including the neighboring part of South America west of the Andes, and the other covering the rest of South America (SA). Sequence divergence between CA and SA clades is about 5-7%, which roughly corresponds to a separation at the time of uplift of the Colombian Andes following formation of the Panama Isthmus before 3.5 Myr Sequence divergence within the SA and CA clades is only 2-3%, suggesting a fairly recent spread of these clades Into their current geographic ranges. Thus, we may not be dealing with taxa with a markedly old evolutionary history. Because juveniles of B. constrictor feed mostly on small rodents, we hypothesized that spread of this species was allowed by a new food source represented by murold rodents that appeared after closure of the Panama portal. With respect to the taxonomy, B. c. imperator may be elevated to full species rank. Within the SA clade, a haplotype of Argentinian B. c. occidentalis is markedly distinct, while the remaining haplotype groups analyzed are distributed throughout large ranges and may all belong to a single nominotypic subspecies.

Cell size but not genome size affects scaling of metabolic rate in eyelid geckos.

The metabolic theory of ecology (MTE) predicts the ubiquity of the of 3/4 scaling exponent relating metabolic rate (MR) to body mass, as well as cell-size invariance coupled with body-size dependence of cellular MR in quickly dividing cells. An alternative prediction is that MR scales interspecifically with a coefficient that is between 2/3 and 1, depending on the cell size and cell MR, which is mostly driven by the cell surface-to-volume ratio. We tested (1) the contribution of cell size to interspecific differences in MR and (2) whether the cell size-MR relationship is mediated by genome size (GS), which usually correlates positively with cell size. We tested (1) and (2) using erythrocyte area as a proxy for cell size in 14 eyelid geckos, which belong to a monophyletic group exhibiting large body-size variation. The scaling of standard MR (SMR) was significantly lower than 3/4, whereas mass-specific SMR correlated with erythrocyte area in both phylogenetically adjusted and conventional analyses. This points to cell-size variation as the factor governing metabolic rate scaling, which questions predictions of the MTE. However, the nonsignificance of the correlation between mass-specific SMR and GS undermines the strength of the relation between GS and cell size, at least in these species.

Cell size does not always correspond to genome size: phylogenetic analysis in geckos questions optimal DNA theories of genome size evolution.

At higher taxonomic levels, a significant correlation between genome size (GS) and erythrocyte size (ES) has been reported for many taxa. Under optimal DNA theories, several mechanisms presuming a causative link between GS and ES have been proposed to explain this seemingly general pattern. The correlation between GS and ES has been rarely tested among closely related organisms within an explicit phylogenetic framework. Eyelid geckos (family Eublepharidae) serve as a proper group to conduct such an analysis. We used flow cytometry to measure GS in 15 forms of eublepharids and conducted a phylogenetic reconstruction of GS and ES to test the successiveness of evolutionary shifts in these traits. Most parsimoniously, there were two independent increases and two decreases in GS during the evolution of eublepharids. Nevertheless, changes in GS and ES were not phylogenetically associated in a manner predicted by optimal DNA theories. Our results question the generality of causative bonds between DNA content and cell size and demonstrate that cell size cannot always serve as a proxy of GS. We suggest there is no need to expect a direct causative link between GS and ES to explain the correlation between GS and cell size at higher taxonomic levels. Such a correlation can be explained by simple mechanistic constraints and a combination of the population-genetic model of genome complexity with cell-size-metabolic rate relationship.