Environmental (and random?) sex determination in endangered and invasive Phelsuma geckos.

INTRODUCTION: Sex is a fundamental characteristic of an individual. It is therefore puzzling why in some systems sex is precisely determined by a genotype, while in others it is influenced by the environment or even subtle, not to say random, factors. Some stochasticity in sex determination would be expected if environmental conditions did not have a large sex-specific effect on fitness. Although data are only available for a small fraction of species, geckos show exceptional variability in sex determination. METHODS: We tested the effects of three incubation temperatures on sex ratio and adult body size in the invasive gecko Phelsuma laticauda and the vulnerable gecko Phelsuma nigristriata. RESULTS: We document temperature-dependent sex determination (TSD) in both species. Only females hatched at a low temperature (24 °C), whereas male production peaked at an intermediate temperature (29 °C) and declined, at least in P. laticauda, again at the highest temperature (31 °C). Interestingly, full siblings hatched from eggs glued together during the incubation at temperatures producing both sexes are often of the opposite sex. We found no significant effect of incubation temperature on adult body length. CONCLUSIONS: Documentation of TSD in the day geckos has implications for conservation practice in environmental management of endangered species or eradication of invasive species. However, it appears that a very subtle (random?) factor may also be involved in their sex determination. In line with this, we found no significant effect of incubation temperature on adult body length, suggesting that, at least in this trait, there is no strong selection against producing females at "male" temperatures.

The fallacy of global comparisons based on per capita measures.

Media, social scientists and public health researchers often present comparisons across countries, and policy makers use such comparisons to take evidence-based action. For a meaningful comparison among countries, one often needs to normalize the measure for differences in population size. To address this issue, the first choice is usually to calculate per capita ratios. Such ratios, however, normalize the measure for differences in population size directly only under the highly restrictive assumption of a proportional increase of the measure with population size. Violation of this assumption frequently leads to misleading conclusions. We compare per capita ratios with an approach based on regression, a widely used statistical procedure that eliminates many of the problems with ratios and allows for straightforward data interpretation. It turns out that the per capita measures in three global datasets (gross domestic product, COVID-19-related mortality and CO2 production) systematically overestimate values in countries with small populations, while countries with large populations tend to have misleadingly low per capita ratios owing to the large denominators. Unfortunately, despite their biases, comparisons based on per capita ratios are still ubiquitous, and they are used for influential recommendations by various global institutions. Their continued use can cause significant damage when employed as evidence for policy actions and should therefore be replaced by a more scientifically substantiated and informative method, such as a regression-based approach.

Pivotal temperature is not for everyone: Evidence for temperature-dependent sex determination in three gecko species.

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.

Differentiated sex chromosomes, karyotype evolution, and spontaneous triploidy in carphodactylid geckos.

Geckos exhibit derived karyotypes without a clear distinction between macrochromosomes and microchromosomes and intriguing diversity in sex determination mechanisms. We conducted cytogenetic analyses in six species from the genera Nephrurus, Phyllurus, and Saltuarius of the gecko family Carphodactylidae. We confirmed the presence of a female heterogametic system with markedly differentiated and heteromorphic sex chromosomes in all examined species, typically with the W chromosome notably larger than the Z chromosome. One species, Nephrurus cinctus, possesses unusual multiple Z1Z1Z2Z2/Z1Z2W sex chromosomes. The morphology of the sex chromosomes, along with repetitive DNA content, suggests that the differentiation or emergence of sex chromosomes occurred independently in the genus Phyllurus. Furthermore, our study unveils a case of spontaneous triploidy in a fully grown individual of Saltuarius cornutus (3n = 57) and explores its implications for reproduction in carphodactylid geckos. We revealed that most carphodactylids retain the putative ancestral gekkotan karyotype of 2n = 38, characterized by predominantly acrocentric chromosomes that gradually decrease in size. If present, biarmed chromosomes emerge through pericentric inversions, maintaining the chromosome (and centromere) numbers. However, Phyllurus platurus is a notable exception, with a karyotype of 2n = 22 chromosomes. Its eight pairs of biarmed chromosomes were probably formed by Robertsonian fusions of acrocentric chromosomes. The family underscores a remarkable instance of evolutionary stability in chromosome numbers, followed by a profound transformation through parallel interchromosomal rearrangements. Our study highlights the need to continue generating cytogenetic data in order to test long-standing ideas about reproductive biology and the evolution of genome and sex determination.

Heteromorphic ZZ/ZW sex chromosomes sharing gene content with mammalian XX/XY are conserved in Madagascan chameleons of the genus Furcifer.

Chameleons are well-known lizards with unique morphology and physiology, but their sex determination has remained poorly studied. Madagascan chameleons of the genus Furcifer have cytogenetically distinct Z and W sex chromosomes and occasionally Z1Z1Z2Z2/Z1Z2W multiple neo-sex chromosomes. To identify the gene content of their sex chromosomes, we microdissected and sequenced the sex chromosomes of F. oustaleti (ZZ/ZW) and F. pardalis (Z1Z1Z2Z2/Z1Z2W). In addition, we sequenced the genomes of a male and a female of F. lateralis (ZZ/ZW) and F. pardalis and performed a comparative coverage analysis between the sexes. Despite the notable heteromorphy and distinctiveness in heterochromatin content, the Z and W sex chromosomes share approximately 90% of their gene content. This finding demonstrates poor correlation of the degree of differentiation of sex chromosomes at the cytogenetic and gene level. The test of homology based on the comparison of gene copy number variation revealed that female heterogamety with differentiated sex chromosomes remained stable in the genus Furcifer for at least 20 million years. These chameleons co-opted for the role of sex chromosomes the same genomic region as viviparous mammals, lacertids and geckos of the genus Paroedura, which makes these groups excellent model for studies of convergent and divergent evolution of sex chromosomes.

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.

Robertsonian fusion triggers recombination suppression on sex chromosomes in Coleonyx geckos.

The classical hypothesis proposes that the lack of recombination on sex chromosomes arises due to selection for linkage between a sex-determining locus and sexually antagonistic loci, primarily facilitated by inversions. However, cessation of recombination on sex chromosomes could be attributed also to neutral processes, connected with other chromosome rearrangements or can reflect sex-specific recombination patterns existing already before sex chromosome differentiation. Three Coleonyx gecko species share a complex X1X1X2X2/X1X2Y system of sex chromosomes evolved via a fusion of the Y chromosome with an autosome. We analyzed synaptonemal complexes and sequenced flow-sorted sex chromosomes to investigate the effect of chromosomal rearrangement on recombination and differentiation of these sex chromosomes. The gecko sex chromosomes evolved from syntenic regions that were also co-opted also for sex chromosomes in other reptiles. We showed that in male geckos, recombination is less prevalent in the proximal regions of chromosomes and is even further drastically reduced around the centromere of the neo-Y chromosome. We highlight that pre-existing recombination patterns and Robertsonian fusions can be responsible for the cessation of recombination on sex chromosomes and that such processes can be largely neutral.

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.

Madagascar Leaf-Tail Geckos (Uroplatus spp.) Share Independently Evolved Differentiated ZZ/ZW Sex Chromosomes.

Geckos are an excellent group to study the evolution of sex determination, as they possess a remarkable variability ranging from a complete absence of sex chromosomes to highly differentiated sex chromosomes. We explored sex determination in the Madagascar leaf-tail geckos of the genus Uroplatus. The cytogenetic analyses revealed highly heterochromatic W chromosomes in all three examined species (Uroplatus henkeli, U. alluaudi, U. sikorae). The comparative gene coverage analysis between sexes in U. henkeli uncovered an extensive Z-specific region, with a gene content shared with the chicken chromosomes 8, 20, 26 and 28. The genomic region homologous to chicken chromosome 28 has been independently co-opted for the role of sex chromosomes in several vertebrate lineages, including monitors, beaded lizards and monotremes, perhaps because it contains the amh gene, whose homologs were repeatedly recruited as a sex-determining locus. We demonstrate that all tested species of leaf-tail geckos share homologous sex chromosomes despite the differences in shape and size of their W chromosomes, which are not homologous to the sex chromosomes of other closely related genera. The rather old (at least 40 million years), highly differentiated sex chromosomes of Uroplatus geckos can serve as a great system to study the convergence of sex chromosomes evolved from the same genomic region.

Cytogenetic Analysis of Seven Species of Gekkonid and Phyllodactylid Geckos.

Geckos (Gekkota), the species-rich clade of reptiles with more than 2200 currently recognized species, demonstrate a remarkable variability in diploid chromosome numbers (2n = 16-48) and mode of sex determination. However, only a small fraction of gekkotan species have been studied with cytogenetic methods. Here, we applied both conventional (karyotype reconstruction and C-banding) and molecular (fluorescence in situ hybridization with probes for rDNA loci and telomeric repeats) cytogenetic analyses in seven species of geckos, namely Blaesodactylus boivini, Chondrodactylus laevigatus, Gekko badenii, Gekko cf. lionotum, Hemidactylus sahgali, Homopholis wahlbergii (Gekkonidae) and Ptyodactylus togoensis (Phyllodactylidae), in order to provide further insights into the evolution of karyotypes in geckos. Our analysis revealed the presence of interstitial telomeric repeats in four species, but we were not able to conclude if they are remnants of previous chromosome rearrangements or were formed by an accumulation of telomeric-like satellite motifs. Even though sex chromosomes were previously identified in several species from the genera Hemidactylus and Gekko by cytogenetic and/or genomic methods, they were not detected by us in any examined species. Our examined species either have poorly differentiated sex chromosomes or, possibly, environmental sex determination. Future studies should explore the effect of temperature and conduct genome-wide analyses in order to identify the mode of sex determination in these species.

XX/XY sex chromosomes in a blind lizard (Dibamidae): Towards understanding the evolution of sex determination in squamates.

The reconstruction of the evolutionary history of sex determination in squamate reptiles (lizards and snakes) is complicated by missing data in many lineages, erroneous reports, and often questionable inferences on state homology. Therefore, despite the large effort, the reconstruction of the ancestral sex determination in squamate reptiles is still controversial. With the hope to shed light on this problem, we aspired to identify the sex chromosome gene content in Dibamus deharvengi, the representative of the family Dibamidae, the putative sister clade to all other squamates. Our analyses revealed XX/XY sex-determination system in D. deharvengi: the X chromosome contains genes with homologues scattered across chicken chromosomes 8, 12, 13, 18, 30, and 33, and the Y chromosome seems to largely degenerate. To the best of our knowledge, this combination has never been reported to form sex chromosomes in any amniote lineage. It suggests that the sex chromosomes can represent an apomorphy of a clade including D. deharvengi. Our findings cover an important gap in the knowledge of sex determination in reptiles and further support multiple independent origins of sex chromosomes in this group.

Meiotic chromosome dynamics and double strand break formation in reptiles.

During meiotic prophase I, tightly regulated processes take place, from pairing and synapsis of homologous chromosomes to recombination, which are essential for the generation of genetically variable haploid gametes. These processes have canonical meiotic features conserved across different phylogenetic groups. However, the dynamics of meiotic prophase I in non-mammalian vertebrates are poorly known. Here, we compare four species from Sauropsida to understand the regulation of meiotic prophase I in reptiles: the Australian central bearded dragon (Pogona vitticeps), two geckos (Paroedura picta and Coleonyx variegatus) and the painted turtle (Chrysemys picta). We first performed a histological characterization of the spermatogenesis process in both the bearded dragon and the painted turtle. We then analyzed prophase I dynamics, including chromosome pairing, synapsis and the formation of double strand breaks (DSBs). We show that meiosis progression is highly conserved in reptiles with telomeres clustering forming the bouquet, which we propose promotes homologous pairing and synapsis, along with facilitating the early pairing of micro-chromosomes during prophase I (i.e., early zygotene). Moreover, we detected low levels of meiotic DSB formation in all taxa. Our results provide new insights into reptile meiosis.

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.

Cytogenetic Analysis of the Members of the Snake Genera Cylindrophis, Eryx, Python, and Tropidophis.

The recent discovery of two independently evolved XX/XY sex determination systems in the snake genera Python and Boa sparked a new drive to study the evolution of sex chromosomes in poorly studied lineages of snakes, where female heterogamety was previously assumed. Therefore, we examined seven species from the genera Eryx, Cylindrophis, Python, and Tropidophis by conventional and molecular cytogenetic methods. Despite the fact that these species have similar karyotypes in terms of chromosome number and morphology, we detected variability in the distribution of heterochromatin, telomeric repeats, and rDNA loci. Heterochromatic blocks were mainly detected in the centromeric regions in all species, although accumulations were detected in pericentromeric and telomeric regions in a few macrochromosomes in several of the studied species. All species show the expected topology of telomeric repeats at the edge of all chromosomes, with the exception of Eryx muelleri, where additional accumulations were detected in the centromeres of three pairs of macrochromosomes. The rDNA loci accumulate in one pair of microchromosomes in all Eryx species and in Cylindrophis ruffus, in one macrochromosome pair in Tropidophis melanurus and in two pairs of microchromosomes in Python regius. Sex-specific differences were not detected, suggesting that these species likely have homomorphic, poorly differentiated sex chromosomes.

Origin and haplotype diversity of the northernmost population of Podarcistauricus (Squamata, Lacertidae): Do lizards respond to climate change and go north?

The northernmost population of the Balkan wall lizards, Podarcistauricus (Pallas, 1814) was recently discovered in the Czech Republic. We studied genetic variability in a mitochondrial marker cytochrome b to shed light on the origin of this remote population. We detected three unique haplotypes, close to those occurring in the populations of Podarcistauricus from central/north Balkans and Hungary. Our data exclude the hypothesis of a single founder (a randomly or intentionally introduced pregnant female or her progeny) of the Czech population and indicate a native, autochthonous origin of the population or recent introduction/range expansion.

Premeiotic endoreplication is essential for obligate parthenogenesis in geckos.

Obligate parthenogenesis evolved in reptiles convergently several times, mainly through interspecific hybridization. The obligate parthenogenetic complexes typically include both diploid and triploid lineages. Offspring of parthenogenetic hybrids are genetic copies of their mother; however, the cellular mechanism enabling the production of unreduced cells is largely unknown. Here, we show that oocytes go through meiosis in three widespread, or even strongly invasive, obligate parthenogenetic complexes of geckos, namely in diploid and triploid Lepidodactylus lugubris, and triploid Hemiphyllodactylus typus and Heteronotia binoei. In all four lineages, the majority of oocytes enter the pachytene at the original ploidy level, but their chromosomes cannot pair properly and instead form univalents, bivalents and multivalents. Unreduced eggs with clonally inherited genomes are formed from germ cells that had undergone premeiotic endoreplication, in which appropriate segregation is ensured by the formation of bivalents made from copies of identical chromosomes. We conclude that the induction of premeiotic endoreplication in reptiles was independently co-opted at least four times as an essential component of parthenogenetic reproduction and that this mechanism enables the emergence of fertile polyploid lineages within parthenogenetic complexes.

Sex-specific growth arrest in a lizard.

(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.

Ratios can be misleading for detecting selection.

Lukás Kratochvíl and Michail Rovatsos respond to the recent study by Thibault Leroy and colleagues on island songbirds.

ZZ/ZW Sex Chromosomes in the Madagascar Girdled Lizard, Zonosaurus madagascariensis (Squamata: Gerrhosauridae).

Scincoidea, the reptilian clade that includes girdled lizards, night lizards, plated lizards and skinks, are considered as a lineage with diversity in sex-determining systems. Recently, the hypothesis on the variability in sex determination in skinks and even more the absence of sex chromosomes in some of them has been rivalling. Homologous, evolutionary stable XX/XY sex chromosomes were documented to be widespread across skinks. However, sex determination in the other scincoidean families is highly understudied. ZZ/ZW sex chromosomes have been identified only in night lizards and a single species of plated lizards. It seems that although there are different sex chromosome systems among scincoidean lineages, they share one common trait: they are mostly poorly differentiated and often undetectable by cytogenetic methods. Here, we report one of the exceptions, demonstrating for the first time ZZ/ZW sex chromosomes in the plated lizard Zonosaurus madagascariensis. Its sex chromosomes are morphologically similar, but the W is clearly detectable by comparative genomic hybridization (CGH), suggesting that the Z and W chromosomes highly differ in sequence content. Our findings confirm the presence of female heterogamety in plated lizards and provides novel insights to expand our understanding of sex chromosome evolution in scincoidean lizards.