Identification of ancestral sex chromosomes in the frog Glandirana rugosa bearing XX-XY and ZZ-ZW sex-determining systems.

Sex chromosomes constantly exist in a dynamic state of evolution: rapid turnover and change of heterogametic sex during homomorphic state, and often stepping out to a heteromorphic state followed by chromosomal decaying. However, the forces driving these different trajectories of sex chromosome evolution are still unclear. The Japanese frog Glandirana rugosa is one taxon well suited to the study on these driving forces. The species has two different heteromorphic sex chromosome systems, XX-XY and ZZ-ZW, which are separated in different geographic populations. Both XX-XY and ZZ-ZW sex chromosomes are represented by chromosome 7 (2n = 26). Phylogenetically, these two systems arose via hybridization between two ancestral lineages of West Japan and East Japan populations, of which sex chromosomes are homomorphic in both sexes and to date have not yet been identified. Identification of the sex chromosomes will give us important insight into the mechanisms of sex chromosome evolution in this species. Here, we used a high-throughput genomic approach to identify the homomorphic XX-XY sex chromosomes in both ancestral populations. Sex-linked DNA markers of West Japan were aligned to chromosome 1, whereas those of East Japan were aligned to chromosome 3. These results reveal that at least two turnovers across three different sex chromosomes 1, 3 and 7 occurred during evolution of this species. This finding raises the possibility that cohabitation of the two different sex chromosomes from ancestral lineages induced turnover to another new one in their hybrids, involving transition of heterogametic sex and evolution from homomorphy to heteromorphy.

The stability investigation of variable viscosity control in the human-robot interaction.

BACKGROUND: For many co-manipulative applications, variable damping is a valuable feature provided by robots. One approach is implementing a high viscosity at low velocities and a low viscosity at high velocities. This, however, is proven to have the possibility to alter human natural motion performance. METHODS: We show that the distortion is caused by the viscosity drop resulting in robot's resistance to motion. To address this, a method for stably achieving the desired behaviour is presented. It involves leveraging a first-order linear filter to slow the viscosity variation down. RESULTS: The proposition is supported by a theoretical analysis using a robotic model. Meanwhile, the user performance in human-robot experiments gets significantly improved, showing the practical efficiency in real applications. CONCLUSIONS: This paper discusses the variable viscosity control in the context of co-manipulation. An instability problem and its solution were theoretically shown and experimentally evidenced through human-robot experiments.

Mass of genes rather than master genes underlie the genomic architecture of amphibian speciation.

The genetic architecture of speciation, i.e., how intrinsic genomic incompatibilities promote reproductive isolation (RI) between diverging lineages, is one of the best-kept secrets of evolution. To directly assess whether incompatibilities arise in a limited set of large-effect speciation genes, or in a multitude of loci, we examined the geographic and genomic landscapes of introgression across the hybrid zones of 41 pairs of frog and toad lineages in the Western Palearctic region. As the divergence between lineages increases, phylogeographic transitions progressively become narrower, and larger parts of the genome resist introgression. This suggests that anuran speciation proceeds through a gradual accumulation of multiple barrier loci scattered across the genome, which ultimately deplete hybrid fitness by intrinsic postzygotic isolation, with behavioral isolation being achieved only at later stages. Moreover, these loci were disproportionately sex linked in one group (Hyla) but not in others (Rana and Bufotes), implying that large X-effects are not necessarily a rule of speciation with undifferentiated sex chromosomes. The highly polygenic nature of RI and the lack of hemizygous X/Z chromosomes could explain why the speciation clock ticks slower in amphibians compared to other vertebrates. The clock-like dynamics of speciation combined with the analytical focus on hybrid zones offer perspectives for more standardized practices of species delimitation.

Sex-chromosome evolution in frogs: what role for sex-antagonistic genes?

Sex-antagonistic (SA) genes are widely considered to be crucial players in the evolution of sex chromosomes, being instrumental in the arrest of recombination and degeneration of Y chromosomes, as well as important drivers of sex-chromosome turnovers. To test such claims, one needs to focus on systems at the early stages of differentiation, ideally with a high turnover rate. Here, I review recent work on two families of amphibians, Ranidae (true frogs) and Hylidae (tree frogs), to show that results gathered so far from these groups provide no support for a significant role of SA genes in the evolutionary dynamics of their sex chromosomes. The findings support instead a central role for neutral processes and deleterious mutations. 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 I)'.

Expanding the classical paradigm: what we have learnt from vertebrates about sex chromosome evolution.

Until recently, the field of sex chromosome evolution has been dominated by the canonical unidirectional scenario, first developed by Muller in 1918. This model postulates that sex chromosomes emerge from autosomes by acquiring a sex-determining locus. Recombination reduction then expands outwards from this locus, to maintain its linkage with sexually antagonistic/advantageous alleles, resulting in Y or W degeneration and potentially culminating in their disappearance. Based mostly on empirical vertebrate research, we challenge and expand each conceptual step of this canonical model and present observations by numerous experts in two parts of a theme issue of Phil. Trans. R. Soc. B. We suggest that greater theoretical and empirical insights into the events at the origins of sex-determining genes (rewiring of the gonadal differentiation networks), and a better understanding of the evolutionary forces responsible for recombination suppression are required. Among others, crucial questions are: Why do sex chromosome differentiation rates and the evolution of gene dose regulatory mechanisms between male versus female heterogametic systems not follow earlier theory? Why do several lineages not have sex chromosomes? And: What are the consequences of the presence of (differentiated) sex chromosomes for individual fitness, evolvability, hybridization and diversification? We conclude that the classical scenario appears too reductionistic. Instead of being unidirectional, we show that sex chromosome evolution is more complex than previously anticipated and principally forms networks, interconnected to potentially endless outcomes with restarts, deletions and additions of new genomic material. 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)'.

Capture and return of sexual genomes by hybridogenetic frogs provide clonal genome enrichment in a sexual species.

Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.

When Sex Chromosomes Recombine Only in the Heterogametic Sex: Heterochiasmy and Heterogamety in Hyla Tree Frogs.

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.

Meiosis reveals the early steps in the evolution of a neo-XY sex chromosome pair in the African pygmy mouse Mus minutoides.

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution.

Hybridization and introgression between toads with different sex chromosome systems.

The growing interest in the lability of sex determination in non-model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of the same clade. Can such lineages hybridize and admix with different sex-determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome-wide markers (RADseq). Adult and sibship data confirmed that the common toad B. bufo is female heterogametic (ZW), while its sister species the spined toad B. spinosus is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively. The geographic extent of introgression is consistent with an expansion of B. spinosus across B. bufo's former ranges in Mediterranean France, as also suggested by species distribution models. However, widespread cyto-nuclear discordances (B. spinosus backrosses carrying B. bufo mtDNA) run against predictions from the dominance effects of Haldane's rule, perhaps because Y and W heterogametologs are not degenerated. Common and spined toads can thus successfully cross-breed despite fundamental differences in their sex determination mechanisms, but remain partially separated by reproductive barriers. Whether and how the interactions of their XY and ZW genes contribute to these barriers shall provide novel insights on the debated role of labile sex chromosomes in speciation.

The effect of phylogeographic history on species boundaries: a comparative framework in Hyla tree frogs.

Because it is indicative of reproductive isolation, the amount of genetic introgression across secondary contact zones is increasingly considered in species delimitation. However, patterns of admixture at range margins can be skewed by the regional dynamics of hybrid zones. In this context, we posit an important role for phylogeographic history: hybrid zones located within glacial refugia (putatively formed during the Late-Pleistocene) should be better defined than those located in post-glacial or introduced ranges (putatively formed during the Holocene and the Anthropocene). We test this hypothesis in a speciation continuum of tree frogs from the Western Palearctic (Hyla), featuring ten identified contacts between species spanning Plio-Pleistocene to Miocene divergences. We review the rich phylogeographic literature of this group and examine the overlooked transition between H. arborea and H. molleri in Western France using a multilocus dataset. Our comparative analysis supports a trend that contacts zones resulting from post-glacial expansions and human translocations feature more extensive introgression than those established within refugial areas. Integrating the biogeographic history of incipient species, i.e. their age since first contact together with their genetic divergence, thus appears timely to draw sound evolutionary and taxonomic inferences from patterns of introgression across hybrid zones.

Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs.

Subdivided Pleistocene glacial refugia, best known as "refugia within refugia", provided opportunities for diverging populations to evolve into incipient species and/or to hybridize and merge following range shifts tracking the climatic fluctuations, potentially promoting extensive cytonuclear discordances and "ghost" mtDNA lineages. Here, we tested which of these opposing evolutionary outcomes prevails in northern Iberian areas hosting multiple historical refugia of common frogs (Rana cf. temporaria), based on a genomic phylogeography approach (mtDNA barcoding and RAD-sequencing). We found evidence for both incipient speciation events and massive cytonuclear discordances. On the one hand, populations from northwestern Spain (Galicia and Asturias, assigned to the regional endemic R. parvipalmata), are deeply-diverged at mitochondrial and nuclear genomes (~4 My of independent evolution), and barely admix with northeastern populations (assigned to R. temporaria sensu stricto) across a narrow hybrid zone (~25 km) located in the Cantabrian Mountains, suggesting that they represent distinct species. On the other hand, the most divergent mtDNA clade, widespread in Cantabria and the Basque country, shares its nuclear genome with other R. temporaria s. s. lineages. Patterns of population expansions and isolation-by-distance among these populations are consistent with past mitochondrial capture and/or drift in generating and maintaining this ghost mitochondrial lineage. This remarkable case study emphasizes the complex evolutionary history that shaped the present genetic diversity of refugial populations, and stresses the need to revisit their phylogeography by genomic approaches, in order to make informed taxonomic inferences.

Integrating hybrid zone analyses in species delimitation: lessons from two anuran radiations of the Western Mediterranean.

Molecular ecologists often rely on phylogenetic evidence for assessing the species-level systematics of newly discovered lineages. Alternatively, the extent of introgression at phylogeographic transitions can provide a more direct test to assign candidate taxa into subspecies or species categories. Here, we compared phylogenetic versus hybrid zone approaches of species delimitation in two groups of frogs from the Western Mediterranean region (Discoglossus and Pelodytes), by using genomic data (ddRAD). In both genera, coalescent analyses recovered almost all nominal taxa as "species". However, the least-diverged pairs D. g. galganoi/jeanneae and P. punctatus/hespericus admix over hundreds of kilometers, suggesting that they have not yet developed strong reproductive isolation and should be treated as conspecifics. In contrast, the comparatively older D. scovazzi/pictus and P. atlanticus/ibericus form narrow contact zones, consistent with species distinctiveness. Due to their complementarity, we recommend taxonomists to combine phylogenomics with hybrid zone analyses to scale the gray zone of speciation, i.e., the evolutionary window separating widely admixing lineages versus nascent reproductively isolated species. The radically different transitions documented here conform to the view that genetic incompatibilities accumulating with divergence generate a weak barrier to gene flow for long periods of time, until their effects multiply and the speciation process then advances rapidly. Given the variability of the gray zone among taxonomic groups, at least from our current abilities to measure it, we recommend to customize divergence thresholds within radiations to categorize lineages for which no direct test of speciation is possible.

No evidence that Y-chromosome differentiation affects male fitness in a Swiss population of common frogs.

The canonical model of sex-chromosome evolution assigns a key role to sexually antagonistic (SA) genes on the arrest of recombination and ensuing degeneration of Y chromosomes. This assumption cannot be tested in organisms with highly differentiated sex chromosomes, such as mammals or birds, owing to the lack of polymorphism. Fixation of SA alleles, furthermore, might be the consequence rather than the cause of recombination arrest. Here we focus on a population of common frogs (Rana temporaria) where XY males with genetically differentiated Y chromosomes (nonrecombinant Y haplotypes) coexist with both XY° males with proto-Y chromosomes (only differentiated from X chromosomes in the immediate vicinity of the candidate sex-determining locus Dmrt1) and XX males with undifferentiated sex chromosomes (genetically identical to XX females). Our study finds no effect of sex-chromosome differentiation on male phenotype, mating success or fathering success. Our conclusions rejoin genomic studies that found no differences in gene expression between XY, XY° and XX males. Sexual dimorphism in common frogs might result more from the differential expression of autosomal genes than from sex-linked SA genes. Among-male variance in sex-chromosome differentiation seems better explained by a polymorphism in the penetrance of alleles at the sex locus, resulting in variable levels of sex reversal (and thus of X-Y recombination in XY females), independent of sex-linked SA genes.

Phylogeography, more than elevation, accounts for sex chromosome differentiation in Swiss populations of the common frog (Rana temporaria).

Sex chromosomes in vertebrates range from highly heteromorphic (as in most birds and mammals) to strictly homomorphic (as in many fishes, amphibians, and nonavian reptiles). Reasons for these contrasted evolutionary trajectories remain unclear, but species such as common frogs with polymorphism in the extent of sex chromosome differentiation may potentially deliver important clues. By investigating 92 common frog populations from a wide range of elevations throughout Switzerland, we show that sex chromosome differentiation strongly correlates with alleles at the candidate sex-determining gene Dmrt1. Y-specific Dmrt1 haplotypes cluster into two main haplogroups, YA and YB , with a phylogeographic signal that parallels mtDNA haplotypes: YA populations, with mostly well-differentiated sex chromosomes, occur primarily south of the main alpine ridge that bisects Switzerland, whereas YB populations, with mostly undifferentiated (proto-)sex chromosomes, occur north of this ridge. Elevation has only a marginal effect, opposing previous suggestions of a major role for climate on sex chromosome differentiation. The Y-haplotype effect might result from differences in the penetrance of alleles at the sex-determining locus (such that sex reversal and ensuing X-Y recombination are more frequent in YB populations), and/or fixation of an inversion on YA (as supported by the empirical observation that YA haplotypes might not recombine in XYA females).

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates).

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species-level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near-East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.

Impact of deleterious mutations, sexually antagonistic selection, and mode of recombination suppression on transitions between male and female heterogamety.

Deleterious mutations accumulating on non-recombining Y chromosomes can drive XY to XY turnovers, as they allow to replace the old mutation-loaded Y by a new mutation-free one. The same process is thought to prevent XY to ZW turnovers, because the latter requires fixation of the ancestral Y, assuming dominance of the emergent feminizing mutation. Using individual-based simulations, we explored whether and how an epistatically dominant W allele can spread in a young XY system that gradually accumulates deleterious mutations. We also investigated how sexually antagonistic (SA) polymorphism on the ancestral sex chromosomes and the mechanism controlling X-Y recombination suppression affect these transitions. In contrast with XY to XY turnovers, XY to ZW turnovers cannot be favored by Y chromosome mutation load. If the arrest of X-Y recombination depends on genotypic sex, transitions are strongly hindered by deleterious mutations, and totally suppressed by very small SA cost, because deleterious mutations and female-detrimental SA alleles would have to fix with the Y. If, however, the arrest of X-Y recombination depends on phenotypic sex, X and Y recombine in XY ZW females, allowing for the purge of Y-linked deleterious mutations and loss of the SA polymorphism, causing XY to ZW turnovers to occur at the same rate as in the absence of deleterious and sex-antagonistic mutations. We generalize our results to other types of turnovers (e.g., triggered by non-dominant sex-determining mutations) and discuss their empirical relevance.

Diversification and speciation in tree frogs from the Maghreb (Hyla meridionalis sensu lato), with description of a new African endemic.

Comparative molecular studies emphasized a new biogeographic paradigm for the terrestrial fauna of North Africa, one of the last uncharted ecoregions of the Western Palearctic: two independent east-west divisions across the Maghreb. Through a comprehensive phylogeography, we assessed how this model suits the genetic diversification documented for the tree frog Hyla meridionalis sensu lato. Analyses of mtDNA variation and thousands of nuclear loci confirmed the old split (low-Pliocene) between Tunisian and Algerian populations. These lineages meet but barely admix in the eastern Maghreb (Algerian-Tunisian border), a sign of putatively advanced reproductive isolation. In the western Maghreb, we report a Pleistocene divergence between Moroccan and Algerian populations. Tree frogs thus follow both predictions: a double east-west break that gave rise to two suture zones characteristic of North-African phylogeography. Moreover, some intraspecific mtDNA variation is not mirrored by the nuclear data, emphasizing that evolutionary units should always be designated by multilocus approaches. Last but not least, we describe the Tunisian lineage as a new species endemic to Africa.

A reciprocal translocation radically reshapes sex-linked inheritance in the common frog.

X and Y chromosomes can diverge when rearrangements block recombination between them. Here we present the first genomic view of a reciprocal translocation that causes two physically unconnected pairs of chromosomes to be coinherited as sex chromosomes. In a population of the common frog (Rana temporaria), both pairs of X and Y chromosomes show extensive sequence differentiation, but not degeneration of the Y chromosomes. A new method based on gene trees shows both chromosomes are sex-linked. Furthermore, the gene trees from the two Y chromosomes have identical topologies, showing they have been coinherited since the reciprocal translocation occurred. Reciprocal translocations can thus reshape sex linkage on a much greater scale compared with inversions, the type of rearrangement that is much better known in sex chromosome evolution, and they can greatly amplify the power of sexually antagonistic selection to drive genomic rearrangement. Two more populations show evidence of other rearrangements, suggesting that this species has unprecedented structural polymorphism in its sex chromosomes.

Swall-E: A robotic in-vitro simulation of human swallowing.

Swallowing is a complex physiological function that can be studied through medical imagery techniques such as videofluoroscopy (VFS), dynamic magnetic resonance imagery (MRI) and fiberoptic endoscopic evaluation of swallowing (FEES). VFS is the gold standard although it exposes the subjects to radiations. In-vitro modeling of human swallowing has been conducted with limited results so far. Some experiments were reported on robotic reproduction of oral and esophageal phases of swallowing, but high fidelity reproduction of pharyngeal phase of swallowing has not been reported yet. To that end, we designed and developed a robotic simulator of the pharyngeal phase of human swallowing named Swall-E. 17 actuators integrated in the robot enable the mimicking of important physiological mechanisms occurring during the pharyngeal swallowing, such as the vocal fold closure, laryngeal elevation or epiglottis tilt. Moreover, the associated computer interface allows a control of the actuation of these mechanisms at a spatio-temporal accuracy of 0.025 mm and 20 ms. In this study preliminary experiments of normal pharyngeal swallowing simulated on Swall-E are presented. These experiments show that a 10 ml thick bolus can be swallowed by the robot in less than 1 s without any aspiration of bolus material into the synthetic anatomical laryngo-tracheal conduit.

Evolutionary and developmental dynamics of sex-biased gene expression in common frogs with proto-Y chromosomes.

BACKGROUND: The patterns of gene expression on highly differentiated sex chromosomes differ drastically from those on autosomes, due to sex-specific patterns of selection and inheritance. As a result, X chromosomes are often enriched in female-biased genes (feminization) and Z chromosomes in male-biased genes (masculinization). However, it is not known how quickly sexualization of gene expression and transcriptional degeneration evolve after sex-chromosome formation. Furthermore, little is known about how sex-biased gene expression varies throughout development. RESULTS: We sample a population of common frogs (Rana temporaria) with limited sex-chromosome differentiation (proto-sex chromosome), leaky genetic sex determination evidenced by the occurrence of XX males, and delayed gonadal development, meaning that XY individuals may first develop ovaries before switching to testes. Using high-throughput RNA sequencing, we investigate the dynamics of gene expression throughout development, spanning from early embryo to froglet stages. Our results show that sex-biased expression affects different genes at different developmental stages and increases during development, reaching highest levels in XX female froglets. Additionally, sex-biased gene expression depends on phenotypic, rather than genotypic sex, with similar expression in XX and XY males; correlates with gene evolutionary rates; and is not localized to the proto-sex chromosome nor near the candidate sex-determining gene Dmrt1. CONCLUSIONS: The proto-sex chromosome of common frogs does not show evidence of sexualization of gene expression, nor evidence for a faster rate of evolution. This challenges the notion that sexually antagonistic genes play a central role in the initial stages of sex-chromosome evolution.