Significance: As in most amphibians, sex chromosomes of the model species Xenopus tropicalis are homomorphic, complicating identification of the heterogametic sex. Using genetic approaches, we have proved the existence of three types of sex chromosomes (Y, W, and Z), defining three kinds of males (YZ, YW, and ZZ) and two kinds of females (ZW and WW). The existence of both male and female heterogametic individuals in one species is an extremely rare situation in nature, because some sex chromosome combinations produce offspring with sex ratios different from 1:1. Thus, parental sex chromosomes must be taken into account when X . tropicalis is used in multigeneration genetic studies or in ecotoxicological assays of endocrine disruptors with gender effects.

The role of environmental factors in sexual differentiation in amphibians is not new. The effect of hormones or hormone-like compounds is widely demonstrated. However, the effect of temperature has traditionally been regarded as something anecdotal that occurs in extreme situations and not as a factor to be considered. The data currently available reveal a different situation. Sexual differentiation in some amphibian species can be altered even by small changes in temperature. On the other hand, although not proven, it is possible that temperature is related to the appearance of sex-reversed individuals in natural populations under conditions unrelated to environmental contaminants. According to this, temperature, through sex reversal (phenotypic sex opposed to genetic sex), could play an important role in the turnover of sex-determining genes and in the maintenance of homomorphic sex chromosomes in this group. Accordingly, and given the expected increase in global temperatures, growth and sexual differentiation in amphibians could easily be affected, altering the sex ratio in natural populations and posing major conservation challenges for a group in worldwide decline. It is therefore particularly urgent to understand the mechanism by which temperature affects sexual differentiation in amphibians.

Sex is determined genetically in amphibians; however, little is known about the sex chromosomes, testis-determining genes, and the genes involved in testis differentiation in this class. Certain inherent characteristics of the species of this group, like the homomorphic sex chromosomes, the high diversity of the sex-determining mechanisms, or the existence of polyploids, may hinder the design of experiments when studying how the gonads can differentiate. Even so, other features, like their external development or the possibility of inducing sex reversal by external treatments, can be helpful. This review summarizes the current knowledge on amphibian sex determination, gonadal development, and testis differentiation. The analysis of this information, compared with the information available for other vertebrate groups, allows us to identify the evolutionarily conserved and divergent pathways involved in testis differentiation. Overall, the data confirm the previous observations in other vertebrates—the morphology of the adult testis is similar across different groups; however, the male-determining signal and the genetic networks involved in testis differentiation are not evolutionarily conserved.

Amphibians have some of the most variable genome sizes among vertebrates. Genome size variation has been attributed to repetitive and noncoding DNA, including satellite repeats, transposable elements, introns, and nuclear insertions of viral and organelle DNA. In vertebrates, satellite DNAs have been widely described in mammals, but few molecular studies have been carried out in amphibians. Here, we provide a detailed characterization of a new family of satellite DNA, present in all 15 examined species of the family Bufonidae. Southern-blot analysis and PCR reveal that this satellite is formed by monomers of 807 bp, is organized in tandem arrays, and has an AT-content of 57.4%. Phylogenetic analyses show that most clades exhibit species-specific variances, indicating that this satellite DNA has evolved by concerted evolution. The homogenization/fixation process is heterogeneous in Bufonidae, where the genera Bufo and Bufotes do not show species-specific differences, while populations from Rhinella marina exhibit population-specific changes. Additionally, variants of this satellite DNA have been identified in Duttaphrynus melanostictus and R. marina, supporting the ‘library hypothesis’ (a set, ‘library’, of satellite DNAs is shared by a species group). Physical mapping in Bufo bufo, Bufo spinosus, Epidalea calamita and Bufotes viridis provides evidence that this repetitive DNA is not dispersed in the karyotype, but accumulated in pericentromeric regions of some chromosomal pairs. This location, together with its presence in the transcriptomes of bufonids, could indicate a role in centromere function or heterochromatin formation and maintenance.

Taxonomy in Bufonidae witnessed notable transformations. Bufotes viridis and Epidalea calamita, previously included in genus Bufo, were relocated in other genera, while the genus Bufo was restricted to members of the earlier Bufo bufo group. On the other hand, Bufo bufo sensu lato now includes four species: Bufo bufo, Bufo spinosus, Bufo verrucosissimus and Bufo eichwaldi. In this study, we examined three species of three Bufonidae genera (B. spinosus, B. viridis and E. calamita) by conventional (C-banding and Ag-NOR staining) and molecular (in situ hybridization with probes for telomeric repeats and rDNA loci, and genomic in situ hybridization (GISH)) cytogenetic methods. C-banding patterns are reported for the first time for B. spinosus and E. calamita populations from Iberian Peninsula and for B. viridis from Greece, and reveal several differences with the reported C-banded karyotypes described for other European populations of these species. Silver staining shows size heteromorphisms of the signals at the Nucleolar Organizing Region (NOR). By contrast, FISH with ribosomal probes only reveal size heteromorphism of rDNA sequences in E. calamita, suggesting that the differences observed after silver staining in B. spinosus and B. viridis should be attributed to differences in chromosomal condensation and/or gene activity rather than to differences in the copy number for ribosomal genes. Regarding telomeric repeats, E. calamita is the only species with interstitial telomeric sequences (ITS) located on centromeric regions, probably originated by accumulation of telomeric sequences in the centromeric heterochromatin. Finally, we analyzed the composition and distribution of repetitive sequences by genome in situ hybridization. These experiments reveal the accumulation of repetitive sequences in centromeric regions of the three species, although these sequences are not conserved when species from different genera are compared.