With or Without W? Molecular and Cytogenetic Markers are Not Sufficient for Identification of Environmentally-Induced Sex Reversal in the Bearded Dragon

Abstract

Transitions from environmental sex determination (ESD) to genotypic sex determination (GSD) require an intermediate step of sex reversal, i.e., the production of individuals with a mismatch between the ancestral genotypic and the phenotypic sex. Among amniotes, the sole well-documented transition in this direction was shown in the laboratory in the central bearded dragon, Pogona vitticeps, where very high incubation temperatures led to the production of females with the male-typical (ZZ) genotype. These sex-reversed females then produced offspring whose sex depended on the incubation temperature. Sex-reversed animals identified by molecular and cytogenetic markers were also reported in the field, and their increasing incidence was speculated as a climate warming-driven transition in sex determination. We show that the molecular and cytogenetic markers normally sex-linked in P. vitticeps are also sex-linked in P. henrylawsoni and P. minor, which points to quite ancient sex chromosomes in this lineage. Nevertheless, we demonstrate, based on a crossing experiment with a male bearded dragon who possesses a mismatch between phenotypic sex and genotype, that the used cytogenetic and molecular markers might not be reliable for the identification of sex reversal. Sex reversal should not be considered as the only mechanism causing a mismatch between genetic sex-linked markers and phenotypic sex, which can emerge also by other processes, here most likely by a rare recombination between regions of sex chromosomes which are normally sex-linked. We warn that sex-linked, even apparently for a long evolutionary time, and sex-specific molecular and cytogenetic markers are not a reliable tool for the identification of sex-reversed individuals in a population and that sex reversal has to be verified by other approaches, particularly by observation of the sex ratio of the progeny.

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

“…Genes can also subsequently re-emerge in the degenerated parts of W and Y. In the latter case, a gene initially lost in the non-recombining region of Y and W can re-appear there owing to a recombination or translocation between sex chromosomes in otherwise non-recombining regions or by gene conversion [107,147,148].…”

“…many times in placental mammals, iguanas and teleosts [143][144][145]) with an autosome, producing multiple neo-sex chromosomes (case 19 in figure 2), where the newly added parts behave as pseudoautosomal regions and can go through subsequent differentiation [133]. Notably, the size of Y and W can be expanded by the accumulation of repetitive sequences [102] (case 11 in figure 2), and repeats from these degenerated chromosomes can 'contaminate' and be amplified on X/Z as well [146,147] (case 12 in figure 2). Genes can also subsequently re-emerge in the degenerated parts of W and Y.…”

Concerning an Article by Ehl et al.: False Premise Leads to False Conclusions

“…We therefore completely reject the claims of Ehl et al [2021] that the sex tests used in our sex reversal work on P. vitticeps were in any way inadequate.…”

“…In their recent paper, entitled "With or without W? Molecular and cytogenetic markers are not sufficient for identification of environmentally-induced sex reversal in the bearded dragon", Ehl et al [2021] demonstrate that a published PCR sex test developed for the bearded dragon (F4-F1 marker) [Quinn et al, 2010] is only partially diagnostic for sex because the sex-linked sequence upon which the test is based is subject to recombination with sex and therefore inappropriate for studies of sex reversal. The authors use the deficiencies of this now superseded PCR F1-F4 sex test developed by Quinn et al [2010] to call into question the more recent and robust sex reversal work of Holleley et al [2015] and subsequent work on this system in Pogona vitticeps [Castelli et al, 2021].…”

“…This criticism is completely misplaced, because none of the sex reversal work criticised by Ehl et al [2021] used the original Quinn F4-F1 PCR sex test.…”