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.

Keywords: Molecular markers; Reversal; Sex chromosomes; Sex linkage; Vertebrates.

Fig. 1

Visualization of the accumulation of AAGG repeats and heterochromatin in selected individuals of P. vitticeps (PVI; A–K), P. henrylawsoni (PHE; M–P), and P. minor (PMI; Q–T). In P. vitticeps, the figure represents successively a standard male ZZ karyotype with no accumulations (A, B), standard female ZW karyotype with AAGG (C) and heterochromatin accumulation (D), karyotype of a ZW^m male with AAGG accumulation (E) but without heterochromatinization (F), karyotype of a sex-reversed ZZ female with no accumulations (G, H), karyotype of a ZW^m male with no accumulations (I, J), and karyotype of a WW^m female with 2 AAGG accumulations (K) and unpaired heterochromatic block (L). In P. henrylawsoni and P. minor, male karyotypes with no accumulations (M, N, Q, R) and female karyotypes with AAGG (O, S) and heterochromatin accumulations (P, T) are shown. W^m in the caption in males reflects the presence of the W-specific marker in PCR, the assignment in females is based on cytogenetics. Boxes in K show W and W^m chromosomes in separated blue channel mode to present their size difference. Scale bars, 10 μm.

Fig. 2

Results of PCR test with primers for the W-specific fragment in P. vitticeps (PVI), P. henrylawsoni (PHE), and P. minor (PMI) visualized by agarose gel electrophoresis. Standard females (F) of PVI, PHE, and PMI display the W-specific fragment (224 bp), whereas in standard males (M) this fragment is not amplified. PVI individuals 1–10 include: 1) ZW^m male; 2) sex-reversed ZZ female; 3, 4) male offspring of sex-reversed females and ZW^m male; 5) offspring of unknown sex from the same cross; 6) standard female used in the ZW × ZW^m cross, i.e., the mother of the offspring 7–10 depicted here; 7, 8) male offspring of standard female and ZW^m male; and 9, 10) female offspring of standard female and ZW^m male.

Fig. 3

Sequences of the W-specific fragment with the male and female reference. P. vitticeps clone C1 sex chromosome anonymous locus genomic sequence (GenBank sequence ID: EU938138.1, compared using BLASTN search), primer F1 used for the amplification of the W-specific fragment, male and female reference sequences differing in 4 SNPs from Quinn et al. [[2010]], and 4 individuals used in the study identical with the female reference sequence are shown. These individuals are a standard ZW female and a male possessing the W-specific marker and the AAGG accumulation involved in ♀ZW × ♂ZW^m cross and their offspring, the male possessing the W-specific marker and the AAGG accumulation. The last depicted sequence represents the male possessing the W-specific marker but without the AAGG accumulation originating from the cross between female sex reversal and the male with the W-specific marker (♀ZZ × ♂ZW^m).