In the viviparous multi-ocellated racerunner lizard Eremias multiocellata, maintaining gravid females at constant temperatures skews offspring sex ratios — female-biased near 25 °C and male-biased near 35 °C — a pattern reported in 2010 and replicated in a high-altitude population in 2012, and originally interpreted as possible temperature-dependent sex determination (TSD). Cytogenetic and genomic work since 2015 has shown that this species in fact carries highly differentiated, range-conserved ZZ/ZW sex chromosomes, so true TSD is no longer tenable; yet the sex-ratio bias itself remains unexplained. The open problem is to identify the mechanism: differential mortality of female (ZW) embryos at temperature extremes, temperature-induced sex reversal overriding genetic sex determination (genotype–phenotype discordance such as ZW males), or an artifact of the morphological sexing method — measuring the hemipenes (the paired copulatory organs of male squamate reptiles), whose size was never validated against genetic sex. Resolution requires gestation-temperature experiments whose offspring and non-viable embryos are genotyped with the now-available W-linked markers, together with field tests of whether maternal thermoregulation can shift offspring sex ratios in natural populations.

Background

Reptiles determine sex through two broad mechanisms: genotypic sex determination (GSD), in which sex chromosomes decide gonad fate — with either male heterogamety (XY males, XX females) or female heterogamety (ZW females, ZZ males) — and temperature-dependent sex determination (TSD), in which the incubation temperature of the egg decides sex in the absence of differentiated sex chromosomes. Viviparity — giving birth to live young after the embryo develops inside the mother, so that embryos experience the mother's body temperature rather than nest temperature — has long been considered difficult to reconcile with TSD, because a mother's relatively stable body temperature offers no thermal cue comparable to a nest. The multi-ocellated racerunner Eremias multiocellata is a small viviparous lacertid lizard of the deserts and steppes of China and Mongolia. Zhang De-Jiu and colleagues maintained gravid females from a low-altitude (about 1400 m) population at constant temperatures during pregnancy and reported in 2010 that warmer gestation temperatures shortened gestation, increased mean litter size, and produced a higher male:female birth ratio, while neonate body mass and snout–vent length (SVL, the standard body-length measure from snout to vent) were unaffected; they concluded the species "might be a TSD species, providing the mothers the opportunity to manipulate the sex ratio of their offspring." A 2012 follow-up on a high-altitude (about 2900 m) population replicated the pattern in weaker form — female-biased litters at 25 °C, male-biased at 35 °C, and near-equal ratios at moderate temperatures and in field-gestating females. In both studies offspring were sexed morphologically, by the size of the hemipenes (the paired copulatory organs of male squamate reptiles, everted or examined in section), a method that was never validated against genetic sex. Three later lines of evidence overturned the TSD interpretation while leaving the bias unexplained. First, comparative genomic hybridization (CGH, a cytogenetic technique that differentially paints chromosome regions by hybridizing differently labeled male and female DNA onto metaphase chromosomes) identified highly differentiated ZZ/ZW sex chromosomes in E. multiocellata, with the differentiated W chromosome present in every female and no male examined. Second, a comparative study of 45 lacertid species showed that all share homologous differentiated ZZ/ZW sex chromosomes inherited from a common ancestor roughly 85 million years ago, and rejected the proposed evolutionary transition to environmental sex determination in this family; it also explicitly criticized the Eremias gestation experiments for never excluding differential embryo mortality between the sexes and for the unvalidated hemipene-based sexing. Third, genotyping-by-sequencing (GBS, a reduced-representation shotgun method that discovers sex-linked DNA markers by their presence in one sex only) yielded 38 W-linked markers for E. multiocellata, eight of them validated by PCR across 15 populations spanning the species' Chinese range without geographic variation, and mapping to the W chromosome of the sand lizard Lacerta agilis. The same 2023 study states that the gestation-temperature experiments "lacked two critical steps — they did not exclude differential mortality between the sexes at a particular temperature and did not consider temperature-mediated sexual reversal," concluding that in this species temperature can interfere with, but not determine, sex ratios. Adjacent viviparous lizards sharpen rather than settle the question: the Tasmanian spotted snow skink Niveoscincus ocellatus, the only reptile known to be polymorphic for sex-determination mode (genetic in highland populations, temperature-influenced sex ratios in lowland ones), retains conserved XY sex-linked markers in both; the Australian water skinks Eulamprus heatwolei and E. tympanum, viviparous and long regarded as TSD, turn out to carry old (79–116 million years) XY chromosomes; and sex reversal demonstrated in the wild in the oviparous bearded dragon Pogona vitticeps proves that temperature can override GSD in reptiles and can even trigger a rapid transition from genetic to temperature-dependent sex determination. Together these results mean every viviparous lizard whose sex ratio responds to gestation temperature nonetheless harbors sex chromosomes, and for none of them — including E. multiocellata — is the mechanism of the thermal sex-ratio bias resolved. In a ZW system, a male-biased brood at high temperature could arise from death of ZW (genetically female) embryos, from genetic females developing as males (sex reversal), or merely from heat-masculinized hemipenes mis-scored as male; distinguishing these requires genotyping every offspring and non-viable embryo from temperature-manipulated pregnancies with the validated W-linked markers.

Problem Statement

Determine the mechanism by which gestation temperature biases offspring sex ratios in the viviparous lizard Eremias multiocellata, whose ZZ/ZW genetic sex determination is now established. Concretely: (1) maintain gravid females across the experimental temperature range (approximately 25–35 °C) established by the source studies, and for every offspring and every non-viable embryo determine both genotype (using the validated W-linked genetic markers) and phenotypic sex by an independent gonadal readout (gonadal histology or a gonad-expressed molecular sex marker), recording the hemipene-based morphological sexing alongside it, each applied blind to genotype, so as to distinguish and decide among the three candidate explanations — (a) differential mortality of genetically female (ZW) embryos at temperature extremes, (b) temperature-induced sex reversal overriding ZZ/ZW genetic sex determination, detectable as ZW individuals whose gonadal phenotype is discordant (male-typed) and varies with gestation temperature, and (c) a sexing artifact arising from temperature-plastic hemipene development, in which gonadal sex matches genotype and the genotypic sex ratio and genotype-specific mortality are unchanged but the hemipene score alone misclassifies individuals; and (2) establish, by combined thermal monitoring of free-living gravid females and genotypic sexing of their neonates across natural habitats, whether maternal behavioral thermoregulation shifts offspring sex ratios in the field. Completion is a mechanistic account — or a genotyping-powered demonstration that no real bias exists — sufficient to decide whether and how gestation temperature can override or merely accompany genetic sex determination in this viviparous species. Both components (1) and (2) are required for completion.

References

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