A ZZ/ZW microchromosome system in the spiny softshell turtle, Apalone spinifera, reveals an intriguing sex chromosome conservation in Trionychidae

Abstract

Reptiles display a wide diversity of sex-determining mechanisms ranging from temperature-dependent sex determination (TSD) to genotypic sex determination (GSD) with either male (XY) or female (ZW) heterogamety. Despite this astounding variability, the origin, structure, and evolution of sex chromosomes remain poorly understood. In turtles, TSD is purportedly ancestral while GSD arose multiple times independently. Here we test whether independent (XY or ZW) or morphologically divergent heterogametic sex chromosome systems evolved in tryonichids (Cryptodira) using the GSD spiny softshell turtle, Apalone spinifera, a species with previously unidentified sex chromosomes. A female-specific signal from comparative genomic hybridization (CGH) was detected in a Giemsa/4',6-diamidino-2-phenylindole faint portion of a microchromosome, indicating the presence of a ZZ/ZW system in A. spinifera. In situ hybridization of a fluorescently labeled 18S rRNA probe identified a large nucleolar organizer region block in the female-specific region of the W (co-localizing with the female-specific CGH signal) and a smaller block on the Z. The heteromorphic ZZ/ZW micro-sex chromosome system detected here is identical to that found in another tryonichid, the Chinese softshell turtle Pelodiscus sinensis, from which A. spinifera diverged ∼95 million years ago. These results reveal a striking sex chromosome conservation in tryonichids, compared to the divergent sex chromosome morphology observed among younger XX/XY systems in pleurodiran turtles. Our findings highlight the need to understand the drivers behind sex chromosome lability and conservation in different lineages and contribute to our knowledge of sex chromosome evolution in reptiles and vertebrates.

Cretaceous park of sex determination: sex chromosomes are conserved across iguanas

“…This stability is comparable to the well-known situation in birds and mammals. Although the stability of sex chromosomes has been demonstrated or inferred for other poikilothermic amniotes [10][11][12], to the best of our knowledge, iguanas represent the case of the oldest sex chromosomes among amniotic poikilothermic vertebrates. The highly unequal rate of turnover of sexdetermining mechanisms among different lineages of lizards makes them a fascinating group for studies of the evolution of sex determination.…”

Molecular Cytogenetic Search for Cryptic Sex Chromosomes in Painted Turtles <b><i>Chrysemys picta</i></b>

“…We used a molecular cytogenetic approach to examine male and female C. picta to determine whether sex chromosomes are present in this species, in which case C. picta would be reclassified as a GSD + EE taxon. This molecular cytogenetic approach has been used by us and others to detect cryptic sex chromosomes in reptiles [Ezaz et al, 2005[Ezaz et al, , 2006Martinez et al, 2008;Kawai et al, 2009;Badenhorst et al, 2013]. Here, we discuss the implications of our findings with regards to our current understanding of the evolution of sex determination.…”

“…Thus, the possibility exists that a cryptic GSD mechanism is present in species previously classified as TSD, which, if true, would require a reclassification of this and potentially many other TSD species as GSD + EE [Valenzuela et al, 2003] and may cause a reevaluation of the evolution of the continuum of sex determination [Valenzuela et al, 2003;Sarre et al, 2004]. One possibility is that cryptic sex chromosomes exist in TSD species that are homomorphic and undetectable by classic cytogenetic techniques but amenable to identification using molecular cytogenetic as in other reptiles [Ezaz et al, 2005[Ezaz et al, , 2006Martinez et al, 2008;Kawai et al, 2009;Badenhorst et al, 2013].…”

Gonadal Differentiation in Reptiles Exhibiting Environmental Sex Determination

“…In reptiles, GSD is more or less inferred from extensive karyotyping as well as assessment of temperature effects on sex determination (or lack thereof). XX/XY, ZW/ZZ chromosome systems have been identified in many groups, although a few remain undefined; that is, homomorphic sex chromosomes or unconventional heteromorphic sex chromosomes could be present [Badenhorst et al, 2013]. Moreover, reptilian sex determination mechanisms are not well understood, and the exact genotype and loci that would prompt sex determination is yet to be elucidated.…”