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

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 similar to 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.