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

Abstract

Sex determination is triggered by factors ranging from genotypic (GSD) to environmental (ESD), or both GSD + EE (GSD susceptible to environmental effects), and its evolution remains enigmatic. The presence/absence of sex chromosomes purportedly separates species at the ESD end of the continuum from the rest (GSD and GSD + EE) because the evolutionary dynamics of sex chromosomes and autosomes differ. However, studies suggest that turtles with temperature-dependent sex determination (TSD) are cryptically GSD and possess sex chromosomes. Here, we test this hypothesis in painted turtles Chrysemys picta (TSD), using comparative-genome-hybridization (CGH), a technique known to detect morphologically indistinguishable sex chromosomes in other turtles and reptiles. Our results show no evidence for the existence of sex chromosomes in painted turtles. While it remains plausible that cryptic sex chromosomes may exist in TSD turtles that are characterized by minor genetic differences that cannot be detected at the resolution of CGH, previous attempts have failed to identify sex-specific markers. Genomic sequencing should prove useful in providing conclusive evidence in this regard. If such efforts uncover sex chromosomes in TSD turtles, it may reveal the existence of a fundamental constraint for the evolution of a full spectrum of sex determination (from pure GSD to pure TSD) that is predicted theoretically. Finding sex chromosomes in ESD organisms would question whether pure ESD mechanisms exist at all in nature, or whether those systems currently considered pure ESD simply await the characterization of an underlying GSD architecture.

Temperature-Dependent Sex Determination Ruled Out in the Chinese Soft-Shelled Turtle <b><i>(Pelodiscus sinensis)</i></b> via Molecular Cytogenetics and Incubation Experiments across Populations

“…In addition to the incubation experiments, the 2 preceding studies were unable to detect the sex chromosome in this turtle, leading to the conclusion that it was a TSD species [Nie et al, 2001]. This conclusion was based on the fact that GSD species mainly have a male/female heterogamety genotype with heteromorphic sex chromosomes, while TSD species generally do not [Ezaz et al, 2009b;Valenzuela et al, 2014]. However, the sex chromosome of some GSD reptiles is extremely cryptic to the extent that it may only be detected using high-resolution cytogenetic techniques, such as CGH [Ezaz et al, 2005[Ezaz et al, , 2006Kawai et al, 2007;Martinez et al, 2008;Badenhorst et al, 2013].…”

“…Transitions from TSD to GSD and vice versa are possible along the continuum of sex determination, and some species possess intermediate mechanisms [Sarre et al, 2011;Valenzuela et al, 2003Valenzuela et al, , 2014, even though these types of sex determinations may be evolutionarily unstable [Bull, 1983]. Recent studies have shown the presence of such intermediate sex-determining systems in some reptiles.…”

Cytogenetic Insights into the Evolution of Chromosomes and Sex Determination Reveal Striking Homology of Turtle Sex Chromosomes to Amphibian Autosomes

“…Cell Culture, Chromosome Preparation, and Chromosome Banding Chromosome preparations were obtained from fibroblast cell cultures established from embryonic or muscle tissue following standard procedures as previously described [Valenzuela, 2009;Badenhorst et al, 2013Badenhorst et al, , 2015Valenzuela et al, 2014;Montiel et al, 2016]. New samples were obtained from private collections or pet trade specimens.…”

Ecotoxicological Perspectives of Sex Determination

“…In TSD systems that lack sex chromosomes [Valenzuela et al, 2014] no consistent genotypic difference distinguishes males and females in contrast to sex chromosomal systems. TSD is present in many vertebrates such as crocodilians, tuatara, most turtles, some lizards, and some fish [Valenzuela and Lance, 2004;Tree of Sex Consortium et al, 2014].…”

“…At the other extreme of the sex determination spectrum lies environmental sex determination (ESD) where the sexual fate is decided based on environmental factors experienced after fertilization [Bull, 1983;Valenzuela and Lance, 2004]. Temperature is the most common environmental factor that affects sex determination in vertebrates (TSD), where the incubation temperature at a time called the thermosensitive period commits the embryo to the male or female fate [Valenzuela and Lance, 2004;Janzen and Phillips, 2006;Bachtrog et al, 2014] in the absence of sex chromosomes [Valenzuela et al, 2014].…”