Sex chromosomes have evolved many times in animals and studying these replicate evolutionary “experiments” can help broaden our understanding of the general forces driving the origin and evolution of sex chromosomes. However this plan of study has been hindered by the inability to identify the sex chromosome systems in the large number of species with cryptic, homomorphic sex chromosomes. Restriction site-associated DNA sequencing (RAD-seq) is a critical enabling technology that can identify the sex chromosome systems in many species where traditional cytogenetic methods have failed. Using newly generated RAD-seq data from 12 gecko species, along with data from the literature, we reinterpret the evolution of sex-determining systems in lizards and snakes and test the hypothesis that sex chromosomes can routinely act as evolutionary traps. We uncovered between 17 and 25 transitions among gecko sex-determining systems. This is approximately one-half to two-thirds of the total number of transitions observed among all lizards and snakes. We find support for the hypothesis that sex chromosome systems can readily become trap-like and show that adding even a small number of species from understudied clades can greatly enhance hypothesis testing in a model-based phylogenetic framework. RAD-seq will undoubtedly prove useful in evaluating other species for male or female heterogamety, particularly the majority of fish, amphibian, and reptile species that lack visibly heteromorphic sex chromosomes, and will significantly accelerate the pace of biological discovery.

Sex in reptiles is determined by genes on sex chromosomes or by incubation temperature. Previously these two modes were thought to be distinct, yet we show that high incubation temperatures reverse genotypic males (ZZ) to phenotypic females in a lizard with ZZ and ZW sex chromosomes. Thus, the W chromosome is not necessary for female differentiation. Sex determination is probably via a dosage-sensitive male-determining gene on the Z chromosome that is inactivated by extreme temperatures. Our data invite a novel hypothesis for the evolution of temperature-dependent sex determination (TSD) and suggest that sex chromosomes may exist in many TSD reptiles.

Significance: Genomes of birds and reptiles, but not mammals, consist of a few large chromosomes and many tiny microchromosomes. Microchromosomes are gene-rich and highly conserved among birds and reptiles and share homology with one or more of the tiny chromosomes of an invertebrate that diverged from the vertebrate lineage 684 Ma. Microchromosomes interact strongly and crowd together at the center of cells, suggesting functional coherence. Many microchromosomes have been lost independently in turtles, snakes, and lizards as they have fused with each other or with larger chromosomes. In mammals they have completely disappeared, yet some chromosomes of the basal platypus line up with several microchromosomes, suggesting that they are the building blocks of the atypically variable chromosomes of mammals.