Although sex determination is ubiquitous in vertebrates, mechanisms of sex determination vary from environmentally to genetically influenced. Within vertebrates, sex determination is typically initiated by loci on sex chromosomes. Sex chromosome systems have evolved independently many dozens of times just within vertebrates, but most foundational sex chromosome research has focused on just a handful of vertebrate lineages, almost exclusively in mammals and birds. Sex chromosomes in mammals and birds have remained (mostly) intact across the entirety of their respective clades, but all other vertebrate groups (fishes, amphibians, and non-avian reptiles) regularly transition between sex chromosome systems (including both XX/XY systems, like mammals; and ZZ/ZW systems, like birds). Historical reliance on these clades has left large gaps in our understanding of sex chromosomes across eukaryotes.

Advances in sequencing technologies have opened the doors to resolve sex chromosomes in ‘non-model’ groups, allowing us to begin filling in these gaps. However, there remain both technical and biological challenges associated with assembly and annotation of sex chromosomes in these speciose vertebrate groups. To address these shortcomings, we turn to the charismatic infraorder of squamate reptiles, Anguimorpha (the clade containing the ‘legendary’ Komodo dragon and the ‘near-mythical’ Gila monster, among many others). To-date, two different ZZ/ZW systems have been identified within the group—one recently evolved and one hypothesized to be as ancient as those of either mammals or birds. We chronicle the evolutionary history of sex chromosomes in this clade by combining state-of-the-art sequencing technologies and developing new ways to better identify and assemble sex chromosomes in ‘non-model’ species. We then demonstrate their utility within anguimorphs by assembling the first, haplotype-resolved genome for—Arizona’s very own—ZW anguimorph the Gila monster to reveal their novel pattern of sex chromosome evolution relative to other amniote lineages.