A reference-based taxonomy reinforces the evolutionary relationships of previously problematic earless lizards (Phyrnosomatidae: Holbrookia)

In the face of anthropogenic change and an urgency to document biodiversity, accurate species delimitation is of tantamount importance. Genome-wide data are powerful for investigating lineage divergence, though whether this reflects species-level differentiation remains a challenge. Here, we develop a reference-based taxonomy for a group of Earless Lizards (Phrynosomatidae: Holbrookia), with a focus on H. lacerata and H. subcaudalis, the latter having potentially imperiled populations. These species have had a taxonomically troubling history, and only recently were H. lacerata and H. subcaudalis elevated to species status using highly conserved morphological data and a few molecular markers. In this study, we used double-digest restriction-site associated DNA sequencing to estimate genetic divergence among species within the genus Holbrookia and to develop a reference-based taxonomy for our focal species. We recovered five populations that corresponded to five well-supported lineages with very little gene flow among them. Our results support the recognition of H. lacerata and H. subcaudalis as two separate species, based on strong phylogenetic support for these lineages and genetic divergence measures that exceed those of currently recognized species within Holbrookia. Reference-based methods for species delimitation offer a promising approach to assess biodiversity in taxonomically confounded taxa or organisms of conservation priority.