Species delineation using Bayesian model-based assignment tests: a case study using Chinese toad-headed agamas (genus Phrynocephalus)

Background: Species are fundamental units in biology, yet much debate exists surrounding how we should delineate species in nature. Species discovery now requires the use of separate, corroborating datasets to quantify independently evolving lineages and test species criteria. However, the complexity of the speciation process has ushered in a need to infuse studies with new tools capable of aiding in species delineation. We suggest that model-based assignment tests are one such tool. This method circumvents constraints with traditional population genetic analyses and provides a novel means of describing cryptic and complex diversity in natural systems. Using toad-headed agamas of the Phrynocephalus vlangalii complex as a case study, we apply model-based assignment tests to microsatellite DNA data to test whether P. putjatia, a controversial species that closely resembles P. vlangalii morphologically, represents a valid species. Mitochondrial DNA and geographic data are also included to corroborate the assignment test results. Results: Assignment tests revealed two distinct nuclear DNA clusters with 95% (230/243) of the individuals being assigned to one of the clusters with > 90% probability. The nuclear genomes of the two clusters remained distinct in sympatry, particularly at three syntopic sites, suggesting the existence of reproductive isolation between the identified clusters. In addition, a mitochondrial ND2 gene tree revealed two deeply diverged clades, which were largely congruent with the two nuclear DNA clusters, with a few exceptions. Historical mitochondrial introgression events between the two groups might explain the disagreement between the mitochondrial and nuclear DNA data. The nuclear DNA clusters and mitochondrial clades corresponded nicely to the hypothesized distributions of P. vlangalii and P. putjatia. Conclusions: These results demonstrate that assignment tests based on microsatellite DNA data can be powerful tools for distinguishing closely related species and support the validity of P. putjatia. Assignment tests have the potential to play a significant role in elucidating biodiversity in the era of DNA data. Nonetheless, important limitations do exist and multiple independent datasets should be used to corroborate results from assignment programs.