Inferring introgression using RADseq and D<inf>FOIL</inf>: Power and pitfalls revealed in a case study of spiny lizards (Sceloporus)

Introgression is now commonly reported in studies across the Tree of Life, aided by recent advancements in data collection and analysis. Nevertheless, researchers working with nonmodel species lacking reference genomes may be stymied by a mismatch between available resources and methodological demands. In this study, we demonstrate a fast and simple approach for inferring introgression using RADseq data, and apply it to a case study involving spiny lizards (Sceloporus) from northeastern México. First, we find evidence for recurrent mtDNA introgression between the two focal species based on patterns of mito-nuclear discordance. We then test for nuclear introgression by exhaustively applying the “five-taxon” D-statistic (D_FOIL) to all relevant individuals sampled for RADseq data. In our case, this exhaustive approach (dubbed “ExD_FOIL”) entails testing up to ~250,000 unique four-taxon combinations of individuals across species. To facilitate use of this ExD_FOIL approach, we provide scripts for many relevant tasks, including the selection of appropriate four-taxon combinations, execution of D_FOILtests in parallel and visualization of introgression results in phylogenetic and geographic space. Using ExD_FOIL, we find evidence for ancient introgression between the focal species. Furthermore, we reveal geographic variation in patterns of introgression that is consistent with patterns of mito-nuclear discordance and with recurrent introgression. Overall, our study demonstrates that the combination of D_FOIL and RADseq data can effectively detect introgression under a variety of sampling conditions (for individuals, populations and loci). Importantly, we also find evidence that batch-specific error and linkage in RADseq data may mislead inferences of introgression under certain conditions.