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Polyploid speciation has played an important role in evolutionary history across the tree of life, yet there remain large gaps in our understanding of how polyploid species form and persist. Although systematic studies have been conducted in numerous polyploid complexes, recent advances in sequencing technology have demonstrated that conclusions from data-limited studies may be spurious and misleading. The North American gray treefrog complex, consisting of the diploid Hyla chrysoscelis and the tetraploid H. versicolor, has long been used as a model system in a variety of biological fields, yet all taxonomic studies to date were conducted with only a few loci from nuclear and mitochondrial genomes. Here, we utilized anchored hybrid enrichment and high-throughput sequencing to capture hundreds of loci along with whole mitochondrial genomes to investigate the evolutionary history of this complex. We used several phylogenetic and population genetic methods, including coalescent simulations and testing of polyploid speciation models with approximate Bayesian computation, to determine that H. versicolor was most likely formed via autopolyploidization from a now extinct lineage of H. chrysoscelis. We also uncovered evidence of significant hybridization between diploids and tetraploids where they co-occur, and show that historical hybridization between these groups led to the re-formation of distinct polyploid lineages following the initial whole-genome duplication event. Our study indicates that a wide variety of methods and explicit model testing of polyploid histories can greatly facilitate efforts to uncover the evolutionary history of polyploid complexes.

The Complex History of Genome Duplication and Hybridization in North American Gray Treefrogs