The origin and eventual loss of biogeographic barriers can create alternating periods of allopatry and secondary contact, facilitating gene flow among distinct metapopulations and generating reticulate evolutionary histories that are not adequately described by a bifurcating evolutionary tree. One such example may exist in the two-lined salamander (Eurycea bislineata) species complex, where discordance among morphological and molecular datasets has created a "vexing taxonomic challenge". Previous phylogeographic analyses of mitochondrial DNA (mtDNA) suggested that the reorganization of Miocene paleodrainages drove vicariance and dispersal, but the inherent limitations of a single-locus dataset precluded the evaluation of subsequent gene flow. Here, we generate triple-enzyme restriction site-associated DNA sequencing (3RAD) data for >100 individuals representing all major mtDNA lineages and use a suite of complementary methods to demonstrate that discordance among earlier datasets is best explained by a reticulate evolutionary history influenced by river drainage reorganization. Systematics of such groups should acknowledge these complex histories and relationships that are not strictly hierarchical.

Funding provided by: American Philosophical SocietyCrossref Funder Registry ID: https://ror.org/04egvf158Award Number: Funding provided by: American Museum of Natural HistoryCrossref Funder Registry ID: https://ror.org/03thb3e06Award Number: Funding provided by: Society of Systematic BiologistsCrossref Funder Registry ID: https://ror.org/0575b2v60Award Number: Funding provided by: University of Tennessee at KnoxvilleCrossref Funder Registry ID: https://ror.org/020f3ap87Award Number: Funding provided by: National Science FoundationCrossref Funder Registry ID: https://ror.org/021nxhr62Award Number: DGE-1452154