Abstract The nature of speciation within subterranean ecosystems following invasions from the surface remains poorly understood. Most proposed examples of in situ subterranean speciation instead appear to reflect multiple independent surface invasions, supporting the classic hypothesis that subterranean ecosystems are evolutionary dead ends. Here, we examine the species diversity within the most widespread subterranean vertebrate species, the Southern Cavefish Typhlichthys subterraneus. Phylogenomic analyses reveal that T. subterraneus as currently recognized is paraphyletic with respect to the Missouri Cavefish T. eigenmanni, as a distinct set of populations is resolved as the sister lineage of a clade formed by T. eigenmanni and T. subterraneus sensu stricto. High-resolution computed tomography (CT) scanning reveals skeletal autapomorphies of this lineage, supporting its recognition as a new species: Typhlichthys styx sp. nov. Ancestral biogeographic reconstructions reveal that speciation in Typhlichthys occurred along aquifer boundaries, with lineages dispersing through widespread karstic aquifer systems across southeastern and central North America. This dispersal facilitated secondary sympatry among cavefish species that last shared common ancestry approximately eight million years ago. Together, these results reveal aquifer geology as a driver of allopatric speciation in obligate cave-dwelling vertebrates, with implications for understanding biodiversity in subterranean ecosystems worldwide.
Brownstein et al. (2026) studied this question.