Secrets of the Spring‐Keepers: Phylogeographic Analyses of a Freshwater Amphipod Uncover Dispersal Pathways in Eastern North America

ABSTRACT Aim While the Nearctic freshwater amphipod Gammarus minus is known for its adaptation to groundwater environments, relatively little is known about other aspects of the evolutionary history of the species throughout its range, which spans much of the eastern United States. As a result, we investigated the phylogeography of the species. Location Eastern United States. Taxon Gammarus minus . Methods Sequence data for two nuclear and two mitochondrial loci were generated for 13 populations of G. minus . These data were combined with sequences from 15 populations obtained from GenBank. From this partitioned dataset, phylogenetic trees were reconstructed using both maximum‐likelihood (IQTREE) and Bayesian Inference (MrBayes/BEAST), and haplotype networks were generated using POPART. BEAST trees were time‐calibrated based on a set of four mixed calibration points. RASP software was used to investigate ancestral areas. Ecological niche models were generated in R using the dismo/ENMeval packages. Results We identified the presence of three clades within G. minus : (I) a “Susquehanna‐Potomac” clade containing populations from central Pennsylvania and northern West Virginia; (II) an “Ozark” clade containing populations from Missouri/Illinois; and (III) a widely distributed “Circum‐Appalachian” clade containing populations from the Appalachians, Interior Plateau, and Piedmont. Time‐calibrated phylogenies place the origin of the G. minus lineage during the Miocene, with clades diverging in the late Miocene to early Pliocene. Phylogenetic reconstructions recovered populations from the Susquehanna‐Potomac clade as basally derived, with other clades likely dispersing southward and westward to reach the Ozarks and Appalachians. Main Conclusions Diversification within G. minus likely was promoted by climatic changes and uplift of the Appalachians during the Miocene. These results suggest a novel model for how drainage reorganizations, climate shifts, and geological processes in eastern North America shaped lineage diversification in freshwater taxa. More broadly, this study demonstrates the value of phylogeographic analyses in revealing hidden patterns of biotic assembly and dispersal across complex landscapes.