The Florida Peninsula as an Engine of Estuarine Evolution: Insights From <i>Gammarus daiberi</i> Bousfield, 1969 (Crustacea: Amphipoda)

ABSTRACT Aim This study investigated how Miocene‐aged vicariance and coastal landscape change have driven estuarine diversification in the southeastern United States. By examining newly documented Gulf Coast populations of Gammarus nr. daiberi from the Wakulla River, Florida, and a new Gammarus sp. from Loving Creek, Louisiana, we resolved their phylogenetic relationships within the G. daiberi – G. tigrinus complex and assessed whether they represent distinct evolutionary lineages. We specifically tested the hypothesis that geological reconfiguration of the Florida peninsula and adjacent coastal plain generated the observed Gulf–Atlantic lineage split. Location Atlantic and Gulf Coasts of the United States of America. Taxon Gammarus daiberi , G. tigrinus . Methods Live amphipods were collected, morphologically identified and preserved in 100% molecular‐grade ethanol. DNA was extracted, and four loci (mitochondrial 16S rRNA, COI; nuclear 18S rRNA, 28S rRNA) were PCR‐amplified and Sanger‐sequenced. Alignments were screened for quality and contamination. Phylogenetic trees were reconstructed from concatenated datasets using maximum likelihood (IQ‐TREE2) and Bayesian inference (MrBayes), with partitioned substitution models. Divergence times were estimated in BEAST under relaxed molecular clocks with four calibration points. Species delimitation employed BPP and STACEY, while ancestral ranges were inferred in RASP. Results The Wakulla River and Loving Creek populations form well‐supported, genetically distinct lineages, separate from Atlantic G. daiberi – G. tigrinus clades. Divergence time estimates indicate a Miocene split approximately 5.7 Ma, coinciding with geological changes in the Florida peninsula. Species delimitation models confirm both Gulf Coast populations as undescribed cryptic taxa. Main Conclusions Genetic, phylogenetic and biogeographic evidence support a Miocene vicariance scenario underlying Gulf–Atlantic lineage divergence in Gammarus daiberi sensu lato . The southeastern coastal plain emerges as a hotspot for diversification of coastal and estuarine taxa, shaped by paleogeographic processes, and highlights the need for expanded sampling to reveal hidden estuarine biodiversity.